Anerosomes for delivering intracellular therapeutic modalities

Abstract

To provide anellosomes for delivering intracellular therapeutic modalities.SOLUTION: The present invention relates generally to anellosomes and compositions and uses thereof. The present disclosure provides an anellosome, e.g., a synthetic anellosome, that can be used as a delivery vehicle, e.g., for delivering genetic material, for delivering an effector, e.g., a payload, or for delivering a therapeutic agent or a therapeutic effector (e.g., an intracellular therapeutic, e.g., an intracellular polypeptide or an intracellular nucleic acid) to a eukaryotic cell (e.g., a human cell or a human tissue).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 778,851 and U.S. Provisional Patent Application No. 62 / 778,866, both filed on December 12, 2018. The contents of the aforementioned applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted as electronic data in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 9 December 2019, titled V2057-7001WO_SL.txt, and its size is 825,829 bytes. [Background technology]

[0003] There remains a need to develop suitable vectors for delivering therapeutic genetic material to patients. [Overview of the project] [Means for solving the problem]

[0004] This disclosure provides anelosomes, such as synthetic anelosomes, that can be used as delivery vehicles to deliver genetic material to eukaryotic cells (e.g., human cells or human tissues), effectors, such as for delivering payloads, or therapeutic drugs or therapeutic effectors (e.g., intracellular therapeutic drugs, such as intracellular polypeptides or intracellular nucleic acids). In some embodiments, the anelosome (e.g., particles, such as viral particles, such as anelovirus particles) comprises a gene element (e.g., a gene element containing therapeutic DNA) encapsulated in a proteinaceous outer layer (e.g., a proteinaceous outer layer containing an anelovirus capsid protein, such as a polypeptide encoded by, for example, an anelovirus ORF1 protein or an anelovirus ORF1 nucleic acid as described herein), which can introduce the gene element into cells (e.g., mammalian cells, such as human cells). In some embodiments, the anestrosome is a particle comprising a proteinous outer layer containing a polypeptide encoded by an anelovirus ORF1 nucleic acid (for example, the ORF1 nucleic acid of an alphatorquevirus, betatorquevirus, or gammatorquevirus, for example, as described herein, for example, the ORF1 nucleic acid of an alphatorquevirus clade 1, an alphatorquevirus clade 2, an alphatorquevirus clade 3, an alphatorquevirus clade 4, an alphatorquevirus clade 5, an alphatorquevirus clade 6, or an alphatorquevirus clade 7).The gene elements of the anestroma of this 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 a protein-containing outer layer, or a polypeptide bound thereto, which can facilitate the containment of the gene element within the protein-containing outer layer and / or the enrichment of the gene element against other nucleic acids within the protein-containing outer layer. In some cases, the gene element is circular or linear. In some cases, the gene element includes or encodes an effector that can be expressed intracellularly (e.g., a nucleic acid effector such as non-coding RNA, or a polypeptide effector, e.g., a protein). In some embodiments, the effector is a therapeutic or therapeutic effector (e.g., an intracellular therapeutic agent, e.g., an intracellular polypeptide or intracellular nucleic acid), as described herein. In some cases, the effector is an endogenous or extrinsic effector against, for example, wild-type anellovirus or target cells. In some embodiments, the effector is extrinsic to wild-type anellovirus or target cells. In some embodiments, an anestronomy can deliver the effector into cells by introducing a gene element encoding the effector into the cells in contact with the cells so that the effector is produced or expressed by the cells. In certain cases, the effector is endogenous (e.g., endogenous to target cells but supplied in increased quantities, e.g., by an anestronomy). In other cases, the effector is extrinsic. In some embodiments, the effector can modulate cellular function or the activity or level of target molecules in cells. For example, the effector can reduce the level of a target protein in cells (e.g., as described in Examples 3 and 4). In other examples, an anestronomy can deliver and express the effector, e.g., an extrinsic protein, in vivo (e.g., as described in Examples 19 and 28).Anerosomes can be used, for example, to deliver genetic material to target cells, tissues, or subjects; to deliver effectors to target cells, tissues, or subjects; or for example, to deliver effectors that can act as therapeutic agents to desired cells, tissues, or subjects, for the treatment of diseases or disorders.

[0005] The present invention further provides synthetic anelosomes. Synthetic anelosomes have at least one structural difference, e.g., deletion, insertion, substitution, or modification (e.g., enzymatic modification), compared to wild-type viruses (e.g., wild-type anelovirus, as described herein). Generally, synthetic anelosomes comprise an exogenous gene element confined within a proteinaceous outer layer, which can be used to deliver the gene element, or the effector encoded therein (e.g., an exogenous or endogenous effector) (e.g., a polypeptide or nucleic acid effector) into a eukaryotic (e.g., human) cell. In several embodiments, anestrom does not induce a detectable and / or unwanted immune or inflammatory response, for example, not causing an increase of more than 1%, 5%, 10%, or 15% in molecular markers of inflammation, such as TNF-α, IL-6, IL-12, IFN, and B-cell responses, such as reactive or neutralizing antibodies. For example, anestrom may be substantially non-immunogenic to target cells, tissues, or subjects.

[0006] In one embodiment, the present invention is characterized by an anestroke comprising (a) a proteinaceous outer layer; (b) a gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector, and a protein-binding sequence (e.g., an external protein-binding sequence); wherein the external effector comprises: (i) an intracellular polypeptide other than nano-luciferase, or (ii) an intracellular nucleic acid (e.g., miRNA or siRNA) other than the external miRNA of a wild-type anelovirus (Anellovirus) as described herein, such as miR-124, miR-518, miR-625, miRNA for n-myc interacting proteins, or TTV-tth8 anelovirus (Anellovirus); wherein the gene element is confined within the proteinaceous outer layer; and the anestroke is configured to deliver the gene element to a eukaryotic cell. Optionally, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to wild-type anellovirus (e.g., as described herein), such as an insertion, substitution, modification (e.g., enzymatic modification), and / or deletion, such as a deletion of a specific domain or portion thereof (e.g., one or more of the following: TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0007] In one embodiment, the present invention is characterized by: (i) a gene element comprising a promoter element, a sequence encoding an effector (e.g., an endogenous or extrinsic effector), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal); and (ii) a synthetic anestronomy comprising a proteinaceous outer layer, wherein the gene element is confined within the proteinaceous outer layer (e.g., a capsid); and the anestronomy is capable of delivering the gene element into a eukaryotic (e.g., mammalian, e.g., human) cell. In some embodiments, the gene element is single-stranded and / or circular DNA. Alternatively, or in combination thereof, the gene element has one, two, three, or all of the following characteristics: it is circular, it is single-stranded, it is incorporated into the cell's genome at a frequency of less than approximately 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements that enter the cell, and / or it is incorporated into the target cell's genome at a frequency of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome. In some embodiments, the incorporation frequency is determined as described in Wang et al. (2004, Gene Therapy 11:711-721, which is incorporated herein by reference in its entirety). In some embodiments, the gene element is confined within a proteinaceous outer layer. In some embodiments, an anerosome can deliver the gene element into a eukaryotic cell.In some embodiments, the gene element is a sequence of wild-type anellovirus (e.g., wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTV)). The nucleic acid sequence (for example, a nucleic acid sequence of 300-4000 nucleotides, for example, a nucleic acid sequence of 300-4000 nucleotides, for example, a nucleic acid sequence of 300-3500 nucleotides, 300-3000 nucleotides, 300-2500 nucleotides, 300-2000 nucleotides, or 300-1500 nucleotides) has at least 75% sequence identity (for example, a nucleic acid sequence of 300-4000 nucleotides, for example, a nucleic acid sequence of 300-3500 nucleotides, 300-3000 nucleotides, 300-2500 nucleotides, 300-2000 nucleotides, or 300-1500 nucleotides) with respect to the (TTMV) or TTMDV sequence, for example, the wild-type anellovirus (ANE) sequence listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17). In some embodiments, the gene element includes a nucleic acid sequence (for example, a nucleic acid sequence of at least 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 2500 nucleotides, or 3000 nucleotides or more) having at least 75% sequence identity (for example, at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) with wild-type anellovirus sequence (for example, wild-type anellovirus sequences listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17 as described herein). In some embodiments, the nucleic acid sequence is codon-optimized for expression in mammalian (e.g., human) cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the nucleic acid sequence are codon-optimized for expression in, for example, mammalian (e.g., human) cells.

[0008] In one embodiment, the present invention features infectious (to human cells) particles containing an anellovirus capsid (e.g., an anellovirus ORF, e.g., a capsid containing ORF1 polypeptide), the capsid enveloping a genetic element comprising a protein-binding sequence that binds to the capsid and a heterologous (to anellovirus) sequence that encodes a therapeutic effector. In some embodiments, the particles can deliver the genetic element into mammalian cells, e.g., human cells. In some embodiments, the genetic element has less than about 6% identity (e.g., less than 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%) relative to wild-type anellovirus. In some embodiments, the gene element has identity of 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6% or less with respect to wild-type anellovirus. In some embodiments, the gene element has identity of at least about 2% to at least about 5.5% (e.g., 2-5%, 3-5%, 4-5%) with respect to wild-type anellovirus. In some embodiments, the gene element has about 2000, 3000, 4000, 4500, or more than 5000 nucleotides of a non-viral sequence (e.g., a non-anelovirus (anellovirus) genome sequence). In some embodiments, the gene element has more than 2000–5000, 2500–4500, 3000–4500, 2500–4500, 3500, or 4000, 4500 (e.g., more than 3000–4500) nucleotides of a non-viral sequence (e.g., a non-anelovirus (anellovirus) genome sequence). In some embodiments, the gene element is single-stranded, circular DNA.Alternatively, or in combination thereof, the gene element has one, two, or three of the following characteristics: it is circular, it is single-stranded, it is incorporated into the cell's genome at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements that enter the cell, it is incorporated into the target cell's genome at a frequency of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome, or it is incorporated at a frequency of less than 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements that enter the cell. In some embodiments, the incorporation frequency is determined as described in Wang et al. (2004, Gene Therapy 11:711-721, which is incorporated herein by reference in its entirety).

[0009] This specification also describes viral vectors and viral particles based on anellovirus, which can be used to deliver drugs (e.g., external or endogenous effectors, e.g., therapeutic effectors) to cells (e.g., cells of a subject to be treated for therapeutic or prophylactic purposes). In some embodiments, anellovirus can be used as an effective delivery vehicle for introducing drugs, such as the effectors described herein, into target cells, e.g., target cells of a subject to be treated for therapeutic or prophylactic purposes.

[0010] In one embodiment, the present invention is characterized by a polypeptide (e.g., a synthetic polypeptide, e.g., ORF1) comprising the following (e.g., sequentially): (i) Arginine-rich region, for example, an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with the arginine-rich region sequence described herein, or a first region comprising a sequence of at least about 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof), (ii) A jelly-roll domain, for example, 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 with the jelly-roll region sequence described herein, or a sequence comprising at least six β-chains. (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 with the 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 with the Anellovirus ORF1 C-terminal domain (CTD) sequence described herein, and (v) Optionally, the polypeptides include amino acid sequences having 100%, 99%, 98%, 95%, 90%, 85%, or less than 80% sequence identity with the wild-type anellovirus ORF1 protein described herein.

[0011] In some embodiments, the polypeptide has at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100% sequence identity with respect to an anelovirus ORF1 molecule as described herein (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). In some embodiments, the sequence identity includes at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100% with respect to a partial sequence of an anelovirus ORF1 molecule described herein (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) (e.g., arginine (Arg) rich domain, jellyroll domain, hypervariable region (HVR), N22 domain, or C-terminal domain (CTD)). In one embodiment, the amino acid sequences of regions (i), (ii), (iii), and (iv) have at least about 90% sequence identity with respect to their respective reference sequences, and the polypeptide comprises amino acid sequences having 100%, 99%, 98%, 95%, 90%, 85%, and less than 80% sequence identity with respect to the wild-type anellovirus ORF1 protein described herein.

[0012] In one embodiment, the present invention is characterized by a complex comprising a polypeptide as described herein (e.g., the Anellovirus ORF1 molecule as described herein), and a gene element comprising 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.

[0013] This disclosure further provides nucleic acid sequences (for example, nucleic acid molecules comprising a gene element as described herein, or nucleic acid molecules comprising a sequence encoding a proteinogenic extraprotein as described herein). The nucleic acid molecules of the present invention may comprise either or both of (a) a gene element as described herein, and (b) a nucleic acid sequence encoding a proteinogenic extraprotein as described herein.

[0014] In one embodiment, the present invention features an isolated nucleic acid molecule comprising a gene element including an effector, for example, a promoter element operably linked to a sequence encoding a payload, and an external protein-binding sequence. In some embodiments, the external protein-binding sequence comprises a sequence that is at least 75% (at least 80%, 85%, 90%, 95%, 97%, 100%) identical to the 5'UTR sequence of an anellovirus disclosed herein. In several embodiments, the gene element is single-stranded DNA, circular, and incorporated at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements entering the cell, and / or incorporated into the genome of the target cell at a frequency of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome, or incorporated at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements entering the cell. In some embodiments, the incorporation frequency is determined as described in Wang et al. (2004, Gene Therapy 11:711-721, which is incorporated herein by reference in its entirety). In several embodiments, the effector is not derived from TTV and is not SV40-miR-S1. In several embodiments, the nucleic acid molecule does not contain the polynucleotide sequence of TTMV-LY2. In several embodiments, the promoter element can instruct the expression of the effector in eukaryotic (e.g., mammalian, e.g., human) cells.

[0015] In some embodiments, the nucleic acid molecule is cyclic. 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 modification, sugar modification, or skeletal modification).

[0016] In some embodiments, the nucleic acid molecule includes a sequence encoding an ORF1 molecule (e.g., Anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the nucleic acid molecule includes a sequence encoding an ORF2 molecule (e.g., Anellovirus ORF2 protein, e.g., as described herein). In some embodiments, the nucleic acid molecule includes a sequence encoding an ORF3 molecule (e.g., Anellovirus ORF3 protein, e.g., as described herein). In one embodiment, the present invention is characterized by a gene element comprising 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 consecutive 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%) sequence identity with respect to a wild-type anellovirus sequence; or at least 72% (e.g., at least 72, (iii) at least 100 consecutive nucleic acids (e.g., at least 300, 500, 1000, 1500) having sequence identity of 73, 74, 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%); and (iii) a protein-binding sequence, e.g., an external protein-binding sequence, where the nucleic acid construct is a single The DNA is stranded; and the nucleic acid constructs are circular and incorporated at frequencies of approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements entering the cell, and / or incorporated into the genome of the target cell in fewer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome. In some embodiments, the gene elements encoding effectors (e.g., extrinsic or endogenous effectors) are codon-optimized.In some embodiments, the gene element is circular. In some embodiments, the gene element is linear. In some embodiments, the gene element includes, for example, an anerovector as described herein. In some embodiments, the gene element described herein includes one or more modified nucleotides (e.g., base modification, sugar modification, or skeletal modification). In some embodiments, the gene element includes a sequence encoding an ORF1 molecule (e.g., Anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the gene element includes a sequence encoding an ORF2 molecule (e.g., Anellovirus ORF2 protein, e.g., as described herein). In some embodiments, the gene element includes a sequence encoding an ORF3 molecule (e.g., Anellovirus ORF3 protein, e.g., as described herein).

[0017] In one embodiment, the present invention is characterized by: (a) a nucleic acid (wherein the nucleic acid is a plasmid, a viral nucleic acid, or incorporated into a helper cell chromosome) comprising a sequence encoding one or more ORF1, ORF2, or ORF3 molecules (e.g., a sequence encoding Anellovirus ORF1 as described herein); and (b) a host cell or helper cell comprising a gene element, wherein the gene element comprises (i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector); and (ii) a protein-binding sequence that binds to the polypeptide of (a), wherein optionally, the gene element does not encode the ORF1 polynucleotide (e.g., the ORF1 protein). For example, a host cell or helper cell comprises (a) and (b) in either cis (both parts of the same nucleic acid molecule) or trans (each part of different nucleic acid molecules). In several embodiments, the gene element of (b) is circular, single-stranded DNA. In some embodiments, the host cells are a production cell line. In some embodiments, the host cells or helper cells are adherent, suspended, or both. In some embodiments, the host cells or helper cells are grown in a microcarrier. In some embodiments, the host cells or helper cells comply with cGMP manufacturing standards. In some embodiments, the host cells or helper cells are grown in a medium suitable for promoting cell proliferation. In certain embodiments, once the host cells or helper cells have grown sufficiently (e.g., to an appropriate cell density), the medium may be replaced with a medium suitable for anesome production by the host cells or helper cells.

[0018] In one embodiment, the present invention is characterized by a pharmaceutical composition comprising an anestronomy (e.g., synthetic anestronomy) as described herein. In several embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In several embodiments, the pharmaceutical composition is approximately 10 per kilogram of the subject. 5 ~10 14The formulation includes a unit dose containing a genomic equivalent. In some embodiments, the pharmaceutical composition containing the preparation is stable at an acceptable period and temperature and / or compatible with the desired route of administration and / or any device required by that route of administration, such as a needle or syringe. In some embodiments, the pharmaceutical composition is formulated for administration as a single dose or multiple doses. In some embodiments, the pharmaceutical composition is formulated at the site of administration, for example, by a healthcare professional. In some embodiments, the pharmaceutical composition contains a desired concentration of anestrosomal genome or genomic equivalent (for example, defined by the number of genomes per volume).

[0019] In one embodiment, the present invention is characterized by a method for treating a disease or disorder in a subject, the method comprising the step of administering an anestronomy, for example, a synthetic anestronomy as described herein, to the subject.

[0020] In one embodiment, the present invention is characterized by a method for delivering an effector or payload (e.g., an endogenous effector or an exogenous effector) to a cell, tissue or subject, the method comprising the step of administering an anestronomy, for example, a synthetic anestronomy as described herein, to the subject, wherein the anestronomy comprises a nucleic acid encoding the effector. In several embodiments, the payload is a nucleic acid. In several embodiments, the payload is a polypeptide.

[0021] In one embodiment, the present invention is characterized by a method for delivering anestrophores to cells, which includes the step of bringing anestrophores, such as synthetic anestrophores as described herein, into contact with cells, such as eukaryotic cells, such as mammalian cells, for example, in vivo or ex vivo.

[0022] One embodiment is characterized by a method for producing anelosomes, such as synthetic anelosomes. This method is as follows: a) Below: (i) an anestroma, for example, a first nucleic acid molecule comprising the nucleic acid sequence of the gene element of a synthetic anestroma described herein, (ii) A first nucleic acid, or a second nucleic acid molecule encoding one or more amino acid sequences having at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as listed in any of Table 16. A step of preparing host cells containing; and b) Incubating host cells under conditions suitable for anestrom production. Includes.

[0023] In some embodiments, the method further includes a step of introducing a first nucleic acid molecule and / or a second nucleic acid molecule into a host cell prior to step (a). In some embodiments, the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. In other embodiments, the second nucleic acid molecule is incorporated into the genome of the host cell. In some embodiments, the second nucleic acid molecule is a helper (e.g., the genome of a helper plasmid or helper virus).

[0024] In another aspect, the present invention is characterized by a method for producing an anestrom composition, which is as follows: a) The procedure includes the step of preparing a host cell to express an anestronomy, for example, comprising one or more components (e.g., all components) of a synthetic anestronomy described herein. For example, the host cell comprises (a) a nucleic acid comprising a sequence encoding Anellovirus ORF1 as described herein (wherein the nucleic acid is a plasmid, a viral nucleic acid, or incorporated into a helper cell chromosome); and (b) a gene element, wherein the gene element comprises (i) a promoter element movably ligated to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous or endogenous effector), and (i) a protein-binding sequence that binds to the polypeptide of (a), wherein the host cell or helper cell comprises (a) and (b) in either cis or trans configuration. In several embodiments, the gene element of (b) is a circular, single-stranded DNA. In some embodiments, the host cell is a production cell line; b) the step of culturing host cells under conditions suitable for producing anestrosomal preparations from host cells (wherein the anestrosomal preparation comprises a proteinoid outer layer (e.g., including an ORF1 molecule) enclosing a genetic element (e.g., as described herein), thereby producing an anestrosomal preparation; and Optionally, c) the step of formulating an anestrom preparation as a pharmaceutical composition suitable for administration to a subject.

[0025] In some embodiments, the anestrosomal components are introduced into host cells at the time of production (e.g., by transient transfection). In some embodiments, the host cells stably express the anestrosomal components (e.g., one or more nucleic acids encoding the anestrosomal components are introduced into the host cell or its progenitor cell, for example, by stable transfection).

[0026] In some embodiments, the method further includes one or more purification steps (e.g., purification by precipitation, chromatography, and / or ultrafiltration). In some embodiments, the purification step includes removing serum, host cell DNA, host cell proteins, particles lacking gene elements, and / or phenol red from the preparation. In some embodiments, the resulting preparation or pharmaceutical composition containing the preparation is stable at an acceptable period and temperature and / or compatible with the desired route of administration and / or any device required by that route of administration, such as a needle or syringe.

[0027] In one embodiment, the present invention is characterized by a method for producing an anestrom composition, which is as follows: a) the step of providing a plurality of anestrom described herein, or a preparation of anestrom described herein; and b) the step of formulating the anestrom or a preparation thereof as a pharmaceutical composition suitable for administration to a subject, for example.

[0028] In one embodiment, the present invention is characterized by a method for producing host cells containing anerosomes, such as first host cells or producer cells (e.g., as shown in Figure 12), for example, a population of first host cells, the method comprising, for example, introducing gene elements into host cells as described herein, and culturing the host cells under conditions suitable for anerosome production. In some embodiments, the method further comprises introducing a helper, such as a helper virus, into host cells. In some embodiments, the introduction step comprises transfection of the host using anerosomes (e.g., chemical transfection) or electroporation.

[0029] In one embodiment, the present invention is characterized by a method for producing anestrophores, the method comprising the steps of providing host cells containing anestrophores, for example, as described herein, such as first host cells or producer cells (for example, as shown in Figure 12), and purifying anestrophores from the host cells. In some embodiments, the method further comprises, prior to the providing step, contacting the host cells with anestrophores, for example, as described herein, and incubating the host cells under conditions suitable for anestrophore production. In some embodiments, the host cells are first host cells or producer cells as described in the host cell production method described above. In some embodiments, the step of purifying anestrophores from the host cells includes a step of lysing the host cells.

[0030] In some embodiments, the method further includes a second step of contacting anestrophores produced by a first host cell or producer cell with a second host cell, for example, a tolerant cell (as shown in Figure 12), for example, a population of second host cells. In some embodiments, the method further includes a step of incubating the second host cells under conditions suitable for anestrophore production. In some embodiments, the method further includes a step of purifying anestrophores from the second host cells, for example, thereby producing a population of anestrophore seeds. In several embodiments, at least about 2 to 100 times more anestrophores are produced from the population of second host cells compared to those from the population of first host cells. In several embodiments, the step of purifying anestrophores from second host cells includes a step of lysing the second host cells. In some embodiments, the method further includes a step of contacting anestrophores produced by the second host cells with a third host cell, for example, a tolerant cell (as shown in Figure 12), for example, a population of third host cells. In some embodiments, the method further includes the step of incubating third host cells under conditions suitable for anestrosomal production. In some embodiments, the method includes the step of purifying anestrosomals from the third host cells, for example, thereby producing an anestrosomal stock population. In several embodiments, the purification of anestrosomals from the third host cells includes the step of lysing the third host cells. In several embodiments, at least about 2 to 100 times more anestrosomals are produced from the population of third host cells compared to those from the population of second host cells.

