AAV production systems with increased packaging efficiencies

Abstract

Disclosed herein are genetically engineered cells for AAV production. The genetically engineered cell comprises molecular systems for temporal control of expression of genes required for AAV production. Also disclosed herein are methods of using genetically engineered cells for AAV production.

Description

[0001] AAV PRODUCTION SYSTEMS WITH INCREASED PACKAGING EFFICIENCIES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119 of U.S. provisional application serial number 63 / 624,170, filed January 23, 2024, the entire contents of which are incorporated by reference herein.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A121070014WO00-SEQ-CRP.xml; Size: 227,570 bytes; and Date of Creation: January 22, 2025) are herein incorporated by reference in its entirety.

[0006] FIELD

[0007] Described herein are Adeno- Associated Virus (AAV) production systems. Also described herein are engineered cells and kits comprising an AAV production system and methods of using the same for AAV production.

[0008] BACKGROUND

[0009] AAV are a promising gene delivery modality for cell and gene therapy. AAV can be modified to carry therapeutic genetic payloads to cells within a subject. The production of AAV normally entails transient transfection of plasmids containing genes required for viral vector production into cell culture. AAV production suffers from a particularly low packaging efficiency. For current manufacturing practices, it is common to observe ten empty capsids for every full capsid. The prevalence of empty capsids means AAV must be manufactured at a much greater scale to achieve the required number of full capsids. Additionally, having so many empty capsids contributes to enhanced immune response compared to a therapy containing lower total number of capsids but higher packaging efficiencies, such as one tenth the total number of capsids but with 100% packaging efficiency.

[0010] SUMMARY

[0011] Described herein are AAV production systems that allow for the co-expression of gene products required for AAV production and one or more additional viral protein(s) and / or one or more viral non-coding RNA(s). The additional viral protein may promote AAV packing efficiency (e.g., by generating conditions for liquid-liquid phase separation when expressed) and / or comprise one or more intrinsically disordered region(s). The viral non-coding RNA may promote AAV replication. Also described herein are engineered cells and kits comprising an AAV production system and methods of using the same for AAV production.

[0012] In some aspects, the disclosure relates to an Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and an additional viral protein. In some embodiments, the one or more polynucleotides of the AAV production system collectively further comprise a nucleotide sequence encoding for E40rf6 and / or VARNA.

[0013] In some embodiments, the additional viral protein is not an AAV protein or an adenoviral El protein.

[0014] In some embodiments, the additional viral protein is capable of generating conditions for liquid-liquid phase separation when expressed in a cell. In some embodiments, the additional viral protein comprises one or more intrinsically disordered region(s).

[0015] In some embodiments, the additional viral protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of any one of SEQ ID NOs: 30-53, 105-109, or 123.

[0016] In some embodiments, the additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of any one of SEQ ID NOs: 1-29, 96-104, or 120, wherein the additional viral protein does not comprise the amino acid sequence of any one of SEQ ID NOs: 11-13.

[0017] In some embodiments, the additional viral protein comprises at least one mutation relative to its wildtype amino acid sequence.

[0018] In some embodiments, the nucleotide sequence encoding the additional viral protein comprises at least one codon that has been optimized, relative to a wild type nucleotide sequence encoding the additional viral protein, for expression in a human cell.

[0019] In some embodiments, the amino acid sequence of Rep52, the amino acid sequence of Rep40, the amino acid sequence of Rep78, the amino acid sequence of Rep68, the amino acid sequence of E2A, the amino acid sequence of E40rf6, the nucleotide sequence of VARNA, the amino acid sequence of VP1, the amino acid sequence of VP2, and / or the amino acid sequence of VP3 comprises one or more mutations relative to its corresponding wild type amino acid sequence.

[0020] In some embodiments, the AAV production system comprises a polynucleotide encoding a LI 52K protein. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of any one of SEQ ID NOs: 30-32. In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-7.

[0021] In some embodiments, the AAV production system comprises a polynucleotide encoding a IV a2 protein. In some embodiments, the IVa2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 33. In some embodiments, the IV a2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 8.

[0022] In some embodiments, the AAV production system comprises a polynucleotide encoding a LI pllla protein. In some embodiments, the LI pllla protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 34. In some embodiments, the LI pllla protein comprises an amino acid sequence having at least 80%%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9.

[0023] In some embodiments, the AAV production system comprises a polynucleotide encoding a L4 100K protein. In some embodiments, the L4 100K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, the L4 100K protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 35 and a second intrinsically disordered region comprising an amino acid sequence having at least 80%%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 36. In some embodiments, the L4 100K protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10.

[0024] In some embodiments, the AAV production system comprises a polynucleotide encoding an E1A protein. In some embodiments, the E1A protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 37. In some embodiments, the El A protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 11.

[0025] In some embodiments, the AAV production system comprises a polynucleotide encoding an E1B 19K protein. In some embodiments, the E1B 19K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 38. In some embodiments, the E1B 19K protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0026] 12.

[0027] In some embodiments, the AAV production system comprises a polynucleotide encoding an E1B 55K protein. In some embodiments, the E1B 55K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 39. In some embodiments, the E1B 55K protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0028] 13.

[0029] In some embodiments, the AAV production system comprises a polynucleotide encoding an E2B protein. In some embodiments, the E2B protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 40. In some embodiments, the E2B protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 14.

[0030] In some embodiments, the AAV production system comprises a polynucleotide encoding an E2B pTP protein. In some embodiments, the E2B pTP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 41. In some embodiments, the E2B pTP protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0031] 15.

[0032] In some embodiments, the AAV production system comprises a polynucleotide encoding an ORF59 protein. In some embodiments, the ORF59 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 42. In some embodiments, the ORF59 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0033] 16.

[0034] In some embodiments, the AAV production system comprises a polynucleotide encoding a LANA protein. In some embodiments, the LANA protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the LANA protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0035] 17.

[0036] In some embodiments, the AAV production system comprises a polynucleotide encoding an EBNA2 protein. In some embodiments, the EBNA2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. In some embodiments, the EBNA2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:

[0037] 18. In some embodiments, the AAV production system comprises a polynucleotide encoding an EBNALP protein. In some embodiments, the EBNALP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45. In some embodiments, the EBNALP protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 19.

[0038] In some embodiments, the AAV production system comprises a polynucleotide encoding a ULI 12-UL113 protein. In some embodiments, the ULI 12-UL113 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46. In some embodiments, the ULI 12- UL113 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 20.

[0039] In some embodiments, the AAV production system comprises a polynucleotide encoding a UL57 protein. In some embodiments, the UL57 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47. In some embodiments, the UL57 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 21.

[0040] In some embodiments, the AAV production system comprises a polynucleotide encoding an ICP4 protein. In some embodiments, the ICP4 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the ICP4 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48 and a second intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 49. In some embodiments, the ICP4 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 22.

[0041] In some embodiments, the AAV production system comprises a polynucleotide encoding an ICP8 protein. In some embodiments, the ICP8 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, the ICP8 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 23.

