Viral vectors for modulating VEGF function

Abstract

This invention relates generally to Anelloviridae family vectors (e.g., anellovectors) and compositions and uses thereof.

Description

[0001] VIRAL VECTORS FOR MODULATING VEGF FUNCTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 575,297, filed April 5, 2024, and U.S. Provisional Application No. 63 / 625,607, filed January 26, 2024. The contents of the aforementioned applications are hereby incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 16, 2025, is named V2057-7037WO_SL.xml and is 311,135 bytes in size.

[0006] BACKGROUND

[0007] There is an ongoing need to develop suitable vectors to deliver therapeutic genetic material to patients.

[0008] SUMMARY

[0009] The present disclosure provides an Anelloviridae family vector (e.g., anelloviral vector), e.g., a synthetic Anelloviridae family vector (e.g., anelloviral vector), that can be used as a delivery vehicle, e.g., for delivering genetic material, for delivering an effector, e.g., a payload, or for delivering a therapeutic agent or a therapeutic effector to a eukaryotic cell (e.g., a human cell or a human tissue).

[0010] Additional features of any of the aforesaid Anelloviridae family vectors (e.g., anelloviral vectors), compositions or methods include one or more of the following enumerated embodiments.

[0011] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.

[0012] Enumerated Embodiments

[0013] 1 . A circular, single stranded DNA genetic element comprising, in order: a) an Anello virus non -coding region (NCR); b) a promoter; and c) an exogenous effector region having a DNA sequence according to SEQ ID NO: 316 or having a DNA sequence encoding a polypeptide of SEQ ID NO: 326, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0014] 2. A circular, single stranded DNA genetic element comprising, in order: a) an Anellovirus non-coding region (NCR); b) a promoter; c) a region having a DNA sequence according to the reverse complement of nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and d) an exogenous effector region.

[0015] 3. A circular, single stranded DNA genetic element comprising, in order: a) an Anellovirus non-coding region (NCR); b) a promoter; c) an exogenous effector region; and d) a region having a DNA sequence according to the reverse complement of nucleotides 2538-2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0016] 4. The genetic element of any of the preceding embodiments, wherein the NCR comprises a DNA sequence according to the reverse complement of nucleotides 64-485 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0017] 5. The genetic element of any of the preceding embodiments, wherein the promoter has a DNA sequence according to SEQ ID NO: 312, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0018] 6. The genetic element of any of the preceding embodiments, wherein the exogenous effector region has a DNA sequence according to SEQ ID NO: 316, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 7. The genetic element of any of the preceding embodiments, wherein the exogenous effector region has a DNA sequence encoding a polypeptide of SEQ ID NO: 326, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0019] 8. The genetic element of any of the preceding embodiments, which comprises a CMV enhancer having a DNA sequence according to SEQ ID NO: 310, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0020] 9. The genetic element of any of the preceding embodiments, which comprises a region having a DNA sequence according to the reverse complement of nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0021] 10. The genetic element of any of the preceding embodiments, wherein the exogenous effector region has a DNA sequence according to SEQ ID NO: 316, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0022] 11. The genetic element of any of the preceding embodiments, which comprises a region having a DNA sequence according to the reverse complement of nucleotides 2538-2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0023] 12. The genetic element of any of the preceding embodiments, which comprises a poly(A) signal having a DNA sequence according to SEQ ID NO: 318, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0024] 13. The genetic element of any of the preceding embodiments, which comprises a region having a DNA sequence according to the reverse complement of nucleotides 2690-2705 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0025] 14. The genetic element of any of the preceding embodiments, which comprises a region having a DNA sequence according to SEQ ID NO: 322, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 15. The genetic element of any of the preceding embodiments, which comprises a loxP site having a DNA sequence according to SEQ ID NO: 324, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0026] 16. A circular, single stranded DNA genetic element comprising, in order: a) a Ring 19 Anellovirus non-coding region (NCR) comprising a DNA sequence according to the reverse complement of nucleotides 64-485 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 310, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 312, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; d) a region having a DNA sequence according to the reverse complement of nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 316, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to the reverse complement of nucleotides 2538-2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 318, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to the reverse complement of nucleotides 2690-2705 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and j) a loxP site having a DNA sequence according to SEQ ID NO: 324, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0027] 17. The genetic element of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 327, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 18. A circular, single stranded DNA genetic element comprising a sequence according to SEQ ID NO: 327, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0028] 19. The genetic element of any of the preceding embodiments, which has a length less than 2800, 2850, 2900, 2950, 3000, 3100, 3200, 3300, 3400, or 3500 nucleotides or between 2500-2600, 2600- 2700, 2700-2750, 2750-2800, 2800-2850, 2850-2900, 2900-2950, 2950-3000, 3000-3100, 3100-3200, 3200-3300, 3300-3400, or 3400-3500 nucleotides; e.g., about 2843 nucleotides.

[0029] 20. A nucleic acid molecule comprising, in order: a) a Ring 19 Anellovirus non-coding region (NCR) comprising a DNA sequence according to nucleotides 64-485 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 309, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 311, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; d) a region having a DNA sequence according to nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 315, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to nucleotides 2538-2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 317, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to nucleotides 2690-2705 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 321, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 21. The nucleic acid molecule of embodiment 20, which further comprises, e.g., 3’ relative to i), j) a loxP site having a DNA sequence according to SEQ ID NO: 323, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0030] 22. A nucleic acid molecule comprising, in order: a) a Ring 19 Anellovirus non-coding region (NCR) comprising a DNA sequence according to nucleotides 529-950 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 309, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 311, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; d) a region having a DNA sequence according to nucleotides 1573-1622 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 315, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to nucleotides 3003-3032 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 317, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to nucleotides 3118-3170 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 321, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0031] 23. The nucleic acid molecule of embodiment 22, which further comprises, e.g., 5’ relative to a), a lox71 site having a DNA sequence according to SEQ ID NO: 330, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0032] 24. The nucleic acid molecule of embodiment 22 or 23, which further comprises, e.g., 3’ relative to i), a lox66 site having a DNA sequence according to SEQ ID NO: 361, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 25. A nucleic acid molecule comprising a sequence according to SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0033] 26. A nucleic acid molecule comprising a sequence according to SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0034] 27. The nucleic acid molecule of any of embodiments 20-26, which is a circular, double stranded DNA molecule.

[0035] 28. An anello viral vector comprising:

[0036] (i) a proteinaceous exterior comprising an Anello virus ORF1 protein, and

[0037] (ii) a genetic element of any of the preceding embodiments, wherein the genetic element is enclosed by the proteinaceous exterior.

[0038] 29. The anelloviral vector of embodiment 28, wherein the ORF1 protein has an amino acid sequence as listed in Table Al, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0039] 30. A pharmaceutical composition comprising the anelloviral vector of embodiment 28 or 29, and a pharmaceutically acceptable carrier and / or excipient.

[0040] 31. A method of delivering an exogenous effector to a subject, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, thereby delivering the exogenous effector to the subject.

[0041] 32. The method of embodiment 31, wherein the subject has: Age-related Macular Degeneration (AMD), e.g., wet AMD.

[0042] 33. The method of embodiment 31, wherein the subject has Macular Edema, e.g., Macular Edema following Retinal Vein Occlusion (RVO).

[0043] 34. The method of embodiment 31, wherein the subject has Diabetic Macular Edema (DME). 35. The method of embodiment 31, wherein the subject has Diabetic Retinopathy (DR).

[0044] 36. The method of embodiment 31, wherein the subject has Retinopathy of Prematurity (ROP).

[0045] 37. A method of treating age-related macular degeneration (AMD) (e.g., wet AMD) in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30.

[0046] 38. A method of treating macular edema (e.g., macular edema following RVO) in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30.

[0047] 39. A method of treating diabetic macular edema in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30.

[0048] 40. A method of treating diabetic retinopathy in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30.

[0049] 41. A method of treating retinopathy of prematurity in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30.

[0050] 42. The method of any of embodiments 31-41, wherein the administration comprises administration to an eye of the subject, e.g., to a photoreceptor, retina, posterior eye cup (PEC), retinal ganglion, optic nerve, optic nerve head, retinal pigmented epithelium (RPE), intravitreal space, or subretinal space of the subject).

[0051] 43. The method of any of embodiments 31-41, wherein the administration comprises administration to the posterior eye cup (PEC). 44. The method of any of embodiments 31-41, wherein the administration comprises administration to the retina.

[0052] 45. The method of any of embodiments 31-44, which results in detectable levels of the genetic element, the exogenous effector region, and / or an mRNA comprising the nucleic acid sequence of the exogenous effector region in an eye of the subject, e.g., to a photoreceptor, retina, posterior eye cup (PEC), retinal ganglion, optic nerve, optic nerve head, retinal pigmented epithelium (RPE), intravitreal space, or subretinal space of the subject).

[0053] 46. The method of any of embodiments 31-45, which results in detectable levels of the genetic element, the exogenous effector region, and / or an mRNA comprising the nucleic acid sequence of the exogenous effector region in the posterior eye cup (PEC).

[0054] 47. The method of any of embodiments 31-46, which results in detectable levels of the genetic element, the exogenous effector region, and / or an mRNA comprising the nucleic acid sequence of the exogenous effector region in the retina.

[0055] 48. The method of any of embodiments 31-47, wherein the Anelloviridae family vector is administered to the subject subretinally, intravitreally, or suprachoroidally.

[0056] 49. The method of any of embodiments 31-48, wherein the Anelloviridae family vector is administered to the subject subretinally.

[0057] 50. The method of any of embodiments 31-48, wherein the Anelloviridae family vector is administered to the subject intravitreally.

[0058] 51. The method of any of embodiments 31-48, wherein the Anelloviridae family vector is administered to the subject suprachoroidally.

[0059] 52. The method of any of embodiments 31-51, further comprising administering a second anelloviral vector of embodiment 28 or 29 or pharmaceutical composition of embodiment 30 to the subject. 53. A method of delivering a genetic element to a subject, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, thereby delivering the genetic element to the subject.

[0060] 54. A method of delivering an mRNA to a subject, the method comprising administering to the subject an anelloviral vector of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element, thereby delivering the mRNA to the subject.

[0061] 55. A method of delivering an exogenous effector to a subject, the method comprising:

[0062] (i) administering to the subject a first plurality of anelloviral vectors of embodiment 28 or 29 or pharmaceutical composition of embodiment 30; and

[0063] (ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the exogenous effector to the subject.

[0064] 56. A method of delivering a genetic element to a subject, the method comprising:

[0065] (i) administering to the subject a first plurality of anelloviral vectors of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30; and

[0066] (ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the genetic element to the subject.

[0067] 57. A method of delivering an mRNA to a subject, the method comprising:

[0068] (i) administering to the subject a first plurality of anelloviral vectors of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element; and

[0069] (ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the mRNA to the subject.

[0070] 58. The method of any of embodiments 55-57, wherein the second plurality comprises anelloviral vectors of embodiment 28 or 29.

[0071] 59. The method of any of embodiments 55-57, wherein the second plurality comprises anelloviral vectors as described herein.

[0072] 60. A method of delivering an exogenous effector to a subject, the method comprising:

[0073] (i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and

[0074] (ii) administering to the subject a second plurality of anelloviral vectors of embodiment 28 or 29 or pharmaceutical composition of embodiment 30, thereby delivering the exogenous effector to the subject.

[0075] 61. A method of delivering a genetic element to a subject, the method comprising:

[0076] (i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and

[0077] (ii) administering to the subject a second plurality of anelloviral vectors of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, thereby delivering the genetic element to the subject.

[0078] 62. A method of delivering an mRNA to a subject, the method comprising:

[0079] (i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and

[0080] (ii) administering to the subject a second plurality of anelloviral vectors of embodiment 28 or 29 or a pharmaceutical composition of embodiment 30, under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element, thereby delivering the mRNA to the subject. 63. The method of any of embodiments 60-62, wherein the first plurality comprises anelloviral vectors of embodiment 28 or 29.

[0081] 64. The method of any of embodiments 60-62, wherein the first plurality comprises anelloviral vectors as described herein.

[0082] 65. The method of any of embodiments 55-64, wherein the anelloviral vectors of the first and second pluralities are the same anelloviral vector.

[0083] 66. The method of any of embodiments 55-64, wherein the anelloviral vectors of the first plurality are different from the anelloviral vectors of the second plurality.

[0084] 67. The method of any of embodiments 55-66, wherein the fust plurality is administered subretinally, e.g., as described in Example 3.

[0085] 68. The method of any of embodiments 55-66, wherein the first plurality is administered intravitreally, e.g., as described in Example 3.

[0086] 69. The method of any of embodiments 55-68, wherein the second plurality is administered subretinally, e.g., as described in Example 3.

[0087] 70. The method of any of embodiments 55-68, wherein the second plurality is administered intravitreally, e.g., as described in Example 3.

[0088] 71. The method of any of embodiments 55-66, wherein the first plurality is administered intravitreally, and the second plurality is administered intravitreally.

[0089] 72. The method of any of embodiments 55-66, wherein the fust plurality is administered subretinally, and the second plurality is administered intravitreally.

[0090] 73. The method of any of embodiments 55-66, wherein the first plurality is administered inUavitreally, and the second plurality is administered subretinally. 74. The method of any of embodiments 55-73, wherein the second plurality is administered at the same dosage as the first plurality.

[0091] 75. The method of any of embodiments 55-73, wherein the second plurality is administered at a different dosage (e.g., a higher dosage or a lower dosage) as the first plurality.

[0092] 76. The method of any of embodiments 55-75, wherein the fust plurality is administered at a dosage of about 3 x 107, 3 x 108, 3 x 109, 3.5 x 109, 3.6 x 109, 4 x 109, 4.5 x 109, 4.8 x 109, 5 x 109, 5.5 x 109, 6 x 109, 3 x IO10, 2.5 x 1011, 2.5 x 1012, or 2.5 x 1013vg / eye.

[0093] 77. The method of any of embodiments 55-76, wherein the fust plur ality is administered at a dosage of 3 x 107- 3 x 108, 3 x 108- 3 x 109, 3 x 109- 3.5 x 109, 3.5 x 109- 3.6 x 109, 3.6 x 109- 4 x 109, 4 x 109- 4.5 x 109, 4.5 x 109- 4.8 x 109, 4.8 x 109- 5 x 109, 5 x 109- 5.5 x 109, 5.5 x 109- 6 x 109, 6 x 109- 2.5 x 10'°, 2.5 x IO10- 2.5 x 10”, 2.5 x 1011- 2.5 x 1012, or 2.5 x 1012- 2.5 x 1013, vg / eye.

[0094] 78. The method of any of embodiments 55-77, wherein the second plurality is administered at a dosage of about 3 x 109, 3.5 x 109, 3.6 x 109, 4 x 109, or 4.5 x 109vg / eye.

[0095] 79. The method of any of embodiments 55-78, wherein the second plurality is administered at a dosage of 3 x 109- 3.5 x 109, 3.5 x 109- 3.6 x 109, 3.6 x 109- 4 x 109, or 4 x 109- 4.5 x 109vg / eye.

[0096] 80. The method of any of embodiments 55-79, wherein the second plurality is administered about 25, 26, 27, 28, 29, or 30 days after the first plurality.

[0097] 81. The method of any of embodiments 55-80, wherein the second plurality is administered about 25-28 or 28-30 days after the first plurality.

[0098] 82. The method of any of embodiments 55-80, wherein the second plurality is administered at least 14 days, 20 days, 25 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or 4 years after the first plurality.

[0099] 83. The method of any of embodiments 31-82, which further comprises administering to the subject one or more of the agents listed in Table E5. 84. The method of any of embodiments 31-83, which further comprises administering to the subject one or more of proparacaine ophthalmic solution, phenylephrine ophthalmic solution, tropicamide ophthalmic solution, atropine ophthalmic solution, dexmedetomidine, atipamezole, ketamine, isoflurane, buprenorphine, methylprednisolone, cefazolin, cefoxitin, ceftiofur, triamcinolone acetonide, or lactated Ringer’s solution.

[0100] 85. The anelloviral vector or method of any of embodiments 28-84, wherein the proteinaceous exterior (e.g., 0RF1 molecule of the proteinaceous exterior) comprises the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829), wherein X" is a contiguous sequence of any n amino acids.

[0101] 86. The ORF1 molecule of embodiment 85, wherein the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829) is comprised in an N22 domain of the ORF1 molecule.

[0102] 87. The ORF1 molecule of embodiment 85 or 86, wherein the ORF1 molecule comprises one or more (e.g., 1, 2, 3, 4, or all 5) of the following Anellovirus ORF1 subdomains: an arginine-rich region, a jelly-roll region, a hypervariable region, an N22 domain, a C-terminal domain (CTD) (e.g., as described herein), e.g., of an Anellovirus ORF1 protein as listed in Table Al (or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto).

[0103] 88. The nucleic acid molecule of any of embodiments 20-27, wherein the nucleic acid molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type Anellovirus genome sequence (e.g., as described herein)

[0104] 89. The nucleic acid molecule of embodiment 88, wherein the at least one difference comprises a deletion (e.g., lacks one or more of: an Anellovirus 5’ UTR conserved domain, an ORF1 gene, ORF2 gene, an Anellovirus GC-rich region, an ORF3 gene, or a functional fragment thereof).

[0105] 90. The nucleic acid molecule of embodiment 88 or 89, wherein the isolated nucleic acid molecule is substantially unable to be enclosed in an Anellovirus capsid (e.g., a proteinaceous exterior of an anelloviral vector as described herein).

[0106] 91. The nucleic acid molecule of any of embodiment 88-90, wherein the nucleic acid molecule encodes an effector (e.g., an exogenous effector or an endogenous effector). 92. The genetic element or nucleic acid molecule of any of the preceding embodiments, which further comprises a promoter.

[0107] 93. The genetic element or nucleic acid molecule of any of the preceding embodiments, which further comprises a poly A sequence.

[0108] 94. The pharmaceutical composition of embodiment 30, wherein the pharmaceutical composition has one or more of the following characteristics: a) the pharmaceutical composition meets a pharmaceutical or good manufacturing practices (GMP) standaid; b) the pharmaceutical composition was made according to good manufacturing practices (GMP); c) the pharmaceutical composition has a pathogen level below a predetermined reference value, e.g., is substantially free of pathogens; d) the pharmaceutical composition has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants; e) the pharmaceutical composition has a predetermined level of non-infectious particles or a predetermined ratio of particles infectious units (e.g., <300:1, < 200:1, <100:1, or <50:1), or f) the pharmaceutical composition has low immunogenicity or is substantially non- immunogenic, e.g., as described herein.

[0109] 95. The pharmaceutical composition of embodiment 94, wherein the pharmaceutical composition has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants.

[0110] 96. The pharmaceutical composition of embodiment 95, wherein the contaminant is selected from the group consisting of: mycoplasma, endotoxin, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replication-competent agents (RCA), e.g., replication-competent virus or unwanted anelloviral vector, free viral capsid protein, adventitious agents, and aggregates.

[0111] 97. The pharmaceutical composition of embodiment 95, wherein the contaminant is host cell DNA and the threshold amount is about 10 ng of host cell DNA per dose of the pharmaceutical composition. 98. The pharmaceutical composition of any one of embodiments 94-97, wherein the pharmaceutical composition comprises less than 10% (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) contaminant by weight.

[0112] 99. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is single-stranded.

[0113] 100. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is circular'.

[0114] 101. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises DNA.

[0115] 102. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is a negative strand DNA.

[0116] 103. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is double-stranded.

[0117] 104. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is linear'.

[0118] 105. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises RNA.

[0119] 106. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises a nucleic acid sequence encoding an Anellovirus ORF1 molecule (e.g., an ORF1 protein as listed in Table Al or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0120] 107. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element does not comprise a nucleic acid sequence encoding an Anellovirus ORF1 molecule (e.g., an ORF1 protein as listed in Table Al or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0121] 108. The genetic element, anello viral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises a nucleic acid sequence encoding an Anellovirus ORF2 molecule (e.g., an ORF2 protein as listed in Table Al, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0122] 109. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element does not comprise a nucleic acid sequence encoding an Anellovirus ORF2 molecule (e.g., an ORF2 protein as listed in Table Al or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0123] 110. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides having a GC content of at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0124] 111. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior comprises the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829), wherein X" is a contiguous sequence of any n amino acids.

[0125] 112. The anelloviral vector, nucleic acid molecule, or method of embodiment 111, wherein the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829) is comprised in an N22 domain.

[0126] 113. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the ORF1 molecule comprises an arginine-rich region (e.g., having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an arginine-rich region sequence of an ORF1 protein listed in Table Al).

[0127] 114. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior comprises an amino acid sequence of at least 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 consecutive nucleotides comprising at least 40% (e.g., at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, or 95%) arginine residues.

[0128] 115. The anello viral vector, nucleic acid molecule, or method of embodiment 113 or 114, wherein the arginine -rich region is located at the N-terminal or C-terminal end of the ORF1 molecule.

[0129] 116. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises one or more of: a TATA box, an initiator element, a cap site, a transcriptional start site, an ORF1 / 1 -encoding sequence, an ORFl / 2-encoding sequence, an ORF2 / 2-encoding sequence, an ORF2 / 3-encoding sequence, an ORF2 / 3t-encoding sequence, a three open-reading frame region, a poly(A) signal, and / or a GC-rich region from an Anellovirus described herein, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0130] 117. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises an Anellovirus 5' UTR conserved domain sequence.

[0131] 118. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises an Anellovirus GC-rich region sequence.

[0132] 119. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises 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 to an Anellovirus 5’ UTR conserved domain sequence as described herein.

[0133] 120. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises 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 to an Anellovirus GC-rich region sequence as described herein.

[0134] 121. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector encodes a therapeutic agent, e.g., a therapeutic peptide or polypeptide or a therapeutic nucleic acid. 122. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector is an exogenous effector.

[0135] 123. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector is an endogenous effector (e.g., wherein the anelloviral vector overexpresses the endogenous effector in a target cell).

[0136] 124. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector modulates expression or activity of a gene or protein, e.g., increases or decreases expression or activity of the gene or protein.

[0137] 125. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is capable of replicating autonomously.

[0138] 126. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is replication-deficient (e.g., incapable of replicating autonomously ).

[0139] 127. The genetic element, anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element integrates into the genome of a eukaryotic cell 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 genetic element that enters the cell.

[0140] 128. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is substantially non-pathogenic, e.g., does not induce a detectable deleterious symptom in a subject (e.g., elevated cell death or toxicity, e.g., relative to a subject not exposed to the anelloviral vector).

[0141] 129. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is substantially non-immunogenic, e.g., does not induce a detectable and / or unwanted immune response. 130. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of at least 1000 of the Anelloviridae family vectors 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 eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0142] 131. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of the Anelloviridae family vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of delivering the genetic element into at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0143] 132. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of the Anelloviridae family vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of delivering at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 8,000, 1 x 104, 1 x 105, 1 x 106, 1 x 107or greater copies of the genetic element per cell to a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0144] 133. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of the Anelloviridae family vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of delivering 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-10, 10-20, 20-50, 50- 100, 100-1000, 1000-104, 1 x 104-l x 105, 1 x 104-l x 106, 1 x 104-l x 107, 1 x 105-l x 106, 1 x 105-l x 107, or 1 x 106-l x 107copies of the genetic element per cell to a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0145] 134. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the target cells into which the genetic element is delivered each receive at least 10, 50, 100, 500, 1000, 10,000, 50,000, 100,000, or more copies of the genetic element.

[0146] 135. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is resistant to degradation by a detergent (e.g., a mild detergent, e.g., a biliary salt, e.g., sodium deoxycholate) relative to a viral particle comprising an external lipid bilayer, e.g., a retrovirus.

[0147] 136. The anello viral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element encapsulated by the proteinaceous exterior is resistant to degradation by a nuclease enzyme (e.g., a DNase).

[0148] 137. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector is capable of infecting mammalian cells, e.g., human cells, e.g., in vitro, in vivo, or ex vivo.

[0149] 138. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae family vector selectively delivers the effector to, or is present at higher levels in (e.g., preferentially accumulates in), a desired cell type, tissue, or organ.

[0150] 139. The anelloviral vector, nucleic acid molecule, or method of embodiment 138, wherein the desired cell type is an eye cell.

[0151] 140. The anelloviral vector, nucleic acid molecule, or method of embodiment 138, wherein the desired tissue is eye tissue.

[0152] 141. The anelloviral vector, nucleic acid molecule, or method of embodiment 138, wherein the desired organ is the eye.

[0153] 142. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-141, wherein the genetic element of the Anelloviridae family vector, or a DNA molecule comprising the nucleic acid sequence of the genetic element, is not detectable in an extraocular tissue (e.g., liver and / or spleen), e.g., according to an assay of Example 2.

[0154] 143. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-141, wherein less than 1, 10, or 100 copies per pg / DNA of the genetic element of the Anelloviridae family vector, or a DNA molecule comprising the nucleic acid sequence of the genetic element, are present in an extraocular tissue (e.g., liver and / or spleen), e.g., as determined according to an assay of Example 2 or 3. 144. The anello viral vector, nucleic acid molecule, or method of embodiment 142 or 143, wherein the extraocular tissue is liver.

[0155] 145. The anello viral vector, nucleic acid molecule, or method of embodiment 142 or 143, wherein the extraocular tissue is spleen.

[0156] 146. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-145, wherein upon administration to the eye of a subject, the subject does not exhibit retinal structural changes, e.g., as determined by Ocular Coherence Tomography (OCT), e.g., as described in Example 2 or 3.

[0157] 147. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-146, wherein upon administration to the eye of a subject, the subject does not exhibit altered intraocular pressure (IOP), e.g., as described in Example 2 or 3.

[0158] 148. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-147, wherein upon administration to the eye of a subject, the subject does not exhibit altered levels of a biomarker as listed in Table E9, e.g., as described in Example 2 or 3.

[0159] 149. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-148, wherein upon administration to the eye of a subject, the subject does not exhibit ocular inflammation, ocular toxicity (e.g., retinal toxicity), and / or an anelloviral vector-related systemic effect, e.g., a parameter as listed in Table E10 or El l.

[0160] 150. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-149, wherein upon administration to the eye of a subject, the subject does not exhibit a substantial change (e.g., increase), e.g., a detectable change (e.g., increase), in red blood cell count, hemoglobin level, and / or hematocrit, e.g., as described in Example 2 or 3.

[0161] 151. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-150, wherein upon administration to the eye of a subject, the subject does not exhibit detectable uveitis (e.g., a uveitis score of 0), e.g., as described in Example 2 or 3. 152. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-150, wherein upon administration to the eye of a subject, the subject has a uveitis score of 0, 1, or 2, e.g., as described in Example 2 or 3.

[0162] 153. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-152, wherein upon administration to the eye of a subject, the subject does not exhibit detectable aqueous flare or Tyndall effect, e.g., as described in Example 2 or 3.

[0163] 154. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-152, wherein upon administration to the eye of a subject, the subject has an aqueous flare grade of 0, 0.5, or 1, e.g., as described in Example 2 or 3.

[0164] 155. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-154, wherein upon administration to the eye of a subject, the subject does not exhibit detectable vitreous haze, e.g., as described in Example 2 or 3.

[0165] 156. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-154, wherein upon administration to the eye of a subject, the subject has a vitreous haze grade of 0, 0.5, or 1, e.g., as described in Example 2 or 3.

[0166] 157. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-156, wherein upon administration to the eye of a subject, the subject does not exhibit detectable retinal perivascular sheathing, e.g., as described in Example 2 or 3.

[0167] 158. The anelloviral vector, nucleic acid molecule, or method of any of embodiments 138-156, wherein upon administration to the eye of a subject, the subject has a retinal perivascular sheathing grade of 0 or 1, e.g., as described in Example 2 or 3.

[0168] 159. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior is provided in cis relative to the genetic element.

[0169] 160. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior is provided in trans relative to the genetic element. 161. The method of any of embodiments 31-85 or 99-160, wherein the Anelloviridae family vector is substantially free of wild-type Anellovirus genomes.

[0170] 162. The method of any of embodiments 31-85 or 99-161, wherein the anelloviral vector is administered to the subject via an SCS microinjector, via a cannula, and / or via a needle.

[0171] 163. An ocular delivery device comprising an anelloviral vector of any of embodiments 28, 29, 85, or 99-162.

[0172] 164. The ocular delivery device of embodiment 163, which is configured for suprachoroidal injection.

[0173] 165. The ocular delivery device of embodiment 163, which is configured for subretinal administration.

[0174] 166. The ocular delivery device of embodiment 165, which comprises a catheter and a needle configured to pass through the catheter (e.g., into the subretinal space of a subject).

[0175] 167. The ocular delivery device of embodiment 163, which is configured for intravitreal administration.

