Compositions containing chloron and their use

Abstract

To provide pharmaceutical compositions and preparations of chlorophene, as well as their use. [Solution] In some embodiments, the Kron comprises particles containing a gene element encapsulated in a proteinaceous outer layer, which can introduce the gene element into a cell (e.g., a human cell). In some cases, the gene element contains a payload, for example, which encodes an extrinsic effector (e.g., a nucleic acid effector such as non-coding RNA, or a polypeptide effector such as a protein) to be expressed in the cell.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 62 / 518,898 filed on 13 June 2017, U.S. Patent Application No. 62 / 597,387 filed on 11 December 2017, and U.S. Patent Application No. 62 / 676,730 filed on 25 May 2018, each of which is incorporated herein by reference as a whole.

[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 13 June 2018, titled V2057-7000WO_SL.txt, and its size is 1,066,292 bytes. [Background technology]

[0003] Existing viral systems for delivering therapeutic drugs can be highly immunogenic because they use viruses that may be associated with the disease or disorder. There is a need for improved delivery vehicles that are substantially non-immunogenic and non-pathogenic. [Overview of the project] [Means for solving the problem]

[0004] The present invention provides a clon, such as a synthetic clon, which can be used as a delivery vehicle to deliver therapeutic agents to, for example, fungal cells. In some embodiments, the clon comprises a particle containing a gene element encapsulated in a proteinaceous outer layer, which can introduce the gene element into a cell (e.g., a human cell). In some cases, the gene element contains a payload, for example, which encodes an extrinsic effector (e.g., a nucleic acid effector such as non-coding RNA, or a polypeptide effector, such as a protein) to be expressed in the cell. For example, the clon can deliver the extrinsic effector into the cell by contacting the cell and introducing the gene element encoding the extrinsic effector into the cell, so that the extrinsic effector is produced or expressed by the cell. In some cases, the extrinsic effector can modulate the function of the cell or modulate the activity or level of a target molecule in the cell. For example, the extrinsic effector can reduce the viability of cancer cells (e.g., as described in Example 22) or reduce the level of a target protein in the cell, such as interferon (e.g., as described in Examples 3 and 4). In another example, the external effector may be a protein expressed by a cell (as described in Example 9, for example).

[0005] Synthetic clons have at least one structural difference compared to wild-type viruses, e.g., deletion, insertion, substitution, or enzymatic modification. Generally, synthetic clons comprise an exogenous gene element confined within a proteinaceous outer layer, which can be used as a substantially non-immunogenic vehicle for delivering the gene element, or the effector encoded therein (e.g., an exogenous or endogenous effector) (e.g., a polypeptide or nucleic acid effector), into eukaryotic cells. Clons can be used for the treatment of diseases and disorders, for example, by delivering therapeutic agents to desired cells or tissues. The gene element of the synthetic clons of this disclosure may be a circular single-stranded DNA molecule, generally comprising a protein-binding sequence, which binds to a proteinaceous outer layer or a polypeptide bound thereto, thereby confining the gene element within the proteinaceous outer layer and / or Within the proteinaceous outer layer, the enrichment of gene elements may be promoted compared to other nucleic acids.

[0006] In one embodiment, the present invention features a synthetic clone comprising a gene element including (i) a promoter element, a sequence encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal). In some embodiments, the gene element is single-stranded DNA. Alternatively, or in combination therewith, the gene element has one or both of the following properties: being circular and / or being 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 gene elements entering the cell; and (ii) a proteinaceous outer layer. In some embodiments, the gene element is confined within the proteinaceous outer layer. In some embodiments, the synthetic clone can deliver the gene element into a eukaryotic cell.

[0007] In one embodiment, the present invention is characterized by: (i) a gene element comprising a promoter element, a sequence encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence); and (ii) a synthetic clone comprising a proteinaceous outer layer, wherein the gene element is confined within the proteinaceous outer layer; and the synthetic clone is capable of delivering the gene element into a eukaryotic cell. In some embodiments, the gene element includes a nucleic acid sequence (for example, a nucleic acid sequence of 300 to 4000 nucleotides, for example, a nucleic acid sequence of 300 to 4000 nucleotides, for example, a nucleic acid sequence of 300 to 3500 nucleotides, 300 to 3000 nucleotides, 300 to 2500 nucleotides, 300 to 2000 nucleotides, or 300 to 1500 nucleotides) 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 wild-type anellovirus sequences (for example, wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequences, for example, wild-type anellovirus sequences listed in any of Tables 1, 3, 5, 7, 9, 11, or 13). 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 respect to a wild-type anellovirus sequence (for example, a wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequence, for example, a wild-type anellovirus sequence listed in any of Tables 1, 3, 5, 7, 9, 11, or 13).

[0008] 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 a clon, for example, a synthetic clon as described herein, to the subject. In some embodiments, the clon comprises a gene element comprising: (i) a promoter element, a sequence encoding an effector (e.g., a payload), and an external protein-binding sequence. In some embodiments, the gene element is single-stranded DNA, wherein the gene element is circular and / or incorporated at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene element entering the cell; and (ii) a proteinoid outer layer, wherein the gene element is confined within the proteinoid outer layer; and the clon can deliver the gene element into a eukaryotic cell.

[0009] In one embodiment, the present invention is characterized by a method for delivering a payload to a cell, tissue or subject, the method comprising the step of administering a clon, e.g., synthetic clon as described herein, to the subject, wherein the clon comprises a nucleic acid sequence encoding the payload. In some embodiments, the clon comprises a gene element comprising: (i) a promoter element, a sequence encoding an effector (e.g., the payload), and an external protein-binding sequence. In some embodiments, the gene element is single-stranded DNA, wherein the gene element is circular and / or incorporated at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene element entering the cell; and (ii) a proteinaceous outer layer; wherein the gene element is confined within the proteinaceous outer layer; and the clon can deliver the gene element into a eukaryotic cell. In some embodiments, the payload is a nucleic acid. In some embodiments, the payload is a protein.

[0010] In one embodiment, the present invention is characterized by a method for delivering synthetic clon to a cell, which includes, for example, one of the embodiments described herein (e.g., the embodiments described above), the step of bringing the synthetic clon into contact with a cell, for example, a eukaryotic cell, for example, a mammalian cell.

[0011] In one embodiment, the present invention comprises a pharmaceutical composition comprising a chloron (e.g., synthetic chloron) as described herein. In several embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In several embodiments, the pharmaceutical composition comprises about 10 per kilogram 5 ~10 14 Includes doses containing genomic equivalents of clones.

[0012] In one embodiment, the present invention features a nucleic acid molecule comprising a gene element including a promoter element, a sequence encoding an effector (e.g., a payload), and an external protein-binding sequence. In several embodiments, the gene element is single-stranded DNA, where the gene element is circular and / or incorporated at a frequency of about 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 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 TTMV-LY polynucleotide sequence. In several embodiments, the promoter element can instruct the expression of the effector in eukaryotic cells.

[0013] In one embodiment, the present invention features a gene element comprising one, two, or three of the following: (i) a promoter element and a sequence encoding an effector (e.g., a payload); where the effector is exogenous with respect to a wild-type anellovirus sequence; and (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%) having sequence identity with respect to a wild-type anellovirus sequence. Or at least 100 (e.g., at least 300, 500, 1000, 1500) consecutive nucleic acids having sequence identity of at least 72% (e.g., at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the sequence of wild-type anellovirus; and (iii) protein-binding sequences, e.g., external protein-binding sequences, where the nucleic acid construct is single-stranded DNA; and the nucleic acid construct is circular and / or incorporated at a frequency of about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the gene elements entering the cell.

[0014] In one embodiment, the present invention is characterized by a method for producing a synthetic chloro composition, which is as follows: a) Providing, for example, a host cell expressing a clones, comprising one or more components (e.g., all components) of a synthetic clones as described herein; b) Producing a preparation of clones from host cells (the synthetic clones of the preparation include a proteinoid outer layer and gene elements comprising a promoter element, a sequence encoding an external effector (e.g., payload), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal)), thereby producing a preparation of synthetic clones; and c) The step of formulating the synthetic chlorine preparation into, for example, a pharmaceutical composition suitable for administration to a subject. Includes.

[0015] In one embodiment, the present invention is characterized by a method for producing a synthetic chloro composition, which is as follows: a) the step of providing a plurality of synthetic clones described herein, or a pharmaceutical composition described herein; and b) the step of formulating the synthetic clones into a pharmaceutical composition suitable for administration to a subject, for example.

[0016] In one embodiment, the present invention is characterized by a method for producing host cells containing synthetic clones, for example, first host cells or producer cells (for example, as shown in Figure 12), for example, a population of first host cells, the method comprising, for example, the steps of introducing a gene element into host cells and culturing the host cells under conditions suitable for the production of synthetic clones. In some embodiments, the method further comprises the step of introducing a helper, for example, a helper virus, into host cells. In some embodiments, the introduction step comprises transfection (e.g., chemical transfection) or electroporation using synthetic clones.

[0017] In one embodiment, the present invention is characterized by a method for producing synthetic clones, the method comprising the steps of providing host cells containing synthetic clones, for example, as described herein, such as first host cells or producer cells (for example, as shown in Figure 12), and purifying the clones from the host cells. In some embodiments, the method further comprises, prior to the providing step, contacting the host cells with synthetic clones, for example, as described herein, and incubating the host cells under conditions suitable for the production of synthetic clones. 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 the clones from the host cells includes a step of lysing the host cells.

[0018] In some embodiments, the method further includes a second step of contacting synthetic clon 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 the production of synthetic clon. In some embodiments, the method further includes a step of purifying the synthetic clon from the second host cells, for example, thereby producing a population of clon seeds. In several embodiments, at least about 2 to 100 times more synthetic clon is produced from the population of second host cells compared to that from the population of first host cells. In several embodiments, the step of purifying clon from second host cells includes a step of lysing the second host cells.

[0019] In some embodiments, the method further includes the step of contacting synthetic clon produced by a second host cell 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 the third host cell under conditions suitable for the production of synthetic clon. In some embodiments, the method includes the step of purifying the synthetic clon from the third host cell, for example, thereby producing a clon stock population. In several embodiments, the purification of clon from the third host cell includes the step of lysing the third host cell. In several embodiments, at least about 2 to 100 times more synthetic clon is produced from the population of third host cells compared to that from the population of second host cells.

[0020] In some embodiments, the method further includes the step of evaluating one or more synthetic clones from a clone seed population or a clone stock population with respect to one or more characteristic control parameters, such as purity, titer, potency (e.g., genomic equivalent per clone particle), and / or nucleic acid sequences (e.g., derived from gene elements contained in the synthetic clones). In some embodiments, the nucleic acid sequences to be evaluated include nucleic acid sequences encoding exogenous effectors.

[0021] In one embodiment, the present invention includes the step of evaluating one or more synthetic krons from a kron seed population or kron stock population with respect to one or more characteristic control parameters, such as purity, potency, potency, and / or nucleic acid sequences (e.g., derived from gene elements contained in the synthetic kron). In some embodiments, the nucleic acid sequences to be evaluated include nucleic acid sequences encoding exogenous effectors.

[0022] In one embodiment, the present invention is characterized by a reaction mixture comprising a synthetic clone 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.

[0023] In some embodiments, the clone (e.g., synthetic clone) is isolated, for example, from host cells and / or from other components in a solution (e.g., supernatant). In some embodiments, the clone (e.g., synthetic clone) is purified, for example, from a solution (e.g., supernatant). In some embodiments, the clone is concentrated in solution relative to other components in the solution.

[0024] In some embodiments of any of the aforementioned clones, compositions, or methods, the gene element comprises a minimal clone genome, as identified, for example, according to the method described in Example 9. In some embodiments, the minimal clone genome comprises a minimal anellovirus genome sufficient for clone replication (e.g., in a host cell). In some embodiments, the minimal clone genome comprises a TTV-tth8 nucleic acid sequence, for example, the TTV-tth8 nucleic acid sequences shown in Table 5, 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. In several embodiments, the minimal clon genome includes a TTMV-LY2 nucleic acid sequence, e.g., the TTMV-LY2 nucleic acid sequences shown in Table 11, 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 of the TTMV-LY2 nucleic acid sequence. In several embodiments, the minimal clon genome is a minimal clon genome capable of self-replication and / or self-amplification. In several embodiments, the minimal clon genome is a minimal clon genome capable of replication or amplification in the presence of a helper, e.g., a helper virus.

[0025] Further features of any of the aforementioned clones, compositions, or methods include one or more of the embodiments listed below.

[0026] 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.

[0027] List of embodiments 1. The following: (i) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal), wherein the gene element is single-stranded DNA and further has one or both of the following characteristics: being circular and / or being 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 gene elements that enter the cell; and (ii) Protein outer layer In a synthetic clone containing, The gene element is confined within a proteinaceous outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0028] 2. Below: (i) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector (e.g., a payload), and a protein binding sequence (e.g., an external protein binding sequence), Genetic elements 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 wild-type anellovirus sequences (e.g., wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequences, e.g., wild-type anellovirus sequences listed in any of Tables 1, 3, 5, 7, 9, 11, or 13); and (ii) Protein outer layer In a synthetic clone containing, The gene element is confined within a proteinaceous outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0029] 3. Below: (i) 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, e.g., an endogenous miRNA), and a protein-binding sequence (e.g., an external protein-binding sequence), The gene element has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity 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 listed in any of Tables 1, 3, 5, 7, 9, 11, or 13); and Genetic elements that are not naturally occurring sequences (for example, wild-type anellovirus sequences (e.g., wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequences, including deletions, substitutions, or insertions to wild-type anellovirus sequences listed in any of Tables 1, 3, 5, 7, 9, 11, or 13); (ii) Protein outer layer In a synthetic clone containing, The gene element is confined within a proteinaceous outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0030] 4. Below: (i) A gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector (e.g., a payload), and a protein binding sequence (e.g., an external protein binding sequence), Gene elements whose protein-binding sequences have 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 the consensus 5'UTR sequences shown in Table 16-1, or the consensus GC-rich sequences shown in Table 16-2; and (ii) Protein outer layer In a synthetic clone containing, The gene element is confined within a proteinaceous outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0031] 5. Below: (i) A gene element comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein binding sequence, wherein the gene element is as follows: (a) A sequence having at least 85% sequence identity with the anelovirus 5'UTR conserved domain nucleotide sequence of nucleotides 323-393 of the nucleic acid sequence in Table 11, or (b) Sequences having at least 85% sequence identity with the anerovirus GC-rich region of nucleotides 2868-2929 of the nucleic acid sequences in Table 11. A gene element containing one or both of the following; and (ii) Protein outer layer In a synthetic clone containing; the gene element is confined within the protein outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0032] 6. Below: (i) A gene element comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein binding sequence, wherein the gene element is as follows: (a) Sequences having at least 85% sequence identity to the anelovirus 5'UTR conserved domain of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13; or (b) Sequences having at least 85% sequence identity to the anelovirus GC-rich region of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13. A gene element containing one or both of the following; and (ii) In a synthetic clone containing a proteinaceous outer layer, the gene element is confined within the proteinaceous outer layer; and A synthetic clone is a synthetic clone that can deliver gene elements into eukaryotic cells.

[0033] 7. A synthetic clone 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 (poll III) promoter having an upstream DNA binding site for an activator protein (such as TetR-VP16, Gal4-VP16, dCas9-VP16, etc.), or a minimal CMV promoter.

[0034] 8. A synthetic chron according to any of the prior embodiments, wherein the promoter element includes a TATA box.

[0035] 9. A synthetic clon according to any of the prior embodiments, wherein the promoter element is endogenous to wild-type anellovirus, for example, any of the wild-type anelloviruses listed in Tables 1, 3, 5, 6, 9, 11, or 13.

[0036] 10. A synthetic clon according to any one of Embodiments 1 to 8, wherein the promoter element is exogenous to wild-type anellovirus.

[0037] 11. A synthetic clone according to any of the prior embodiments, wherein the external effector encodes a therapeutic agent, such as a therapeutic peptide or polypeptide or a therapeutic nucleic acid.

[0038] 12. A synthetic clone according to any of the prior embodiments, wherein the external effector comprises regulatory nucleic acids, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or markers, antigens, peptides, 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, 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.

[0039] 13. A synthetic clone according to any of the prior embodiments, wherein the external effector includes miRNA.

[0040] 14. A synthetic clone 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.

[0041] 15. A synthetic clone according to any of the prior embodiments, wherein the external effector comprises miRNA and reduces the expression of a host gene.

[0042] 16. A synthetic clone 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, or 60-120 nucleotides.

[0043] 17. A synthetic clone 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, or 60-120 nucleotides long.

[0044] 18. A synthetic clone according to any of the prior embodiments, wherein the sequence encoding an external effector has a size of at least about 100 nucleotides.

[0045] 19. A synthetic clone according to any of the prior embodiments, wherein the sequence encoding an external effector has a size of at least about 100 to about 5000 nucleotides.

[0046] 20. A synthetic clone according to any of the prior embodiments, wherein the sequence encoding the external 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.

[0047] 21. A synthetic clon according to any of the prior embodiments, wherein the sequence encoding an external 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.

[0048] 22. The synthetic clone according to Embodiment 21, wherein the sequence encoding the external effector is located between the poly-A region and the GC-rich region of the gene element.

[0049] 23. A synthetic clone according to any of the prior embodiments, comprising (for example, within a proteinaceous outer layer) one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 of Table 12, or amino acid sequences having at least 85% sequence identity thereto.

[0050] 24. A synthetic clone according to any of the prior embodiments, comprising (for example, within a proteinaceous outer layer) one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, ORF1, ORF1 / 1, or ORF1 / 2 of any of Tables 2, 4, 6, 8, 10, or 14, or amino acid sequences having at least 85% sequence identity thereto.

[0051] 25. A synthetic clone according to any of the prior embodiments, wherein the protein-binding sequence comprises a nucleic acid sequence having at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with wild-type anellovirus, for example, the 5'UTR-conserved domain or GC-rich domain of a wild-type anellovirus sequence listed in any of Tables 1, 3, 5, 6, 9, 11, 13, A, or B.

[0052] 26. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the consensus 5'UTR nucleic acid sequence shown in Table 16-1.

[0053] 27. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the exemplary TTV5'UTR nucleic acid sequence shown in Table 16-1.

[0054] 28. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-CT30F5'UTR nucleic acid sequence shown in Table 16-1.

[0055] 29. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-HD23a5'UTR nucleic acid sequence shown in Table 16-1.

[0056] 30. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-JA20 5'UTR nucleic acid sequence shown in Table 16-1.

[0057] 31. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-TJN02 5'UTR nucleic acid sequence shown in Table 16-1.

[0058] 32. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-tth8 5'UTR nucleic acid sequence shown in Table 16-1.

[0059] 33. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the consensus GC-rich region shown in Table 16-2.

[0060] 34. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the exemplary TTV GC-rich region shown in Table 16-2.

[0061] 35. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with respect to the TTV-CT30F GC-rich region shown in Table 16-2.

[0062] 36. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with respect to the TTV-HD23a GC-rich region shown in Table 16-2.

[0063] 37. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-JA20 GC-rich region shown in Table 16-2.

[0064] 38. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with respect to the TTV-TJN02 GC-rich region shown in Table 16-2.

[0065] 39. A synthetic clone according to any of the prior embodiments, wherein the gene element, for example, the protein binding sequence of the gene element, contains at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-tth8 GC-rich region shown in Table 16-2.

[0066] 40. A synthetic clone 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.

[0067] 41. A synthetic clone 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 about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%.

[0068] 42. A synthetic clone according to any of the prior embodiments, wherein the gene element comprises a sequence having at least 85% sequence identity with respect to the anelovirus 5'UTR conserved domain nucleotide sequence of nucleotides 1-393 of the nucleic acid sequence in Table 11, and a sequence having at least 85% sequence identity with respect to the anelovirus GC-rich region of nucleotides 2868-2929 of the nucleic acid sequence in Table 11.

[0069] 43. A synthetic clone according to any of the prior embodiments, wherein the protein-binding sequence can bind to an external protein, for example, a capsid protein, for example, an anelovirus capsid protein, for example, a capsid protein containing an amino acid sequence 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 any of the sequences listed in Tables 1 to 14, 16, or 18.

[0070] 44. A synthetic clone according to any of the prior embodiments, wherein the gene element has at least 75% identity with the nucleic acid sequence in Table 11.

[0071] 45. A synthetic clone according to any of the prior embodiments, wherein the protein-binding sequence binds to an arginine-rich region of the proteinaceous outer layer.

[0072] 46. ​​A synthetic clone 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.

[0073] 47. The synthetic clone according to Embodiment 46, wherein the external protein comprises an amino acid sequence having at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with a capsid protein, for example, an anelovirus capsid protein, for example, one of the sequences listed in Tables 1 to 14, 16, or 18, or a capsid protein comprising an amino acid sequence encoded by one of the sequences listed in Tables 1 to 14, 15, 17, or 19 or a fragment thereof.

[0074] 48. A synthetic clone 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, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal polyarginine sequence, a variable region, a C-terminal polyglutamine / glutamic acid sequence, and one or more disulfide crosslinks.

[0075] 49. A synthetic clone 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.

[0076] 50. A synthetic clone 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.

[0077] 51. A synthetic clone 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 4 kb), or at least 100 nucleotides (e.g., at least 1 kb).

[0078] 52. A synthetic clone according to any of the prior embodiments, wherein the gene element is single-stranded.

[0079] 53. A synthetic clone according to any of the prior embodiments, wherein the gene element is circular.

[0080] 54. A synthetic clone according to any of the prior embodiments, wherein the gene element is DNA.

[0081] 55. A synthetic clone according to any of the prior embodiments, wherein the gene element is a negative-strand DNA.

[0082] 56. A synthetic clone according to any of the prior embodiments, wherein the gene element comprises an episome.