[0042] In some embodiments, the AAV production system comprises a polynucleotide encoding a ULI 1 protein. In some embodiments, the ULI 1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 51. In some embodiments, the UL11 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 24.

[0043] In some embodiments, the AAV production system comprises a polynucleotide encoding an El protein. In some embodiments, the El protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 52. In some embodiments, the El protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 25.

[0044] In some embodiments, the AAV production system comprises a polynucleotide encoding an E2 protein. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 105. In some embodiments, the E2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 96.

[0045] In some embodiments, the AAV production system comprises a polynucleotide encoding an mSK_082 E2 protein. In some embodiments, the mSK_082 E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the mSK_082 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 97.

[0046] In some embodiments, the AAV production system comprises a polynucleotide encoding a UL12 protein. In some embodiments, the UL12 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108. In some embodiments, the UL12 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 107 and a second intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the ULI 2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the UL12 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 99.

[0047] In some embodiments, the AAV production system comprises a polynucleotide encoding an UL12.5 protein. In some embodiments, the UL12.5 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the UL12.5 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the UL12.5 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 101.

[0048] In some embodiments, the AAV production system comprises a polynucleotide encoding an NS2 protein. In some embodiments, the NS2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 109. In some embodiments, the NS2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the NS2 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the NS2 protein is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of SEQ ID NO: 119. In some embodiments, the nucleotide sequence encoding NS2 has been modified to remove potential splice sites. In some embodiments, the nucleotide sequence encoding NS2 comprises, relative to the nucleotide sequence of SEQ ID NO: 119, one or more of (two or more of, three or more of, four or more of, five or more of, six or more of, seven or more of, eight or more of, nine or more of, or all ten of) a T at position 759, a C at position 769, a G at position 771, T at position 922, a C at position 923, a C at position 924, a C at position 1,110, a T at position, 1,111, a C at position 1,112, and an A at position 1,419 (positions of SEQ ID NO: 119 determined sequentially). In some embodiments, the nucleotide sequence encoding NS2 comprises, relative to the nucleotide sequence of SEQ ID NO: 119, a T at position 759, a C at position 769, a G at position 771, T at position 922, a C at position 923, a C at position 924, a C at position 1,110, a T at position, 1,111, a C at position 1,112, and an A at position 1,419 (positions of SEQ ID NO: 119 determined sequentially).

[0049] In some embodiments, the AAV production system comprises a polynucleotide encoding an E6 protein. In some embodiments, the E6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the E6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the AAV production system comprises a polynucleotide encoding an LI 13.6K protein. In some embodiments, the LI 13.6K protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 28.

[0050] In some embodiments, the AAV production system comprises a polynucleotide encoding a ORF6 protein. In some embodiments, the ORF6 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 29.

[0051] In some embodiments, the AAV production system comprises a polynucleotide encoding an NP1 protein. In some embodiments, the NP1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 123. In some embodiments, the NP1 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the NP1 protein is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the nucleotide sequence encoding NP1 has been modified to remove potential splice sites. In some embodiments, the nucleotide sequence encoding NP1 comprises, relative to the nucleotide sequence of SEQ ID NO: 121, an A at position 585 and / or a C at position 588 (positions of SEQ ID NO: 121 determined sequentially). In some embodiments, the nucleotide sequence encoding NP1 comprises, relative to the nucleotide sequence of SEQ ID NO: 121, an A at position 585 and a C at position 588 (positions of SEQ ID NO: 121 determined sequentially) .

[0052] In some embodiments, the one or more polynucleotides of the AAV production system further comprise a nucleotide sequence encoding for a non-coding RNA comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 122.

[0053] In some embodiments, the AAV production system comprises a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the nucleotide sequence of one or more of SEQ ID NOs: 54-82, 110-119, or 121.

[0054] In some embodiments, the AAV production system is capable of producing helper virus-free AAV. In some embodiments, the one or more polynucleotides of the AAV production system do not encode a viral capsid protein other than VP1, VP2, and VP3.

[0055] In some embodiments, the AAV production system comprises a first polynucleotide comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3. In some embodiments, the AAV production system comprises a first polynucleotide comprising a nucleotide sequence encoding for: Rep52; Rep40; Rep78; Rep68; VP1; VP2; and VP3.

[0056] In some embodiments, the AAV production system comprises a second polynucleotide comprising a nucleotide sequence encoding for: E2A; E40rf6; and VARNA.

[0057] In some embodiments, the AAV production system comprises a third polynucleotide comprising a nucleotide sequence encoding the additional viral protein.

[0058] In some embodiments, the AAV production system comprises a transfer plasmid.

[0059] In some aspects, the disclosure relates to an Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; a viral noncoding RNA comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 122; a first additional viral protein; and a second additional viral protein; wherein the AAV production system comprises a polynucleotide comprising a sequence encoding a polycistronic RNA, wherein the polycistronic RNA comprises the sequence encoding for the viral noncoding RNA, the sequence encoding for the first additional viral protein, and the sequence encoding for the second additional viral protein. In some embodiments, the one or more polynucleotides of the AAV production system further comprise a nucleotide sequence encoding for E40rf6.

[0060] In some embodiments, the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the first additional viral protein; and the sequence encoding the second additional viral protein. In some embodiments, the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the first additional viral protein; a sequence encoding a viral 2a peptide; and the sequence encoding the second additional viral protein.

[0061] In some embodiments, the first additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26 or 96- 103. In some embodiments, the second additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26 or 96- 103.

[0062] In some embodiments, the first additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 120.

[0063] In some embodiments, the second additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 103.

[0064] In some embodiments, the AAV production system comprises: a first polynucleotide comprising the sequence encoding for the polycistronic RNA; a second polynucleotide comprising a sequence of encoding for: Rep52 or Rep40; VP1; VP2; VP3; E2A; and E40rf6; and a third polynucleotide comprising a sequence encoding for: Rep78 or Rep68; VP1; VP2; VP3; E2A; and E40rf6.

[0065] In some embodiments, the AAV production system does not encode Rep68.

[0066] In some aspects, the disclosure relates to engineered cells for Adeno-Associated Virus (AAV) production, where the engineered cells comprise an AAV production system described herein. In some embodiments, one or more polynucleotides of the AAV production system are stably integrated into the genome of the engineered cell.

[0067] In some embodiments, an engineered cell comprises: a first plasmid comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; a second plasmid comprising a second polynucleotide comprising a nucleotide sequence encoding for E2A, optionally wherein the second polynucleotide further encodes for E40rf6 and / or VARNA; and a third plasmid comprising a nucleotide sequence encoding an additional viral protein, optionally wherein the additional viral protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26 or 96-103.

[0068] In some embodiments, an engineered cell comprises: a genomic integration of a first nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; optionally wherein the first nucleotide sequence encodes for Rep52, Rep40, Rep78, Rep68, VP1, VP2, and VP3; a genomic integration of a second nucleotide sequence encoding for: E2A, optionally wherein the second nucleotide sequence further encodes for E40rf6 and / or VARNA; and a genomic integration of a third nucleotide sequence encoding for the additional viral protein.