[0176] 168. The ocular delivery device of any of embodiments 163-167, which comprises a microinjector (e.g., comprising a microneedle), a cannula (e.g., a fine bore cannula), and / or a syringe.

[0177] 169. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the nucleic acid molecule comprises the DNA sequence according to SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0178] 170. The anelloviral vector, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises the DNA sequence according to the reverse complement of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 171. A nucleic acid molecule comprising the DNA sequence according to SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0179] 172. A circular, single-stranded DNA genetic element comprising the DNA sequence according to the reverse complement of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0180] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0181] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0182] BRIEF DESCRIPTION OF THE DRAWINGS

[0183] Figure 1 shows a flatmount diagram of how retinal and posterior eye cup (PEC) punches were collected for subretinally injected eyes in Group 1 (OS) and Group 3 (OD). The procedure is as follows: Separated the retina from the RPE / choroid and snap froze in individual collection tubes. Noted the punch number on each tube so the retina and RPE / choroid could be matched. Subretinal bleb: collected an 8mm punch and cut into 2 pieces (regions 2 and 3 in the figure) and separated retina and RPE / choroid into separate tubes. Periphery: Collected regions 4, 5, 6, and 7 as 8 mm punches. Collected optic nerve (region 1) as a 6mm punch. Collected remaining tissue from the retina and RPE / choroid into 2-3 tubes as needed.

[0184] Figure 2 shows a flatmount diagram of how retinal and posterior eye cup (PEC) punches were collected for intravitreally injected eyes in Group 2 (OS) and Group 3 (OS). The procedure is as follows: collected 8 mm punches as described in the figure. Separated retina from RPE / choroid and snap froze in individual collection tubes. Noted the punch number on each tube so the retina and RPE / choroid could be matched. Collected regions 2, 3, 4, 5, as 8 mm punches. Collected optic nerve (region 1) as a 6 mm punch. Collected remaining tissue from retina and RPE / Choroid into 2-3 tubes as needed.

[0185] Figure 3 shows a flatmount diagram of how retinal and posterior eye cup (PEC) punches were collected for subretinally injected eyes (OD). Scale: optic nerve = 1.5 mm; inferior to superior arcade = 6-8 mm; ora serrata to ora serrata = 25 mm. Figure 4 shows a flatmount diagram of how retinal and posterior eye cup (PEC) punches were collected for intravitreally injected eyes (OS). Scale: optic nerve = 1.5 mm; inferior to superior arcade = 6-8 mm; ora serrata to ora serrata = 25 mm.

[0186] Figure 5A shows the aflibercept genome copies detected in the PEC at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected either subretinally (SR) or intravitreally (IVT) with 7.4E+8 vg of either AN VI 9. Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. In SR columns, the data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region of the dissected eye. n = 1 eye / group, 5 or 6 punches / eye.

[0187] Figure 5B shows the aflibercept genome copies detected in the retina at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected either subretinally (SR) or intravitreally (IVT) with 7.4E+8 vg of either AN VI 9. Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. In SR columns, the data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region of the dissected eye. n = 1 eye / group, 5 or 6 punches / eye.

[0188] Figure 5C shows the mRNA copies detected in the PEC at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected either subretinally (SR) or intravitreally (IVT) with 7.4E+8 vg of either ANV19. Aflibercept or A AV2. Aflibercept. Each column represents one treatment eye. In SR columns, the data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region of the dissected eye. n = 1 eye / group, 5 or 6 punches / eye.

[0189] Figure 5D shows the mRNA copies detected in the retina at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected either subretinally (SR) or intravitreally (IVT) with 7.4E+8 vg of either ANV19. Aflibercept or A AV2. Aflibercept. Each column represents one treatment eye. In SR columns, the data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region of the dissected eye. n = 1 eye / group, 5 or 6 punches / eye.

[0190] Figure 6A shows aflibercept genome copies in punches collected from the liver 8 weeks following subretinal (SR) or intravitreal (IVT) administration with 7.4E+8 vg of either AN VI 9. Aflibercept or AAV2. Aflibercept. Each column represents one treatment animal. N=1 animal / group, 2 punches / tissue.

[0191] Figure 6B shows aflibercept genome copies in punches collected from the spleen 8 weeks following subretinal (SR) or intravitreal (IVT) administration with 7.4E+8 vg of either ANV 19.Aflibercept or AAV2.Aflibercept. Each column represents one treatment animal. N=1 animal / group, 2 punches / tissue.

[0192] Figure 7A shows the optical coherence tomography (OCT) images of the right eye (OD) and left eye (OS) of a Group 1 animal treated with 7.4E+8 vg of ANV19.Aflibercept by subretinal (SR) injection. Images were taken before administration (pretest), after administration (postdose), and at Days 14, 28, and 56 after administration.

[0193] Figure 7B shows the optical coherence tomography (OCT) images of the right eye (OD) and left eye (OS) of a Group 2 animal treated with 7.4E+8 vg of ANV 19.Aflibercept by intravitreal (IVT) injection. Images were taken before administration (pretest) and at Days 14, 28, and 56 after administration.

[0194] Figure 7C shows the optical coherence tomography (OCT) images of the right eye (OD) of a Group 3 animal treated with 7.4E+8 vg of AAV2.Aflibercept by subretinal (SR) injection and left eye (OS) of a Group 3 animal treated with 7.4E+8 vg of AAV2.Aflibercept by intravitreal (IVT) injection. Images of the OD eye shown are before administration (pretest), after administration (postdose), and at Days 14, 28, and 56 after administration, and images of the OS eye shown are before administration (pretest) and at Days 14, 28, and 56 after administration.

[0195] Figure 8A shows the average aflibercept genome copies detected in the PEC at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected subretinally (SR) with 4.8E+9 vg of either ANV 19. Aflibercept or A A V2. Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OD eye, second ANV 19 column is from Group 4 OD eye, and third ANV 19 column is from Group 4 OS eye, while the AAV2 column is from Group 2 OD eye. Data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region and optic nerve of the dissected eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 9 punches / eye.

[0196] Figure 8B shows the average aflibercept genome copies detected in the PEC at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected intr vitreally (IVT) with 3.6E+9 vg of either ANV 19. Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. First ANV 19 column is from Group 1 OS eye, second ANV19 column is from Group 3 OD eye, and the ANV 19+ column is from Group 3 OS redosed eye, while the AAV2 column is from Group 2 OS eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05 and marked with * or ****. “ns” = not significant, n = 1 eye / group, 6 punches / eye. Figure 8C shows the average aflibercept genome copies detected in the retina at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected subretinally (SR) with 4.8E+9 vg of either ANV 19. Aflibercept or A A V2. Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OD eye, second ANV19 column is from Group 4 OD eye, and third ANV 19 column is from Group 4 OS eye, while the AAV2 column is from Group 2 OD eye. For better visual representation, the data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region and optic nerve of the dissected eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 9 punches / eye.

[0197] Figure 8D shows the average aflibercept genome copies detected in the retina at 8 weeks postadministration as assessed by qPCR of the harvested DNA from NHP eyes injected intravitreally (IVT) with 3.6E+9 vg of either ANV 19.Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. First ANV 19 column is from Group 1 OS eye, second ANV19 column is from Group 3 OD eye, and the ANV19+ column is from Group 3 OS redosed eye, while the AAV2 column is from Group 2 OS eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05 and marked with *. “ns” = not significant, n — 1 eye / group, 6 punches / eye.

[0198] Figure 9A shows the mRNA copies detected in the PEC at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected subretinally (SR) with 4.8E+9 vg of either ANV 19. Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OD eye, second ANV 19 column is from Group 4 OD eye, and third ANV 19 column is from Group 4 OS eye, while the AAV2 column is from Group 2 OD eye. Data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region and optic nerve of the dissected eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 9 punches / eye.

[0199] Figure 9B shows the mRNA copies detected in the PEC at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected intravitreally (IVT) with 3.6E+9 vg of either AN VI 9. Aflibercept or AAV2. Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OS eye, second ANV 19 column is from Group 3 OD eye, and the AN V 19+ column is from Group 3 OS redosed eye, while the AAV2 column is from Group 2 OS eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 6 punches / eye. Figure 9C shows the mRNA copies detected in the retina at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected subretinally (SR) with 4.8E+9 vg of either ANV19.Aflibercept or AAV2.Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OD eye, second ANV 19 column is from Group 4 OD eye, and third ANV 19 column is from Group 4 OS eye, while the AAV2 column is from Group 2 OD eye. The data points in hatching represent punches from the subretinal bleb and the black data points represent punches from the peripheral region and optic nerve of the dissected eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 9 punches / eye.

[0200] Figure 9D shows the mRNA copies detected in the retina at 8 weeks post-administration as assessed by RT-ddPCR of mRNA extracted from the eyes injected intravitreally (IVT) with 3.6E+9 vg of either ANV19.Aflibercept or AAV2.Aflibercept. Each column represents one treatment eye. First ANV19 column is from Group 1 OS eye, second ANV 19 column is from Group 3 OD eye, and the ANV19+ column is from Group 3 OS redosed eye, while the AAV2 column is from Group 2 OS eye. One way ANOVA was performed to measure statistical significance. Groups were considered significantly different if p<0.05. “ns” = not significant, n = 1 eye / group, 6 punches / eye.

[0201] Figure 10A shows aflibercept genome copies in punches collected from liver 8 weeks following subretinal (SR) administration with 4.8E+9 vg or intra vitreal (IVT) administration with 3.6E+9 vg of either ANV 19. Aflibercept or AAV2. Aflibercept. Each column represents one treatment animal. ANV19 SR / IVT is from Group 1, ANV19 SR / SR is from Group 4, ANV19 IVT / IVT+ is from Group 3, and AAV2 SR / IVT is from Group 2. n=l animal / group, 2 punches / tissue.

[0202] Figure 10B shows aflibercept genome copies in punches collected from spleen 8 weeks following subretinal (SR) administration with 4.8E+9 vg or intravitreal (IVT) administration with 3.6E+9 vg of either ANV 19. Aflibercept or AAV2. Aflibercept. Each column represents one treatment animal. ANV 19 SR / IVT is from Group 1 , ANV 19 SR / SR is from Group 4, ANV19 IVT / IVT+ is from Group 3, and AAV2 SR / IVT is from Group 2. n=l animal / group, 2 punches / tissue.

[0203] Figure 11 A shows the optical coherence tomography (OCT) images of the right eye (OD) of a Group 1 animal treated with 4.8E+9 vg of AN VI 9. Aflibercept by subretinal (SR) injection and images of the left eye (OS) of a Group 1 animal treated with 3.6E+9 vg of ANV19. Aflibercept by intravitreal (IVT) injection. Images were taken before administration (pretest). Scale bar = 200pm.

[0204] Figure 11B shows the optical coherence tomography (OCT) images of the right eye (OD) of a Group 2 animal treated with 4.8E+9 vg of AAV2. Aflibercept by subretinal (SR) injection and left eye (OS) of a Group 2 animal treated with 3.6E+9 vg of AAV2. Aflibercept by intravitreal (IVT) injection. Images were taken before administration (pretest) and on Days 14, 28, and 56 after administration. Scale bar = 200pm.

[0205] Figure 11C shows the optical coherence tomography (OCT) images of the right eye (OD) of Group 3 animal treated with 3.6E+9 vg of ANV19.Aflibercept by intravitreal (IVT) injection and left eye (OS) of Group 3 animal treated with 3.6E+9 vg of ANV 19.Aflibercept by intravitreal (IVT) injection and redosed on Day 28 with 3.6E+9 vg of ANV 19.Aflibercept by intravitreal (IVT) injection. Images were taken before administration (pretest) and on Days 14, 28 (after redosing for OS eye), and 56. Scale bar = 200pm.

[0206] Figure 11D shows the optical coherence tomography (OCT) images of the right eye (OD) and left eye (OS) of a Group 4 animal treated with 4.8E+9 vg of ANV19.Aflibercept by subretinal (SR) injection. Images were taken before administration (pretest) and on Days 14, 28, and 56 after administration. Scale bar = 200pm.

[0207] The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments that are presently exemplified. It should be understood, however, that the invention is not limited to the precise arrangement and instrumentalities of the embodiments shown in the drawings.

[0208] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0209] Definitions

[0210] The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

[0211] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is to be understood to preferably also disclose a group which consists only of these embodiments.

[0212] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

[0213] The wording “compound, composition, product, etc. for treating, modulating, etc.” is to be understood to refer a compound, composition, product, etc. per se which is suitable for the indicated purposes of treating, modulating, etc. The wording “compound, composition, product, etc. for treating, modulating, etc.” additionally discloses that, as an embodiment, such compound, composition, product, etc. is for use in treating, modulating, etc.

[0214] The wording “compound, composition, product, etc. for use in .. .”, “use of a compound, composition, product, etc. in the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for . . or “compound, composition, product, etc. for use as a medicament. . .” indicates that such compounds, compositions, products, etc. are to be used in therapeutic methods which may be practiced on the human or animal body. They are considered as an equivalent disclosure of embodiments and claims pertaining to methods of treatment, etc. If an embodiment or a claim thus refers to “a compound for use in treating a human or animal being suspected to suffer from a disease”, this is considered to be also a disclosure of a “use of a compound in the manufacture of a medicament for treating a human or animal being suspected to suffer from a disease” or a “method of treatment by administering a compound to a human or animal being suspected to suffer from a disease”. The wording “compound, composition, product, etc. for treating, modulating, etc.” is to be understood to refer a compound, composition, product, etc. per se which is suitable for the indicated purposes of treating, modulating, etc.

[0215] If hereinafter examples of a term, value, number, etc. are provided in parentheses, this is to be understood as an indication that the examples mentioned in the parentheses can constitute an embodiment. For example, if it is stated that “in 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 to the Anellovirus ORFl-encoding nucleotide sequence of Table 1 (e.g., nucleotides 571 - 2613 of the nucleic acid sequence of Table 1)”, then some embodiments relate to nucleic acid molecules comprising a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to nucleotides 571 - 2613 of the nucleic acid sequence of Table 1.

[0216] As used herein, the term “Anelloviridae family vector” refers to a vehicle derived from or similar to a virus of the Anelloviridae family (e.g., an Alphatorque virus, Betatorque virus, Gammatorquevirus, or chicken anemia virus), wherein the vehicle comprises a genetic element encapsulated in a proteinaceous exterior (e.g., the genetic element is substantially protected from digestion with DNase I by a proteinaceous exterior). In some embodiments, an Anelloviridae family vector comprises a genetic element derived from or highly similar to (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) that of an Alphatorquevirus, Betatorquevirus, or Gammatorque virus. In some embodiments, an Anelloviridae family vector comprises a proteinaceous exterior comprising a protein derived from or similar to (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) a capsid protein of an Alphatorquevirus, Betatorquevirus, or Gammatorquevirus (e.g., an Alphatorquevirus 0RF1, Betatorquevirus 0RF1, or Gammatorquevirus ORF1). In some embodiments, encapsulated within a proteinaceous exterior encompasses 100% coverage by a proteinaceous exterior, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. For example, gaps or discontinuities (e.g., that render the proteinaceous exterior permeable to water, ions, peptides, or small molecules) may be present in the proteinaceous exterior, so long as the genetic element is retained in the proteinaceous exterior or protected from digestion with DNase I, e.g., prior to entry into a host cell. In some embodiments, the Anelloviridae family vector is purified, e.g., it is separated from its original source and / or substantially free (>50%, >60%, >70%, >80%, >90%) of other components. In some embodiments, the Anelloviridae family vector is capable of introducing the genetic element into a target cell (e.g., via infection). In some embodiments, the Anelloviridae family vector is an infective synthetic viral particle.

[0217] As used herein, the terms “anelloviral vector” and “anellovirus vector” are used interchangeably herein to refer to a vehicle comprising a genetic element, e.g., circular DNA, encapsulated in a proteinaceous exterior. A “synthetic anelloviral vector” or “synthetic anellovirus vector,” as used herein, generally refers to an anelloviral vector that is not naturally occurring, e.g., has a sequence that is different relative to a wild-type virus (e.g., a wild-type Anellovirus as described herein). In some embodiments, the synthetic anelloviral vector is engineered or recombinant, e.g., comprises a genetic element that comprises a difference or modification relative to a wild-type viral genome (e.g., a wild-type Anellovirus genome as described herein). In some embodiments, encapsulated within a proteinaceous exterior encompasses 100% coverage by a proteinaceous exterior, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. For example, gaps or discontinuities (e.g., that render the proteinaceous exterior permeable to water, ions, peptides, or small molecules) may be present in the proteinaceous exterior, so long as the genetic element is retained in the proteinaceous exterior, e.g., prior to entry into a host cell. In some embodiments, the anelloviral vector is purified, e.g., it is separated from its original source and / or substantially free (>50%, >60%, >70%, >80%, >90%) of other components. In some embodiments, the anelloviral vector is an ANELLOVECTOR™ viral vector. In some embodiments, the synthetic anelloviral vector is a synthetic ANELLOVECTOR™ viral vector.

[0218] An anelloviral vector may, in some embodiments, comprise a nucleic acid vector that comprises sufficient nucleic acid sequence derived from or highly similar to (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) an Anellovirus genome sequence or a contiguous portion thereof to allow packaging into a proteinaceous exterior (e.g., a capsid), and further comprises a heterologous sequence. In some embodiments, the nucleic acid vector is a viral vector or a naked nucleic acid. In some embodiments, the nucleic acid vector comprises at least about 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 3500 consecutive nucleotides of a native Anellovirus sequence or a sequence highly similar (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) thereto. In some embodiments, the anelloviral vector further comprises one or more of an Anellovirus ORF1, ORF2, or ORF3. In some embodiments, the heterologous sequence comprises a multiple cloning site, comprises a heterologous promoter, comprises a coding region for a therapeutic protein, or encodes a therapeutic nucleic acid. In some embodiments, the capsid is a wild-type Anellovirus capsid. In embodiments, an anelloviral vector comprises a genetic element described herein, e.g., comprises a genetic element comprising a promoter, a sequence encoding a therapeutic effector, and a capsid binding sequence.

[0219] As used herein, the term “Anellovirus non-coding region (NCR)” refers to a sequence of an untranslated region present in an Anellovirus genome sequence that extends from just upstream of the ORF2 start codon to the untranslated region of the Anellovirus genome sequence just downstream of the ORF3 stop codon in a circular genome. The Anellovirus NCR may comprise the “Anellovirus 5’ NCR sequence” and “Anellovirus 3’ NCR sequence.” In some embodiments, the Anellovirus NCR sequence is contiguous. In some embodiments, the Anellovirus NCR sequence is non-contiguous. In some embodiments, the portions of a non-contiguous Anellovirus NCR sequence are separated by a heterologous insertion (e.g., comprising a recombinase hybrid site, e.g., as described herein). In some embodiments, the Anellovirus NCR sequence comprises a sequence found in a wild-type Anellovirus, and in other embodiments, the Anellovirus NCR comprises one or more mutations relative to the closest Anellovirus sequence. In some embodiments, the Anellovirus NCR is comprised by a nucleic acid molecule that does not comprise Anellovirus ORF2 or ORF3 coding sequences. In some instances, the Anellovirus NCR comprises a negative strand sequence corresponding to a portion of positive strand Anelloviridae family virus or Anelloviridae family vector sequence described herein.

[0220] As used herein, the term “Anellovirus 5’ NCR” refers to a sequence of an untranslated region present in an Anellovirus genome sequence that extends from just upstream of the ORF2 start codon through the 5’ UTR conserved domain to the Anellovirus 3’ NCR sequence, and sequences with homology thereto. In some embodiments, an Anellovirus 5' NCR sequence comprises origin of replication activity. In some embodiments, the Anellovirus 5' NCR sequence is contiguous. In some embodiments, the Anellovirus 5’ NCR sequence is non-contiguous. In some embodiments, the portions of a non-contiguous Anellovirus 5’ NCR sequence are separated by a heterologous insertion (e.g., comprising a recombinase hybrid site, e.g., as described herein). In some embodiments, the Anellovirus 5’ NCR sequence comprises a sequence found in a wild-type Anellovirus, and in other embodiments, the Anellovirus 5’ NCR comprises one or more mutations relative to the closest Anellovirus sequence. In some instances, the Anellovirus 5' NCR comprises a negative strand sequence corresponding to a portion of positive strand Anelloviridae family virus or Anelloviridae family vector sequence described herein. In a circular genetic element, the Anellovirus 5’ NCR and Anellovirus 3’ NCR may be directly adjacent to each other (e.g., to form an Anellovirus NCR). Exemplary dividing points between the Anellovirus 5’ NCR and the Anellovirus 3’ NCR are shown, e.g., as described herein.

[0221] As used herein, the term “Anellovirus 3’ NCR” refers to a sequence of an untranslated region present in an Anellovirus genome sequence that extends from just downstream of the 0RF3 stop codon through the GC-rich region to the Anellovirus 5’ NCR sequence, and sequences with homology thereto. In some embodiments, the Anellovirus 3’ NCR sequence is contiguous. In some embodiments, the Anellovirus 3’ NCR sequence is non-contiguous. In some embodiments, the portions of a non-contiguous Anellovirus 3' NCR sequence are separated by a heterologous insertion (e.g., comprising a recombinase hybrid site, e.g., as described herein). In some embodiments, the Anellovirus 3' NCR sequence comprises a sequence found in a wild-type Anellovirus, and in other embodiments, the Anellovirus 3’ NCR comprises one or more mutations relative to the closest Anellovirus sequence. In some instances, the Anellovirus 3’ NCR comprises a negative strand sequence corresponding to a portion of positive strand Anelloviridae family virus or Anelloviridae family vector sequence described herein.

[0222] As used herein, the term “Anellovirus GC-rich region” refers to a wild-type or engineered sequence that has an activity and a structural feature of a GC-rich region present in a wild-type Anellovirus, or a functional fragment thereof. In some embodiments, the functional fragment has a length of at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. Typically, the negative strand comprising the Anellovirus GC-rich region is packaged into a particle (e.g., an Anelloviridae family vector) as described herein. In some embodiments, the Anellovirus GC-rich region is a wild-type Anellovirus GC-rich region. In some embodiments, the Anellovirus GC-rich region is an engineered Anellovirus GC-rich region having a nucleic acid sequence with at least one difference relative to the closest wild-type Ancllovii us GC-rich region sequence. In some embodiments, the Anellovirus GC-rich region comprises the reverse complement of a sequence annotated as a GC-rich region in Table Nl.

[0223] As used herein, the term “Anellovirus 5’ UTR conserved domain” refers to a wild-type or engineered sequence that has an activity and a structural feature of an Anellovirus 5’ UTR conserved domain present in a wild-type Anellovirus, or a functional fragment thereof. In some embodiments, the functional fragment has a length of at least 15, 20, 30, 40, 50, 60, or 70 nucleotides. Typically, the negative strand comprising the Anellovirus 5’ UTR conserved domain is packaged into a particle (e.g., an Anelloviridae family vector) as described herein. In some embodiments, the Anellovirus 5’ UTR conserved domain is a wild-type Anellovirus 5’ UTR conserved domain. In some embodiments, the Anellovirus 5’ UTR conserved domain is an engineered Anellovirus 5’ UTR conserved domain having a nucleic acid sequence with at least one difference relative to the closest wild-type Anellovirus 5’ UTR conserved domain sequence. In some embodiments, the Anellovirus 5’ UTR conserved domain comprises the reverse complement of a sequence annotated as a 5’ UTR conserved domain in Table Nl.

[0224] As used herein, the term “antibody molecule” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “antibody molecule” encompasses full-length antibodies and antibody fragments (e.g., scFvs). In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., the antibody molecule comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody molecule is generally characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0225] The term “m vitro assembly,” as used herein with respect to an Anelloviridae family vector or an anelloVLP, refers to the formation of a proteinaceous exterior comprising an ORF1 molecule, wherein the formation does not take place inside of a cell (e.g., takes place in a cell-free system such as a cell-free suspension, a lysate, or a supernatant). In some instances, in vitro assembly of an Anelloviridae family vector comprises encapsulation, outside of a cell, of a genetic element (e.g., as described herein) within the proteinaceous exterior. In some instances, in vitro assembly of an anelloVLP comprises association, outside of a cell, of an effector (e.g., an exogenous effector, e.g., as described herein) with the proteinaceous exterior (e.g., encapsulated within the proteinaceous exterior). In vitro assembly of a proteinaceous exterior may occur, in some instances, under conditions suitable for multimerization of a plurality of ORF1 molecules (e.g., nondenaturing conditions), e.g., to form a multimer of more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ORF1 molecules. In some instances, in vitro assembly results in the formation of a proteinaceous exterior comprising at least about 20, 30, 40, 50, or 60 ORF1 molecules, or about 20-30, 30-40, 40-50, 50-60, or 60-70 ORF1 molecules). In some instances, the proteinaceous exterior is formed from ORF1 molecules that were produced in a cell and then purified therefrom. In some instances, the in vitro assembly takes place in a solution free of cells or constituents thereof. In other instances, the in vitro assembly takes place in a solution comprising cell debris (e.g., from lysed cells). In some instances, the in vitro assembly takes place in a solution substantially free of cellular nucleic acid molecules (e.g., genomic DNA, mitochondrial DNA, mRNA, and / or noncoding RNA from a cell).

[0226] As used herein, a nucleic acid “encoding” refers to a nucleic acid sequence encoding an amino acid sequence or a functional polynucleotide (e.g., a non-coding RNA, e.g., an siRNA or miRNA). An “exogenous” agent (e.g., an effector, a nucleic acid (e.g., RNA), a gene, payload, protein) as used herein refers to an agent that is either not comprised by, or not encoded by, a corresponding wildtype virus, e.g., an Anellovirus as described herein. In some embodiments, the exogenous agent does not naturally exist, such as a protein or nucleic acid that has a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein or nucleic acid. In some embodiments, the exogenous agent does not naturally exist in the host cell. In some embodiments, the exogenous agent exists naturally in the host cell but is exogenous to the virus. In some embodiments, the exogenous agent exists naturally in the host cell, but is not present at a desired level or at a desired time.

[0227] A “heterologous” agent or element (e.g., an effector, a nucleic acid sequence, an amino acid sequence), as used herein with respect to another agent or element (e.g., an effector, a nucleic acid sequence, an amino acid sequence), refers to agents or elements that are not naturally found together, e.g., in a wild-type virus, e.g., an Anellovirus. In some embodiments, a heterologous nucleic acid sequence may be present in the same nucleic acid as a naturally occurring nucleic acid sequence (e.g., a sequence that is naturally occurring in the Anellovirus). In some embodiments, a heterologous agent or element is exogenous relative to an Anellovirus from which other (e.g., the remainder of) elements of the anelloviral vector are based.

[0228] As used herein, the term “genetic element” refers to a nucleic acid sequence, generally in an anelloviral vector. It is understood that the genetic element can be produced as naked DNA and optionally further assembled into a proteinaceous exterior. It is also understood that an anelloviral vector can insert its genetic element into a cell, resulting in the genetic element being present in the cell and the proteinaceous exterior not necessarily entering the cell.

[0229] As used herein, the term “ORF1 molecule” refers to a polypeptide having an activity and / or a structural feature of an Anellovirus 0RF1 protein (e.g., an Anellovirus 0RF1 protein as described herein, e.g., as listed in Table Al), or a functional fragment thereof. An 0RF1 molecule may, in some instances, comprise one or more of (e.g., 1, 2, 3 or 4 of): a first region comprising at least 60% basic residues (e.g., at least 60% arginine residues), a second region comprising at least about six beta strands (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands), a third region comprising a structure or an activity of an Anellovirus N22 domain (e.g., as described herein, e.g., an N22 domain from an Anellovirus ORF1 protein as described herein), and / or a fourth region comprising a structure or an activity of an Anellovirus C -terminal domain (CTD) (e.g., as described herein, e.g., a CTD from an Anellovirus ORF1 protein as described herein). In some instances, the ORF1 molecule comprises, in N-terminal to C-terminal order, the first, second, third, and fourth regions. In some instances, an anelloviral vector comprises an ORF1 molecule comprising, in N-terminal to C-terminal order, the first, second, third, and fourth regions. An ORF1 molecule may, in some instances, comprise a polypeptide encoded by an Anellovirus ORF1 nucleic acid (e.g., as listed in Table Nl). An 0RF1 molecule may, in some instances, further comprise a heterologous sequence, e.g., a hypervariable region (HVR), e.g., an HVR from an Anellovirus ORF1 protein, e.g., as described herein. An “Anellovirus ORF1 protein,” as used herein, refers to an ORF1 protein encoded by an Anellovirus genome (e.g., a wild-type Anellovirus genome, e.g., as described herein), e.g., an ORF1 protein having the amino acid sequence as listed in Table Al, or as encoded by the ORF1 gene as listed in Table Nl.