[0031] In some embodiments, host cells are grown in a medium suitable for promoting cell proliferation. In certain embodiments, once the host cells or helper cells have grown sufficiently (e.g., to an appropriate cell density), the medium may be replaced with a medium suitable for anestrosomal production by the host cells or helper cells. In some embodiments, anestrosomals produced by the host cells are separated from the host cells (e.g., by lysing the host cells) before contact with the second host cells. In some embodiments, anestrosomals produced by the host cells are contacted with the second host cells without the intervention of a purification step.

[0032] In one embodiment, the present invention is characterized by a method for preparing a pharmaceutical anestroma preparation. This method includes (a) preparing an anestroma preparation as described herein; (b) evaluating the preparation (e.g., a pharmaceutical anestroma preparation, an anestroma seed population, or an anestroma stock population) with respect to one or more pharmaceutical quality control parameters, e.g., identity, purity, potency, efficacy (e.g., in genomic equivalents per anestroma particle), and / or nucleic acid sequences from genetic elements contained in the anestroma; and (c) formulating the preparation for a pharmaceutical use of evaluation that meets predetermined criteria, e.g., pharmaceutical specifications. In some embodiments, the step of evaluating identity includes evaluating (e.g., verifying) the sequences of genetic elements of the anestroma, e.g., sequences encoding effectors. In some embodiments, the step of evaluating purity includes evaluating the amount of impurities, e.g., mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived impurities (e.g., serum albumin or trypsin), replicable factors (RCAs), e.g., replicable viruses or unwanted anestrophores (e.g., desired anestrophores, e.g., anestrophores other than the synthetic anestrophores described herein), free viral capsid proteins, incidental materials, and aggregates. In some embodiments, the step of evaluating titer includes evaluating the ratio of functional to non-functional (e.g., infectious and non-infectious) anestrophores in the preparation (e.g., evaluation by HPLC). In some embodiments, the step of evaluating potency includes evaluating the level of detectable anestrosomal function in the preparation (e.g., expression and / or function of the effectors encoded therein or genomic equivalents).

[0033] In several embodiments, the formulated preparation is substantially free of pathogens, host cell contaminants, or impurities; it contains a predetermined level of non-infectious particles or particles in a predetermined ratio: infectious units (e.g., <300:1, <200:1, <100:1, or <50:1). In some embodiments, multiple anesomes can be produced in a single batch. In several embodiments, the levels of anesomes produced in a batch can be evaluated (e.g., individually or together).

[0034] In one embodiment, the present invention is as follows: (i) a first nucleic acid molecule comprising the nucleic acid sequence of the anestrom gene element described herein, and (ii) A second nucleic acid molecule that optionally encodes one or more amino acid sequences selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 listed in any of Table 16, or one or more amino acid sequences having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity thereto. It is characterized by host cells containing [the specified element].

[0035] In one embodiment, the present invention is characterized by a reaction reaction comprising an anestroma and a helper virus as described herein, wherein the helper virus comprises polynucleotides, for example, polynucleotides encoding an external protein (e.g., an external protein that can bind to an external protein binding sequence, and optionally, a lipid envelope), polynucleotides encoding a replication protein (e.g., polymerase), or any combination thereof.

[0036] In some embodiments, anelosomes (e.g., synthetic anelosomes) are isolated, for example, from host cells and / or from other components in a solution (e.g., supernatant). In some embodiments, anelosomes (e.g., synthetic anelosomes) are purified, for example, from a solution (e.g., supernatant). In some embodiments, anelosomes are concentrated in solution relative to other components in the solution.

[0037] In some embodiments of the anestrom, anelovector, composition, or method described above, the gene element includes an anestrom genome, such as one identified according to the method described in Example 9. In some embodiments, the anestrom genome includes a TTV-tth8 nucleic acid sequence having deletions of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of nucleotides 3436-3707 of the TTV-tth8 nucleic acid sequence, such as the TTV-tth8 nucleic acid sequences shown in Table 5. In several embodiments, the anestrosomal genome comprises a TTMV-LY2 nucleic acid sequence having deletions of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of nucleotides 574-1371, 1432-2210, 574-2210, and / or 2610-2809, for example, the TTMV-LY2 nucleic acid sequences shown in Table 15. In several embodiments, the anestrosomal genome is capable of self-replication and / or self-amplification. In several embodiments, the anestrosomal genome is not capable of self-replication and / or self-amplification. In several embodiments, the anestrosomal genome can be replicated and / or amplified in trans, for example, in the presence of a helper, for example, a helper virus.

[0038] Further features of any of the aforementioned anelosomes, anelovectors, compositions, or methods include one or more of the embodiments listed below.

[0039] Those skilled in the art will recognize, or confirm by common experimentation, various equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the embodiments listed below.

[0040] List of embodiments 1. The following: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., DNA sequence) encoding an external effector, and a protein-binding sequence (e.g., an external protein-binding sequence). In anesomes containing; External effects pedals are as follows: (i) intracellular polypeptides other than nano-luciferase, (ii) Intracellular nucleic acids other than miR-124, miR-518, miR-625, miRNA, or miRNAs for n-myc interacting proteins (e.g., miRNAs or siRNAs), or wild-type anellovirus, for example, as described herein, including endogenous miRNAs of TTV-tth8 anellovirus; The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements to eukaryotic cells; An anestroma in which, optionally selected, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0041] 2. Below: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., DNA sequence) encoding an external effector, and a protein-binding sequence (e.g., an external protein-binding sequence). In anesomes containing; The external effector comprises an intracellular polypeptide or intracellular nucleic acid; The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements to eukaryotic cells; An anestroma in which a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0042] 3. Below: (a) Protein outer layer; (b) Gene elements including a promoter element operably linked to a heterogeneous nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an endogenous effector or an exogenous effector) In anesomes containing; External effects pedals are as follows: (i) intracellular polypeptides other than nano-luciferase, (ii) Intracellular nucleic acids other than miR-124, miR-518, miR-625, miRNA, or miRNAs for n-myc interacting proteins (e.g., miRNAs or siRNAs), or wild-type anellovirus, for example, as described herein, including endogenous miRNAs of TTV-tth8 anellovirus; The gene elements are confined within a proteinaceous outer layer; An anestroma in which, optionally selected, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0043] 4. A method for treating a disease or disorder in a subject, the method comprising the step of administering an effective amount of an anestrosomal composition to the subject, wherein the anestrosomal composition is as follows: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector), and a protein-binding sequence (e.g., an external protein-binding sequence). It contains multiple anestrosomes, including; The effects pedals are as follows: (i) intracellular polypeptides other than nano-luciferase, (ii) intracellular nucleic acids other than miR-124, miR-518, miR-625, and miRNAs for n-myc interacting proteins, or wild-type anellovirus, for example, endogenous miRNAs of TTV-tth8 anellovirus, as described herein; The gene elements are confined within a proteinaceous outer layer; A method wherein, optionally, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0044] 5. The method according to Embodiment 4, wherein the disease or disorder is a neurodegenerative disorder, metabolic disorder, developmental disorder, or gastrointestinal disorder.

[0045] 6. A method for delivering an effector to a subject, the method comprising the step of administering an effective amount of an anestrom composition to the subject. Here, the anestrom composition is as follows: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector), and a protein-binding sequence (e.g., an exogenous protein-binding sequence). It contains multiple anestrosomes, including; External effects pedals are as follows: (i) intracellular polypeptides other than nano-luciferase, (ii) intracellular nucleic acids other than miR-124, miR-518, miR-625, and miRNAs for n-myc interacting proteins, or wild-type anellovirus, for example, endogenous miRNAs of TTV-tth8 anellovirus, as described herein; Optionally, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; This is a method for delivering an effects pedal to a subject.

[0046] 7. A method for modulating, for example, inhibiting or enhancing the biological function of a subject, for example, a subject having a disease or disorder that can be treated by modulating the subject's biological function, the method comprising the step of administering an effective amount of an anestrosomal composition (for example, as described herein) to the subject. Here, the anestrom composition is as follows: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector), and a protein-binding sequence (e.g., an exogenous protein-binding sequence). It contains multiple anestrosomes, including; The effects pedals are as follows: (i) intracellular polypeptides other than nano-luciferase, (ii) intracellular nucleic acids other than miR-124, miR-518, miR-625, and miRNAs for n-myc interacting proteins, or wild-type anellovirus, for example, endogenous miRNAs of TTV-tth8 anellovirus, as described herein; Optionally, a gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; A method for thereby regulating, for example inhibiting or enhancing, the biological functions of a subject.

[0047] 8. An anestrome or method according to any of the prior embodiments, wherein the miRNA comprises miR-34a, miR-506, or Let7a.

[0048] 9. An anestronomy or method according to any of the prior embodiments, wherein the siRNA is configured to downregulate KRAS G12D, CpG(B)-STAT3, cyclin D1, C-myc, or C-myb.

[0049] 10. An anestronomy or method according to any one of Embodiments 3, 4, 6, or 7, wherein the miRNA or siRNA is endogenous to the anestronomy.

[0050] 11. An anestronomy or method according to any of the prior embodiments, wherein the therapeutic cytoplasmic peptide is a DDP-4 inhibitor, a GLP-1 signaling activator, or a neutrophil elastase inhibitor.

[0051] 12. A method for treating a disease or disorder in a subject, comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject, wherein the anestrosomal composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) (both as described herein). In some embodiments, (i) The disease or disorder is cancer or includes cancer, and the effector reduces the levels or activity of EGFR, IDH1, IDH2, LRP5, DKK2, KRAS, VEGF, IGF receptor, FGF receptor, or TGF-β receptor; (ii) The disease or disorder is liver dysfunction, and the effector reduces the level or activity of pathogenic α-1 antitrypsin protein, the level or activity of non-pathogenic α-1 antitrypsin protein, and / or reduces the activity of neutrophil elastase; or (iii) The disease or disorder is a developmental disorder, and the effector is an increase in the level or activity of a growth factor or its receptor (e.g., FGF receptor, e.g., FGFR3); This is used to treat the subject's disease or disorder.

[0052] 13. A method for treating a disease or disorder of a subject, the method comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject, wherein the anestrosomal composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) (both as described herein), (i) The disease or disorder is or includes polycythemia vera, and the effector includes an inhibitor of n-myc interaction protein activity (e.g., an n-myc interaction protein inhibitor); (ii) The disease or disorder is or includes ovarian cancer, and the effector includes an inhibitor of EGFR activity (e.g., an EGFR inhibitor); (iii) The disease or disorder is or includes a hematological malignancy (e.g., leukocyte or lymphoma), and the effector includes an inhibitor of IDH1 and / or IDH2 activity (e.g., an IDH1 inhibitor and / or an IDH2 inhibitor); (iv) The disease or disorder is or includes colon cancer, and the effector includes an inhibitor of LRP5 and / or DKK2 activity (e.g., an LRP5 and / or DKK2 inhibitor); (v) The disease or disorder is cancer or includes cancer, and the effector includes miR-34a, Let7a miRNA, or miR-506; (vi) The disease or disorder is or includes pancreatic cancer, and the effector includes an inhibitor of KRAS activity (e.g., KRAS G12siRNA); (vii) The disease or disorder is or includes α-1 antitrypsin deficiency, and the effector includes an inhibitor of neutrophil elastase activity (e.g., a neutrophil elastase inhibitor); (viii) The disease or disorder is or includes achondroplasia, and the effector includes an activator of FGFR3 activity (e.g., FGFR3 or an FGFR3 agonist); (ix) The disease or disorder is Huntington's disease or includes Huntington's disease, and the effector includes an activator of HTT activity (e.g., wild-type or non-pathogenic HTT) or an inhibitor of pathogenic mutant HTT (e.g., siRNA or miRNA that binds to pathogenic mutant HTT); or (x) The disease or disorder is type 2 diabetes mellitus, and the effector is an inhibitor of DPP-4 activity (e.g., a DPP-4 inhibitor); A method for treating a subject's disease or disorder.

[0053] 14. A method for treating a disease or disorder of a subject, comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject, wherein the anestrosomal composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) (both as described herein), wherein the effector is an immune activator (e.g., a neutrophil elastase inhibitor), m A method comprising iR-367, miR-302a, miR-302b, miR-302c, miR-302d, viral proteins such as influenza proteins such as influenza NP or NS proteins, inhibitors of RSV proteins, siRNA against CpG(B)-STAT3, cyclins such as siRNA against cyclin D1, siRNA against C-myc, siRNA against C-myb, EGFR inhibitors, IDH1 inhibitors, IDH2 inhibitors, LRP5 inhibitors, DKK2 inhibitors, miR-34a, Let7a miRNA, miR-506, KRAS inhibitors, FGFR3 activators, HTT activators, inhibitors of pathogenic mutant HTT, or DPP-4 inhibitors; thereby treating a disease or disorder of a subject.

[0054] 15. A method for treating a disease or disorder in a subject, the method comprising the step of administering an effective amount of an anestrosomal composition to the subject, Here, the anestroma composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector) (both as described herein), wherein the disease or disorder is or includes polycythemia vera, ovarian cancer, hematological malignancies, colon cancer, pancreatic cancer, α-1 antitrypsin deficiency, achondroplasia, Huntington's disease, or diabetes mellitus (e.g., type 2 diabetes mellitus); A method for treating a subject's disease or disorder.

[0055] 16. A method for delivering an effector to a subject having a disease or disorder, the method comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject. Here, the anestroma composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector) (both as described herein), and in this case, (i) The disease or disorder is cancer or includes cancer, and the effector reduces the levels or activity of EGFR, IDH1, IDH2, LRP5, DKK2, KRAS, VEGF, IGF receptor, FGF receptor, or TGF-β receptor; (ii) The disease or disorder is liver dysfunction, and the effector reduces the level or activity of pathogenic α-1 antitrypsin protein, increases the level or activity of non-pathogenic α-1 antitrypsin protein, and / or reduces the activity of neutrophil elastase; or (iii) The disease or disorder is a developmental disorder, and the effector is an increase in the level or activity of a growth factor or its receptor (e.g., FGF receptor, e.g., FGFR3); This is a method for delivering an effector to a subject.

[0056] 17. A method for delivering an effector to a subject having a disease or disorder, the method comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject. Here, the anestroma composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector) (both as described herein), and in this case, (i) The disease or disorder is or includes polycythemia vera, and the effector includes an inhibitor of n-myc interaction protein activity (e.g., an n-myc interaction protein inhibitor); (ii) The disease or disorder is or includes ovarian cancer, and the effector includes an inhibitor of EGFR activity (e.g., an EGFR inhibitor); (iii) The disease or disorder is or includes a hematological malignancy (e.g., leukocyte or lymphoma), and the effector includes an inhibitor of IDH1 and / or IDH2 activity (e.g., an IDH1 inhibitor and / or an IDH2 inhibitor); (iv) The disease or disorder is or includes colon cancer, and the effector includes an inhibitor of LRP5 and / or DKK2 activity (e.g., an LRP5 and / or DKK2 inhibitor); (v) The disease or disorder is cancer or includes cancer, and the effector includes miR-34a, Let7a miRNA, or miR-506; (vi) The disease or disorder is or includes pancreatic cancer, and the effector includes an inhibitor of KRAS activity (e.g., KRAS G12siRNA); (vii) The disease or disorder is or includes α-1 antitrypsin deficiency, and the effector includes an inhibitor of neutrophil elastase activity (e.g., a neutrophil elastase inhibitor); (viii) The disease or disorder is or includes achondroplasia, and the effector includes an activator of FGFR3 activity (e.g., FGFR3 or an FGFR3 agonist); (ix) The disease or disorder is Huntington's disease or includes Huntington's disease, and the effector includes an activator of HTT activity (e.g., wild-type or non-pathogenic HTT) or an inhibitor of pathogenic mutant HTT (e.g., siRNA or miRNA that binds to pathogenic mutant HTT); or (x) The disease or disorder is type 2 diabetes mellitus, and the effector is an inhibitor of DPP-4 activity (e.g., a DPP-4 inhibitor); A method for delivering an effector to a subject.

[0057] 18. A method for delivering an effector to a subject having a disease or disorder, the method comprising the step of administering an effective amount of an anestrosomal composition or isolated nucleic acid molecule (e.g., an expression vector) to the subject. Here, the anestroma composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector) (both as described herein), In this case, the effector includes an immune activator (e.g., a neutrophil elastase inhibitor), miR-367, miR-302a, miR-302b, miR-302c, miR-302d, an influenza NP or NS protein inhibitor, an RSV protein inhibitor, siRNA against CpG(B)-STAT3, siRNA against cyclin D1, siRNA against C-myc, siRNA against C-myb, EGFR inhibitor, IDH1 inhibitor, IDH2 inhibitor, LRP5 inhibitor, DKK2 inhibitor, miR-34a, Let7a miRNA, miR-506, KRAS inhibitor, FGFR3 activator, HTT activator, an inhibitor against pathogenic mutant HTT, or a DPP-4 inhibitor; A method for delivering an effector to a subject.

[0058] 19. A method for delivering an effector to a subject having a disease or disorder, the method comprising the step of administering an effective amount of an anestrom composition to the subject, Here, the anestroma composition or isolated nucleic acid molecule contains a gene element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector) (both as described herein); In this case, the disease or disorder is or includes polycythemia vera, ovarian cancer, hematological malignancies, colon cancer, pancreatic cancer, alpha-1 antitrypsin deficiency, achondroplasia, Huntington's disease, or diabetes mellitus (e.g., type 2 diabetes mellitus); A method for delivering an effector to a subject.

[0059] 20. A method for producing an anelosome composition, the method comprising the following: a) preparing a host cell comprising one or more nucleic acid molecules encoding the components of the anelosome according to any of the preceding embodiments; b) maintaining (e.g., culturing) the host cell under conditions that allow the cell to produce one or more anelosomes, thereby producing an anelosome; and c) formulating a preparation of the anelosome as a pharmaceutical composition suitable for administration to a subject A method comprising:

[0060] 21. A method for producing an anelosome composition, the method comprising the following: a) preparing a plurality of anelosomes according to any of the preceding embodiments; b) optionally, evaluating the plurality of anelosomes for one or more of the following: contaminants described herein, optical density measurements (e.g., OD260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio); and c) formulating the plurality of anelosomes as a pharmaceutical composition suitable for administration to a subject, e.g., if one or more of the parameters in (b) meet a specified threshold A method comprising:

[0061] 22. The method according to embodiment 21, wherein the anelosome composition comprises at least 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 or 10 anelosomes.

[0062] 23. The method according to Embodiment 21 or 22, wherein the anestrom composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0063] 24. Effects pedals are as follows: (i) Therapeutic intracellular polypeptides (e.g., inhibitors of IDH1, IDH2, LRP5, or DPP-4, or non-pathogenic huntingtin polypeptides); (ii) Regulatory intracellular polypeptides (e.g., intracellular polypeptides that reduce the activity of IDH1, IDH2, LRP5, DKK2, KRAS, neutrophil elastase inhibitors, FGFR3, HTT, or DPP-4); (iii) Intracellular polypeptides that bind to endogenous proteins or polypeptides (e.g., endogenous IDH1, IDH2, LRP5, DKK2, KRAS, neutrophil elastase inhibitors, FGFR3, HTT, or DPP-4); (iv) an intracellular ligand for an endogenous protein or polypeptide (for example, the endogenous protein is selected from IDH1, IDH2, LRP5, DKK2, KRAS, a neutrophil elastase inhibitor, FGFR3, HTT, or DPP-4); and / or (v) Non-enzymatic intracellular polypeptides (e.g., non-pathogenic huntingtin polypeptides) An anestrom or method according to any of the prior embodiments, including

[0064] 25. Effects pedals are as follows: (i) Therapeutic siRNAs, such as siRNAs that reduce enzyme levels, or siRNAs that reduce levels of intracellular polypeptides (e.g., IDH1, IDH2, LRP5, DPP-4, or pathogenic huntingtin polypeptide); (ii) Hairpin RNA molecule; (iii) pre-miRNA molecules (e.g., pre-miRNAs containing miR-34a, Let7a miRNA, or miR-506); or (iv) miRNAs that reduce enzyme levels, or miRNAs that reduce intracellular polypeptide levels An anestrom or method according to any of the prior embodiments, including

[0065] 26. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an inhibitor comprising nucleic acid (e.g., siRNA or miRNA).

[0066] 27. An anestrome or method according to any of the prior embodiments, wherein the effector comprises a polypeptide, for example, an antibody molecule (for example, a monoclonal antibody or antibody fragment, for example, scFv), a GPCR-binding molecule, an ion channel-binding molecule, or an inhibitor comprising a kinase inhibitor.

[0067] 28. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an immune activator (e.g., a neutrophil elastase inhibitor).

[0068] 29. An anestronomy or method according to any of the prior embodiments, wherein the effector comprises miR-34a, Let7a miRNA, miR-506, miR-367, miR-302a, miR-302b, miR-302c, and miR-302d.

[0069] 30. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an inhibitor of a viral protein, such as an influenza protein, such as an influenza NP or NS protein.

[0070] 31. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an inhibitor of the RSV protein.

[0071] 32. An anestrome or method according to any of the prior embodiments, wherein the effector comprises CpG(B)-STAT3, siRNA against cyclin D1, siRNA against C-myc, and siRNA against C-myb.

[0072] 33. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an EGFR inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, an LRP5 inhibitor, a DKK2 inhibitor, miR-34a, Let7a miRNA, miR-506, a KRAS inhibitor, an FGFR3 activator, an HTT activator, an inhibitor of pathogenic mutant HTT, or a DPP-4 inhibitor.

[0073] 34. An anelosome or method according to any of the prior embodiments, wherein the gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration), e.g., insertion, substitution, enzymatic modification, and / or deletion, e.g., deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) compared to the wild-type anellovirus genome sequence (e.g., as described herein).

[0074] 35. Gene elements are nucleic acid sequences: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(Sequence ID 160); (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. An anestrome or method according to any of the prior embodiments, comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides.

[0075] 36. An anestrome or method according to any of the prior embodiments, wherein the gene element comprises a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0076] 37. An anestrome or method according to Embodiment 36, wherein the gene element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 80%.

[0077] 38. An anestrome or method according to Embodiment 36, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0078] 39. The anelosome or method according to Embodiment 36, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 80%.

[0079] 40. An anestrome or method according to any of the prior embodiments, wherein the neutrophil elastase inhibitor is siRNA.

[0080] 41. Effectors include EGFR activity inhibitors (e.g., EGFR inhibitors), IDH1 and / or IDH2 activity inhibitors (e.g., IDH1 inhibitors and / or IDH2 inhibitors), LRP5 and / or DKK2 activity inhibitors (e.g., LRP5 and / or DKK2 inhibitors), KRAS activity inhibitors, neutrophil elastase activity inhibitors (e.g., neutrophil elastase inhibitors), FGFR3 activity activators (e.g., FGFR3 or FGFR3 agonists), HTT activity activators (e.g., An anestronomy or method according to any of the prior embodiments, comprising (biotype or non-pathogenic HTT), DPP-4 activity (e.g., DPP-4 inhibitor), immune activator (e.g., neutrophil elastase inhibitor), miR-367, miR-302a, miR-302b, miR-302c, miR-302d, an inhibitor of influenza NP or NS protein, an inhibitor of RSV protein, an inhibitor of CpG(B)-STAT3, an inhibitor of cyclin D1, an inhibitor of C-myc, or an inhibitor of C-myb.