[0069] In some aspects, the disclosure relates to kits comprising an Adeno-Associated Virus (AAV) production system or an engineered cell described herein. In some embodiments, a kit comprises a transfer plasmid, wherein the transfer plasmid comprises, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat.

[0070] In some aspects, the disclosure relates to methods for Adeno-Associated Virus (AAV) production. In some embodiments, a method comprises culturing an engineered cell described herein such that it expresses: Rep52 or Rep40; Rep78 or Rep68; VARNA; VP1; VP2; VP3; and the additional viral protein. In some embodiments, a method comprises culturing the engineered cell such that it expresses: Rep52; Rep40; Rep78; Rep68; E2A; VP1; VP2; VP3; and the additional viral protein.

[0071] In some aspects, the disclosure relates to methods for generating an engineered cell for Adeno-Associated Virus (AAV) production. In some embodiments, a method comprises introducing an AAV production system described herein into the cell. In some embodiments, a method comprises integrating a polynucleotide of the AAV production system into the genome of the cell.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 shows schematics for exemplary plasmids for co-expressing AAV production genes with an additional viral protein (e.g., Ad5 LI 52K) or a control (e.g., SUPT5H). From top to bottom the plasmids depicted are: pHelper, pRepCap, pAAV-EGFP, and the supplemented Ad LI 52K or SUPT5H plasmid.

[0074] FIG. 2 shows that co-expression of AAV production genes with Ad5 LI 52K boosts AAV titers.

[0075] FIG. 3 shows results from titration experiments using Ad5 LI 52K. Ad5 LI 52K boosts AAV titers in a concentration dependent manner.

[0076] FIG. 4 shows that co-expression of AAV production genes with various viral proteins boosts AAV titers. Dotted line indicates approximate titer of the wt sample containing no additional viral genes besides pHelper, pRepCap, and pAAV.

[0077] FIG. 5 shows that co-expression of AAV production genes with various HSV and Bocavirus proteins boosts AAV titers. Dotted line indicates approximate titer of the wt sample containing no additional viral genes besides pHelper, pRepCap, and pAAV. FIG. 6 shows that co-expression of Bocavirus non-coding RNA BocaSR and Bocavirus proteins NP1 and NS2 boost AAV titers. Shown are AAV titers in transducing units per mL without and with doxycycline.

[0078] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0079] AAV are a promising gene delivery modality for cell and gene therapy. Current AAV manufacturing methods utilize four main adenovirus genes (El, E2A, E4, and VARNA). These manufacturing methods suffer from particularly low packaging efficiencies.

[0080] Without being bound to any particular theory, the inventors of the instant application have discovered that by promoting conditions for liquid-liquid phase separation (LLPS) during AAV production (e.g., by co-expressing AAV gene products with a sufficient amount of a protein of Table 1 or Table 2), one can harness the formation of viral biomolecular condensates to produce AAV at higher titers, purities, and with better functionality.

[0081] LLPS is a phenomenon in which proteins, RNAs, and / or DNAs can form membraneless organelles or biomolecular condensates or agglomerates. See e.g., Wang et al., Signal Transduct Target Ther. 2021 Aug 2;6(l):290. These biomolecular condensates increase the localized concentration of these components while excluding other factors, allowing for more complex and efficient functions in the cell. LLPS can occur when proteins containing intrinsically disordered regions (IDRs) and nucleic acids are present at high enough concentration, resulting in transition from suspension in solution to localized agglomeration, loosely analogous to oil droplets suspended in water.

[0082] Viruses use LLPS to concentrate factors required for viral particle replication and assembly and to avoid the cellular immune response. See e.g., Wei et al., Pront Immunol. 2022 Aug 9:13:985622. Lor instance, Herpes simplex virus ICP4 has been reported to form liquid droplets in cells (Seyffert et al., Sci. 2021 Apr 24;22(9):4447), while human cytomegalovirus has been shown to use ULI 12-113 as scaffolding to form biomolecular condensates (Caragliano et al., Cell Rep. 2022 Mar 8;38(10): 110469).

[0083] The inventors of the instant application have also discovered that production of AAV can be promoted by co-expressing a viral non-coding RNA. The viral non-coding RNA may promote AAV replication.

[0084] I. Adeno-Associated Virus Production Systems

[0085] In some aspects, the disclosure relates to adeno-associated virus (AAV) production systems. The AAV production systems described herein allow for the co-expression of gene products required for AAV production and one or more additional viral protein(s) and / or one or more viral non-coding RNA(s). An “additional viral protein,” as used herein, may be capable of promoting AAV packing efficiency (e.g., by generating conditions for liquidliquid phase separation when expressed) and / or comprise one or more intrinsically disordered region(s). A “viral non-coding RNA,” as used herein, may be capable of promoting AAV replication.

[0086] An AAV production system, as described herein, comprises one or more polynucleic acids collectively comprising: (a) an AAV production component and (b) additional viral protein component and / or a viral non-coding RNA component. In some embodiments, an AAV production system comprises and additional viral protein component. In some embodiments, an AAV production system comprises a viral non-coding RNA component. In some embodiments, an AAV production system comprises an additional viral protein component and a viral non-coding RNA component.

[0087] In some embodiments, an AAV production system disclosed herein is capable of producing helper virus-free AAV. An AAV production system is “capable of producing helper virus-free AAV,” as described herein, when the AAV production system does not meaningfully produce AAV helper- virus when the AAV production system is comprised within a cell (i.e., at least 90%, at least 95%, at least 96%, at least 97% at least 98%, at least 99%, or at least 99.9% of the viral titer produced in the cell comprising the AAV production system is AAV).

[0088] A. AAV Production Component

[0089] The AAV production systems described herein comprise an AAV production component. As used herein, the term “AAV production component” refers to one or more polynucleotides that collectively encode the gene products required to generate an AAV vector in a recombinant host cell. Exemplary AAV gene products (a subset of which are required to generate AAV, as expounded upon below) include Rep52, Rep40, Rep78, Rep68, E2A, E4ORF6, VARNA, Cap (VP1, VP2, VP3), AAP, L4 100K, and MAAP.

[0090] Each of the AAV productions systems described herein encodes an AAV long Rep (Rep78 and / or Rep68), an AAV short Rep (Rep52 and / or Rep40), and AAV Cap (encoding VP1, VP2, and VP3) (collectively, the AAV gene products required to generate an AAV vector in a recombinant host cell). The Rep gene products (comprising Rep52, Rep40, Rep78 and Rep68) are involved in AAV genome replication. The Cap gene products (comprising VP1, VP2, VP3) encode viral capsid proteins. In some embodiments, an AAV production system encodes Rep78, but not Rep68. In some embodiments, an AAV production system encodes Rep40, but not Rep52. In some embodiments, an AAV production system encodes Rep52, but not Rep40. In some embodiments, and AAV production system encodes Rep78 and Rep 52, but not Rep68 or Rep40. In some embodiments, an AAV production system encodes Rep78 and Rep 40, but not Rep68 or Rep52.