[0230] The term “ORF1 domain,” as used herein with respect to an ORF1 molecule, refers to the portion of the ORF1 molecule having the structure or function of an Anellovirus ORF1 protein. The ORF1 domain is generally capable of forming a multimer with other copies of the ORF1 domain (e.g., in other ORF1 molecules), or with other ORF1 molecules, e.g., to form a proteinaceous exterior (e.g., of an anelloviral vector or anelloVLP as described herein). In some instances, the ORF1 molecule may comprise one or more additional domains other than the ORF1 domain (for example, a domain comprising or attached to a surface effector). In some instances, the amino acid sequence of an ORF1 domain comprises an insertion (e.g., an insertion encoding a surface moiety or a domain capable of binding to a surface moiety), e.g., between the N-terminal end and C-terminal end of the ORF1 domain. In certain instances, the insertion does not substantially disrupt the structure and / or function of the ORF1 domain, e.g., such that the ORF1 domain remains capable of forming a multimer with other ORF1 domains or ORF1 molecules. The position within the ORF1 domain sequence into which the insertion is made is referred to herein as the “insertion point.” An insertion can be made into an ORF1 domain by any genetic or polypeptide engineering method known in the art. In some embodiments, an ORF1 molecule consists of an ORF1 domain. In other embodiments, an ORF1 molecule comprises an ORF1 domain and a heterologous domain (e.g., a surface moiety as described herein). In some embodiments, an ORF1 domain is connected to a surface moiety by a polypeptide linker region.

[0231] As used herein, the term “ORF2 molecule” refers to a polypeptide having an activity and / or a structural feature of an Anellovirus ORF2 protein (e.g., an Anellovirus ORF2 protein as described herein, e.g., as listed in Table Al), or a functional fragment thereof. An “Anellovirus ORF2 protein,” as used herein, refers to an ORF2 protein encoded by an Anellovirus genome (e.g., a wild-type Anellovirus genome, e.g., as described herein), e.g., an ORF2 protein having the amino acid sequence as listed in Table Al, or as encoded by the ORF2 gene as listed in Table Nl.

[0232] As used herein, the term “particle” refers to a vehicle having a diameter of less than 100 nm (e.g., about 20-25, 25-30, 30-35, or 35-40 nm) comprising a proteinaceous exterior. In some instances, the particle comprises a plurality of ORF1 molecules. The proteinaceous exterior of the particle generally forms an encapsulation capable of limiting or preventing movement of certain molecules between the inside and outside of the proteinaceous exterior. In some embodiments, gaps or discontinuities (e.g., that render the proteinaceous exterior permeable to water, ions, peptides, or small molecules) may be present in the proteinaceous exterior. In certain embodiments, the gaps or discontinuities are of a sufficiently small size (e.g., diameter) that the proteinaceous exterior limits or prevents one or more large macromolecules (e.g., peptides, polypeptides, polynucleotides, lipids, or polysaccharides) from passing through the proteinaceous exterior.

[0233] As used herein, the term “proteinaceous exterior” refers to an exterior component that is predominantly (e.g., >50%, >60%, > 70%, >80%, > 90%) protein.

[0234] As used herein, the term “regulatory nucleic acid” refers to a nucleic acid sequence that modifies expression, e.g., transcription and / or translation, of a DNA sequence that encodes an expression product. In embodiments, the expression product comprises RNA or protein.

[0235] As used herein, the term “regulatory sequence” refers to a nucleic acid sequence that modifies transcription of a target gene product. In some embodiments, the regulatory sequence is a promoter or an enhancer.

[0236] As used herein, the term “replication protein” refers to a protein, e.g., a viral protein, that is utilized during infection, viral genome replication / expression, viral protein synthesis, and / or assembly of the viral components.

[0237] As used herein, a “substantially non-pathogenic” organism, particle, or component, refers to an organism, particle (e.g., a virus or an anelloviral vector, e.g., as described herein), or component thereof that does not cause or induce a detectable disease or pathogenic condition, e.g., in a host organism, e.g., a mammal, e.g., a human. In some embodiments, administration of an anelloviral vector to a subject can result in minor reactions or side effects that are acceptable as part of standard of care.

[0238] As used herein, the term “non-pathogenic” refers to an organism or component thereof that does not cause or induce a detectable disease or pathogenic condition, e.g., in a host organism, e.g., a mammal, e.g., a human.

[0239] As used herein, a “substantially non-integrating” genetic element refers to a genetic element, e.g., a genetic element in a virus or anelloviral vector, e.g., as described herein, wherein less than about 0.01%, 0.05%, 0.1%, 0.5%, or 1% of the genetic element that enter into a host cell (e.g., a eukaryotic cell) or organism (e.g., a mammal, e.g., a human) integrate into the genome. In some embodiments the genetic element does not delectably integrate into the genome of, e.g., a host cell. In some embodiments, integration of the genetic element into the genome can be detected using techniques as described herein, e.g., nucleic acid sequencing, PCR detection and / or nucleic acid hybridization.

[0240] As used herein, a “substantially non-immunogenic” organism, particle, or component, refers to an organism, particle (e.g., a virus or anelloviral vector, e.g., as described herein), or component thereof, that does not cause or induce an undesired or untargeted immune response, e.g., in a host tissue or organism (e.g., a mammal, e.g., a human). In some embodiments, the substantially non-immunogenic organism, particle, or component does not produce a detectable immune response. In some embodiments, the substantially non-immunogenic anelloviral vector does not produce a detectable immune response against a protein comprising an amino acid sequence or encoded by a nucleic acid sequence shown in Table Nl. In some embodiments, an immune response (e.g., an undesired or untargeted immune response) is detected by assaying antibody presence or level (e.g., presence or level of an anti-anelloviral vector antibody, e.g., presence or level of an antibody against an anelloviral vector as described herein) in a subject, e.g., 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 levels described in Kakkola et al. (2008; Virology 382: 182-189; incorporated herein by reference). Antibodies against an Anellovirus or an anelloviral vector based thereon can also be detected by methods in the art for detecting anti-viral antibodies, e.g., methods of detecting anti-AAV antibodies, e.g., as described in Calcedo et al. (2013; Front. Immunol. 4(341): 1-7; incorporated herein by reference).

[0241] A “subsequence” as used herein refers to a nucleic acid sequence or an amino acid sequence that is comprised in a larger nucleic acid sequence or amino acid sequence, respectively. In some instances, a subsequence may comprise a domain or functional fragment of the larger sequence. In some instances, the subsequence may comprise a fragment of the larger sequence capable of forming secondary and / or tertiary structures when isolated from the larger sequence similar to the secondary and / or tertiary structures formed by the subsequence when present with the remainder of the larger sequence. In some instances, a subsequence can be replaced by another sequence (e.g., a subsequence comprising an exogenous sequence or a sequence heterologous to the remainder of the larger sequence, e.g., a corresponding subsequence from a different Anellovirus).

[0242] As used herein, the term “surface moiety” refers to a moiety for which at least a portion is exposed on the exterior surface of a particle (e.g., exposed to the solution surrounding the particle). The surface moiety is generally attached, directly or indirectly, to a component of the proteinaceous exterior of the particle (e.g., an ORF1 molecule). In some instances, the surface moiety is covalently attached to the component of the proteinaceous exterior of the particle (e.g., the ORF1 molecule). In some instances, the surface moiety is noncovalently attached to the component of the proteinaceous exterior of the particle (e.g., the ORF1 molecule). In some instances, the surface moiety is bound to a binding moiety that is in turn attached (e.g., covalently or noncovalently) to the component of the proteinaceous exterior of the particle (e.g., the ORF1 molecule). In some instances, the surface moiety is comprised in an ORF1 molecule (e.g., is a heterologous domain of an ORF1 molecule). In some instances, a surface moiety is exogenous relative to an Anellovirus (e.g., the Anellovirus from which the ORF1 molecule was derived and / or an Anellovirus for which the ORF1 protein has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the ORF1 molecule). In some instances, a surface moiety is exogenous relative a target cell (e.g., a mammalian cell, e.g., a human cell) to be infected by the particle.

[0243] As used herein, “treatment”, "treating" and cognates thereof refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to preventing, minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy).

[0244] This invention relates generally to Anelloviridae family vectors (e.g., anelloviral vectors), e.g., synthetic Anelloviridae family vectors (e.g., anelloviral vectors), and uses thereof. The present disclosure provides Anelloviridae family vectors (e.g., anelloviral vectors), compositions comprising Anelloviridae family vectors (e.g., anelloviral vectors), and methods of making or using Anelloviridae family vectors (e.g., anelloviral vectors). Anelloviridae family vectors (e.g., anelloviral vectors) are generally useful as delivery vehicles, e.g., for delivering a therapeutic agent to a eukaryotic cell. Generally, an Anelloviridae family vector (e.g., anelloviral vector) will include a genetic element comprising a nucleic acid sequence (e.g., encoding an effector, e.g., an exogenous effector or an endogenous effector) encapsulated within a proteinaceous exterior. An Anelloviridae family vector (e.g., anelloviral vector) may include one or more deletions of sequences (e.g., regions or domains as described herein) relative to an Anellovirus sequence (e.g., as described herein). Anelloviridae family vectors (e.g., anelloviral vectors) can be used as a substantially non-immunogenic vehicle for delivering the genetic element, or an effector encoded therein (e.g., a polypeptide or nucleic acid effector, e.g., as described herein), into eukaryotic cells, e.g., to treat a disease or disorder in a subject comprising the cells.

[0245] TABLE OF CONTENTS

[0246] I. Anelloviridae Family Vectors (e.g., anelloviral vectors)

[0247] A. Anelloviridae Family Viruses (e.g., Anelloviruses) i. Nucleic acid sequences ii. Amino acid sequences encoded by nucleic acid sequences iii. Proteins comprising amino acid sequences iv. Polypeptides comprising amino acid sequences B. Capsid Proteins (e.g., 0RF1 molecules) i. Conserved ORF1 motif in N22 domain ii. Exemplary ORF1 sequences iii. Identification of ORF1 protein sequences

[0248] C. ORF2 molecules i. Conserved ORF2 motif

[0249] D. Genetic elements

[0250] E. Protein binding sequence

[0251] F. 5’ UTR Conserved Domains

[0252] G. GC-rich regions

[0253] H. Effectors

[0254] I. Regulatory Sequences

[0255] J. Surface Moieties

[0256] K. Aflibercept genetic elements and genetic element constructs

[0257] L. AAV-Anellovirus hybrid vectors

[0258] II. Compositions and Methods for Making Anelloviridae Family Vectors

[0259] A. Genetic Element Constructs i. Tandem constructs ii. Cis / trans constructs

[0260] B. Recombinase-based production of genetic elements and anello viral vectors i. Self-replicating rescue (SRR) constructs (e.g., SRR plasmids) ii. Exemplary site-specific recombinases and recombinase recognition sites

[0261] C. Host Cells and methods of using host cells for producing an anelloviral vector i. Introduction of genetic elements into host cells ii. Exemplary cell types

[0262] D. Culture Conditions

[0263] E. Harvest

[0264] F. In vitro assembly methods

[0265] G. In vitro circularization

[0266] H. Enrichment and Purification

[0267] III. Pharmaceutical Compositions

[0268] IV. Methods of use

[0269] A. Ocular Delivery Methods

[0270] B. Ocular Delivery Systems C. Dosing

[0271] V. Redosing

[0272] I. Anelloviridae family vectors (e.g., anelloviral vectors)

[0273] In some aspects, the invention described herein comprises compositions and methods of using and making an Anelloviridae family vector (e.g., anelloviral vector), Anelloviridae family vector (e.g., anelloviral vector) preparations, and therapeutic compositions. In some embodiments, the anelloviral vector has a sequence, structure, and / or function that is based on an Anelloviridae virus (e.g., an Anellovirus as described herein). It is understood that applicable embodiments described herein with respect to anelloviral vectors may also be applied o Anelloviridae family vectors (e.g., a vector based on or derived from a chicken anemia virus (CAV), e.g., as described herein). In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) comprises a nucleic acid or polypeptide comprising a sequence as shown in Table Al; or Table Nl, or fragments or portions thereof, or a reverse complement thereof, or other substantially non-pathogenic virus, e.g., a symbiotic virus, commensal virus, native virus. In some embodiments, an Anelloviridae family virus-based vector comprises at least one element exogenous to that Anelloviridae family virus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector disposed within a genetic element of the vector. In some embodiments, an Anelloviridae family virus-based vector comprises at least one element heterologous to another element from that Anelloviridae family virus, e.g., an effector-encoding nucleic acid sequence that is heterologous to another linked nucleic acid sequence, such as a promoter element. In some embodiments, an Anelloviridae family vector comprises a genetic element (e.g., circular DNA, e.g., single stranded DNA), which comprise at least one element that is heterologous relative to the remainder of the genetic element and / or the proteinaceous exterior (e.g., an exogenous element encoding an effector, e.g., as described herein). An Anelloviridae family vector may be a delivery vehicle (e.g., a substantially non- pathogenic delivery vehicle) for a payload into a host, e.g., a human. In some embodiments, the Anelloviridae family vector is capable of replicating in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the Anelloviridae family vector is substantially non-pathogenic and / or substantially non-integrating in the mammalian (e.g., human) cell. In some embodiments, the Anelloviridae family vector is substantially non-immunogenic in a mammal, e.g., a human. In some embodiments, the Anelloviridae family vector is replication-deficient. In some embodiments, the Anelloviridae family vector is replication-competent.

[0274] In one aspect, the invention includes an Anelloviridae family vector comprising: a) a genetic element comprising (i) a sequence encoding an exterior protein (e.g., a non- pathogenic exterior protein), (ii) an exterior protein binding sequence that binds the genetic element to the non-pathogenic exterior protein, and (iii) a sequence encoding an effector (e.g., an endogenous or exogenous effector); and b) a proteinaceous exterior that is associated with, e.g., envelops or encapsulates, the genetic element.

[0275] In some embodiments, the Anelloviridae family vector (e.g. anelloviral vector) includes sequences or expression products from (or having >70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology to) a non-enveloped, circular, single-stranded DNA virus. Animal circular singlestranded DNA viruses generally refer to a subgroup of single strand DNA (ssDNA) viruses, which infect eukaryotic non-plant hosts, and have a circular genome. Thus, animal circular ssDNA viruses are distinguishable from ssDNA viruses that infect prokaryotes (i.e. Microviridae and Inoviridae) and from ssDNA viruses that infect plants (i.e. Geminiviridae and Nanoviridae). They are also distinguishable from linear ssDNA viruses that infect non-plant eukaryotes (i.e. Parvoviridiae).

[0276] In some embodiments, the genetic element comprises a promoter element. In some embodiments, the promoter element is selected from an RNA polymerase Il-dependent promoter, an RNA polymerase Ill-dependent promoter, a PGK promoter, a CMV promoter, an EF-la promoter, an SV40 promoter, a CAGG promoter, or a UBC promoter, TTV viral promoters, Tissue specific, U6 (pollIII), minimal CMV promoter with upstream DNA binding sites for activator proteins (TetR-VP16, Gal4-VP16, dCas9-VP16, etc. ). In some embodiments, the promoter element comprises a TATA box. In some embodiments, the promoter element is endogenous to a wild-type Anelloviridae family virus (e.g., Anellovirus), e.g., as described herein.

[0277] In some embodiments, the genetic element comprises one or more of the following characteristics: single-stranded, circular', negative strand, and / or DNA. In some embodiments, the portions of the genetic element excluding the effector have a combined size of about 2.5-5 kb (e.g., about 2.8-4kb, about 2.8-3.2kb, about 3.6-3.9kb, or about 2.8-2.9kb), less than about 5kb (e.g., less than about 2.9kb, 3.2 kb, 3.6kb, 3.9kb, or 4kb), or at least 100 nucleotides (e.g., at least Ikb).

[0278] In some embodiments, a replication deficient, replication defective, or replication incompetent genetic element does not encode all of the necessary machinery or components required for replication of the genetic element. In some embodiments, a replication defective genetic element does not encode a replication factor. In some embodiments, a replication defective genetic element does not encode one or more ORFs (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF21 / 3 e.g., as described herein). In some embodiments, the machinery or components not encoded by the genetic element may be provided in trans (e.g., using a helper, e.g., a helper virus or helper plasmid, or encoded in a nucleic acid comprised by the host cell, e.g., integrated into the genome of the host cell), e.g., such that the genetic element can undergo replication in the presence of the machinery or components provided in trans. In some embodiments, a packaging deficient, packaging defective, or packaging incompetent genetic element cannot be packaged into a proteinaceous exterior (e.g., wherein the proteinaceous exterior comprises a capsid or a portion thereof, e.g., comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., as described herein). In some embodiments, a packaging deficient genetic element is packaged into a proteinaceous exterior at an efficiency less than 10% (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) compared to a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein). In some embodiments, the packaging defective genetic element cannot be packaged into a proteinaceous exterior even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that would permit packaging of the genetic element of a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein). In some embodiments, a packaging deficient genetic element is packaged into a proteinaceous exterior at an efficiency less than 10% (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) compared to a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein), even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that would permit packaging of the genetic element of a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein).

[0279] In some embodiments, a packaging competent genetic element can be packaged into a proteinaceous exterior (e.g., wherein the proteinaceous exterior comprises a capsid or a portion thereof, e.g., comprising a polypeptide encoded by an ORFlnucleic acid, e.g., as described herein). In some embodiments, a packaging competent genetic element is packaged into a proteinaceous exterior at an efficiency of at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or higher) compared to a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein). In some embodiments, the packaging competent genetic element can be packaged into a proteinaceous exterior in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that would permit packaging of the genetic element of a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein). In some embodiments, a packaging competent genetic element is packaged into a proteinaceous exterior at an efficiency of at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or higher) compared to a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein) in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that would permit packaging of the genetic element of a wild-type Anelloviridae family virus (e.g., Anellovirus) (e.g., as described herein). Anelloviridae Family Viruses (e.g., Anelloviruses)

[0280] In some embodiments, an Anelloviridae family vector, e.g., as described herein, comprises sequences or expression products derived from an Anellovirus . In some embodiments, an Anelloviridae family vector includes one or more sequences or expression products that are exogenous relative to the Anellovirus. In some embodiments, an Anelloviridae family vector includes one or more sequences or expression products that are endogenous relative to the Anellovirus. In some embodiments, an Anelloviridae family vector includes one or more sequences or expression products that are heterologous relative to one or more other sequences or expression products in the Anelloviridae family vector. Anelloviridae family viruses (e.g., Anellovirus) generally have single-stranded circular DNA genomes with negative polarity.

[0281] In some embodiments, the genetic element comprises a nucleotide sequence encoding an amino acid sequence or a functional fragment thereof or a sequence having at least about 60%, 70% 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences described herein, e.g., an Anellovirus amino acid sequence.

[0282] In some embodiments, an Anelloviridae family vector as described herein comprises one or more nucleic acid molecules (e.g., a genetic element as described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus sequence, e.g., as described herein, or a fragment thereof. In embodiments, the Anelloviridae family vector comprises a nucleic acid sequence selected from a sequence as shown in Table Nl, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, or a reverse complement thereof. In embodiments, the Anelloviridae family vector comprises a polypeptide comprising a sequence as shown in Table Al, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0283] In some embodiments, an Anelloviridae family vector as described herein comprises one or more nucleic acid molecules (e.g., a genetic element as described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of a TATA box, cap site, initiator element, transcriptional start site, 5’ UTR conserved domain, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, three open-reading frame region, poly(A) signal, GC-rich region, or any combination thereof, of any of the Anelloviridae family viruses (e.g., Anellovirus) described herein (e.g., an Anelloviridae family virus (e.g., Anellovirus) sequence as annotated, or as encoded by a sequence listed, in Table Nl, or a reverse complement thereof. In some embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein, e.g., an ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3 sequence of any of the Anelloviruses described herein (e.g., an Anelloviridae family virus (e.g., Anellovirus) sequence as annotated, or as encoded by a sequence listed, in Table Nl). In embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae family virus (e.g., Anellovirus) ORF1 protein (e.g., an ORF1 amino acid sequence as shown in Table Al, or an ORF1 amino acid sequence encoded by a nucleic acid sequence as shown in Table Nl).

[0284] Nucleic acid sequences

[0285] In some 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF1 nucleotide sequence of Table Nl. In some 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF2 nucleotide sequence of Table Nl. In some 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF3 nucleotide sequence of Table Nl. In some 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 to the Anelloviridae family virus (e.g., Anellovirus) GC-rich region nucleotide sequence of Table Nl , or a reverse complement thereof. In some 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 to the Anelloviridae family virus (e.g., Anellovirus) 5’ UTR conserved domain nucleotide sequence of Table Nl, or a reverse complement thereof.

[0286] In the field of Anellovirus research, the sequences of particular Anellovirus strains are commonly provided as a positive strand sequence for convenience of indicating the protein-coding regions. Consequently, unless specified otherwise, this disclosure also adopts the convention of providing the positive strand sequence corresponding to a particular Anellovirus genome or elements thereof. However, as known in the art and as described herein, a genetic element of an anelloviral vector is typically a negative strand, such that the negative strand is enclosed into the proteinaceous exterior. The positive strand sequences of Table Nl, for example, can readily be converted to the corresponding negative strand sequence, for example, using tools such as the Reverse Complement tool on Bioinformatics.org available on the world wide web at www.bioinformatics.org / sms / rev_comp.html. Consequently, a 5’ UTR conserved domain of a genetic element as described herein may comprise the reverse complement of a sequence annotated as a 5’ UTR conserved domain (e.g., in Table Nl). Similarly, a GC-rich region of a genetic element as described herein may comprise the reverse complement of a sequence annotated as a GC-rich region (e.g., in Table Nl). Likewise, an Anellovirus NCR of a genetic element may comprise the reverse complement of the relevant portion of a sequence of Table Nl.

[0287] In some embodiments, the Anellovirus is a Betatorquevirus.

[0288] Amino acid sequences encoded by nucleic acid sequences

[0289] In 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF1 amino acid sequence of Table Al. In 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF2 amino acid sequence of Table Al. In 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 to the Anelloviridae family virus (e.g., Anellovirus) ORF3 amino acid sequence of Table Al. In some embodiments, the nucleic acid is a genetic element construct or a construct for providing the polypeptide (e.g., an ORF1 molecule and / or an ORF2 molecule) in trans.

[0290] Proteins comprising amino acid sequences

[0291] In some embodiments, the Anelloviridae family vector described herein comprises an Anellovirus ORF or ORF molecule (e.g., an Anellovirus ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2) includes a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a corresponding Anellovirus ORF sequence, e.g., as described herein). In embodiments, the Anelloviridae family vector described herein comprises a protein having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF1 amino acid sequence of Table Al. In embodiments, the Anelloviridae family vector described herein comprises a protein having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF2 amino acid sequence of Table Al. In embodiments, the Anelloviridae family vector described herein comprises a protein having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF3 amino acid sequence of Table Al.

[0292] In some embodiments, an ORF1 molecule (e.g., comprised in the Anelloviridae family vector) comprises a polypeptide encoded by the Anelloviridae family virus (e.g., Anellovirus) ORF1 nucleic acid sequence of Table Nl. In some embodiments, the ORF1 molecule (e.g., comprised in the Anelloviridae family vector) comprises an Anelloviridae family virus (e.g., Anellovirus) ORF1 protein of Table Al or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof. In some embodiments, an ORF2 molecule (e.g., comprised in the Anelloviridae family vector) comprises a polypeptide encoded by the Anelloviridae family virus (e.g., Anellovirus) ORF2 nucleic acid sequence of Table Nl. In some embodiments, the ORF2 molecule (e.g., comprised in the Anelloviridae family vector) comprises an Anelloviridae family virus (e.g., Anellovirus) ORF2 protein of Table Al or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof. In some embodiments, an ORF3 molecule (e.g., comprised in the Anelloviridae family vector) comprises a polypeptide encoded by the Anelloviridae family virus (e.g., Anellovirus) ORF3 nucleic acid sequence of Table Nl. In some embodiments, the ORF3 molecule (e.g., comprised in the Anelloviridae family vector) comprises an Anelloviridae family virus (e.g., Anellovirus) ORF3 protein of Table Al or a splice variant or post- translationally processed (e.g., proteolytically processed) variant thereof.

[0293] Polypeptides comprising amino acid sequences

[0294] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae family virus (e.g., Anellovirus) ORF1 amino acid sequence described herein. In embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF1 amino acid sequence of Table Al.

[0295] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF1 nucleic acid described herein. In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF1 nucleic acid as listed in Table Nl.

[0296] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae family virus (e.g., Anellovirus) 0RF2 amino acid sequence described herein. In embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF2 amino acid sequence of Table Al.

[0297] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF2 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF2 nucleic acid described herein. In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF2 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF2 nucleic acid as listed in Table Nl.

[0298] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae family virus (e.g., Anellovirus) ORF3 amino acid sequence described herein. In embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anelloviridae family virus (e.g., Anellovirus) ORF3 amino acid sequence of Table Al.

[0299] In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF3 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF3 nucleic acid described herein. In some embodiments, the polypeptide described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF3 molecule encoded by an Anelloviridae family virus (e.g., Anellovirus) ORF3 nucleic acid as listed in Table Nl.

[0300] In some embodiments, the polypeptide comprises an amino acid sequence (e.g., an ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3 sequence) as shown in Table Al, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0301] Ringl9 is an Anellovirus that was isolated from RPE cells. In some embodiments, a method described herein comprises delivering an anelloviral vector (e.g., an anelloviral vector having sequence similarity to Ringl9) to eye tissue (e.g., to the eye of a subject), for example, to retinal tissue and / or RPE cells. Table Nl. Exemplary Anellovirus nucleic acid sequence (Betatorquevirus). Table Al. Exemplary Anellovirus amino acid sequence (Betatorquevirus) Capsid Proteins (e.g., ORF1 molecules)

[0302] In some embodiments, the anello viral vector comprises an ORF1 molecule and / or a nucleic acid encoding an ORF1 molecule.

[0303] Generally, an ORF1 molecule comprises a polypeptide having the structural features and / or activity of an Anellovirus ORF1 protein (e.g., an Anellovirus ORF1 protein as described herein, e.g., as listed in Table Al), or a functional fragment thereof. In some embodiments, the ORF1 molecule comprises a truncation relative to an Anellovirus ORF1 protein (e.g., an Anellovirus ORF1 protein as described herein, e.g., as listed in Table Al). In some embodiments, the ORF1 molecule is truncated by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids of the Anellovirus ORF1 protein. In some embodiments, an ORF1 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anello virus 0RF1 protein sequence as shown in Table Al. In some embodiments, an ORF1 molecule comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an Betatorquevirus ORF1 protein, e.g., as described herein. An ORF1 molecule can generally bind to a nucleic acid molecule, such as DNA (e.g., a genetic element, e.g., as described herein). In some embodiments, an ORF1 molecule localizes to the nucleus of a cell. In certain embodiments, an ORF1 molecule localizes to the nucleolus of a cell. In some embodiments, an ORF1 molecule is encoded by an ORF1 nucleic acid. In some embodiments, the ORF1 nucleic acid comprises an antisense strand, which can be directly transcribed to produce rnRNA encoding the ORF1 molecule. In some embodiments, the ORF1 nucleic acid comprises a sense strand.

[0304] Without wishing to be bound by theory, an ORF1 molecule may be capable of binding to other ORF1 molecules, e.g., to form a proteinaceous exterior (e.g., as described herein). Such an ORF1 molecule may be described as having the capacity to form a capsid. In some embodiments, the proteinaceous exterior may encapsulate a nucleic acid molecule (e.g., a genetic element as described herein). In some embodiments, a plurality of ORF1 molecules may form a multimer, e.g., to produce a proteinaceous exterior. In some embodiments, the multimer may be a homomultimer. In other embodiments, the multimer may be a heteromultimer (e.g., comprising a plurality of distinct ORF1 molecules). It is also contemplated that an ORF1 molecule may have replicase activity.

[0305] An ORF1 molecule may, in some embodiments, comprise one or more of: a first region comprising an arginine rich region, e.g., a region having at least 60% basic residues (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% basic residues; e.g., between 60%-90%, 60%-80%, 70%-90%, or 70-80% basic residues), and a second region comprising jelly-roll domain, e.g., at least six beta strands (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands).

[0306] Arginine-rich region

[0307] An arginine rich region (e.g., comprised an ORF1 molecule as described herein) has at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity to an arginine -rich region sequence described herein or a sequence of at least about 40 amino acids comprising at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof).