[0081] 41a. An anestronomy or method according to any of the prior embodiments, wherein the effector comprises an IDH1 inhibitor, an IDH2 inhibitor, an EGFR inhibitor, an LRP5 inhibitor, a DKK2 inhibitor, a DPP-4 inhibitor, a KRAS inhibitor, a neutrophil elastase inhibitor, an FGFR3 activator, or an HTT activator.

[0082] 41b. An anestrome or method according to any of the prior embodiments, wherein the effector comprises an IDH1 inhibitor or an IDH2 inhibitor.

[0083] 42. An anestronomy or method according to any of the prior embodiments, wherein the disease or disorder is selected from the list consisting of: polycythemia vera, cancer (e.g., ovarian cancer, hematological malignancies, colon cancer, or pancreatic cancer), α-1 antitrypsin deficiency, achondroplasia, Huntington's disease, and diabetes mellitus (e.g., type 2 diabetes mellitus).

[0084] 1000 or less: (a) Arginine-rich region sequences described herein (for example, MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO: 216) or [ka] , or as listed in Tables A2, A4, A6, A8, A10, A12, C1~C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20~37, or D1~D10) an amino acid sequence having at least 70% sequence identity (e.g., at least approximately 70, 80, 90, 95, 96, 97, 98, 99, or 100%), or a first region comprising a sequence of at least 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof), (b) Jelly roll region arrays described herein (for example, [ka] A second region containing an amino acid sequence having at least 30% sequence identity (e.g., at least approximately 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) with any of the sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a sequence containing at least six (e.g., at least 6, 7, 8, 9, 10, 11, or 12) β-chains; (c) N22 domain sequences described herein (for example, [ka] A third region containing amino acid sequences having at least 30% (for example, at least about 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to, or 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); and (d) An anerovirus ORF1 C-terminal domain (CTD) sequences described herein (for example, [ka] The fourth region includes amino acid sequences having at least 30% (for example, at least approximately 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the following: , or as listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10) A polypeptide comprising one or more of the following, for example, an ORF1 molecule, A polypeptide wherein the ORF1 molecule has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a specific domain (e.g., one or more of the arginine-rich region, jellyroll domain, HVR, N22, or CTD, as described herein).

[0085] The polypeptide according to Embodiment 1000, wherein the amino acid sequences of regions (a), (b), (c), and (d) have at least 90% sequence identity with respect to their respective reference sequences.

[0086] 1001. Polypeptides are as follows: (i) Area 1 and Area 2; (ii) Area 1 and Area 3; (iii) Area 1 and Area 4; (iv) Second and third areas; (v) Second and fourth regions; (vi) Third and fourth regions; (vii) The first region, the second region, and the third region; (viii) Area 1, Area 2, and Area 4; (ix) first area, third area, and fourth area; (x) Second area, third area, and fourth area A polypeptide according to embodiment 1000, including the polypeptide described above.

[0087] 1002. Below: (a) Arginine-rich region sequences described herein (for example, MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO: 216) or [ka] , or as listed in Tables A2, A4, A6, A8, A10, A12, C1~C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20~37, or D1~D10) an amino acid sequence having at least 70% sequence identity (e.g., at least approximately 70, 80, 90, 95, 96, 97, 98, 99, or 100%), or a first region comprising a sequence of at least 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof), (b) Jelly roll region arrays described herein (for example, [ka] A second region containing an amino acid sequence having at least 30% sequence identity (for example, at least approximately 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) with respect to any of the sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a sequence containing at least six β-chains; (c) N22 domain sequences described herein (for example, [ka] A third region containing amino acid sequences having at least 30% (for example, at least about 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to, or 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); and (d) An anerovirus ORF1 C-terminal domain (CTD) sequences described herein (for example, [ka] The fourth region includes amino acid sequences having at least 30% (for example, at least approximately 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the following: , or as listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10) Polypeptides containing, for example, the ORF1 molecule, A polypeptide wherein the ORF1 molecule has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a specific domain (e.g., one or more of the arginine-rich region, jellyroll domain, HVR, N22, or CTD, as described herein).

[0088] The polypeptide according to Embodiment 1002, wherein the amino acid sequences of regions (a), (b), (c), and (d) have at least 90% sequence identity with respect to their respective reference sequences.

[0089] 1003. The first region contains an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with amino acids 1-38 of the ORF1 sequence listed in Table 16; The second region includes an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with amino acids 39-246 of the ORF1 sequence listed in Table 16; The third region includes an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with amino acids 375-537 of the ORF1 sequence listed in Table 16; and / or A polypeptide according to any of the prior embodiments, wherein the fourth region includes an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with amino acids 538-666 of the ORF1 sequence listed in Table 16.

[0090] 1003A. The polypeptide according to Embodiment 1003, wherein the amino acid sequences of the first, second, third, and fourth regions have at least 90% sequence identity with respect to their respective reference sequences.

[0091] 1004. The first region contains an amino acid sequence having at least 70% (e.g., at least approximately 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the arginine-rich region sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10; The second region contains an amino acid sequence having at least 70% (e.g., at least approximately 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the jelly roll region sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10; The third region contains an amino acid sequence having at least 70% (e.g., at least approximately 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the N22 domain sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10; A polypeptide according to any of the prior embodiments, wherein the fourth region contains an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with any of the CTD sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

[0092] 1004A. The polypeptide according to Embodiment 1004, wherein the amino acid sequences of the first, second, third, and fourth regions have at least 90% sequence identity with respect to their respective reference sequences.

[0093] 1005. The polypeptide according to any of the prior embodiments, wherein the polypeptide comprises a first region, a second region, a third region, and a fourth region in the order from the N-terminus to the C-terminus.

[0094] 1006. A polypeptide according to any of the prior embodiments, wherein at least one difference is present in the first region compared to the arginine-rich region of the wild-type ORF1 protein.

[0095] 1007. The polypeptide according to any of the prior embodiments, wherein the first region is the polypeptide described above, or the portion excluding the first region, comprises an arginine-rich region derived from the ORF1 protein of an anellovirus other than the wild-type anellovirus having the greatest sequence identity.

[0096] 1008. Region 1 is a polypeptide according to any of the prior embodiments, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with respect to an arginine-rich region of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0097] 1009. The first region is a polypeptide having less than 15% sequence identity (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%) to the wild-type anerovirus genome (e.g., as described herein), or a polypeptide according to any of the prior embodiments, comprising a portion thereof having the same amino acid length as the first region.

[0098] 1010. A polypeptide according to any of the prior embodiments, wherein the first region has DNA-binding activity and / or nuclear localization activity.

[0099] 1011. A polypeptide according to any of the prior embodiments, wherein the first region has a DNA-binding region and / or a nuclear localization sequence.

[0100] 1012. A polypeptide according to any of the prior embodiments, wherein at least one difference is present in the second region compared to the jelly roll region of the wild-type ORF1 protein.

[0101] 1013. The polypeptide according to any of the prior embodiments, wherein the second region is the polypeptide described above, or the portion excluding the second region, comprises a jellyroll region derived from the ORF1 protein of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0102] 1014. Region 2 is the polypeptide according to any of the prior embodiments, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with respect to the jelly roll region of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0103] 1015. The second region is a polypeptide having less than 15% sequence identity (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%) to the wild-type anerovirus genome (e.g., as described herein), or a polypeptide according to any of the prior embodiments, comprising a portion thereof having the same amino acid length as the second region.

[0104] 1016. A polypeptide according to any of the prior embodiments, wherein at least one difference is in the third region compared to the N22 domain of the wild-type ORF1 protein.

[0105] 1017. The polypeptide according to any of the prior embodiments, wherein the third region is the polypeptide described above, or the portion excluding the third region, includes an N22 domain region derived from the ORF1 protein of an anellovirus other than the wild-type anellovirus having the greatest sequence identity.

[0106] 1018. Region 3 is the polypeptide according to any of the prior embodiments, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with respect to the N22 region of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0107] 1019. The third region is a polypeptide having less than 15% sequence identity (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%) to the wild-type anerovirus genome (e.g., as described herein), or a polypeptide according to any of the prior embodiments, comprising a portion thereof having the same amino acid length as the third region.

[0108] 1020. A polypeptide according to any of the prior embodiments, wherein at least one difference is in the fourth region compared to the CTD domain of the wild-type ORF1 protein.

[0109] 1021. The polypeptide according to any of the prior embodiments, wherein the fourth region, or the portion excluding the fourth region, includes a CTD domain region derived from the ORF1 protein of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0110] 1022. Region 4 is the polypeptide according to any of the prior embodiments, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with respect to the CTD region of an anelovirus other than the wild-type anelovirus having the greatest sequence identity.

[0111] 1023. The fourth region is a polypeptide having less than 15% sequence identity (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1%) to the wild-type anerovirus genome (e.g., as described herein), or a polypeptide according to any of the prior embodiments, comprising a portion thereof having the same amino acid length as the fourth region.

[0112] 1024. A polypeptide according to any of the prior embodiments, further comprising an amino acid sequence, for example, a hypervariable region (HVR) sequence (for example, the HVR sequence of the Anellovirus ORF1 molecule described herein), wherein the amino acid sequence comprises at least about 55 (for example, at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (for example, about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55-140, 60-140 amino acids).

[0113] 1025. The polypeptide according to embodiment 1024, wherein the HVR sequence is located between the second and third regions.

[0114] 1026. The polypeptide according to Embodiment 1024 or 1025, wherein the HVR sequence comprises an amino acid sequence in which the ORF1 protein has at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with an anellovirus HVR other than the wild-type anellovirus having the greatest sequence identity.

[0115] 1027. The polypeptide according to any one of embodiments 1024 to 1026, wherein the HVR sequence is heterogeneous with respect to one or more of the first, second, third, and / or fourth regions.

[0116] 1028. A polypeptide according to any of embodiments 1024 - 1027, wherein at least one difference is included in the HVR sequence as compared to the sequence of the HVR of a wild-type ORF1 protein (e.g., derived from a wild-type anellovirus genome as described herein).

[0117] 1029. A polypeptide according to any of embodiments 1024 - 1028, wherein the HVR sequence, or a portion thereof excluding the HVR sequence, includes an HVR derived from an ORF1 protein of an anellovirus other than the wild-type anellovirus with which the polypeptide has the highest sequence identity.

[0118] 1030. A polypeptide according to any of embodiments 1024 - 1029, wherein the HVR sequence includes an amino acid sequence having at least 70% sequence identity to an HVR derived from an anellovirus other than the wild-type anellovirus with which the polypeptide has the highest sequence identity.

[0119] 1031. A polypeptide according to any of embodiments 1024 - 1030, wherein the HVR includes 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 HVR sequence listed in any of Table A2, A4, A6, A8, A10, A12, C1 - C5, 2,  4, 6, 8, 10, 12, 14, 16, 18, 20 - 37, or D1 - D10.

[0120] 1032. A polypeptide according to any of embodiments 1024 - 1031, wherein the HVR sequence includes at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity to amino acids 247 - 374 of the ORF1 sequence listed in Table 16.

[0121] 1033. A polypeptide according to any of the preceding embodiments, further comprising a heterologous polypeptide that is heterologous to one or more of the first region, the second region, the third region, and / or the fourth region and / or is exogenous to the anellosome comprising the polypeptide.

[0122] 1034. The polypeptide according to embodiment 1033, wherein the polypeptide lacks the anellovirus HVR sequence.

[0123] 1035. The polypeptide according to embodiment 1033, wherein the heterologous polypeptide is present on the outer layer of the anellosome.

[0124] 1036. The polypeptide according to embodiment 1033, wherein the heterologous polypeptide is present on the inner layer of the anellosome.

[0125] 1037. The polypeptide according to any of embodiments 1033 - 1036, wherein the heterologous polypeptide has a functional group that is exogenous to the anellosome or wild - type anellovirus.

[0126] 1038. The polypeptide according to any of embodiments 1033 - 1037, wherein the heterologous polypeptide is composed of about 140 or fewer amino acids (e.g., 100, 110, 120, 125, 130, 135, 136, 137, 138, 139, 140, 145, 150, 155, or 160 or fewer amino acids).

[0127] 1039. The polypeptide according to any of embodiments 1033 - 1038, wherein the size of the heterologous polypeptide is, for example, 50 - 150% compared to the wild - type HVR region of the anellovirus described herein.

[0128] 1039A. The polypeptide according to any of embodiments 1033 - 1039, wherein the heterologous polypeptide is located between the second region and the third region.

[0129] 1040. A polypeptide according to any of the prior embodiments, further comprising one or more amino acids between the first and second regions, one or more amino acids between the second and third regions, and / or one or more amino acids between the third and fourth regions.

[0130] 1041. A polypeptide according to any of the prior embodiments, further comprising one or more amino acids located N-terminal to the first region.

[0131] 1042. A polypeptide according to any of the prior embodiments, further comprising one or more amino acids located N-terminal to the fourth region.

[0132] 1043. A polypeptide according to any of the prior embodiments, comprising a plurality of subsequences consisting of at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) consecutive amino acids having 100% sequence identity with the corresponding subsequence of the wild-type anellovirus ORF1 amino acid sequence 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.

[0133] 1044. A polypeptide according to any of the prior embodiments, comprising, for example, a plurality of subsequences consisting of at least 10 (e.g., 10, 15, 20, 25, 30, 40, or 50) consecutive amino acids having at least 80% sequence identity with the corresponding subsequence of the wild-type anellovirus ORF1 amino acid sequence 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.

[0134] 1045. A polypeptide according to any of the prior embodiments, comprising, for example, a plurality of subsequences consisting of at least 20 (e.g., 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100) consecutive amino acids having at least 60% sequence identity with the corresponding subsequence of the wild-type anellovirus ORF1 amino acid sequence 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.

[0135] 1046. A polypeptide according to any one of embodiments 1043 to 1045, wherein multiple subsequences are located within a first region, a second region, a third region, and / or a fourth region.

[0136] 1047. A polypeptide according to any of the prior embodiments, wherein the first region comprises at least 40 amino acids (for example, at least about 50, 60, 70, 80, 90, or 100 amino acids, for example, about 40-100, 40-90, 40-80, 40-70, 50-100, 50-70, 60-100, 60-90, 60-80, or 60-70 amino acids).

[0137] 1048. A polypeptide according to any of the prior embodiments, wherein the first region comprises at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%) basic residues (e.g., arginine, lysine, or a combination thereof).

[0138] 1049. A polypeptide according to any of the prior embodiments, wherein the first region comprises at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%) arginine residues.

[0139] 1050. A polypeptide according to any of the prior embodiments, wherein the polypeptide forms a homomultimer with additional copies of the polypeptide.

[0140] 1051. The polypeptide according to embodiment 1050, wherein the first region binds to a corresponding first region on an additional copy of the polypeptide.

[0141] 1052. The polypeptide according to embodiment 1050, wherein the homomultimer forms a capsid that encapsulates, for example, a nucleic acid, such as a gene element or an anellovirus genome, or a portion thereof.

[0142] 1053. The polypeptide according to any of the preceding embodiments, wherein the polypeptide is a capsid protein or can form a part of the capsid.

[0143] 1054. The polypeptide according to any of the preceding embodiments, wherein the polypeptide has replicase activity.

[0144] 1055. The polypeptide according to any of the preceding embodiments, wherein the polypeptide binds to a nucleic acid (e.g., DNA).

[0145] 1056. The following: (a) A polypeptide according to any of the preceding embodiments, and (b) A gene element comprising 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. A complex comprising.

[0146] 1057. The following: (a) An ORF1 molecule, and (b) A gene element comprising 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. A complex comprising, wherein the ORF1 molecule binds (e.g., by non-covalent binding) to the gene element. A complex comprising an ORF1 molecule, a gene element, or both an ORF1 molecule and a gene element, having at least one difference (e.g., mutation, chemical modification, or epigenetic alteration), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a specific domain (e.g., one or more of the arginine-rich region, jelly roll region, HVR, N22, or CTD, as described herein) or a genomic region (e.g., one or more of the TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, as described herein).

[0147] 1058. The complex according to Embodiment 1056 or 1057, wherein the complex is in vitro, for example, the complex is substantially in a cell-free composition.

[0148] 1059. The complex according to any one of embodiments 1056 to 1058, wherein the complex is present in a cell, for example, a host cell, for example, a helper cell, for example, in the nucleus of a cell.

[0149] The complex according to any one of embodiments 1056 to 1059, wherein the 1060.ORF1 molecule is part of the proteinaceous outer layer.

[0150] 1061. A complex according to any one of embodiments 1056 to 1060, wherein the gene elements have undergone replication.

[0151] 1062. The complex according to any one of embodiments 1056 to 1061, wherein the complex is an anestroma.

[0152] 1063. The complex according to any one of embodiments 1056 to 1062, wherein the gene element further comprises a nucleic acid encoding a polypeptide.

[0153] 1064. A complex according to any one of embodiments 1056 to 1063, wherein the gene element does not contain nucleic acid encoding a polypeptide.

[0154] 1065. The complex according to any one of embodiments 1056 to 1064, wherein the gene element includes, for example, a GC-rich region as described herein.

[0155] 1066.GC-rich areas are as follows: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172) Any of the following nucleic acid sequences; or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. The complex according to Embodiment 1065, comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides.

[0156] 1067. Below: (a) Protein outer layer; (b) polypeptide or complex described in any of the prior embodiments; (c) A gene element comprising a promoter operably linked to a nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an endogenous effector or an exogenous effector, as described herein). Includes; Here, the genetic element is enclosed within a proteinaceous outer layer in an anestromorph.

[0157] 1068. Below: (a) Protein outer layer; (b) Below: (i) A promoter operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector, as described herein), and (ii) A gene element comprising a nucleic acid encoding a polypeptide as described in any of the prior embodiments. This includes; where the gene element is confined within a proteinous outer layer, forming an anestroma.

[0158] 1069. Below: (a) Protein outer layer; (b) ORF1 molecule or nucleic acid encoding an ORF1 molecule; (c) A gene element containing a promoter operably linked to a heterogeneous nucleic acid sequence (e.g., DNA sequence) that codes for an effector. Includes; Here, the genetic element is enclosed within a proteinaceous outer layer in an anestromorph.

[0159] 1070. Below: (a) Protein outer layer; (b) ORF1 molecule or nucleic acid encoding an ORF1 molecule; (c) Promoter element and nucleic acid sequence (e.g., DNA sequence) encoding an effector (e.g., an external effector or an endogenous effector), and nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. A gene element comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides. In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; optionally, the gene elements are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0160] 1071. Below: (a) Protein outer layer; (b) ORF1 molecule or nucleic acid encoding an ORF1 molecule; (c) A gene element comprising a promoter element, a nucleic acid (e.g., a DNA sequence) encoding an effector (e.g., an exogenous or endogenous effector), and a sequence containing at least 20 (e.g., at least 20, 25, 30, 31, 32, 33, 34, 35, or 36) consecutive nucleotides having a GC content of at least 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%) In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; optionally, the gene elements are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0161] 1072. Below: (a) Protein outer layer; (b) An ORF1 molecule or a nucleic acid that encodes an ORF1 molecule, (i) At least 30% of the amino acids of the ORF1 molecule (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) are part of the β-chain; (ii) The secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β-chains; (iii) The secondary structure of the ORF1 molecule contains at least a β-chain:α-helix ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. ORF1 molecules or nucleic acids encoding ORF1 molecules; and (c) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector), and a protein-binding sequence. In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; optionally, the gene elements are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0162] 1073. Below: (a) Protein outer layer; (b) ORF1 molecule or nucleic acid encoding an ORF1 molecule; (c) A gene element comprising a promoter element, a nucleic acid (e.g., a DNA sequence) encoding an effector (e.g., an external or endogenous effector), and a protein-binding sequence. In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; optionally, the gene elements are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0163] 1074. Below: (a) Protein outer layer; (b) A promoter element and a nucleic acid (e.g., a DNA sequence) that encodes an effector (e.g., an external effector or an endogenous effector), and the nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. A gene element comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides. In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; The gene elements are optional and are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0164] 1075. Below: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid (e.g., a DNA sequence) encoding an effector (e.g., an exogenous or endogenous effector), and a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%. In anesomes containing; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; Anerosomes are configured to deliver gene elements into eukaryotic cells; The gene elements are optional and are as follows: (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) An anestroma that, for example, does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence, as described herein.

[0165] 1076. Below: (a) Protein outer layer; (b) In an anestroma comprising a gene element comprising a promoter element and a nucleic acid (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic effector or an endogenous effector); The gene elements are as follows: Nucleic acid sequence: CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) It includes a region having at least 95% (e.g., at least 95, 96, 97, 98, 99, or 100%) sequence identity with respect to the nucleic acid sequence encoding the effector (e.g., a packaging region located 3' to the effector); The gene elements are confined within a proteinaceous outer layer; Here, an anestronomy is an anestronomy configured to deliver genetic elements into eukaryotic cells.

[0166] 1076A. Below: (i) a promoter element and a gene element comprising a nucleic acid sequence encoding a therapeutic external effector (the gene element comprising a sequence having at least 95% sequence identity to a 5'UTR nucleotide sequence derived from an anerovirus described herein (e.g., as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17); and / or (ii) A protein outer layer comprising a polypeptide having at least 95% sequence identity with the polypeptide encoded by the ORF1 gene of an anelovirus described herein (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17). In anesomes containing; The gene elements are confined within a proteinaceous outer layer; An anestronomy is a type of anestronomy that, by choice, can deliver genetic elements into mammalian cells.

[0167] 1076B. and below: (I) Below: (a) a promoter element; (b) a nucleic acid sequence encoding an external effector (e.g., an external effector as described herein), wherein the nucleic acid sequence is operably linked to the promoter element; (c) the following: (c)(i) The nucleic acid sequence of nucleotides 323-393 of Sequence ID No. 54, or a nucleic acid sequence that is at least 85% identical thereto; (c)(ii) Any nucleic acid sequence of sequence number 113, sequence number 114, sequence number 115, sequence number 116, sequence number 117, sequence number 118, or sequence number 119, or a nucleic acid sequence that is at least 85% identical to it; or (c)(iii) The nucleic acid sequence of nucleotides 117-187 of sequence number 61, or a nucleic acid sequence that is at least 85% identical thereto. A gene element containing a 5'UTR domain; (II) Protein outer layer containing ORF1 molecule In anesomes containing, The gene element is confined within a proteinaceous outer layer; and Synthetic anestrosomes are anestrosomes that can deliver genetic elements into mammalian cells, such as human cells.

[0168] 1077. An anestrome according to any of the prior embodiments, wherein the proteinaceous outer layer contains an ORF1 molecule.

[0169] 1078. An anestrome according to any of the prior embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the protein in the proteinate outer layer comprises an ORF1 molecule.

[0170] 1079. An anestrome according to any of the prior embodiments, wherein 1% or less of the protein in the proteinaceous outer layer (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or less) comprises ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules.

[0171] An anestrome according to any of the prior embodiments, wherein the 1080.ORF1 molecule contains amino acids that have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the ORF1 protein encoded by any of the sequences listed in Table A1-A12, B1-B5, C1-C5, 1-18, 20-37, or D1-D10.

[0172] 1081. An anestrome according to any of the prior embodiments, wherein the ORF1 molecule comprises the polypeptide described in any of the prior embodiments.

[0173] 1082. An anestrome according to any of the prior embodiments, wherein the gene element further comprises a nucleic acid sequence encoding an ORF1 molecule.