[0091] In some embodiments, an AAV production system described herein further encodes E2A, E4ORF6, VARNA, AAP, L4 100K, and / or MAAP. The E2A gene product is involved in aiding DNA synthesis processivity during AAV replication. The E4ORF6 gene product supports AAV replication. The VARNA gene product plays a role in regulating translation. The AAP and L4 100K gene products play a role in capsid assembly. The MAAP gene product supports viral secretion.

[0092] In some embodiments, a nucleotide sequence encoding an AAV gene product is operably linked to a promoter. As used herein, the term “promoter” refers to a nucleotide sequence that is bound by proteins to initiate transcription of RNA from DNA. A promoter may be a constitutive promoter (i.e., an unregulated promoter that allows for continual transcription). Examples of constitutive promoters are known in the art and include, but are not limited to, cytomegalovirus (CMV) promoters, elongation factor 1 a (EFla) promoters, simian vacuolating virus 40 (SV40) promoters, ubiquitin-C (UBC) promoters, U6 promoters, p5 promoters, pl9 promoters, p40 promoters, E2A promoters, E4 promoters and phosphoglycerate kinase (PGK) promoters. See e.g., Ferreira et al. Proc. Natl. Acad. Sci. U.S.A. 2013 Jul; 110(28): 11284-89; Pub. No.: US 2014 / 377861 Al; Qin et al. PLoS one 5.5 (2010): el0611. - the entireties of which are incorporated herein by reference. Alternatively, a promoter may be an inducible promoter (i.e., only activates transcription under specific circumstances). An inducible promoter may be a chemically inducible promoter, a temperature inducible promoter, or a light inducible promoter. Additional types of inducible promoters are known to those having ordinary skill in the art. Examples of inducible promoters are known in the art and include, but are not limited to, tetracycline / doxycycline inducible promoters, cumate inducible promoters, ABA inducible promoters, CRY2-CIB1 inducible promoters, DAPG inducible promoters, pTRE3G promoters, pTREtight promoters, the Gal4 UAS operator sequences and mifepristone inducible promoters, and promoters containing at least one of VanR, TtgR, PhlF, or CymR operator sequences. See e.g., Stanton et al., ACS Synth. Biol. 2014 Dec 19; 3(12): 880-91; Liang et al., Sci. Signal. 2011 Mar 15; 4(164): rs2; Patent No.: US 7,745,592 B2; Patent No.: US 7,935,788 B2 - the entireties of which are incorporated herein by reference. In some embodiments, an AAV production component comprises a nucleotide sequence encoding a Rep52 protein. In some embodiments, the Rep52 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 83, wherein the Rep52 is capable of functioning in AAV genome replication. In some embodiments, the Rep52 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a Rep52 protein comprising the amino acid sequence of SEQ ID NO: 83 operably linked to a promoter (as described herein).

[0093] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a Rep40 protein. In some embodiments, the Rep40 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 84, wherein Rep40 is capable of functioning in AAV genome replication. In some embodiments, the Rep40 protein comprises an amino acid sequence having 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a Rep40 protein comprising the amino acid sequence of SEQ ID NO: 84 operably linked to a promoter (as described herein).

[0094] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a Rep78 protein. In some embodiments, the Rep78 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 85, wherein the Rep78 protein is capable of functioning in AAV genome replication. In some embodiments, the Rep78 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a Rep78 protein comprising the amino acid sequence of SEQ ID NO: 85 operably linked to a promoter (as described herein).

[0095] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a Rep68 protein. In some embodiments, the Rep68 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 86, wherein the Rep68 protein is capable of functioning in AAV genome replication. In some embodiments, the Rep68 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 86. In some embodiments, an AAV production component comprises a nucleotide sequence encoding a Rep68 protein comprising the amino acid sequence of SEQ ID NO: 86 operably linked to a promoter (as described herein).

[0096] In some embodiments, an AAV production component comprises a nucleotide sequence encoding an E2A protein. In some embodiments, the E2A protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 87, wherein the E2A protein is capable of aiding DNA synthesis processivity during AAV replication. In some embodiments, the E2A protein comprises an amino acid sequence having 1-50, 1-45, 1- 40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the AAV production component comprises a nucleotide sequence encoding an E2A protein comprising the amino acid sequence of SEQ ID NO: 87 operably linked to a promoter (as described herein).

[0097] In some embodiments, an AAV production component comprises a nucleotide sequence encoding an E4ORF6 protein. In some embodiments, the E4ORF6 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 88, wherein the E4ORF6 protein is capable of supporting AAV replication. In some embodiments, the E4ORF6 protein comprises an amino acid sequence having 1-50, 1-45, 1- 40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the AAV production component comprises a nucleotide sequence encoding an E4ORF6 protein comprising the amino acid sequence of SEQ ID NO: 88 operably linked to a promoter (as described herein).

[0098] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a VARNA. In some embodiments, the VARNA comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the nucleotide sequence of SEQ ID NO: 89, wherein the VARNA is capable regulating translation. In some embodiments, the VARNA protein comprises a nucleotide sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective nucleic acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a VARNA of SEQ ID NO: 89 operably linked to a promoter (as described herein).

[0099] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a VP1 protein. In some embodiments, the VP1 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 90, wherein the VP1 protein is capable of being incorporated into the AAV capsid. In some embodiments, the VP1 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1- 35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a VP1 protein comprising the amino acid sequence of SEQ ID NO: 90 operably linked to a promoter (as described herein).

[0100] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a VP2 protein. In some embodiments, the VP2 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 91, wherein the VP2 protein is capable of being incorporated into the AAV capsid. In some embodiments, the VP2 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1- 35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a VP2 protein comprising the amino acid sequence of SEQ ID NO: 91 operably linked to a promoter (as described herein).

[0101] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a VP3 protein. In some embodiments, the VP3 protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 92, wherein the VP3 protein is capable of being incorporated into the AAV capsid. In some embodiments, the VP3 protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1- 35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the AAV production component comprises a nucleotide sequence encoding a VP3 protein comprising the amino acid sequence of SEQ ID NO: 92 operably linked to a promoter (as described herein).

[0102] In some embodiments, an AAV production component comprises a nucleotide sequence encoding an AAP protein. In some embodiments, the AAP protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 93, wherein the AAP protein is capable of regulating AAV capsid assembly. In some embodiments, the AAP protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1- 20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the AAV production component comprises a nucleotide sequence encoding an AAP protein comprising the amino acid sequence of SEQ ID NO: 93 operably linked to a promoter (as described herein).

[0103] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a L4 100K protein. In some embodiments, the L4 100K protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 94, wherein the L4 100K protein is capable of regulating capsid assembly. In some embodiments, the L4 100K protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1- 20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the AAV production component comprises a nucleotide sequence encoding an L4 100K protein comprising the amino acid sequence of SEQ ID NO: 94 operably linked to a promoter (as described herein).

