Anellosomes for delivering secreted therapeutic modalities

The synthetic anellosome of any of the preceding claims, wherein the ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO:

• 54.

The synthetic anellosome of any of the preceding claims, wherein the genetic element comprises the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID NO:

• 54, or a nucleic acid sequence having at least 85% sequence identity thereto.

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 16, 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 of SEQ ID NO:

• 58, or a nucleic acid sequence having at least 85% sequence 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, ORF1/1, or ORF1/2 as listed in Table 16, 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, ORF1/1, or ORF1/2 as listed in Table 16, 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 16, 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, ORF1/1, or ORF1/2 as listed in Table 16, 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 (SEQ ID NO:

• 829), 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 claims, 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 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 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., blood cells, skin cells, muscle cells, nerve cells, adipose cells, endothelial cells, immune cells, liver cells, lung epithelial cells, e.g., in vitro.

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.

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 Table 16, 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 Table 16, or an amino acid sequence having at least 85% sequence identity thereto.