[0174] 1083. An anestrome according to any of the prior embodiments, wherein the gene element does not contain a nucleic acid sequence encoding the ORF1 molecule.

[0175] 1084. An anestrome according to any of the prior embodiments, wherein the gene element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 80%.

[0176] 1085. An anestrome according to any of the prior embodiments, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0177] 1086. An anestrome according to any of the prior embodiments, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 80%.

[0178] 1087. In an isolated nucleic acid composition comprising nucleic acids encoding a polypeptide described in any of the prior embodiments (for example, comprising one, two, or more nucleic acid molecules), Optionally, an isolated nucleic acid composition may have at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0179] 1088. In an isolated nucleic acid composition (for example, containing one, two, or more nucleic acid molecules), the isolated nucleic acid composition contains a gene element that encodes an ORF1 molecule; Here, (i) At least 30% of the amino acids in the ORF1 molecule (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) are part of the β-sheet; (ii) The secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β-sheets; (iii) The secondary structure of the ORF1 molecule includes at least a β-sheet:α-helix ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; A gene element comprises a promoter element, a nucleic acid sequence encoding an effector (e.g., an external or endogenous effector), and a proteinoid outer layer; The gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0180] 1089. Below: (a) Genetic elements that encode the ORF1 molecule; (b) Nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them; and (c) At least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), e.g., insertion, substitution, enzymatic modification, and / or deletion, e.g., deletion of one or more of the following domains (e.g., TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) In an isolated nucleic acid composition containing (for example, containing one, two, or more nucleic acid molecules); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0181] 1090. Below: (a) Genetic elements that encode the ORF1 molecule; (b) an isolated nucleic acid composition having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6% and containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides (for example, containing 1, 2, or more nucleic acid molecules); The isolated nucleic acid composition has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration), such as insertion, substitution, enzymatic modification, and / or deletion, such as deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region), compared to the wild-type anellovirus genome sequence (e.g., as described herein); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0182] 1090A. An isolated nucleic acid composition (for example, comprising 1, 2, or more nucleic acid molecules), wherein the isolated nucleic acid composition comprises a gene element containing a 5'UTR nucleotide sequence derived from an anellovirus (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17) as described herein.

[0183] An isolated nucleic acid composition according to any of embodiments 1089 to 1090, wherein 1091.(a) and (b) are part of the same nucleic acid.

[0184] An isolated nucleic acid composition according to any of embodiments 1089 to 1091, wherein 1092.(a) and (b) are parts of different nucleic acids.

[0185] 1093. The gene elements include: TATA box, initiator element, cap region, transcription start site, 5'UTR conserved domain, ORF1 coding sequence, ORF1 / 1 coding sequence, ORF1 / 2 coding sequence, ORF2 coding sequence, ORF2 / 2 coding sequence, ORF2 / 3 coding sequence, ORF2 / 3t coding sequence, 3 open reading frame region, poly(A) signal, and / or anerovirus as described herein. An isolated nucleic acid composition according to any of the prior embodiments, comprising one or more sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an anellovirus (as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17) GC-rich regions, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0186] 1094. An isolated nucleic acid composition according to any of the prior embodiments, wherein the gene element further comprises an anellovirus genome sequence (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, as described herein), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0187] 1095. The isolated nucleic acid composition according to Embodiment 1094, further comprising at least one additional copy (e.g., 1, 2, 3, 4, 5, or 6 copies) of an anellovirus genome sequence or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0188] 1096. An isolated nucleic acid composition according to any of the prior embodiments, further comprising at least one additional copy of a gene element (e.g., a total of 1, 2, 3, 4, 5, or 6 copies).

[0189] 1097. Nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. At least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides; and At least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as insertions, substitutions, enzymatic modifications, and / or deletions, such as deletions of one or more of a specific domain (e.g., TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signaling, or GC-rich region). In an isolated nucleic acid composition containing (for example, containing one, two, or more nucleic acid molecules); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0190] In an isolated nucleic acid composition having a GC content of 1098.70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%, comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides (for example, comprising 1, 2, or more nucleic acid molecules); The isolated nucleic acid composition comprises at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); Optionally, the nucleic acid molecules are as follows: (i) For example, deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence, as described herein; (ii) For example, deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence, as described herein; and / or (iii) For example, as described herein, at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence An isolated nucleic acid composition that does not contain any of the above.

[0191] 1099.ORF1 molecule is an isolated nucleic acid composition according to any of the prior embodiments, comprising the polypeptide described in any of the prior embodiments.

[0192] 1100. An isolated nucleic acid composition according to any of the prior embodiments, comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 80%.

[0193] An isolated nucleic acid composition according to any of the prior embodiments, comprising at least 36 consecutive nucleotides having a GC content of 1101.70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0194] 1102. An isolated nucleic acid composition according to any of the prior embodiments, comprising at least 36 consecutive nucleotides having a GC content of at least 80%.

[0195] 1103. An isolated nucleic acid composition according to any of the prior embodiments, further comprising one or more nucleic acid sequences encoding promoter elements, effectors (e.g., external effectors or endogenous effectors), and / or protein-binding sequences (e.g., external protein-binding sequences).

[0196] 1104. An isolated nucleic acid composition according to any of the prior embodiments, comprising at least 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides of a nucleic acid sequence having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto, or a nucleic acid sequence having at least 100, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto.

[0197] 1105. Nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); An isolated nucleic acid molecule (e.g., an expression vector) containing a nucleic acid sequence having at least 95% sequence identity (e.g., at least 95, 96, 97, 98, 99, or 100%) to [the specified target].

[0198] 1106. An isolated nucleic acid composition according to any of the prior embodiments, wherein the isolated nucleic acid molecule is cyclic.

[0199] 1107. Below: (a) a nucleic acid encoding a polypeptide described in any of the prior embodiments (the nucleic acid is a plasmid, a viral nucleic acid, or is incorporated into a cell chromosome), and (b) A gene element comprising 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, wherein the gene element optionally encodes an ORF1 polypeptide (e.g., an ORF1 protein). Isolated cells containing these cells.

[0200] 1108. Below: (a) a nucleic acid that encodes the ORF1 molecule (the nucleic acid is either a plasmid, a viral nucleic acid, or integrated into a cell chromosome), and (b) A gene element comprising 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. Isolated cells containing, for example, host cells.

[0201] 1109. Below: (a) a nucleic acid that encodes the ORF1 molecule (the nucleic acid is either a plasmid, a viral nucleic acid, or integrated into a cell chromosome), and (b) A gene element that does not encode an ORF1 molecule, wherein the gene element comprises 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. Isolated cells containing, for example, host cells.

[0202] 1109A. Below: (i) a nucleic acid molecule (e.g., a first nucleic acid molecule) containing the nucleic acid sequence of an anestrom gene element (e.g., a gene element that does not encode the ORF1 molecule) as described herein, and (ii) A nucleic acid molecule, e.g., a second nucleic acid molecule, which optionally encodes one or more amino acid sequences having at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence selected from, for example, any of the sequences listed in Table 16, or one or more amino acid sequences having at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) such amino acid sequences, e.g., a second nucleic acid molecule. Isolated cells containing, for example, host cells.

[0203] 1110. An isolated cell according to any of the prior embodiments, wherein a gene element that does not encode the ORF1 molecule encodes a fragment of the ORF1 molecule, for example, a fragment that does not form a capsid, for example, a fragment of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 20, or less than 10 nucleotides.

[0204] 1111. Isolated cells containing nucleic acids encoding the ORF1 molecule (for example, the nucleic acid is a plasmid, a viral nucleic acid, or is incorporated into a cell chromosome), for example, in a host cell, the isolated cells do not contain one or more of the ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules.

[0205] 1112. Isolated cells, such as host cells, comprising the nucleic acid composition described in any of the prior embodiments.

[0206] 1113. A helper nucleic acid (e.g., plasmid or viral nucleic acid) that encodes an ORF1 molecule, wherein isolated cells do not contain one or more of the ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules.

[0207] 1114. Below: (a) Isolated cells as described herein, and (b) An anestroms described herein A composition containing the following:

[0208] 1115. Below: (a) A cell containing nucleic acid that encodes the ORF1 molecule (for example, the nucleic acid is a plasmid, a viral nucleic acid, or is incorporated into a cell chromosome), and (b) A gene element that does not encode an ORF1 molecule (e.g., intracellular or extracellular, e.g., in a cell culture medium) wherein the gene element comprises a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic effector or an endogenous effector), and a protein-binding sequence. A composition containing the following:

[0209] 1116. A pharmaceutical composition comprising a polypeptide, complex, anestroma, or isolated nucleic acid described in any of the prior embodiments, and a pharmaceutically acceptable carrier and / or excipient.

[0210] 1117. A method for producing ORF1 molecules, wherein the method is as follows: (a) the step of preparing a host cell (e.g., a host cell as described herein) containing a nucleic acid encoding a polypeptide as described in any of the prior embodiments, and (b) A step of maintaining host cells under conditions that allow cells to produce polypeptides, thereby producing the ORF1 molecule. A method that includes this.

[0211] 1118. A method for producing ORF1 molecules, wherein the method is as follows: (a) the step of preparing a host cell containing a nucleic acid composition described in any of the prior embodiments (for example, a host cell as described herein), and (b) A step of maintaining host cells under conditions that allow cells to produce polypeptides, thereby producing the ORF1 molecule. A method that includes this.

[0212] 1119. The method according to embodiment 1117 or 1118, wherein the host cell is a helper cell.

[0213] 1120. The method according to Embodiment 1119, wherein the helper cells include one or more additional nucleic acids encoding one or more additional ORFs of wild-type anellovirus (e.g., ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules), as described herein.

[0214] 1121. The method according to any one of embodiments 1117 to 1120, wherein nucleic acids are incorporated into the genome of a host cell.

[0215] 1122. The method according to any one of embodiments 1117 to 1121, wherein the host cell produces at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 copies (e.g., at least about 60 copies) of polypeptide per host cell.

[0216] 1123. The method according to any one of embodiments 1117 to 1122, wherein a host cell produces at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 10,000, or 100,000 copies (e.g., at least about 60 copies) of polypeptide per anestronomy produced by the host cell.

[0217] 1124. The method according to any one of embodiments 1117 to 1123, wherein the method comprises the steps of preparing a plurality of host cells and maintaining the host cells under conditions that enable the production of at least 1,000 copies of polypeptide per cell.

[0218] 1125. Multiple host cells, at least approximately 1 × 10⁻¹⁶ 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 9×10 8 , 1 x 10 9, 1 x 10 10 , 1 x 10 11 , or 1 × 10 12 The method according to Embodiment 1124, for producing a copy of the polypeptide.

[0219] 1126. A method for producing an anestrom composition, wherein the method is as follows: (a) A step of preparing helper cells, for example, the helper cells described herein; (b) The step of introducing a gene element into helper cells under conditions that enable the cells to produce anestrom, (c) For example, the step of formulating anestromis as a pharmaceutical composition suitable for administration to a subject. Includes, This provides a method for preparing an anestrom composition.

[0220] 1127. A method for producing an anestrom composition, wherein the method is as follows: (a) Step of preparing host cells; (b) Step of introducing helper cells into host cells; (c) The step of introducing a gene element into a host cell under conditions that enable the cell to produce an anestroma (for example, before, after, or simultaneously with (b)), and (d) For example, the step of formulating anestromis as a pharmaceutical composition suitable for administration to a subject. Includes, This provides a method for preparing an anestrom composition.

[0221] 1128. A method for producing an anestrom composition, wherein the method is as follows: (a) A step of preparing helper cells containing nucleic acids that encode the ORF1 molecule (for example, the nucleic acid is a plasmid, a viral nucleic acid, or is incorporated into the helper cell chromosome); (b) A step of introducing helper cells into host cells under conditions that enable the cells to produce anestrom, wherein the gene element does not encode an ORF1 molecule, and the gene element comprises a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector), and a protein binding sequence; and (c) For example, the step of formulating anestromis as a pharmaceutical composition suitable for administration to a subject. Includes, This provides a method for preparing an anestrom composition.

[0222] 1129. A method for producing an anestrom composition, wherein the method is as follows: (a) Step of preparing host cells; (b) The step of introducing a helper nucleic acid encoding the ORF1 molecule (for example, the nucleic acid is a plasmid or a viral nucleic acid) into a host cell; and (b) A step of introducing helper cells into host cells under conditions that enable the cells to produce anestrom, wherein the gene element does not encode an ORF1 molecule, and the gene element comprises a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector), and a protein binding sequence. Includes, This is a method for producing anesomes.

[0223] 1130. The method according to any of the prior embodiments, further comprising the step of separating anestrophores from helper cells or host cells.

[0224] 1131. The method according to any of the prior embodiments, wherein the step of preparing helper cells includes the step of introducing a helper nucleic acid into host cells, for example, the helper nucleic acid encoding an ORF1 molecule (for example, the nucleic acid is a plasmid or viral nucleic acid).

[0225] 1132. The method according to any of the prior embodiments, wherein the helper cells contain the ORF1 molecule.

[0226] 1133. Nucleic acids include: TATA box, initiator element, cap site, transcription start site, 5'UTR conserved domain, ORF1 coding sequence, ORF1 / 1 coding sequence, ORF1 / 2 coding sequence, ORF2 coding sequence, ORF2 / 2 coding sequence, ORF2 / 3 coding sequence, ORF2 / 3t coding sequence, 3 open reading frame, poly(A) signal, and / or anerovirus as described herein. The method according to any of the prior embodiments, comprising one or more sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an anellovirus (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17) GC-rich regions, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0227] 1134. The method according to any of the prior embodiments, wherein the nucleic acid comprises an anellovirus genome sequence (for example, as described herein, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0228] 1135. The method according to any of the prior embodiments, wherein the nucleic acid comprises at least one additional copy (e.g., a total of 1, 2, 3, 4, 5, or 6 copies) of an anellovirus genome sequence or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0229] 1136. The method according to any of the prior embodiments, wherein the host cell or helper cell contains at least one additional copy of nucleic acid (e.g., a total of 1, 2, 3, 4, 5, or 6 copies).

[0230] 1137. The method according to any of the prior embodiments, wherein the nucleic acid is cyclic.

[0231] 1137A. Anerosomes, for example, a method for producing synthetic anesomes, the following: a) Below: (i) An anestroma, for example, a nucleic acid molecule comprising the nucleic acid sequence of the gene element of a synthetic anestroma described herein, for example, a first nucleic acid molecule, (ii) For example, a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more amino acid sequences having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as listed in any of Table 16; A step of preparing host cells containing; and b) The step of culturing host cells under conditions suitable for producing anestrom A method that includes this.

[0232] 1137B. The method according to Embodiment 1137A, further comprising the step of introducing a first nucleic acid molecule and / or a second nucleic acid molecule into a host cell prior to step (a).

[0233] 1137C. The method according to Embodiment 1137A or 1137B, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

[0234] 1137D. The method according to embodiment 1137C, wherein a second nucleic acid molecule is incorporated into the genome of a host cell.

[0235] 1137E. The method according to Embodiment 1137C, wherein the second nucleic acid molecule is a helper (e.g., a helper plasmid or the genome of a helper virus).

[0236] 1137F. The method according to any one of Embodiments 1137A to 1137E, wherein the first nucleic acid comprises one or more sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, of a TATA box, initiator element, cap region, transcription start site, 5'UTR conserved domain, and / or GC-rich region derived from an anellovirus described herein (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1 to B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

[0237] 1138. A method for delivering an effector to a subject, the following: (a) Protein outer layer containing ORF1 molecule; (b) A promoter element and a nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an external effector or an endogenous effector), and a nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. A gene element comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides. The step includes administering an anestroma containing the anestroma to a subject; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This is a method for delivering an effects unit to a subject.

[0238] 1139. A method for delivering an effector to a subject, the following: (a) Protein outer layer containing ORF1 molecule; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous or endogenous effector), and a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%. The step includes administering an anestroma containing the anestroma to a subject; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This is a method for delivering an effects unit to a subject.

[0239] 1140. A method for delivering an effector to a subject, the following: (a) Protein outer layer containing ORF1 molecule; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector), and a protein-binding sequence. The step includes administering an anestroma containing the anestroma to a subject; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This is a method for delivering an effects unit to a subject.

[0240] 1141. A method for delivering an effector to target cells, the following: (a) Protein outer layer containing ORF1 molecule; (b) A promoter element and a nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an external effector or an endogenous effector), and a nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. A gene element comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides. The step includes bringing anestrom containing the target cell into contact with the target cell; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This provides a method for delivering effectors to target cells.

[0241] 1142. A method for delivering an effector to target cells, the following: (a) Protein outer layer containing ORF1 molecule; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous or endogenous effector), and a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%. The step includes bringing anestrom containing the target cell into contact with the target cell; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This provides a method for delivering effectors to target cells.

[0242] 1143. A method for delivering an effector to target cells, the following: (a) Protein outer layer containing ORF1 molecule; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an extrinsic or endogenous effector), and a protein-binding sequence. The step includes bringing anestrom containing the target cell into contact with the target cell; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This provides a method for delivering effectors to target cells.

[0243] 1143A. A method for delivering an effector to target cells, the following: (i) A gene element comprising a promoter element and a nucleic acid sequence encoding a therapeutic external effector (the gene element comprises a sequence having at least 95% sequence identity to a 5'UTR nucleotide sequence derived from an anellovirus described herein (e.g., as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17); and / or (ii) A protein outer layer comprising a polypeptide having at least 95% sequence identity with the polypeptide encoded by the ORF1 gene of an anelovirus described herein (for example, as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17). The step includes bringing anestrom containing the target cell into contact with the target cell; Here, the gene element is confined within a proteinaceous outer layer; The gene elements are selected at will. (i) For example, as described herein, it does not contain deletions of nucleotides 3436-3607 compared to the wild-type TTV-tth8 genome sequence; (ii) For example, as described herein, it does not contain deletions of nucleotides 1432-2210 compared to the wild-type TTMV-LY2 genome sequence; and / or (iii) For example, as described herein, it does not contain at least 10¹ nucleotide deletions compared to the wild-type TTMV-LY2 genome sequence; This provides a method for delivering effectors to target cells.

[0244] 1144. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element does not encode the amino acid sequence of NCBI accession number A7XCE8.1.

[0245] 1145. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the ORF1 molecule contains an amino acid sequence having at least 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any of the ORF1 sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

[0246] 1146. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) of the amino acids of the ORF1 molecule are part of a β-sheet.

[0247] 1147. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β-sheets.

[0248] 1148. A polypeptide, complex, anelocome, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the secondary structure of the ORF1 molecule includes at least a β-sheet:α-helix ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0249] 1149. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the ORF1 molecule contains an arginine-rich region (for example, having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the arginine-rich region sequences listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10).

[0250] 1150. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to Embodiment 1149, wherein the arginine-rich region comprises at least 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 consecutive nucleotides containing 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%) arginine residues.

[0251] 1151. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to Embodiment 1149 or 1150, wherein the arginine-rich region is located at the N-terminus or C-terminus of the ORF1 molecule.

[0252] 1152. The arginine-rich region is the amino acid sequence TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (Sequence ID 808), RRRYARPYRRRHIRRYRRRRRHFRRRR (Sequence ID 809), MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (Sequence ID 216), or [ka] A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of Embodiments 1149 to 1151, having at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to [the specified value].

[0253] 1153. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of Embodiments 1149 to 1152, wherein the arginine-rich region has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any arginine-rich region sequence listed in Table A2, A4, A6, A8, A10, A12, C1 to C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 to 37, or D1 to D10.

[0254] 1154. The ORF1 molecule is the jelly roll domain of the ORF1 molecule described herein, for example, the amino acid sequence [ka] A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising a jellyroll domain 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 with a jellyroll domain sequence 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.

[0255] 1155. The ORF1 molecule is the N22 domain of the ORF1 molecule described herein, for example, the amino acid sequence [ka] A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising an N22 domain having an amino acid sequence of an N22 domain having, or an N22 domain 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 with an N22 domain sequence 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.

[0256] 1156. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the ORF1 molecule is localized to the nucleus of a cell.

[0257] 1157. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less sequence identity with respect to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of the wild-type anellovirus genome sequence described herein.

[0258] 1158. Genetic elements or isolated nucleic acid molecules, for example, of approximately 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 330 of the genome sequence of wild-type alpha-torquevirus (e.g., clade 1, 2, or 3 alpha-torquevirus) as described herein. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less sequence identity with respect to 0, 3400, 3450, 3460, 3470, 3480, 3490, 3500, 3510, 3520, 3530, 3540, 3550, 3560, 3570, or 3580 consecutive nucleotides.

[0259] 1159. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less sequence identity with respect to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of the wild-type beta-torquevirus genome sequence described herein.

[0260] 1160. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less sequence identity with respect to approximately 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3120, 3130, 3140, 3141, or 3142 consecutive nucleotides of the wild-type gamma-torquevirus genome sequence described herein.

[0261] 1161. Genetic elements or isolated nucleic acid molecules, for example, of approximately 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3450, 3460, 3470, 3480, 3490, 3500 of the genome sequence of wild-type alpha-torque virus (e.g., clade 1, 2, or 3 alpha-torque virus) as described herein. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to 3510, 3520, 3530, 3540, 3550, 3560, 3570, or 3580 consecutive nucleotides (e.g., approximately 500-3580, 1000-3580, 1500-3580, 2000-3580, or 3000-3580 consecutive nucleotides).

[0262] 1162. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains at least 500, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to, for example, about 500-1000, 500-1100, 500-1200, 500-1219, 1000-1100, 1000-1200, or 1000-1219 consecutive nucleotides of the wild-type beta-torquevirus genome sequence described herein.

[0263] 1163. A gene element or isolated nucleic acid molecule is, for example, approximately 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3120, 3130, 3140, 3141, or 3142 consecutive nucleotides of the wild-type gamma-torquevirus genome sequence described herein. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence (for example, approximately 500-3142, 1000-3142, 1500-3142, 2000-3142, or 2500-3142 consecutive nucleotides).

[0264] 1164. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or less sequence identity to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of the wild-type TTMV-LY2 genome sequence described herein.

[0265] 1165. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains, for example, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or less sequence identity with respect to about 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3550, 3560, 3570, 3580, or 3581 consecutive nucleotides of the wild-type TTV-tth8 genome sequence described herein.

[0266] 1166. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains deletions of at least 1578, 1579, 1580, 1590, 1600, 1650, 1700, 1750, or 2000 nucleotides compared to, for example, the wild-type anellovirus genome sequence described herein.

[0267] 1167. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains deletions of 1-99, 1-90, 1-80, 1-70, 1-60, 1-50, 10-99, 10-90, 10-80, 10-70, 10-60, 10-50, 20-99, 20-90, 20-80, 20-70, 20-60, 20-50, 30-99, 30-90, 30-80, 30-70, 30-60, 30-50, 40-99, 40-90, 40-80, 40-70, 40-60, or 40-50 nucleotides compared to, for example, the wild-type anellovirus genome sequence described herein.

[0268] 1168. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule does not contain a 100-nucleotide deletion, a 172-nucleotide deletion, or a 1577-nucleotide deletion compared to, for example, the wild-type anellovirus genome sequence described herein.

[0269] 1169. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains three or more deletions compared to, for example, the wild-type anellovirus genome sequence described herein.

[0270] 1170. Gene element or isolated nucleic acid molecule, nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising a region having at least 75% (for example, at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to a given.

[0271] 1171. Gene element or isolated nucleic acid molecule, nucleic acid sequence: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising a region having at least 95% (e.g., at least 95, 96, 97, 98, 99, or 100%) sequence identity with respect to.