[0104] In some embodiments, an AAV production component comprises a nucleotide sequence encoding a MAAP protein. In some embodiments, the MAAP protein comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 95, wherein the MAAP protein is capable of regulating AAV capsid assembly. In some embodiments, the MAAP protein comprises an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the AAV production component comprises a nucleotide sequence encoding an MAAP protein comprising the amino acid sequence of SEQ ID NO: 95 operably linked to a promoter (as described herein).

[0105] In some embodiments, an AAV production component is (i.e., the gene products of the AAV component are) encoded on a single polynucleotide. In other embodiments, multiple polynucleotides collectively comprise the AAV production component (i.e., at least two of the gene products of the AAV production component are encoded on different polynucleotides). For example, an AAV production component may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 polynucleotides. In some embodiments, an AAV production component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 polynucleotides.

[0106] In some embodiments, an AAV production component comprises one or more polynucleotides that collectively encode the gene products: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; and VP3. In some embodiments, an AAV production component comprises one or more polynucleotides that collectively encode the gene products: Rep52 or Rep40; Rep78 or Rep68; E2A; E4ORF6; VARNA; VP1; VP2; and VP3. In some embodiments, an AAV production component comprises one or more polynucleotides that collectively encode the gene products: Rep52 or Rep40; Rep78 or Rep68; E2A; E4ORF6; VARNA; VP1; VP2; VP3; AAP; and MAAP. In some embodiments, a AAV production component comprises one or more polynucleotides that collectively encode the gene products: Rep52, Rep40, Rep78, Rep68, E2A, E4ORF6, VARNA, VP1, VP2, and VP3. In some embodiments, a AAV production component comprises one or more polynucleotides that collectively encode the gene products: Rep52, Rep40, Rep78, Rep68, E2A, E4ORF6, VARNA, VP1, VP2, VP3, AAP, and MAAP. In some embodiments, the AAV production component comprises a polynucleotide encoding L4 100K.

[0107] In some embodiments, the one or more polynucleotides of the AAV production system do not encode a viral capsid protein other than VP1, VP2, and VP3.

[0108] In some embodiments, an AAV production system described herein further encodes a transactivator capable of inducing expression of a gene product required to generate an AAV vector in a recombinant host cell.

[0109] In some embodiments, an AAV production system described herein further comprises a transfer polynucleic acid molecule comprising a recombinant AAV genome.

[0110] B. Additional Viral Protein Component

[0111] The AAV production systems described herein may comprise an additional viral protein component. As used herein, the term “additional viral protein component” refers to a polynucleotide that encodes an additional viral protein (i.e., beyond the AAV gene products required for AAV production). The sequence encoding the additional viral protein may be operably linked to a promoter (as described herein).

[0112] In some embodiments, the additional viral protein may be capable of promoting AAV packing efficiency (e.g., when co-expressed in sufficient amounts with the gene products required for AAV production in a cell). In some embodiments, the additional viral protein component comprises a polynucleotide encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 viral proteins that are capable of promoting packaging efficiency. In some embodiments, the additional viral protein component comprises a polynucleotide encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 viral proteins that are capable of promoting packaging efficiency.

[0113] In some embodiments, the additional viral protein is capable of generating conditions for liquid-liquid phase separation (e.g., when expressed in sufficient amounts in a cell). In some embodiments, the additional viral protein component comprises a polynucleotide encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 viral proteins that are capable of generating conditions for liquid-liquid phase separation. In some embodiments, the additional viral protein component comprises a polynucleotide encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 viral proteins that are capable of generating conditions for liquid-liquid phase separation.

[0114] In some embodiments, the additional viral protein comprises one or more intrinsically disordered region(s). In some embodiments, the additional viral protein component comprises a polynucleotide encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 viral proteins that comprise one or more intrinsically disordered region(s). In some embodiments, the additional viral protein component comprises a polynucleotide encoding 2, 3, 4, 5, 6, 7, 8, 9, or 10 viral proteins that comprise one or more intrinsically disordered region(s).

[0115] As used herein, an “intrinsically disordered region” refers to a polypeptide region that lacks a fixed or ordered three-dimensional structure, such as in the absence of an additional structure promoting factor. Amino acid sequences of exemplary intrinsically disordered regions are provided in TABLE 1.

[0116] In some embodiments, an additional viral protein comprises at least one (e.g., one, two, or more) intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of any one of SEQ ID NOs: 30-53, 105-109, or 123. In some embodiments, an additional viral protein comprises at least one (e.g., one, two, or more) intrinsically disordered region comprising an amino acid sequence having 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of any one of SEQ ID NOs: 30-53, 105-109, or 123. In some embodiments, an additional viral protein comprises at least one (e.g., one, two, or more) intrinsically disordered region comprising the amino acid sequence of any one of SEQ ID NOs: 30-53, 105-109, or 123.

[0117] In some embodiments, the additional viral protein comprises a polynucleotide that encodes an additional viral protein comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of any one of SEQ ID NOs: 1-29, 96-104, or 120.

[0118] In some embodiments, the additional viral protein comprises at least one mutation relative to its wildtype amino acid sequence.

[0119] In some embodiments, the nucleotide sequence encoding the additional viral protein comprises at least one codon that has been optimized, relative to a wild type nucleotide sequence encoding the additional viral protein, for expression in a human cell.

[0120] In some embodiments, the additional viral protein is an adenoviral protein. In some embodiments, adenoviral protein is an Ad2 protein, and Ad4 protein, or an Ad5 protein.

[0121] In some embodiments, the additional viral protein is a Kaposi's sarcoma-associated herpesvirus (KSHV) protein.

[0122] In some embodiments, the additional viral protein is an Epstein-Barr virus (EBV) protein.

[0123] In some embodiments, the additional viral protein is a human cytomegalovirus (MCMV) protein.

[0124] In some embodiments, the additional viral protein is a herpes simplex virus- 1 (HSV- 1) protein.

[0125] In some embodiments, the additional viral protein is a human papillomavirus (HPV) protein.

[0126] In some embodiments, the additional viral protein is a LI 52K protein. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 30. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 30.

[0127] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 54. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 54.

[0128] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 55. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 55.

[0129] In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 31. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 31.

[0130] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 56. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 56.

[0131] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 57. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 57.

[0132] In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 32. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 58. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 58.

[0133] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 59. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 59.

[0134] In some embodiments, the LI 52K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the LI 52K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 60. In some embodiments, the nucleotide sequence encoding the LI 52K protein comprises the sequence of SEQ ID NO: 60.

[0135] In some embodiments, the additional viral protein is a IVa2 protein. In some embodiments, the IVa2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 33. In some embodiments, the IVa2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the IVa2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 33.

[0136] In some embodiments, the IVa2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the IVa2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the IVa2 protein comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the IV a2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 61. In some embodiments, the nucleotide sequence encoding the IVa2 protein comprises the sequence of SEQ ID NO: 61.

[0137] In some embodiments, the additional viral protein is a LI pllla protein.

[0138] In some embodiments, the LI pllla protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 34. In some embodiments, the LI pllla protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the LI pllla protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 34.

[0139] In some embodiments, the LI pllla protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the LI pllla protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the LI pllla protein comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the LI pllla protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 62. In some embodiments, the nucleotide sequence encoding the LI pllla protein comprises the sequence of SEQ ID NO: 62.