[0083] 57. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon has a lipid content of 10%, 5%, 2%, or less than 1% by weight, and does not contain, for example, a lipid bilayer.

[0084] 58. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon 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.

[0085] 59. The synthetic chloron 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 synthetic chloron is not decomposed after incubation with a surfactant (e.g., 0.5% by weight of a surfactant) at 37°C for 30 minutes.

[0086] 60. A synthetic clone according to any of the prior embodiments, wherein the gene element has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with respect to a wild-type Circoviridae sequence or wild-type Anellovirus sequence, such as wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequence, such as one of the sequences listed in any of Tables 1, 3, 5, 7, 9, 11, or 13.

[0087] 61. The synthetic clone according to Embodiment 60, wherein the gene element comprises the deletion of at least one element, for example, one element listed in any of Tables 1, 3, 5, 7, 9, 11, or 13, relative to a wild-type anellovirus sequence, such as a wild-type TTV sequence or a wild-type TTMV sequence.

[0088] 62. The synthetic clone according to Embodiment 61, wherein the gene element includes a deletion containing a nucleic acid sequence corresponding to nucleotides 3436-3607 of the TTV-tth8 sequence, for example, the nucleic acid sequence shown in Table 5.

[0089] 63. The synthetic clone according to Embodiment 61, wherein the gene element includes a deletion comprising nucleic acid sequences corresponding to nucleotides 574-1371 and / or nucleotides 1432-2210 of the TTMV-LY2 sequence, for example, the nucleic acid sequences shown in Table 11.

[0090] 64. A synthetic clone according to Embodiment 61 or 62, wherein the gene element includes a deletion comprising a nucleic acid sequence corresponding to nucleotides 1372-1431 of the TTMV-LY2 sequence, for example, the nucleic acid sequence shown in Table 11.

[0091] 65. A synthetic clone according to Embodiments 61, 63, or 64, wherein the gene element includes a deletion comprising a nucleic acid sequence corresponding to nucleotides 2610-2809 of the TTMV-LY2 sequence, for example, the nucleic acid sequence shown in Table 11.

[0092] 66. A synthetic clone 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, such as wild-type Torque Tenovirus (TTV), Torque Teno minivirus (TTMV), or TTMDV sequence, such as one sequence listed in any of Tables 1, 3, 5, 7, 9, 11, or 13.

[0093] 67. A synthetic clone 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.

[0094] 68. A synthetic clone according to any of the prior embodiments, wherein the synthetic clone further comprises a second gene element, for example, a second gene element confined within a proteinaceous outer layer.

[0095] 69. The synthetic clone according to embodiment 68, wherein the genetic element comprises a protein binding sequence, for example an exogenous protein binding sequence, for example a packaging signal, for example as described herein, for example a 5'UTR conserved domain or a GC-rich region.

[0096] 70. The synthetic clone according to any one of the preceding embodiments, wherein the synthetic clone does not infect bacterial cells to a detectable extent, for example infects less than 1%, 0.5%, 0.1%, or 0.01% of bacterial cells.

[0097] 71. The synthetic clone according to any one of the preceding embodiments, wherein the synthetic clone is capable of infecting mammalian cells, for example human cells, for example immune cells, hepatocytes, or epithelial cells, for example in vitro.

[0098] 72. The synthetic clone according to any one of the preceding embodiments, wherein the genetic element integrates at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the clones that enter a cell, for example the synthetic clone is non-integrating.

[0099] 73. The synthetic clone according to any one of the preceding embodiments, wherein the genetic element is capable of replicating, for example producing, at least 10 2 , 2×10 2 , 5×10 2 , 10 3 , 2×10 3 , 5×10 3 , or 10 4 genome equivalents of the genetic element per cell, for example as measured by a quantitative PCR assay.

[0100] 74. The synthetic clone, wherein as measured for example by a quantitative PCR assay, the genetic element has at least 10 2 , 2×10 2 , 5×10 2 , 10 3 , 2×10 3 , 5×10 3, or 10 4 A synthetic clone according to any of the prior embodiments, which can replicate, for example, produce a large genome equivalent of a gene element.

[0101] 75. A synthetic clone 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.

[0102] 76. A synthetic clone 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 a host cell genome.

[0103] 77. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon 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 clon).

[0104] 78. A synthetic clon 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.

[0105] 79. The synthetic clon according to Embodiment 78, wherein the substantially non-immunogenic clon 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.

[0106] 80. The synthetic clon according to Embodiment 78 or 79, wherein the immune response comprises one or more of the following: an antibody specific to clon; a cellular response to clon or cells containing clon (immune effector cell (e.g., T cell or NK cell) response); or macrophage phagocytosis of cells against clon or cells containing clon.

[0107] 81. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon 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., an antibody positivity rate of about 60%, 50%, 40%, 30%, 20%, or less than 10%) when measured by the assay described herein, or is substantially non-immunogenic.

[0108] 82. A synthetic clone according to any of the prior embodiments, wherein a population of at least 1000 synthetic clones can deliver at least 100 copies of a gene element into one or more eukaryotic cells.

[0109] 83. A synthetic clone according to any of the prior embodiments, wherein the population of synthetic clones 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.

[0110] 84. The population of synthetic chloroplasm is present in the population of eukaryotic cells at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 8,000, and 1 × 10¹⁶ cells per cell. 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 A synthetic clone according to any of the prior embodiments, capable of delivering copies of the above-mentioned gene elements.

[0111] 85. A population of synthetic chloroplasm is present in a population of eukaryotic cells at a rate of 1 × 10⁶ cells per cell. 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 × 107 A synthetic clone according to any of the prior embodiments, which can deliver copies of the gene elements.

[0112] 86. A synthetic cron according to any of the prior embodiments, wherein the synthetic cron is present after at least two passages.

[0113] 87. A synthetic clones according to any of the prior embodiments, wherein the synthetic clones are produced by a process comprising at least two passages.

[0114] 88. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon selectively delivers an external effector to a desired cell type, tissue, or organ (e.g., retinal photoreceptors, endothelial layer, or pancreas).

[0115] 89. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon exhibits higher selectivity in vitro for a fetal kidney cell line (e.g., HEK293T) than for a lung epithelial cancer cell line (e.g., A549).

[0116] 90. A synthetic clon according to any of the prior embodiments, wherein the synthetic clon is present in a high level (e.g., preferentially accumulates) in a desired organ or tissue compared to other organs or tissues.

[0117] 91. The synthetic clone according to Embodiment 90, wherein the desired organ or tissue is bone marrow, blood, heart, gastrointestinal tract, or skin.

[0118] 92. A synthetic clone according to any of the prior embodiments, wherein the eukaryotic cell is a mammalian cell, such as a human cell.

[0119] 93. A synthetic clon, or a copy thereof, that 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) according to any of the prior embodiments.

[0120] 94. When synthetic clons are 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 synthetic clons 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 A synthetic clone according to any of the prior embodiments, produced in the cell pellet and supernatant at a genome equivalent of (times) per mL.

[0121] 95. A composition comprising a synthetic chloron as described in any of the prior embodiments.

[0122] 96. A pharmaceutical composition comprising a synthetic clon described in any of the prior embodiments, and a pharmaceutically acceptable carrier or excipient.

[0123] 97. The composition or pharmaceutical composition according to Embodiment 95 or 96, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of chlorofluoropolymers, such as synthetic chlorofluoropolymers.

[0124] 98. 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 95 to 97, comprising a synthetic chloron.

[0125] 99. Below: a)(i) A gene element as described herein, for example, a gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal), wherein the gene element is single-stranded DNA and further has one or both of the following characteristics: being circular and / or being 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 gene elements that enter the cell; and (ii) Protein outer layer including at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The individual clones (e.g., synthetic clones as described herein) (the gene elements are confined within a proteinaceous outer layer; and Synthetic chlorofluorocarbons can deliver gene elements into eukaryotic cells. b) Excipients for formulations, and optionally, c) Mycoplasma in amounts less than the target amount, 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 clon, free viral capsid proteins, exogenous contaminants, and / or aggregates. A pharmaceutical composition containing the following:

[0126] 100.a)(i) A gene element described herein, for example, a gene element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence), Genetic elements having at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity 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 listed in any of Tables 1, 3, 5, 7, 9, 11, or 13); and (ii) Protein outer layer including at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 individual clones (for example, the synthetic clones described herein) (The gene element is confined within a proteinaceous outer layer; and Synthetic chlorofluorocarbons can deliver gene elements into eukaryotic cells. b) Excipients for formulations, and optionally, c) Mycoplasma in amounts less than the target amount, 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 clon, free viral capsid proteins, exogenous contaminants, and / or aggregates. A pharmaceutical composition containing the following:

[0127] 101. 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 95 to 100, having one or more of the above.

[0128] 102. A composition or pharmaceutical composition according to any of Embodiments 95 to 101, wherein the pharmaceutical composition has a level of impurities below a predetermined standard value, for example, substantially free of impurities.

[0129] 103. The composition or pharmaceutical composition according to Embodiment 102, 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 clon (desired clon, e.g., clon other than synthetic clon as described herein), free viral capsid proteins, exogenous contaminants, and aggregates.

[0130] 104. The composition or pharmaceutical composition according to Embodiment 103, 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.

[0131] 105. A composition or pharmaceutical composition according to any of Embodiments 95 to 104, 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%).

[0132] 106. Use of a synthetic clone, composition, or pharmaceutical composition described in any of the prior embodiments for the purpose of treating a disease or disorder of a subject.

[0133] 107. Use according to Embodiment 106, wherein the disease or disorder is selected from immunodeficiency, interferonosis (e.g., type I interferonosis), infection, inflammatory disorder, autoimmune disease, cancer (e.g., solid tumor, e.g., lung cancer), and gastrointestinal disorder.

[0134] 108. A synthetic clone, composition, or pharmaceutical composition described in any of the prior embodiments for use in treating a disease or disorder of a subject.

[0135] 109. A method for treating a disease or disorder of a subject, the method comprising the step of administering to the subject a synthetic chloroform described in any of the prior embodiments or a pharmaceutical composition described in any of embodiments 95 to 105.

[0136] 110. The method according to Embodiment 109, wherein the disease or disorder is selected from immunodeficiency, interferonosis (e.g., type I interferonosis), infection, inflammatory disorder, autoimmune disease, cancer (e.g., solid tumor, e.g., lung cancer), and gastrointestinal disorder.

[0137] 111. A method for modulating, for example, enhancing the biological function of a subject, the method comprising the step of administering to the subject a synthetic chloroform described in any of the prior embodiments or a pharmaceutical composition described in any of embodiments 95 to 105.

[0138] 112. A method for treating a disease or disorder in a subject, wherein the method provides the subject with the following: (i) A gene element comprising a promoter element, a sequence encoding an effector (e.g., a payload), and an external protein binding sequence; Genetic elements that are single-stranded DNA, where the genetic element is circular, and / 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 genetic elements that enter a cell; and (ii) Protein outer layer The step includes administering a clone, for example, a synthetic clone, Here, the gene element is confined within a proteinaceous outer layer; and A method for delivering gene elements into eukaryotic cells, such as synthetic clons.

[0139] 113. The method according to Embodiment 112, wherein the disease or disorder is selected from immunodeficiency, interferonosis (e.g., type I interferonosis), infection, autoimmune disease, cancer (e.g., solid tumors, e.g., lung cancer), and gastrointestinal disorder.

[0140] 114. The method according to any of embodiments 109 to 113, wherein the effector is not SV40-miR-S1, but for example, the effector is a protein-encoded payload.

[0141] 115. The method according to any one of embodiments 109 to 114, wherein the chromium does not include an external effector.

[0142] 116. The method according to any one of embodiments 109 to 115, wherein the clones include a wild-type circovirus or a wild-type anellovirus, such as TTV or TTMV.

[0143] 117. The method according to any one of embodiments 109 to 116, wherein delivery of gene elements to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a target cell population of a subject is achieved by administration of a clone, for example, a synthetic clone.

[0144] 118. The method according to any one of embodiments 109 to 117, wherein the administration of a clo, for example, synthetic clo, achieves delivery of an external effector to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the target cell population of the subject.

[0145] 119. The method according to Embodiment 117 or 118, wherein the target cells include, for example, mammalian cells, such as human cells, such as immune cells, hepatocytes, and lung epithelial cells, in vitro.

[0146] 120. The method according to any one of embodiments 117 to 119, wherein the target cells are located in the liver or lungs.

[0147] 121. The method according to any one of Embodiments 117 to 120, 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.

[0148] 122. The method according to any one of Embodiments 109 to 121, wherein the effector includes miRNA, and the miRNA reduces, for example, the level of a target protein or RNA in the cell or population of cells to which the clone is delivered by, for example, at least 10%, 20%, 30%, 40%, or 50%.

[0149] 123. A method for delivering a synthetic clon to a cell, comprising the step of bringing a synthetic clon described in any of the prior embodiments into contact with a cell, for example, a eukaryotic cell, for example, a mammalian cell.

[0150] 124. The method according to Embodiment 123, 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.

[0151] 125. The method according to Embodiment 124, wherein a helper virus is brought into contact with cells before, simultaneously with, or after the step of bringing a synthetic clone into contact with cells.

[0152] 126. The method according to embodiment 123, further comprising the step of bringing a helper polynucleotide into contact with a cell.

[0153] 127. The method according to Embodiment 126, 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.

[0154] 128. The method according to Embodiment 126, wherein the helper polynucleotide is RNA (e.g., mRNA), DNA, plasmid, viral polynucleotide, or any combination thereof.

[0155] 129. The method according to any one of embodiments 126 to 128, wherein the helper polynucleotide is brought into contact with the cell before, simultaneously with, or after the step of bringing the synthetic clones into contact with the cell.

[0156] 130. The method according to any one of embodiments 123 to 129, further comprising the step of bringing a helper protein into contact with a cell.

[0157] 131. The method according to Embodiment 130, wherein the helper protein comprises a viral replication protein or a capsid protein.

[0158] 132. A host cell comprising a synthetic clon as described in any of the prior embodiments.

[0159] 133. 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.

[0160] 134. A nucleic acid molecule comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein-binding sequence, wherein the gene element is as follows: (a) A sequence having at least 85% sequence identity with the anelovirus 5'UTR conserved domain nucleotide sequence of nucleotides 323-393 of the nucleic acid sequence in Table 11, or (b) A nucleic acid molecule containing one or both of the sequences having at least 85% sequence identity to the anellovirus GC-rich region between nucleotides 2868 and 2929 of the nucleic acid sequences in Table 11.

[0161] 135. A nucleic acid molecule comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein-binding sequence, wherein the gene element is as follows: (a) A sequence having at least 85% sequence identity to the anelovirus 5'UTR conserved domain of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13, or (b) Sequences having at least 85% sequence identity to the anelovirus GC-rich region of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13. A nucleic acid molecule containing one or both of the following.

[0162] 136. Below: (i) A sequence encoding a promoter element and an effector (e.g., payload) (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.

[0163] 137. A method for producing a synthetic chloro composition, the following: a) A step of providing a host cell comprising a synthetic clone, for example, one or more nucleic acid molecules encoding a component of the synthetic clone described herein, wherein the synthetic clone comprises a proteinaceous outer layer and a gene element, for example, a promoter element and a sequence encoding an external effector (e.g., a payload) and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal); b) A step of producing synthetic clones from host cells, thereby creating synthetic clones; and c) The step of formulating the synthetic clones into a pharmaceutical composition suitable for administration to a subject, for example. Methods that include...

[0164] 138. A method for producing a synthetic chloro composition, the following: a) A step of providing a plurality of synthetic clones described in any of the prior embodiments, or a composition or pharmaceutical composition described in any of embodiments 95 to 105; b) Optionally, a step of evaluating multiple synthetic clones with respect to one or more of the following: inclusions described herein, optical density measurement (e.g., OD260), particle count (e.g., by HPLC), infectivity (e.g., particle:infectivity ratio); and c) For example, if one or more of the parameters in (b) meet a specified threshold, the step of formulating multiple synthetic clones into a pharmaceutical composition suitable for administration to a subject. Methods that include...

[0165] 139. The synthetic chloro 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 method according to Embodiment 138, comprising a synthetic chloron.

[0166] 140. The method according to Embodiment 138 or 139, wherein the synthetic chloro 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.

[0167] 141. A reaction mixture comprising a synthetic clone and a helper virus as described in any of the prior embodiments, 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 sequence), and optionally a lipid envelope.

[0168] 142. A reaction mixture comprising a synthetic clone as described in any of the prior embodiments, and a second nucleic acid sequence encoding one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 of Table 12, or amino acid sequences having at least 85% sequence identity thereto.

[0169] 143. A reaction mixture comprising a synthetic clone as described in any of the prior embodiments, and a second nucleic acid sequence encoding one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, ORF1, ORF1 / 1, or ORF1 / 2 as shown in any of Tables 2, 4, 6, 8, 10, or 14, or amino acid sequences having at least 85% sequence identity thereto.

[0170] 144. The reaction mixture according to Embodiment 142 or 143, wherein the second nucleic acid sequence is part of a gene element.

[0171] 145. The reaction mixture according to Embodiment 144, 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.

[0172] 146. 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 clone comprising, for example, a proteinaceous outer layer that encloses or encapsulates a gene element.

[0173] 147. Below: a) 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 clone comprising, for example, a proteinaceous outer layer that encloses or surrounds a gene element; and b) Excipients for pharmaceutical formulations A pharmaceutical composition containing the following:

[0174] 148. Below: a) 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 gene element is bound to it, for example, a proteinaceous outer layer that encloses or encapsulates it, at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 individual clones (for example, the synthetic clones described herein) b) Excipients for formulations, and optionally, c) Mycoplasma in amounts less than the target amount, 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 clon, free viral capsid proteins, exogenous contaminants, and / or aggregates. A pharmaceutical composition containing the following:

[0175] 149. A cron 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 cron is non-integrated; the cron has a sequence, structure and / or function based on an anelovirus or other non-pathogenic virus; and the cron is non-pathogenic.

[0176] 150. A clone or composition according to any one of the prior embodiments, wherein the proteinaceous outer layer comprises a non-pathogenic external protein.

[0177] 151. A clone or composition according to any one of the prior embodiments, wherein the proteinaceous outer layer comprises one or more glycosylated proteins, a hydrophilic DNA-binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal polyarginine sequence, a variable region, a C-terminal polyglutamine / glutamic acid sequence, and one or more disulfide crosslinks.

[0178] 152. A clone 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, pH and temperature stability, surfactant resistance, and non-immunogenicity or non-pathogenicity in host cells.

[0179] 153. A clone 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 15.

[0180] 154. A clone or composition according to any one of the prior embodiments, wherein the non-pathogenic external protein contains at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequences or fragments thereof listed in Table 16 or Table 17.

[0181] 155. A clone 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-immunogenicity and / or tolerance), pharmacokinetics, endocytosis and / or cell adhesion, nuclear entry, intracellular regulation and localization, exocytosis regulation, proliferation, and nucleic acid protection.

[0182] 156. 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). A clone or composition according to any one of the prior embodiments, comprising: a resonating agent; 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.

[0183] 157. A clone or composition according to any one of the prior embodiments, wherein the effector contains sequences that are identical to at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of one or more miRNA sequences listed in Table 18.

[0184] 158. A clone or composition according to a prior embodiment, wherein an effector, for example, miRNA, targets a host gene and, for example, regulates gene expression.

[0185] 159. A clone or composition according to a prior embodiment, wherein the miRNA has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences listed in Table 16, for example.

[0186] 160. A clone 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.

[0187] 161. A clone 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 approximately 1-10 kb, being located in the cell nucleus, being able to bind to endogenous proteins, and producing microRNA that targets host genes.

[0188] 162. A clone 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 one or more sequences listed in Table 19 or Table 20.

[0189] 163. The viral sequence is either single-stranded DNA (e.g., Anellovirus, Bidnavirus, Circovirus, Geminivirus, Genomovirus, Inovirus, Microvirus, Nanovirus, Parvovirus, and Spiravirus), or double-stranded DNA (e.g., Adenovirus, Ampullavirus, Ascovirus, A Asfarvirus, baculovirus, fusellovirus, globulovirus, guttavirus, hypnothovirus, herpesvirus, iridovirus, lipospirixvirus, nimavirus, and poxvirus), RNA viruses (e.g., alphavirus, furovirus, hepatitis virus) A clone or composition according to a prior embodiment, derived from at least one of the following: virus, Hordeivirus, Tobamovirus, Tobravirus, Tricornavirus, Rubivirus, Birnavirus, Cystovirus, Partitivirus, and Reovirus.

[0190] 164. A clone or composition according to a prior embodiment, 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.

[0191] 165. A clone 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.

[0192] 166. A clone or composition according to any one of the prior embodiments, wherein the clone is replicable in mammalian cells, such as human cells.

[0193] 167. A clone or composition according to a prior embodiment, wherein the clone is nonpathogenic and / or nonintegrative in host cells.

[0194] 168. A clone or composition according to a prior embodiment, wherein the clone is non-immunogenic in the host.

[0195] 169. A clon or composition according to any one of the prior embodiments, wherein the clon inhibits / enhances one or more viral properties in a host or host cell, e.g., selectivity, e.g., infectivity, e.g., immunosuppression / activation.

[0196] 170. The kron or composition according to the preceding embodiment, wherein the kron is present in an amount sufficient to modulate (e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more) phenotype, virus level, gene expression, other viruses, competition with a disease state, etc.).

[0197] 171. The composition according to any one of the preceding embodiments, further comprising at least one virus or vector comprising a genome of a virus, e.g., a variant of a kron, e.g., a commensal / native virus.

[0198] 172. The composition according to any one of the preceding embodiments, further comprising a heterologous moiety, at least one small molecule, antibody, polypeptide, nucleic acid, targeting agent, imaging agent, nanoparticle, and combinations thereof.

