Anellosomes and methods of use

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises the Anellovirus 5′ UTR conserved domain nucleotide sequence of nucleotides 185-254 of SEQ ID NO:

• 886, or a nucleic acid sequence at least 95% identical thereto.

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule is encoded by nucleotides 512-2545 of SEQ ID NO:

• 878.

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule is encoded by nucleotides 501-2489 of SEQ ID NO:

• 886.

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule comprises an amino acid sequence comprising one or more of the amino acid sequences of an arg-rich region, jelly-roll domain, hypervariable domain, N22 domain, and/or C-terminal domain as listed in Table D2 or D4, or an amino acid sequence having at least 85% identity thereto.

The synthetic anellosome of any of the preceding claims, further comprising a polypeptide comprising the amino acid sequence of an ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 as listed in Table C1 or C2, or an amino acid sequence having at least 85% identity thereto.

The synthetic anellosome of any of the preceding claims, wherein the genetic element encodes the amino acid sequence of an ORF1, ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 as listed in Table C1 or C2, or an amino acid sequence having at least 85% identity thereto.

The synthetic anellosome of any of the preceding claims, wherein the synthetic anellosome does not comprise a polypeptide comprising the amino acid sequence of an ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 as listed in Table C1 or C2, or an amino acid sequence having at least 85% identity thereto.

The synthetic anellosome of any of the preceding claims, wherein the genetic element does not encode the amino acid sequence of an ORF1, ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 as listed in Table C1 or C2, or an amino acid sequence having at least 85% identity thereto.

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule comprises the amino acid sequence YNPX2DXGX2N, wherein Xn is each independently a contiguous sequence of any n amino acids.

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule comprises, in order in the N-terminal to C-terminal direction, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and a ninth beta strand.

The synthetic anellosome of any of the preceding claims, wherein the genetic element is capable of being amplified by rolling circle replication in a host cell, e.g., to produce at least 8 copies.

The synthetic anellosome of any of the preceding claims, wherein the genetic element is single-stranded.

The synthetic anellosome of any of the preceding claims, wherein the genetic element is circular.

The synthetic anellosome of any of the preceding claims, wherein the genetic element is DNA.

The synthetic anellosome of any of the preceding claims, wherein the genetic element is a negative strand DNA.

The synthetic anellosome of any of the preceding claims, wherein the genetic element integrates at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of the anellosomes that enters the cell, e.g., wherein the synthetic anellosome is non-integrating.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises a sequence of the Consensus 5′ UTR nucleic acid sequence shown in Table 16-1.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises a sequence of the Consensus GC-rich region shown in Table 16-2.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises a sequence of at least 100 nucleotides in length, which consists of G or C at at least 70% (e.g., about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%) of the positions.

The synthetic anellosome of any of the preceding dims, wherein the genetic element comprises the nucleic acid sequence of SEQ ID NO:

• 120.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises a sequence having at least 85% sequence identity to the Anellovirus 5′ UTR conserved domain nucleotide sequence of nucleotides 1-393 of the nucleic acid sequence of Table 11 and a sequence having at least 85% sequence identity to the Anellovirus GC-rich region of nucleotides 2868-2929 of the nucleic acid sequence of Table 11.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises at least 75% identity to the nucleotide sequence of Table 11.

The synthetic anellosome of any of the preceding claims, wherein the promoter element is exogenous to wild-type Anellovirus.

The synthetic anellosome of any of the preceding claims, wherein the promoter element is endogenous to wild-type Anellovirus.

The synthetic anellosome of any of the preceding claims, wherein the exogenous effector encodes a therapeutic agent, e.g., a therapeutic peptide or polypeptide or a therapeutic nucleic acid.

The synthetic anellosome of any of the preceding claims, wherein the exogenous effector comprises a regulatory nucleic acid, e.g., an miRNA, siRNA, mRNA, lncRNA, RNA, DNA, an antisense RNA, gRNA;

• a fluorescent tag or marker, an antigen, a peptide, a synthetic or analog peptide from a naturally-bioactive peptide, an agonist or antagonist peptide, an anti-microbial peptide, a pore-forming peptide, a bicyclic peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, a small molecule, an immune effector (e.g., influences susceptibility to an immune response/signal), a death protein (e.g., an inducer of apoptosis or necrosis), a non-lytic inhibitor of a tumor (e.g., an inhibitor of an oncoprotein), an epigenetic modifying agent, an epigenetic enzyme, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand, an antibody, a receptor, or a CRISPR system or component.

The synthetic anellosome of any of the preceding claims, wherein the exogenous effector comprises an miRNA, and decreases expression of a host gene.

The synthetic anellosome of any of the preceding claims, wherein the exogenous effector comprises a nucleic acid sequence about 20-200, 30-180, 40-160, 50-140, 60-120, 200-2000, 200-500, 500-1000, 1000-1500, or 1500-2000 nucleotides in length.

The synthetic anellosome of any of the preceding claims, wherein the nucleic acid sequence encoding the exogenous effector is about 20-200, 30-180, 40-160, 50-140, 60-120, 200-2000, 200-500, 500-1000, 1000-1500, or 1500-2000 nucleotides in length.

The synthetic anellosome of any of the preceding claims, which comprises (e.g., in the proteinaceous exterior) one or more of an amino acid sequence chosen from ORF2, ORF2/2, ORF2/3, ORF1, ORF1/1, or ORF1/2 as listed in any of Tables C1-C5, or an amino acid sequence having at least 85% sequence identity thereto.

The synthetic anellosome of any of the preceding claims, wherein the genetic element has a length of about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0 kb.

The synthetic anellosome of any of the preceding claims, wherein the synthetic anellosome is capable of infecting human cells, e.g., immune cells, liver cells, or lung epithelial cells.

The synthetic anellosome of any of the preceding claims, which is substantially non-immunogenic, e.g., does not induce a detectable and/or unwanted immune response, e.g., as detected according to the method described in Example 4.

The synthetic anellosome of any of the preceding claims, wherein a population of at least 1000 of the anellosomes is capable of delivering at least about 100 copies (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies) of the genetic element into one or more human cells.

The synthetic anellosome of any of the preceding claims, which comprises (e.g., in the proteinaceous exterior) one or more of an amino acid sequence chosen from ORF2, ORF2/2, ORF2/3, ORF2t/3, ORF1, ORF1/1, or ORF1/2 as listed in any of Tables C1-C5, or an amino acid sequence having at least 85% sequence identity thereto.

A pharmaceutical composition comprising the synthetic anellosome of any of the preceding claims, and a pharmaceutically acceptable carrier or excipient.

A reaction mixture comprising:

• (i) a first nucleic acid (e.g., a double-stranded or single-stranded circular DNA) comprising the sequence of the genetic element of the synthetic anellosome of any of the preceding claims, and
• (ii) a second nucleic acid sequence encoding one or more of an amino acid sequence chosen from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2, e.g., as listed in any of Tables C1-C5, or an amino acid sequence having at least 85% sequence identity thereto.

A host cell comprising:

• (i) a first nucleic acid molecule comprising the nucleic acid sequence of a genetic element of a synthetic anellosome of any of the preceding claims, and
• (ii) optionally, a second nucleic acid molecule encoding one or more of an amino acid sequence chosen from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as listed in any of Tables C1-C5, or an amino acid sequence having at least 85% sequence identity thereto.