[0272] 1172. Gene element or isolated nucleic acid molecule, nucleic acid sequence: [ka] A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising a nuclear region having at least 75% (for example, at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to a given.

[0273] 1173. Gene element or isolated nucleic acid molecule, nucleic acid sequence: [ka] A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising a region having at least 75% (for example, at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to a given.

[0274] 1174. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 80%.

[0275] 1175. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule comprises at least 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0276] 1176. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule comprises at least 36 consecutive nucleotides having a GC content of at least 80%.

[0277] 1177. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, further comprising nucleic acid sequences encoding an anelovirus, for example, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of a wild-type anelovirus described herein.

[0278] 1178. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the nucleic acid sequence encoding a promoter element, effector, or protein binding sequence 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 with respect to, for example, the nucleic acid sequence encoding a promoter element, effector, or protein binding sequence of any of the anelloviruses listed in Tables A1-A12, B1-B5, C1-C5, or 1-18 described herein.

[0279] 1179. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule includes a packaging region located 3' to the nucleic acid sequence encoding an effector.

[0280] 1180. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule includes a packaging region located 5' to the nucleic acid sequence encoding an effector.

[0281] 1181. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule comprises a nucleic acid sequence encoding an anellovirus protein having 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 with respect to, for example, the amino acid sequences of ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of the anellovirus described herein.

[0282] 1182. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule comprises single-stranded DNA.

[0283] 1183. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein a gene element or isolated nucleic acid molecule is incorporated into the genome of a eukaryotic cell at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements that enter the cell.

[0284] 1184. The gene element or isolated nucleic acid molecule is a wild-type anellovirus sequence (e.g., wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTV)). Anellovirus (TTMV) or TTMDV sequences, for example, wild-type anellovirus sequences (as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or approximately 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1 A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, having at least 75% (for example, at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to a portion thereof consisting of 700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 consecutive nucleotides.

[0285] 1185. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the protein-binding sequence has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with the consensus 5'UTR sequence shown in Table 20.

[0286] 1186. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the protein-binding sequence has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with the consensus GC-rich sequences shown in Table 21.

[0287] 1187. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the protein-binding sequence has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with the consensus 5'UTR sequence shown in Table 38 and the GC-rich sequence shown in Table 39.

[0288] 1188. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains a sequence having at least 85% sequence identity to the anellovirus 5'UTR conserved domain of any of the nucleic acid sequences in Table A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0289] 1189. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element or isolated nucleic acid molecule contains a sequence having at least 85% sequence identity with respect to the anellovirus GC-rich region of any of the nucleic acid sequences in Table A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0290] 1190. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the promoter element 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 virus promoter, a tissue-specific, U6(pollIII), or a minimal CMV promoter having an upstream DNA binding site for an activator protein (such as TetR-VP16, Gal4-VP16, or dCas9-VP16).

[0291] 1191. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the effector encodes a therapeutic agent, such as a therapeutic peptide or polypeptide, or a therapeutic nucleic acid.

[0292] 1192. Effectors include regulatory nucleic acids, such as miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or markers, antigens, peptides, synthetic or analogous peptides derived from naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degrading or self-destructive peptides, small molecules, immune effectors (e.g., those that affect sensitivity to immune responses / signals), and cell death-inducing proteins (apoptosis). A polypeptide, complex, anestrophy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, comprising (or a necrosis inducer), a non-soluble tumor inhibitor (e.g., an oncoprotein inhibitor), an epigenetic modifier, an epigenetic enzyme, a transcription factor, a DNA or protein modifying enzyme, a DNA insertion agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor of a single 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.

[0293] 1193. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the anestronomy is capable of autonomous replication.

[0294] 1194. An isolated nucleic acid molecule according to any of the prior embodiments, wherein the expression vector is selected from the group consisting of plasmids, cosmids, artificial chromosomes, phages, and viruses.

[0295] 1195. Isolated cells comprising isolated nucleic acids or anestrophores as described in any of the prior embodiments.

[0296] 1196. Isolated cells according to Embodiment 195, further comprising an anelovirus, for example, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of the ananelovirus described herein.

[0297] 1197. A method for delivering an effector to a subject, comprising the step of administering to the subject a polypeptide, complex, anestroma, isolated nucleic acid, isolated cell, or composition described in any of the prior embodiments; wherein the gene element or isolated nucleic acid molecule encodes an effector, and the effector is expressed in the subject.

[0298] 1198. A method for treating a disease or disorder of a subject in need thereof, comprising the step of administering to the subject a polypeptide, complex, anestroma, isolated nucleic acid, isolated cell, or composition described in any of the prior embodiments; wherein the gene element or isolated nucleic acid molecule encodes a therapeutic agent, and the therapeutic agent is expressed in the subject.

[0299] 1199. A method for delivering an effector to ex vivo cells or a population of cells (e.g., cells or a population of cells taken from a subject), comprising the step of introducing a polypeptide, complex, anestroma, isolated nucleic acid, isolated cell, or composition described in any of the prior embodiments into the cells or population of cells; wherein the gene element or isolated nucleic acid molecule encodes the effector, and the effector is expressed in the cells or population of cells.

[0300] 1200. An anestrome according to any of the prior embodiments, wherein the gene element is single-stranded DNA and has one or both of the following characteristics: it is circular, and / or is incorporated into the genome of a eukaryotic cell at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements that enter the cell.

[0301] 1201. An anestrome according to any of the prior embodiments, wherein the gene elements have at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with wild-type anellovirus sequences (e.g., wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequences, e.g., wild-type anellovirus sequences, e.g., as listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

[0302] 1202. An anestrome according to any of the prior embodiments, wherein the protein-binding sequence has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with the consensus 5'UTR sequence shown in Table 38, or the consensus GC-rich sequence shown in Table 39, or both.

[0303] 1203. An anestrome according to any of the prior embodiments, wherein the promoter element includes 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 virus promoter, a tissue-specific, U6(pollIII), or a minimal CMV promoter having an upstream DNA binding site for activator proteins (such as TetR-VP16, Gal4-VP16, or dCas9-VP16).

[0304] 1204. An anestrome according to any of the prior embodiments, wherein the promoter element comprises a TATA box.

[0305] 1205. An anestrome according to any of the prior embodiments, wherein the promoter element is endogenous to wild-type anellovirus, for example, wild-type anellovirus sequences listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17.

[0306] 1206. An anestrome according to any of the prior embodiments, wherein the promoter element is exothermic to wild-type anellovirus, for example, wild-type anellovirus sequences listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17.

[0307] 1207. An anestrome according to any of the prior embodiments, wherein the effector encodes a therapeutic agent, such as a therapeutic peptide or polypeptide, or a therapeutic nucleic acid.

[0308] 1208. An anestronomy according to any of the prior embodiments, wherein the effector comprises regulatory nucleic acids, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or markers, antigens, peptides, synthetic or analogous peptides derived from naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degrading or self-destructive peptides, small molecules, immune effectors (e.g., those that affect sensitivity to immune responses / signals), cell death-inducing proteins (inducers of apoptosis or necrosis), non-soluble tumor inhibitors (e.g., inhibitors of oncoproteins), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein-modifying enzymes, DNA insertion agents, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors of one enzyme, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands, antibodies, receptors, or CRISPR systems or components.

[0309] 1209. An anestrome according to any of the prior embodiments, wherein the effector contains miRNA.

[0310] 1210. An anestrome according to any of the prior embodiments, wherein the effector, for example, miRNA, targets a host gene, for example, regulates gene expression, for example, increases or decreases gene expression.

[0311] 1211. An anestrome according to any of the prior embodiments, wherein the effector contains miRNA and reduces the expression of a host gene.

[0312] 1212. An anestrome according to any of the prior embodiments, wherein the effector comprises a nucleic acid sequence having a length of approximately 20-200, 30-180, 40-160, 50-140, or 60-120 nucleotides.

[0313] 1213. An anestrome according to any of the prior embodiments, wherein the nucleic acid sequence encoding the effector is approximately 20-200, 30-180, 40-160, 50-140, or 60-120 nucleotides long.

[0314] 1214. An anestrome according to any of the prior embodiments, wherein the sequence encoding the effector has a size of at least about 100 nucleotides.

[0315] 1215. An anestrome according to any of the prior embodiments, wherein the sequence encoding the effector has a size of at least about 100 to about 5000 nucleotides.

[0316] 1216. An anestrome according to any of the prior embodiments, wherein the sequence encoding the effector has a size of approximately 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, or 1500-2000 nucleotides.

[0317] 1217. An anestrome according to any of the prior embodiments, wherein the sequence encoding the effector is located inside or adjacent to (for example, on the 5' or 3' side of) one or more of the following: the ORF1 locus of a gene element (e.g., the C-terminus of the ORF1 locus), the miRNA locus, the 5' non-coding region upstream of the TATA box, the 5' UTR, the 3' non-coding region downstream of the poly-A region, or the non-coding region upstream of the GC-rich region.

[0318] 1218. An anestrome according to Embodiment 1217, wherein the sequence encoding the effector is located between the poly-A region and the GC-rich region of the gene element.

[0319] 1219. An anestrome according to any of the prior embodiments, wherein the protein-binding sequence includes a nucleic acid sequence having sequence identity of 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%) with respect to the 5'UTR conserved domain or GC-rich domain of wild-type anellovirus, for example, any of the wild-type anelloviruses listed in Table A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17 of the wild-type anellovirus sequence.

[0320] 1220. Gene elements, for example, the protein binding sequence of a gene element, is as follows: (i) Consensus 5'UTR nucleic acid sequences shown in Table 38; (ii) Exemplary TTV 5'UTR nucleic acid sequences shown in Table 38; (iii) TTV-CT30F 5'UTR nucleic acid sequence shown in Table 38; (iv) TTV-HD23a 5'UTR nucleic acid sequence shown in Table 38; (v) TTV-JA20 5'UTR nucleic acid sequence shown in Table 38; (vi) The TTV-TJN02 5'UTR nucleic acid sequence shown in Table 38; (vii) TTV-tth8 5'UTR nucleic acid sequence shown in Table 38; (viii) Consensus GC-rich regions shown in Table 39; (ix) Exemplary TTV GC-rich regions shown in Table 39; (x) TTV-CT30F GC-rich region shown in Table 39; (xi) TTV-JA20 GC-rich regions shown in Table 39; (xii) TTV-TJN02 GC-rich region shown in Table 39; (xiii) TTV-HD23a GC-rich region shown in Table 39; (xiv) TTV-tth8 GC-rich regions shown in Table 39 An anestrome according to any of the prior embodiments, having at least about 75% identity (for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the above.

[0321] 1221. An anestrome according to any of the prior embodiments, wherein the proteinaceous outer layer comprises an external protein capable of specifically binding to a protein-binding sequence.

[0322] 1222. An anestrome according to any of the prior embodiments, wherein the proteinaceous outer layer comprises one or more of the following: one or more glycosylated proteins, a hydrophilic DNA-binding 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 crosslinks.

[0323] 1223. An anestrome according to any of the prior embodiments, wherein the proteinaceous outer layer comprises one or more of the following features: icosahedral symmetry, recognition and / or binding to molecules that interact with one or more host cells and mediate entry into the host cells, deletion of lipid molecules, deletion of carbohydrates, pH and temperature stability, surfactant resistance, and being substantially non-immunogenic or substantially non-pathogenic in host cells.

[0324] 1224. An anestrome according to any of the prior embodiments, wherein the proteinaceous outer layer comprises at least one functional domain that provides one or more functions, e.g., species and / or tissue and / or cell targeting, gene element binding and / or packaging, immune evasion (substantially non-immunogenic and / or tolerant), pharmacokinetics, endocytosis and / or cell adhesion, nuclear entry, intracellular regulation and localization, exocytosis regulation, proliferation, and nucleic acid protection.

[0325] 1225. An anestrome according to any of the prior embodiments, wherein the portion of the gene element excluding the effector has a total size of approximately 2.5–5 kb (e.g., approximately 2.8–4 kb, approximately 2.8–3.2 kb, approximately 3.6–3.9 kb, or approximately 2.8–2.9 kb), less than approximately 5 kb (e.g., approximately 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).

[0326] 1226. An anestrome according to any of the prior embodiments, wherein the gene element is single-stranded.

[0327] 1227. An anestrome according to any of the prior embodiments, wherein the gene element is circular.

[0328] 1228. An anestrome according to any of the prior embodiments, wherein the gene element is DNA.

[0329] 1229. An anestrome according to any of the prior embodiments, wherein the gene element is a minus-strand DNA.

[0330] 1230. An anestrome according to any of the prior embodiments, wherein the gene element comprises an episome.

[0331] 1231. An anestrome according to any of the prior embodiments, wherein the anestrome has a lipid content of 10%, 5%, 2%, or less than 1% by weight, and for example, does not contain a lipid bilayer.

[0332] 1232. An anestrosome according to any of the prior embodiments, wherein the anestrosome is resistant to degradation by surfactants (e.g., neutral detergents, e.g., bile salts, e.g., sodium deoxycholate) compared to viral particles containing an outer lipid bilayer, e.g., retroviruses.

[0333] 1233. An anestrome according to Embodiment 58, wherein at least about 50% (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%) of the anestrome remains undegraded after incubation with a surfactant (e.g., 0.5% by weight of the surfactant) at 37°C for 30 minutes.

[0334] 1234. An anestrome according to any of the prior embodiments, wherein the gene element includes a deletion of at least one element, for example, an element listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, compared to a wild-type anellovirus sequence, for example, a wild-type TTV sequence or a wild-type TTMV sequence.

[0335] 1235. The gene elements are as follows: (i) TTV-tth8 sequences, e.g., nucleotides 3436-3607 of the nucleic acid sequences shown in Table 5; (ii) TTMV-LY2 sequences, e.g., nucleotides 574-1371 and / or nucleotides 1432-2210 of the nucleic acid sequences shown in Table 15; (iii) TTMV-LY2 sequence, e.g., nucleotides 1372-1431 of the nucleic acid sequence shown in Table 15; or (iv) TTMV-LY2 sequence, for example, nucleotides 2610-2809 of the nucleic acid sequence shown in Table 15 An anestrome according to Embodiment 1234, comprising a deletion containing a nucleic acid sequence corresponding to [the specified value].

[0336] 1236. An anestrome according to any of the prior embodiments, wherein the gene element comprises at least 72 nucleotides (e.g., at least 73, 74, 75 nt, etc., optionally less than the full length of the genome) of a wild-type anellovirus sequence, for example, wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequence, for example, one sequence listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0337] 1237. An anestrome according to any of the prior embodiments, wherein the gene element further comprises one or more sequences from the following: sequences encoding one or more miRNAs, sequences encoding one or more replication proteins, sequences encoding extragenic genes, sequences encoding therapeutic agents, regulatory sequences (e.g., promoters, enhancers), sequences encoding one or more regulatory sequences targeting endogenous genes (siRNA, lncRNA, shRNA), sequences encoding therapeutic mRNA or proteins, and sequences encoding cytolytic / cytotoxic RNA or proteins.

[0338] 1238. An anestrome according to any of the prior embodiments, wherein the anestrome further comprises a second gene element, for example, a second gene element confined within a proteinaceous outer layer.

[0339] 1239. An anestrome according to Embodiment 1238, wherein the gene element comprises a protein-binding sequence, e.g., an external protein-binding sequence, e.g., a packaging signal, e.g., as described herein, e.g., a 5'UTR-conserved domain or a GC-rich region.

[0340] 1240. An anestrosome according to any of the prior embodiments, wherein the anestrosome does not infect bacterial cells to a detectable extent, for example, less than 1%, 0.5%, 0.1%, or 0.01% of bacterial cells.

[0341] 1241. An anestrosome according to any of the prior embodiments, which can infect mammalian cells, such as human cells, such as immune cells, hepatocytes, and epithelial cells, for example, in vitro.

[0342] 1242. An anestroke according to any of the prior embodiments, wherein the gene elements are incorporated at frequencies of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, and 0.1% of the anestrokes entering the cell, and for example, the anestrokes are non-integrated.

[0343] 1243. When gene elements are measured, for example by quantitative PCR assay, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10 per cell. 2 , 2×10 2 , 5×10 2 , 10 3 , 2×10 3 , 5×10 3 , or 10 4 An anestrosome according to any of the prior embodiments, which can replicate (for example, by rolling circle replication) a genome equivalent of a gene element, for example, which can be produced.

[0344] 1244. When gene elements are measured, for example by a quantitative PCR assay, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10 in one cell compared to those present in the anestroma before delivery of the gene element into the cell. 2 , 2×10 2 , 5×10 2 , 10 3 , 2×10 3 , 5×10 3 , or 10 4 An anestrosome according to any of the prior embodiments, which can replicate a large genome equivalent of a gene element (for example, by rolling circle replication), for example, which can be produced.

[0345] 1244A. An anestrome according to embodiment 1243 or 1244, wherein the protein outer layer is supplied to the gene element in a cis and / or trans configuration.

[0346] 1244B. An anestrome according to any one of embodiments 1243 to 1244A, wherein a helper nucleic acid (e.g., a helper virus) in the cell encodes a proteinaceous outer layer or a portion thereof (e.g., an ORF1 molecule).

[0347] 1244C. An anestrome according to any of embodiments 1243 to 1244B, wherein one or more replication factors (e.g., replicases) are supplied to the gene element in a cis and / or trans configuration.

[0348] 1244D. An anestrome according to Embodiment 1244C, wherein a helper nucleic acid (e.g., a helper virus) in the cell encodes one or more replication factors.

[0349] 1245. An anestrome according to any of the prior embodiments, wherein the gene element is non-replicable, for example, the gene element is modified at the origin of replication or does not have an origin of replication.

[0350] 1246. An anestrome according to any of the prior embodiments, wherein the gene element is non-self-replicating and can be replicated without, for example, being incorporated into the host cell genome.

[0351] 1247. An anestrosomal according to any of the prior embodiments, wherein the anestrosomal is substantially nonpathogenic and does not induce, for example, any detectable adverse symptoms in a subject (e.g., increased cell death or toxicity compared to a subject not exposed to the anestrosomal).

[0352] 1248. An anestrosome according to any of the prior embodiments, which is substantially non-immunogenic and, for example, does not induce a detectable and / or unwanted immune response when detected according to the method described in Example 4.

[0353] 1249. The anerosome according to Embodiment 1248, wherein the substantially non-immunogenic anerosome has an efficacy in a subject that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the efficacy in a control subject lacking an immune response.

[0354] 1250. An annealosome according to Embodiment 1248 or 1249, wherein the immune response comprises one or more of the following: an antibody or portion thereof specific to an annealosome, or a product encoded by the nucleic acid thereof; a cellular response to an annealosome or an annealosome-containing cell (e.g., an immune effector cell (e.g., a T cell or NK cell) response); or macrophage phagocytosis of an annealosome or an annealosome-containing cell.

[0355] 1251. An anestrosome according to any of the prior embodiments, wherein the anestrosome is less immunogenic than AAV, for example, inducing an immune response lower than that detected for an equivalent amount of AAV when measured by the assay described herein, and inducing an antibody positivity rate of less than 70% (e.g., about 60%, 50%, 40%, 30%, 20%, or less than 10%) when measured by the assay described herein, or is substantially non-immunogenic.

[0356] 1252. An anestrome according to any of the prior embodiments, wherein a population of at least 1000 anestromes can deliver at least 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 gene element into one or more eukaryotic cells.

[0357] 1253. An anestrosomal population (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 gene elements per cell) can deliver gene elements to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a population of eukaryotic cells, for example, the eukaryotic cells being HEK293T cells as described in Example 22, as described in any of the prior embodiments.

[0358] 1254. Populations of anelosomes (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 gene elements per cell) in a population of eukaryotic cells, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 8,000, 1×10 4 , 1 x 10 5 , 1 x 106 , 1 x 10 7 The above copies of the gene element can be delivered, for example, the eukaryotic cell being a HEK293T cell as described in Example 22, as described in any of the prior embodiments of the anestronomy.

[0359] 1255. Populations of anelosomes (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 gene elements per cell) in a population of eukaryotic cells, with 1-3, 1-4, 1-5, 1-7, 1-8, 1-9, 1-10, 5-10, 10-20, 20-50, 50-100, 100-1000, 1000-10 4 , 1 x 10 4 ~1 × 10 5 , 1 x 10 4 ~1 × 10 6 , 1 x 10 4 ~1 × 10 7 , 1 x 10 5 ~1 × 10 6 , 1 x 10 5 ~1 × 10 7 , or 1 × 10 6 ~1 × 10 7 An anestroma can deliver a copy of a gene element, for example, in which case the eukaryotic cell is a HEK293T cell, as described in Example 22, as described in any of the prior embodiments.

[0360] 1256. An anestronomy according to any of the prior embodiments, wherein the anestronomy is present after at least two passages.

[0361] 1257. An anestrosome according to any of the prior embodiments, wherein the anestrosome is produced by a process comprising at least two passages.

[0362] 1258. An anestrosome according to any of the prior embodiments, wherein the anestrosome selectively delivers an effector to a desired cell type, tissue, or organ (e.g., bone marrow, blood, heart, GI, skin, retinal photoreceptors, endothelial layer, or pancreas) or is present there at a higher level (preferably accumulates there).

[0363] 1259. An anestronomy according to any of the prior embodiments, wherein the eukaryotic cell is a mammalian cell, such as a human cell.

[0364] 1260. An anestrome according to any of the prior embodiments, wherein the anestrome, or a copy thereof, is detectable 24 hours after delivery into the cell (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 30 days, or 1 month).

[0365] 1261. When an anestronomy is used, for example, in an infectivity assay, for example, the assay described in Example 7, 3 to 4 days after infection, for example, at least about 10 times the amount of anestronomy used to infect cells. 8 times (for example, about 10 5 double, 10 6 double, 10 7 double, 10 8 double, 10 9 double, or 10 10 An anestrosome according to any of the prior embodiments, produced in the cell pellet and supernatant at a genome equivalent / mL of (1 / 2) times.

[0366] 1262. A composition comprising an anestronomy as described in any of the prior embodiments.

[0367] 1263. A pharmaceutical composition comprising an anestrome described in any of the prior embodiments, and a pharmaceutically acceptable carrier or excipient.

[0368] 1264. A composition or pharmaceutical composition according to Embodiment 1262 or 1263, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more anerosomes, for example, anerosomes.

[0369] 1265. At least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 A composition or pharmaceutical composition according to any one of embodiments 1262 to 1264, comprising anestrosomes.

[0370] 1266. The following characteristics: a) The pharmaceutical composition must meet pharmaceutical or Good Manufacturing Practice (GMP) standards; b) The pharmaceutical composition is manufactured in accordance with Good Manufacturing Practices (GMP); c) The pharmaceutical composition has pathogen levels below predetermined standard values, for example, it is substantially free of pathogens; d) The pharmaceutical composition has a level of impurities below a predetermined standard value, for example, it is substantially free of impurities; e) The pharmaceutical composition has a predetermined level of non-infectious particles, or a predetermined particle:infectious unit ratio (e.g., <300:1, ≤200:1, ≤100:1, or <50:1), or f) The pharmaceutical composition is low immunogenic or substantially non-immunogenic, for example, as described herein. A composition or pharmaceutical composition according to any one of embodiments 1262 to 1265, having one or more of the above.

[0371] 1267. A composition or pharmaceutical composition according to any of embodiments 1262 to 1266, wherein the pharmaceutical composition has a level of impurities below a predetermined standard value, for example, substantially free of impurities.