[0199] 173. A vector comprising a genetic element, comprising: (i) a sequence encoding a non-pathogenic outer protein; (ii) an outer protein binding sequence that binds the genetic element to the non-pathogenic outer protein; and (iii) a sequence encoding an effector, e.g., a regulatory nucleic acid.

[0200] 174. The vector according to the preceding embodiment, wherein the genetic element cannot integrate into the genome of a host cell.

[0201] 175. The vector according to any one of the preceding embodiments, wherein the genetic element is replicable in a mammalian cell, e.g., a human cell.

[0202] 176. The vector according to any one of the preceding embodiments, further comprising an exogenous nucleic acid sequence selected to modulate expression of a gene, e.g., a human gene.

[0203] 177. A pharmaceutical composition comprising the vector according to any one of the preceding embodiments and a pharmaceutically acceptable excipient.

[0204] 178. The composition according to the prior embodiment, wherein the vector is nonpathogenic and / or nonintegrative in host cells.

[0205] 179. The composition according to any one of the prior embodiments, wherein the vector is non-immunogenic in the host.

[0206] 180. 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).

[0207] 181. The composition according to any one of the prior embodiments, further comprising at least one virus or vector, e.g., a variant of a virus, a commensal / native virus, a helper virus, or a non-anerovirus genome.

[0208] 182. 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.

[0209] 183. A method for producing, propagating, and recovering a clone as described in any one of the prior embodiments.

[0210] 184. A method for designing and fabricating a vector as described in any one of the prior embodiments.

[0211] 185. A method comprising the step of administering to a subject an effective amount of a composition described in any one of the prior embodiments.

[0212] 186. A method for identifying dysvirosis in a subject, the following: In the step of analyzing genetic information from a sample obtained from a subject who requires it, the viral genetic information is isolated from the subject's genetic information and other microorganisms; The steps include comparing viral genetic information to a reference standard, such as a control or healthy subject; If an imbalance or abnormal ratio of the subject's viral gene information is obtained by comparing the viral gene information, the step is to identify the subject's dysvirosis. A method that includes this.

[0213] 187. 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 1, 3, 5, 7, 9, 11, or 13, 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 the ORFs listed in Tables 2, 4, 6, 8, 10, 12, or 14, 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 14-1. The present invention also provides, for example, the following items: (Item 1) below: (i) A gene element comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein binding sequence, wherein the gene element is as follows: (a) a sequence having at least 85% sequence identity to the Anellovirus 5' UTR conserved domain nucleotide sequence at nucleotides 323 to 393 of the nucleic acid sequence in Table 11, or (b) a sequence having at least 85% sequence identity to the Anellovirus GC-rich region at nucleotides 2868 to 2929 of the nucleic acid sequence in Table 11 a genetic element comprising one or both of; and (ii) a proteinaceous outer layer a synthetic ciron comprising; wherein said genetic element is enclosed within said proteinaceous outer layer; and wherein said synthetic ciron is capable of delivering said genetic element into a eukaryotic cell. (Item 2) The synthetic ciron according to Item 1, wherein said genetic element is single-stranded. (Item 3) The synthetic ciron according to any one of Items 1 to 2, wherein said genetic element is DNA. (Item 4) The synthetic ciron according to Item 3, wherein said genetic element is minus-strand DNA. (Item 5) The synthetic ciron according to any one of Items 1 to 4, wherein the genetic element is incorporated at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of said crons entering the cell, for example, the synthetic ciron is non-integrating. (Item 6) The synthetic ciron according to any one of Items 1 to 5, wherein said genetic element comprises the sequence of the consensus 5' UTR nucleic acid sequence shown in Table 16-1. (Item 7) The synthetic ciron according to any one of Items 1 to 6, wherein said genetic element comprises the sequence of the consensus GC-rich region shown in Table 16-2. (Item 8) The synthetic clone according to any one of items 1 to 7, wherein the gene element comprises a sequence of at least 100 nucleotides in length, which consists of G or C at at least 70% of the position (e.g., about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%). (Item 9) A synthetic clone according to any one of items 1 to 8, wherein the gene element comprises a sequence having at least 85% sequence identity with the anelovirus 5'UTR conserved domain nucleotide sequence of nucleotides 1 to 393 of the nucleic acid sequence in Table 11, and a sequence having at least 85% sequence identity with the anelovirus GC-rich region of nucleotides 2868 to 2929 of the nucleic acid sequence in Table 11. (Item 10) A synthetic clone according to any one of items 1 to 9, wherein the gene element has at least 75% identity with the nucleotide sequence in Table 11. (Item 11) The synthetic clones described in any one of items 1 to 10, wherein the promoter element is exogenous to wild-type anellovirus. (Item 12) The synthetic clones described in any one of items 1 to 10, wherein the promoter element is endogenous with respect to wild-type anellovirus. (Item 13) The synthetic clone according to any one of items 1 to 12, wherein the external effector encodes a therapeutic agent, such as a therapeutic peptide or polypeptide or a therapeutic nucleic acid. (Item 14) The aforementioned external effector includes regulatory nucleic acids, such as miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or markers, antigens, peptides, 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, 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 enzymes, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands, antibodies, receptors, or CRISPR systems or components, as described in any one of items 1 to 13. (Item 15) The aforementioned external effector is a synthetic clone according to any one of items 1 to 14, comprising miRNA and reducing the expression of a host gene. (Item 16) The synthetic clone according to any one of items 1 to 15, wherein the external effector comprises a nucleic acid sequence having a length of approximately 20 to 200, 30 to 180, 40 to 160, 50 to 140, or 60 to 120 nucleotides. (Item 17) A synthetic clone according to any one of items 1 to 16, wherein the nucleic acid sequence encoding the external effector is approximately 20 to 200, 30 to 180, 40 to 160, 50 to 140, or 60 to 120 nucleotides in length. (Item 18) The synthetic clone according to any one of items 1 to 17, wherein the nucleic acid sequence encoding the exogenous effector is located inside or adjacent to (for example, on the 5' or 3' side of) one or more 3' noncoding regions downstream of the ORF1 locus, e.g., the C-terminus of the ORF1 locus, or the poly-A region. (Item 19) The synthetic clone according to any one of items 1 to 18, wherein the nucleic acid sequence encoding the external effector is located between the poly-A region and the GC-rich region of the gene element. (Item 20) A synthetic clone according to any one of items 1 to 19, comprising (for example, within the proteinaceous outer layer) one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 of Table 12, or amino acid sequences having at least 85% sequence identity thereto. (Item 21) A synthetic clone according to any one of items 1 to 20, wherein the portion of the gene element excluding the effector has a total size of about 2.5 to 5 kb (e.g., about 2.8 to 4 kb, about 2.8 to 3.2 kb, about 3.6 to 3.9 kb, or about 2.8 to 2.9 kb), less than about 5 kb (e.g., about 2.9 kb, 3.2 kb, 3.6 kb, 3.9 kb, or 4 kb), or at least 100 nucleotides (e.g., at least 1 kb). (Item 22) The synthetic clones described above are synthetic clones according to any one of items 1 to 21, wherein the synthetic clones do not contain a lipid bilayer. (Item 23) The synthetic clon described in any one of items 1 to 22 is capable of infecting mammalian cells, such as human cells, such as immune cells, hepatocytes, or lung epithelial cells. (Item 24) If the aforementioned gene element is measured, for example, by a quantitative PCR assay, then at least 10 per cell 2 , 2×10 2 , 5×10 2 , 10 3, 2×10 3 , 5×10 3 , or 10 4 A synthetic clone as described in any one of items 1 to 23, which can replicate, for example, produce a genome equivalent of a gene element. (Item 25) A synthetic clone as described in any one of items 1 to 24, which is substantially nonpathogenic and does not induce any detectable adverse symptoms in a subject (e.g., increased cell death or toxicity compared to a subject not exposed to the clone). (Item 26) A synthetic clone according to any one of items 1 to 25 that 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. (Item 27) The synthetic clon described in item 26, wherein the substantially non-immunogenic clon 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. (Item 28) The synthetic clon described in item 26 or 27, wherein the immune response comprises one or more of the following: an antibody specific to the clon; a cellular response to the clon or a cell containing the clon (e.g., an immune effector cell (e.g., a T cell or NK cell) response); or macrophage phagocytosis of the cell or a cell containing the clon. (Item 29) A synthetic clone according to any one of items 1 to 28, wherein a population of at least 1000 of the synthetic clones can deliver at least 100 copies of the gene element into one or more eukaryotic cells. (Item 30) below: (i) A gene element comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein binding sequence, wherein the gene element is as follows: (a) Sequences having at least 85% sequence identity to the anelovirus 5'UTR conserved domain of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13; or (b) Sequences having at least 85% sequence identity to the anelovirus GC-rich region of the nucleic acid sequences in Tables 1, 3, 5, 7, 9, or 13. Genetic elements including one or both of the following; and (ii) Protein outer layer In a synthetic clone containing; the gene element is confined within a proteinaceous outer layer; and The synthetic clone is capable of delivering the gene element into a eukaryotic cell. (Item 31) A synthetic clone according to item 30, comprising (for example, within the proteinaceous outer layer) one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 of any of Tables 2, 4, 6, 8, 10, or 14, or one or more amino acid sequences having at least 85% sequence identity thereto. (Item 32) A nucleic acid molecule comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein-binding sequence, wherein the gene element is as follows: (a) A sequence having at least 85% sequence identity with the anelovirus 5'UTR conserved domain nucleotide sequence of nucleotides 323-393 of the nucleic acid sequence in Table 11, or (b) A nucleic acid molecule containing one or both of the sequences having at least 85% sequence identity to the anellovirus GC-rich region between nucleotides 2868 and 2929 of the nucleic acid sequences in Table 11. (Item 33) In a nucleic acid molecule comprising a promoter element, a nucleic acid sequence encoding an external effector, and a protein-binding sequence, the gene element is as follows: (a) a sequence having at least 85% sequence identity to the Anellovirus 5'UTR conserved domain of the nucleic acid sequence of Tables 1, 3, 5, 7, or 13, or (b) a sequence having at least 85% sequence identity to the Anellovirus GC-rich region of the nucleic acid sequence of Tables 1, 3, 5, 7, or 13 A nucleic acid molecule comprising one or both of (Item 34) A pharmaceutical composition comprising the synthetic clone according to any one of Items 1 to 31 and a pharmaceutically acceptable carrier or excipient. (Item 35) at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The pharmaceutical composition according to Item 34, comprising 10 synthetic clones. (Item 36) A reaction mixture comprising the synthetic clone according to any one of Items 1 to 31, and a second nucleic acid sequence encoding one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF1, ORF1 / 1, or ORF1 / 2 in Table 12, or an amino acid sequence having at least 85% sequence identity thereto. (Item 37) A reaction mixture comprising the synthetic clone according to any one of Items 1 to 31, and a second nucleic acid sequence encoding one or more amino acid sequences selected from ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, ORF1, ORF1 / 1, or ORF1 / 2 of any of Tables 2, 4, 6, 8, 10, or 14, or an amino acid sequence having at least 85% sequence identity thereto. (Item 38) The reaction mixture according to Item 36 or 37, wherein the second nucleic acid sequence is part of a genetic element. (Item 39) The reaction mixture according to item 36 or 37, 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 or helper virus. (Item 40) Use of a synthetic clone as described in any one of items 1 to 31 or a pharmaceutical composition as described in any one of items 34 to 35, for the purpose of delivering the aforementioned gene element to a host gene. (Item 41) Use of a synthetic chloron described in any one of items 1 to 31 or a pharmaceutical composition described in any one of items 34 to 35, for the purpose of treating a disease or disorder of a subject. (Item 42) The use described in item 41, wherein the disease or disorder is selected from immunodeficiency, interferonosis (e.g., type I interferonosis), infection, autoimmune disease, cancer (e.g., solid tumors, e.g., lung cancer), and gastrointestinal disorder. (Item 43) A synthetic chloroform described in any one of items 1 to 31 or a pharmaceutical composition described in any one of items 34 to 35, intended to treat a disease or disorder of a subject. (Item 44) A method for treating a disease or disorder in a subject, the method comprising the step of administering to the subject a synthetic clon described in any one of items 1 to 31 or a pharmaceutical composition described in any one of items 34 to 35, wherein the disease or disorder is selected from immunodeficiency, interferonosis (e.g., type I interferonosis), infection, autoimmune disease, cancer (e.g., solid tumors, e.g., lung cancer), and gastrointestinal disorder. (Item 45) A method for producing a synthetic chloro composition, the following: a) A step of providing a plurality of synthetic clones as described in any one of items 1 to 31 or a pharmaceutical composition as described in any one of items 34 to 35; b) Optionally, a step of evaluating multiple synthetic clones with respect to one or more of the following: inclusions described herein, optical density measurement (e.g., OD260), particle count (e.g., by HPLC), infectivity (e.g., particle:infectivity ratio); and c) For example, if one or more of the parameters in (b) meet a specified threshold, the step of formulating multiple synthetic clones into a pharmaceutical composition suitable for administration to a subject. Methods that include...

[0214] Other features, purposes, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0216] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show embodiments illustrated herein. However, it should be understood that the present invention is not limited to the exact arrangement and usefulness of the embodiments shown in the drawings. [Brief explanation of the drawing]

[0217] [Figure 1] Figure 1A shows the sequence similarity (%) of the amino acid regions of the capsid protein sequence. Figure 1B shows the sequence similarity (%) of the capsid protein sequence. [Figure 2] This figure shows one embodiment of the Kron. [Figure 3] A schematic diagram of the kanamycin vector ("Cron1") that encodes the LY1 strain of TTMiniV is drawn. [Figure 4]A schematic diagram of the kanamycin vector ("Cron2") encoding the LY2 strain of TTMiniV is drawn. [Figure 5] The transfection efficiency of synthetic chlorophenate in 293T and A549 cells is depicted. [Figure 6A] This image shows quantitative PCR results demonstrating successful infection of 293T cells by synthetic clones. [Figure 6B] This image shows quantitative PCR results demonstrating successful infection of 293T cells by synthetic clones. [Figure 7A] This image shows quantitative PCR results demonstrating successful infection of A549 cells by synthetic clones. [Figure 7B] This image shows quantitative PCR results demonstrating successful infection of A549 cells by synthetic clones. [Figure 8A] This image shows quantitative PCR results demonstrating successful infection of Raji cells by synthetic clones. [Figure 8B] This image shows quantitative PCR results demonstrating successful infection of Raji cells by synthetic clones. [Figure 9A] This image shows quantitative PCR results demonstrating successful infection of Jurkat cells by synthetic clones. [Figure 9B] This image shows quantitative PCR results demonstrating successful infection of Jurkat cells by synthetic clones. [Figure 10A] This image shows quantitative PCR results demonstrating successful infection of Chang cells by synthetic clones. [Figure 10B] This image shows quantitative PCR results demonstrating successful infection of Chang cells by synthetic clones. [Figure 11A-11B] This is a series of graphs showing luciferase expression from cells transfected or infected with TTMV-LY2Δ574-1371,Δ1432-2210,2610::nLuc. Luminescence was observed in the infected cells, indicating the achievement of replication and packaging. [Figure 11C]This diagram illustrates the phylogenetic tree of alphatorquevirus (Torque Teno Virus; TTV), highlighting the clades. At least 100 anellovirus strains are shown, divided into five clades. Exemplary sequences from each of the five clades are provided, for example, in Tables 1-14 of this specification. Top box = Clade 1; Upper center box = Clade 2; Middle box = Clade 3; Lower center box = Clade 4; Bottom box = Clade 5 [Figure 12] This is a schematic diagram illustrating an exemplary workflow for the production of krons (e.g., replicable or non-replicable krons as described herein). [Figure 13] This graph shows the primer specificity for primer sets designed for the quantification of TTV and TTMV genome equivalents. Quantitative PCR based on SYBR green chemistry shows, as indicated, one distinct peak for each amplification product using TTMV or TTV-specific primers for each plasmid encoding the respective genome. [Figure 14] This is a series of graphs showing the PCR efficiency in the quantification of TTV genome equivalents by qPCR. Primer increment concentrations and a constant concentration (250 nM) of hydrolysis probe were used with two different commercially available qPCR master mixes. Error propagation during the quantification process was minimal, with efficiencies ranging from 90–110%. [Figure 15] This graph shows exemplary amplification plots of linear amplification of TTMV (target 1) or TTV (target 2) over 7 log10 of genome equivalent concentrations. Genomic equivalents were quantified for 7-10 dilutions with high PCR efficiency and linearity (R2TTMV: 0.996; R2TTV: 0.997). [Figure 16A]This is a series of graphs showing the quantification of TTMV genomic equivalents in PCR stocks. (A) Amplification plots of two stocks, each diluted 1:10 and performed in two replicates. (B) The same two samples shown in panel A, shown here with respect to the linear range. Upper and lower limits are shown for two representative samples. PCR efficiency: 99.58%, R2: 0988. [Figure 16B] This is a series of graphs showing the quantification of TTMV genomic equivalents in PCR stocks. (A) Amplification plots of two stocks, each diluted 1:10 and performed in two replicates. (B) The same two samples shown in panel A, shown here with respect to the linear range. Upper and lower limits are shown for two representative samples. PCR efficiency: 99.58%, R2: 0988. [Figure 17A] This is a series of graphs showing the functional effects of synthetic clones containing the extramiRNA, miR-625. (A) Effects on the cell viability of non-small cell lung cancer (NSCLC) cells when infected with clones expressing miR-625 in three different NSCLC cell lines (A549 cells, NCI-H40 cells, and SW900 cells). (B) Effects of clones expressing miR-625 on YFP reporter expression by HEK293T cells. [Figure 17B] This is a series of graphs showing the functional effects of synthetic clones containing the extramiRNA, miR-625. (A) Effects on the cell viability of non-small cell lung cancer (NSCLC) cells when infected with clones expressing miR-625 in three different NSCLC cell lines (A549 cells, NCI-H40 cells, and SW900 cells). (B) Effects of clones expressing miR-625 on YFP reporter expression by HEK293T cells. [Figure 17C] This graph shows the quantitative analysis of p65 immunoblot analysis normalized to the total protein of SW900 cells, either in contact with the indicated clons or left untreated. [Figure 18]This figure shows the pairwise identity of viral DNA sequence alignments within five alpha-torque virus clades. Viral DNA sequences from each TTV clade were aligned. Pairwise identity (%) across a 50 bp sliding window is shown along the alignment length for each clade. The average pairwise identity is displayed. [Figure 19] This figure shows the pairwise identity of representative sequence alignments from each alpha-torque virus clade. DNA sequences from TTV-CT30F, TTV-TJN02, TTV-tth8, TTV-JA20, and TTV-HD23a were aligned. Pairwise identity (%) across a 50 bp sliding window is shown along the alignment length. The upper brackets indicate non-coding and coding regions with the indicated pairwise identity. The lower brackets indicate regions with high sequence conservation. [Figure 20] This figure shows the pairwise identity of amino acid alignments for putative proteins between five alpha-torque virus clades. The amino acid sequences of putative proteins from TTV-CT30F, TTV-TJN02, TTV-tth8, TTV-JA20, and TTV-HD23a were aligned. Pairwise identity (%) across a 50aa sliding window is shown along the length of each alignment. Pairwise identity of both open reading frame DNA sequences and protein amino acid sequences is shown. [Figure 21] This figure shows that the domain within the 5'UTR is highly conserved across five alpha-torque virus clades. The 71bp 5'UTR conserved domain sequences of each representative alpha-torque virus were aligned. The sequences exhibit 96.6% pairwise identity across the five clades. The sequences shown in Figure 21 (sequence numbers 703–708, in order of appearance, respectively) are also listed, for example, in Table 16-1 of this specification. [Figure 22]This figure shows the alignment of GC-rich domains from five alpha-torque virus clades. Each anellovirus has a region with a GC content of over 70% downstream of the ORF. Alignment of GC-rich regions from TTV-CT30F, TTV-TJN02, TTV-tth8, TTV-JA20, and TTV-HD23a is shown. Although these regions vary in length, when aligned they exhibit 81.8% pairwise identity. The sequences shown in Figure 22 (sequence numbers 709-714, in order of appearance, respectively) are also listed in Table 16-2 of this specification, for example. [Modes for carrying out the invention]

[0218] definition The phrase "compounds, compositions, products, etc. intended for therapeutic, regulatory, etc." should be understood to mean, in itself, compounds, compositions, products, etc. suitable for the explicitly stated purpose of therapeutic, regulatory, etc. The phrase "compounds, compositions, products, etc. intended for therapeutic, regulatory, etc." further discloses, as embodiments, that such compounds, compositions, products, etc. may be used for therapeutic, regulatory, etc.

[0219] The phrases "compounds, compositions, products, etc. for use in..." or "use of compounds, compositions, products, etc. in the manufacture of drugs, pharmaceutical compositions, veterinary compositions, diagnostic compositions, etc. for..." indicate that such compounds, compositions, products, etc. are intended to be used in therapeutic methods that may be performed on human or animal bodies. They can be considered equivalent disclosures of embodiments and claims relating to therapeutic methods, etc. If an embodiment or claim refers to "compounds for use in the treatment of a person or animal suspected of having a disease," this is also considered a disclosure of "use of compounds in the manufacture of drugs intended for the treatment of a person or animal suspected of having a disease" or "a therapeutic method by administering a compound to a person or animal suspected of having a disease." The phrase "compounds, compositions, products, etc. for therapeutic, modulatory, etc." should be understood to mean compounds, compositions, products, etc. suitable for the explicitly stated purpose of therapeutic, modulatory, etc.

[0220] Hereafter, where examples of terms, values, numbers, etc. are given in parentheses, this should be understood as indicating that the examples described in parentheses may constitute embodiments. For example, if it is stated that "In some embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 1 (e.g., nucleotides 571-2613 of the nucleic acid sequence of Table 1)," then some embodiments relate to a nucleic acid molecule containing a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with nucleotides 571-2613 of the nucleic acid sequence of Table 1.