[0308] In some embodiments, an ORF1 molecule as described herein comprises a deletion or truncation of an arginine -rich region. In some embodiments, the entire arginine-rich region is deleted. In some embodiments, a portion of the arginine-rich region (e.g., a N-terminal portion of the structural arginine- rich region) is deleted. In embodiments, the ORF1 molecule does not comprise an Anellovirus ORF1 arginine-rich region, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, an ORF1 molecule having a deletion or truncation of the arginine -rich region further comprises a deletion or truncation of at least a portion of a C-terminal domain (e.g., as described herein).

[0309] Jelly Roll domain

[0310] A jelly-roll domain or region (e.g., comprised an ORF1 molecule as described herein) comprises (e.g., consists of) a polypeptide (e.g., a domain or region comprised in a larger polypeptide) comprising one or more (e.g., 1, 2, or 3) of the following characteristics:

[0311] (i) at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) of the amino acids of the jelly-roll domain are part of one or more P-sheets;

[0312] (ii) the secondary structure of the jelly-roll domain comprises at least four (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12) -strands; and / or

[0313] (iii) the tertiary structure of the jelly-roll domain comprises at least two (e.g., at least 2, 3, or 4) P- sheets; and / or

[0314] (iv) the jelly-roll domain comprises a ratio of P-sheets to a-helices of at least 2:1, 3:1, 4:1, 5:1, 6:l, 7:l, 8:l, 9:l, or l0:l.

[0315] In certain embodiments, a jelly-roll domain comprises two p-sheets.

[0316] In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the P-sheets comprises about eight (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12) P-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the P-sheets comprises eight P-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the P-sheets comprises seven P-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the P-sheets comprises six P-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the p-sheets comprises five p-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the P-sheets comprises four P- strands.

[0317] In some embodiments, the jelly-roll domain comprises a first P-sheet in antiparallel orientation to a second P-sheet. In certain embodiments, the first P-sheet comprises about four (e.g., 3, 4, 5, or 6) P- strands. In certain embodiments, the second P-sheet comprises about four (e.g., 3, 4, 5, or 6) P-strands. In embodiments, the first and second p-sheet comprise, in total, about eight (e.g., 6, 7, 8, 9, 10, 11, or 12) P-strands.

[0318] In certain embodiments, a jelly-roll domain is a component of a capsid protein (e.g., an ORF1 molecule as described herein). In certain embodiments, a jelly-roll domain has self-assembly activity. In some embodiments, a polypeptide comprising a jelly-roll domain binds to another copy of the polypeptide comprising the jelly-roll domain. In some embodiments, a jelly-roll domain of a first polypeptide binds to a jelly-roll domain of a second copy of the polypeptide.

[0319] Other Subdomains

[0320] An 0RF1 molecule may also include a third region comprising the structure or activity of an Anellovirus N22 domain (e.g., as described herein, e.g., an N22 domain from an Anellovirus ORF1 protein as described herein).

[0321] An ORF1 molecule may also include a fourth region comprising the structure or activity of an Anellovirus C-terminal domain (CTD) (e.g., as described herein, e.g., a CTD from an Anellovirus ORF1 protein as described herein).

[0322] In some embodiments, an ORF1 molecule as described herein comprises a deletion or truncation of a C-terminal domain (CTD). In some embodiments, the entire CTD is deleted. In some embodiments, a portion of the CTD (e.g., a C-terminal portion of the CTD) is deleted. In embodiments, the ORF1 molecule does not comprise an Anellovirus ORF1 CTD, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, an ORF1 molecule having a deletion or truncation of the CTD further comprises a deletion or truncation of at least a portion of an arginine -rich region (e.g., as described herein).

[0323] In some embodiments, the ORF1 molecule comprises, in N-terminal to C-terminal order, the first, second, third, and fourth regions.

[0324] The ORF1 molecule may, in some embodiments, further comprise a hypervariable region (HVR), e.g., an HVR from an Anellovirus ORF1 protein, e.g., as described herein. In some embodiments, the HVR is positioned between the second region and the third region. In some embodiments, the HVR comprises at least about 55 (e.g., at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (e.g., about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55- 140, or 60-140 amino acids).

[0325] In some embodiments, the first region can bind to a nucleic acid molecule (e.g., DNA). In some embodiments, the basic residues are selected from arginine, histidine, or lysine, or a combination thereof. In some embodiments, the first region comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% arginine residues (e.g., between 60%-90%, 60%-80%, 70%-90%, or 70-80% arginine residues). In some embodiments, the first region comprises about 30-120 amino acids (e.g., about 40-120, 40-100, 40- 90, 40-80, 40-70, 50-100, 50-90, 50-80, 50-70, 60-100, 60-90, or 60-80 amino acids). In some embodiments, the first region comprises the structure or activity of a viral ORF1 arginine-rich region (e.g., an arginine-rich region from an Anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the first region comprises a nuclear localization signal. In some embodiments, the second region comprises a jelly-roll domain, e.g., the structure or activity of a viral ORF1 jelly-roll domain (e.g., a jelly -roll domain from an Anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the second region is capable of binding to the second region of another ORF1 molecule, e.g., to form a proteinaceous exterior (e.g., capsid) or a portion thereof.

[0326] In some embodiments, the fourth region is exposed on the surface of a proteinaceous exterior (e.g., a proteinaceous exterior comprising a multimer of ORF1 molecules, e.g., as described herein).

[0327] In some embodiments, the first region, second region, third region, fourth region, and / or HVR each comprise fewer than four (e.g., 0, 1, 2, or 3) beta sheets.

[0328] In some embodiments, one or more of the first region, second region, third region, fourth region, and / or HVR may be replaced by a heterologous amino acid sequence (e.g., the corresponding region from a heterologous ORF1 molecule). In some embodiments, the heterologous amino acid sequence has a desired functionality, e.g., as described herein.

[0329] In some embodiments, the ORF1 molecule comprises a plurality of conserved motifs (e.g., motifs comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids). In some embodiments, the conserved motifs may show 60, 70, 80, 85, 90, 95, or 100% sequence identity to an ORF1 protein of one or more wild-type Anellovirus clades (e.g., Betatorquevirus). In some embodiments, the conserved motifs each have a length between 1-1000 (e.g., between 5-10, 5-15, 5-20, 10-15, 10-20, 15-20, 5-50, 5-100, 10-50, 10-100, 10-1000, 50-100, 50-1000, or 100-1000) amino acids. In certain embodiments, the conserved motifs consist of about 2-4% (e.g., about 1-8%, 1-6%, 1-5%, 1-4%, 2-8%, 2-6%, 2-5%, or 2-4%) of the sequence of the ORF1 molecule, and each show 100% sequence identity to the corresponding motifs in an ORF1 protein of the wild-type Anellovirus clade. In certain embodiments, the conserved motifs consist of about 5-10% (e.g., about 1- 20%, 1-10%, 5-20%, or 5-10%) of the sequence of the ORF1 molecule, and each show 80% sequence identity to the corresponding motifs in an ORF1 protein of the wild-type Anellovirus clade. In certain embodiments, the conserved motifs consist of about 10-50% (e.g., about 10-20%, 10-30%, 10-40%, 10- 50%, 20-40%, 20-50%, or 30-50%) of the sequence of the ORF1 molecule, and each show 60% sequence identity to the corresponding motifs in an ORF1 protein of the wild-type Anellovirus clade.

[0330] In some embodiments, an ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF1 protein, e.g., as described herein (e.g., as shown in Table Al).

[0331] Conserved ORF1 Motif in N22 Domain

[0332] In some embodiments, a polypeptide (e.g., an ORF1 molecule) described herein comprises the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829), wherein X" is a contiguous sequence of any n amino acids. For example, X2indicates a contiguous sequence of any two amino acids. In some embodiments, the YNPX2DXGX2N (SEQ ID NO: 829) is comprised within the N22 domain of an ORF1 molecule, e.g., as described herein. In some embodiments, a genetic element described herein comprises a nucleic acid sequence (e.g., a nucleic acid sequence encoding an ORF1 molecule, e.g., as described herein) encoding the amino acid sequence YNPX2DXGX2N (SEQ ID NO: 829), wherein X" is a contiguous sequence of any n amino acids.

[0333] In some embodiments, a polypeptide (e.g., an ORF1 molecule) comprises a conserved secondary structure, e.g., flanking and / or comprising a portion of the YNPX2DXGX2N (SEQ ID NO: 829) motif, e.g., in an N22 domain. In some embodiments, the conserved secondary structure comprises a first beta strand and / or a second beta strand. In some embodiments, the first beta strand is about 5-6 (e.g., 3, 4, 5, 6, 7, or 8) amino acids in length. In some embodiments, the first beta strand comprises the tyrosine (Y) residue at the N-terminal end of the YNPX2DXGX2N (SEQ ID NO: 829) motif. In some embodiments, the YNPX2DXGX2N (SEQ ID NO: 829) motif comprises a random coil (e.g., about 8-9 amino acids of random coil). In some embodiments, the second beta strand is about 7-8 (e.g., 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second beta strand comprises the asparagine (N) residue at the C-terminal end of the YNPX2DXGX2N (SEQ ID NO: 829) motif.

[0334] Exemplary ORF1 Sequences

[0335] In some embodiments, a polypeptide (e.g., an ORF1 molecule) described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anellovirus ORF1 subsequences, e.g., as described herein). In some embodiments, an Anelloviridae family vector (e.g., anelloviral vector) described herein comprises an ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anellovirus ORF1 subsequences, e.g., as described herein. In some embodiments, an anelloviral vector described herein comprises a nucleic acid molecule (e.g., a genetic element) encoding an ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anellovirus ORF1 subsequences, e.g., as described herein.

[0336] In some embodiments, the one or more Anellovirus ORF1 subsequences comprises one or more of an arginine (Arg)-rich domain, a jelly-roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD) (e.g., as listed herein), or sequences having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the ORF1 molecule comprises a plurality of subsequences from different Anelloviruses. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an N22 domain, and a CTD from one Anelloviridae family vims (e.g., Anellovirus), and an HVR from another. In some embodiments, the ORF1 molecule comprises one or more of a jelly-roll domain, an HVR, an N22 domain, and a CTD from one Anelloviridae family vims (e.g., Anellovirus), and an Argrich domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Argrich domain, an HVR, an N22 domain, and a CTD from one Anelloviridae family virus (e.g., Anellovirus), and a jelly-roll domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an HVR, and a CTD from one Anelloviridae family virus (e.g., Anellovirus), and an N22 domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an HVR, and an N22 domain from one Anelloviridae family virus (e.g., Anellovirus), and a CTD from another.

[0337] Identification of ORF1 protein sequences

[0338] In some embodiments, an ORF1 protein sequence, or a nucleic acid sequence encoding an ORF1 protein, can be identified from the genome of an Anelloviridae family virus, e.g., an Anellovirus (e.g., a putative Anelloviridae family virus genome identified, for example, by nucleic acid sequencing techniques, e.g., deep sequencing techniques). In some embodiments, an ORF1 protein sequence is identified by one or more (e.g., 1, 2, or all 3) of the following selection criteria:

[0339] (i) Length Selection: Protein sequences (e.g., putative ORF1 sequences passing the criteria described in (ii) or (iii) below) may be size-selected for those greater than about 600 amino acid residues to identify putative ORF1 proteins. In some embodiments, an ORF1 protein sequence is at least about 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acid residues in length. In some embodiments, an Alphatorquevirus ORF1 protein sequence is at least about 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 900, or 1000 amino acid residues in length. In some embodiments, a Betatorque virus ORF1 protein sequence is at least about 650, 660, 670, 680, 690, 700, 750, 800, 900, or 1000 amino acid residues in length. In some embodiments, a Gammatorquevirus ORF1 protein sequence is at least about 650, 660, 670, 680, 690, 700, 750, 800, 900, or 1000 amino acid residues in length. In some embodiments, a nucleic acid sequence encoding an ORF1 protein is at least about 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 nucleotides in length. In some embodiments, a nucleic acid sequence encoding an Alphatorquevirus ORF1 protein sequence is at least about 2100, 2150, 2200, 2250, 2300, 2400, or 2500 nucleotides in length. In some embodiments, a nucleic acid sequence encoding a Betatorquevirus ORF1 protein sequence is at least about 1900, 1950, 2000, 2500, 2100, 2150, 2200, 2250, 2300, 2400, or 2500 or 1000 nucleotides in length. In some embodiments, a nucleic acid sequence encoding a Gammatorquevirus ORF1 protein sequence is at least about 1900, 1950, 2000, 2500, 2100, 2150, 2200, 2250, 2300, 2400, or 2500 or 1000 nucleotides in length. (ii) Presence of ORFl motif: Protein sequences (e.g., putative 0RF1 sequences passing the criteria described in (i) above or (iii) below) may be filtered to identify those that contain the conserved 0RF1 motif in the N22 domain described above. In some embodiments, a putative Anellovirus ORF1 sequence comprises the sequence YNPXXDXGXXN (SEQ ID NO: 829). In some embodiments, a putative Anellovirus ORF1 sequence comprises the sequence Y[NCS]PXXDX[GASKR]XX[NTSVAK].

[0340] (Hi) Presence of arginine-rich region: Protein sequences (e.g., putative ORF1 sequences passing the criteria described in (i) and / or (ii) above) may be filtered for those that include an arginine-rich region (e.g., as described herein). In some embodiments, a putative ORF1 sequence comprises a contiguous sequence of at least about 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids that comprises at least 30% (e.g., at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50%) arginine residues. In some embodiments, a putative ORF1 sequence comprises a contiguous sequence of about 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 amino acids that comprises at least 30% (e.g., at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50%) arginine residues. In some embodiments, the arginine-rich region is positioned at least about 30, 40, 50, 60, 70, or 80 amino acids downstream of the start codon of the putative ORF1 protein. In some embodiments, the arginine-rich region is positioned at least about 50 amino acids downstream of the start codon of the putative ORF1 protein.

[0341] In some embodiments, an ORF1 protein is identified in an Anellovirus genome sequence as described in Example 36 of PCT Publication No. WO2020 / 123816 (incorporated herein by reference in its entirety).

[0342] ORF2 molecules

[0343] In some embodiments, the anelloviral vector comprises an ORF2 molecule and / or a nucleic acid encoding an ORF2 molecule. Generally, an ORF2 molecule comprises a polypeptide having the structural features and / or activity of an Anellovirus ORF2 protein (e.g., an Anellovirus ORF2 protein as described herein, e.g., as listed in Table Al), or a functional fragment thereof. In some embodiments, an ORF2 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF2 protein sequence as shown in Table Al. In some embodiments, an ORF2 molecule is encoded by an ORF2 nucleic acid. In some embodiments, the ORF2 nucleic acid comprises an antisense strand, which can be directly transcribed to produce mRNA encoding the ORF2 molecule. In some embodiments, the ORF2 nucleic acid comprises a sense strand.

[0344] In some embodiments, an ORF2 molecule has a length of 250 or fewer amino acids (e.g., about 150-200 amino acids). In some embodiments, an ORF2 molecule has a length of about 50-150 amino acids. In some embodiments, an ORF2 molecule has a length of about 100-200 amino acids (e.g., about 100-150 amino acids). In some embodiments, the ORF2 molecule comprises a helix-turn-helix motif (e.g., a helix-turn-helix motif comprising two alpha helices flanking a turn region). In some embodiments, the ORF2 molecule does not comprise the amino acid sequence of the ORF2 protein of TTV isolate TA278 or TTV isolate SANBAN. In some embodiments, an ORF2 molecule has protein phosphatase activity. In some embodiments, an ORF2 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF2 protein, e.g., as described herein (e.g., as shown in Table Al).

[0345] Conserved ORF2 Motif

[0346] In some embodiments, a polypeptide (e.g., an ORF2 molecule) described herein comprises the amino acid sequence [W / F]X7HX3CX'CXSH (SEQ ID NO: 949), wherein X" is a contiguous sequence of any n amino acids. In embodiments, X7indicates a contiguous sequence of any seven amino acids. In some embodiments, X3indicates a contiguous sequence of any three amino acids. In some embodiments, X1indicates any single amino acid. In some embodiments, X5indicates a contiguous sequence of any five amino acids. In some embodiments, the [W / F] can be either tryptophan or phenylalanine. In some embodiments, the [W / F]X7HX3CX'CX5H (SEQ ID NO: 949) is comprised within the N22 domain of an ORF2 molecule, e.g., as described herein. In some embodiments, a genetic element described herein comprises a nucleic acid sequence (e.g., a nucleic acid sequence encoding an ORF2 molecule, e.g., as described herein) encoding the amino acid sequence [W / F]X7HX3CX*CX5H (SEQ ID NO: 949), wherein X” is a contiguous sequence of any n amino acids.

[0347] Genetic Elements

[0348] In some embodiments, the Anelloviridae family vector (e.g., anello viral vector) comprises a genetic element. In some embodiments, the genetic element has one or more of the following characteristics: is substantially non-integrating with a host cell’s genome, is an episomal nucleic acid, is a single stranded DNA, is circular, is about 1 to 10 kb, exists within the nucleus of the cell, can be bound by endogenous proteins, produces an effector, such as a polypeptide or nucleic acid (e.g., an RNA, iRNA, microRNA) that targets a gene, activity, or function of a host or target cell. In one embodiment, the genetic element is a substantially non-integrating DNA. In some embodiments, the genetic element comprises a packaging signal, e.g., a sequence that binds a capsid protein. In some embodiments, outside of the packaging or capsid-binding sequence, the genetic element has less than 70%. 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to a wild type Anellovirus nucleic acid sequence, e.g., has less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to an Anellovirus nucleic acid sequence, e.g., as described herein. In some embodiments, outside of the packaging or capsid-binding sequence, the genetic element has less than 500, 450, 400, 350, 300, 250, 200, 150, or 100 contiguous nucleotides that are at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an Anellovirus nucleic acid sequence. In certain embodiments, the genetic element is a circular, single stranded DNA that comprises a promoter sequence, a sequence encoding a therapeutic effector, and a capsid binding protein. In some embodiments, the genetic element may comprise other sequences that include DNA, RNA, or artificial nucleic acids.

[0349] In some embodiments, the genetic element has at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus nucleic acid sequence, e.g., as described herein (e.g., as described in Table Nl), or a fragment thereof, or a reverse complement thereof, or encodes an amino acid sequence having at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus amino acid sequence (e.g., as described Table Al), or a fragment thereof. In some embodiments, the genetic element comprises a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a payload), e.g., a polypeptide effector (e.g., a protein) or nucleic acid effector (e.g., a non-coding RNA, e.g., a miRNA, siRNA, mRNA, IncRNA, RNA, DNA, an antisense RNA, gRNA).

[0350] In some embodiments, the genetic element has a length less than 20kb (e.g., less than about 19kb, 18kb, 17kb, 16kb, 15kb, 14kb, 13kb, 12kb, l lkb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, Ikb, or less). In some embodiments, the genetic element has, independently or in addition to, a length greater than 1000b (e.g., at least about l.lkb, 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, 2.8kb, 2.9kb, 3kb, 3. Ikb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4. Ikb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5kb, or greater). In some embodiments, the genetic element has a length of about 2.5-4.6, 2.8-4.0, 3.0-3.8, or 3.2-3.7 kb. In some embodiments, the genetic element has a length of about 1.5-2.0, 1.5-2.5, 1.5-3.0, 1.5-3.5, 1.5-3.8, 1.5-3.9, 1.5-4.0, 1.5-4.5, or 1.5-5.0 kb. In some embodiments, the genetic element has a length of about 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-3.8, 2.0-3.9, 2.0-4.0, 2.0-4.5, or 2.0-5.0 kb. In some embodiments, the genetic element has a length of about 2.5-3.0, 2.5-3.5, 2.5-3.8, 2.5-3.9, 2.5-4.0, 2.5-4.5, or 2.5-5.0 kb. In some embodiments, the genetic element has a length of about 3.0-5.0, 3.5-5.0, 4.0-5.0, or 4.5-5.0 kb. In some embodiments, 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.

[0351] In some embodiments, the genetic element comprises one or more of the features described herein, e.g., a sequence encoding a substantially non-pathogenic protein, a protein binding sequence, one or more sequences encoding a regulatory nucleic acid, one or more regulatory sequences, one or more sequences encoding a replication protein, and other sequences. In some embodiments, the substantially non-pathogenic protein comprises 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 to any one of the amino acid sequences described herein, an Anellovirus amino acid sequence, e.g., as listed in any of Table Al.

[0352] In some embodiments, the double-stranded circular DNA and / or the genetic element does not comprise one or more plasmid elements (e.g., an origin of replication or a selectable marker, e.g., a resistance gene). In some embodiments, the double-stranded circular DNA and / or the genetic element does not comprise a plasmid backbone. In some embodiments, the double-stranded circular DNA and / or the genetic element does not comprise one or more bacterial plasmid elements (e.g., a bacterial origin of replication or a selectable marker, e.g., a bacterial resistance gene). In some embodiments, the doublestranded circular- DNA and / or the genetic element does not comprise a bacterial plasmid backbone. In some embodiments, the double-stranded circular- DNA and / or the genetic element does not comprise one or more mammalian plasmid elements (e.g., a mammalian origin of replication or a selectable marker, e.g., a mammalian resistance gene). In some embodiments, the double-stranded circular DNA and / or the genetic element does not comprise a mammalian plasmid backbone. In some embodiments, the doublestranded circular DNA and / or the genetic element does not comprise one or more insect plasmid elements (e.g., an insect origin of replication or a selectable marker, e.g., an insect resistance gene). In some embodiments, the double-stranded circular DNA and / or the genetic element does not comprise an insect plasmid backbone.

[0353] In some embodiments, a genetic element comprises a sequence encoding an effector (e.g., an exogenous effector). In some embodiments, the effector-encoding sequence is inserted into an Anellovirus genome sequence (e.g., as described herein). In some embodiments, the effector-encoding sequence replaces a contiguous sequence (e.g., of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides) from the Anellovirus genome sequence. In some embodiments, the effector-encoding sequence replaces a TATA box, cap site, transcriptional start site, 5’ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region sequence, or a portion thereof (e.g., a portion consisting of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides), e.g., as described herein, or a sequence having at least 70% 80%, 85%, 90% 95%, 96%, 97%, 98% and 99% nucleotide sequence identity thereto.

[0354] In some embodiments, the sequence of a first nucleic acid element comprised in a genetic element (e.g., a TATA box, cap site, transcriptional start site, 5’ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) overlaps with the sequence of a second nucleic acid element (e.g., a TATA box, cap site, transcriptional start site, 5’ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region), e.g., by at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides. In some embodiments, the sequence of a first nucleic acid element comprised in a genetic element (e.g., a TATA box, cap site, transcriptional start site, 5’ UTR, open reading frame (ORF), poly(A) signal, or GC- rich region) does not overlap with the sequence of a second nucleic acid element (e.g., a TATA box, cap site, transcriptional start site, 5’ UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0355] Protein Bindins Sequence

[0356] In some embodiments, the genetic element encodes a protein binding sequence that binds to the substantially non-pathogenic protein. In some embodiments, the protein binding sequence facilitates packaging the genetic element into the proteinaceous exterior. In some embodiments, the protein binding sequence specifically binds an arginine-rich region of the substantially non-pathogenic protein. In some embodiments, the genetic element comprises a protein binding sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a 5’ UTR conserved domain or GC-rich domain of an Anellovirus sequence (e.g., to the reverse complement of the sequence annotated in Table Nl).

[0357] In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of the sequence annotated as the 5’ UTR conserved domain nucleotide sequence of Table Nl. In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of the sequence annotated as the GC-rich domain nucleotide sequence of Table Nl.

[0358] 5’ UTR Conserved Domains

[0359] A genetic element may include an Anellovirus 5’ UTR conserved domain. Typically, the negative strand comprising the Anellovirus 5’ UTR conserved domain is packaged into a particle (e.g., an Anelloviridae family vector as described herein. In some embodiments, the Anellovirus 5’ UTR conserved domain is a wild-type Anellovirus 5’ UTR conserved domain. In some embodiments, the Anellovirus 5’ UTR conserved domain is an engineered Anellovirus 5’ UTR conserved domain having a nucleic acid sequence with at least one difference relative to the closest wild-type Anellovirus 5’ UTR conserved domain sequence. In some embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of the sequence annotated as 5’ UTR conserved domain nucleotide sequence of Table Nl or Table 38. Table 38. Exemplary 5’ UTR sequences from Anelloviruses.

[0360] Identification of 5' UTR sequences

[0361] In some embodiments, an Anelloviridae family virus (e.g., Anellovirus) 5’ UTR sequence can be identified within the genome of an Anelloviridae family virus (e.g., Anellovirus) (e.g., a putative Anelloviridae family virus genome identified, for example, by nucleic acid sequencing techniques, e.g., deep sequencing techniques). In some embodiments, an Anelloviridae family virus (e.g., Anellovirus) 5’ UTR sequence is identified by one or both of the following steps:

[0362] (i) Identification of circularization junction point: In some embodiments, a 5’ UTR will be positioned near a circularization junction point of a full-length, circularized Anelloviridae family virus (e.g., Anellovirus) genome. A circularization junction point can be identified, for example, by identifying overlapping regions of the sequence. In some embodiments, an overlapping region of the sequence can be trimmed from the sequence to produce a full-length Anelloviridae family virus (e.g., Anellovirus) genome sequence that has been circularized. In some embodiments, a genome sequence is circularized in this manner using software. Without wishing to be bound by theory, computationally circularizing a genome may result in the start position for the sequence being oriented in a non-biological. Landmarks within the sequence can be used to re -orient sequences in the proper direction. For example, landmark sequence may include sequences having substantial homology to one or more elements within an Anelloviridae family virus (e.g., Anellovirus) genome as described herein (e.g., one or more of a TATA box, cap site, initiator element, transcriptional start site, 5' UTR conserved domain, ORF1, ORF1 / 1, 0RF1 / 2, 0RF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, three open-reading frame region, poly(A) signal, or GC-rich region of an Anelloviridae family virus (e.g., Anellovirus), e.g., as described herein).

[0363] (ii) Identification of 5’ UTR sequence: Once a putative Anelloviridae family virus (e.g., Anellovirus) genome sequence has been obtained, the sequence (or portions thereof, e.g., having a length between about 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides) can be compared to one or more Anelloviridae family virus (e.g., Anellovirus) 5’ UTR sequences (e.g., as described herein) to identify sequences having substantial homology thereto. In some embodiments, a putative Anelloviridae family virus (e.g., Anellovirus) 5’ UTR region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae family virus (e.g., Anellovirus) 5’ UTR sequence as described herein.

[0364] GC-Rich Regions

[0365] A genetic element may include an Anellovirus GC-rich region. Typically, the negative strand comprising the Anellovirus GC-rich region is packaged into a particle (e.g., an Anelloviridae family vector as described herein. In some embodiments, the Anellovirus GC-rich region is a wild-type Anellovirus GC-rich region. In some embodiments, the Anellovirus GC-rich region is an engineered Anellovirus GC-rich region having a nucleic acid sequence with at least one difference relative to the closest wild-type Anellovirus GC-rich region sequence. In some embodiments, the Anellovirus GC-rich region comprises a contiguous sequence of at least 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides having a GC content of at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse complement of the sequence annotated as GC-rich region nucleotide sequence of Table N1 or Table 39.

[0366] Table 39. Exemplary GC-rich sequences from Anelloviruses. Effectors

[0367] In some embodiments, the genetic element encodes an effector, e.g., an exogenous effector. In some embodiments, the genetic element comprises a therapeutic expression sequence, e.g., a sequence that encodes an effector such as a therapeutic peptide or polypeptide, e.g., an intracellular peptide or intracellular polypeptide, a secreted polypeptide, or a protein replacement therapeutic. In some embodiments, the exogenous effector is an inhibitor of vascular endothelial growth factor (VEGF). In some embodiments, the inhibitor of VEGF is a fusion protein comprising VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2, fused to an Fc domain. In some embodiments, the Fc domain is the Fc portion of a human IgG immunoglobulin, e.g., the human IgGl immunoglobulin. In an embodiment, the exogenous effector is aflibercept.

[0368] Regulatory Sequences

[0369] In some embodiments, the genetic element comprises a regulatory sequence, e.g., a promoter or an enhancer, operably linked to the sequence encoding the effector.

[0370] In some embodiments, a promoter includes a DNA sequence that is located adjacent to a DNA sequence that encodes an expression product. A promoter may be linked operatively to the adjacent DNA sequence. A promoter typically increases an amount of product expressed from the DNA sequence as compared to an amount of the expressed product when no promoter exists. A promoter from one organism can be utilized to enhance product expression from the DNA sequence that originates from another organism. For example, a vertebrate promoter may be used for the expression of jellyfish GFP in vertebrates. In addition, one promoter element can increase an amount of products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more products. Multiple promoter elements are well-known to persons of ordinary skill in the art.