[0372] 1268. The composition or pharmaceutical composition according to Embodiment 1267, wherein the contaminants are selected from the group consisting of mycoplasma, exotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replicatable organisms (RCAs), e.g., replicatable viruses or unwanted anestrophores (desired anestrophores, e.g., anestrophores other than those described herein), free viral capsid proteins, exogenous contaminants, and aggregates.

[0373] 1269. The composition or pharmaceutical composition according to Embodiment 1268, wherein the impurity is host cell DNA, and the threshold amount is approximately 10 ng of host cell DNA per dose of the pharmaceutical composition.

[0374] 1270. A composition or pharmaceutical composition according to any of Embodiments 1262 to 1269, wherein the pharmaceutical composition contains less than 10% by weight of impurities (for example, about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less than 0.1%).

[0375] 1271. Use of an anestroma, composition, or pharmaceutical composition described in any of the prior embodiments for the purpose of treating a disease or disorder of a subject (e.g., as described herein).

[0376] 1272. An anestroma, composition, or pharmaceutical composition according to any of the prior embodiments for use in treating a disease or disorder of a subject (e.g., as described herein).

[0377] 1273. A method for treating a disease or disorder of a subject (e.g., as described herein), the method comprising the step of administering to the subject an anestronomy (e.g., synthetic anestronomy) or pharmaceutical composition described in any of the prior embodiments.

[0378] 1274. A method for modulating, for example, enhancing or inhibiting the biological function of a subject (as described herein), the method comprising the step of administering to a subject an anestroke (e.g., a synthetic anestroke) or pharmaceutical composition described in any of the prior embodiments.

[0379] 1275. The method according to any one of embodiments 1273 to 1274, wherein the anestroma does not contain an external effector.

[0380] 1276. The method according to any one of Embodiments 1273 to 1275, wherein the anestroma comprises, for example, a wild-type anellovirus as described herein.

[0381] 1277. The method according to any one of embodiments 1273 to 1276, wherein delivery of gene elements to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a population of target cells in a subject is achieved by administration of anelosomes, for example, synthetic anelosomes.

[0382] 1278. The method according to any one of embodiments 1273 to 1277, wherein the administration of anelosomes, for example synthetic anelosomes, achieves the delivery of an effector to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the target cell population of a subject.

[0383] 1279. The method according to Embodiment 1277 or 1278, wherein the target cells include, for example, mammalian cells, such as human cells, such as adipocytes, ovarian cells, chondrocytes, neurons, blood cells, skin cells, muscle cells, or epithelial cells, in vitro.

[0384] 1280. The method according to any one of embodiments 1277 to 1279, wherein the target cells are located in adipose tissue, ovaries, cartilage, or the central nervous system.

[0385] 1281. The method according to any one of embodiments 1273 to 1280, wherein each target cell to which the gene element is delivered receives at least 10, 50, 100, 500, 1,000, 10,000, 50,000, 100,000, or more copies of the gene element.

[0386] 1282. The method according to any one of Embodiments 1273 to 1281, wherein the effector comprises miRNA, and the miRNA reduces, for example, the level of a target protein or RNA in a cell or population of cells to which an anestronomy is delivered by, for example, at least 10%, 20%, 30%, 40%, or 50%.

[0387] 1283. A method for delivering an anestronomy, for example, a synthetic anestronomy, to a cell, comprising the step of bringing an anestronomy described in any of the prior embodiments into contact with a cell, for example, a eukaryotic cell, for example, a mammalian cell.

[0388] 1284. The method according to Embodiment 1283, further comprising the step of bringing a helper virus into contact with a cell, wherein the helper virus comprises a polynucleotide, for example, a polynucleotide encoding an external protein, for example, an external protein that can bind to an external protein binding sequence, and optionally, a lipid envelope.

[0389] 1285. The method according to Embodiment 1284, wherein a helper virus is brought into contact with the cell before, simultaneously with, or after the step of bringing the anestroma into contact with the cell.

[0390] 1286. The method according to embodiment 1283, further comprising the step of bringing a helper polynucleotide into contact with a cell.

[0391] 1287. The method according to Embodiment 1286, wherein the helper polynucleotide comprises a sequence polynucleotide encoding an external protein, for example, an external protein that can bind to an external protein binding sequence, and a lipid envelope.

[0392] 1288. The method according to Embodiment 1286, wherein the helper polynucleotide is RNA (e.g., mRNA), DNA, plasmid, viral polynucleotide, or any combination thereof.

[0393] 1289. The method according to any one of embodiments 1286 to 1288, wherein a helper polynucleotide is brought into contact with the cell before, simultaneously with, or after the step of bringing the anestroma into contact with the cell.

[0394] 1290. The method according to any one of embodiments 123 to 129, further comprising the step of bringing a helper protein (e.g., a growth factor) into contact with a cell.

[0395] 1291. The method according to Embodiment 1290, wherein the helper protein comprises a viral replication protein or a capsid protein.

[0396] 1292. A host cell containing an anestronomy as described in any of the prior embodiments.

[0397] 1293. A nucleic acid molecule comprising a promoter element, a sequence encoding an effector (e.g., payload), and an external protein-binding sequence, The nucleic acid molecule is single-stranded DNA, and the nucleic acid molecule is circular, and / or is incorporated at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the nucleic acid molecules that enter the cell; The effects pedal is not from TTV, and it's not the SV40-miR-S1; The nucleic acid molecule does not contain the TTMV-LY polynucleotide sequence; Promoter elements are nucleic acid molecules that can instruct the expression of effectors in eukaryotic cells.

[0398] 1294. Below: (i) A sequence encoding the promoter element and the effector (e.g., payload) (optionally, the effector is exogenous to the wild-type anellovirus sequence), (ii) at least 72 consecutive nucleotides having at least 75% sequence identity to the wild-type anellovirus sequence (e.g., at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, or 150 nucleotides); or at least 100 consecutive nucleotides having at least 72% sequence identity to the wild-type anellovirus sequence (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%) and: (iii) Protein binding sequences, for example, an external protein binding sequence and In a gene element containing, The nucleic acid construct is single-stranded DNA; A gene element in which a nucleic acid construct is incorporated at a frequency of less than 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene element that enters the cell and / or is circular.

[0399] 1295. A method for producing an anestrom composition, the following: a) A step of preparing a host cell comprising one or more nucleic acid molecules encoding components of an anestronomy, for example, a synthetic anestronomy as described herein, wherein the anestronomy comprises a proteinaceous outer layer and a gene element comprising a promoter element, a sequence encoding an effector (e.g., an endogenous or extrinsic effector), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal); b) A step of producing anesomes from host cells, thereby creating anesomes; and c) The step of formulating the anestrom into a pharmaceutical composition suitable for administration to a subject, for example. Methods that include...

[0400] 1296. A method for producing a synthetic anestroma composition, the following: a) A step of preparing a plurality of anestromes, compositions, or pharmaceutical compositions as described in any of the prior embodiments; b) Optionally, the step of evaluating multiple anestrophores with respect to one or more of the following: contaminants described herein, optical density measurement (e.g., OD260), particle count (e.g., by HPLC), infectivity (e.g., particle:infectivity ratio, e.g., determined by fluorescence and / or ELISA); and c) For example, if one or more of the parameters in (b) meet a specified threshold, the step of formulating multiple anestromes into a pharmaceutical composition suitable for administration to a subject. Methods that include...

[0401] 1297. The anestrom composition contains at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 The anestroke composition contains at least 10 anestrokes per mL. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 The method according to Embodiment 1296, comprising an anestrosomal genome.

[0402] 1298. The method according to Embodiment 1296 or 1297, wherein the anestrom composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0403] 1299. A reaction mixture comprising an anestroma and a helper virus as described in any of the prior embodiments, wherein the helper virus comprises polynucleotides, for example, polynucleotides encoding an external protein (for example, an external protein that can bind to an external protein sequence), and optionally a lipid envelope.

[0404] 1300. A reaction mixture comprising an anestrome as described in any of the prior embodiments, and a second nucleic acid molecule encoding one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, ORF1, ORF1 / 1, or ORF1 / 2 from 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, or one or more amino acid sequences having at least 75% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto.

[0405] 1301. The reaction mixture according to Embodiment 1300, wherein the second nucleic acid sequence is part of a gene element.

[0406] 1302. The reaction mixture according to Embodiment 1301, wherein the second nucleic acid sequence is not part of a gene element, but for example, the second nucleic acid sequence is contained in a helper cell or a helper virus.

[0407] 1303. Below: A gene element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence for binding the gene element to the non-pathogenic external protein, and (iii) a sequence encoding an effector, such as a regulatory nucleic acid; and A synthetic anelosome that binds to a genetic element and includes a proteinaceous outer layer that, for example, envelopes or encapsulates it.

[0408] 1304. The following: a) (i) A sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds a genetic element to the non-pathogenic external protein, and (iii) an effector, for example, a sequence encoding a regulatory nucleic acid; and An anelosome that binds to the genetic element and includes a proteinaceous outer layer that, for example, envelopes or encapsulates it; and b) An excipient for a pharmaceutical formulation A pharmaceutical composition comprising.

[0409] 1305. The following: a) (i) A sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds a genetic element to the non-pathogenic external protein, and (iii) an effector, for example, a sequence encoding a regulatory nucleic acid; and An anelosome that binds to the genetic element and includes a proteinaceous outer layer that, for example, envelopes or encapsulates it, at least 10 3 10 4 10 5 10 6 10 7 10 8 or 10 9 individual anelosomes (for example, the synthetic anelosomes described herein) b) An excipient for a pharmaceutical formulation, and optionally, c) An amount less than a predetermined amount of the following: mycoplasma, exotoxin, host cell nucleic acid (for example, host cell DNA and / or host cell RNA), process impurities of animal origin (for example, serum albumin or trypsin), replication-competent organisms (RCA), for example, replication-competent viruses or unwanted anelosomes, free virus capsid proteins, adventitious substances, and / or aggregates A pharmaceutical composition comprising.

[0410] 1306. An anestroke or composition according to any one of the prior embodiments, further comprising at least one of the following characteristics: the gene element is single-stranded DNA; the gene element is circular; the anestroke is non-integrated; the anestroke has a sequence, structure and / or function based on an anelovirus or other non-pathogenic virus; and the anestroke is non-pathogenic.

[0411] 1307. An anestrome or composition according to any one of the prior embodiments, wherein the proteinaceous outer layer comprises a non-pathogenic external protein.

[0412] 1308. An anelosome or composition according to any one of the prior embodiments, wherein the proteinaceous outer layer comprises one or more glycosylated proteins, hydrophilic DNA-binding regions, arginine-rich regions, threonine-rich regions, glutamine-rich regions, N-terminal polyarginine sequences, variable regions, C-terminal polyglutamine / glutamic acid sequences, and one or more disulfide crosslinks.

[0413] 1309. An anestronomy or composition according to any one of the prior embodiments, wherein the proteinaceous outer layer comprises one or more of the following characteristics: icosahedral symmetry, recognition and / or binding to molecules that interact with one or more host cell molecules to mediate entry into host cells, deletion of lipid molecules, deletion of carbohydrates, inclusion of one or more desired carbohydrates (e.g., glycosylation), pH and temperature stability, surfactant resistance, and non-immunogenicity or non-pathogenicity in the host.

[0414] 1310. An anestrome or composition according to any one of the prior embodiments, wherein the sequence encoding a non-pathogenic external protein contains at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of one or more sequences or fragments thereof listed in Table 19.

[0415] 1311. An anelosome or composition according to any one of the prior embodiments, wherein the nonpathogenic external protein comprises at least one functional domain that provides one or more functions, e.g., species and / or tissue and / or cell targeting, viral genome binding and / or packaging, immune evasion (non-immunogenic and / or tolerance), pharmacokinetics, endocytosis and / or cell adhesion, nuclear entry, intracellular regulation and localization, exocytosis regulation, proliferation, and nucleic acid protection.

[0416] 1312. Effectors include regulatory nucleic acids, e.g., miRNA, siRNA, mRNA, IncRNA, RNA, DNA, antisense RNA, gRNA; therapeutic agents, e.g., fluorescent tags or markers, antigens, peptide therapeutic agents, synthetic or analog peptides derived from naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degrading or self-destructive peptides, and multiple degrading or self-destructive peptides, small molecules, and immune effectors (e.g., those that affect sensitivity to immune responses / signals). An anestronomy or composition according to any one of the prior embodiments, comprising: a cell death-inducing protein (e.g., an inducer of apoptosis or necrosis); a non-soluble tumor inhibitor (e.g., an inhibitor of oncoproteins); an epigenetic modifier; an epigenetic enzyme; a transcription factor; a DNA or protein modifying enzyme; a DNA insertion agent; an efflux pump inhibitor; a nuclear receptor activator or inhibitor; a proteasome inhibitor; a competitive inhibitor of a single enzyme; a protein synthesis effector or inhibitor; a nuclease; a protein fragment or domain; a ligand or receptor; and a CRISPR system or constituent.

[0417] 1312a. An anestrome or composition according to any one of the prior embodiments, wherein the effector comprises an IDH1 inhibitor, an IDH2 inhibitor, an EGFR inhibitor, an LRP5 inhibitor, a DKK2 inhibitor, a DPP-4 inhibitor, a KRAS inhibitor, a neutrophil elastase inhibitor, an FGFR3 activator, or an HTT activator.

[0418] 1312b. An anestrome or composition according to any one of the prior embodiments, wherein the effector comprises an IDH1 inhibitor or an IDH2 inhibitor.

[0419] 1313. An anestrome or composition according to any one of the prior embodiments, wherein the effector comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the miRNA sequences listed in Table 40.

[0420] 1314. An anestrome or composition according to a prior embodiment, wherein an effector, for example, miRNA, targets a host gene, for example, regulates gene expression.

[0421] 1315. An anestrome or composition according to a prior embodiment, wherein the miRNA contains a sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the miRNA sequences listed in Table 40.

[0422] 1316. An anestronomy or composition according to any one of the prior embodiments, wherein the gene element further comprises one or more sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic agent, a regulatory sequence (e.g., promoter, enhancer), a sequence encoding one or more regulatory sequences targeting an endogenous gene (siRNA, lncRNA, shRNA), a sequence encoding therapeutic mRNA or protein, or one or more cytolytic and / or cytotoxic RNA or protein.

[0423] 1317. An anestrome or composition according to any one of the prior embodiments, wherein the gene element has one or more of the following characteristics: being non-integrated with the host cell genome, being an episomal nucleic acid, being single-stranded DNA, being about 1-10 kb, being located in the cell nucleus, being able to bind to endogenous proteins, and producing microRNA that targets host genes.

[0424] 1318. An anestrome or composition according to any one of the prior embodiments, wherein the gene element has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with at least one viral sequence or at least one or more sequences listed in Table 23, or a fragment thereof (e.g., a fragment encoding ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules, and / or a fragment including one or more TATA boxes, cap sites, transcription start sites, 5'UTR, open reading frames (ORF), poly(A) signals, or GC-rich regions).

[0425] 1319. The viral sequence is that of single-stranded DNA viruses (e.g., Anellovirus, Bidnavirus, Circovirus, Geminivirus, Genomovirus, Inovirus, Microvirus, Nanovirus, Parvovirus, and Spiravirus), double-stranded DNA viruses (e.g., Adenovirus, Ampullavirus, Ascovirus, Asfarvirus, baculovirus, fusellovirus, globulovirus, guttavirus, hypnothavirus, herpesvirus, iridovirus, lipospirixvirus, nimavirus, and poxvirus), RNA viruses (e.g., alphavirus, furovirus, hepatitis virus) An anestronomy or composition according to the prior embodiments, derived from at least one of the following: virus, Hordeivirus, Tobamovirus, Tobravirus, Tricornavirus, Rubivirus, Birnavirus, Cystovirus, Partitivirus, and Reovirus.

[0426] 1320. An anestronomy or composition according to the prior embodiments, wherein the viral sequence is derived from one or more non-aneroviruses, such as adenovirus, herpesvirus, poxvirus, vaccinia virus, SV40, papillomavirus, retrovirus, such as lentivirus, an RNA virus, a single-stranded RNA virus, such as hepatitis virus, or a double-stranded DNA virus, such as rotavirus.

[0427] 1321. An anelosome or composition according to any one of the prior embodiments, wherein a protein-binding sequence interacts with an arginine-rich region of a proteinaceous outer layer.

[0428] 1322. An anestrosome or composition according to any one of the prior embodiments, wherein the anestrosome is replicable in mammalian cells, such as human cells.

[0429] 1323. An anestrosome or composition according to a prior embodiment, wherein the anestrosome is nonpathogenic and / or nonintegrative in host cells.

[0430] 1324. An anestroke or composition according to a prior embodiment, wherein the anestroke is non-immunogenic in the host.

[0431] 1325. An anestrosome or composition according to any one of the prior embodiments, wherein the anestrosome inhibits / enhances one or more viral properties in a host or host cell, e.g., selectivity, e.g., infectivity, e.g., immunosuppression / activation.

[0432] 1326. An anestrosome or composition according to a prior embodiment, wherein the anestrosome is present in an amount sufficient to regulate (for example, phenotype, viral level, gene expression, competition with other viruses, disease states, etc.) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.

[0433] 1327. The composition according to any one of the prior embodiments, further comprising at least one virus or vector, e.g., a virus, e.g., a variant of an anestrom, e.g., a genome of a commensal / native virus.

[0434] 1328. A composition according to any one of the prior embodiments, further comprising a heterogeneous portion, at least one small molecule, an antibody, a polypeptide, a nucleic acid, a targeting agent, an imaging agent, nanoparticles, and combinations thereof.

[0435] 1329. A vector comprising a gene element, the gene element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence for binding the gene element to the non-pathogenic external protein, and (iii) a sequence encoding an effector, such as a regulatory nucleic acid.

[0436] 1330. A vector according to a prior embodiment, wherein the gene element cannot be integrated with the genome of a host cell.

[0437] 1331. A vector according to any one of the prior embodiments, wherein the gene element is replicable in mammalian cells, such as human cells.

[0438] 1332. A vector according to any one of the prior embodiments, further comprising, for example, an exogenous nucleic acid sequence selected to regulate the expression of a gene, for example, a human gene.

[0439] 1333. A pharmaceutical composition comprising a vector and a pharmaceutical excipient as described in any one of the prior embodiments.

[0440] 1334. The composition according to the prior embodiment, wherein the vector is non-pathogenic and / or non-integrative in host cells.

[0441] 1335. A composition according to any one of the prior embodiments, wherein the vector is non-immunogenic in the host.

[0442] 1336. The composition according to the prior embodiment, wherein the vector is present in an amount sufficient to regulate (phenotype, viral level, gene expression, competition with other viruses, disease states, etc., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more).

[0443] 1337. A composition according to any one of the prior embodiments, further comprising at least one virus or vector, e.g., a virus, including the genome of an anestrom variant, a commensal / native virus, a helper virus, or a non-anerovirus.

[0444] 1338. A composition according to any one of the prior embodiments, further comprising a heterogeneous portion, at least one small molecule, an antibody, a polypeptide, a nucleic acid, a targeting agent, an imaging agent, nanoparticles, and combinations thereof.

[0445] 1339. A method for producing, growing, and recovering anestromas as described in any one of the prior embodiments.

[0446] 1340. A method for designing and fabricating a vector as described in any one of the prior embodiments.

[0447] 1341. A method comprising the step of administering to a subject an effective amount of a composition described in any one of the prior embodiments.

[0448] 1342. A method for delivering a nucleic acid or protein payload to target cells, tissues or a subject, the method comprising the step of bringing a target cell, tissue or subject into contact with a nucleic acid composition comprising (a) a primary DNA derived from a virus (the primary DNA being sufficient to enable the production of particles that can infect target cells, tissues or a subject) and (a) a secondary DNA sequence encoding a nucleic acid or protein payload, the improvements being as follows: The first DNA sequence contains at least 500 (at least 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000) nucleotides having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity with the corresponding sequence listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, or The first DNA sequence encodes a sequence that has at least 80% (for example, at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity with the ORFs listed in Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10, or A method wherein the first DNA sequence includes a sequence having at least 90% (e.g., at least 95%, 97%, 99%, 100%) sequence identity with the consensus sequences listed in Table 19.

[0449] 1343. A method for delivering a nucleic acid or protein effector to target cells, tissues or a subject, the method comprising the step of bringing a target cell, tissue or subject into contact with a nucleic acid composition comprising (a) a virus-derived first DNA sequence (the first DNA sequence is sufficient to enable the production of an anestrom described in any of the prior embodiments that can infect target cells, tissues or a subject) and (a) a second DNA sequence encoding a nucleic acid or protein effector.

[0450] 1344. Codon-optimized nucleic acid molecules that encode an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of wild-type anellovirus ORF1, ORF2, or ORF3.

[0451] 1345. For example, a codon-optimized nucleic acid molecule according to Embodiment 1344 that codes for an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the wild-type anellovirus ORF1 amino acid sequence 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.

[0452] 1346. Below: (a) Anerosomes, for example, the anesome described in any of the prior embodiments, and (b) A carrier selected from vesicles, lipid nanoparticles (LNPs), erythrocytes, exosomes (e.g., mammalian or plant exosomes), or fusosomes. A pharmaceutical composition containing the above.

[0453] 2001. Below: (a) Protein outer layer; (b) An anestroma comprising a gene element including a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an extrinsic effector), and a protein-binding sequence (e.g., an external protein-binding sequence), The gene elements are as follows: (i) Sequence identity of at least 72.2% (e.g., at least 72.2, 72.3, 72.4, 72.5, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A1; (ii) Sequence identity of at least 68.4% (e.g., at least 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) with respect to the anellovirus sequences listed in Table A3; (iii) Sequence identity of at least 81.7% (e.g., at least 81.7, 81.8, 81.9, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A5; (iv) Sequence identity of at least 92.6% (e.g., at least 92.6, 92.7, 92.8, 92.9, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A7; (v) Sequence identity of at least 65% (e.g., at least 65, 66, 67, 68, 69, 70, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A9; or (vi) Sequence identity of at least 65% (e.g., at least 65, 66, 67, 68, 69, 70, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A11. Having; Optionally, a gene element may have at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; An anestrosome is a type of anestrosome configured to deliver genetic elements to eukaryotic cells.

[0454] 2002. Below: (a) Protein outer layer; (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector), and a protein-binding sequence (e.g., an exogenous protein-binding sequence). An anesome containing, The gene elements are as follows: (i) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017 or fewer nucleotides compared to the anellovirus sequences listed in Table A1, e.g., substitutions, insertions, or deletions; (ii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 11160, 1170, 1171, 1172, 1173, or 1174 or fewer nucleotides compared to the anellovirus sequences listed in Table A3, e.g., substitutions, insertions, or deletions; (iii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 or fewer nucleotides compared to the anellovirus sequences listed in Table A5, e.g., substitutions, insertions, or deletions; (iv) Differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280 nucleotides or less compared to an anerovirus sequence listed in Table A7, e.g., substitution, insertion, or deletion; (v) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or fewer nucleotides compared to an anelovirus sequence listed in Table A9, e.g., substitution, insertion or deletion; or (vi) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or less nucleotides compared to the anellovirus sequences listed in Table A11, e.g., substitution, insertion or deletion. Includes; Optionally, a gene element may have at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; An anestrosome is a type of anestrosome configured to deliver genetic elements to eukaryotic cells.