[0221] As used herein, the term “cron” refers to a vehicle containing a genetic element confined within a proteinaceous outer layer, such as an episome, such as circular DNA. “Synthetic cron” as used herein generally refers to a cron having a modified sequence relative to a wild-type virus (e.g., the wild-type anellovirus described herein), which is not naturally occurring. In some embodiments, the synthetic cron is engineered or recombinant and includes, for example, a genetic element with modifications relative to a wild-type viral genome (e.g., the wild-type anellovirus genome described herein). In some embodiments, confined within a proteinaceous outer layer includes 100% coverage by the proteinaceous outer layer, as well as less than 100% coverage, such as 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. For example, gaps or discontinuities (e.g., those that make the protein outer layer permeable to water, ions, peptides, or small molecules) may be present in the protein outer layer, as long as the gene element is retained within the protein outer layer, for example, before entering a host cell. In some embodiments, the chlorine is purified, for example, by being separated from the original source and / or substantially free of other components (>50%, >60%, >70%, >80%, >90%).

[0222] As used herein, “coding” nucleic acid refers to a nucleic acid sequence that codes for an amino acid sequence or a functional polynucleotide (e.g., non-coding RNA, e.g., siRNA or miRNA).

[0223] As used herein, "dysvirosis" refers to a dysregulation of the viral population in a subject.

[0224] As used herein, “exogenous” substance (e.g., effector, nucleic acid (e.g., RNA), gene, payload, protein) refers to a substance that is not contained in or encoded by the corresponding wild-type virus, e.g., an anelovirus described herein. In some embodiments, the exogenous substance is not naturally occurring, such as a protein or nucleic acid having a modified sequence (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein or nucleic acid. In some embodiments, the exogenous substance is not naturally occurring in host cells. In some embodiments, the exogenous substance is naturally occurring in host cells but is exogenous with respect to the virus. In some embodiments, the exogenous substance is naturally occurring in host cells but is not present at a desired level or at a desired time.

[0225] As used herein, the term “genetic element” generally refers to a nucleic acid sequence within a clone. It is understood that a genetic element can be produced as naked DNA and optionally assembled into a proteinaceous outer layer. It is also understood that a clone can insert its genetic element into a cell, thereby ensuring that the genetic element is present within the cell and thus the proteinaceous outer layer does not necessarily enter the cell.

[0226] As used herein, “substantially non-pathogenic” organisms, particles, or constructs refer to organisms, particles (e.g., viruses or cron as described herein) or constructs thereof that do not induce or cause a detectable disease or pathological condition in a host organism, e.g., a mammal, e.g., a human. In some embodiments, administration of cron to a subject may result in some acceptable reactions or side effects as part of standard treatment.

[0227] As used herein, “non-pathogenic” means an organism or its constituents that does not induce or cause a detectable disease or pathological condition in a host organism, such as a mammal, such as a human.

[0228] As used herein, “substantially non-integrating” gene elements refer to gene elements that are integrated into the genome, such as viruses or clones (as described herein), where about 0.01%, 0.05%, 0.1%, 0.5%, or less than 1% of the gene elements that enter a host cell (e.g., a eukaryotic cell) or organism (e.g., a mammal, e.g., a human) are integrated. In some embodiments, gene elements are not integrated into the genome of the host cell to a detectable extent, for example. In some embodiments, the integration of gene elements into the genome can be detected using techniques described herein, such as nucleic acid sequencing, PCR detection, and / or nucleic acid hybridization.

[0229] As used herein, “substantially non-immunogenic” organisms, particles, or constructs refer to organisms, particles (e.g., viruses or cron as described herein) or constructs thereof that do not induce or stimulate an unwanted or non-targeted immune response in a host tissue or organism (e.g., a mammal, e.g., a human). In some embodiments, substantially non-immunogenic organisms, particles, or constructs do not produce a detectable immune response. In some embodiments, substantially non-immunogenic cron do not produce a detectable immune response to proteins containing amino acid sequences encoded by nucleic acid sequences shown in any of Tables 1 to 14. In several embodiments, an immune response (e.g., an unwanted or non-targeted immune response) is determined by testing the presence or level of an antibody in a subject (e.g., the presence or level of an anti-Cron antibody, e.g., the presence or level of an antibody against synthetic Cron as described herein) according to, for example, a method for detecting anti-TTV antibodies described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or a method for measuring anti-TTV IgG levels described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies against anellovirus or Cron based thereon are also detected by methods of the art for detecting antiviral antibodies, e.g., Calcedo It can also be detected by the method for detecting anti-AAV antibodies described in et al. (2013;Front.Immunol.4(341):1-7; incorporated herein by reference).

[0230] As used herein, the term "proteinic outer layer" refers to an external structure that is primarily protein.

[0231] As used herein, the term “regulatory nucleic acid” refers to a nucleic acid sequence that modifies the expression of a DNA sequence encoding an expression product, e.g., transcription and / or translation. In some embodiments, the expression product includes RNA or a protein.

[0232] As used herein, the term “regulatory sequence” refers to a nucleic acid sequence that modifies the transcription of a target gene product. In some embodiments, the regulatory sequence is a promoter or enhancer.

[0233] As used herein, the term “replicating protein” refers to a protein used in infection, viral genome replication / expression, viral protein synthesis, and / or assembly of viral components, such as a viral protein.

[0234] As used herein, “treatment,” “to treat,” and their equivalents refer to the medical management of a subject intended to improve, alleviate, stabilize, prevent, or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment aimed at improving a disease, pathological condition, or disorder), causal treatment (treatment aimed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to alleviate symptoms), preventive treatment (treatment aimed at preventing, minimizing, or partially or completely inhibiting the onset of the associated disease, pathological condition, or disorder); and supportive treatment (treatment used to adjunct another treatment).

[0235] As used herein, “viral community (virome)” refers to a virus in a specific environment, such as a part of the body, such as an organism, such as a cell, such as a tissue.

[0236] This invention relates, in general, to clones, such as synthetic clones, and their uses. This disclosure provides synthetic clones, compositions comprising synthetic clones, and methods for manufacturing or using synthetic clones. Synthetic clones are generally useful as delivery vehicles, for example, for delivering therapeutic agents to eukaryotic cells. Generally, synthetic clones include a gene element containing an extracellular nucleic acid sequence (e.g., encoding an extracellular effector) confined within a proteinaceous outer layer. Synthetic clones can be used, for example, as substantially non-immunogenic vehicles for delivering the gene element, or the effector encoded therein (e.g., polypeptides or nucleic acid effectors as described herein), to eukaryotic cells to treat diseases or disorders of subjects, including cells.

[0237] Kron In some embodiments, the present invention as described herein includes compositions and methods for the use and production of synthetic clones. In some embodiments, the clone comprises a gene element (e.g., circular DNA, e.g., single-stranded DNA) which comprises at least one extrinsic element (e.g., an extrinsic element encoding an effector, as described herein) relative to the rest of the gene element and / or proteinaceous outer layer. The clone may be a payload delivery vehicle to a host, e.g., a human (e.g., a substantially non-immunogenic delivery vehicle). In some embodiments, the clone is replicable in eukaryotic cells, e.g., mammalian cells, e.g., human cells. In some embodiments, the clone is substantially non-pathogenic and / or substantially non-integrating in mammalian (e.g., human) cells. In some embodiments, the clone is substantially non-immunogenic in mammals, e.g., humans. In some embodiments, the clons have sequences, structures, and / or functions based on an anelovirus (e.g., an anelovirus described herein, e.g., an anelovirus comprising nucleic acids or polypeptides containing sequences shown in any of Tables 1 to 14) or other substantially nonpathogenic viruses, e.g., symbiotic viruses, commensal viruses, or native viruses. Generally, an anelovirus-based clons include at least one extrinsic element to the anelovirus, e.g., an extrinsic effector or a nucleic acid sequence encoding an extrinsic effector located within the clon's genetic element. In some embodiments, the clons are replication defects. In some embodiments, the clons are replicable.

[0238] In one embodiment, the present invention includes: (i) a gene element comprising a promoter element, a sequence encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal), wherein the gene element is single-stranded DNA and further has one or both of the following characteristics: being circular, and / or being 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 gene elements that enter the cell; and (ii) a synthetic clone comprising a proteinaceous outer layer, wherein the gene element is confined within the proteinaceous outer layer; and the synthetic clone is capable of delivering the gene element into a eukaryotic cell.

[0239] In some embodiments of the synthetic clones described herein, gene elements are incorporated at a frequency of less than approximately 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, less than approximately 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of gene elements from multiple synthetic clones administered to a subject are incorporated into the genome of one or more host cells of the subject. In some embodiments, for example, as described herein, gene elements of a population of synthetic clones are incorporated into the genome of the host cell at a frequency lower than that of an equivalent population of AAV viruses, for example, at a frequency of about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than that of an equivalent population of AAV viruses.

[0240] In one embodiment, the present invention relates to a gene element comprising: (i) a promoter element, a sequence encoding an external effector (e.g., a payload), and a protein-binding sequence (e.g., an external protein-binding sequence), wherein the gene element is a wild-type anellovirus sequence (e.g., wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTV) (ii) a synthetic clon comprising a gene element having at least 75% sequence identity (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to a wild-type anellovirus (TTMV) or TTMDV sequence, for example, any wild-type anellovirus sequence listed in Table 1, 3, 5, 7, 9, 11, or 13; and (ii) a synthetic clon comprising a proteinoid outer layer, wherein the gene element is confined within the proteinoid outer layer; and the synthetic clon is capable of delivering the gene element into a eukaryotic cell.

[0241] In one embodiment, the present invention is as follows: a) A gene element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence for binding a gene element to the non-pathogenic external protein, and (iii) a sequence encoding a regulatory nucleic acid; and b) A protein outer layer that binds to the gene element, for example, an enveloping or encapsulating it. Includes synthetic chloromone.

[0242] In some embodiments, the clons include sequences or expression products derived from (or having >70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology to) non-enveloping, circular, single-stranded DNA viruses. Animal circular single-stranded DNA viruses generally refer to a subgroup of single-stranded DNA (ssDNA) viruses that infect eukaryotic non-plant hosts and have a circular genome. Thus, animal circular ssDNA viruses can be distinguished from ssDNA viruses that infect prokaryotes (i.e., Microviridae and Inoviridae) and ssDNA viruses that infect plants (i.e., Geminiviridae and Nanoviridae). They can also be distinguished from linear ssDNA viruses that infect non-plant eukaryotic cells (i.e., Parvoviridae).

[0243] In some embodiments, the cloning modulates the function of a host cell, for example, transiently or for an extended period. In some embodiments, the cellular function is stably altered, for example, the modulation lasting for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer, or any time in between.

[0244] In some embodiments, the function of cells is transiently altered, and for example, the regulation lasts for about 30 minutes to about 7 days or less, or for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days or less, or any time in between.

[0245] In some embodiments, the gene element includes a promoter element. In several embodiments, the promoter element is selected from RNA polymerase II-dependent promoters, RNA polymerase III-dependent promoters, PGK promoters, CMV promoters, EF-1α promoters, SV40 promoters, CAGG promoters, or UBC promoters, TTV virus promoters, tissue-specific U6 (pollIII), minimal CMV promoters having upstream DNA binding sites for activator proteins (such as TetR-VP16, Gal4-VP16, dCas9-VP16, etc.). In several embodiments, the promoter element includes a TATA box. In several embodiments, the promoter element is endogenous to, for example, wild-type anellovirus as described herein.

[0246] In some embodiments, the gene element comprises one or more of the following characteristics: single-stranded, circular, minus-stranded, and / or DNA. In some embodiments, the gene element comprises an episome. In some embodiments, the portion of the gene element excluding the effector has a total size of about 2.5–5 kb (e.g., about 2.8–4 kb, about 2.8–3.2 kb, about 3.6–3.9 kb, or about 2.8–2.9 kb), less than about 5 kb (e.g., about 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).

[0247] The clones, compositions containing clones, and methods of using such clones described herein are, in some cases, based in examples illustrating how to produce clones by combining different effectors, such as miRNA (e.g., for IFN or miR-625), shRNA, etc., with a protein-binding sequence, such as a DNA sequence that binds to a capsid protein, such as Q99153, with a protein-based outer layer, such as a capsid disclosed in Arch Virol (2007) 152:1961-1975 (which can then be used to deliver the extrinsic effector to cells (e.g., animal cells, such as human cells, or non-human cells such as pig or mouse cells)). In several embodiments, the extrinsic effector can suppress the expression of factors such as interferons. The examples further illustrate how clones can be produced, for example, by inserting the extrinsic effector into a sequence derived from an anelovirus. Based on these examples, the following description takes into account various variations of specific findings and combinations considered in the examples. For example, those skilled in the art will understand from the examples that a particular miRNA is used as just one example of an extrinsic effector, and that other extrinsic effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, a substantially non-pathogenic protein, as described herein, may be used instead of the particular capsid used in this example. Also, an anelovirus sequence described herein may be used instead of the particular anellovirus sequence described in this example. These considerations also apply to regulatory sequences such as protein-binding sequences and promoters. Independently of these, those skilled in the art will particularly consider such embodiments that are closely related to this example.

[0248] In some embodiments, Kron, or a gene element contained therein, is introduced into a cell (e.g., a human cell). In some embodiments, for example as described in Example 19, once Kron or a gene element is introduced into a cell, an extrinsic effector (e.g., RNA, miRNA) encoded by the gene element of Kron is expressed in the cell (e.g., a human cell). In some embodiments, the introduction of Kron, or a gene element contained therein, into a cell modulates (e.g., increases or decreases) the level of a target molecule (e.g., a target nucleic acid, e.g., RNA, or a target polypeptide) in the cell, for example, by altering the expression level of the target molecule by the cell (e.g., as described in Example 22). In several embodiments, the introduction of Kron, or a gene element contained therein, reduces the level of interferon produced by the cell, as described in Examples 3 and 4. In some embodiments, the introduction of Kron, or a gene element contained therein, into a cell modulates (e.g., increases or decreases) the function of the cell. In several embodiments, the introduction of Kron, or the genetic elements contained therein, into cells modulates (e.g., increases or decreases) the viability of the cells. In several embodiments, the introduction of Kron, or the genetic elements contained therein, into cells reduces the viability of cells (e.g., cancer cells), as described, for example, in Example 22.

[0249] In some embodiments, the clones described herein (e.g., synthetic clones) induce antibody positivity rates of less than 70% (e.g., antibody positivity rates of about 60%, 50%, 40%, 30%, 20%, or less than 10%). In some embodiments, the antibody positivity rate is measured according to methods known in the art. In some embodiments, the antibody positivity rate is measured by detecting antibodies against anellovirus (e.g., as described herein) or clones based thereon in a biological sample, for example, according to the anti-TTV antibody detection method described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or the method for determining anti-TTV IgG serum positivity rates described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Furthermore, antibodies against anellovirus or clones based thereon can also be detected by methods of the art for detecting antiviral antibodies, such as the method for detecting anti-AAV antibodies described in Calcedo et al. (2013; Front.Immunol.4(341):1-7; incorporated herein by reference).

[0250] Anellovirus In some embodiments, for example, the synthetic clones described herein include sequences or expression products derived from an anelovirus. Generally, synthetic clones include one or more sequences or expression products that are external to an anelovirus. The genus Anellovirus was formerly classified as a clade of the family Circoviridae, but has recently been classified as a separate family. Anelloviruses generally have a negatively polarized single-stranded circular DNA genome. Anelloviruses are not associated with human diseases. However, attempts to link anellovirus infection to human disease have been hampered by the high incidence of asymptomatic anellovirus viremia in control cohorts, the remarkable genomic diversity of the anellovirus viridae family, the inability to propagate this virus in vitro to date, and the lack of animal models for anellovirus disease (Yzebe et al., Panminerva Med. (2002) 44:167-177; Biagini, P., Vet. Microbiol. (2004) 98:95-101).

[0251] Anellovirus is thought to be transmitted orally, nasally, or fecal-oral, and by mother-to-child and / or uterine transmission (Gerner et al., Ped. Infect. Dis. J. (2000) 19:1074-1077). Infected individuals exhibit characteristics of anellovirus viremia that persist for a long period (several months to several years). Humans may be co-infected with two or more gene groups or strains (Saback, et al., Scad. J. Infect. Dis. (2001) 33:121-125). These gene groups have been suggested to be able to recombine in infected individuals (Rey et al., Infect. (2003) 31:226-233). Double-stranded isotype (replicated) intermediates have been found in several tissues, including the liver, peripheral blood mononuclear cells, and bone marrow (Kikuchi et al., J. Med. Virol. (2000) 61:165-170; Okamoto et al., Biochem. Biophys. Res. Commun. (2002) 270:657-662; Rodriguez-Ilnigo et al., Am. J. Pathol. (2000) 156:1227-1234).

[0252] In some embodiments, the clones described herein include, for example, one or more nucleic acid molecules (e.g., gene elements described herein) that have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an anellovirus sequence or fragment thereof as described herein. In some embodiments, the anellovirus sequence is selected from the sequences shown in any of Tables 1, 3, 5, 7, 9, 11, or 13. In some embodiments, the clones described herein include one or more nucleic acid molecules (e.g., gene elements described herein) having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the anelloviruses described herein (e.g., an anellovirus sequence annotated in any of Tables 1-16 or 19, or encoded by one of the sequences listed therein). In some embodiments, the nucleic acid molecule includes ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3 sequences of any of the anelloviruses described herein (e.g., an anellovirus sequence annotated in any of Tables 1-16 or 19, or encoded by one of the sequences listed therein).In several embodiments, the nucleic acid molecule includes a sequence encoding a capsid protein that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an anelovirus ORF1 or ORF2 protein (for example, an ORF1 or ORF2 amino acid sequence shown in any of Tables 2, 4, 6, 8, 10, 12, 14, or 16, or an ORF1 or ORF2 amino acid sequence encoded by a nucleic acid sequence shown in any of Tables 1, 3, 5, 7, 9, 11, 13, 15, or 19).

[0253] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 1 (e.g., nucleotides 571-2613 of the nucleic acid sequence in Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 1 (e.g., nucleotides 571-587 and / or 2137-2613 of the nucleic acid sequence in Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence of Table 1 (e.g., nucleotides 571-687 and / or 2339-2659 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence of Table 1 (e.g., nucleotides 299-691 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 1 (e.g., nucleotides 299-687 and / or 2137-2659 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 1 (e.g., nucleotides 299-687 and / or 2339-2831 of the nucleic acid sequence of Table 1).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 nucleotide sequence of Table 1 (e.g., nucleotides 299-348 and / or 2339-2831 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence of Table 1 (e.g., nucleotides 84-90 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus Cap site nucleotide sequence in Table 1 (e.g., nucleotides 107-114 of the nucleic acid sequence in Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 5'UTR conserved domain nucleotide sequence of Table 1 (e.g., nucleotides 177-247 of the nucleic acid sequence in Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 3 open reading frame region nucleotide sequence of Table 1 (e.g., nucleotides 2325-2610 of the nucleic acid sequence in Table 1).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus poly(A) signaling nucleotide sequence of Table 1 (e.g., nucleotides 2813-2818 of the nucleic acid sequence of Table 1). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence of Table 1 (e.g., nucleotides 3415-3570 of the nucleic acid sequence of Table 1).

[0254] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 3 (e.g., nucleotides 599-2839 of the nucleic acid sequence in Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 3 (e.g., nucleotides 599-727 and / or 2381-2839 of the nucleic acid sequence in Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence of Table 3 (e.g., nucleotides 599-727 and / or 2619-2813 of the nucleic acid sequence of Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence of Table 3 (e.g., nucleotides 357-731 of the nucleic acid sequence of Table 3). In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 3 (e.g., nucleotides 357-727 and / or 2381-2813 of the nucleic acid sequence of Table 3). In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 3 (e.g., nucleotides 357-727 and / or 2619-3021 of the nucleic acid sequence of Table 3).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 nucleotide sequence of Table 3 (e.g., nucleotides 357-406 and / or 2619-3021 of the nucleic acid sequence of Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence of Table 3 (e.g., nucleotides 89-90 of the nucleic acid sequence of Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus Cap site nucleotide sequence in Table 3 (e.g., nucleotides 107-114 of the nucleic acid sequence in Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 3 (e.g., nucleotides 174-244 of the nucleic acid sequence in Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 3 open reading frame region nucleotide sequence in Table 3 (e.g., nucleotides 2596-2810 of the nucleic acid sequence in Table 3).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus poly(A) signaling nucleotide sequence in Table 3 (e.g., nucleotides 3017-3022 in the nucleic acid sequence in Table 3). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 3 (e.g., nucleotides 3691-3794 in the nucleic acid sequence in Table 3).

[0255] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 5 (e.g., nucleotides 599-2830 of the nucleic acid sequence in Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 5 (e.g., nucleotides 599-715 and / or 2363-2830 of the nucleic acid sequence in Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence of Table 5 (e.g., nucleotides 599-715 and / or 2565-2789 of the nucleic acid sequence of Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence of Table 5 (e.g., nucleotides 336-719 of the nucleic acid sequence of Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 5 (e.g., nucleotides 336-715 and / or 2363-2789 of the nucleic acid sequence of Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 5 (e.g., nucleotides 336-715 and / or 2565-3015 of the nucleic acid sequence of Table 5).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 nucleotide sequence of Table 5 (e.g., nucleotides 336-388 and / or 2565-3015 of the nucleic acid sequence of Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence of Table 5 (e.g., nucleotides 83-88 of the nucleic acid sequence of Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus Cap site nucleotide sequence in Table 5 (e.g., nucleotides 104-111 of the nucleic acid sequence in Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 5'UTR conserved domain nucleotide sequence shown in Table 5 (e.g., nucleotides 170-240 of the nucleic acid sequence shown in Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 3 open reading frame region nucleotide sequence shown in Table 5 (e.g., nucleotides 2551-2786 of the nucleic acid sequence shown in Table 5).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus poly(A) signaling nucleotide sequence in Table 5 (e.g., nucleotides 3011-3016 of the nucleic acid sequence in Table 5). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus GC-rich nucleotide sequence in Table 5 (e.g., nucleotides 3632-3753 of the nucleic acid sequence in Table 5).