[0371] In some embodiments, a native promoter for a gene or nucleic acid sequence of interest is used. The native promoter may be used when it is desired that expression of the gene or the nucleic acid sequence should mimic the native expression. The native promoter may be used when expression of the gene or other nucleic acid sequence must be regulated temporally or developmentally, or in a tissuespecific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0372] In one embodiment, high-level constitutive expression is desired. Examples of such promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter / enhancer, the cytomegalovirus (CMV) immediate early promoter / enhancer (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic beta-actin promoter and the phosphoglycerol kinase (PGK) promoter.

[0373] In another embodiment, inducible promoters may be desired. Inducible promoters are those which are regulated by exogenously supplied compounds, either in cis or in trans. Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, or in replicating cells only.

[0374] In some embodiments, the genetic element comprises a gene operably linked to a tissue-specific promoter.

[0375] The genetic element may include an enhancer, e.g., a DNA sequence that is located adjacent to the DNA sequence that encodes a gene. Enhancer elements are typically located upstream of a promoter element or can be located downstream of or within a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence that encodes the product. Enhancer elements can increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.

[0376] Surface Moieties

[0377] An Anelloviridae family vector as described herein may, in some instances, include one or more moieties attached to its surface (e.g., a surface moiety that can act as an effector and / or a targeting agent). In some instances, an Anelloviridae family vector comprises more than one distinct surface moiety (e.g., a first surface moiety having an effector function as described herein and a second surface moiety that targets the Anelloviridae family vector to a cell or tissue of interest). In some instances, the surface moiety is covalently attached to the surface of the Anelloviridae family vector. For example, the surface moiety may be covalently attached to the proteinaceous exterior or a component thereof (e.g., covalently attached to an ORF1 molecule of the proteinaceous exterior). In certain embodiments, the surface moiety is fused to an ORF1 molecule. In some instances, the surface moiety is noncovalently attached to the surface of the Anelloviridae family vector. For example, the surface moiety may be noncovalently bound to the proteinaceous exterior or a component thereof (e.g., noncovalently bound to an ORF1 molecule of the proteinaceous exterior). In certain embodiments, the surface moiety comprises a region that specifically binds to a cognate moiety on or attached to the ORF1 molecule. In an embodiment, the ORF1 molecule comprises a binding moiety (e.g., an antibody molecule) that specifically recognizes an epitope on the region on the surface moiety. In an embodiment, the surface moiety comprises a binding moiety (e.g., an antibody molecule) that specifically recognizes an epitope on the ORF1 molecule. The surface moiety can, in some instances, comprise a polypeptide. The surface moiety may, in some instances, comprise a nucleic acid molecule (e.g., DNA and / or RNA). The surface moiety may, in some instances, comprise a small molecule. In some instances, a surface moiety comprises an antigen (e.g., an antigen recognized by the immune system of a subject to be delivered the Anelloviridae family vector). In some instances, a surface moiety as described herein comprises a ligand (e.g., a ligand that binds specifically to a receptor on a target cell).

[0378] In some instances, the surface moiety comprises an effector function (e.g., as described herein). For example, the surface moiety may modulate a biological activity, e.g., of a target cell or organ. In some instances, the surface moiety induces modulation of the biological activity via binding to a cognate moiety on a target cell. For example, the surface moiety may comprise a ligand that binds to a receptor on the surface of the target cell, e.g., wherein binding of the surface moiety to the receptor initiates a downstream signaling cascade of interest. In some instances, the effector activity comprises increasing or decreasing enzymatic activity, gene expression, cell signaling, and / or cellular or organ function within a target cell or organ. Effector activities may also include binding regulatory proteins to modulate activity of the regulator, such as transcription or translation. Effector activities also may include activator or inhibitor functions.

[0379] In some instances, the surface moiety can target the Anelloviridae family vector to a target cell. For example, the surface moiety may specifically bind to a cognate moiety on the surface of the target cell. The cognate moiety on the surface of the target cell may be, for example, a molecule specifically expressed or preferentially expressed by the target cell. The cognate moiety may be, for example, a polypeptide, lipid, sugar', or small molecule. In certain embodiments, the cognate moiety is a transmembrane protein (e.g., comprising an extracellular domain that binds to the surface moiety of the Anelloviridae family vector). In certain embodiments, the cognate moiety is tethered to the surface of the cell (e.g., via a GPI anchor). In some instances, the surface moiety provides a tropism (e.g., to a target tissue or target cell type) for the Anelloviridae family vector.

[0380] In an aspect, the disclosure provides an 0RF1 molecule comprising: (i) the amino acid sequence of an Anellovirus ORF1 protein, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and (ii) a click handle (e.g., an NHS click handle or a maleimide click handle, e.g., as described herein). In certain embodiments, the click handle is covalently attached to the ORF1 molecule. In certain embodiments, the click handle is noncovalently attached to the ORF1 molecule. In certain embodiments, the click handle is used to attach the ORF1 molecule to a surface moiety, e.g., via a click reaction, e.g., as described herein.

[0381] A “click handle,” as that term is used herein, refers to a chemical moiety that is capable of reacting with a second click handle in a click reaction. In some embodiments, a click handle comprises an NHS moiety and / or a maleimide moiety. In certain embodiments, a click handle comprises a DBCO moiety. In certain embodiments, a click handle comprises an azide moiety. In some embodiments, a click handle is attached to a polypeptide (e.g., an ORF1 molecule). In other embodiments, a click handle comprises a reactive group capable of forming a covalent bond with a polypeptide (e.g., an ORF1 molecule). A “click reaction”, as that term is used herein, refers to a range of reactions used to covalently link a first and a second moiety, for convenient production of linked products. It typically has one or more of the following characteristics: it is fast, is specific, is high-yield, is efficient, is spontaneous, does not significantly alter biocompatibility of the linked entities, has a high reaction rate, produces a stable product, favors production of a single reaction product, has high atom economy, is chemoselective, is modular', is stereoselective, is insensitive to oxygen, is insensitive to water, is high purity, generates only inoffensive or relatively non-toxic byproducts that can be removed by nonchromatographic methods (e.g., crystallization or distillation), needs no solvent or can be performed in a solvent that is benign or physiologically compatible, e.g., water, stable under physiological conditions. Examples include an alkyne / azide reaction, a diene / dienophile reaction, or a thiol / alkene reaction. Other reactions can be used.

[0382] Aflibercept genetic elements and genetic element constructs

[0383] In some embodiments, a genetic element described herein comprises an Aflibercept payload. In some embodiments, a genetic element described herein comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of Table X. In some embodiments, a genetic element described herein comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of Table XI.

[0384] Table X. pRTx-4321 post recombination (positive strand)

[0385] Annotations:

[0386] Region / Element Base range

[0387] GC-rich region 64-175

[0388] Initiator Element 348-363

[0389] 5' UTR conserved domain 415-485

[0390] Intron 1 427-514

[0391] CMV enhancer 524-903

[0392] CMV promoter 904-1107

[0393] Kozak Sequence 1158-1167

[0394] Aflibercept coding sequence 1164-2537

[0395] SV40 poly(A) signal 2568-2689

[0396] SV40 polyA late regulatory (terminator) 2615-2689 lOObp random stuffer 2706-2805 loxP site 2810-2843

[0397] Exemplary Aflibercept amino acid sequence (e.g., encoded by nucleotides 1164-2537 of Table X):

[0398] In some embodiments, a genetic element described herein comprises one or more regions set forth in Table X2. In the table, the negative strand sequence of each element (shown in the rightmost column) is the reverse complement of the positive strand sequence of that element. The location (in the leftmost column) refers to the nucleotide positions in the positive strand sequence. Table X2. Annotated regions of pRTx-4321 post recombination plasmid

[0399]

[0400] In some embodiments, a genetic element construct described herein comprises an Aflibercept payload. In some embodiments, a genetic element construct described herein comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of Table X3 In some embodiments, a genetic element described herein comprises a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of Table X4.

[0401] Table X3. pRTx-4321 pre recombination (positive strand) Initiator Element 813-828

[0402] 5' UTR conserved domain 880-950

[0403] Intron 1 892-979

[0404] CMV enhancer 989-1368

[0405] CMV promoter 1369-1572

[0406] Kozak Sequence 1623-1632

[0407] Aflibercept coding sequence 1629-3002

[0408] SV40 poly(A) signal 3033-3154

[0409] SV40 polyA late regulatory (terminator) 3098-3117 lOObp random stuffer 3171-3270

[0410] Iox66 loxp site 3275-3308 pUC57-Kan EcoRV Linearized 3309-5456, 1-431

[0411] Lac operon operator 3390-3406

[0412] Lac operon promoter (complement) 3414-3444

[0413] C-tag 3422-3433

[0414] E. coli catabolite activator protein binding site (complement) 3459-3480 pUC origin of replication 3709-4382

[0415] High-copy-number ColEl / pMBl / pBR322 / pUC origin of replication (complement) 3768-4356

[0416] Kanamycin resistance (KanR) coding sequence

[0417] (complement) 4534-5343

[0418] Table X4. pRTx-4321 pre recombination (negative strand)

[0419] Name pRTx-4321

[0420] Type Plasmid

[0421] Length 5456 bp

[0422]

[0423] In some embodiments, a genetic element construct described herein comprises one or more regions set forth in Table X5. In the table, the negative strand sequence of each element (shown in the rightmost column) is the reverse complement of the positive strand sequence of that element. The location (in the leftmost column) refers to the nucleotide positions in the positive strand sequence.

[0424] Table X5. Annotated regions of pRTx-4321 pre recombination plasmid

[0425] In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of 40-120 nucleotides, 50-110 nucleotides, or 60-100 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322 or SEQ ID NO: 321, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, or about 120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322 or SEQ ID NO: 321, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, or 110-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322 or SEQ ID NO: 321, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of 55-65, 65-75, 75-85, 85-95, 95-105, or 105-115 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322 or SEQ ID NO: 321, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of 61 nucleotides. In some embodiments, a genetic element or nucleic acid molecule described herein (e.g., a genetic element construct) comprises a region having a length of 100 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322 or SEQ ID NO: 321, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0426] AAV-Anello virus hybrid vectors

[0427] In some embodiments, a vector described herein comprises at least one anellovirus element and at least one AAV element. For instance, in some embodiments, a genetic element described herein comprises an Anellovirus 5’ NCR and / or Anellovirus 3’ NCR, and further comprises one or more (e.g., two) AAV inverted terminal repeat. In some embodiments, the genetic element comprises all of an Anellovirus 5’ NCR, Anellovirus 3’ NCR, a first AAV inverted terminal repeat, and a second AAV inverted terminal repeat. In some embodiments, the AAV inverted terminal repeat (e.g., the first AAV ITR) has a sequence according to the reverse complement of nucleotides 269-409 of SEQ ID NO: 401. In some embodiments, the AAV inverted terminal repeat (e.g., the second AAV ITR) has a sequence according to the reverse complement of nucleotides 3252-3392 of SEQ ID NO: 401.

[0428] In some embodiments, the genetic element encodes an exogenous effector as described herein. In some embodiments, the exogenous effector has an amino acid sequence of SEQ ID NO: 326, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the genetic element is produced from a plasmid having a nucleotide sequence of SEQ ID NO: 401, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0429] In some embodiments, the genetic element is enclosed in an AAV capsid. In some embodiments, the AAV is AAV2.

[0430] AAV-Anellovirus hybrid vectors, and methods of making them, are also described in International Application WO / 2022 / 170195, which is hereby incorporated by reference in its entirety.

[0431] II. Compositions and Methods for Making Anelloviridae Family Vectors

[0432] The present disclosure provides, in some aspects, Anelloviridae family vectors (e.g., anelloviral vectors) and methods thereof for delivering effectors. In some embodiments, the Anelloviridae family vectors (e.g., anelloviral vectors) or components thereof can be made as described below. In some embodiments, the compositions and methods described herein can be used to produce a genetic element or a genetic element construct. In some embodiments, the compositions and methods described herein can be used to produce one or more Anelloviridae family virus capsid proteins (e.g., Anellovirus ORF) molecules (e.g., an ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORFl / 2molecule, or a functional fragment or splice variant thereof). In some embodiments, the compositions and methods described herein can be used to produce a proteinaceous exterior or a component thereof (e.g., an ORF1 molecule), e.g., in a host cell. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) or components thereof can be made using a tandem construct, e.g., as described in PCT Publication No. WO 2021252955, which is incorporated herein by reference in its entirety. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) or components thereof (e.g., an Anelloviridae family polypeptide, e.g., an ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecule, or a functional fragment or splice variant thereof) can be made using a bacmid / insect cell system, e.g., as described as described in PCT Publication No. WO 2021 / 252943, which is incorporated herein by reference in its entirety. Methods of producing an anelloviral vector using a host cell are described, for example, below in the section entitled “Host Cells and Methods of Using Host Cells for Producing an anelloviral vector”. Method of producing an anelloviral vector in a cell -free system are described, for example, below in the section entitled “In vitro assembly methods”.

[0433] Without wishing to be bound by theory, rolling circle amplification may occur via Rep protein binding to a Rep binding site (e.g., comprising a 5’ UTR, e.g., comprising a hairpin loop and / or an origin of replication, e.g., as described herein) positioned 5’ relative to (or within the 5’ region of) the genetic element region. The Rep protein may then proceed through the genetic element region, resulting in the synthesis of the genetic element. The genetic element may then be circularized and then encapsulated within a proteinaceous exterior to form an Anelloviridae family vector (e.g., anelloviral vector).

[0434] Genetic Element Constructs, e.g., for assembly of Anelloviridae family vectors

[0435] In some methods of making as described herein, the genetic element is made using a genetic element construct, wherein the genetic element construct can act as a template for the production of the genetic element. In some embodiments, a genetic element construct comprises a genetic element region and optionally other sequence such as vector backbone. A genetic element construct may be any nucleic acid construct suitable for delivery of the sequence of the genetic element into a host cell in which the genetic element can be encapsulated within a proteinaceous exterior. In some embodiments, the genetic element construct comprises a promoter. In some embodiments, the genetic element construct is a linear nucleic acid molecule. In some embodiments, the genetic element construct is a circular- nucleic acid molecule (e.g., a plasmid, viral nucleic acid, bacmid, artificial chromosome, or a minicircle, e.g., as described herein). In some embodiments, a step of in vitro circularization may be used in producing a genetic element, e.g., as described herein. In embodiments, the double-stranded circular nucleic acid construct (e.g., minicircle) can be introduced into a host cell, in which it can be converted into or used as a template for generating single-stranded circular- genetic elements, e.g., as described herein. In some embodiments, the genetic element is generated by a polymerase based on a template sequence in the nucleic acid construct. In some embodiments, the polymerase produces a single-stranded copy of the genetic element sequence, which can optionally be circularized to form a genetic element as described herein.

[0436] The genetic element construct may, in some embodiments, be double-stranded. In some embodiments, the genetic element construct comprises RNA. In some embodiments, the genetic element construct comprises one or more modified nucleotides.

[0437] Tandem Constructs

[0438] In some embodiments, a genetic element construct comprises a first copy of a genetic element sequence (e.g., the nucleic acid sequence of a genetic element, e.g., as described herein) and at least a portion of a second copy of a genetic element sequence (e.g., the nucleic acid sequence of the same genetic element, or the nucleic acid sequence of a different genetic element), arranged in tandem. Genetic element constructs having such a structure are generally referred to herein as tandem constructs. Such tandem constructs are used for producing an Anelloviridae family vector (e.g., anelloviral vector) genetic element. The first copy of the genetic element sequence and the second copy of the genetic element sequence may, in some instances, be immediately adjacent to each other on the genetic acid construct. In other instances, the first copy of the genetic element sequence and the second copy of the genetic element sequence may be separated, e.g., by a spacer sequence. Without being bound by theory, a tandem construct described herein may, in some embodiments, replicate by rolling circle replication. In some embodiments, a tandem construct is a plasmid. In some embodiments, a tandem construct is circular. In some embodiments, a tandem construct is linear. In some embodiments, a tandem construct is singlestranded. In some embodiments, a tandem construct is double-stranded. In some embodiments, a tandem construct is DNA.

[0439] Additional descriptions of tandem constructs that can be used with the invention are described, for example, PCT Publication No. WO 2021252955, incorporated herein by reference in its entirety.

[0440] Cis / T runs Constructs

[0441] In some embodiments, a genetic element construct as described herein comprises one or more sequences encoding one or more Anelloviridae family virus ORFs, e.g., proteinaceous exterior components (e.g., polypeptides encoded by an Anellovirus ORF1 nucleic acid, e.g., as described herein). For example, the genetic element construct may comprise a nucleic acid sequence encoding an Anellovirus ORF1 molecule. Such genetic element constructs can be suitable for introducing the genetic element and the Anelloviridae family virus ORF(s) into a host cell in cis. In other embodiments, a genetic element construct as described herein does not comprise sequences encoding one or more Anelloviridae family virus ORFs, e.g., proteinaceous exterior components (e.g., polypeptides encoded by an Anellovirus ORF1 nucleic acid, e.g., as described herein). For example, the genetic element construct may not comprise a nucleic acid sequence encoding an Anellovirus ORF1 molecule. Such genetic element constructs can be suitable for introducing the genetic element into a host cell, with the one or more Anelloviridae family virus ORFs to be provided in trans (e.g., via introduction of a second nucleic acid construct encoding one or more of the Anelloviridae family virus ORFs, or via an Anelloviridae family virus ORF cassette integrated into the genome of the host cell). In some embodiments, an ORF1 molecule is provided in trans, e.g., as described herein. In some embodiments, an ORF2 molecule is provided in trans, e.g., as described herein. In some embodiments, an ORF1 molecule and an ORF2 molecule are both provided in trans, e.g., as described herein.

[0442] In some embodiments, the genetic element construct comprises a sequence encoding an Anellovirus ORF1 molecule, or a splice variant or functional fragment thereof (e.g., a jelly-roll region, e.g., as described herein). In embodiments, the portion of the genetic element that does not comprise the sequence of the genetic element comprises the sequence encoding the Anellovirus ORF1 molecule, or splice variant or functional fragment thereof (e.g., in a cassette comprising a promoter and the sequence encoding the Anellovirus ORF1 molecule, or splice variant or functional fragment thereof). In further embodiments, the portion of the construct comprising the sequence of the genetic element comprises a sequence encoding an Anellovirus ORF1 molecule, or a splice variant or functional fragment thereof (e.g., a jelly-roll region, e.g., as described herein). In embodiments, encapsulation of such a genetic element in a proteinaceous exterior (e.g., as described herein) produces a replication-component Anelloviridae family vector (e.g., anelloviral vector) (e.g., an Anelloviridae family vector that upon infecting a cell, enables the cell to produce additional copies of the anelloviral vector without introducing further nucleic acid constructs, e.g., encoding one or more Anelloviridae family virus ORFs as described herein, into the cell).

[0443] In other embodiments, the genetic element does not comprise a sequence encoding an Anellovirus 0RF1 molecule, or a splice variant or functional fragment thereof (e.g., a jelly-roll region, e.g., as described herein). In embodiments, encapsulation of such a genetic element in a proteinaceous exterior (e.g., as described herein) produces a replication-incompetent Anelloviridae family vector (e.g., anelloviral vector) (e.g., an Anelloviridae family vector that, upon infecting a cell, does not enable the infected cell to produce additional Anelloviridae family vector, e.g., in the absence of one or more additional constructs, e.g., encoding one or more Anellovirus ORFs as described herein).

[0444] Recombinase-based production of genetic elements and anelloviral vectors

[0445] A genetic element for an Anelloviridae family vector (e.g., an anelloviral vector) may be produced via site-specific recombination of a genetic element construct to produce a circular nucleic acid molecule comprising the genetic element sequence. In some embodiments, the circular nucleic acid molecule is a double-stranded DNA minicircle. In some embodiments, the circular nucleic acid molecule is in turn converted to a circular' single-stranded DNA molecule, which can in turn serve as the genetic element of an Anelloviridae family vector (e.g., an anelloviral vector) as described herein. Generally, the genetic element construct comprises a set of recombinase recognition sequences flanking the sequence of a genetic element of an Anelloviridae family vector. The recombinase recognition sites may be recognized by a site-specific recombinase. The remainder of the genetic element construct may, in some instances, comprise a vector backbone comprising elements for replication of the construct in a cell, such as a mammalian cell.

[0446] Contacting the genetic element construct with the site-specific recombinase (e.g., in a host cell) may result in excision of the genetic element sequence from the remainder of the construct and the formation of two circular nucleic acid molecules, one comprising the genetic element sequence and the other comprising the remainder of the vector backbone. In some embodiments, the circular nucleic acid molecule (e.g., a minicircle) comprising the genetic element sequence is converted to cssDNA in the host cell (e.g., a mammalian host cell), and is then encapsulated in a proteinaceous exterior comprising 0RF1 molecules (e.g., as described herein) to produce an Anelloviridae family vector.

[0447] In some embodiments, a site specific recombinase-based system for producing genetic elements comprises three plasmids: (1) a first plasmid (e.g., a vector plasmid) comprising the sequence of the genetic element, flanked by a pair of recombinase recognition sites; (2) an expression plasmid comprising a cassette encoding a site-specific recombinase (e.g., as described herein) capable of recognizing the recombinase recognition sites; and (3) a plasmid (e.g., a self-replicating rescue (SRR) plasmid) providing one or more Anelloviridae family viral proteins (e.g., Anellovirus ORF1, ORF3, and / or ORF3 molecules), including a capsid protein (e.g., an 0RF1 molecule, e.g., as described herein).

[0448] In some embodiments, a site specific recombinase-based system for producing genetic elements comprises two plasmids: (1) a first plasmid (e.g., a vector plasmid) comprising the sequence of the genetic element, flanked by a pair of recombinase recognition sites; and (2) a second plasmid (e.g., a selfreplicating rescue (SRR) plasmid) providing one or more Anelloviridae family viral proteins (e.g., Anellovirus ORF1, ORF3, and / or ORF3 molecules), including a capsid protein (e.g., an ORF1 molecule , e.g., as described herein).

[0449] Self-replicating rescue (SRR) constructs (e.g., SRR plasmids)

[0450] In some embodiments, a rescue construct can be used to provide one or more Anelloviridae family viral proteins, or functional fragments or variants thereof (e.g., one or more of Anellovirus ORF1, ORF2, and / or ORF3 molecules). In some embodiments, the rescue construct is capable of self-replicating in a host cell (e.g., a self-replicating rescue (SRR) plasmid). In some embodiments, the SRR plasmid can be used in the site-specific recombinase based systems.

[0451] In some embodiments, the rescue construct includes an expression cassette comprising the protein coding sequence of an Anelloviridae family virus (e.g., an Anellovirus as described herein), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the rescue construct includes a sequence encoding a replication protein (e.g., a large T antigen, a PCV Rep, or a PCV Rep’), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the rescue construct includes an exogenous origin of replication (e.g., a viral origin, e.g., an SV40, PCV, or AAV origin). In certain embodiments, the replication protein coding sequence is downstream of an internal ribosome entry site (IRES ) positioned downstream of the expression cassette comprising the protein coding sequence of the Anelloviridae family virus. In certain embodiments, the replication protein coding sequence is comprised in a separate cassette from the expression cassette comprising the protein coding sequence of the Anelloviridae family virus. In some embodiments, the rescue construct further comprises one or more additional expression cassettes, e.g., encoding a site-specific recombinase, an Anelloviridae family viral protein (e.g., an Anellovirus ORF1 molecule), a replication protein and / or viral origin (e.g., as described herein), or another transgene of interest.

[0452] In some embodiments, the rescue construct comprises a single expression cassette (e.g., as described above). In some embodiments, the rescue construct comprises two expression cassettes. In some embodiments, the rescue construct comprises three expression cassettes. In some embodiments, the rescue construct comprises four expression cassettes. In some embodiments, the rescue construct comprises five or more expression cassettes. The exemplary expression cassettes described herein can be positioned in any order within the rescue construct. In some embodiments, one or more of the expression cassettes is in the opposite orientation relative to one or more of the other expression cassettes. In other embodiments, all of the expression cassettes in a rescue construct are in the same orientation relative to each other.

[0453] In some embodiments, the rescue construct does not contain an Anellovirus NCR sequence (e.g. does not contain an Anellovirus 5’ NCR sequence and / or an Anellovirus 3’ NCR sequence). In some embodiments, the rescue construct does not comprise an Anellovirus 5’ UTR conserved domain. In some embodiments, the rescue construct does not comprise an Anellovirus GC-rich region. In some embodiments, the rescue construct does not contain Anellovirus sequences homologous to the vector plasmid (e.g., a contiguous sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, or 500 nucleotides having sequence identity to any Anellovirus sequence of the same length in the vector plasmid).

[0454] Exemplary SV40 Large T Antigen amino acid sequence: Exemplary site-specific recombinases and recombinase recognition sites

[0455] The recombinase-based systems described herein utilize site-specific recombinases to induce recombination of vector plasmids at recombinase recognition sites, thereby producing double-stranded DNA molecules (e.g., minicircles) comprising the sequence between the recombinase recognition sites (e.g., comprising the sequence of a genetic element for an Anelloviridae family vector as described herein).

[0456] In some embodiments, the site-specific recombinase comprises a Cre recombinase (e.g., as described herein, e.g., in Table V2), or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the site-specific recombinase comprises the amino acid sequence of Cre as listed in Table V2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the sitespecific recombinase comprises the amino acid sequence of SV40-NLS-iCre as listed in Table V2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0457] In certain embodiments, at least one (e.g., one or both) of the recombinase recognition sites comprises a lox66 site as listed in Table V3, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, at least one (e.g., one or both) of the recombinase recognition sites comprises a lox71 site as listed in Table V3, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, a circular double-stranded nucleic acid molecule (e.g., minicircle) produced after a Cre recombination event (e.g., as described herein) comprises a loxP site as listed in Table V3, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, a circular double-stranded nucleic acid molecule (e.g., minicircle) produced after a Cre recombination event (e.g., as described herein) comprises a lox72 site as listed in Table V3, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0458] In some embodiments, the site-specific recombinase comprises a Bxbl recombinase (e.g., as described herein, e.g., in Table V2), or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the site-specific recombinase comprises the amino acid sequence of Bxbl as listed in Table V2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the site-specific recombinase comprises the amino acid sequence of SV40-NLS-HA_Bxbl as listed in Table N2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0459] In certain embodiments, at least one of the recombinase recognition sites comprises an attB site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, at least one of the recombinase recognition sites comprises an attP site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, one recombinase recognition site comprises an attB site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the other recombinase recognition site comprises an attP site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, a circular double-stranded nucleic acid molecule (e.g., minicircle) produced after a Bxbl recombination event (e.g., as described herein) comprises an attL site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In certain embodiments, a circular double-stranded nucleic acid molecule (e.g., minicircle) produced after a Bxbl recombination event (e.g., as described herein) comprises an attR site as listed in Table V4, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0460] Table V2. Exemplary site-specific recombinase polypeptides

[0461] Table V3. Exemplary recombinase recognition sequences and recombinase hybrid sites for Cre recombinases Table V4. Exemplary recombinase recognition sequences for Bxbl recombinases

[0462] As used herein, the term “recombinase hybrid site” refers to a DNA site having a sequence that, when in double stranded form, is capable of being produced by a site-specific recombinase that recombines two recombinase recognition sites. No particular process of making is implied: a recombinase hybrid site can be produced by a site specific recombinase or another method, such as DNA replication of an existing sequence. In some embodiments, a single stranded DNA that comprises a recombinase hybrid site was produced by a method wherein a site -specific recombinase generated a double stranded DNA comprising a recombinase hybrid site, followed by conversion of the double stranded DNA to a single stranded DNA. In some embodiments (e.g., with Cre recombinase) the recombinase hybrid site has the same sequence as one of the corresponding recombinase recognition sites. In some embodiments (e.g., with Bxbl recombinase), the recombinase hybrid site has a different site from either of the two corresponding recombinase recognition sites. In some embodiments, the recombinase hybrid site is a loxP site, an attL site, or an attR site.

[0463] As used herein, the term “recombinase recognition site” refers to a DNA site having a sequence that is capable of being recognized by a site-specific recombinase and recombined with a second recombinase recognition site, thereby producing a recombinase hybrid site. In some embodiments, the two recombinase recognition sites recognized by the recombinase have the same sequence, and in other embodiments, they have different sequences. In some embodiments, the recombinase recognition site is a loxP site, an attB site, or an attP site.

