Anellovectors and methods of use

An anellovector comprising:

• (i) a proteinaceous exterior comprising an Anellovirus ORF1 protein comprising an amino acid sequence of SEQ ID NO:
• 2466, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF 1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid sequence according to nucleotides 338-2305 of SEQ ID NO:
• 2465, or a polypeptide encoded by a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Anellovirus ORF1 nucleic acid sequence, and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises:
• (a) a 5′ UTR conserved domain according to nucleotides 1-64 of SEQ ID NO:
• 2465, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector.

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%,
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises:
• (a) a 5′ UTR conserved domain according to nucleotides 1-64 of SEQ ID NO:
• 2465, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and wherein the genetic element has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus genome sequence of SEQ ID NO:
• 2465;
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

A method of manufacturing an anellovector composition, the method comprising:

• (a) providing a cell, e.g., a host cell as described herein;
• (b) introducing a nucleic acid molecule encoding an ORF1 polypeptide comprising a sequence of SEQ ID NO:
• 2466 or ORF2 polypeptide comprising a sequence of SEQ ID NO:
• 2467 (or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto) into the cell;
• (c) introducing a genetic element construct into the cell (e.g., before, after, or simultaneously with (b)),
• (d) incubating the cell under conditions that allow the cell to produce anellovector;
• and
• (e) formulating the anellovectors, e.g., as a pharmaceutical composition suitable for administration to a subject,
• thereby making the anellovector composition.

A method of manufacturing an anellovector composition, the method comprising:

• (a) providing a cell, e.g., a host cell as described herein;
• (b) introducing a nucleic acid molecule encoding an ORF1 or ORF2 polypeptide into the cell;
• (c) introducing a genetic element construct comprising a 5′ UTR conserved domain according to nucleotides 1-64 of SEQ ID NO:
• 2465 (or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
• or 99% sequence identity thereto) into the cell (e.g., before, after, or simultaneously with (b)),
• (d) incubating the cell under conditions that allow the cell to produce anellovector;
• and
• (e) formulating the anellovectors, e.g., as a pharmaceutical composition suitable for administration to a subject,
• thereby making the anellovector composition.

A method of making an anellovector, e.g., a synthetic anellovector, comprising:

• (a) providing a host cell comprising:
• (i) a nucleic acid molecule, e.g., a first nucleic acid molecule, comprising the nucleic acid sequence of a Anellovirus genome comprising a sequence of SEQ ID NO:
• 2465 (or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more of an amino acid sequence chosen from an ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 encoded by SEQ ID NO:
• 2465, or an amino acid sequence having at least 70% 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto;
• and
• (b) culturing the host cell under conditions suitable to make the anellovector.

An anellovector comprising:

• (i) a proteinaceous exterior comprising an Anellovirus ORF1 protein comprising an amino acid sequence of SEQ ID NO:
• 268, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid sequence according to nucleotides 407-2617 of SEQ ID NO:
• 267, or a polypeptide encoded by a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Anellovirus ORF1 nucleic acid sequence, and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises:
• (a) a 5′ UTR conserved domain according to nucleotides 1-70 of SEQ ID NO:
• 267, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector.

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%,
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises:
• (a) a 5′ UTR conserved domain according to nucleotides 1-70 of SEQ ID NO:
• 267, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

An anellovector comprising:

• (i) a proteinaceous exterior (e.g., comprising an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a genetic element enclosed by the proteinaceous exterior, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and wherein the genetic element has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus genome sequence of SEQ ID NO:
• 267;
• wherein the proteinaceous exterior and/or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus ORF1 protein and/or wild-type Anellovirus genome, respectively (e.g., as described herein), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, jelly-roll domain, HVR, N22, or CTD, e.g., as described herein) or genomic region (e.g., one or more of a TATA box, cap site, transcriptional start site, 5′ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region, e.g., as described herein).

A method of manufacturing an anellovector composition, the method comprising:

• (f) providing a cell, e.g., a host cell as described herein;
• (g) introducing a nucleic acid molecule encoding an ORF1 polypeptide comprising a sequence of SEQ ID NO:
• 268 or ORF2 polypeptide comprising a sequence of SEQ ID NO:
• 269 (or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto) into the cell;
• (h) introducing a genetic element construct into the cell (e.g., before, after, or simultaneously with (b)),
• (i) incubating the cell under conditions that allow the cell to produce anellovector;
• and
• (j) formulating the anellovectors, e.g., as a pharmaceutical composition suitable for administration to a subject,
• thereby making the anellovector composition.

A method of manufacturing an anellovector composition, the method comprising:

• (f) providing a cell, e.g., a host cell as described herein;
• (g) introducing a nucleic acid molecule encoding an ORF1 or ORF2 polypeptide into the cell;
• (h) introducing a genetic element construct comprising a 5′ UTR conserved domain according to nucleotides 1-70 of SEQ ID NO:
• 267 (or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto) into the cell (e.g., before, after, or simultaneously with (b)),
• (i) incubating the cell under conditions that allow the cell to produce anellovector;
• and
• (j) formulating the anellovectors, e.g., as a pharmaceutical composition suitable for administration to a subject,
• thereby making the anellovector composition.

A method of making an anellovector, e.g., a synthetic anellovector, comprising:

• (a) providing a host cell comprising:
• (i) a nucleic acid molecule, e.g., a first nucleic acid molecule, comprising the nucleic acid sequence of a Anellovirus genome comprising a sequence of SEQ ID NO:
• 267 (or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and
• (ii) a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more of an amino acid sequence chosen from an ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 encoded by SEQ ID NO:
• 267, or an amino acid sequence having at least 70% 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto;
• and
• (b) culturing the host cell under conditions suitable to make the anellovector.