[0256] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 7 (e.g., nucleotides 590-2899 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 7 (e.g., nucleotides 590-712 and / or 2372-2899 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence in Table 7 (e.g., nucleotides 590-712 and / or 2565-2873 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence in Table 7 (e.g., nucleotides 354-716 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 7 (e.g., nucleotides 354-712 and / or 2372-2873 of the nucleic acid sequence of Table 7). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 7 (e.g., nucleotides 354-712 and / or 2565-3075 of the nucleic acid sequence of Table 7).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 nucleotide sequence in Table 7 (e.g., nucleotides 354-400 and / or 2565-3075 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence in Table 7 (e.g., nucleotides 86-90 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus Cap site nucleotide sequence in Table 7 (e.g., nucleotides 107-114 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus 5'UTR conserved domain nucleotide sequence in Table 7 (e.g., nucleotides 174-244 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus 3 open reading frame region nucleotide sequence in Table 7 (e.g., nucleotides 2551-2870 of the nucleic acid sequence in Table 7).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus poly(A) signaling nucleotide sequence in Table 7 (e.g., nucleotides 3071-3076 of the nucleic acid sequence in Table 7). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 7 (e.g., nucleotides 3733-3853 of the nucleic acid sequence in Table 7).

[0257] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 9 (e.g., nucleotides 577-2787 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 9 (e.g., nucleotides 577-699 and / or 2311-2787 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence of Table 9 (e.g., nucleotides 577-699 and / or 2504-2806 of the nucleic acid sequence of Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence of Table 9 (e.g., nucleotides 341-703 of the nucleic acid sequence of Table 9). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 9 (e.g., nucleotides 341-699 and / or 2311-2806 of the nucleic acid sequence of Table 9). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 9 (e.g., nucleotides 341-699 and / or 2504-2978 of the nucleic acid sequence of Table 9).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 nucleotide sequence in Table 9 (e.g., nucleotides 341-387 and / or 2504-2978 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence in Table 9 (e.g., nucleotides 83-87 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus Cap site nucleotide sequence in Table 9 (e.g., nucleotides 104-111 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 9 (e.g., nucleotides 171-241 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 3 open reading frame region nucleotide sequence in Table 9 (e.g., nucleotides 2463-2784 of the nucleic acid sequence in Table 9).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus poly(A) signaling nucleotide sequence in Table 9 (e.g., nucleotides 2974-2979 of the nucleic acid sequence in Table 9). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 9 (e.g., nucleotides 3644-3758 of the nucleic acid sequence in Table 9).

[0258] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence of Table 11 (e.g., nucleotides 612-2612 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence of Table 11 (e.g., nucleotides 612-719 and / or 2274-2612 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence of Table 11 (e.g., nucleotides 612-719 and / or 2449-2589 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence of Table 11 (e.g., nucleotides 424-723 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence of Table 11 (e.g., nucleotides 424-719 and / or 2274-2589 of the nucleic acid sequence of Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence of Table 11 (e.g., nucleotides 424-719 and / or 2449-2812 of the nucleic acid sequence of Table 11).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus TATA box nucleotide sequence of Table 11 (e.g., nucleotides 237-243 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus Cap site nucleotide sequence of Table 11 (e.g., nucleotides 260-267 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus transcription start site nucleotide sequence of Table 11 (e.g., nucleotide 267 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus 5'UTR conserved domain nucleotide sequence of Table 11 (e.g., nucleotides 323-393 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 3 open reading frame region nucleotide sequence of Table 11 (e.g., nucleotides 2441-2586 of the nucleic acid sequence in Table 11). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus poly(A) signal nucleotide sequence of Table 11 (e.g., nucleotides 2808-2813 of the nucleic acid sequence in Table 11).In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus GC-rich nucleotide sequence of Table 11 (e.g., nucleotides 2868-2929 of the nucleic acid sequence of Table 11).

[0259] In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 nucleotide sequence in Table 13 (e.g., nucleotides 432-2453 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 1 nucleotide sequence in Table 13 (e.g., nucleotides 432-584 and / or 1977-2453 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF1 / 2 nucleotide sequence in Table 13 (e.g., nucleotides 432-584 and / or 2197-2388 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 nucleotide sequence in Table 13 (e.g., nucleotides 283-588 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 2 nucleotide sequence in Table 13 (e.g., nucleotides 283-584 and / or 1977-2388 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2 / 3 nucleotide sequence in Table 13 (e.g., nucleotides 283-584 and / or 2197-2614 of the nucleic acid sequence in Table 13).In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus TATA box nucleotide sequence in Table 13 (e.g., nucleotides 21-25 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus Cap site nucleotide sequence in Table 13 (e.g., nucleotides 42-49 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus transcription start site nucleotide sequence in Table 13 (e.g., nucleotide 49 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule contains a nucleic acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus 5'UTR conserved domain nucleotide sequence in Table 13 (e.g., nucleotides 117-187 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus 3 open reading frame region nucleotide sequence in Table 13 (e.g., nucleotides 2186-2385 of the nucleic acid sequence in Table 13). In several embodiments, the nucleic acid molecule includes a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus poly(A) signal nucleotide sequence in Table 13 (e.g., nucleotides 2676-2681 of the nucleic acid sequence in Table 13).In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus GC-rich nucleotide sequence in Table 13 (for example, nucleotides 3054-3172 of the nucleic acid sequence in Table 13).

[0260] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 2. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 2. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 2. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 2. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 2. In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 2.

[0261] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 4. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 4. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 4. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 4. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 4. In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 4.

[0262] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 6. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 6. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 6. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 6. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 6. In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 6.

[0263] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 8. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 8. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 8. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 8. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 8. In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 8.

[0264] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 10. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 10. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 10. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 10. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 10. In several embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 10.

[0265] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 12. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 12. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 12. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 12. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 12.

[0266] In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 14. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 14. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 14. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 14. In several embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding an amino acid sequence having at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 14.

[0267] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 2. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 2. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 2. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 2. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 2.In several embodiments, the clons described herein include proteins comprising amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 2.

[0268] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence of Table 4. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 4. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 4. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence of Table 4. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence of Table 4.In several embodiments, the clons described herein include proteins comprising amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 4.

[0269] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 6. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 6. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 6. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 6. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 6.In several embodiments, the clons described herein include proteins comprising amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 6.

[0270] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence shown in Table 8. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 8. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 8. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence shown in Table 8. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence shown in Table 8.In several embodiments, the clons described herein include proteins comprising amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence shown in Table 8.

[0271] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence of Table 10. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence of Table 10. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence of Table 10. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence of Table 10. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence of Table 10.In several embodiments, the clons described herein include proteins comprising amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Anellovirus ORF2t / 3 amino acid sequence of Table 10.

[0272] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence of Table 12. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence of Table 12. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence of Table 12. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence of Table 12. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence of Table 12.

[0273] In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 amino acid sequence of Table 14. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF1 / 2 amino acid sequence shown in Table 14. In several embodiments, the clons described herein include a protein containing an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 amino acid sequence shown in Table 14. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 2 amino acid sequence of Table 14. In several embodiments, the clons described herein include a protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the Anellovirus ORF2 / 3 amino acid sequence of Table 14.

[0274] [Table 1-1]

[0275] Table 1-2

[0276] Table 1-3

[0277] Table 2-1

[0278] Table 3-1

[0279] Table 3-2

[0280] Table 3-3

[0281] Table 4-1

[0282] Table 4-2

[0283] Table 5-1

[0284] Table 5-2

[0285] Table 5-3

[0286] Table 6-1

[0287] Table 6-2

[0288] Table 7-1

[0289] Table 7-2

[0290] Table 7-3

[0291] Table 8-1

[0292] Table 8-2

[0293] Table 9-1

[0294] Table 9-2

[0295] Table 9-3

[0296] Table 10-1

[0297] Table 10-2

[0298] Table 11-1

[0299] Table 11-2

[0300] Table 11-3

[0301] Table 12-1

[0302] Table 12-2

[0303] Table 13-1

[0304] Table 13-2

[0305] Table 13-4

[0306] [Table 14-1]

[0307] [Table 14-2]

[0308] In some embodiments, the synthetic clones include a minimal anellovirus genome, as identified, for example, by the method described in Example 9. In some embodiments, the synthetic clones include an anellovirus sequence, or a portion thereof, as described in Example 13.

[0309] In some embodiments, the synthetic chromosome includes a gene element containing a consensus anellovirus (Anellovirus) motif, for example, as shown in Table 14-1. In some embodiments, the synthetic chromosome includes a gene element containing a consensus anellovirus (Anellovirus) ORF1 motif, for example, as shown in Table 14-1. In some embodiments, the synthetic chromosome includes a gene element containing a consensus anellovirus (Anellovirus) ORF1 / 1 motif, for example, as shown in Table 14-1. In some embodiments, the synthetic chromosome includes a gene element containing a consensus anellovirus (Anellovirus) ORF1 / 1 motif, for example, as shown in Table 14-1. The synthetic chromium includes a gene element containing the Anellovirus ORF1 / 2 motif. In some embodiments, the synthetic chromium includes a gene element containing the Consensus Anellovirus ORF2 / 2 motif, for example, as shown in Table 14-1. In some embodiments, the synthetic chromium includes a gene element containing the Consensus Anellovirus ORF2 / 3 motif, for example, as shown in Table 14-1. In some embodiments, X shown in Table 14-1 represents any amino acid. In some embodiments, Z shown in Table 14-1 represents glutamic acid or glutamine. In some embodiments, B shown in Table 14-1 represents aspartic acid or asparagine. In some embodiments, J shown in Table 14-1 represents leucine or isoleucine.

[0310] [Table 14-3]

[0311] [Table 14-4]

[0312] Genetic elements In some embodiments, the clone includes a gene element. In some embodiments, the gene element has one or more of the following characteristics: substantially non-integrating with the host cell genome, being an episomal nucleic acid, single-stranded DNA, circular, about 1 to 10 kb, located in the cell nucleus, capable of binding to endogenous proteins, and producing microRNA that targets host genes. In one embodiment, the gene element is substantially non-integrating DNA. In some embodiments, the gene element has at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to, for example, an anellovirus sequence or fragment thereof described herein (for example, listed in any of Tables 1 to 14). In several embodiments, the gene element includes a sequence that encodes an exogenous effector (e.g., a payload), such as a polypeptide effector (e.g., a protein) or a nucleic acid effector (e.g., non-coding RNA, such as miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, or gRNA).

[0313] In some embodiments, the gene elements have a length of less than 20kb (e.g., less than approximately 19kb, 18kb, 17kb, 16kb, 15kb, 14kb, 13kb, 12kb, 11kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb). In some embodiments, the gene elements are independently or in addition to more than 1000b (e.g., at least about 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb). The gene elements have lengths of kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, and 5kb or more. In some embodiments, the gene elements have lengths of approximately 2.5–4.6, 2.8–4.0, 3.0–3.8, or 3.2–3.7kb.

[0314] In some embodiments, the gene element includes one or more of the features described herein, such as a sequence encoding a substantially non-pathogenic protein, a protein-binding sequence, one or more sequences encoding a regulatory sequence, one or more regulatory sequences, one or more sequences encoding a replication protein, and other sequences.

[0315] In one embodiment, the present invention includes a gene element comprising (i) a substantially nonpathogenic external protein, (ii) an external protein-binding sequence for binding the substantially nonpathogenic external protein to the gene element, and (iii) a regulatory nucleic acid, comprising a nucleic acid sequence (e.g., a DNA sequence) encoding a regulatory nucleic acid. In such embodiments, the gene element may comprise one or more sequences having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity with respect to any one of the nucleotide sequences compared to a native viral sequence.

[0316] Proteins, for example, substantially non-pathogenic proteins In some embodiments, the gene element includes a sequence encoding a protein, for example, a substantially non-pathogenic protein. In several embodiments, the substantially non-pathogenic protein is the main component of the proteinaceous outer layer of the clone. Multiple substantially non-pathogenic protein molecules can self-assemble into an icosahedral structure, which constitutes the proteinaceous outer layer. In several embodiments, the protein resides in the proteinaceous outer layer.

[0317] In some embodiments, the protein, for example, a substantially nonpathogenic protein and / or proteinaceous outer layer protein, comprises one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosylated amino acids.

[0318] In some embodiments, the protein, for example, a substantially non-pathogenic protein and / or proteinaceous outer layer, comprises at least one hydrophilic DNA-binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal polyarginine sequence, a variable region, a C-terminal polyglutamine / glutamic acid sequence, and one or more disulfide crosslinks.

[0319] In some embodiments, the gene element includes a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence encoding a capsid protein or a fragment of a capsid protein, or with any one of the nucleotide sequences encoding a capsid protein as described herein, for example, listed in Tables 1 to 16 or 19. In some embodiments, the substantially non-pathogenic protein includes a capsid protein or a functional fragment of a capsid protein, encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with any one of the nucleotide sequences listed in, for example, Tables 1, 3, 5, 7, 9, 11, 13, or 15 described herein.

[0320] [Table 15-1]

[0321] [Table 15-2]

[0322] [Table 15-3]

[0323] Table 15-4

[0324] Table 15-5

[0325] Table 15-6

[0326] Table 15-7

[0327] Table 15-8

[0328] Table 15-9

[0329] Table 15-10

[0330] Table 15-11

[0331] Table 15-12

[0332] Table 15-13

[0333] Table 15-14

[0334] Table 15-15

[0335] Table 15-16

[0336] Table 15-17

[0337] Table 15-18

[0338] Table 15-19

[0339] Table 15-20

[0340] Table 15-21

[0341] Table 15-22

[0342] Table 15-23

[0343] Table 15-24

[0344] Table 15-25

[0345] Table 15-26

[0346] Table 15-27

[0347] Table 15-28

[0348] Table 15-29

[0349] Table 15-30

[0350] Table 15-31

[0351] Table 15-32

[0352] Table 15-33

[0353] Table 15-34

[0354] Table 15-35

[0355] Table 15-36

[0356] Table 15-37

[0357] Table 15-38

[0358] Table 15-39

[0359] Table 15-40

[0360] Table 15-41

[0361] Table 15-42

[0362] Table 15-43

[0363] Table 15-44

[0364] Table 15-45

[0365] Table 15-46

[0366] Table 15-47

[0367] Table 15-48

[0368] Table 15-49

[0369] Table 15-50

[0370] Table 15-51

[0371] Table 15-52

[0372] Table 15-53

[0373] Table 15-54

[0374] Table 15-55

[0375] Table 15-56

[0376] Table 15-57

[0377] Table 15-58

[0378] Table 15-59

[0379] Table 15-60

[0380] Table 15-61

[0381] Table 15-62

[0382] Table 15-63

[0383] Table 15-64

[0384] Table 15-65

[0385] Table 15-66

[0386] Table 15-67

[0387] Table 15-68

[0388] Table 15-69

[0389] Table 15-70

[0390] Table 15-71

[0391] Table 15-72

[0392] Table 15-73

[0393] Table 15-74

[0394] Table 15-75

[0395] Table 15-76

[0396] Table 15-77

[0397] Table 15-78

[0398] Table 15-79

[0399] Table 15-80

[0400] Table 15-81

[0401] Table 15-82

[0402] Table 15-83

[0403] Table 15-84

[0404] Table 15-85

[0405] Table 15-86

[0406] Table 15-87

[0407] Table 15-88

[0408] Table 15-89

[0409] Table 15-90

[0410] Table 15-91

[0411] Table 15-92

[0412] Table 15-93

[0413] Table 15-94

[0414] Table 15-95

[0415] Table 15-96

[0416] Table 15-97

[0417] Table 15-98

[0418] Table 15-99

[0419] Table 15-100

[0420] Table 15-101

[0421] Table 15-102

[0422] Table 15-103

[0423] Table 15-104

[0424] [Table 15-105]

[0425] [Table 15-106]

[0426] [Table 15-107]

[0427] [Table 15-108]

[0428] In some embodiments, the gene element includes a nucleotide sequence encoding a capsid protein or a functional fragment of a capsid protein, or a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the amino acid sequences listed in, for example, Tables 2, 4, 6, 8, 10, 12, 14, or 16. In some embodiments, the substantially non-pathogenic protein includes a capsid protein or a functional fragment of a capsid protein, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the amino acid sequences listed in, for example, Tables 2, 4, 6, 8, 10, 12, 14, or 16.

[0429] [Table 16-1]

[0430] [Table 16-2]

[0431] Table 16-3

[0432] Table 16-4

[0433] Table 16-5

[0434] Table 16-6

[0435] Table 16-7

[0436] Table 16-8

[0437] Table 16-9

[0438] Table 16-10

[0439] Table 16-11

[0440] Table 16-12

[0441] Table 16-13

[0442] Table 16-14

[0443] Table 16-15

[0444] Table 16-16

[0445] Table 16-17

[0446] Table 16-18

[0447] Table 16-19

[0448] Table 16-20

[0449] Table 16-21

[0450] Table 16-22

[0451] Table 16-23

[0452] Table 16-24

[0453] Table 16-25

[0454] Table 16-26

[0455] Table 16-27

[0456] Table 16-28

[0457] Table 16-29

[0458] Table 16-30

[0459] Table 16-31

[0460] Table 16-32

[0461] In some embodiments, the gene element includes an amino acid sequence or a functional fragment thereof, or a nucleotide sequence encoding a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the amino acid sequences listed in Table 17, for example. In some embodiments, the substantially non-pathogenic protein includes an amino acid sequence or a functional fragment thereof, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the amino acid sequences listed in Tables 2, 4, 6, 8, 10, 12, 14, 16, or 17, for example.

[0462] In some embodiments, the gene element is an amino acid sequence listed in this specification, for example, Tables 2, 4, 6, 8, 10, 12, 14, 16, or 17, or shown in Figure 1, or a functional fragment thereof, from about position 1 to about position 150 (e.g., about position 20 to about position 35, about position 25 to about position 30, about position 26 to about position 30, or for example, any subset of amino acids within each range), from about position 150 to about position 390 (e.g., about position 200 to about position 380, about position 205 to about position 375, about position 205 to about position 371, or for example). It includes a nucleotide sequence that encodes an amino acid sequence having, for example, any subset of amino acids within each range, approximately 390 to approximately 525, approximately 525 to approximately 850 (for example, approximately 530 to approximately 840, approximately 545 to approximately 830, approximately 550 to approximately 820, or for example, any subset of amino acids within each range), approximately 850 to approximately 950 (for example, approximately 860 to approximately 940, approximately 870 to approximately 930, approximately 880 to approximately 923, or for example, any subset of amino acids within each range). In some embodiments, substantially non-pathogenic proteins include an amino acid sequence or a functional fragment thereof, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 60%,

[0463] In some embodiments, substantially non-pathogenic proteins are defined as amino acid sequences or functional fragments thereof, or any one of the amino acid sequences or ranges of amino acids listed herein, for example in Tables 2, 4, 6, 8, 10, 12, 14, 16, or 17, or shown in Figure 1, with at least about 60%, 65%, 70%, 75%, 80%, 85%, and 90% of them. The sequences include sequences having 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, wherein the sequences are functional domains or provide functions such as species and / or tissue and / or cell targeting, viral genome 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, nucleic acid protection, and combinations thereof. In some embodiments, diverse amino acids with low sequence identity may provide one or more of the properties described herein, as well as differences in cell / tissue / species specificity (e.g., targeting).

[0464] Protein binding sequence A strategy used by many viruses is for the viral capsid protein to recognize specific protein-binding sequences within its genome. For example, in viruses with non-segmented genomes, such as yeast LA virus, there is a secondary structure (stem-loop) and a specific sequence at the 5' end of the genome, both of which are used for binding to the viral capsid protein. However, viruses with segmented genomes, such as those in the Reoviridae, Orthomyxoviridae (influenza), Bunyaviruses, and Arenaviruses, need to package each of their genomic segments. Some viruses use complementary regions of segments to facilitate the virus containing one of the genomic molecules. Other viruses have specific binding sites for each of the various segments. (e.g., Curr Opin Struct Biol. 2010) See Feb;20(1):114-120;Journal of Virology(2003),77(24),13036-13041.

[0465] In some embodiments, the gene element encodes a protein-binding sequence that binds to a substantially non-pathogenic protein. In some embodiments, the protein-binding sequence facilitates the packaging of the gene element into a proteinaceous outer layer. In some embodiments, the protein-binding sequence specifically binds to an arginine-rich region of a substantially non-pathogenic protein. In some embodiments, the gene element includes the protein-binding sequence described in Example 8. In some embodiments, the gene element includes a protein-binding sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the 5'UTR conserved domain or GC-rich region of an anellovirus sequence (e.g., as shown in any of Tables 1, 3, 5, 7, 9, 11, or 13). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 1 (e.g., nucleotides 177-247 of the nucleic acid sequence in Table 1). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 1 (e.g., nucleotides 3415-3570 of the nucleic acid sequence in Table 1). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 3 (e.g., nucleotides 174-244 of the nucleic acid sequence in Table 3). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 3 (e.g., nucleotides 3691-3794 of the nucleic acid sequence in Table 3).In some embodiments, the protein-binding sequence is the anerovirus (Anellov) shown in Table 5. The protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the irus)5'UTR conserved domain nucleotide sequence (e.g., nucleotides 170-240 of the nucleic acid sequence in Table 5). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the anellovirus GC-rich nucleotide sequence (e.g., nucleotides 3632-3753 of the nucleic acid sequence in Table 5). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 7 (e.g., nucleotides 174-244 of the nucleic acid sequence in Table 7). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 7 (e.g., nucleotides 3733-3853 of the nucleic acid sequence in Table 7). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 9 (e.g., nucleotides 171-241 of the nucleic acid sequence in Table 9). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 9 (e.g., nucleotides 3644-3758 of the nucleic acid sequence in Table 9).In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 11 (e.g., nucleotides 323-393 of the nucleic acid sequence in Table 11). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 11 (e.g., nucleotides 2868-2929 of the nucleic acid sequence in Table 11). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus 5'UTR conserved domain nucleotide sequence in Table 13 (e.g., nucleotides 117-187 of the nucleic acid sequence in Table 13). In some embodiments, the protein-binding sequence has at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the anellovirus GC-rich nucleotide sequence in Table 13 (e.g., nucleotides 3054-3172 of the nucleic acid sequence in Table 13).