[0464] Host Cells and Methods of Using Host Cells for Producing an anelloviral vector

[0465] The Anelloviridae family vector (e.g., anelloviral vector) described herein can be produced, for example, in a host cell. Generally, a host cell is provided that comprises an Anelloviridae family vector (e.g., anelloviral vector) genetic element and the components of an Anelloviridae family vector (e.g., anelloviral vector) proteinaceous exterior (e.g., a polypeptide encoded by an Anellovirus ORF1 nucleic acid, or an Anellovirus ORF1 molecule). For example, in some embodiments, the host cell comprises a nucleic acid sequence encoding an Anellovirus ORF1 molecule, e.g., a splice variant or a functional fragment of an Anellovirus ORF1 polypeptide (e.g., a wild-type Anellovirus ORF1 protein or a polypeptide encoded by a wild-type Anellovirus ORF1 nucleic acid, e.g., as described herein). In embodiments, the nucleic acid sequence encoding the Anellovirus ORF1 molecule is comprised in a nucleic acid construct (e.g., a plasmid, viral vector, virus, minicircle, bacmid, or artificial chromosome) comprised in the host cell. In embodiments, the nucleic acid sequence encoding the Anellovirus ORF1 molecule is integrated into the genome of the host cell.

[0466] Producing an Anelloviridae family vector (e.g. anelloviral vector) using the compositions or methods described herein may also involve expression of an Anellovirus ORF2 molecule (e.g., as described herein), or a splice variant or functional fragment thereof. In some embodiments, the anelloviral vector does not comprise an ORF2 molecule, or a splice variant or functional fragment thereof, and / or a nucleic acid encoding an 0RF2 molecule, or a splice variant or functional fragment thereof. In some embodiments, producing the anellovir l vector comprises expression of an 0RF2 molecule, or a splice variant or functional fragment thereof, but the 0RF2 molecule is expressed from a nucleic acid other than Anelloviridae family vector.

[0467] The host cell is then incubated under conditions suitable for enclosure of the genetic element within the proteinaceous exterior (e.g., culture conditions as described herein). In some embodiments, the host cell is further incubated under conditions suitable for release of the Anelloviridae family vector (e.g., anelloviral vector) from the host cell, e.g., into the surrounding supernatant. In some embodiments, the host cell is lysed for harvest of Anelloviridae family vector (e.g., anelloviral vector) from the cell lysate. In some embodiments, an Anelloviridae family vector (e.g., anelloviral vector) may be introduced to a host cell line grown to a high cell density. In some embodiments, a host cell is an Expi-293 cell.

[0468] In an aspect, the present disclosure provides a host cell (e.g., as described herein). In some embodiments, the host or host cell is a plant, insect, bacteria, fungus, vertebrate, mammal (e.g., human), or other organism or cell.

[0469] Introduction of genetic elements into host cells

[0470] The genetic element, or a nucleic acid construct comprising the sequence of a genetic element, may be introduced into a host cell. In some embodiments, the genetic element itself is introduced into the host cell. In some embodiments, a genetic element construct comprising the sequence of the genetic element (e.g., as described herein) is introduced into the host cell. A genetic element or genetic element construct can be introduced into a host cell, for example, using methods known in the ai t. For example, a genetic element or genetic element construct can be introduced into a host cell by transfection (e.g., stable transfection or transient transfection). In embodiments, the genetic element or genetic element construct is introduced into the host cell by lipofectamine transfection. In embodiments, the genetic element or genetic element construct is introduced into the host cell by calcium phosphate transfection. In some embodiments, the genetic element or genetic element construct is introduced into the host cell by electroporation. In some embodiments, the genetic element or genetic element construct is introduced into the host cell using a gene gun. In some embodiments, the genetic element or genetic element construct is introduced into the host cell by nucleofection. In some embodiments, the genetic element or genetic element construct is introduced into the host cell by PEI transfection. In some embodiments, the genetic element is introduced into the host cell by contacting the host cell with an Anelloviridae family vector (e.g., anelloviral vector) comprising the genetic element. In some embodiments, cells are suspended in 2S Chica buffers. In embodiments, the genetic element construct is capable of replication once introduced into the host cell. In embodiments, the genetic element can be produced from the genetic element construct once introduced into the host cell. In some embodiments, the genetic element is produced in the host cell by a polymerase, e.g., using the genetic element construct as a template.

[0471] In some embodiments, the genetic elements or vectors comprising the genetic elements are introduced (e.g., transfected) into cell lines that express a viral polymerase protein in order to achieve expression of the Anelloviridae family vector (e.g., anelloviral vector). To this end, cell lines that express an Anelloviridae family vector (e.g., anelloviral vector) polymerase protein may be utilized as appropriate host cells. Host cells may be similarly engineered to provide other viral functions or additional functions.

[0472] To prepare the Anelloviridae family vector (e.g., anelloviral vector) disclosed herein, a genetic element construct may be used to transfect cells that provide Anelloviridae family vector (e.g., anelloviral vector) proteins and functions required for replication and production. Alternatively, cells may be transfected with a second construct (e.g., a virus) providing Anelloviridae family vector (e.g., anelloviral vector ) proteins and functions before, during, or after transfection by the genetic element or vector comprising the genetic element disclosed herein. In some embodiments, the second construct may be useful to complement production of an incomplete viral particle. The second construct (e.g., virus) may have a conditional growth defect, such as host range restriction or temperature sensitivity, e.g., which allows the subsequent selection of transfectant viruses. In some embodiments, the second construct may provide one or more replication proteins utilized by the host cells to achieve expression of the Anelloviridae family vector (e.g., anelloviral vector). In some embodiments, the host cells may be transfected with vectors encoding viral proteins such as the one or more replication proteins. In some embodiments, the second construct comprises an antiviral sensitivity.

[0473] The genetic element or vector comprising the genetic element disclosed herein can, in some instances, be replicated and produced into Anelloviridae family vectors (e.g., anelloviral vectors) using techniques known in the art. For example, various viral culture methods are described, e.g., in U.S. Pat. No. 4,650,764; U.S. Pat. No. 5,166,057; U.S. Pat. No. 5,854,037; European Patent Publication EP 0702085A1; U.S. patent application Ser. No. 09 / 152,845; International Patent Publications PCT WO97 / 12032; WO96 / 34625; European Patent Publication EP-A780475; WO 99 / 02657; WO 98 / 53078; WO 98 / 02530; WO 99 / 15672; WO 98 / 13501; WO 97 / 06270; and EPO 780 47SA1, each of which is incorporated by reference herein in its entirety.

[0474] Exemplary cell types

[0475] Exemplary host cells suitable for production of Anelloviridae family vector (e.g., anelloviral vector) include, without limitation, mammalian cells, e.g., human cells and insect cells. In some embodiments, the host cell is a human cell or cell line. In some embodiments, the cell is an immune cell or cell line, e.g., a T cell or cell line, a cancer cell line, a hepatic cell or cell line, a neuron, a glial cell, a skin cell, an epithelial cell, a mesenchymal cell, a blood cell, an endothelial cell, an eye cell (e.g., a photoreceptor cell, a retinal cell, a cell of the posterior eye cup (PEC), retinal ganglion cell, a cell of the optic nerve, a cell of the optic nerve head, or a retinal pigmented epithelium (RPE) cell), a gastrointestinal cell, a progenitor cell, a precursor cell, a stem cell, a lung cell, a cardiac cell, or a muscle cell. In some embodiments, the host cell is an animal cell (e.g., a mouse cell, rat cell, rabbit cell, or hamster cell, or insect cell).

[0476] In some embodiments, the host cell is a human cell. In embodiments, the host cell is a HEK293T cell, HEK293F cell, A549 cell, Jurkat cell, Raji cell, Chang cell, HeLa cell Phoenix cell, MRC-5 cell, NCI-H292 cell, or Wi38 cell. In some embodiments, the host cell is a non-human primate cell (e.g., a Vero cell, CV-1 cell, or LLCMK2 cell). In some embodiments, the host cell is a murine cell (e.g., a McCoy cell). In some embodiments, the host cell is a hamster cell (e.g., a CHO cell or BHK 21 cell). In some embodiments, the host cell is a MARC-145, MDBK, RK-13, or EEL cell. In some embodiments, the host cell is an epithelial cell (e.g., a cell line of epithelial lineage).

[0477] In some embodiments, the host cell is a lymphoid cell. In some embodiments, the host cell is a T cell or an immortalized T cell. In embodiments, the host cell is a Jurkat cell. In embodiments, the host cell is a MOLT cell (e.g., a MOLTA or a MOLT-3 cell). In embodiments, the host cell is a MOLTA cell. In embodiments, the host cell is a MOLT-3 cell. In some embodiments, the host cell is an acute lymphoblastic leukemia (ALL) cell, e.g., a MOLT cell, e.g., a MOLTA or MOLT-3 cell. In some embodiments, the host cell is a B cell or an immortalized B cell. In some embodiments, the host cell comprises a genetic element construct (e.g., as described herein).

[0478] In some embodiments, the host cell is a MOLT cell (e.g., a MOLTA or a MOLT-3 cell).

[0479] In some embodiments, the host cell is an acute lymphoblastic leukemia (ALL) cell, e.g., a MOLT cell, e.g., a MOLTA or MOLT-3 cell.

[0480] In some embodiments, the host cell is a 293 cell (e.g., a HEK293 cell, a HEK293T cell, or an Expi-293 cell). In some embodiments, the host cell is an Expi-293F cell.

[0481] In an aspect, the present disclosure provides a method of manufacturing an Anelloviridae family vector (e.g., anelloviral vector) comprising a genetic element enclosed in a proteinaceous exterior, the method comprising providing a Expi-293 cell comprising an Anelloviridae family vector (e.g., anelloviral vector) genetic element, and incubating the Expi-293 cell under conditions that allow the Anelloviridae family vector (e.g., anelloviral vector) genetic element to become enclosed in a proteinaceous exterior in the Expi-293 cell. In some embodiments, the Expi-293 cell further comprises one or more Anellovirus proteins (e.g., an Anellovirus ORF1 molecule) that form part or all of the proteinaceous exterior. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) genetic element is produced in the Expi-293 cell, e.g., from a genetic element construct (e.g., as described herein). In some embodiments, the method further comprises introducing the Anelloviridae family vector (e.g., anelloviral vector) genetic element construct into the Expi-293 cell.

[0482] In an aspect, the present disclosure provides a method of manufacturing an Anelloviridae family vector (e.g., anelloviral vector) comprising a genetic element enclosed in a proteinaceous exterior, the method comprising providing a MOLT-4 cell comprising an Anelloviridae family vector (e.g., anelloviral vector) genetic element, and incubating the MOLT-4 cell under conditions that allow the Anelloviridae family vector (e.g., anelloviral vector) genetic element to become enclosed in a proteinaceous exterior in the MOLT-4 cell. In some embodiments, the MOLT-4 cell further comprises one or more Anellovirus proteins (e.g., an Anellovirus ORF1 molecule) that form part or all of the proteinaceous exterior. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) genetic element is produced in the MOLT-4 cell, e.g., from a genetic element construct (e.g., as described herein). In some embodiments, the method further comprises introducing the Anelloviridae family vector (e.g., anelloviral vector) genetic element constr uct into the MOLT-4 cell.

[0483] In an aspect, the present disclosure provides a method of manufacturing an Anelloviridae family vector (e.g., anelloviral vector) comprising a genetic element enclosed in a proteinaceous exterior, the method comprising providing a MOLT-3 cell comprising an Anelloviridae family vector (e.g., anelloviral vector) genetic element, and incubating the MOLT-3 cell under conditions that allow the Anelloviridae family vector (e.g., anelloviral vector) genetic element to become enclosed in a proteinaceous exterior in the MOLT-3 cell. In some embodiments, the MOLT-3 cell further comprises one or more Anellovirus proteins (e.g., an Anellovirus ORF1 molecule) that form part or all of the proteinaceous exterior. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) genetic element is produced in the MOLT-3 cell, e.g., from a genetic element construct (e.g., as described herein). In some embodiments, the method further comprises introducing the Anelloviridae family vector (e.g., anelloviral vector) genetic element construct into the MOLT-3 cell.

[0484] In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) is cultivated in a continuous animal cell line (e.g., immortalized cell lines that can be serially propagated).

[0485] Culture Conditions

[0486] Host cells comprising a genetic element and components of a proteinaceous exterior can be incubated under conditions suitable for enclosure of the genetic element within the proteinaceous exterior, thereby producing an Anelloviridae family vector (e.g., anelloviral vector). In some embodiments, the host cells are incubated in liquid media (e.g., Grace's Supplemented (TNM-FH), IPL-41, TC-100, Schneider’s Drosophila, SF-900 II SFM, or and EXPRESS-FIVE™ SFM). In some embodiments, the host cells are incubated in adherent culture. In some embodiments, the host cells are incubated in suspension culture. In some embodiments, the host cells are incubated in a tube, bottle, microcarrier, or flask. In some embodiments, the host cells are incubated in a dish or well (e.g., a well on a plate). In some embodiments, the host cells are incubated under conditions suitable for proliferation of the host cells. In some embodiments, the host cells are incubated under conditions suitable for the host cells to release Anelloviridae family vectors (e.g., anelloviral vectors) produced therein into the surrounding supernatant.

[0487] The production of Anelloviridae family vector (e.g., anelloviral vector)-containing cell cultures according to the present invention can be carried out in different scales (e.g., in flasks, roller bottles or bioreactors). The media used for the cultivation of the cells to be infected generally comprise the standard nutrients required for cell viability, but may also comprise additional nutrients dependent on the cell type. Optionally, the medium can be protein-free and / or serum-free. Depending on the cell type the cells can be cultured in suspension or on a substrate. In some embodiments, different media is used for growth of the host cells and for production of Anelloviridae family vectors (e.g., anelloviral vectors).

[0488] Harvest

[0489] Anelloviridae family vectors (e.g., anelloviral vectors) produced by host cells can be harvested, e.g., according to methods known in the art. For example, Anelloviridae family vectors (e.g., anelloviral vectors) released into the surrounding supernatant by host cells in culture can be harvested from the supernatant. In some embodiments, the supernatant is separated from the host cells to obtain the Anelloviridae family vectors (e.g., anelloviral vectors). In some embodiments, the host cells are lysed before or during harvest. In some embodiments, the Anelloviridae family vectors (e.g., anelloviral vectors) are harvested from the host cell lysates. In some embodiments, the Anelloviridae family vectors (e.g., anelloviral vectors) are harvested from both the host cell lysates and the supernatant. In some embodiments, the purification and isolation of Anelloviridae family vectors (e.g., anelloviral vectors) is performed according to known methods in virus production, for example, as described in Rinaldi, et al., DNA Vaccines: Methods and Protocols (Methods in Molecular' Biology), 3rd ed. 2014, Humana Press (incorporated herein by reference in its entirety). In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) may be harvested and / or purified by separation of solutes based on biophysical properties, e.g., ion exchange chromatography or tangential flow filtration, prior to formulation with a pharmaceutical excipient. In vitro assembly methods

[0490] An Anelloviridae family vector (e.g., anelloviral vector) may be produced, e.g., by in vitro assembly, e.g., in a cell-free suspension or in a supernatant. In some embodiments, the genetic element is contacted to an 0RF1 molecule in vitro, e.g., under conditions that allow for assembly.

[0491] In some embodiments, baculovirus constructs are used to produce Anelloviridae family virus (e.g., Anellovirus) proteins. These proteins may then be used, e.g., for in vitro assembly to encapsulate a genetic element, e.g., a genetic element comprising RNA. In some embodiments, a polynucleotide encoding one or more Anelloviridae family virus (e.g., Anellovirus) protein is fused to a promoter for expression in a host cell, e.g., an insect or animal cell. In some embodiments, the polynucleotide is cloned into a baculovirus expression system. In some embodiments, a host cell, e.g., an insect cell is infected with the baculovirus expression system and incubated for a period of time. In some embodiments, an infected cell is incubated for about 1, 2, 3, 4, 5, 10, 15, or 20 days. In some embodiments, an infected cell is lysed to recover the Anelloviridae family virus (e.g., Anellovirus) protein.

[0492] In some embodiments, an isolated Anelloviridae family virus (e.g., Anellovirus) protein is purified. In some embodiments, an Anellovirus protein is purified using purification techniques including but not limited to chelating purification, heparin purification, gradient sedimentation purification, and / or SEC purification. In some embodiments, a purified Anelloviridae family virus (e.g., Anellovirus) protein is mixed with a genetic element to encapsulate the genetic element, e.g., a genetic element comprising RNA. In some embodiments, a genetic element is encapsulated using an ORF1 protein, ORF2 protein, or modified version thereof. In some embodiments two nucleic acids are encapsulated. For instance, the first nucleic acid may be an mRNA e.g., chemically modified mRNA, and the second nucleic acid may be DNA.

[0493] In some embodiments, DNA encoding Anellovirus (AV) ORF1 (e.g., wildtype ORF1 protein, ORF1 proteins harboring mutations, e.g., to improve assembly efficiency, yield or stability, chimeric ORF1 protein, or fragments thereof) are expressed in insect cell lines (e.g., Sf9 and / or HighFive), animal cell lines (e.g., chicken cell lines (MDCC)), bacterial cells (e.g., E. coli) and / or mammalian cell lines (e.g., 293expi and / or MOLT4). In some embodiments, DNA encoding AV ORF1 may be untagged. In some embodiments, DNA encoding AV ORF1 may contain tags fused N-terminally and / or C-terminally. In some embodiments, DNA encoding AV ORF1 may harbor mutations, insertions or deletions within the ORF1 protein to introduce a tag, e.g., to aid in purification and / or identity determination, e.g., through immunostaining assays (including but not limited to ELISA or Western Blot). In some embodiments, DNA encoding AV ORF1 may be expressed alone or in combination with any number of helper proteins. In some embodiments, DNA encoding AV ORF1 is expressed in combination with AV ORF2 and / or ORF3 proteins.

[0494] In some embodiments, ORF1 proteins harboring mutations to improve assembly efficiency may include, but are not limited to, ORF1 proteins that harbor mutations introduced into the N-terminal Arginine Arm (ARG arm) to alter the pl of the ARG arm permitting pH sensitive nucleic acid binding to trigger particle assembly. In some embodiments, 0RF1 proteins harboring mutations that improve stability may include mutations to an interprotomer contacting beta strands F and G of the canonical jellyroll beta-barrel to alter hydrophobic state of the protomer surface and improve thermodynamic favorability of capsid formation.

[0495] In some embodiments, the present disclosure describes a method of making an anelloviral vector, the method comprising: (a) providing a mixture comprising: (i) a genetic element, and (ii) an 0RF1 molecule and (b) incubating the mixture under conditions suitable for encapsulating the genetic element within a proteinaceous exterior comprising the 0RF1 molecule, thereby making an anelloviral vector; optionally wherein the mixture is not comprised in a cell. In some embodiments, the method further comprises, prior to the providing of (a), expressing the 0RF1 molecule, e.g., in a host cell (e.g., an insect cell or a mammalian cell). In some embodiments, the expressing comprises incubating a host cell (e.g., an insect cell or a mammalian cell) comprising a nucleic acid molecule (e.g., a baculovirus expression vector) encoding the ORF1 molecule under conditions suitable for producing the ORF1 molecule. In some embodiments, the method further comprises, prior to the providing of (a), purifying the ORF1 molecule expressed by the host cell. In some embodiments, the method is performed in a cell-free system. In some embodiments, the present disclosure describes a method of manufacturing an anelloviral vector composition, comprising: (a) providing a plurality of anelloviral vectors or compositions according to any of the preceding embodiments; (b) optionally evaluating the plurality for one or more of: a contaminant described herein, an optical density measurement (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle:infectious unit ratio, e.g., as determined by fluorescence and / or ELISA); and (c) formulating the plurality of anelloviral vectors, e.g., as a pharmaceutical composition suitable for administration to a subject, e.g., if one or more of the parameters of (b) meet a specified threshold.

[0496] In vitro circularization

[0497] In some instances, the genetic element to be packaged into a proteinaceous exterior is a single stranded circular DNA. In some embodiments, double-stranded circular DNA is produced by in vitro circularization, e.g., as described in Example 35 of PCT Publication No. WO 2020 / 123816, incorporated by reference herein in its entirety. Generally, in vitro circularized DNA constructs can be produced by digesting a plasmid comprising the sequence of a genetic element to be packaged, such that the genetic element sequence is excised as a linear DNA molecule. The resultant linear DNA can then be ligated, e.g., using a DNA ligase, to form a double-stranded circular DNA. In some embodiments, the doublestranded DNA construct is smaller than a plasmid. In some embodiments, the double-stranded DNA construct is excised from a plasmid and then circularized, e.g., by in vitro circularization.

[0498] In some embodiments, a genetic element to be used for in vitro assembly of a particle (e.g., an anelloviral vector) as described herein is produced in a cell. In some embodiments, a cell is transfected with a construct (e.g., a plasmid, tandem construct, and / or an in vitro circularized nucleic acid molecule, e.g., as described herein) comprising the sequence of a genetic element. In embodiments, the cell is incubated under conditions suitable for replication of the construct. In embodiments, the cell is incubated under conditions suitable for production and / or replication of the genetic element (e.g., from the construct). In embodiments, the cell is lysed to recover the genetic element. In some embodiments, a genetic element to be used for in vitro assembly is a DNA, e.g., a single-stranded DNA (ssDNA). In embodiments, the genetic element is a negative sense ssDNA. In some embodiments, a genetic element to be used for in vitro assembly comprises RNA (e.g., an mRNA).

[0499] Enrichment and purification

[0500] Harvested Anelloviridae family vectors can be purified and / or enriched, e.g., to produce an anelloviral vector preparation. In some embodiments, the harvested anelloviral vectors are isolated from other constituents or contaminants present in the harvest solution, e.g., using methods known in the art for purifying viral particles (e.g., purification by sedimentation, chromatography, and / or ultrafiltration). In some embodiments, the purification steps comprise removing one or more of serum, host cell DNA, host cell proteins, particles lacking the genetic element, and / or phenol red from the preparation. In some embodiments, the harvested Anelloviridae family vectors are enriched relative to other constituents or contaminants present in the harvest solution, e.g., using methods known in the art for enriching viral particles.

[0501] In some embodiments, the resultant preparation or a pharmaceutical composition comprising the preparation will be stable over an acceptable period of time and temperature, and / or be compatible with the desired route of administration and / or any devices this route of administration will require, e.g., needles or syringes.

[0502] III. Pharmaceutical Compositions

[0503] The Anelloviridae family vector, anelloviral vector, or other vector described herein may also be included in pharmaceutical compositions with a pharmaceutical excipient, e.g., as described herein. In some embodiments, the pharmaceutical composition comprises at least 105, 106, 107, 108, 109, IO10, 1011, 1012, IO13, 1014, or 1015Anelloviridae family vectors. In some embodiments, the pharmaceutical composition comprises about 105-1015, 1O5-1O10, or 1010-1015Anelloviridae family vectors. In some embodiments, the pharmaceutical composition comprises about 108(e.g., about 105, 106, 107, 108, 109, or IO10) genomic equivalents / mL of the Anelloviridae family vector. In some embodiments, the pharmaceutical composition comprises 1O5-1O10, 1O6-1O10, 1O7-1O10, 1O8-1O10, 1O9-1O10, 105-106, 105-107, 105-108, 105-109, 1051011, 105-1012, 105-1013, 105-1014, 105-1015, or 1010-1015genomic equivalents / mL of the Anelloviridae family vector. In some embodiments, the pharmaceutical composition comprises sufficient Anelloviridae family vectors to deliver at least 1, 2, 5, or 10, 100, 500, 1000, 2000, 5000, 8,000, 1 x 104, 1 x 105, 1 x 106, 1 x 107or greater copies of a genetic element comprised in the Anelloviridae family vectors per cell to a population of the eukaryotic cells. In some embodiments, the pharmaceutical composition comprises sufficient Anelloviridae family vectors to deliver at least about 1 x 104, 1 x 105, 1 x 106, 1 x or 107, or about 1 x 104-l x 105, 1 x 104-l x 106, 1 x 104-l x 107, 1 x 105-l x 106, 1 x 105-l x 107, or 1 x 106-l x 107copies of a genetic element comprised in the Anelloviridae family vectors per cell to a population of the eukaryotic cells.

[0504] In some embodiments, the pharmaceutical composition has one or more of the following characteristics: the pharmaceutical composition meets a pharmaceutical or good manufacturing practices (GMP) standard; the pharmaceutical composition was made according to good manufacturing practices (GMP); the pharmaceutical composition has a pathogen level below a predetermined reference value, e.g., is substantially free of pathogens; the pharmaceutical composition has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants; or the pharmaceutical composition has low immunogenicity or is substantially non-immunogenic, e.g., as described herein.

[0505] In some embodiments, the pharmaceutical composition comprises below a threshold amount of one or more contaminants. Exemplary contaminants that are desirably excluded or minimized in the pharmaceutical composition include, without limitation, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal -derived components (e.g., serum albumin or trypsin), replication- competent viruses, non-infectious particles, free viral capsid protein, adventitious agents, and aggregates. In embodiments, the contaminant is host cell DNA. In embodiments, the composition comprises less than about 10 ng of host cell DNA per dose. In embodiments, the level of host cell DNA in the composition is reduced by filtration and / or enzymatic degradation of host cell DNA. In embodiments, the pharmaceutical composition consists of less than 10% (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) contaminant by weight.

[0506] In one aspect, the invention described herein includes a pharmaceutical composition comprising: a) an Anelloviridae family vector (e.g., anelloviral vector) comprising a genetic element comprising (i) a sequence encoding a non-pathogenic exterior protein, (ii) an exterior protein binding sequence that binds the genetic element to the non-pathogenic exterior protein, and (iii) a sequence encoding a regulatory nucleic acid; and a proteinaceous exterior that is associated with, e.g., envelops or encapsulates, the genetic element; and b) a pharmaceutical excipient.

[0507] IV. Methods of Use

[0508] The Anelloviridae family vectors, e.g., anelloviral vectors, and compositions comprising Anelloviridae family vectors, e.g., anelloviral vectors, described herein may be used in methods of treating a disease, disorder, or condition, e.g., in a subject (e.g., a mammalian subject, e.g., a human subject) in need thereof. Administration of a pharmaceutical composition described herein may be, for example, by way of parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration. In some embodiments, an Anelloviridae family vector, e.g., anelloviral vector, or pharmaceutical composition as described herein is administered subretinally. In some embodiments, an Anelloviridae family vector, e.g., anelloviral vector, or pharmaceutical composition as described herein is administered intravitreally. In some embodiments, an Anelloviridae family vector, e.g., anelloviral vector, or pharmaceutical composition as described herein is administered suprachoroidally. The anelloviral vectors may be administered alone or formulated as a pharmaceutical composition.

[0509] The Anelloviridae family vector (e.g., anelloviral vector) may be administered in the form of a unit-dose composition, such as a unit dose parenteral composition. Such compositions are generally prepared by admixture and can be suitably adapted for parenteral administration. Such compositions may be, for example, in the form of injectable and infusible solutions or suspensions or suppositories or aerosols.

[0510] In some embodiments, administration of an Anelloviridae family vector (e.g., anelloviral vector) or composition comprising same, e.g., as described herein, may result in delivery of a genetic element comprised by the Anelloviridae family vector (e.g., anelloviral vector) to a target cell, e.g., in a subject.

[0511] An Anelloviridae family vector (e.g., anelloviral vector) or composition thereof described herein, e.g., comprising an effector (e.g., an endogenous or exogenous effector), may be used to deliver the effector to a cell, tissue, or subject. In some embodiments, the effector is a therapeutic effector. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) or composition thereof is used to deliver the effector to the eye of a subject, e.g., a mammalian subject, e.g., a human subject. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) or composition thereof is used to deliver the effector to a cell of the eye of a subject, e.g., a mammalian subject, e.g., a human subject. In certain embodiments, the cell of the eye is a photoreceptor cell, a retinal cell, a cell of the posterior eye cup (PEC), retinal ganglion cell, a cell of the optic nerve, a cell of the optic nerve head, or a retinal pigmented epithelium (RPE) cell. In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector) or composition thereof is used to deliver the effector to bone marrow, blood, heart, GI or skin. Delivery of an effector by administration of an Anelloviridae family vector (e.g., anelloviral vector) composition described herein may modulate (e.g., increase or decrease) expression levels of a noncoding RNA or polypeptide in the cell, tissue, or subject. Modulation of expression level in this fashion may result in alteration of a functional activity in the cell to which the effector is delivered. In some embodiments, the modulated functional activity may be enzymatic, structural, or regulatory in nature.