[0140] In some embodiments, the additional viral protein is a L4 100K protein.

[0141] In some embodiments, the L4 100K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, the L4 100K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, the L4 100K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0142] In some embodiments, the L4 100K protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 35, and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 36. In some embodiments, the first intrinsically disordered region comprises 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second intrinsically disordered region comprises 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the L4 100K protein comprises a first intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 35 and a second intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 36.

[0143] In some embodiments, the L4 100K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the L4 100K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the L4 100K protein comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the L4 100K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 63. In some embodiments, the nucleotide sequence encoding the L4 100K protein comprises the sequence of SEQ ID NO: 63.

[0144] In some embodiments, the additional viral protein is an E1A protein.

[0145] In some embodiments, the E1A protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 37. In some embodiments, the E1A protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the El A protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 37.

[0146] In some embodiments, the E1A protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the El A protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the El A protein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the E1A protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 64. In some embodiments, the nucleotide sequence encoding the E1A protein comprises the sequence of SEQ ID NO: 64.

[0147] In some embodiments, the additional viral protein is an E1B 19K protein.

[0148] In some embodiments, the E1B 19K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 38. In some embodiments, the E1B 19K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the E1B 19K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the E1B 19K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the E1B 19K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1- 10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the E1B 19K protein comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the nucleotide sequence encoding the E1B 19K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the nucleotide sequence encoding the E1B 19K protein comprises the sequence of SEQ ID NO: 65.

[0149] In some embodiments, the additional viral protein is an E1B 55K protein.

[0150] In some embodiments, the E1B 55K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 39. In some embodiments, the E1B 55K protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the E1B 55K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 39.

[0151] In some embodiments, the E1B 55K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the E1B 55K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1- 10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the E1B 55K protein comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the E1B 55K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 66. In some embodiments, the nucleotide sequence encoding the E1B 55K protein comprises the sequence of SEQ ID NO: 66.

[0152] In some embodiments, the additional viral protein is an E2B protein.

[0153] In some embodiments, the E2B protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 40. In some embodiments, the E2B protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the E2B protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 40.

[0154] In some embodiments, the E2B protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 14. In some embodiments, the E2B protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the E2B protein comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding the E2B protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 67. In some embodiments, the nucleotide sequence encoding the E2B protein comprises the sequence of SEQ ID NO: 67.

[0155] In some embodiments, the additional viral protein is an E2B pTP protein.

[0156] In some embodiments, the E2B pTP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 41. In some embodiments, the E2B pTP protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the E2B pTP protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 41.

[0157] In some embodiments, the E2B pTP protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 15. In some embodiments, the E2B pTP protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1- 10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the E2B pTP protein comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the E2B pTP protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 68. In some embodiments, the nucleotide sequence encoding the E2B pTP protein comprises the sequence of SEQ ID NO: 68.

[0158] In some embodiments, the additional viral protein is a ORF59 protein.

[0159] In some embodiments, the ORF59 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 42. In some embodiments, the ORF59 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the ORF59 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 42.

[0160] In some embodiments, the ORF59 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 16. In some embodiments, the ORF59 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the ORF59 protein comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the ORF59 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 69. In some embodiments, the nucleotide sequence encoding the ORF59 protein comprises the sequence of SEQ ID NO: 69.

[0161] In some embodiments, the additional viral protein is a LANA protein.

[0162] In some embodiments, the LANA protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the LANA protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the LANA protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 43.

[0163] In some embodiments, the LANA protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 17. In some embodiments, the LANA protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the LANA protein comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the nucleotide sequence encoding the LANA protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the nucleotide sequence encoding the LANA protein comprises the sequence of SEQ ID NO: 70.

[0164] In some embodiments, the additional viral protein is an EBNA2 protein.

[0165] In some embodiments, the EBNA2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 44. In some embodiments, the EBNA2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the EBNA2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 44.

[0166] In some embodiments, the EBNA2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 18. In some embodiments, the EBNA2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the EBNA2 protein comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the EBNA2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the nucleotide sequence encoding the EBNA2 protein comprises the sequence of SEQ ID NO: 71.

[0167] In some embodiments, the additional viral protein is an EBNALP protein.

[0168] In some embodiments, the EBNALP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 45. In some embodiments, the EBNALP protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the EBNALP protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 45.

[0169] In some embodiments, the EBNALP protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the EBNALP protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1- 15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the EBNALP protein comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the EBNALP protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the nucleotide sequence encoding the EBNALP protein comprises the sequence of SEQ ID NO: 72.

[0170] In some embodiments, the additional viral protein is a ULI 12-UL113 protein.

[0171] In some embodiments, the UL112-UL113 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 46. In some embodiments, the ULI 12-UL113 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the ULI 12-UL113 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 46.

[0172] In some embodiments, the UL112-UL113 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the UL112-UL113 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the ULI 12- UL113 protein comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the nucleotide sequence encoding the UL112-UL113 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the nucleotide sequence encoding the UL112-UL113 protein comprises the sequence of SEQ ID NO: 73.

[0173] In some embodiments, the additional viral protein is a UL57 protein.

[0174] In some embodiments, the UL57 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 47. In some embodiments, the UL57 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the UL57 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 47.

[0175] In some embodiments, the UL57 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the UL57 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the UL57 protein comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the nucleotide sequence encoding the UL57 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the nucleotide sequence encoding the UL57 protein comprises the sequence of SEQ ID NO: 74.

[0176] In some embodiments, the additional viral protein is an ICP4 protein.

[0177] In some embodiments, the ICP4 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the ICP4 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the ICP4 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49.

[0178] In some embodiments, the ICP4 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 48, and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 49. In some embodiments, the first intrinsically disordered region comprises 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the second intrinsically disordered region comprises 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the ICP4 protein comprises a first intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 48 and a second intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 49.

[0179] In some embodiments, the ICP4 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 22. In some embodiments, the ICP4 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the ICP4 protein comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding the ICP4 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 75. In some embodiments, the nucleotide sequence encoding the ICP4 protein comprises the sequence of SEQ ID NO: 75.

[0180] In some embodiments, the additional viral protein is an ICP8 protein.

[0181] In some embodiments, the ICP8 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 50. In some embodiments, the ICP8 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the ICP8 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 50.

[0182] In some embodiments, the ICP8 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ICP8 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ICP8 protein comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding the ICP8 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 76. In some embodiments, the nucleotide sequence encoding the ICP8 protein comprises the sequence of SEQ ID NO: 76.

[0183] In some embodiments, the additional viral protein is a ULI 1 protein.

[0184] In some embodiments, the ULI 1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 51. In some embodiments, the UL11 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the ULI 1 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 51.

[0185] In some embodiments, the UL11 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 24. In some embodiments, the ULI 1 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the ULI 1 protein comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the nucleotide sequence encoding the ULI 1 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 77. In some embodiments, the nucleotide sequence encoding the ULI 1 protein comprises the sequence of SEQ ID NO: 77.