[0466] In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with the nucleic acid sequences shown in Figure 16-1 and / or Figure 21. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence of the consensus 5'UTR sequence shown in Figure 16-1, where X1, X2, X3, X4, and X5 are each independently any nucleotide, for example, X1=G or T, X2=C or A, X3=G or A, X4=T or C, and X5=A, C, or T). In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the consensus 5'UTR sequence shown in Figure 16-1. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes an exemplary TTV shown in Figure 16-1. The nucleotide sequence includes a nucleic acid sequence having at least approximately 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the 5'UTR sequence. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least approximately 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the TTV-CT30F 5'UTR sequence shown in Figure 16-1. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the TTV-HD23a 5'UTR sequence shown in Figure 16-1. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with the TTV-TJN02 5'UTR sequence shown in Figure 16-1.

[0467] [Table 16-33]

[0468] In some embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleic acid sequence set forth in Figure 16-2 and / or Figure 22. In some embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises the nucleic acid sequence of the consensus GC-rich sequence shown in Figure 16-1, wherein X4, X5, X6, X7, X 12 , X 13 , X 14 , X 15 , X 20 , X 21 , X 22 , X 26 , X 29、 X 30 , and X 33 are each independently any nucleotide, and X2, X3, X8, X9, X 10 , X 11 , X 16 , X 17 , X 18 , X 19 , X 23 , X 24 , X 25 , X 27 , X 28 , X 31 , X 32 , and X 34 are each independently absent or any nucleotide. In some embodiments, X1 to X 34One or more (e.g., all) of these are independently the nucleotides (or absences) described in Figure 16-2. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the consensus GC-rich sequence shown in Figure 16-1. In some embodiments, the gene element (e.g., the protein-binding sequence of the gene element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the exemplary TTV GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, or any combination thereof, e.g., fragments 1-3 in order). In several embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-CT30F GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1 to 7 in order). In several embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the TTV-HD23a GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, or any combination thereof, e.g., fragments 1-6 in order).In several embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-JA20 GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, or any combination thereof, e.g., fragments 1 and 2 in order). In several embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-TJN02 GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1-8 in order). In several embodiments, the gene element (e.g., the protein-binding sequence of the gene element) includes a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the TTV-tth8 GC-rich sequence shown in Figure 16-1 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, or any combination thereof, e.g., fragments 1-6 in order).

[0469] [Table 16-34]

[0470] [Table 16-35]

[0471] [Table 16-36]

[0472] [Table 16-37]

[0473] effector In some embodiments, the gene element may include one or more sequences encoding a functional nucleic acid, such as an exogenous effector, such as a therapeutic agent, such as a regulatory nucleic acid, such as cytotoxic or cytolytic RNA or protein. In some embodiments, the functional nucleic acid is non-coding RNA.

[0474] In some embodiments, the sequence encoding the external effector is inserted within a gene element at an insertion site, for example, as described in Examples 10, 12, or 22. In some embodiments, the sequence encoding the external effector is inserted into a non-coding region within a gene element, for example, a non-coding region located on the 3' side of the open reading frame and the 5' side of the GC-rich region of the gene element, a 5' non-coding region upstream of the TATA box, the 5' UTR, downstream of the poly-A signal 3' non-coding region, or a 3' non-coding region upstream of the GC-rich region. In some embodiments, the sequence encoding the external effector is inserted within a gene element, for example, near nucleotide 3588 of the TTV-tth8 plasmid as described herein, or, for example, near nucleotide 2843 of the TTMV-LY2 plasmid as described herein. In several embodiments, the sequence encoding the external effector is inserted within a gene element, for example, between nucleotides 336 and 3015 of the TTV-tth8 plasmid or within that range, as described herein, or between nucleotides 242 and 2812 of the TTV-LY2 plasmid or within that range, as described herein. In some embodiments, the sequence encoding the external effector replaces part or all of the open reading frame (for example, the ORFs described herein, e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3 shown in any of Tables 1 to 14).

[0475] In some embodiments, the sequence encoding the external effector includes 100-2000, 100-1000, 100-500, 100-200, 200-2000, 200-1000, 200-500, 500-1000, 500-2000, or 1000-2000 nucleotides. In some embodiments, the external effector is a nucleic acid or protein payload, as described in Example 11, for example.

[0476] regulatory nucleic acids In some embodiments, the regulatory nucleic acid modifies the expression of endogenous and / or exogenous genes. In one embodiment, the regulatory nucleic acid targets host genes. Examples of regulatory nucleic acids, but not limited to, include nucleic acids that hybridize with exogenous genes (e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA as described elsewhere herein), nucleic acids that hybridize with exogenous nucleic acids such as viral DNA or RNA, nucleic acids that hybridize with RNA, nucleic acids that interfere with gene transcription, nucleic acids that interfere with RNA translation, nucleic acids that stabilize or destabilize RNA by targeting degradation, and nucleic acids that regulate DNA or RNA binding factors. In several embodiments, the regulatory nucleic acid encodes miRNA.

[0477] In some embodiments, the regulatory nucleic acid typically comprises RNA or an RNA-like structure containing 5 to 500 base pairs (depending on the specific RNA structure, for example, 5 to 30 bp for miRNA and 200 to 500 bp for lncRNA), and may have a nucleic acid base sequence that is identical (or complementary) or nearly identical (or substantially complementary) to the coding sequence in the target gene expressed in the cell, or a sequence that codes for the target gene expressed in the cell.

[0478] In some embodiments, the regulatory nucleic acid includes a nucleic acid sequence, such as a guide RNA (gRNA). In some embodiments, the DNA targeting portion includes the guide RNA or a nucleic acid encoding the guide RNA. A short synthetic RNA of gRNA may consist of a “scaffold” sequence necessary to bind to an incomplete effector portion and a user-defined targeting sequence of approximately 20 nucleotides for the genomic target. In practice, guide RNA sequences are generally designed to have a length of 17–24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to the targeting nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in designing effective guide RNAs. Gene editing has also been achieved using chimeric “single guide RNA” (“sgRNA”), which is an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and contains both tracrRNA (which binds to a nuclease) and at least one crRNA (which induces the nuclease to the sequence targeted for editing). Furthermore, chemically modified sgRNAs have also been shown to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991.

[0479] Regulatory nucleic acids include gRNAs that recognize specific DNA sequences (e.g., sequences adjacent to or within promoters, enhancers, silencers, or repressors of a gene).

[0480] Some regulatory nucleic acids can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules typically include RNA or RNA-like structures containing 15–50 base pairs (e.g., about 18–25 base pairs) and having a nucleic acid base sequence identical (complementary) or nearly identical (substantially complementary) to the coding sequence in the target gene expressed in the cell. Examples of RNAi molecules, but not limited to, include: small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), meloduplexes, and Dicer substrates (U.S. Patents No. 8,084,599, 8,349,809, and 8,513,207).

[0481] Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 100 nucleotides. This somewhat arbitrary restriction distinguishes lncRNAs from smaller regulatory RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and other small RNAs. Generally, the majority of lncRNAs (about 78%) are characterized as tissue-specific. Branched lncRNAs (which account for a significant proportion of all lncRNAs in the mammalian genome, about 20%) that transcribe in the opposite direction to adjacent protein-coding genes are thought to regulate the transcription of adjacent genes.

[0482] A gene element encodes a regulatory nucleic acid having a sequence that is substantially or completely complementary to all or a fragment of an endogenous gene or gene product (e.g., mRNA). The regulatory nucleic acid can complement the sequence at the intron-exon boundary to prevent the naenonuclear RNA transcript of a particular gene from maturing into mRNA for transcription. A regulatory nucleic acid complementary to a particular gene hybridizes with the gene's mRNA to inhibit its translation. The antisense regulatory nucleic acid may be DNA, RNA, or a derivative or hybrid thereof.

[0483] The length of the regulatory nucleic acid that hybridizes with the target transcript may be 5–30 nucleotides, approximately 10–30 nucleotides, or approximately 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides. The percentage of identity of the regulatory nucleic acid with respect to the target transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0484] The gene element may encode a microRNA (miRNA) molecule identical to a regulatory nucleic acid, such as approximately 5 to 25 consecutive nucleotides of a target gene. In some embodiments, the miRNA sequence targets mRNA, starts with dinucleotide AA, contains approximately 30-70% (approximately 30-60%, 40-60%, or 45-55%) GC content, and, as determined, for example by standard BLAST search, does not have high identity (%) with any nucleotide sequence other than the target in the mammalian genome into which it is to be introduced.

[0485] In some embodiments, the regulatory nucleic acid is at least one, for example, 2, 3, 4, 5, 6, or more miRNAs. In some embodiments, the gene element includes a sequence encoding a miRNA having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide identity with respect to any of the nucleotide sequences, or a sequence complementary to one of the sequences described herein, for example, in Table 18.

[0486] [Table 18-1]

[0487] [Table 18-2]

[0488] [Table 18-3]

[0489] Table 18-4

[0490] Table 18-5

[0491] Table 18-6

[0492] Table 18-7

[0493] Table 18-8

[0494] Table 18-9

[0495] siRNA and shRNA are analogous to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNA can function as miRNA and vice versa (Zeng et al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). MicroRNAs like siRNA downregulate target genes using RISC, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein production by translational repression or poly(A) removal and mRNA degradation (Wu et al., Proc Natl Acad Sci USA 103:4034-4039, 2006). Known miRNA binding sites are located within the mRNA 3'UTR; miRNAs appear to target sites that have nearly complete complementarity to nucleotides 2-8 from the 5' end of the miRNA (Rajewsky, Nat Genet 38 Suppl:S8-13,2006; Lim (et al., Nature 433:769-773, 2005). This region is known as the seed region. Since siRNA and miRNA are replaceable, extracellular siRNA downregulates mRNA that has seed complementarity with siRNA (Birmingham et al., Nat Methods 3:199-204, 2006). Multiple target sites within the 3'UTR confer stronger downregulation (Doench). et al., Genes Dev 17:438-442, 2003).

[0496] Known miRNA sequence listings can be found in databases maintained by research institutions such as the Wellcome Trust Sanger Institute, the Penn Center for Bioinformatics, the Memorial Sloan Kettering Cancer Center, and the European Molecular Biology Laboratory. Known effective siRNA sequences and cognitive binding sites are also well described in the relevant literature. RNAi molecules can be easily designed and fabricated using techniques known in the art. In addition, there are computational tools that increase the likelihood of finding effective and specific sequence motifs (Lagana et al., Methods Mol. Bio., 2015, 1269:393-412).

[0497] Regulatory nucleic acids can regulate the expression of RNA encoded by genes. In some embodiments, since multiple genes share a certain degree of sequence homology with one another, regulatory nucleic acids can be designed to target a class of genes with sufficient sequence homology. In some embodiments, regulatory nucleic acids may include sequences that are shared among various gene targets or that are complementary to sequences unique for a particular gene target. In some embodiments, regulatory nucleic acids can be designed to target conserved regions of RNA sequences that are homologous among several genes, thereby targeting several genes within a gene family (e.g., different gene isotypes, splice variants, mutant genes, etc.). In some embodiments, regulatory nucleic acids can be designed to target sequences unique to a specific RNA sequence of a single gene.

[0498] In some embodiments, the gene element may include one or more sequences that encode regulatory nucleic acids that regulate the expression of one or more genes.

[0499] In some embodiments, gRNAs described elsewhere in this specification are used as part of a CRISPR system for gene editing. For gene editing purposes, a clone may be designed to contain one or more guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. Generally, at least about 16 or 17 nucleotides of the gRNA enable Cas9-mediated DNA cleavage; in the case of Cpf1, at least about 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage.

[0500] Therapeutic peptides or polypeptides In some embodiments, the gene element includes a sequence encoding a therapeutic peptide or polypeptide. Such therapeutic agents include, but are not limited to, low molecular weight peptides, peptide mimes (e.g., peptoids), amino acids, and amino acid analogs. These therapeutic agents generally have molecular weights of less than approximately 5,000 grams per mole, less than approximately 2,000 grams per mole, less than approximately 1,000 grams per mole, less than approximately 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Such therapeutic agents include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viruses or microbial particles, synthetic particles, and their agonists or antagonists.

[0501] In some embodiments, the gene element includes a peptide, such as a therapeutic peptide. The peptide may be linear or branched. The peptide has a length of about 5 to about 500 amino acids, about 15 to about 400 amino acids, about 20 to about 325 amino acids, about 25 to about 250 amino acids, about 50 to about 150 amino acids, or in the range between these.

[0502] Some examples of peptides, but not limited to, include fluorescent tags or markers, antigens, peptide therapeutics, synthetic or analog peptides derived from naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, targeting or cytotoxic peptides, degrading or self-destructive peptides, and degrading or self-destructive peptides. Also, peptides useful in the present invention as described herein include antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, and nanobodies (see, for example, Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-113). Such antigen-binding peptides can bind to cytosolic antigens, nuclear antigens, or intraorganelle antigens.

[0503] In some embodiments, the gene element includes a sequence that encodes a protein, such as a therapeutic protein. Some examples of therapeutic proteins, but not limited to, include hormones, cytokines, enzymes, antibodies, transcription factors, receptors (e.g., membrane receptors), ligands, membrane transporters, secreted proteins, peptides, carrier proteins, structural proteins, nucleases, or components thereof.

[0504] In some embodiments, the compositions or clones described herein include polypeptides that bind to ligands capable of targeting specific locations, tissues, or cells.

[0505] Regulatory array In some embodiments, the gene element includes a regulatory sequence, such as a promoter or enhancer.

[0506] In some embodiments, the promoter includes a DNA sequence located adjacent to the DNA encoding the expression product. The promoter may be operably ligated to the adjacent DNA sequence. Typically, the promoter increases the amount of product expressed from the DNA sequence compared to the amount of product expressed in the absence of the promoter. A promoter from one organism can be used to increase the expression of a product from a DNA sequence derived from another organism. For example, a vertebrate promoter can be used for the expression of jellyfish GFP in vertebrates. In addition, a single promoter element can increase the amount of product expressed from multiple tandem-ligated DNA sequences. Thus, a single promoter element can increase the expression of one or more products. Multiple promoter elements are well known to those skilled in the art.

[0507] In one embodiment, a high level of constitutive expression is desired. Examples of such promoters, though not limited to them, include retroviral Rous sarcoma virus (RVS) long-terminal repeat (LTR) promoters / enhancers and cytomegalovirus (CMV) intermediate early promoters / enhancers (e.g., Boshart et al.). Examples include the SV40 promoter, dihydrofolate reductase promoter, cytoplasmic β-actin promoter, and phosphoglycerol kinase (PGK) promoter (see al., Cell, 41:521-530 (1985)).

[0508] In other embodiments, inducible promoters may be desired. These inducible promoters are either cis or trans in configuration and are regulated by an externally supplied compound, and include, but are not limited to, a zinc-inducible sheep metallothionine (MT) promoter; a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; a T7 polymerase promoter system (International Publication No. 98 / 10088); a tetracycline inhibitory system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); a tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); also see Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)); and a RU486-inducible system (Wang et al. Examples include al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997); and rapamycin-inducible systems (Magari et al., J. Clin. Invest., 100:2865-2872 (1997); Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other types of inducible promoters that may be useful in relation to the present invention are those that are regulated only by specific physiological conditions, such as temperature, acute phase, or in replicating cells.

[0509] In some embodiments, a native promoter of the gene or nucleic acid sequence of interest is used. A native promoter can be used when it is desired that the expression of the gene or nucleic acid sequence should mimic native expression. A native promoter can be used when the expression of a gene or other nucleic acid sequence must be regulated transiently or progressively, tissue-specifically, or in response to a specific transcriptional stimulus. In other embodiments, other native expression regulatory elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may be used to mimic native expression.

[0510] In some embodiments, the gene element includes a gene operably linked to a tissue-specific promoter. For example, if expression in skeletal muscle is desired, a promoter active in muscle can be used. Examples include promoters derived from genes encoding skeletal α-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle promoters having higher activity than native promoters. Li et al. See al., Nat. Biotech., 17:241-245 (1999). Examples of tissue-specific promoters include the following: liver albumin, Miyatake et al. J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996); bone (osteocalcin, Stein et al., Mol. Biol. Rep., 24:185-96 (1997); bone sialoprotein, Chen et al., J. Bone Miner. Res. 11:654-64 (1996)); lymphocytes (CD2, Hansal et al. al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor α chain), nerve (neuron-specific enolase (NSE) promoter, Andersen et al. Cell.Mol.Neurobiol., 13:503-15 (1993); neurofilament light chain gene, Piccioli et al., Proc.Natl.Acad.Sci.USA, 88:5611-5 (1991); neuron-specific vgf gene, Piccioli et al., Neuron, 15:373-84 (1995)].

[0511] The gene element may include an enhancer, for example, a DNA sequence located adjacent to the DNA sequence encoding the gene. The enhancer element may typically be located upstream of the promoter element, or downstream of the coding DNA sequence (e.g., a DNA sequence transcribed or translated into one or more products), or within it. Thus, the enhancer element can be located 100, 200, 300 base pairs, or more upstream or downstream of the DNA sequence encoding the product. The enhancer element can increase the amount of recombinant product expressed from the DNA sequence beyond the expression increase provided by the promoter element. Multiple enhancer elements are readily available to those skilled in the art.

[0512] In some embodiments, the gene element includes one or more reverse-terminal repeats (ITRs) flanking to a sequence encoding the expression product described herein. In some embodiments, the gene element includes one or more long-terminal repeats (LTRs) flanking to a sequence encoding the expression product described herein. Examples of promoter sequences that can be used include, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, and Rous sarcoma virus promoter.

[0513] replication factors In some embodiments, the gene elements of a clone, such as a synthetic clone, may include sequences encoding one or more replication proteins. In some embodiments, the clone can be replicated by a rolling circle replication method, such as the synthesis of a leading strand, without the binding of a lagging strand. In these embodiments, the clone includes three additional elements: i) a gene encoding an initiator protein, ii) a double-stranded origin, and iii) a single-stranded origin. A rolling circle replication (RCR) protein complex containing the replication proteins binds to the leading strand, destabilizing the origin. The RCR complex cleaves the genome to generate a free 3'OH end. Cellular DNA polymerase initiates viral DNA replication from the free 3'OH end. After the genome has been replicated, the RCR complex covalently closes the loop. This results in the release of a circular positive single-stranded parental DNA molecule and a circular double-stranded DNA molecule consisting of the parental negative strand and a newly synthesized positive strand. The single-stranded DNA molecule can be enveloped or participate in a second round of replication. For example, see Virology Journal 2009, 6:60 doi:10.1186 / 1743-422X-6-60.

[0514] The gene element may include a sequence that encodes a polymerase, such as RNA polymerase or DNA polymerase.

[0515] Other arrays In some embodiments, the gene element further includes nucleic acids that encode a product (e.g., ribozymes, therapeutic mRNA encoding proteins, exogenous genes).

[0516] In some embodiments, the gene element includes one or more sequences that affect the species and / or tissue and / or cell-specificity of the clone in the host or host cell (e.g., capsid protein sequence), infectivity (e.g., capsid protein sequence), immunosuppression / activation (e.g., regulatory nucleic acid), viral genome binding and / or packaging, immune evasion (non-immunogenicity and / or tolerance), pharmacokinetics, endocytosis and / or cell adhesion, nuclear entry, intracellular regulation and localization, exocytosis regulation, proliferation, and nucleic acid protection.

[0517] In some embodiments, the gene element may include other sequences, including DNA, RNA, or artificial nucleic acids. Other sequences, but not limited to, include genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecules. In one embodiment, the gene element includes a sequence encoding siRNA to target a different locus of the same gene expression product as the regulatory nucleic acid. In another embodiment, the gene element includes a sequence encoding siRNA to target a different gene expression product than the regulatory nucleic acid.

[0518] In some embodiments, the gene element includes one or more sequences that encode one or more miRNAs, sequences that encode one or more replication proteins, sequences that encode exogenous genes, sequences that encode therapeutic agents, regulatory sequences (e.g., promoters, enhancers), sequences that encode one or more regulatory sequences that target endogenous genes (siRNA, lncRNA, shRNA), and sequences that encode therapeutic mRNA or proteins.

[0519] Other sequences may have lengths of approximately 2 to 5000 nt, approximately 10 to 100 nt, approximately 50 to 150 nt, approximately 100 to 200 nt, approximately 150 to 250 nt, approximately 200 to 300 nt, approximately 250 to 350 nt, approximately 300 to 500 nt, approximately 10 to 1000 nt, approximately 50 to 1000 nt, approximately 100 to 1000 nt, approximately 1000 to 2000 nt, approximately 2000 to 3000 nt, approximately 3000 to 4000 nt, approximately 4000 to 5000 nt, or any range in between.

[0520] Exogenous genes For example, a gene element may include genes related to signaling biochemical pathways, such as signaling biochemical pathway-related genes or polynucleotides. Examples include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from diseased tissue compared to non-disease control tissue or cells. This may be a gene expressed at an abnormally high level; or a gene expressed at an abnormally low level, in which case the altered expression correlates with the onset and / or progression of the disease. Disease-related genes are also called gene processing mutations or gene mutations, and they are either the direct cause of the disease or are in a state of association disequilibrium with the pathogenic gene.