[0455] 2002. Below: (a) Protein outer layer; (b) An anestroma comprising a gene element including a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an extrinsic effector), and a protein-binding sequence (e.g., an external protein-binding sequence), The gene elements are as follows: (i) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017 or fewer nucleotides compared to the anellovirus sequences listed in Table B1, e.g., substitutions, insertions, or deletions; (ii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 11160, 1170, 1171, 1172, 1173, or 1174 or fewer nucleotides compared to the anellovirus sequences listed in Table B2, e.g., substitutions, insertions, or deletions; (iii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 or fewer nucleotides compared to the anellovirus sequences listed in Table B3, e.g., substitutions, insertions, or deletions; (iv) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280 nucleotides or less compared to an anelovirus sequence listed in Table B4, e.g., substitution, insertion or deletion; or (v) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or less compared to the anellovirus sequences listed in Table B5, e.g., substitutions, insertions, or deletions; Includes; Optionally, a gene element may have at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region); The gene elements are confined within a proteinaceous outer layer; An anestrosome is a type of anestrosome configured to deliver genetic elements to eukaryotic cells.

[0456] 2003. The gene element is not a naturally occurring sequence (for example, wild-type anellovirus sequence (for example, wild-type Torque Tenovirus (TTV), Torque Tenomivirus (TTV) An anestrosome as described in any of the prior embodiments, which is at least one difference (e.g., mutation, chemical modification, or epigenetic alteration), e.g., insertion, substitution, enzymatic modification, and / or deletion, e.g., deletion of a specific domain (e.g., one or more of the TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region), compared to a Teno minivirus (TTMV) or TTMDV sequence, e.g., a wild-type anestrovirus sequence, e.g., as listed in any of Tables B1-B5, A1, A3, A5, A7, A9, A11, 1, 3, 5, 7, 9, 11, or 13), e.g., insertion, substitution, enzymatic modification, and / or deletion, e.g., deletion of a specific domain (e.g., one or more of the TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0457] An anestronomy according to any of the prior embodiments, comprising a polypeptide having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with the amino acid sequence of an anestrovirus ORF1 molecule (e.g., an anestrovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12).

[0458] 2005. An anestrome according to Embodiment 2004, wherein the proteinaceous outer layer comprises a polypeptide.

[0459] 2006. An anestrome according to Embodiment 2005, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the protein in the proteinate outer layer comprises a polypeptide.

[0460] 2007. An anestrome according to any of the prior embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the protein in the proteinate outer layer comprises an ORF1 molecule.

[0461] An anestrome according to any of the prior embodiments, comprising a nucleic acid molecule (e.g., in a gene element) that encodes an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to the amino acid sequence of an anelovirus ORF1 molecule (e.g., an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12).

[0462] 2009. Gene elements are nucleic acid sequences: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(Sequence ID 160); (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. An anestrome according to any of the prior embodiments, comprising a region containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides.

[0463] 2010. An anestrome according to any of the prior embodiments, wherein the gene element comprises a 5'UTR region and / or a GC-rich region as described herein (e.g., listed in Table 38 or 39, respectively).

[0464] 2011. Below: (i) Sequence identity of at least 72.2% (e.g., at least 72.2, 72.3, 72.4, 72.5, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A1; (ii) Sequence identity of at least 68.4% (e.g., at least 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) with respect to the anellovirus sequences listed in Table A3; (iii) Sequence identity of at least 81.7% (e.g., at least 81.7, 81.8, 81.9, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A5; (iv) Sequence identity of at least 92.6% (e.g., at least 92.6, 92.7, 92.8, 92.9, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A7; (v) Sequence identity of at least 65% (e.g., at least 65, 66, 67, 68, 69, 70, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A9; or (vi) Sequence identity of at least 65% (e.g., at least 65, 66, 67, 68, 69, 70, 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the anellovirus sequences listed in Table A11. An isolated nucleic acid molecule (e.g., an expression vector) containing a gene element having; An isolated nucleic acid molecule in which the gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0465] 2012. Below: (i) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017 or fewer nucleotides compared to the anellovirus sequences listed in Table A1, e.g., substitutions, insertions, or deletions; (ii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 11160, 1170, 1171, 1172, 1173, or 1174 or fewer nucleotides compared to the anellovirus sequences listed in Table A3, e.g., substitutions, insertions, or deletions; (iii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 or fewer nucleotides compared to the anellovirus sequences listed in Table A5, e.g., substitutions, insertions, or deletions; (iv) Differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280 nucleotides or less compared to an anerovirus sequence listed in Table A7, e.g., substitution, insertion, or deletion; (v) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or fewer nucleotides compared to an anelovirus sequence listed in Table A9, e.g., substitution, insertion or deletion; or (vi) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or less nucleotides compared to the anellovirus sequences listed in Table A11, e.g., substitution, insertion or deletion. An isolated nucleic acid molecule (e.g., an expression vector) containing a gene element containing; An isolated nucleic acid molecule in which the gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0466] 2012A. Below: (i) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017 or fewer nucleotides compared to the anellovirus sequences listed in Table B1, e.g., substitutions, insertions, or deletions; (ii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 11160, 1170, 1171, 1172, 1173, or 1174 or fewer nucleotides compared to the anellovirus sequences listed in Table B2, e.g., substitutions, insertions, or deletions; (iii) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 or fewer nucleotides compared to the anellovirus sequences listed in Table B3, e.g., substitutions, insertions, or deletions; (iv) A difference of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280 nucleotides or less compared to an anelovirus sequence listed in Table B4, e.g., substitution, insertion or deletion; or (v) Nucleotide differences of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or less compared to the anellovirus sequences listed in Table B5, e.g., substitutions, insertions, or deletions; An isolated nucleic acid molecule (e.g., an expression vector) containing a gene element containing; An isolated nucleic acid molecule in which the gene element has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type anellovirus genome sequence (e.g., as described herein), such as an insertion, substitution, enzymatic modification, and / or deletion, such as a deletion of one or more of a specific domain (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0467] 2013. The gene element is not a naturally occurring sequence (for example, wild-type anellovirus sequence (for example, wild-type Torque Tenovirus (TTV), Torque Tenomivirus (TTV) An isolated nucleic acid molecule as described in any of the prior embodiments, having at least one difference (e.g., mutation, chemical modification, or epigenetic alteration), such as insertion, substitution, enzymatic modification, and / or deletion, such as deletion of a specific domain (e.g., one or more of the TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region), compared to a Teno minivirus (TTMV) or TTMDV sequence, for example, a wild-type anellovirus sequence, as listed in any of Tables B1-B5, A1, A3, A5, A7, A9, A11, 1, 3, 5, 7, 9, 11, or 13).

[0468] 2014. The isolated nucleic acid molecule contains a gene element encoding an ORF1 molecule (for example, an ORF1 molecule listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto); Here, (i) At least 30% of the amino acids in the ORF1 molecule (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) are part of the β-sheet; (ii) The secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β-sheets; (iii) An isolated nucleic acid molecule according to any of the prior embodiments, wherein the secondary structure of the ORF1 molecule includes at least a β-sheet:α-helix ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0469] 2015. Nucleic acid sequences: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(Sequence ID 160); (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. An isolated nucleic acid molecule according to any of the prior embodiments, comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides.

[0470] 2016. An isolated nucleic acid molecule according to any of the prior embodiments, comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0471] 2017. An isolated nucleic acid molecule according to any of the prior embodiments, wherein the gene elements further comprise one or more sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, derived from an anelovirus described herein (for example, as listed in any of Tables B1-B5, A1, A3, A5, A7, A9, or A11): a TATA box, an initiator element, a cap region, a transcription start site, a 5'UTR conserved domain, an ORF1 coding sequence, an ORF1 / 1 coding sequence, an ORF1 / 2 coding sequence, an ORF2 coding sequence, an ORF2 / 2 coding sequence, an ORF2 / 3 coding sequence, an ORF2 / 3t coding sequence, a 3 open reading frame region, a poly(A) signal, and / or a GC-rich region.

[0472] 2018. An isolated nucleic acid molecule according to any of the prior embodiments, wherein the gene element further comprises an anellovirus genome sequence (for example, as described herein and listed, for example, in any of Tables B1-B5, A1, A3, A5, A7, A9, A11, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or at least one or two copies (e.g., 1, 2, 3, 4, 5, or 6 copies) of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0473] 2019. An isolated nucleic acid molecule according to any of the prior embodiments, further comprising at least one additional copy of a gene element (e.g., a total of 1, 2, 3, 4, 5, or 6 copies).

[0474] 2020. An isolated nucleic acid molecule according to any of the prior embodiments, wherein the isolated nucleic acid molecule is cyclic.

[0475] 2021. An isolated nucleic acid composition (for example, comprising one, two, or more nucleic acid molecules) comprising an isolated nucleic acid molecule described in any of the prior embodiments.

[0476] 2022. An isolated nucleic acid according to any of the prior embodiments, wherein the gene element further comprises a nucleic acid sequence (e.g., a DNA sequence) encoding a promoter element, an effector (e.g., an extrinsic effector or an endogenous effector), and / or a protein-binding sequence (e.g., an external protein-binding sequence).

[0477] 2022A. An isolated nucleic acid molecule according to any of the prior embodiments, wherein a gene element is inserted into or substituted into the hypervariable domain (HVD) of ORF1.

[0478] 2023. An anestrome or isolated nucleic acid molecule according to any of the prior embodiments, wherein the gene element comprises, for example, one or more of the following: TATA box, initiator element, 5'UTR conserved domain, ORF1, ORF2, ORF2 downstream sequence, ORF2, ORF3, and / or GC-rich region, or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0479] An anestroma or isolated nucleic acid according to any of the prior embodiments, comprising (e.g., in a proteinaceous outer layer) or encoding one or more polypeptides comprising an amino acid sequence selected from ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0480] 2025. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element comprises a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0481] 2026. An anestrome or isolated nucleic acid according to Embodiment 2025, wherein the gene element comprises a sequence containing at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 80%.

[0482] 2027. An anestrome or isolated nucleic acid according to Embodiment 2025, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

[0483] 2028. An anestrome or isolated nucleic acid according to Embodiment 2025, wherein the gene element comprises at least 36 consecutive nucleotides having a GC content of at least 80%.

[0484] 2029. Gene elements, nucleic acid sequences: (i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (Sequence ID 160) (ii) GCGCTX1CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (Sequence No. 164) (X1 is selected from T, G, or A) (iii) GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG (Sequence No. 165); (iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(Sequence No. 166); (v)GCGCTGCGCGCGCGGCCCCCCCGGGGGAGGCATTGCCT(Sequence ID 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(Sequence No. 169); (viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix)GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(Sequence No. 171); or (x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(Sequence ID 172); or nucleic acid sequences having at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them. An anestroma or isolated nucleic acid according to any of the prior embodiments, comprising a region (e.g., a packaging region) containing at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides.

[0485] 2030. An anestrome or isolated nucleic acid according to Embodiment 2029, wherein the packaging region is located 3' to the nucleic acid sequence encoding the effector.

[0486] 2031. Below: (a) A first region comprising an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with the arginine-rich region sequence of an anelovirus ORF1 molecule described herein (e.g., an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); (b) 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 with the jelly roll region sequence of an anelovirus ORF1 molecule described herein (e.g., an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); (c) A third region comprising an amino acid sequence having at least 30% (e.g., at least about 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with the N22 domain sequence of an anelovirus ORF1 molecule described herein (e.g., an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); and / or (d) A fourth region comprising an amino acid sequence having at least 30% (e.g., at least about 30, 35, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity with the Anellovirus ORF1 C-terminal domain (CTD) sequence of an Anellovirus ORF1 molecule described herein (e.g., an Anellovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12). A polypeptide comprising one or more of the following: A polypeptide wherein the ORF1 molecule exhibits at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as the deletion of a specific domain (e.g., one or more of the arginine-rich region, jellyroll domain, HVR, N22, or CTD, as described herein).

[0487] 2031A. Below: (a) A first region comprising an amino acid sequence having at least 90% sequence identity with the arginine-rich region sequence of an anelovirus ORF1 molecule described herein (for example, an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); (b) A second region comprising an amino acid sequence having at least 90% sequence identity with the jelly roll region sequence of an anelovirus ORF1 molecule described herein (for example, an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); (c) A third region comprising an amino acid sequence having at least 90% sequence identity with the N22 domain sequence of an anelovirus ORF1 molecule described herein (for example, an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12); and / or (d) A fourth region comprising an amino acid sequence having at least 90% sequence identity with the C-terminal domain (CTD) sequence of an anelovirus ORF1 molecule described herein (for example, an anelovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12). A polypeptide comprising one or more of the following: The polypeptide according to Embodiment 2031, wherein the ORF1 molecule has at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) compared to the wild-type ORF1 protein (e.g., as described herein), e.g., insertion, substitution, chemical or enzymatic modification, and / or deletion, e.g., deletion of a specific domain (e.g., one or more of the arginine-rich region, jellyroll domain, HVR, N22, or CTD, as described herein).

[0488] 2032. Polypeptide is as follows: (i) Area 1 and Area 2; (ii) Area 1 and Area 3; (iii) Area 1 and Area 4; (iv) Second and third areas; (v) Second and fourth regions; (vi) Third and fourth regions; (vii) The first region, the second region, and the third region; (viii) Area 1, Area 2, and Area 4; (ix) first area, third area, and fourth area; (x) Second area, third area, and fourth area A polypeptide according to Embodiment 2031, including the following:

[0489] 2033. The polypeptide according to Embodiment 2031 or 2032, wherein the polypeptide comprises a first region, a second region, a third region, and a fourth region in the order from the N-terminus to the C-terminus.

[0490] 2034. A polypeptide according to any of the prior embodiments, further comprising an amino acid sequence, for example, a hypervariable region (HVR) sequence (for example, the HVR sequence of the Anellovirus ORF1 molecule as described herein), wherein the amino acid sequence comprises at least about 55 (for example, at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (for example, 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).

[0491] The polypeptide according to Embodiment 2034, wherein 2035.HVR comprises 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 with the Anellovirus ORF1 HVR sequence of the Anellovirus ORF1 molecule described herein (e.g., the Anellovirus ORF1 sequence listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12).

[0492] The polypeptide according to embodiment 2034 or 2035, wherein the 2036.HVR sequence is located between the second and third regions.

[0493] 2037. A polypeptide according to any of Embodiments 2034 to 2036, wherein the HVR comprises one or more features of the HVR described herein.

[0494] 2038. A polypeptide containing the amino acid sequence of ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, wherein the polypeptide is wild-type anerovirus (Anellovir A polypeptide having at least one difference (e.g., mutation or chemical modification), e.g., conjugation, addition, insertion, substitution, and / or deletion, e.g., deletion of a specific domain, compared to the ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 sequences (e.g., as listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, as described herein), e.g., conjugation, addition, insertion, substitution, and / or deletion, e.g., deletion of a specific domain.

[0495] 2039. A polypeptide comprising the amino acid sequence of ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0496] Polypeptides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98%, and 99% or less sequence identity with an amino acid sequence selected from ORF1, ORF2, ORF2, or ORF3 of any of Tables C1-C5, A2, A4, A6, A8, A10, or A12.

[0497] 2041. A polypeptide having at least one amino acid difference of 2, 5, 10, 20, 50, or 100 or less, e.g., substitution, insertion, or deletion, compared to an amino acid sequence selected from ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 of any of Tables C1-C5, A2, A4, A6, A8, A10, or A12.

[0498] 2042. A polypeptide according to any of the prior embodiments, wherein the polypeptide is an isolated polypeptide.

[0499] 2043. Below: (a) A polypeptide described in any of the prior embodiments, and (b) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an external or endogenous effector), and a protein-binding sequence. A complex containing this.

[0500] 2044. The composite according to Embodiment 2043, wherein the composite includes one or more features of the composite described herein.

[0501] 2045. A fusion protein comprising a first amino acid sequence selected from any of the ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules in Table C1-C5, A2, A4, A6, A8, A10, or A12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and a heterologous portion.

[0502] 2046. A fusion protein comprising a first amino acid sequence selected from any ORF1 molecule in Table C1-C5, A2, A4, A6, A8, A10, or A12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and a heterologous portion.

[0503] 2047. A fusion protein according to any of the prior embodiments, wherein the heterogeneous portion includes a targeting portion.

[0504] 2048. A fusion protein according to any of the prior embodiments, wherein the first amino acid sequence is at least one difference (e.g., mutation or chemical modification), e.g., conjugation, addition, insertion, substitution, and / or deletion, e.g., deletion of a specific domain, compared to the wild-type anellovirus ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 sequences (e.g., as listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12, as described herein), e.g., conjugation, addition, insertion, substitution, and / or deletion, e.g., deletion of a specific domain.

[0505] 2049. A host cell comprising an anestroma, isolated nucleic acid, fusion protein, or polypeptide as described in any of the prior embodiments.

[0506] 2050. A reaction mixture comprising an anestrome according to any of the prior embodiments and a helper virus, wherein the helper virus comprises polynucleotides, for example, an external protein, for example, an external protein-binding sequence, and optionally, polynucleotides encoding an external protein that binds to a lipid envelope.

[0507] 2051. A method for treating a disease or disorder of a subject, comprising the step of administering to the subject an anestroma, isolated nucleic acid molecule, fusion protein, or polypeptide described in any of the prior embodiments, or a pharmaceutical composition described in any of the prior embodiments.

[0508] 2052. The method according to Embodiment 2051, wherein the disease or disorder is selected from immune disorders, infections, inflammatory disorders, autoimmune diseases, cancer (e.g., solid tumors), and gastrointestinal disorders.

[0509] 2053. Use of an anestroma, isolated nucleic acid molecule, fusion protein, or polypeptide described in any of the prior embodiments for the purpose of treating a disease or disorder of a subject.

[0510] 2054. Use according to Embodiment 2053, wherein the disease or disorder is selected from immune disorders, infections, inflammatory disorders, autoimmune diseases, cancer (e.g., solid tumors, e.g., lung cancer), and gastrointestinal disorders.

[0511] 2055. An anestroma, isolated nucleic acid molecule, composition, or pharmaceutical composition as described in any of the prior embodiments for use in the treatment of a disease or disorder of a subject.

[0512] 2055A. An anestroma, isolated nucleic acid molecule, composition, or pharmaceutical composition as described in any of the prior embodiments for use as a pharmaceutical agent.

[0513] 2056. A method for modulating, for example inhibiting or enhancing the biological function of a subject, the method comprising the step of administering to a subject an anestroke, isolated nucleic acid molecule, fusion protein, or polypeptide described in any of the prior embodiments, or a pharmaceutical composition described in any of the prior embodiments.

[0514] 2057. A method for delivering anestrophores to cells, comprising the step of contacting an anestrophor, isolated nucleic acid molecule, fusion protein, or polypeptide described in any of the prior embodiments with a cell, eukaryotic cell, mammalian cell, etc.

[0515] 2058. The method according to Embodiment 2057, further comprising the step of bringing a helper virus into contact with a cell, wherein the helper virus comprises polynucleotides, for example, an extrinsic protein, for example, an extrinsic protein that binds to an extrinsic protein binding sequence, and optionally, polynucleotides that encode a lipid envelope.

[0516] 2059. The method according to Embodiment 2058, wherein a helper virus is brought into contact with a cell before, simultaneously with, or after contact between the anestroma and the cell.

[0517] 2060. The method according to Embodiment 2057, further comprising the step of bringing a helper polynucleotide into contact with a cell.

[0518] 2061. The method according to Embodiment 2060, wherein the helper polynucleotide comprises a sequence polynucleotide that encodes an external protein, for example, an external protein binding sequence and a lipid envelope.

[0519] 2062. The method according to Embodiment 2060, wherein the helper polynucleotide is RNA (e.g., mRNA), DNA, plasmid, viral polynucleotide, or any combination thereof.

[0520] 2063. The method according to any one of embodiments 2060 to 2062, wherein a helper polynucleotide is brought into contact with the cell before, simultaneously with, or after contact between the anestroma and the cell.

[0521] 2064. The method according to any one of embodiments 2057 to 2063, further comprising the step of bringing a helper protein into contact with a cell.

[0522] 2065. The method according to Embodiment 2064, wherein the helper protein comprises a viral replication protein or a capsid protein.

[0523] 2066. A method for delivering nucleic acids or protein effectors to target cells, tissues or subjects, the method comprising the step of bringing a nucleic acid composition comprising (a) a primary DNA sequence derived from a virus (the primary DNA sequence being sufficient to enable the production of particles that can infect target cells, tissues or subjects) and (a) a secondary DNA sequence encoding a nucleic acid or protein effector into contact with target cells, tissues or subjects, the improvements being as follows: The first DNA sequence contains at least 500 (at least 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000) nucleotides having at least 80% (at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity with the corresponding sequence listed in any of Tables B1-B5, A1, A3, A5, A7, A9, or A11, or The first DNA sequence encodes a sequence that has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity with anellovirus ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, and / or ORF3 molecules (e.g., listed in any of Tables C1-C5, A2, A4, A6, A8, A10, or A12). Methods that include...

[0524] 2067. A method for producing an anestrom composition, the following: a) A step of preparing a host cell comprising one or more nucleic acid molecules comprising sequences encoding components of an anestroma as described in any of the prior embodiments, wherein the anestroma comprises a gene element comprising a proteinoid outer layer, a gene element such as a promoter element, sequences encoding an effector (e.g., an endogenous effector or an extrinsic effector), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal); b) A step of producing anesomes from host cells and thereby constructing anesomes; and c) For example, as a pharmaceutical composition suitable for administration to a subject, the step of formulating an anelosome comprising; Optionally, a method in which one or more nucleic acid molecules encode a helper protein.

[0525] 2068. In a method for producing an anelosome composition, the following: a) The step of preparing a plurality of anelosomes according to any of the preceding embodiments; b) Optionally, the following: evaluating the plurality of anelosomes for one or more of the contaminants, optical density measurements (e.g., OD260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio) described herein; and c) For example, when one or more of the parameters in (b) meet the specified threshold, formulating a plurality of anelosomes as a pharmaceutical composition suitable for administration to a subject comprising.

[0526] 2069. The method according to embodiment 2068, wherein the anelosome composition comprises at least 10 5 10 6 10 7 10 9 10 10 10 11 10 12 10 13 10 14 or 10 15 anelosomes.

[0527] 2070. The method according to embodiment 2068 or 2069, wherein the anelosome composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0528] 2071. The anelosome or isolated nucleic acid according to any of the preceding embodiments, wherein the gene element is configured to replicate mammalian cells, such as human cells.

[0529] 2072. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element further comprises, for example, an exogenous nucleic acid sequence selected to regulate the expression of a gene, for example, a human gene.

[0530] 2073. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the protein-binding sequence is composed of G or C.

[0531] 2074. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element comprises a sequence of at least 80, 90, 100, 110, 120, 130, or 140 nucleotides in length, which consists of G or C at positions of at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) or approximately 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%.

[0532] 2075. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the protein-binding sequence binds to an arginine-rich region of the proteinaceous outer layer.

[0533] 2076. An anestroma or isolated nucleic acid according to any of the prior embodiments, wherein the proteinaceous outer layer comprises an external protein capable of specifically binding to a protein-binding sequence.

[0534] 2077. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the portion of the gene element excluding the effector has a total size of approximately 2.5 to 5 kb (e.g., approximately 2.8 to 4 kb, approximately 2.8 to 3.2 kb, approximately 3.6 to 3.9 kb, or approximately 2.8 to 2.9 kb), less than approximately 5 kb (e.g., approximately 2.9 kb, 3.2 kb, 3.6 kb, 3.9 kb, or less than 4 kb), or at least 100 nucleotides (e.g., at least 1 kb).