[0186] In some embodiments, the additional viral protein is an El protein.

[0187] In some embodiments, the El protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 52. In some embodiments, the El protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the El protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 52.

[0188] In some embodiments, the El protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the El protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the El protein comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the nucleotide sequence encoding the El protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the nucleotide sequence encoding the El protein comprises the sequence of SEQ ID NO: 78.

[0189] In some embodiments, the additional viral protein is an E2 protein.

[0190] In some embodiments, the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 53.

[0191] In some embodiments, the E2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 26. In some embodiments, the E2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the E2 protein comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the nucleotide sequence encoding the E2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 79. In some embodiments, the nucleotide sequence encoding the E2 protein comprises the sequence of SEQ ID NO: 79. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 105. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the E2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 105.

[0192] In some embodiments, the E2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 96. In some embodiments, the E2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the E2 protein comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the nucleotide sequence encoding the E2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 110. In some embodiments, the nucleotide sequence encoding the E2 protein comprises the sequence of SEQ ID NO: 110.

[0193] In some embodiments, the additional viral protein is an mSK_082 E2 protein.

[0194] In some embodiments, the mSK_082 E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the mSK_082 E2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the mSK_082 E2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 106.

[0195] In some embodiments, the mSK_082 E2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 97. In some embodiments, the mSK_082 E2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the mSK_082 E2 protein comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the nucleotide sequence encoding the mSK_082 E2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 111. In some embodiments, the nucleotide sequence encoding the mSK_082 E2 protein comprises the sequence of SEQ ID NO: 111.

[0196] In some embodiments, the additional viral protein is a UL12 protein.

[0197] In some embodiments, the UL12 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108. In some embodiments, the UL12 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108. In some embodiments, the UL12 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108.

[0198] In some embodiments, the UL12 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 107, and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first intrinsically disordered region comprises 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the second intrinsically disordered region comprises 1-15, 1- 10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the UL12 protein comprises a first intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 107 and a second intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 108.

[0199] In some embodiments, the UL12 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 98. In some embodiments, the UL12 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the UL12 protein comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the nucleotide sequence encoding the UL12 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 112. In some embodiments, the nucleotide sequence encoding the UL12 protein comprises the sequence of SEQ ID NO: 112.

[0200] In some embodiments, the UL12 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 99. In some embodiments, the UL12 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the UL12 protein comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the nucleotide sequence encoding the UL12 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 113. In some embodiments, the nucleotide sequence encoding the UL12 protein comprises the sequence of SEQ ID NO: 113.

[0201] In some embodiments, the additional viral protein is a UL12.5 protein.

[0202] In some embodiments, the UL12.5 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the UL12.5 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the UL12.5 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 108.

[0203] In some embodiments, the UL12.5 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 100. In some embodiments, the UL12.5 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the ULI 2.5 protein comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the nucleotide sequence encoding the UL12.5 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 114. In some embodiments, the nucleotide sequence encoding the UL12.5 protein comprises the sequence of SEQ ID NO: 114.

[0204] In some embodiments, the UL12.5 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 101. In some embodiments, the UL12.5 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the ULI 2.5 protein comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the nucleotide sequence encoding the UL12.5 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 115. In some embodiments, the nucleotide sequence encoding the UL12.5 protein comprises the sequence of SEQ ID NO: 115.

[0205] In some embodiments, the additional viral protein is an NS2 protein.

[0206] In some embodiments, the NS2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 109. In some embodiments, the NS2 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the NS2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 109.

[0207] In some embodiments, the NS2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 102. In some embodiments, the NS2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the NS2 protein comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the nucleotide sequence encoding the NS2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 116. In some embodiments, the nucleotide sequence encoding the NS2 protein comprises the sequence of SEQ ID NO: 116. In some embodiments, the NS2 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 103. In some embodiments, the NS2 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the NS2 protein comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the nucleotide sequence encoding the NS2 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of any one of SEQ ID NOs: 117 or 119. In some embodiments, the nucleotide sequence encoding the NS2 protein comprises the sequence of SEQ ID NO: 117. In some embodiments, the nucleotide sequence encoding the NS2 protein comprises the sequence of SEQ ID NO: 119.

[0208] In some embodiments, the nucleotide sequence encoding NS2 comprises, relative to the nucleotide sequence of SEQ ID NO: 119, one or more of (two or more of, three or more of, four or more of, five or more of, six or more of, seven or more of, eight or more of, nine or more of, or all ten of) a T at position 759, a C at position 769, a G at position 771, T at position 922, a C at position 923, a C at position 924, a C at position 1,110, a T at position, 1,111, a C at position 1,112, and an A at position 1,419 (positions of SEQ ID NO: 119 determined sequentially). In some embodiments, the nucleotide sequence encoding NS2 comprises, relative to the nucleotide sequence of SEQ ID NO: 119, a T at position 759, a C at position 769, a G at position 771, T at position 922, a C at position 923, a C at position 924, a C at position 1,110, a T at position, 1,111, a C at position 1,112, and an A at position 1,419 (positions of SEQ ID NO: 119 determined sequentially).

[0209] In some embodiments, the additional viral protein is an E6 protein.

[0210] In some embodiments, the E6 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 27. In some embodiments, the E6 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the E6 protein comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding E6 comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 80. In some embodiments, the nucleotide sequence encoding the E6 protein comprises the sequence of SEQ ID NO: 80.

[0211] In some embodiments, the E6 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 104. In some embodiments, the E6 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the E6 protein comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the nucleotide sequence encoding E6 comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 118. In some embodiments, the nucleotide sequence encoding the E6 protein comprises the sequence of SEQ ID NO: 118.

[0212] In some embodiments, the additional viral protein is a LI 13.6K protein.

[0213] In some embodiments, the LI 13.6K protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 28. In some embodiments, the LI 13.6K protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1- 10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the LI 13.6K protein comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the nucleotide sequence encoding the LI 13.6K protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 81. In some embodiments, the nucleotide sequence encoding the LI 13.6K protein comprises the sequence of SEQ ID NO: 81.

[0214] In some embodiments, the additional viral protein is an ORF6 protein.

[0215] In some embodiments, the ORF6 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 29. In some embodiments, the ORF6 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the ORF6 protein comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the nucleotide sequence encoding the ORF6 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 82. In some embodiments, the nucleotide sequence encoding the ORF6 protein comprises the sequence of SEQ ID NO: 82.

[0216] In some embodiments, the additional viral protein is an NP1 protein.

[0217] In some embodiments, the NP1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 123. In some embodiments, the NP1 protein comprises an intrinsically disordered region comprising 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the NP1 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 123.

[0218] In some embodiments, the NP1 protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 120. In some embodiments, the NP1 protein comprises an amino acid sequence having 1-40, 1-35, 1-30 1-25, 1-20, 1-15, 1-10, or 1-5 mutations (collective amino acid deletions, insertions, and / or substitutions) relative to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the ORF6 protein comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the nucleotide sequence encoding the ORF6 protein comprises a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) with the amino acid sequence of SEQ ID NO: 122. In some embodiments, the nucleotide sequence encoding the ORF6 protein comprises the sequence of SEQ ID NO: 121.