[0512] In some embodiments, the Anelloviridae family vector (e.g., anelloviral vector), or copies thereof, are detectable in a cell 24 hours (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) after delivery into a cell. In embodiments, an Anelloviridae family vector (e.g., anelloviral vector) or composition thereof mediates an effect on a target cell, and the effect lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months. In some embodiments (e.g., wherein the Anelloviridae family vector (e.g., anelloviral vector) or composition thereof comprises a genetic element encoding an exogenous protein), the effect lasts for less than 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months.

[0513] Ocular Delivery Methods

[0514] In some aspects, the present disclosure provides a method of modulating, e.g., enhancing or inhibiting, a biological function in an eye of the subject, the method comprising administering an anelloviral vector or pharmaceutical composition described herein to the eye of the subject. In some embodiments, the biological function comprises one or more of: best corrected visual acuity (BCVA) retinal sensitivity to light (e.g., as measured by perimetry or microperimetry, e.g., in the dark and light- adapted states, full-field, multi-focal, focal or pattern electroretinography ERG), contrast sensitivity, reading speed, and / or color vision. In some embodiments, the biological function is measured using clinical biomicroscopic examination, fundus photography, optical coherence tomography (OCT), fundus auto-fluorescence (FAF), infrared and / or multicolor imaging, fluorescein or ICG angiography, and / or adoptive optics.

[0515] In some embodiments, a diseases, disorder, or condition that can be treated with an anelloviral vector described herein, or a composition comprising the anelloviral vector, is a disease of the eye. In some embodiments, the disease of the eye is selected from the group consisting of: macular degeneration, such as neovascular age-related macular degeneration (nAMD) (also known as wet AMD or WAMD), retinal vein occlusion (RVO), macular edema following retinal vein occlusion, diabetic macular edema (DME), or diabetic retinopathy (DR), or retinopathy of prematurity (ROP). In some embodiments, the disease, disorder, or condition that can be treated with the anelloviral vector described herein, or a composition comprising the anelloviral vector, is a VEGF-associated disorder (e.g., a macular edema). The method of treatment may comprise administering to the intravitreal space, suprachoroidal space, subretinal space, or outer surface of the sclera in the eye of said human subject (e.g., by suprachoroidal injection (for example, via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via transvitreal approach (a surgical procedure), subretinal administration via the suprachoroidal space (for example, a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted and tunneled through the suprachoroidal space toward the posterior pole, where a small needle injects into the subretinal space), or a posterior juxtascleral depot procedure (for example, via a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and kept in direct apposition to the scleral surface)) the anelloviral vector described herein. The method may comprise delivering to the retina or posterior eye cup (PEC) of said human subject the anelloviral vector described herein, by the use of a suprachoroidal drug delivery device such as a microinjector. The method may comprise delivering to the retina or posterior eye cup (PEC) of said human subject the anelloviral vector described herein, wherein the human subject has a Best- Corrected Visual Acuity (BCVA) that is <20 / 20 and >20 / 400.

[0516] In some aspects, the present disclosure provides a method for prophylactic treatment of AMD or ocular neovascular diseases as described herein, comprising administering a pharmaceutically effective amount of the pharmaceutical compositions provided herein to a human subject in need of such treatment. The present disclosure may be used to treat patients at risk of developing AMD, or presenting early symptoms of the disease. This may include treatment of eyes either simultaneously or sequentially. Simultaneous treatment may mean that the treatment is administered to each eye at the same time or that both eyes are treated during the same visit to a treating physician or other healthcare provider. It has been documented that patients have a higher risk of developing AMD in a healthy fellow eye of an eye that presents symptoms of AMD, or in patients who have a genetic predisposition toward developing AMD. Compositions of the present disclosure can be used as a prophylactic treatment in prevention of AMD in the fellow eye.

[0517] In some aspects, the human subject treated with an anelloviral vector described herein shows no clinically significant retinal toxicity as assessed by serial ophthalmic examinations over at least about a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month period. In some aspects, the human subject shows no clinically significant retinal toxicity as assessed by serial ophthalmic examinations over at most about a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month period. In some aspects, no superficial, anterior segment or vitreous inflammatory signs are present in the human subject over at least a two month period. In some cases, no superficial, anterior segment or vitreous inflammatory signs are present in the human subject at 1 week or at 3, 6, 9 or 12 months after administration of the pharmaceutical composition. In some aspects, there is no evidence of visual acuity loss, TOP elevation, retinal detachment, or any intraocular or systemic immune response in said human subject at least 120 days post administration.

[0518] In some aspects, the invention features a method of delivering an anelloviral vector to a subject, e.g., to an eye of a subject (e.g., to a photoreceptor, retina, posterior eye cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigmented epithelium (RPE) of the subject). The method includes administering a pharmaceutical composition comprising an anelloviral vector as described herein to the subject, e.g., to an eye of a subject.

[0519] In some embodiments, the method of delivering an anelloviral vector to a subject comprises contacting the anelloviral vector to any suitable ocular cell. Ocular cells associated with age-related macular degeneration include, but are not limited to, cells of neural origin, cells of all layers of the retina, especially retinal pigment epithelial cells, glial cells, and pericytes. Other ocular cells that can be contacted as a result of the inventive method include, for example, endothelial cells, iris epithelial cells, corneal cells, ciliary epithelial cells, Mueller cells, astrocytes, muscle cells surrounding and attached to the eye (e.g., cells of the lateral rectus muscle), fibroblasts (e.g., fibroblasts associated with the episclera), orbital fat cells, cells of the sclera and episclera, connective tissue cells, muscle cells, and cells of the trabecular meshwork. Other cells linked to various ocular-related diseases include, for example, fibroblasts and vascular endothelial cells.

[0520] Generally, the anelloviral vector can be delivered in the form of a suspension injected intraocularly (e.g., subretinally) under direct observation using an operating microscope. This procedure may involve vitrectomy followed by injection of vector suspension using a fine cannula through one or more small retinotomies into the subretinal space.

[0521] Briefly, an infusion cannula can be sutured in place to maintain a normal globe volume by infusion (of e.g. saline) throughout the operation. A vitrectomy is performed using a cannula of appropriate bore size (for example 20 to 27 gauge), wherein the volume of vitreous gel that is removed is replaced by infusion of saline or other isotonic solution from the infusion cannula. The vitrectomy is advantageously performed because (1) the removal of its cortex (the posterior hyaloid membrane) facilitates penetration of the retina by the cannula; (2) its removal and replacement with fluid (e.g. saline) creates space to accommodate the intraocular injection of vector, and (3) its controlled removal reduces the possibility of retinal tears and unplanned retinal detachment.

[0522] In some embodiments, the anelloviral vector is directly injected into the subretinal space outside the central retina, by utilizing a cannula of the appropriate bore size (e.g. 27-45 gauge), thus creating a bleb in the subretinal space. In other embodiments, the subretinal injection of vector suspension is preceded by subretinal injection of a small volume (e.g. about 0.1 to about 0.5 ml) of an appropriate fluid (such as saline or Ringer's solution) into the subretinal space outside the central retina. This initial injection into the subretinal space establishes an initial fluid bleb within the subretinal space, causing localized retinal detachment at the location of the initial bleb. This initial fluid bleb can facilitate targeted delivery of vector suspension to the subretinal space (by defining the plane of injection prior to vector delivery), and minimize possible vector administration into the choroid and the possibility of vector injection or reflux into the vitreous cavity. In some embodiments, this initial fluid bleb can be further injected with fluids comprising one or more vector suspensions and / or one or more additional therapeutic agents by administration of these fluids directly to the initial fluid bleb with either the same or additional fine bore cannulas.

[0523] Intraocular administration of the anello viral vector and / or the initial small volume of fluid can be performed using a fine bore cannula (e.g. 21-A5 gauge) attached to a syringe. In some embodiments, the plunger of this syringe may be driven by a mechanized device, such as by depression of a foot pedal. The fine bore cannula is advanced through the sclerotomy, across the vitreous cavity and into the retina at a site pre -determined in each subject according to the area of retina to be targeted (but outside the central retina). Under direct visualization the vector suspension is injected mechanically under the neurosensory retina causing a localized retinal detachment with a self-sealing non-expanding retinotomy. As noted above, the anelloviral vector can be either directly injected into the subretinal space creating a bleb outside the central retina or the vector can be injected into an initial bleb outside the central retina, causing it to expand (and expanding the area of retinal detachment). In some embodiments, the injection of vector suspension is followed by injection of another fluid into the bleb.

[0524] Without wishing to be bound by theory, the rate and location of the subretinal injection(s) can result in localized shear forces that can damage the macula, fovea and / or underlying RPE cells. The subretinal injections may be performed at a rate that minimizes or avoids shear forces. In some embodiments, the anelloviral vector is injected over about 15-17 minutes. In some embodiments, the anelloviral vector is injected over about 17-20 minutes. In some embodiments, the anelloviral vector is injected over about 20-22 minutes. In some embodiments, the anelloviral vector is injected at a rate of about 35 to about 65 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 35 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 40 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 45 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 50 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 55 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 60 pl / ml. In some embodiments, the anelloviral vector is injected at a rate of about 65 pl / ml. One of ordinary skill in the art would recognize that the rate and time of injection of the bleb may be directed by, for example, the volume of the composition comprising the anelloviral vector or size of the bleb necessary to create sufficient retinal detachment to access the cells of central retina, the size of the cannula used to deliver the anelloviral vector, and the ability to safely maintain the position of the canula of the invention.

[0525] One or multiple (e.g. 2, 3, or more) blebs can be created. Generally, the total volume of bleb or blebs created by the methods and systems of the invention cannot exceed the fluid volume of the eye, for example about 4 ml in a typical human subject. The total volume of each individual bleb is preferably at least about 0.3 ml, and more preferably at least about 0.5 ml in order to facilitate a retinal detachment of sufficient size to expose the cell types of the central retina and create a bleb of sufficient dependency for optimal manipulation. One of ordinary skill in the ai t will appreciate that in creating the bleb according to the methods and systems of the invention that the appropriate intraocular pressure must be maintained in order to avoid damage to the ocular structures. The size of each individual bleb may be, for example, about 0.5 to about 1.2 ml, about 0.8 to about 1.2 ml, about 0.9 to about 1.2 ml, about 0.9 to about 1.0 ml, about 1.0 to about 2.0 ml, about 1.0 to about 3.0 ml. Thus, in one example, to inject a total of 3 ml of vector suspension, 3 blebs of about 1 ml each can be established. The total volume of all blebs in combination may be, for example, about 0.5 to about 3.0 ml, about 0.8 to about 3.0 ml, about 0.9 to about 3.0 ml, about 1.0 to about 3.0 ml, about 0.5 to about 1.5 ml, about 0.5 to about 1.2 ml, about 0.9 to about 3.0 ml, about 0.9 to about 2.0 ml, about 0.9 to about 1.0 ml.

[0526] In order to safely and efficiently transduce areas of target retina (e.g. the central retina) outside the edge of the original location of the bleb, the bleb may be manipulated to reposition the bleb to the target area for transduction. Manipulation of the bleb can occur by the dependency of the bleb that is created by the volume of the bleb, repositioning of the eye containing the bleb, repositioning of the head of the human with an eye or eyes containing one or more blebs, and / or by means of a fluid-air exchange. This is particularly relevant to the central retina since this area typically resists detachment by subretinal injection. In some embodiments fluid-air exchange is utilized to reposition the bleb; fluid from the infusion cannula is temporarily replaced by air, e.g. from blowing air onto the surface of the retina. As the volume of the air displaces vitreous cavity fluid from the surface of the retina, the fluid in the vitreous cavity may flow out of a cannula. The temporar y lack of pressure from the vitreous cavity fluid causes the bleb to move and gravitate to a dependent part of the eye. By positioning the eye globe appropriately, the bleb of subretinal vector is manipulated to involve adjacent areas (e.g. the macula and / or fovea). In some cases, the mass of the bleb is sufficient to cause it to gravitate, even without use of the fluid-air exchange. Movement of the bleb to the desired location may further be facilitated by altering the position of the subject's head, so as to allow the bleb to gravitate to the desired location in the eye. Once the desired configuration of the bleb is achieved, fluid is returned to the vitreous cavity. The fluid is an appropriate fluid, e.g., fresh saline. Generally, the subretinal vector may be left in situ without retinopexy to the retinotomy and without intraocular tamponade, and the retina will spontaneously reattach within about 48 hours.

[0527] The composition can be administered directly to the eye of a mammal, such as, for example, a mouse, a rat, a non-human primate, or a human. Any administration route is appropriate so long as the composition contacts an appropriate ocular cell. The composition can be appropriately formulated and administered in the form of an injection, eye lotion, ointment, implant, and the like. The composition can be administered, for example, topically, intracamerally, subconjunctivally, intraocularly, retrobulbarly, periocularly (e.g., subtenon delivery), subretinally, or suprachoroidally. Topical formulations are well known in the art. Patches, corneal shields (see, e.g., U.S. Pat. No. 5,185,152), ophthalmic solutions (see, e.g., U.S. Pat. No. 5,710,182), and ointments also are known in the art and can be used in the context of the inventive method. The composition also can be administered non-invasively using a needleless injection device, such as the Biojector 2000 Needle -Free Injection Management System™ available from Bioject Medical Technologies Inc. (Tigard, Oreg.).

[0528] Alternatively, the composition can be administered using invasive procedures, such as, for instance, intravitreal injection or subretinal injection, optionally preceded by a vitrectomy, or periocular (e.g., subtenon) delivery. The composition can be injected into different compartments of the eye, e.g., the vitreal cavity or anterior chamber. Preferably, the composition is administered intravitreally, most preferably by intravitreal injection.

[0529] In some embodiments, the composition may be administered using an ocular delivery system comprising the use of a microneedle (USPN 8,808, 225, incorporated herein in its entirety).

[0530] Ocular Delivery Systems

[0531] In some embodiments, a composition or method described herein involves an ocular- delivery system, such as the Orbit Subretinal Delivery System. Briefly, such a delivery system may comprise a cannula to be inserted into the eye for delivering the anelloviral vector into the eye, a device body for delivering saline solution or anelloviral vector to the cannula, a first line for delivering the saline solution to the device body, and a second line for delivering the anelloviral vector to the device body. More particularly, in some embodiments, the delivery system is provided as three “sets”. The first set is a subretinal injection device set that comes with a subretinal injection device, which comprises a cannula tip / needle, a needle advancement knob, a subretinal injection device body (with a magnet), a dose line luer, and a BSS line luer. The system can also comprise a magnetic pad and an ophthalmic marker. The magnet provides stabilization during injection. The second set (which can be referred to as the tubing set) includes tubing assembly, a BSS syringe, two syringe snap collars, and a CPC adapter. The third set (which can be referred to as the dosing set) comprises a dose syringe and a tubing clamp.

[0532] For the subretinal injection device, the internal needle is connected to the needle advancement knob, which is connected to the subretinal injection device body. This has two lines, each attaching to either the BSS line luer or the dose line luer.

[0533] Accordingly, in some embodiments, an anelloviral vector described herein is situated in an ocular delivery system. The ocular delivery system may comprise, for example: a) a cannula comprising a first end and a second end, wherein the first end of the cannula optionally comprises a needle, b) a device body which optionally comprises a magnet, wherein the second end of the cannula is operably connected to the device body, c) needle advancement knob situated on the device body, wherein the needle advancement knob can be adjusted by a user to advance the needle to extend beyond the first end of the cannula, e.g., extend into the subretinal space; d) a first line operably attached to the connected to device body (e.g., attached with a luer), wherein the first line may be used to deliver a wash solution, e.g., a saline solution, e.g., BSS, wherein optionally the first line is operably connected to a first syringe (e.g., connected using a syringe snap collar-); e) a second line operably attached to the connected to device body (e.g., attached with a luer), wherein the second line may be used to deliver a composition (e.g., an Anelloviridae family vector (e.g., anelloviral vector) or pharmaceutical composition) described herein, wherein optionally the second line is operably connected to a second syringe (e.g., connected using a syringe snap collar).

[0534] In some embodiments, the ocular delivery system is part of a kit. The kit may further comprise one or both of a magnetic pad and an ophthalmic marker.

[0535] In some embodiments, a method described herein comprises administering a pharmaceutical composition described herein using an ocular delivery system, e.g., the ocular delivery system described above. In some embodiments, the method comprises surgically preparing the eye for administration of the composition, e.g., by exposing the sclera (e.g., by conjunctival peritomy), optionally transferring ink to the sclera to create a suturing template, creating a suture loop, and creating a sclerotomy. The cannula may be inserted into the sclerotomy. The ocular delivery system may be placed. For instance, the magnetic pad may be placed on the subject’s forehead, and the device body may be placed on the magnetic pad, e.g., in the same meridian as the sclerotomy. The first end of the cannula may be positioned directly above the opposite edge of the cornea. Cannulation may be performed. For instance, the suture loops may be lifted and the cannula may be passed through the suture loops. The device body may be slid toward the eye. The cannula may be inserted into the sclerotomy. The needle may be advanced into the subretinal space using the needle advancement knob. A saline solution (e.g., BSS) may be administered, e.g., using the syringe connected to the first line. The saline solution may form a visible bleb. The pharmaceutical composition may be administered, e.g., using the syringe connected to the second line. The pharmaceutical composition may be released into the bleb. The needle may then be retracted. The ocular delivery system may be removed from the eye.

[0536] The delivery method may also comprise one or more of the following steps.

[0537] The BSS syringe of the tubing set is attached to the delivery system via the BSS line of the subretinal injection device. A plunger is inserted into the dose syringe, followed by attachment of a sterile needle to the dose syringe.

[0538] A plunger is also inserted into the dose syringe, and a sterile needle is attached to the dose syringe. This needle is then inserted into the vial and is used to aspirate the subretinal infusate into the syringe. The needle is then removed.

[0539] The tab is then rotated into the latched position in order to prime the dose line. The dose syringe is attached to the dose line of the Subretinal injection device. The dose syringe plunger is then advanced slowly until it reaches a hard stop and a tactile click is reached. This primes the dose line and the dose syringe assures the correct subretinal dose volume is ready for injection into the subretinal area.

[0540] If using pneumatic injection, the plunger is to be rotated counterclockwise to remove the threaded rod from the BSS syringe and leave the seal in the BSS syringe. The tubing is to be inserted in the open barrel of the BSS syringe and secured in place by sliding the syringe snap collar over both components. The tubing assembly is then attached to the pneumatic source of choice (e.g., a vitrectomy machine) using the CPC adaptor, if necessary. The viscous fluid control injection pressure is to be set to 36 psi.

[0541] The provided tubing claims supplied with the subretinal delivery system are only to be used with an alternate dose syringe (not supplied in the set) that is validated for use with the Orbit SDS. The alternate syringe’s labeling must indicate that it is validated for use with the Orbit SDS and include instructions for use with the Orbit SDS. If using an alternate dose syringe, the tubing clamp is placed on the dose line following priming to prevent potential backflow into the alternate dose syringe during BSS syringe use. Immediately before injecting the infusate, the tubing clamp is to be removed.

[0542] Exemplary Surgical Steps:

[0543] The site is prepared by inserting the lid speculum, and inserting a valved port for the chandelier. The eye is rotated inferonasally to expose the superotemporal quadrant, and a conjunctival peritomy is performed to expose the sclera. A cannulation path that does not interfere with identified vortex veins or long posterior ciliary neurovascular bundles is selected. Ink is applied to the tips of the ophthalmic marker with the limbus, and gently press it against the sclera to transfer the ink. After drying the scleral surface, the marker is aligned with the limbus and is gently pressed against the sclera to transfer ink, and thereby creating the suturing template (about 10 ink dots). The suture loop is created. A sclerotomy is performed.

[0544] Exemplary Device Placement:

[0545] The adhesive backing is removed from the magnetic pad, and the pad is placed over the sterile fenestrated drape, on top of the patient’s forehead. The primed subretinal injection device body on top of the magnetic pad is placed in the same meridian as the sclerotomy. The distal tip of the subretinal injection cannula is positioned directly above the opposite edge of the cornea to ensure sufficient slack for advancement. The needle is advanced and the flow of BSS or BSS PLUS is checked. The needle is fully retracted.

[0546] Exemplary Cannulation:

[0547] Using smooth forceps, the flexible cannula is grasped, approximately 10 mm from the distal tip. Using toothed forceps on the eye to help with insertion, the suture loops are lifted and then the posterior lip of the sclerotomy is grasped. The cannula is passed through the suture loops. Prior to insertion, the subretinal injection device body is slid toward the eye to provide additional slack and maintain a tangential path to the eye’ s curvature. While grasping the center of the posterior lip of the sclerotomy and pulling away from the eye, the flexible cannula is inserted into the sclerotomy. The eye is rotated back to the neutral axis.

[0548] Exemplary method for using Clearside Biomedical SCS microinjector:

[0549] In some embodiments, a pharmaceutical composition or method described herein involves an ocular delivery system, such as an SCS microinjector. Briefly, such a delivery system may comprise a needle sized appropriately to deliver an anello viral vector to the suprachoroidal space, a chamber to contain the anelloviral vector, and a plunger to administer the anelloviral vector. In some embodiments, the microinjector is comprised of a needle of various lengths (needle length is printed on the needle - either 900 um or 1100 um). The needle is a 30 gauge needle. The needle is connected to a conjunctiva compressing hub, which is connected to a chamber (e.g., a barrel), which has a 100 uL capacity and has indicators in increments of 25 uL. The barrel is connected to a plunger and plunger handle to inject the drug. The microinjector also comes with a needle safety cap with integrated fixed length calipers of 4.5 mm. The Clearside SCS microinjector is designed for suprachoroidal drug delivery.

[0550] Accordingly, in some embodiments, a pharmaceutical composition described herein is situated in an ocular delivery system. The ocular delivery system may comprise: a) a needle, e.g., a 30 gauge needle, wherein optionally the needle is 800-1200 um (e.g., about 900 um or 1100 um in length); b) optionally, a conjunctiva compressing hub connected to the needle; c) a chamber connected to one or both of the conjunctiva compressing hub and the needle; and d) optionally, a plunger connected to the chamber.

[0551] In some embodiments, the ocular delivery system is part of a kit. The kit may further comprise, one or both of a needle safety cap and calipers.

[0552] In some embodiments, a method described herein comprises administering a pharmaceutical composition described herein using an ocular delivery system, e.g., the ocular delivery system described above. In some embodiments, the method comprises inserting the needle into the suprachoroidal space and administering the pharmaceutical composition into the suprachoroidal space.

[0553] Dosing

[0554] Therapeutically effective doses of the composition or anelloviral vector as described herein may be administered subretinally and / or intraretinally (e.g., by subretinai injection via the transvitreal approach (a surgical procedure), or subretinai administration via the suprachoroidal space) in a volume ranging from 0.1 mL to 0.5 mL, preferably in 0.1 to 0.30 ml.. (100-300 pl), and most preferably, in a volume of 0.25 mL (250 pl). Therapeutically effective doses of the anelloviral vector should be administered suprachoroidally (e.g., by suprachoroidal injection) in a volume of 100 pl or less, for example, in a volume of 50-100 μl. Therapeutically effective doses of the anelloviral vector should be administered to the outer surface of the sclera (e.g., by a posterior juxtascleral depot procedure) in a volume of 500 μl or less, for example, in a volume of 10-20 pl. 20-50 pl. 50-100 pl, 100-200 μl, 200-300 pl, 300-400 pl, or 400-500 pl. Subretinai injection is a surgical procedure performed by trained retinal surgeons that involves a vitrectomy with the subject under local anesthesia, and subretinai injection of the gene therapy into the retina (see, e.g., Campochiaro et al., 2017, Hum Gen Ther 28(1 ):99-l 11, which is incorporated by reference herein in its entirety). In a specific embodiment, the subretinai administration is performed via the suprachoroidal space using a suprachoroidal catheter which injects drug into the subretinai space, such as a subretinai drug delivery device that comprises a catheter which can be inserted and tunneled through the suprachoroidal space to the posterior pole, where a small needle injects into the subretinai space (see, e.g., Baldassarre et al.. 2017, Subretinai Deli very of Cells via the Suprachoroidal Space: Janssen Trial. In: Schwartz et al. (eds) Cellular Therapies for Retinal Disease, Springer, Cham; International Patent Application Publication No. WO 2016 / 040635 Al; each of which is incorporated by reference herein in its entirety). Suprachoroidal administration procedures involve administration to the suprachoroidal space of the eye, and are normally performed using a suprachoroidal drug delivery device such as a microinjector with a microneedle (see, e.g., Hariprasad, 2016, Retinal Physician 13: 20-23; Goldstein, 2014. Retina Today 9(5): 82-87; each of which is incorporated by reference herein in its entirety ). The suprachoroidal drug delivery devices that can be used to deposit the anelloviral vector in the suprachoroidal space according to the invention described herein include, but are not limited to, suprachoroidal drug delivery devices manufactured by Clearside® Biomedical, Inc. (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23) and MedOne suprachoroidal catheters. The subretinal drug delivery devices that can be used to deposit the anelloviral vector in the subretinal space via the suprachoroidal space according to the invention described herein include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals, Inc. (see, for example, International Patent Application Publication No. WO 2016 / 040635 Al). In a specific embodiment, administration to the outer surface of the sclera is performed by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and kept in direct apposition to the scleral surface. See Section 5.3.2 for more details of the different modes of administration. Suprachoroidal, subretinal, juxtascleral and / or intraretinal administration should result in deli very of the soluble transgene product to the retina, the vitreous humor, and / or the aqueous humor. The expression of the exogenous effector by retinal cells, e.g., rod. cone, retinal pigment epithelial, horizontal, bipolar, amacrine, ganglion, and / or Muller cells, results in delivery and maintenance of the transgene product in the retina, the vitreous humor, and / or the aqueous humor. Doses that maintain a concentration of the transgene product at a Cmin of at least 0.330 pg / mL in the Vitreous humour, or 0.110 pg / mL in the Aqueous humour (the anterior chamber of toe eye) for three months are desired; thereafter, Vitreous Cmin concentrations of the transgene product ranging from 1.70 to 6.60 pg / mL, and / or Aqueous Crain concentrations ranging from 0.567 to 2.20 jtg / mL should be maintained. However, because the transgene product is continuously produced, maintenance of lower concentrations can be effective. The concentration of toe transgene product can be measured in patient samples of the vitreous humour and / or aqueous from the anterior chamber of the treated eye.

[0555] Alternati vely, vitreous humour concentrations can be estimated and / or monitored by measuring the patient's serum concentrations of the transgene product — the ratio of systemic to vi treat exposure to the transgene product is about 1 :90,000. (E.g., see, vitreous humor and serum concentrations of ranibizumab reported in Xu L. et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, at p, 1621 and Table 5 at p. 1623. which is incorporated by reference herein in its entirety).

[0556] Anelloviral vectors can be delivered to the eye by intraocular injection into the vitreous. In this application, the injection volume of the anelloviral vector could be substantially larger, as the volume is not constrained by the anatomy of the subretinal space. Acceptable dosages in this instance can range from 25 ul to 1000 ul. In this application, the target cells to be transduced include the retinal ganglion cells, which are the retinal cells primarily affected in glaucoma. V. Redosing

[0557] The Anelloviridae family vector (e.g., anelloviral vector) as described herein can, in some instances, be used as a delivery vehicle that can be administered in multiple doses (e.g., doses administered separately). While not wishing to be bound by theory, in some embodiments, an Anelloviridae family vector (e.g., anelloviral vector) (e.g., as described herein) induces a relatively low immune response (as measured, for example, as 50% GMT values), e.g., allowing for repeated dosing of a subject with one or more Anelloviridae family vectors (e.g., anelloviral vectors) (e.g., multiple doses of the same Anelloviridae family vector (e.g., anelloviral vector) or different Anelloviridae family vectors (e.g., anelloviral vectors)). In an aspect, the invention provides a method of delivering an effector, comprising administering to a subject a fust plurality of Anelloviridae family vectors (e.g., anelloviral vectors) and then a second plurality of Anelloviridae family vectors (e.g., anelloviral vectors). In some embodiments, the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors) comprise the same proteinaceous exterior as the Anelloviridae family vectors (e.g., anelloviral vectors) of the first plurality. In another aspect, the invention provides a method of selecting a subject (e.g., a human subject) to receive an effector, wherein the subject previously received, or was identified as having received, a first plurality of Anelloviridae family vectors (e.g., anelloviral vectors) comprising a genetic element encoding an effector, in which the method involves selecting the subject to receive a second plurality of Anelloviridae family vectors (e.g., anelloviral vectors) comprising a genetic element encoding an effector (e.g., the same effector as that encoded by the genetic element of the first plurality of Anelloviridae family vectors (e.g., anelloviral vectors), or a different effector as that encoded by the genetic element of the first plurality of Anelloviridae family vectors (e.g., anelloviral vectors)). In another aspect, the invention provides a method of identifying a subject (e.g., a human subject) as suitable to receive a second plurality of Anelloviridae family vectors (e.g., anelloviral vectors), the method comprising identifying the subject has having previously received a first plurality of Anelloviridae family vectors (e.g., anelloviral vectors) comprising a genetic element encoding an effector, wherein the subject being identified as having received the first plurality of Anelloviridae family vectors (e.g., anelloviral vectors) is indicative that the subject is suitable to receive the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors). In some embodiments, the first plurality of Anelloviridae family vectors (e.g., anelloviral vectors) is administered by the same route of administration as the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors), e.g. intravitreal administration for both the first plurality and second plurality of Anelloviridae family vectors (e.g., anelloviral vectors). In some embodiments, the fust plurality of Anelloviridae family vectors (e.g., anelloviral vectors) is administered by a different route of administration as the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors), e.g. subretinal administration for the first plurality and intravitreal administration for the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors), or intravitreal administration for the first plurality and subretinal administration for the second plurality of Anelloviridae family vectors (e.g., anelloviral vectors).