[0521] Disease-related genes and polynucleotides are studied at the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins. Information is available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.). Examples of disease-related genes or polynucleotides are listed in Tables A and B of U.S. Patent No. 8,697,359, which are incorporated herein by reference in their entirety. Disease-specific information is available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine. These are available from the Library of Medicine (Bethesda, Md.). Examples of signal transduction biochemical pathway-related genes and polynucleotides are listed in Tables A-C of U.S. Patent No. 8,697,359, which are incorporated herein by reference in their entirety.

[0522] Furthermore, the gene element can encode a targeting moiety, as described elsewhere in this specification. This can be achieved, for example, by inserting polynucleotides that encode proteins such as sugars, glycolipids, or antibodies. Those skilled in the art are well aware of further methods for preparing targeting moieties.

[0523] Virus sequence In some embodiments, the gene element comprises at least one viral sequence. In some embodiments, these sequences are homologous or identical to one or more sequences derived from single-stranded DNA viruses, such as anellovirus, bidnavirus, circovirus, geminivirus, genomovirus, inovirus, microvirus, nanovirus, parvovirus, and spiravirus. In some embodiments, the sequence is homologous or identical to one or more sequences derived from double-stranded DNA viruses, such as adenovirus, ampullavirus, ascovirus, asfarvirus, baculovirus, fusellovirus, globulovirus, guttavirus, hytrosavirus, herpesvirus, iridovirus, lipospirixvirus, nimavirus, and poxvirus. In some embodiments, the sequence is homologous or identical to one or more sequences derived from RNA viruses, such as alphavirus, furovirus, hepatitis virus, hordeivirus, tobamovirus, tobravirus, tricornavirus, rubivirus, birnavirus, cystovirus, partitivirus, and reovirus.

[0524] In some embodiments, the gene element may include one or more sequences derived from non-pathogenic viruses, such as symbiotic viruses, such as commensal viruses, such as native viruses, such as anelloviruses. Recently, a renaming has been made, and three anelloviruses capable of infecting human cells have been classified into the genera Alphatorquevirus (TT), Betatorquevirus (TTM), and Gammatorquevirus (TTMD) of the Anelloviridae family. To date, anelloviruses have not been associated with human diseases. In some embodiments, the gene element may include sequences homologous or identical to Torque Teno Virus (TT), a non-enveloping single-stranded DNA virus with a circular minus-sense genome. In some embodiments, the gene elements may include sequences homologous or identical to SEN viruses, sentinel viruses, TTV-like miniviruses, and TT viruses. Various types of TT viruses are described, including TT virus genotype 6, TT virus group, TTV-like virus DXL1, and TTV-like virus DXL2. In some embodiments, the gene elements may include sequences homologous or identical to a third virus having an intermediate genome size between TTV and TTMV, called a smaller virus, Torque Teno-like Mini Virus (TTM), or Torque Teno-like Midi Virus (TTMD). In some embodiments, the gene element may include one or more sequences or a fragment of a sequence derived from a nonpathogenic virus having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity with respect to any one of the nucleotide sequences listed in Table 19, for example.

[0525] Table 19-1

[0526] Table 19-2

[0527] Table 19-3

[0528] Table 19-4

[0529] Table 19-5

[0530] Table 19-6

[0531] Table 19-7

[0532] Table 19-8

[0533] Table 19-9

[0534] Table 19-10

[0535] Table 19-11

[0536] Table 19-12

[0537] Table 19-13

[0538] Table 19-14

[0539] Table 19-15

[0540] Table 19-16

[0541] Table 19-17

[0542] Table 19-18

[0543] Table 19-19

[0544] Table 19-20

[0545] [Table 19-21]

[0546] [Table 19-22]

[0547] [Table 19-23]

[0548] [Table 19-24]

[0549] [Table 19-25]

[0550] [Table 19-26]

[0551] [Table 19-27]

[0552] [Table 19-28]

[0553] In some embodiments, the gene element may include one or more sequences or a fragment of a substantially nonpathogenic virus having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity with respect to any one of the nucleotide sequences listed in Table 20, for example.

[0554] [Table 20-1]

[0555] [Table 20-2]

[0556] [Table 20-3]

[0557] [Table 20-4]

[0558] In some embodiments, the gene element is one or more non-aneroviruses, such as adenovirus, herpesvirus, poxvirus, and vaccinia virus. The recombinant retrovirus includes RNA viruses such as SV40, papillomavirus, and retroviruses, such as lentivirus, single-stranded RNA viruses such as hepatitis virus, or double-stranded DNA viruses such as rotavirus, and one or more sequences having homology or identity to one or more sequences derived from SV40, papillomavirus, and retrovirus. In some embodiments, the recombinant retrovirus is deficient and can provide assistance for producing infectious particles. Such assistance can be provided, for example, by using helper cells containing plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable cell lines for replicating the clones described herein include cell lines known in the art, such as A549, which can be modified as described herein. The gene elements may further include genes encoding selection markers so that the desired gene elements can be identified.

[0559] In some embodiments, the gene element is either identical in sequence to the polypeptide encoded by the initial nucleotide sequence by at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or otherwise includes non-silent mutations, such as base substitutions, deletions, or additions that result in an amino acid change in the encoded polypeptide, as long as they are useful for carrying out the invention. In this regard, several conservative amino acid substitutions can be performed, and these are generally recognized not to inactivate the overall protein function, for example, with respect to positively charged amino acids (and vice versa), i.e., lysine, arginine, and histidine; negatively charged amino acids (and vice versa), i.e., aspartic acid and glutamic acid; and several groups of neutrally charged amino acids (and in all cases, and vice versa), i.e., (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine ​​and serine, (4) glycine and proline, (5) isoleucine, leucine, and valine, (6) methionine, leucine, and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and, for example, tyrosine, tryptophan, and phenylalanine. Amino acids can be classified according to their physical properties and contributions to secondary and tertiary protein structures. Conservative substitution is recognized in the art as a one-amino acid substitution with another amino acid having similar properties.

[0560] The identity of two or more nucleic acid or polypeptide sequences having identical or specified percentages of nucleotides, or identical amino acid residues (approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity across a specified region when compared and aligned for maximum match across a comparison window or specified region), can be measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, for example, the NCBI website www.ncbi.nlm.nih.gov / BLAST / ). Identity may also refer to or apply to the complement of the test sequence. Identity also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described herein, the algorithm takes gaps, etc. Identity may exist over regions of at least approximately 10 amino acids or nucleotide lengths, approximately 15 amino acids or nucleotide lengths, approximately 20 amino acids or nucleotide lengths, approximately 25 amino acids or nucleotide lengths, approximately 30 amino acids or nucleotide lengths, approximately 35 amino acids or nucleotide lengths, approximately 40 amino acids or nucleotide lengths, approximately 45 amino acids or nucleotide lengths, approximately 50 amino acids or nucleotide lengths, or more.

[0561] In some embodiments, the gene element includes a nucleotide sequence having at least about 75% nucleotide sequence identity, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity, with respect to, for example, one of the nucleotide sequences listed in Table 19 or Table 20. Since the gene code is degenerate, homologous nucleotide sequences can include any number of "silent" base changes, i.e., nucleotide substitutions that also encode the same amino acids.

[0562] Gene editing constructs The gene element of the synthetic clone may contain one or more genes that encode one component of the gene editing system. Exemplary gene editing systems include the clustered regulatory interspaced short palindromic repeat (CRISPR) system, zinc finger nucleases (ZFNs), and transcriptional activator-like effector-based nucleases (TALENs). Methods based on ZFN, TALEN, and CRISPR are described, for example, in Gaj et al. Trends Biotechnol. 31.7 (2013):397-405; the CRISPR method for gene editing is described, for example, in Guan et al., Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model. DNA Repair 2016 Oct;46:1-8.doi:10.1016 / j.dnarep.2016.07.004; and Zheng et al., Precise gene deletion and replacement using the CRISPR / Cas9 system in human cells. BioTechniques, Vol.57, No.3, September 2014, pp.115-124.

[0563] The CRISPR system is an adaptive defense system initially discovered in bacteria and archaea. The CRISPR system uses RNA-induced nucleases, called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1), to cleave foreign DNA. In a typical CRISPR / Cas system, the endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome to be edited) by a sequence-specific, non-coding "guide RNA" that targets single-stranded or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). Some Class II CRISPR systems include type II Cas endonucleases such as Cas9, CRISPR RNA ("crRNA"), and transactivating crRNA ("tracrRNA"). The crRNA contains a "guide RNA," typically a roughly 20-nucleotide RNA sequence corresponding to the target DNA sequence. crRNA also contains a region that binds to tracrRNA to form a partially double-stranded structure, which is cleaved by RNase III to form a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave a target DNA sequence. The target DNA sequence must generally be adjacent to a “protospacer fringe motif” (“PAM”) specific to a given Cas endonuclease; however, PAM sequences appear throughout a given genome.

[0564] In some embodiments, the clone contains a gene for a CRISPR endonuclease. For example, several CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases, such as Cas9 endonucleases, bind to a G-rich PAM site, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA at a position 3 nucleotides upstream from the PAM site (its 5' end). Another class II CRISPR system includes the smaller V-type endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (derived from Acidaminococcus sp.) and LbCpf1 (derived from Lachnospiraceae sp.). Cpf1 endonucleases bind to T-rich PAM sites, such as 5'-TTN. Cpf1 can also recognize the 5'-CTA PAM motif. Cpf1 cleaves target DNA by introducing offset or staggered double-strand breaks with 4- or 5-nucleotide 5' overhangs, for example, by cleaving the target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from the PAM site (its 3' end) on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand; the 5-nucleotide overhangs resulting from these offset breaks enable precise genome editing by homologous recombination DNA insertion compared to insertions with blunt-end cuts. See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[0565] Various CRISPR-binding (Cas) genes may be included in the clone. Specific examples of genes include those encoding Cas proteins from class II systems, such as Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, the clone includes a gene encoding a Cas protein, such as the Cas9 protein, which may originate from any of the diverse prokaryotic species. In some embodiments, the clone includes a gene encoding a specific Cas protein, such as a specific Cas9 protein, which is selected to recognize a specific protospacer adjacent motif (PAM) sequence. In some embodiments, the clone includes nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, and the clone may be introduced into cells, zygotes, embryos, or animals to enable recognition and modification of sites containing the same, similar, or different PAM motifs, for example. In some embodiments, the clone includes a gene encoding an inactivated nuclease, such as a modified Cas protein including nuclease-deficient Cas9.

[0566] The wild-type Cas9 protein generates double-strand breaks (DSBs) at specific DNA sequences targeted by gRNA; however, several CRISPR endonucleases with modified functionality are known. For example, the "nickase" version of Cas9 generates only single-strand breaks; and the non-catalytic Cas9 ("dCas9") does not cleave target DNA. The gene encoding dCas9 can be fused with a gene encoding an effector domain to repress (CRISPRi) or activate (CRISPRa) the expression of a target gene. For example, the gene may encode a transcriptional silencer (e.g., the KRAB domain) or a transcriptional activator (e.g., the dCas9-VP64 fusion). A gene encoding non-catalytic Cas9 (dCas9) fused to a FokI nuclease ("dCas9-FokI") can be incorporated to generate DSBs at homologous target sequences for two gRNAs. For example, see the numerous CRISPR / Cas9 plasmids disclosed in the Addgene repository (Addgene, 75 Sidney St., Suite 550A, Cambridge, MA 02139; addgene.org / crispr / ) and available from there. "Double nickase" Cas9, which introduces two separate double-strand breaks, each directed by a separate guide RNA, has been described by Ran et al. (2013) Cell, 154:1380-1389 as achieving more precise genome editing.

[0567] CRISPR technology for editing genes in eukaryotic cells is disclosed in U.S. Patent Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1, as well as U.S. Patent Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. The Cpf1 endonuclease, along with its corresponding guide RNA and PAM site, is disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1.

[0568] In some embodiments, the clone comprises polypeptides described herein, e.g., genes encoding targeted nucleases, e.g., Cas9, e.g., wild-type Cas9, nickase Cas9 (e.g., Cas9 D10A), inactive Cas9 (dCas9), eSpCas9, Cpf1, C2C1, or C2C3, and gRNAs. The selection of the nuclease-encoding gene and gRNA is determined by whether the targeted mutation is a nucleotide deletion, substitution, or addition, e.g., a nucleotide deletion, substitution, or addition to a targeted sequence. A gene encoding a non-catalytic endonuclease, e.g., inactive Cas9 (dCas9, e.g., D10A; H840A), anchored to all or part of (e.g., its bioactive portion) of one or more effector domains (e.g., VP64), produces a chimeric protein that can regulate the activity and / or expression of one or more target nucleic acid sequences.

[0569] As used herein, “bioactive portion of effector domain” is a protein that maintains (e.g., completely, partially, or minimally) the function of an effector domain (e.g., “minimal” or “core” domain). In some embodiments, cloning includes a gene encoding a fusion of one or more effector domains, in whole or in part, with dCas9 in order to produce a chimeric protein useful for the methods described herein. Thus, in some embodiments, cloning includes a gene encoding a dCas9-methylase fusion. In other embodiments, cloning includes a gene encoding a dCas9-enzyme fusion containing a site-specific gRNA that targets an endogenous gene.

[0570] In other embodiments, the clonn comprises a gene encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (all or bioactive portions) fused with dCas9.

[0571] Protein outer layer In some embodiments, a clone, such as a synthetic clone, includes a proteinaceous outer layer that confines the genetic elements. The proteinaceous outer layer may include substantially non-pathogenic external proteins that cannot induce an immune response in mammals. In some embodiments, the synthetic clone is lipid-free in the proteinaceous outer layer. In some embodiments, the synthetic clone is lipid-free, such as a viral envelope. In some embodiments, the interior of the synthetic clone is completely (e.g., 100%) covered by the proteinaceous outer layer. In some embodiments, the interior of the synthetic clone is covered by external proteins with coverage of less than 100%, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. In some embodiments, the proteinaceous outer layer includes gaps or discontinuities (e.g., allowing permeability to water, ions, peptides, or small molecules) as long as the genetic elements are retained within the clone.

[0572] In some embodiments, the proteinaceous outer layer includes one or more proteins or polypeptides, such as complementary proteins or polypeptides, that specifically recognize and / or bind to host cells in order to mediate the entry of gene elements into host cells.

[0573] In some embodiments, the proteinaceous outer layer includes one or more of the following: 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.

[0574] In some embodiments, the proteinaceous outer layer includes 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, lipid molecule deletion, carbohydrate deletion, pH and temperature instability, surfactant resistance, and being substantially non-immunogenic or substantially non-pathogenic in host cells.

[0575] vector The gene elements described herein may be incorporated into vectors. Suitable vectors, as well as methods for their manufacture and use, are known in the prior art.

[0576] In one embodiment, the present invention includes a vector comprising 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 a regulatory nucleic acid.

[0577] Either a gene element or a sequence within a gene element can be obtained by any suitable method. For example, various recombination methods are known in the art, such as screening libraries from cells containing viral sequences using standard techniques, inducing sequences from vectors known to contain such sequences, or direct isolation from cells and tissues containing them. Alternatively, or in combination therewith, some or all of the gene elements can be produced by synthesis rather than cloning.

[0578] In some embodiments, the vector includes a regulatory element, a nucleic acid sequence homologous to the target gene, and various reporter constructs for inducing the expression of a reporter molecule within a living cell and / or when the intracellular molecule is present in the target cell.

[0579] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are absent or unexpressed in the recipient organism or tissue, and whose expression is associated with some easily detectable characteristic, such as a polypeptide manifested by enzymatic activity. Reporter gene expression is tested at an appropriate time after the DNA is introduced into the recipient cell. Suitable reporter genes include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, constructs with the smallest 5' flanking region exhibiting the highest levels of reporter gene expression are identified as promoters. These promoter regions can be ligated to reporter genes and used to evaluate the substance's ability to regulate promoter-driven transcription.

[0580] In some embodiments, the vector is substantially nonpathogenic and / or substantially nonintegrative in host cells, or substantially nonimmunogenic in host cells.

[0581] In some embodiments, the vector is present in an amount sufficient to regulate one or more of the following by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more: phenotype, viral level, gene expression, competition with other viruses, or disease states.

[0582] composition The synthetic clones or vectors described herein may also be incorporated into a pharmaceutical composition together with pharmaceutical excipients, for example, as described herein. In some embodiments, the pharmaceutical 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 It contains 10 synthetic clones. In some embodiments, the pharmaceutical composition is 10 5 ~10 15 , 10 5 ~10 10 , or 10 10 ~10 15 It contains several synthetic clones. In some embodiments, the pharmaceutical composition contains about 10 8 (For example, about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 ) Contains synthetic clones in genome equivalents / mL. In some embodiments, the pharmaceutical composition is determined, for example, according to the method of Example 18, 10 5 ~10 10 , 10 6 ~10 10 , 10 7 ~10 10 , 10 8~10 10 , 10 9 ~10 10 , 10 5 ~10 6 , 10 5 ~10 7 , 10 5 ~10 8 , or 10 5 ~10 9 The synthetic clones contain genome equivalents / mL. In some embodiments, the pharmaceutical composition contains at least 1, 2, 5, or 10, 100, 500, 1000, 2000, 5000, 8,000, or 1 × 10¹⁶ of clones per cell. 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 The synthetic cron contains enough copies of, or more, of the gene elements per cell to deliver to a population of eukaryotic cells. In some embodiments, the pharmaceutical composition contains at least about 1 × 10⁶ per cell in the cron. 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , or approximately 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 It contains enough synthetic clones to deliver copies of the gene element to a population of eukaryotic cells.

[0583] In some embodiments, the pharmaceutical composition has one or more of the following characteristics: the pharmaceutical composition meets pharmaceutical or Good Manufacturing Practices (GMP); the pharmaceutical composition is manufactured in accordance with Good Manufacturing Practices (GMP); the pharmaceutical composition has a pathogen level below a predetermined threshold, e.g., substantially free of pathogens; the pharmaceutical composition has a contaminant level below a predetermined threshold, e.g., substantially free of contaminants; or the pharmaceutical composition is low immunogenic, or substantially non-immunogenic, e.g., as described herein.

[0584] In some embodiments, the pharmaceutical composition contains one or more types of contaminants in amounts below a threshold. Examples of contaminants that are desirable to be excluded or minimized in the pharmaceutical composition include, but are not limited to, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived components (e.g., serum albumin or trypsin), replicable viruses, non-infectious particles, free viral capsid proteins, exogenous contaminants, and aggregates. In some embodiments, the contaminant is host cell DNA. In some embodiments, the composition contains less than about 500 ng of host cell DNA per dose. In some embodiments, the pharmaceutical composition consists of less than 10% by weight of a contaminant (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%).

[0585] In one aspect, the present invention as described herein is as follows: a) (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence for binding a gene element to the non-pathogenic external protein, and (iii) a sequence encoding a regulatory nucleic acid; and Synthetic clones comprising gene elements, for example, gene elements comprising a proteinaceous outer layer that encloses or confines them, and b) Excipients for pharmaceutical formulations Includes a pharmaceutical composition containing the following:

[0586] Small vesicles In some embodiments, the composition further comprises a carrier structure, such as microparticles, liposomes, vesicles, or exosomes. In some embodiments, the liposome comprises a spherical vesicle structure consisting of a single or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. The liposome may be anionic, neutral, or cationic. Liposomes are generally biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their cargo across biological membranes (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679).

[0587] Vesicles can be produced from several different lipids; however, phospholipids are most commonly used for the preparation of liposomes as drug carriers. Examples of vesicles, but not limited to, include DOTMA, DOTAP, DOTIM, DDAB (alone or with cholesterol to obtain DOTMA), as well as cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparing multi-membrane vesicle lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, the teachings relating to the preparation of multi-membrane vesicle lipids are incorporated herein by reference). Vesicular formation can occur spontaneously when lipid membranes are mixed with aqueous solutions, but it can also be promoted by applying force in the form of shaking using a homogenizer, ultrasonic generator, or extruder (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679). Extruded lipids can be produced by extrusion through a gradually decreasing size filter, as described in Templeton et al., Nature Biotech, 15:647-652, 1997 (the teachings on the preparation of extruded lipids are incorporated herein by reference).

[0588] As described herein, additives may be added to the vesicles to modify their structure and / or properties. For example, cholesterol or sphingomyelin may be added to the mixture to help stabilize the structure and prevent leakage of the internal cargo. Furthermore, the vesicles may also be prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679). In addition, the vesicles may be surface-modified during or after synthesis to contain reactive groups complementary to the reactive groups on the recipient cell. Such reactive groups include, but are not limited to, maleimide groups. For example, the vesicles may be synthesized to contain, but are not limited to, maleimide-conjugated phospholipids such as DSPE-MaL-PEG2000.

[0589] Vesicular preparations can be composed primarily of natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialogangliosides. Preparations composed solely of phospholipids have low stability in plasma. However, the rapid release of the encapsulated cargo can be suppressed by manipulating the lipid membrane with cholesterol, or its stability can be enhanced with 1,2-dioleioyl-sn-glycero-3-phosphoethanolamine (DOPE) (for example, see Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679).

[0590] In several embodiments, lipids can be used to form lipid microparticles. Examples of lipids, but not limited to, include DLin-KC2-DMA4, C12-200, and colipidodisteroylphosphatidylcholine, cholesterol, and PEG-DMG, which can be formulated using a spontaneous vesicle formation procedure (see, for example, Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi: 10.1038 / mtna. 2011.3). The molar ratio of the constituents may be approximately 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / disteroylphosphatidylcholine / cholesterol / PEG-DMG). Tekmira has a portfolio of approximately 95 patent families in the United States and overseas, which relate to various forms of lipid microparticles and lipid microparticle formulations (for example, U.S. Patent Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,80 See Patent No. 3,397; No. 8,101,741; No. 8,188,263; No. 7,915,399; No. 8,236,943 and No. 7,838,658, as well as European Patent No. 1766035; No. 1519714; No. 1781593 and No. 1664316), all of which can be used and / or applied to the present invention.