[0535] 2078. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element is single-stranded.

[0536] 2079. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element is circular.

[0537] 2080. An anestroma or isolated nucleic acid according to any of the prior embodiments, wherein the gene element is DNA.

[0538] 2081. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element is a negative-strand DNA.

[0539] 2082. An anestrome or isolated nucleic acid according to any of the prior embodiments, wherein the gene element comprises an episome.

[0540] 2083. An anestrosomal or isolated nucleic acid according to any of the prior embodiments, wherein the anestrosomal is present in a higher level (e.g., preferably accumulates) in a desired organ or tissue compared to other organs or tissues.

[0541] 2084. An anestroma or isolated nucleic acid according to any of the prior embodiments, wherein the eukaryotic cell is a mammalian cell, such as a human cell.

[0542] 2085. A composition comprising an anestroma or isolated nucleic acid as described in any of the prior embodiments.

[0543] 2086. A pharmaceutical composition comprising an anestroma or isolated nucleic acid as described in any of the prior embodiments, and a pharmaceutically acceptable carrier or excipient.

[0544] 2087. Below: a) At least 10 as described in any of the prior embodiments 3 , 10 4 , 10 5 , 10 6 , 10 7 , 108 , or 10 9 individual anestrosomes; b) Excipients for formulations, and optionally, c) Less than predetermined amounts of mycoplasma, exotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replicable organisms (RCAs), e.g., replicable viruses or unwanted anestrophores, free viral capsid proteins, incidental materials, and / or aggregates. A pharmaceutical composition containing the following:

[0545] 2088. The composition or pharmaceutical composition according to Embodiment 2085 or 2086, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more anestrosomes, such as synthetic anestrosomes.

[0546] 2089. At least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 A composition or pharmaceutical composition according to any one of embodiments 2085 to 2088, comprising anestrosomes.

[0547] 2090. Below: a) At least 10 as described in any of the prior embodiments 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 individual anestrosomes; b) Excipients for formulations, and optionally, c) Less than predetermined amounts of mycoplasma, exotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replicable organisms (RCAs), e.g., replicable viruses or unwanted anestrophores, free viral capsid proteins, incidental materials, and / or aggregates. A pharmaceutical composition containing the following:

[0548] 2091. The following characteristics: a) The pharmaceutical composition must meet pharmaceutical or Good Manufacturing Practice (GMP) standards; b) The pharmaceutical composition is manufactured in accordance with Good Manufacturing Practices (GMP); c) The pharmaceutical composition has pathogen levels below predetermined standard values, for example, it is substantially free of pathogens; d) The pharmaceutical composition has a level of impurities below a predetermined standard value, for example, it is substantially free of impurities; e) The pharmaceutical composition has a predetermined level of non-infectious particles, or a predetermined particle:infectious unit ratio (e.g., <300:1, ≤200:1, ≤100:1, or <50:1), or f) The pharmaceutical composition is low immunogenic or substantially non-immunogenic, for example, as described herein. A composition or pharmaceutical composition according to any one of embodiments 2085 to 2090, having one or more of the above.

[0549] 2092. A composition or pharmaceutical composition according to any of Embodiments 1085 to 2091, wherein the pharmaceutical composition has a level of impurities below a predetermined standard value, for example, substantially free of impurities.

[0550] 2093. The composition or pharmaceutical composition according to Embodiment 92, wherein the contaminants are selected from the group consisting of mycoplasma, exotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replicatable organisms (RCAs), e.g., replicatable viruses or unwanted anestrophores (desired anestrophores, e.g., anestrophores other than those described herein), free viral capsid proteins, incidental substances, and aggregates.

[0551] 2094. The composition or pharmaceutical composition according to Embodiment 2093, wherein the impurity is host cell DNA, and the threshold amount is approximately 500 ng of host cell DNA per dose of the pharmaceutical composition.

[0552] 2095. A composition or pharmaceutical composition according to any of Embodiments 2085 to 2094, wherein the pharmaceutical composition contains less than 10% by weight of impurities (for example, about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less than 0.1%).

[0553] 2096. The method according to any of the prior embodiments, wherein the anestroma does not contain an external effector.

[0554] 2097. The method according to any of the prior embodiments, wherein delivery of gene elements to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a population of target cells in a subject is achieved by administration of anelosomes, for example, synthetic anelosomes.

[0555] 2098. The method according to any of the prior embodiments, wherein the administration of anelosomes, for example synthetic anelosomes, achieves delivery of an external effector to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a subject's target cell population.

[0556] 2099. The method according to Embodiment 2097 or 2098, wherein the target cells include, for example, mammalian cells, such as human cells, such as immune cells, hepatocytes, and lung epithelial cells, in vitro.

[0557] 2100. The method according to any one of embodiments 2097 to 2099, wherein the target cells are located in the liver or lungs.

[0558] 2101. The method according to any one of embodiments 2097 to 2100, wherein each target cell to which the gene element is delivered receives at least 10, 50, 100, 500, 1,000, 10,000, 50,000, 100,000, or more copies of the gene element.

[0559] 2102. The method according to any of the prior embodiments, wherein the effector comprises a miRNA, and optionally the miRNA reduces, for example, the level of a target protein or RNA in a cell or population of cells to which an anestronomy is delivered by, for example, at least 10%, 20%, 30%, 40%, or 50%.

[0560] 2103. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein a gene element (e.g., the 5'UTR of a gene element) physically associates (e.g., binds) with a proteinaceous outer layer (e.g., an ORF1 molecule in the proteinaceous outer layer).

[0561] 2104. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element confined within the proteinaceous outer layer is resistant to endonuclease digestion, as determined, for example, by the method described in Martin et al. (2013, Hum. Gene Ther. Methods 24(4):253-269; the whole thereof is incorporated herein by reference); and optionally, the amount of DNase used is about 60 U / ml or about 300 U.

[0562] 2105. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element comprises a sequence of at least 100 nucleotides in length, and is composed of G or C at at least 80% of the positions.

[0563] 2106. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element is circular, single-stranded DNA.

[0564] 2107. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element does not include one or more bacterial plasmid elements (e.g., bacterial origin of replication or selection marker, e.g., bacterial resistance gene).

[0565] 2108. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element is incorporated at a frequency of less than 1% of the anestromas entering a mammalian cell.

[0566] 2109. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the promoter element is either exogenous or endogenous with respect to wild-type anellovirus.

[0567] 2110. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the external effector is a therapeutic external effector, such as a therapeutic peptide, therapeutic polypeptide, or therapeutic nucleic acid (e.g., miRNA).

[0568] 2111. A population of at least 1000 (e.g., at least 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 50,000, 75,000, 100,000, 200,000, 500,000, 1,000,000 or more) anestrosomes contains at least 100 (e.g., at least 100, 1) gene elements. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, for delivering copies of 50, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 50,000, or more copies to one or more mammalian cells.

[0569] 2112. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the anestronomy comprises one or more polypeptides comprising one or more amino acid sequences selected from Anellovirus ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 (for example, as described herein), or amino acid sequences having at least 95% sequence identity thereto.

[0570] 2113. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element comprises an amino acid sequence selected from an anelovirus ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 (for example, as described herein), or a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity thereto.

[0571] 2114. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the anestronomy does not contain a polynucleotide encoding one or both of a replication factor and / or a capsid protein, or the anestronomy is replication-deficient.

[0572] 2115. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, for contacting an anestronomy with a cell in vitro or in vivo.

[0573] 2116. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the anestronomy does not contain a polypeptide having at least 95% sequence identity with Anellovirus ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 (for example, as described herein).

[0574] 2117. A polypeptide, complex, anestronomy, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein a gene element can be amplified by rolling circle replication (e.g., in a cell, e.g., a host cell, e.g., a mammalian cell, e.g., a human cell, e.g., HEK293T or A549 cell) to produce, for example, at least 2, 4, 8, 16, 32, 64, 128, 256, 518, or 1024 copies.

[0575] 2118. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element is produced from a double-stranded circular DNA molecule.

[0576] 2119. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein a double-stranded circular DNA molecule is produced by in vitro cyclization.

[0577] 2118. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein the gene element is produced from a DNA molecule containing two copies of the nucleic acid sequence of the gene element.

[0578] 2119. A polypeptide, complex, anestroma, isolated nucleic acid, cell, composition, or method according to any of the prior embodiments, wherein two copies of the nucleic acid sequence of a gene element are arranged in tandem within a DNA molecule.

[0579] 2120. A nucleic acid molecule comprising two copies of a nucleic acid sequence including the 5'UTR of an anestrom gene element (e.g., a gene element described in any of the prior embodiments).

[0580] 2121. A nucleic acid molecule comprising: a promoter element; a nucleic acid sequence encoding an external effector; a 5'UTR listed in any of Tables B1 to B5, or a nucleic acid sequence having at least 85% identity thereto (e.g., at least 85%, 90%, 95%, 96%, 97%, 99%, or 100%); and a GC-rich region listed in any of Tables B1 to B5, or a nucleic acid sequence having at least 85% identity thereto (e.g., at least 85%, 90%, 95%, 96%, 97%, 99%, or 100%).

[0581] 2122. The nucleic acid according to Embodiment 2121, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0582] 2123. The nucleic acid according to Embodiment 2121, wherein the nucleic acid molecule is cyclic.

[0583] 2124. Genetic elements, [ka] ( Sequence ID 1001; X1 is either C or does not exist. nucleic acid sequence, or nucleic acid sequences having at least 95% identity with respect to the same. A polypeptide, complex, anerosome, isolated nucleic acid, cell, composition, or method according to any of the preceding embodiments, comprising a 5’UTR containing .

[0584] 3001. The following: (i) The following: (a) A promoter element and (b) A nucleic acid sequence encoding an exogenous effector, wherein the nucleic acid sequence is operably linked to the promoter element, and the exogenous effector is one of the following: An IDH1 inhibitor; An IDH2 inhibitor; An EGFR inhibitor; An LRP5 inhibitor; A DKK2 inhibitor; A DPP-4 inhibitor; A KRAS inhibitor; A neutrophil elastase inhibitor; An FGFR3 activator, or An HTT activator A nucleic acid sequence that is an intracellular therapeutic agent selected from the above, and (c) The following: The nucleic acid sequence of nucleotides 323-393 of SEQ ID NO: 54, or a nucleic acid sequence at least 85% identical thereto; Any of the nucleic acid sequences of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or a nucleic acid sequence at least 85% identical thereto; or The nucleic acid sequence of nucleotides 117-187 of SEQ ID NO: 61, or a nucleic acid sequence at least 85% identical thereto A 5’UTR domain containing , and A gene element containing ; <​​​​​​​​

[0585] 3002. The synthetic anestronomy according to Embodiment 3001, wherein the intracellular therapeutic agent is an intracellular polypeptide or an intracellular nucleic acid.

[0586] 3003. The synthetic anestroma according to Embodiment 3001, wherein the ORF1 molecule contains the amino acid sequence of SEQ ID NO: 217, or an amino acid sequence having at least 90% identity thereto.

[0587] A synthetic anestronomy according to any of the prior embodiments, wherein the 3004.ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO: 54.

[0588] 3005. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element comprises the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID NO: 54, or a nucleic acid sequence having at least 85% identity thereto.

[0589] A synthetic anestrome according to any of the prior embodiments, wherein the 3006.ORF1 molecule comprises an amino acid sequence including one or more of the amino acid sequences of the arg-rich region, jelly-roll domain, hypervariable domain, N22 domain, and / or C-terminal domain listed in Table 16, or an amino acid sequence having at least 85% identity thereto.

[0590] A synthetic anestroma according to any of the prior embodiments, wherein the 3007.ORF1 molecule contains the amino acid sequence of SEQ ID NO: 58, or a nucleic acid sequence having at least 85% identity thereto.

[0591] 3008. A synthetic anestronomy according to any of the prior embodiments, further comprising a polypeptide containing the amino acid sequence of ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto.

[0592] 3009. A synthetic anestrome according to any of the prior embodiments, wherein the gene element codes for the amino acid sequence of ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto.

[0593] 3010. A synthetic anestronomy according to any of the prior embodiments, wherein the synthetic anestronomy does not contain a polypeptide comprising the amino acid sequence of ORF2, ORF2 / 2, ORF2 / 3, TAIP, ORF1 / 1, or ORF1 / 2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto.

[0594] 3011. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element does not encode the amino acid sequence of ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto.

[0595] The 3012.ORF1 molecule has the amino acid sequence YNPX 2 DXGX 2 N(Sequence ID 829)(X n A synthetic anestrome according to any of the prior embodiments, comprising (where each is independently a sequence of any n amino acids).

[0596] The 3013.ORF1 molecule has the amino acid sequence YNPX 2 DXGX 2 It further comprises a first β-chain and a second β-chain flanking with N (sequence number 829), for example, the first β-chain having the amino acid sequence YNPX 2 DXGX 2 It contains the tyrosine (Y) residue of N (SEQ ID NO: 829) and / or the second β chain has the amino acid sequence YNPX 2 DXGX 2 A synthetic anestrome according to Embodiment 3012, comprising the second asparagine (N) residue (N to C) of N (Sequence ID 829).

[0597] A synthetic anestronomy according to any of the prior embodiments, wherein the 3014.ORF1 molecule comprises, in order from the N-terminus to the C-terminus, a 1st β-chain, a 2nd β-chain, a 1st α-helix, a 3rd β-chain, a 4th β-chain, a 5th β-chain, a 2nd α-helix, a 6th β-chain, a 7th β-chain, an 8th β-chain, and a 9th β-chain.

[0598] 3015. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element can be amplified by rolling circle replication within a host cell to produce, for example, at least 8 copies.

[0599] 3016. A synthetic anestrome according to any of the prior embodiments, wherein the gene element is single-stranded.

[0600] 3017. A synthetic anestrome according to any of the prior embodiments, wherein the gene element is circular.

[0601] 3018. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element is DNA.

[0602] 3019. A synthetic anestrome according to any of the prior embodiments, wherein the gene element is a negative-strand DNA.

[0603] 3020. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element is incorporated at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, and 0.1% of the anestronomy entering human cells.

[0604] 3021. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element comprises a sequence of the consensus 5'UTR nucleic acid sequence shown in Table 16-1.

[0605] 3022. A synthetic anestrome according to any of the prior embodiments, wherein the gene element comprises a sequence of a consensus GC-rich region shown in Table 16-2.

[0606] 3023. A synthetic anestrome according to any of the prior embodiments, wherein the gene element comprises a sequence of at least 100 nucleotides in length, which consists of G or C at at least 70% (e.g., about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%).

[0607] 3024. A synthetic anestrome according to any of the prior embodiments, wherein the gene element comprises the nucleic acid sequence of SEQ ID NO: 120.

[0608] 3025. A synthetic anestronomy according to any of the prior embodiments, wherein the promoter element is exogenous with respect to wild-type anellovirus.

[0609] 3026. A synthetic anestronomy according to any of the prior embodiments, wherein the promoter element is endogenous with respect to wild-type anellovirus.

[0610] 3027. A synthetic anestronomy according to any of the prior embodiments, wherein the external effector comprises a regulatory nucleic acid, such as miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA, peptide, synthetic or analogous peptide derived from naturally occurring bioactive peptides, agonist or antagonist peptide, a competitive inhibitor of one enzyme, ligand, antibody, receptor, or CRISPR system or component.

[0611] 3028. A synthetic anestronomy according to any of the prior embodiments, wherein the external effector contains miRNA and reduces the expression of a host gene.

[0612] 3029. A synthetic anestronomy according to any of the prior embodiments, wherein the external effector comprises a nucleic acid sequence having a length of approximately 20-200, 30-180, 40-160, 50-140, 60-120, 200-2000, 200-500, 500-1000, 1000-1500, or 1500-2000 nucleotides.

[0613] 3030. A synthetic anestronomy according to any of the prior embodiments, wherein the nucleic acid sequence encoding an external effector is approximately 20-200, 30-180, 40-160, 50-140, 60-120, 200-2000, 200-500, 500-1000, 1000-1500, or 1500-2000 nucleotides in length.

[0614] 3031. A synthetic anestronomy according to any of the prior embodiments, wherein the gene element has a length of approximately 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, or 4.5-5.0 kb.

[0615] 3032. A synthetic anestronomy according to any of the prior embodiments, wherein the synthetic anestronomy can infect human cells, such as immune cells, hepatocytes, or lung epithelial cells.

[0616] 3033. A synthetic anestronomy according to any of the prior embodiments that is substantially non-immunogenic, for example, detectable and / or does not induce an unwanted immune response when detected according to the method described in Example 4.

[0617] 3034. A synthetic anestrosomal according to Embodiment 3033, wherein the substantially non-immunogenic anestrosomal has an efficacy in a subject of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the efficacy in a control subject lacking an immune response.

[0618] 3035. A synthetic anestronomy according to any of the prior embodiments, wherein a population of at least 1000 anestroms can deliver 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 gene element into one or more human cells.

[0619] 3036. A pharmaceutical composition comprising a synthetic anestroma described in any of the prior embodiments, and a pharmaceutically acceptable carrier or excipient.

[0620] 3037. At least 10 3 , 10 4 , 105, 10 6 , 10 7 , 10 8 , or 10 9 A synthetic anesome according to Embodiment 3036, comprising the synthetic anesome.

[0621] 3038. The pharmaceutical composition according to Embodiment 3036 or 3037, wherein the pharmaceutical composition has a predetermined ratio of particles:infectious units (e.g., <300:1, <200:1, <100:1, or <50:1).

[0622] 3039. Below: (i) A first nucleic acid molecule (e.g., double-stranded or single-stranded circular DNA) comprising the sequence of gene elements of the synthetic anestroma described in any of the prior embodiments, and (ii) A second nucleic acid molecule that encodes, for example, one or more amino acid sequences selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 listed in Table 16, or amino acid sequences having at least 85% sequence identity thereto. A reaction mixture containing the following:

[0623] 3040. The reaction mixture according to Embodiment 3039, wherein the first nucleic acid and the second nucleic acid are in the same nucleic acid molecule.

[0624] 3041. The reaction mixture according to Embodiment 3039, wherein the first nucleic acid and the second nucleic acid are different ...

Claims

1. below: (I) Below: (a) Promoter element and (b) A nucleic acid sequence encoding an external effector, wherein the nucleic acid sequence is operably linked to the promoter element, and the external effector is an intracellular polypeptide or intracellular nucleic acid; Genetic elements including; and (II) Protein outer layer containing an anelovirus ORF1 molecule with the amino acid sequence of SEQ ID NO: 58 Synthetic particles containing, The gene element is confined within the proteinaceous outer layer; and The synthetic particles are capable of delivering the gene elements into human cells.

2. The synthetic particle according to claim 1, further comprising a polypeptide having an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs. 55-57, 59, or 60, or an amino acid sequence that is at least 90% identical thereto.

3. The synthetic particle according to claim 1 or 2, wherein the gene element encodes one of the amino acid sequences of sequence numbers 55 to 60, or an amino acid sequence having at least 90% identity thereto.

4. The synthetic particle according to claim 1, wherein the synthetic particle does not contain a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs. 55-57, 59, or 60, or an amino acid sequence having at least 90% identity thereto.

5. The synthetic particle according to claim 1 or 4, wherein the gene element does not encode any one of the amino acid sequences of sequence numbers 55 to 60, or an amino acid sequence having at least 90% identity thereto.

6. The aforementioned gene element is: (i) It can be amplified by rolling circle replication within the host cell; (ii) Single stranded; (iii) It is circular; (iv) DNA; and / or (v) It is a negative strand DNA, Synthetic particles according to any one of claims 1 to 5.

7. The aforementioned gene element is: (i) incorporated at a frequency of less than 10% of the particles that enter the human cells; (ii) A sequence comprising at least 100 nucleotides in length, which consists of G or C at at least 70% of the positions; and / or (iii) Having a length of 1.5 to 5.0 kb, Synthetic particles according to any one of claims 1 to 6.

8. The synthetic particle according to any one of claims 1 to 7, wherein the promoter element is either external or endogenous to wild-type anelovirus (Anellovirus).

9. The synthetic particle according to any one of claims 1 to 8, wherein the external effector contains miRNA and reduces the expression of host genes.

10. The synthetic particles according to any one of claims 1 to 9, wherein the synthetic particles can infect human cells, such as immune cells, hepatocytes, or lung epithelial cells.

11. Synthetic particles according to any one of claims 1 to 10, which do not induce a detectable and / or unwanted immune response.

12. The synthetic particle according to claim 11, wherein the synthetic particle has an efficacy in a subject that is at least about 10% of the efficacy in a control subject lacking an immune response.

13. The synthetic particle according to any one of claims 1 to 12, wherein a population of at least 1,000 of the particles can deliver at least about 100 copies of the gene element into one or more human cells.

14. The synthetic particle according to any one of claims 1 to 13, wherein the external effector is a polypeptide for intracellular therapeutic use or a nucleic acid for intracellular therapeutic use.

15. The intracellular polypeptide or intracellular nucleic acid is as follows: IDH1 inhibitors; IDH2 inhibitors; EGFR inhibitors; LRP5 inhibitors; DKK2 inhibitors; DPP-4 inhibitors; KRAS inhibitors; Neutrophil elastase inhibitors; FGFR3 Activator, or HTT Activator A synthetic particle according to any one of claims 1 to 14, comprising:

16. The synthetic particle according to any one of claims 1 to 15, wherein the external effector comprises an intracellular polypeptide other than nano-luciferase.

17. The synthetic particle according to any one of claims 1 to 16, wherein the external effector comprises intracellular nucleic acids other than miR-124, miR-518, miR-625, miRNA for n-myc interacting proteins, or endogenous miRNA of wild-type anelovirus (Anellovirus).

18. A pharmaceutical composition comprising synthetic particles according to any one of claims 1 to 17, and a pharmaceutically acceptable carrier or excipient.

19. at least 10 ３ The pharmaceutical composition according to claim 18, comprising synthetic particles.

20. The pharmaceutical composition according to claim 18 or 19, wherein the pharmaceutical composition has a predetermined ratio of particles:infectious units.

21. A method for producing a synthetic particle preparation, wherein the method is as follows: a) the step of preparing a plurality of synthetic particles according to any one of claims 1 to 17, or a pharmaceutical composition according to any one of claims 18 to 20; and b) The step of formulating the plurality of synthetic particles as a pharmaceutical composition. Methods that include...

22. An in vitro method for delivering an external effector to a mammalian cell, wherein the method is as follows: (a) the step of preparing synthetic particles according to any one of claims 1 to 17; and (b) The step of bringing mammalian cells into contact with the synthetic particles. Including; Here, the synthetic particles can deliver the gene elements to the mammalian cells; The intracellular polypeptide or intracellular nucleic acid is a therapeutic intracellular polypeptide or therapeutic intracellular nucleic acid, A method for delivering the external effector to the mammalian cell.

23. A composition comprising synthetic particles according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 20 for use in delivering the aforementioned gene element to a host cell.

24. The use of synthetic particles according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 20 in the manufacture of a drug for delivering the aforementioned gene element to a host cell.

25. A composition comprising synthetic particles according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 20, for use in the treatment of a disease or disorder of a subject.

26. Use of synthetic particles according to any one of claims 1 to 17 or a pharmaceutical composition according to any one of claims 18 to 20 in the manufacture of a drug for the treatment of a disease or disorder of a subject.

27. The composition according to claim 25 or the use according to claim 26, wherein the disease or disorder is cancer.

Images