[0558] All references and publications cited herein are hereby incorporated by reference.

[0559] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0560] EXAMPLES

[0561] Table of Contents

[0562] Example 1 : Production of an anelloviral vector with Aflibercept as payload for in vivo administration Example 2: In Vivo Administration of Anelloviral vector Expressing Aflibercept in Non-Human Primates Example 3: In Vivo Administration of an Anelloviral vector Expressing Aflibercept in Non-Human Primates at a Higher Dose and a Redosing Arm

[0563] EXAMPLE 1 - Production of an anelloviral vector with Aflibercept as payload for in vivo administration

[0564] This example describes the production of an anelloviral vector with Aflibercept as payload (ANV 19. Aflibercept) for in vivo administration.

[0565] MOLT-4 cells were transfected with the three plasmids listed in table EO (pRTx-3525; pRTx-2848; pRTx-4321) via electroporation. Transfected cells were harvested 72 hours postelectroporation and centrifuged. The supernatant was discarded, and the pellet was frozen at - 80°C.

[0566] Table EO. Plasmids transfected to produce ANV19. Aflibercept Table El. Exemplary Cre recombinase expression plasmid (pRTx-2848) 6421 ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTC ( SEQ ID

[0567] NO : 503 )

[0568] Annotations:

[0569] Region / Element Base range

[0570] CMV enhancer 235-614

[0571] CMV promoter 615-818

[0572] T7 RNA polymerase promoter 863-880 iCre coding sequence 964-2019 bGHpA terminator sequence 2070-2293 Origin of replication 2962-3097

[0573] SV40 poly A sequence 4146-4267

[0574] Lac operon operator (lacO) 4340-4356

[0575] Lac operon promoter (complement) 4364-4394

[0576] C-tag 4372-4383

[0577] Catabolite activator protein binding site 4409-4430 Origin of replication (complement) 4718-5306 Ampicillin resistance gene promoter 6338-6442 (complement) Table E2. Ringl9 SRR plasmid (pRTx-3525)

[0578] Annotations:

[0579] Region / Element Base range pHEfl A promoter 436-1609 EF-1 -alpha core promoter 457-668

[0580] EF-1 -alpha intron A 669-1607

[0581] Initiator element 1614-1618

[0582] 5’ UTR conserved domain 1670-1740

[0583] Intron 1 1682-1769

[0584] ORF2 / 3 coding sequence 1770-2056, 3756-4131

[0585] ORF2 / 2 coding sequence 1770-2056, 3629-3902

[0586] ORF2 coding sequence 1770-2060

[0587] ORF1 coding sequence 1952-3919

[0588] ORF1 / 1 coding sequence 1952-2056, 3629-3919

[0589] ORF1 / 2 coding sequence 1952-2056, 3756-3902

[0590] IRES-SV Large T Antigen-SV40 ori-pUC57-Kan 4131-9391, 1-435 concatenated sequence 1

[0591] EMCV internal ribosome entry site (IRES) 4203-4789

[0592] SV40 Large T Antigen coding sequence 4790-6916

[0593] SV40 poly A sequence (complement) 6947-7068

[0594] SV40 origin of replication 7108-7243

[0595] Lad repressor protein binding site (complement ) 7325-7341

[0596] Lac operon promoter (complement) 7349-7379

[0597] CAP binding site (complement) 7394.7415 pUC origin of replication (complement) 7644-8317

[0598] ColEl / pMBl / pBR322 / pUC origin of replication 7703-8291 Aminoglycoside phosphotransferase (Kan / G418 8469-9278 resistance protein) coding sequence (complement)

[0599] Eight transfected MOLT-4 cell pellets representing 16L of production cell culture fluid were thawed and resuspended in buffer, resulting in a 1.6L pool. The lysate was initially clarified by centrifugation, then sterile f iltered.

[0600] The clarified harvest pool was buffer exchanged using tangential flow filtration (TFF). After buffer exchange, the pool was sterile filtered. The buffer-exchanged clarified harvest pool was divided into 2 equal volume pools to perform 2 chromatography cycles on a CIMultus DEAE Monolith (Sartorius AG). The monolith was equilibrated and the first pool was loaded onto the monolith. After elution of the vector, the column was stripped and the chromatography process was then repeated with the second half of harvest pool, and each resulting elution was pooled for further processing.

[0601] Triton phase separation was performed on the above elution pool to remove endotoxin from the pool using a Triton buffer. The pool was centrifuged and the top aqueous phase was collected from each tube via pipetting and pooled.

[0602] The post-triton phase separation pool was further purified via heparin affinity chromatography. Vector was eluted and the resulting 50 mL eluate pool was collected for further processing.

[0603] The heparin eluate pool was loaded onto a 10OkDa TFF cartridge (Formulatrix) and buffer exchange was performed. A final 2.5x concentration was then performed to a 4 mL final volume.

[0604] The buffer exchanged TFF pool was sterile filtered and aliquoted for storage at -80°C.

[0605] EXAMPLE 2 - In Vivo Administration of Anelloviral vector Expressing Aflibercept in Non-Human Primates

[0606] This Example describes the administration of an anelloviral vector comprising Aflibercept as payload into non-human primates (Cynomolgus monkeys).

[0607] Materials & Methods:

[0608] Vectors:

[0609] AAV2. Aflibercept (AAV2.pRTx2533), an adeno-associated virus based on the plasmid pRTx-2533 (Table E3, SEQ ID NO: 401) with a payload comprising Aflibercept was prepared and was diluted in sterile 1XPBS and 0.001% pluronic.

[0610] Table E3. pRTx-2533

[0611] Name pRTx-2533 5641 GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC

[0612] 5701 CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG

[0613] 5761 CTCGCCGCAG CCGAACGACC GAGCGCAGCG A ( SEQ ID NO : 401 )

[0614] Annotations:

[0615] Region / Element Base range

[0616] E. coli catabolite activator protein binding site 130-151

[0617] Lac operon promoter 166-196

[0618] Lac operon operator 204-220

[0619] AAV2 inverted terminal repeat 269-409

[0620] Ring 2 5' NCR 421-844

[0621] CMV enhancer 881-1260

[0622] CMV promoter 1261-1464

[0623] Kozak Sequence 1515-1524

[0624] Aflibercept coding sequence 1521-2894

[0625] SV40 poly(A) signal 2925-3046

[0626] Ring2 3' NCR 3074-3240

[0627] AAV2 inverted terminal repeat 3252-3392

[0628] AmpR promoter 3909-4013

[0629] Beta-lactamase ampicillin resistance coding sequence 4014-4874

[0630] High-copy-number ColEl / pMBl / pBR322 / pUC origin of replication 5045-5633

[0631] The anelloviral vector ANV19. Aflibercept was prepared by the method described in the previous Example.

[0632] Care and use of animals This study complied with all applicable sections of the Final Rules of the Animal Welfare Act regulations (Code of Federal Regulations, Title 9), the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare (OLAW, current edition), and the Guide for the Care and Use of Laboratory Animals (NRC, current edition). The Protocol and any amendments or procedures involving the care or use of animals in this study was reviewed and approved by the Testing Facility Institutional Animal Care and Use Committee before the initiation of such procedures.

[0633] If an animal was determined to be in overt pain / distress or appeared moribund and beyond the point where recovery appears reasonable, the animal was euthanized for humane reasons in accordance with the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia (AVMA, current edition) and with the procedures outlined in the Protocol.

[0634] Institutional Animal Care and Use Committee Approval

[0635] The protocol and amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by Charles River Laboratories, Mattawan Institutional Animal Care and Use Committee (IACUC) before conduct. During the study, the care and use of animals was conducted with guidance from the guidelines of the USA National Research Council.

[0636] This study was conducted in non-naive adult Cynomolgus monkeys of female sex aged 2-5 years old. All animals were used in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were in the normal range at baseline ophthalmic screening, including tonometry, slit-lamp biomicroscopy, fundoscopy, and optical coherence tomography (OCT). All animals recruited in the study were prescreened for AAV2 neutralizing antibodies (Nab) prior to animal aii ival / sclcction for study. Only animals deemed acceptable were assigned to the treatment groups. Animals were assigned to groups based on Nab screening results. Animals not assigned to groups based on Nab screening results were randomized into treatment groups using a standard, randomization procedure.

[0637] Experimental Design

[0638] Table E4. Study Design OU = both eyes; OD = right eye; OS = left eye

[0639] Administration of Test Materials (Ocular)

[0640] Route of Ocular / Intravitreal (IVT) (OU- Groups 2) (OS only- Group 3)

[0641] Administration / Ocular / Subretinal (SR) (OU- Group 1) (OD only- Group 3)

[0642] Application:

[0643] Treatment Frequency: Once

[0644] Study Duration: 4 weeks in-life

[0645] Surgical Animal Preparation Pre-operative Procedures:

[0646] Pre-operative procedures were performed in accordance with Testing Facility SOP.

[0647] Anesthesia was induced and maintained as indicated in the Scheduled Medications and Dosages table below (Table E5). Table E5. Scheduled Medications and Dosages

[0648] Surgical Dosing Procedure

[0649] Intra vitreal:

[0650] Animals were sedated / anesthetized to effect and placed in dorsal recumbency. Topical Proparacaine was applied to the eye. The conjunctival fornices were flushed with a 1:50 dilution of betadine solution / saline and the eyelid margins swabbed with undiluted 5% betadine solution. The eye was draped and a wire eyelid speculum placed. A caliper was used to mark a spot 3.0 mm posterior to the limbus on the inferotemporal bulbar conjunctiva. The conjunctiva at the marked spot was swabbed with undiluted 5% betadine solution. A 31 g needle was then inserted tangentially through the limbus and into the anterior chamber to collect 50pL aqueous humor. A sample of vitreous (maximum volume obtainable up to 50pL) was obtained using a 27 g needle. Predose aqueous and vitreous humor samples were collected from both eyes for animals in Groups 2 and from Group 3 OS. Conjunctival forceps were used to fixate the globe position while a StaClear syringe with 31 g needle was inserted at the marked spot, through the sclera and advanced into the vitreous humor. The injection needle was positioned to face the posterior axis of the globe and 150 pL delivered into the vitreous by slowly depressing the syringe plunger. The needle was held in place for approximately 10 seconds to lessen reflux of the injected material. Subsequently, the needle was removed and the episcleral tissues approximated to the site of insertion were grasped with the conjunctival forceps to further lessen reflux of the injected material.

[0651] The contralateral eye had an identical injection procedure performed, with the exception of Group 3.

[0652] Subretinal:

[0653] Animals were sedated / anesthetized and placed in dorsal recumbency. Topical Proparacaine was applied to the eye. The conjunctival fornices were flushed with a 1:50 dilution of betadine solution / saline and the eyelid margins swabbed with undiluted 5% betadine solution. The eye was draped and a wire eyelid speculum placed. A lateral canthotomy was performed using Stevens tenotomy scissors. A caliper was used to mark spots 3.0 mm posterior to the limbus on the superotemporal and inferotemporal sclera. Bipolar' cautery was used to cauterize the sclera under the marked spots, followed by topical application of undiluted 5% betadine solution. Scleral fixation forceps were used to fix the globe position while a microvitreoretinal blade with a valved cannula was inserted at each marked spot, through the conjunctiva and sclera, and advanced into the vitreous humor. The trocar was positioned to face the posterior axis of the globe and then retracted to leave the scleral port in place. A 31 g needle was then inserted tangentially through the limbus and into the anterior chamber to collect 50 pL aqueous humor for Group 1 OU, and Group 3 OD only. A sample of vitreous (maximum volume obtainable up to 50 pL) was obtained using either a 25 or 27 g needle inserted through one of the ports. Vitreous humor was collected for Group 1 OU, and Group 3 OD only. A direct contact surgical lens was placed on the cornea with sterile coupling gel. An endoilluminator probe was inserted through one of the scleral ports to facilitate direct visualization of the posterior segment through the microscope. A subretinal injection cannula was inserted through the second port and advanced into the mid- vitreous. The small diameter injection cannula was then advanced until it contacted the retinal surface. The test articles were then slowly delivered to induce and fill a subretinal bleb. When appropriate bleb formation was visualized, the injection was continued to deliver the entire dose volume, 150 pL, into the subretinal space. If bleb formation was not visualized, the small diameter injection cannula was repositioned, and the injection was attempted again at the same location or an alternative location depending on surgeon preference. Once the injection dose was delivered, the injection cannula and endoilluminator probe were removed from the scleral ports, and the contact lens removed from the cornea. The scleral ports were then removed. The lateral canthotomy site, if performed, was closed using 7-0 Vicryl suture.

[0654] The contralateral eye had an identical injection procedure performed, with the exception of Group 3.

[0655] Ocular humor collections:

[0656] Intervals: Predose (aqueous and vitreous humor. Groups 1 to 3, OU): during surgical dosing procedure. See Section 10.2.

[0657] In -life (aqueous and vitreous humor, OU): Day 28(±1), Groups 1 to 3 Terminal (aqueous and vitreous humor, OU): during necropsy procedure

[0658] Sample Target Aqueous humor (AH): 50 pL (predose and Day 28(±1) in-life collection), 100 Volume: pL OS and 50 pL OD (terminal collection)

[0659] Vitreous humor (VH): 50 pL (predose and Day 28(±1) in-life collection). Maximum volume obtainable of at least 1 mL OS and 50 pL OD (terminal collection)

[0660] In-life Collection Samples were collected by an ophthalmologist. Animals were Details: sedated / anesthetized to effect.

[0661] Ocular humor sample collections were conducted in compliance with Testing Facility SOP.

[0662] Initial Handling and After collection, tared weights were collected, and samples placed directly on Storage: dry ice and frozen.

[0663] Terminal Procedures:

[0664] Method of Euthanasia

[0665] Euthanasia was by euthanasia solution administration, under sedation, if necessary (e.g., ketamine), followed by a Testing Facility SOP approved method to ensure death, e.g., exsanguination. Necropsy: eyes and optic nerve collected as detailed below. Tissue was collected for DNA, RNA, and protein analysis.

[0666] Ocular Tissue Dissection;

[0667] Following termination, eyes were marked with ink for orientation at the 12 o’clock position. Eyes with proximal optic nerves were enucleated. After enucleated orientation was marked again. Care was given to ensure that cross contamination between tissues did not occur. Gloves and disposable collection tools were changed between collection and dissection of each tissue. The cutting board and non-disposable instruments were wiped down with a 10% bleach solution, rinsed with water, followed by a wipe down of 70% or greater ethanol between each of the specified tissues; sterile trays were used.

[0668] Eyes from each animal in Groups 1 to 3 were enucleated and processed for DNA and RNA. Flatmount diagrams describing how retinal and posterior eye cup (PEC) punches are collected are shown in FIG. 1 for SR and FIG. 2 for IVT injected eyes. Ocular humor samples (maximum volume obtainable, of at least 10OpL OS and 50 pL OD for AH and at least 1.0 mL OS and 50 pL OD for VH) were collected. AH was collected with a 29-31 G needle and VH was collected with a 16-18 G needle.

[0669] Two approximately 5 mm long contiguous samples of the optic nerve were taken from each eye in the experiment and were allocated for ocular dissection. One sample was for DNA analysis and one sample was for RNA analysis. The following samples were collected fresh from eyes (Table E6).

[0670] Table E6. Ocular Tissue Samples Collected

[0671] All tissues were collected in 2 mL reinforced tubes (SPEX Sample Prep) containing 5 mm stainless steel beads (Qiagen, LLC) and flash-frozen immediately. They were stored at -80°C until ready for homogenization.

[0672] DNA Isolation:

[0673] Frozen tissue samples were lysed with an automated tissue homogenizer (Geno / Grinder SPEX Sample Prep) in Buffer ATL (Qiagen, USA) and proteinase K (Qiagen, USA) at 1250 rpm for (2) 30 second rounds. Homogenized tissues were digested on a heat block at 56 C for ~4 hours. Genomic DNA was precipitated with Buffer AL (Qiagen, USA) and Ethanol, then isolated with Qiagen DNeasy 96 Blood & Tissue Kit. Isolated DNA was quantified using a NanoDrop 8000 Spectrophotometer (Thermofisher, USA). RNA Isolation:

[0674] Frozen tissue samples were lysed with an automated tissue homogenizer (Geno / Grinder SPEX Sample

[0675] Prep, USA) in QIAzol lysis reagent (Qiagen, USA) at 1250 rpm for (2) 30 second rounds. RNA was isolated in the aqueous phase by addition of Phenol Chloroform (Thermofisher, USA) and centrifugation at 6000 rpm for 15 minutes at 4C. The upper aqueous phase was transferred into a fresh S-block (Qiagen, USA). RNA was precipitated with the addition of 1 volume of 70% Ethanol and then isolated with the use of a Qiagen RNeasy 96 kit. RNA concentrations were quantified via the Qubit RNA High Sensitivity Assay Kit (Thermofisher, USA) qPCR:

[0676] Genomic DNA was assayed by qPCR on the QuantStudio 5 - Real-Time PCR System (Thermo Fisher, USA) using TaqMan Gene Expression Master Mix (Thermofisher, USA). The sequence detection primers and FAM custom probes that were used in this study were synthesized by Integrated DNA Technologies, USA. Aflibercept primer / probe sequences are described in Table E7.

[0677] One-Step RT-ddPCR:

[0678] RNA was diluted in nuclease-free water and combined with the reagents from the One Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, USA; Catalog#! 864022) and an Aflibercept primer / probe set with final primer concentrations of 900nM and probe concentrations of 250nM, to measure transgene expression. After the RT-ddPCR reaction setup, each reaction was converted to droplets using the Automated Droplet Generator (Bio-Rad, USA) according to the manufacturer’s instructions. After the droplet generation, the droplets were subjected to endpoint PCR thermocycling, with the following cycling conditions: 1 cycle of 48 C for 1 hour for reverse transcription, followed by 1 cycle of 95 C for 10 mins; 40 cycles of 95 C for 30 sec, 60 C for 1 min; and 1 cycle of 98 C for 10 min and finally a 4°C hold. The cycled plate was then transferred to the QX200 Droplet Reader (Bio-Rad, USA) and analyzed using the QX Manager Software (Bio-Rad, USA).

[0679] Table E7. Primer and probes designed to quantify Aflibercept. ELISA:

[0680] The concentration of intraocular unbound Aflibercept protein was measured using a commercially available Aflibercept-specific ELISA (Eagle Biosciences, Amhearst, NH). Until processing, all of the Aqueous Humor and Vitreous Humor samples collected at week 4 and week 8 timepoints were stored at - 80C. All of the samples were diluted at 1:10 dilution in assay dilution buffer. The assay was then performed according to the manufacturer’s instructions. Recombinant human (rh) VEGF-A was used as the immobilized molecule on the surface of a 96-well plate. Horseradish peroxidase-conjugated antihuman IgG, which binds to the Fc portion of aflibercept in the sample, was used as the detection antibody. The lower limit of quantification of this assay was 5ng / mL. The standard curve of Aflibercept was constructed directly proportional to the assay dynamic range (6-200ng / mL). The optical density (OD) was measured with a photometer at 450nm.

[0681] Western Blot:

[0682] Western blot analyses of the Aqueous Humor and Vitreous Humor were performed as described: Proteins were separated by electrophoresis in a 10% Bolt Bis-Tris Mini Protein Gel (ThermoFisher Scientific, USA) under reducing conditions and transferred by electroblotting in transfer buffer (192 mM glycine, 25mM Tris, pH8.4) to a nitrocellulose membrane (BioRad, USA). Antigen-bound primary antibodies were detected using appropriate IRDye labelled secondary antibodies (Li-Cor, USA). Specific detection of Aflibercept was done by probing with an anti-human IgG antibody that does not cross-react with NHP IgG (Biorad, USA). Typically, aflibercept will give a band at ~70kDa which is due to glycosylation of the protein.

[0683] Results:

[0684] 8 weeks after injection, DNA from the posterior eye cup (PEC) and the retina were collected and processed separately. At the dose of 7.4E+8 viral genomes (vg) per eye, aflibercept genomes were detected by qPCR in the PEC and in the retina following either subretinal (SR) or intravitreal (IVT) administration of either ANV 19. Aflibercept or AAV2. Aflibercept (Figures 5A and 5B). Greater aflibercept genome copies were detected in the PEC and similar' levels in the retina following subretinal administration of AN VI 9. Aflibercept as compared to the A A V2. Aflibercept dose-matched control (Figures 5A and 5B). In the intravitreal delivery groups, low levels of genome copies were detected in the PEC and retina in the ANV19.Aflibercept-treated eye (Figures 5A and 5B).

[0685] To characterize whether ANV19. Aflibercept infection could induce aflibercept expression in eye tissue, RNA was collected from the PEC and the retina and processed separately at 8 weeks after transduction and assessed by RT-ddPCR. Aflibercept mRNA was detected in PEC and in the retina following SR administration of ANV19.Aflibercept (Figure 5C and 5D). However, at the low dose of 7.4E+8 vg of ANV 19.Aflibercept, no aflibercept mRNA was detected in the PEC or retina when administered intravitreally (Figures 5C and 5D). As expected, aflibercept protein expression was also not detected by Elisa or Western blot in ocular fluid at the low dose of 7.4E+8 vg of ANV 19. Aflibercept administered via SR or IVT (data not shown).

[0686] In addition to the vector transduction efficiency, vector safety was also assessed, Biopsy samples from extra ocular tissues such as liver (Figure 6A) and spleen (Figure 6B ) were assessed for vector biodistribution. There were no aflibercept genome copies detected in either tissue collected from the ANV 19. Aflibercept or AAV2. Aflibercept treatment groups, whether administered via SR or IVT.

[0687] Ophthalmoscopic examinations including Ocular Coherence Tomography (OCT) was performed to assess retinal health (Figures 7A-7C). Procedure-related OCT findings including transient segmental loss of the hyperreflective ellipsoid zone, irregular thickening of the perifoveal outer plexiform layer, presence of minimal hyperreflective subretinal debris, and / or minimal thinning of the outer nuclear layer were present within the subretinal dosing site of most eyes on Day 14 following subretinal dose administration (Figures 7A and 7C). In all cases, partial or complete recovery of the ellipsoid zone was present by study termination, as expected following subretinal dose administration. Group 1 animal OD and Group 3 OD both demonstrated multifocal minimal to mild accumulation of hyperreflective material within the inferior portion of the subretinal dosing site on Day 14, which reduced in severity by Day 28 and nearly or completely resolved by Day 56 (Figures 7A and 70). Overall, all OCT findings were consistent with expected retinal structural changes following the subretinal dosing procedure, there were no test article effects identified. All eyes that received an intravitreal dose of ...

Claims

CLAIMSWhat is claimed is:

1. A circular, single stranded DNA genetic element comprising, in order: a) an Anello virus non-coding region (NCR); b) a promoter; and c) an exogenous effector region having a DNA sequence according to SEQ ID NO: 316 or having a DNA sequence encoding a polypeptide of SEQ ID NO: 326, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

2. A circular, single stranded DNA genetic element comprising, in order: a) an Anellovirus non-coding region (NCR); b) a promoter; c) a region having a DNA sequence according to the reverse complement of nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and d) an exogenous effector region.

3. A circular, single stranded DNA genetic element comprising, in order: a) an Anellovirus non -coding region (NCR); b) a promoter; c) an exogenous effector region; and d) a region having a DNA sequence according to the reverse complement of nucleotides 2538- 2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

4. A circular, single stranded DNA genetic element comprising, in order: a) a Ringl9 Anellovirus non-coding region (NCR) comprising a DNA sequence according to the reverse complement of nucleotides 64-485 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 310, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 312, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto;d) a region having a DNA sequence according to the reverse complement of nucleotides 1108- 1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 316, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to the reverse complement of nucleotides 2538- 2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 318, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to the reverse complement of nucleotides 2690- 2705 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 322, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and j) a loxP site having a DNA sequence according to SEQ ID NO: 324, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

5. A circular, single stranded DNA genetic element comprising a sequence according to SEQ ID NO: 327, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

6. A nucleic acid molecule comprising, in order: a) a Ringl9 Anellovirus non-coding region (NCR) comprising a DNA sequence according to nucleotides 64-485 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 309, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 311, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; d) a region having a DNA sequence according to nucleotides 1108-1157 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto;e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 315, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to nucleotides 2538-2567 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 317, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to nucleotides 2690-2705 of SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 321, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

7. A nucleic acid molecule comprising, in order: a) a Ring 19 Anellovirus non-coding region (NCR) comprising a DNA sequence according to nucleotides 529-950 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a CMV enhancer having a DNA sequence according to SEQ ID NO: 309, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) a CMV promoter having a DNA sequence according to SEQ ID NO: 311, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; d) a region having a DNA sequence according to nucleotides 1573-1622 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) an exogenous effector region having a DNA sequence according to SEQ ID NO: 315, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) a region having a DNA sequence according to nucleotides 3003-3032 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; g) a poly(A) signal having a DNA sequence according to SEQ ID NO: 317, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; h) a region having a DNA sequence according to nucleotides 3118-3170 of SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and i) a region having a length of 50-120 nucleotides, wherein optionally the region has a DNA sequence according to SEQ ID NO: 321, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

8. A nucleic acid molecule comprising a sequence according to SEQ ID NO: 325, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

9. A nucleic acid molecule comprising a sequence according to SEQ ID NO: 328, or a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

10. An anelloviral vector comprising:(i) a proteinaceous exterior comprising an Anello virus ORF1 protein, and(ii) a genetic element of any of the preceding claims, wherein the genetic element is enclosed by the proteinaceous exterior.

11. A pharmaceutical composition comprising the anelloviral vector of claim 10, and a pharmaceutically acceptable carrier and / or excipient.

12. A method of delivering an exogenous effector to a subject, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11, thereby delivering the exogenous effector to the subject.

13. A method of delivering a genetic element to a subject, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11, thereby delivering the genetic element to the subject.

14. A method of delivering an mRNA to a subject, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11, under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element, thereby delivering the mRNA to the subject.

15. A method of treating age-related macular degeneration (AMD) (e.g., wet AMD) in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11.

16. A method of treating macular edema (e.g., macular edema following RVO) in a subject in need thereof, the method comprising administering to the subject an anello viral vector of claim 10 or a pharmaceutical composition of claim 11.

17. A method of treating diabetic macular edema in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11.

18. A method of treating diabetic retinopathy in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11.

19. A method of treating retinopathy of prematurity in a subject in need thereof, the method comprising administering to the subject an anelloviral vector of claim 10 or a pharmaceutical composition of claim 11.

20. A method of delivering an exogenous effector to a subject, the method comprising:(i) administering to the subject a first plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11 ; and(ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the exogenous effector to the subject.

21. A method of delivering a genetic element to a subject, the method comprising:(i) administering to the subject a first plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11 ; and(ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the genetic element to the subject.

22. A method of delivering an mRNA to a subject, the method comprising:(i) administering to the subject a first plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11 , under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element; and(ii) administering to the subject (a) a second plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors, thereby delivering the mRNA to the subject.

23. A method of delivering an exogenous effector to a subject, the method comprising:(i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and(ii) administering to the subject a second plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11 , thereby delivering the exogenous effector to the subject.

24. A method of delivering a genetic element to a subject, the method comprising:(i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and(ii) administering to the subject a second plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11, thereby delivering the genetic element to the subject.

25. A method of delivering an mRNA to a subject, the method comprising:(i) administering to the subject (a) a first plurality of anelloviral vectors comprising a genetic element that encodes an exogenous effector, e.g., aflibercept, or (b) a pharmaceutical composition comprising the second plurality of anelloviral vectors; and(ii) administering to the subject a second plurality of anelloviral vectors of claim 10 or a pharmaceutical composition of claim 11, under conditions that allow for the production of an mRNA encoding the exogenous effector from the genetic element, thereby delivering the mRNA to the subject.

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