[0591] In some embodiments, the microparticles comprise one or more solid polymers arranged in a random manner. The microparticles may be biodegradable. Biodegradable microparticles can be synthesized using methods known in the art, including, but not limited to, solvent evaporation, hot-melt microencapsulation, solvent removal, and spray drying. Exemplary methods for synthesizing microcapsules are described in Bershteyn et al., Soft Matter 4:1787-1787, 2008 and U.S. Patent Application Publication No. 2008 / 0014144 A1, and specific teachings relating to microcapsule synthesis are incorporated herein by reference.

[0592] Examples of synthetic polymers that can be used to form biodegradable microparticles include, but are not limited to, aliphatic polyesters, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL), polyanhydride, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactidoco-caprolactone), as well as natural polymers such as albumin, alginates, and other polysaccharides such as dextran and cellulose, collagen (including chemical derivatives thereof, such as substitution, addition, hydroxylation, oxidation of chemical groups such as alkyl and alkylene, and other modifications commonly carried out by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. Generally, these materials degrade by enzymatic hydrolysis or exposure to water, surface or bulk erosion.

[0593] The diameter of the microparticles is in the range of 0.1 to 1000 micrometers (μm). In some embodiments, their diameters are in the range of 1 to 750 μm, or 50 to 500 μm, or 100 to 250 μm. In some embodiments, their diameters are in the range of 50 to 1000 μm, 50 to 750 μm, 50 to 500 μm, or 50 to 250 μm. In some embodiments, their diameters are in the range of 0.05 to 1000 μm, 10 to 1000 μm, 100 to 1000 μm, or 500 to 1000 μm. In some embodiments, their diameters are approximately 0.5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1000 μm. When used in reference to microparticles, the term "approximately" means + / - 5% of the stated absolute value.

[0594] In some embodiments, ligands are conjugated to the surface of microparticles via functional chemical groups (carboxylic acids, aldehydes, amines, sulfhydryls, and hydroxyls) present on the surface of the particles and on the ligand to be bound. For example, functional groups can be introduced into microparticles by incorporating stabilizers together with the functional chemical groups during the emulsion preparation process of the microparticles.

[0595] Another example of introducing functional groups to microparticles is by directly crosslinking the particles and ligands using homo- or hetero-functional crosslinking agents during a post-production process. This procedure may use suitable chemistry and Class 1 crosslinking agents (such as CDI, EDAC, glutaraldehyde, as described in more detail below) or any other crosslinking agents that attach ligands to the particle surface by chemical modification of the particles after production. This also includes a process in which amphiphilic molecules such as fatty acids, lipids, or functional stabilizers are passively adsorbed and attached to the particle surface, thereby introducing functional end groups for ligand anchoring.

[0596] In some embodiments, microparticles may be synthesized to include one or more targeting groups on their outer surface in order to target a specific cell or tissue type (e.g., cardiomyocytes). These targeting groups may include, but are not limited to, receptors, ligands, and antibodies. These targeting groups bind to their partners on the cell surface. In some embodiments, microparticles are incorporated into a lipid bilayer including the cell surface to deliver mitochondria to the cell.

[0597] Microparticles may also contain a lipid bilayer on their outermost surface. This bilayer may consist of one or more lipids of the same or different types. Examples include, but are not limited to, phospholipids such as phosphocholine and phosphoinositol. Specific examples include, but are not limited to, various other lipids such as DMPC, DOPC, DSPC, and those described herein for use with liposomes.

[0598] In some embodiments, the carrier includes, for example, nanoparticles as described herein.

[0599] In some embodiments, the vesicles or microparticles described herein are functionalized with a diagnostic agent. Examples of diagnostic agents include, but are not limited to, commercially available imaging agents used in positron emission tomography (PET), computed tomography (CAT), single-photon emission computed tomography, X-ray, fluorescence analysis, and magnetic resonance imaging (MRI); as well as contrast agents. Examples of materials suitable for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium.

[0600] Transmembrane polypeptide In some embodiments, the composition further comprises transmembrane polypeptides (MPPs) for transporting the constituents into cells or across membranes, such as the cellular or nuclear membrane. Transmembrane polypeptides that can facilitate the transport of substances across membranes include, but are not limited to, transcellular peptides (CPPs) (see, e.g., U.S. Patent No. 8,603,966), fusion peptides for intracellular delivery in plants (see, e.g., Ng et al., PLoS One, 2016, 11:e0154081), protein transduction domains, Trojan peptides, and membrane transposition signals (MTS) (see, e.g., Tung et al., Advanced Drug Delivery Reviews 55:281-294 (2003)). Some MPPs are rich in amino acids such as arginine and have positively charged side chains.

[0601] Transmembrane polypeptides have the ability to induce transmembrane transmembrane movement of their constituents, enabling macromolecular translocation within cells of multiple tissues in vivo when administered systemically. Transmembrane polypeptides, sometimes also called peptides, pass through cells in significantly higher quantities than would be achieved by passive diffusion when brought into contact with cells under appropriate conditions, from the external environment to the cytoplasm, organelles such as mitochondria, or the intracellular environment, including the cell nucleus.

[0602] The components transported across the membrane may be reversibly or irreversibly linked to the transmembrane polypeptide. The linker may be a chemical bond, such as one or more covalent or non-covalent bonds. In some embodiments, the linker is a peptide linker. Such linkers may be 2 to 30 amino acids long or longer. Examples of linkers include flexible, rigid, or cleavable linkers.

[0603] combination In one embodiment, the synthetic clone or composition containing the synthetic clone described herein may further contain one or more heterologous parts. In one embodiment, the synthetic clone or composition containing the synthetic clone described herein may also contain one or more heterologous parts in the fusion. In some embodiments, the heterologous parts may be linked to a gene element. In some embodiments, the heterologous parts may be encapsulated within a protein outer layer as part of the clone. In some embodiments, the heterologous parts may be administered together with the synthetic clone.

[0604] In one embodiment, the present invention comprises a cell or tissue containing one of the synthetic clones and a heterogeneous part described herein.

[0605] In another embodiment, the present invention includes a pharmaceutical composition comprising a synthetic clon and a heterogeneous portion described herein.

[0606] In some embodiments, the heterologous portion may be a virus (e.g., an effector (e.g., a drug, a small molecule), a targeting agent (e.g., a DNA targeting agent, an antibody, a receptor ligand), a tag (e.g., a fluorophore, a photosensitive agent such as KillerRed), or any of the editing or targeting portions described herein. In some embodiments, the membrane transposition polypeptide described in the present invention is linked to one or more heterologous portions. In one embodiment, the heterologous portion may be a small molecule (e.g., a peptide mime or an organic small molecule with a molecular weight of less than 2000 daltons), a peptide or polypeptide (e.g., an antibody or its antigen-binding fragment), a nanoparticle, an aptamer, or a drug (pharmacoagent).

[0607] virus In some embodiments, the composition may further include viruses as heterogeneous parts, such as single-stranded DNA viruses, such as anellovirus, bidnavirus, circovirus, geminivirus, genomovirus, inovirus, microvirus, nanovirus, parvovirus, and spiravirus. In some embodiments, the composition may further include double-stranded DNA viruses, such as adenovirus, ampullavirus, ascovirus, asfarvirus, baculovirus, fusellovirus, globulovirus, guttavirus, hytrosavirus, herpesvirus, iridovirus, lipospirixvirus, nimavirus, and poxvirus. In some embodiments, the composition may further include RNA viruses, such as alphavirus, furovirus, hepatitis virus, hordeivirus, tobamovirus, tobravirus, tricornavirus, rubivirus, birnavirus, cystovirus, partitivirus, and reovirus. In some embodiments, Chron is administered together with the virus as a heterologous portion.

[0608] In some embodiments, the heterogeneous portion may include nonpathogenic viruses, such as symbiotic viruses, commensal viruses, or native viruses. In some embodiments, the nonpathogenic virus is one or more species of anellovirus, such as the genera Alphatorquevirus (TT), Betatorquevirus (TTM), and Gammatorquevirus (TTMD). In some embodiments, the anellovirus may include Torque Teno Virus (TT), SEN virus, Sentinel virus, TTV-like minivirus, TT virus, TT virus genotype 6, TT virus group, TTV-like virus DXL1, TTV-like virus DXL2, Torque Teno-like Mini Virus (TTM), or Torque Teno-like Midi Virus (TTMD). In some embodiments, the nonpathogenic virus comprises one or more sequences having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity with respect to any one of the nucleotide sequences listed in Table 19 or Table 20, for example.

[0609] In some embodiments, the heterogeneous portion may include one or more viruses identified as being deleted in the subject. For example, a subject identified as having dyvirosis may be administered a composition containing clonn and one or more viral compositions or viruses that are disproportionate in the subject or have a different ratio than the baseline, e.g., in a healthy subject.

[0610] In some embodiments, the heterogeneous portion may include one or more non-aneroviruses, such as adenovirus, herpesvirus, poxvirus, vaccinia virus, SV40, papillomavirus, retrovirus, RNA viruses such as lentivirus, single-stranded RNA viruses such as hepatitis virus, or double-stranded DNA viruses such as rotavirus. In some embodiments, the cron or virus is deficient or requires assistance to produce infectious particles. Such assistance can be provided, for example, by using helper cells containing nucleic acids, such as plasmids or DNA integrated into the genome, that encode one or more (e.g., all) of the structural genes of the replication-deficient cron or virus under the control of a regulatory sequence within the LTR. Suitable cell lines for replicating the clones described herein include cell lines known in the art, such as A549 cells, which can be modified as described herein.

[0611] effector In some embodiments, the composition or synthetic chlorine may further include effectors having effector activity. Effectors can increase or decrease biological activity, such as enzyme activity, gene expression, cell signaling, and cell or organ function. Effector activity may also include binding regulatory proteins to modulate the activity of regulatory factors such as transcription or translation. Effector activity may further include activator or inhibitory functions. For example, an effector can induce enzyme activity by triggering increased substrate affinity of an enzyme; for example, fructose 2,6-bisphosphate activates phosphofructokinase 1 to increase the rate of glycolysis in response to insulin. In another example, an effector can inhibit the activation of a receptor by inhibiting substrate binding to it; for example, naltrexone and naloxone bind to opioid receptors without activating them, thereby blocking the ability of these receptors to bind opioids. Effector activity may also include the regulation of protein stability / degradation and / or transcript stability / degradation. For example, proteins can be targeted for degradation by polypeptide cofactors, such as ubiquitin, which mark them on the protein for the purpose of degradation. In another example, effectors inhibit enzyme activity by blocking the active site of the enzyme; for example, methotrexate is a structural analog of tetrahydrofolate, i.e., a coenzyme of dihydrofolate reductase, an enzyme that inhibits nucleotide base synthesis by binding to dihydrofolate reductase more than 1000 times more strongly than its native substrate.

[0612] Targeting section In some embodiments, the compositions or synthetic clones described herein may further include a targeting moiety, for example, a targeting moiety that specifically binds to a target molecule present on a target cell. The targeting moiety can modulate a specific function of the target molecule or cell, modulate a specific molecule (e.g., an enzyme, protein, or nucleic acid), for example, a specific molecule downstream of the target molecule in a pathway, or specifically bind to a target to localize the clone or gene element. For example, the targeting moiety may include a therapeutic agent that interacts with a specific molecule of interest to increase, decrease, or otherwise modulate its function.

[0613] Tagging or monitoring section In some embodiments, the compositions or synthetic clones described herein may further include tags for labeling or monitoring the compositions or synthetic clones described herein. The tagging or monitoring portion may be removable by chemical or enzymatic cleavage, e.g., proteolysis or intent splicing. Affinity tags may be useful for purifying tagged polypeptides using affinity techniques. Some examples include chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), and poly(His) tags. Solubilization tags may be useful in helping recombinant proteins expressed in chaperone-deficient species such as Escherichia coli (E. coli) to assist in proper protein folding and prevent precipitation. Some examples include thioredoxin (TRX) and poly(NANP). The tagging or monitoring portion may include photosensitive tags, e.g., fluorescent tags. Fluorescent tags are useful for visualization. GFP and its variants are some examples of commonly used fluorescent tags. Protein tags can undergo specific enzymatic modifications (such as biotinylation by biotin ligases) or chemical modifications (such as reaction with FlAsH-EDT2 for fluorescence imaging). The tagging or monitoring portion is often attached to link the protein to multiple other components. The tagging or monitoring portion can also be removed by specific proteolytic or enzymatic cleavage (e.g., by TEV proteases, thrombin, factor Xa, or enteropeptidases).

[0614] nanoparticles In some embodiments, the compositions or synthetic chlorofluorocarbons described herein may further include nanoparticles. Examples of nanoparticles include inorganic materials having sizes of about 1 to about 1000 nanometers, about 1 to about 500 nanometers, about 1 to about 100 nm, about 50 to about 300 nm, about 75 to about 200 nm, and about 100 to about 200 nm, as well as any range in between. Nanoparticles generally have composite structures with dimensions in the nanoscale. In some embodiments, nanoparticles are typically spherical, but various forms are possible depending on the nanoparticle composition. The portion of the nanoparticle that comes into contact with the external environment is generally identified as the surface of the nanoparticle. In the case of the nanoparticles described herein, the size limit can be restricted to two dimensions, so that the nanoparticles include composite structures having a diameter of about 1 to about 1000 nm, where the specific diameter varies depending on the nanoparticle composition and the intended use of the nanoparticles according to the experimental design. For example, nanoparticles used in therapeutic applications typically have a size of about 200 nm or less.

[0615] Additional desirable properties of nanoparticles, such as surface charge and steric stabilization, may also vary depending on the specific intended application. Exemplary properties considered desirable for clinical applications are described in Davis et al, Nature 2008 vol.7, pp. 771-782; Duncan, Nature 2006 vol.6, pp. 688-701; and Allen, Nature 2002 vol.2, pp. 750-763, each of which is incorporated herein by reference in whole. Additional properties can be confirmed by reading this disclosure by those skilled in the art. Exemplary techniques for detecting the dimensions and properties of nanoparticles include, but are not limited to, dynamic light scattering (DLS) and various microscopy methods such as transmission electron microscopy (TEM) and atomic force microscopy (AFM). Exemplary techniques for detecting particle morphology include, but are not limited to, TEM and AFM. Exemplary techniques for detecting the surface charge of nanoparticles include, but are not limited to, zeta potential spectroscopy. Other techniques for detecting other chemical properties include, 1 H, 11 B, and13 C and 19 This includes methods using FNMR, UV / Vis and infrared / Raman spectroscopy, and fluorescence spectroscopy (when nanoparticles are used in combination with fluorescent labels), as well as other techniques that can be verified by those skilled in the art.

[0616] small molecule In some embodiments, the compositions or synthetic chlorines described herein may further include small molecules. Small molecules include, but are not limited to, low molecular weight peptides, peptide mimes (e.g., peptoids), amino acids, amino acid analogs, synthetic polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heteroorganic and organometallic compounds), for example, organic or inorganic compounds with a molecular weight of less than about 2,000 grams per mole, for example, organic or inorganic compounds with a molecular weight of less than about 1,000 grams per mole, for example, organic or inorganic compounds with a molecular weight of less than about 500 grams per mole, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. Small molecules may also include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viruses or microbial particles, synthetic molecules, and agonists or antagonists.

[0617] Examples of suitable molecules are listed below: “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autoacoids; Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Examples of small molecules include those described in Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology (all incorporated herein by reference). Some examples of small molecules, but not limited to, include prion drugs such as tacrolimus, HECT ligase inhibitors such as ubiquitin ligase or heclin, histone modifiers such as sodium butyrate, enzyme inhibitors such as 5-azacytidine, anthracyclines such as doxorubicin, β-lactams such as penicillin, antibacterial agents, chemotherapeutic agents, antiviral agents, modulators from other organisms such as VP64, and chemotherapeutic agents with pharmacokinetic deficiencies, as well as drugs with insufficient bioavailability.

[0618] In some embodiments, the small molecule is an epigenetic modifier, such as those described in de Groote et al. Nuc. Acids Res. (2012):1-18. Exemplary small molecule epigenetic modifiers are described in Lu et al. J. Biomolecular Screening 17.5 (2012):555-71, e.g., Table 1 or 2, which are incorporated herein by reference. In some embodiments, the epigenetic modifier includes vorinostat or romidepsin. In some embodiments, the epigenetic modifier includes an inhibitor of class I, II, III, and / or IV histone deacetylase (HDAC). In some embodiments, the epigenetic modifier includes an activator of SirTI. In some embodiments, the epigenetic modifiers are garcinol, Lys-CoA, C646, (+)-JQI, I-BET, BICI, MS120, DZNep, UNC0321, EPZ004777, AZ505, AMI-I, pyrazoleamide 7b, benzo[d]imidazole 17b, acylated dapsone derivatives (e.g., PRMTI), methylstat, 4,4'-dicarboxy-2,2'-bipyridine, SID8573633 1. Hydroxamate analogs 8, tranylcypromine (tanylcypromie), bisguanidine and biguanide polyamine analogs, UNC669, vidaza, decitabine, sodium phenylbutyrate (SDB), lipoic acid (LA), quercetin, valproic acid, hydrazine, bactrim, green tea extract (e.g., epigallocatechin gallate (EGCG)), curcumin, sulforphan and / or allicin / diallyl disulfide. In some embodiments, the epigenetic modifier inhibits DNA methylation, for example, an inhibitor of DNA methyltransferase (e.g., 5-azacitidine and / or decitabine). In some embodiments, the epigenetic modifier modifies histone modifications, such as histone acetylation, histone methylation, histone smoylation and / or histone phosphorylation. In some embodiments, the epigenetic modifier is a histone deacetylase inhibitor (e.g., vorinostat and / or trichostatin A).

[0619] In some embodiments, the small molecule is a pharmaceutically effective agent. In one embodiment, the small molecule is an inhibitor of metabolic activity or a component. Useful classes of pharmaceutically effective agents include, but are not limited to, antibiotics, anti-inflammatory drugs, angiogenic or vasoactive agents, growth factors, and chemotherapeutic (antitum) agents (e.g., tumor suppressants). One or a combination of molecules from the categories and examples described herein, or from (Orme-Johnson 2007, Methods Cell Biol. 2007;80:813-26), may be used. In one embodiment, the present invention includes a composition comprising an antibiotic, an anti-inflammatory drug, an angiogenic or vasoactive agent, a growth factor, or a chemotherapeutic agent.

[0620] Peptides or proteins In some embodiments, the compositions or synthetic clones described herein may further comprise peptides or proteins. Examples of peptide portions include, but are not limited to, peptide ligands or antibody fragments (e.g., antibody fragments that bind to receptors such as extracellular receptors), neuropeptides, hormone peptides, peptide drugs, toxic peptides, viral or microbial peptides, synthetic peptides, and agonist or antagonist peptides.

[0621] The peptide portion may be linear or branched. The peptide may have a length of approximately 5 to 200 amino acids, approximately 15 to 150 amino acids, approximately 20 to 125 amino acids, approximately 25 to 100 amino acids, or any range in between.

[0622] Some examples of peptides, but not limited to, include fluorescent tags or markers, antigens, antibody fragments such as single-domain antibodies, ligands and receptors such as glucagon-like peptide-1 (GLP-1), GLP-2 receptor 2, cholecystokinin B (CCKB), and somatostatin receptors, peptide therapeutics such as those that bind to specific cell surface receptors such as G protein-binding receptors (GPCRs) or ion channels, synthesized or analog peptides from naturally occurring bioactive peptides, antimicrobial peptides, pore-forming peptides, tumor-targeting or cytotoxic peptides, and degrading or self-destructive peptides such as apoptosis-inducing peptide signals or photosensitizer peptides.

[0623] The peptides useful for the present invention as described herein also include small molecule-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies and nanobodies (see, for example, Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-113). Such small molecule antigen-binding peptides can bind to cytosolic antigens, nuclear antigens, and intraorganelle antigens.

[0624] In some embodiments, the compositions or clones described herein include polypeptides linked to ligands that can target specific locations, tissues, or cells.

[0625] Oligonucleotide aptamers In some embodiments, the compositions or synthetic clones described herein may further comprise oligonucleotide aptamers. The aptamer portion is an oligonucleotide or a peptide aptamer. Oligonucleotide aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind with high affinity and specificity to pre-selected targets, including proteins and peptides.

[0626] Oligonucleotide aptamers are nucleic acid species that can be manipulated by iterative round in vitro selection or similarly by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers provide clear molecular recognition and can be produced by chemical synthesis. In addition, aptamers possess desirable conservation properties and will induce little to no immunogenicity in therapeutic applications.

[0627] Both DNA and RNA aptamers can exhibit robust binding affinity to a variety of targets. For example, DNA and RNA aptamers have been selected for t-lysozyme, thrombin, human immunodeficiency virus trans-responsive element (HIV TAR) (see en.wikipedia.org / wiki / Aptamer - cite_note-10), hemin, interferon-gamma, vascular endothelial growth factor (VEGF), prostate-specific antigen (PSA), dopamine, and non-classical oncogenes, including heat shock factor 1 (HSF1).

[0628] Peptide aptamer In some embodiments, the compositions or synthetic clones described herein may further comprise a peptide aptamer. The peptide aptamer comprises a low molecular weight peptide of 12-14 kDa and has one (or more) short variable peptide domains. The peptide aptamer can be designed to specifically bind to and inhibit intracellular protein-protein interactions.

[0629] Peptide aptamers are artificial proteins selected or engineered to bind to specific target molecules. These proteins contain one or more peptide loops of variable sequences. They are typically isolated from combinatorial libraries and then often impro...

Claims

[Claim 1] The composition and method described in the specification.

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