[0219] In some embodiments, the nucleotide sequence encoding NP1 comprises, relative to the nucleotide sequence of SEQ ID NO: 121, an A at position 585 and / or a C at position 588 (positions of SEQ ID NO: 121 determined sequentially). In some embodiments, the nucleotide sequence encoding NP1 comprises, relative to the nucleotide sequence of SEQ ID NO: 121, an A at position 585 and a C at position 588 (positions of SEQ ID NO: 121 determined sequentially) .

[0220] C. Additional Viral Non-Coding RNA Component

[0221] The AAV production systems described herein may comprise a viral non-coding RNA component. As used herein, the term “a viral non-coding RNA component” refers to a polynucleotide that encodes a viral non-coding RNA. The sequence encoding the viral noncoding RNA may be operably linked to a promoter (as described herein). In some embodiments, the viral non-coding RNA may be capable of promoting AAV replication.

[0222] In some embodiments, the non-coding RNA comprising a nucleotide sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the nucleotide sequence of SEQ ID NO: 122. In some embodiments, the non-coding RNA comprises the nucleotide sequence of SEQ ID NO: 122.

[0223] D. Exemplary AAV Production Systems

[0224] In some embodiments, an AAV production system comprises: (i) a first polynucleotide comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; a second polynucleotide comprising a nucleotide sequence encoding for E2A, optionally further comprising a nucleotide sequence encoding for E40rf6 and / or VARNA; and a third polynucleotide comprising a nucleotide sequence encoding for the additional viral protein (e.g., of the additional viral protein component described above).

[0225] In some embodiments, the first polynucleotide further comprises a first promoter operably linked to the nucleotide sequence encoding for: Rep52 or Rep40, a second promoter operably linked to the nucleotide sequence encoding for Rep78 or Rep68, and a third promoter operably linked to the nucleotide sequence encoding for VP1, VP2, and VP3.

[0226] In some embodiments, the second polynucleotide further comprises a first promoter operably linked to the nucleotide sequence encoding for E2A and a second promoter operably linked to the nucleotide sequence encoding for E40rf6 and VARNA.

[0227] In some embodiments, the third polynucleotide further comprises a promoter operably linked to the nucleotide sequence encoding for the additional viral protein.

[0228] In some embodiments, an AAV production system is as depicted in FIG. 1.

[0229] In some embodiments, an Adeno-Associated Virus (AAV) production system comprises one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; E40rf6; VP1; VP2; VP3; a viral noncoding RNA comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 122; a first additional viral protein; and a second additional viral protein; wherein the AAV production system comprises a polynucleotide comprising a sequence encoding a polycistronic RNA, wherein the polycistronic RNA comprises the sequence encoding for the viral noncoding RNA, the sequence encoding for the first additional viral protein, and the sequence encoding for the second additional viral protein. In some embodiments, the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the first additional viral protein; and the sequence encoding the second additional viral protein. In some embodiments, the polycistronic mRNA comprises, 5’ to 3’: the sequence encoding the first additional viral protein; the sequence encoding the viral noncoding RNA; and the sequence encoding the second additional viral protein. In some embodiments, the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the first additional viral protein; the sequence encoding the second additional viral protein; and the sequence encoding the viral noncoding RNA.

[0230] In some embodiments, the polycistronic RNA further comprises a sequence encoding a viral 2A peptide. The amino acid sequences of various viral 2A peptides are known in the prior art, as well as their corresponding coding sequences. In some embodiments, the nucleotide sequence encoding the viral 2A peptide separates the nucleotide sequence encoding for the first additional viral protein and the nucleotide sequence encoding the second additional viral protein. In some embodiments, the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the first additional viral protein; a sequence encoding a viral 2a peptide; and the sequence encoding the second additional viral protein.

[0231] In some embodiments, the sequence encoding the polycistronic RNA is operably linked to a promoter. In some embodiments, the promoter is an inducible promoter (e.g., a tetracycline / doxycycline inducible promoter).

[0232] In some embodiments, the first additional viral protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of any one of SEQ ID NOs: 1-26, 96-104, or 120. In some embodiments, the second additional viral protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of any one of SEQ ID NOs: 1-26, 96-104, or 120.

[0233] In some embodiments, the first additional viral protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the first additional protein comprises the amino acid sequence of SEQ ID NO: 120.

[0234] In some embodiments, the second additional viral protein comprises an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the second additional protein comprises the amino acid sequence of SEQ ID NO: 103.

[0235] In some embodiments, the AAV production system comprises: a first polynucleotide comprising the sequence encoding for the polycistronic RNA; a second polynucleotide comprising a sequence of encoding for: Rep52 or Rep40; VP1; VP2; VP3; E2A; and E40rf6; and a third polynucleotide comprising a sequence encoding for: Rep78 or Rep68; VP1; VP2; VP3; E2A; and E40rf6.

[0236] In some embodiments, the AAV production system does not encode Rep68.

[0237] E. Percent Identity

[0238] As used herein, the term “percent identity” refers to a relationship between the sequences of two polynucleotides, as determined by sequence comparison (alignment). In some embodiments, identity is determined across the entire length of a sequence. In some embodiments, identity is determined over a region of a sequence.

[0239] Identity of related nucleic acid sequences can be readily calculated by those having ordinary skill in the art.

[0240] In some embodiments, the percent identity of two sequences is determined using the algorithm of Karlin and Altschul 1990 Proc. Natl. Acad. Sci. U.S.A. 87:2264-68, modified as in Karlin and Altschul 1993 Proc. Natl. Acad. Sci. U.S.A. 90:5873-77. This algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al. 1990 J. Mol. Biol. 215:403-10. BLAST® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the disclosure. Where gaps exist between two sequences, Gapped BLAST® can be utilized, for example, as described in Altschul et al. 1997 Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST® and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

[0241] In some embodiments, the percent identity of two sequences is determined using the algorithm of Smith-Waterman (Smith, T.E. & Waterman, M.S. 1981 J. Mol. Biol. 147:195- 197).

[0242] In some embodiments, the percent identity of two sequences is determined using the algorithm of Needleman-Wunsch (Needleman, S.B. & Wunsch, C.D. 1970 J. Mol. Biol. 48:443-453).

[0243] In some embodiments, the percent identity of two sequences is determined using the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0244] In some embodiments, the percent identity of two sequences is determined using Clustal Omega (Sievers et al. 2011 Mol Syst Biol. 7:539), using default parameters.

[0245] In some embodiments, the identity of two nucleic acid sequences is determined by aligning the two sequences, calculating the number of identical nucleotides, and dividing by the length of one of the nucleic acid sequences. In some embodiments, the identity of two nucleic acid sequences is determined by aligning the two sequences, calculating the number of identical nucleotides, and dividing by the length of the longer nucleic acid sequence. In some embodiments, the identity of two nucleic acid sequences is determined by aligning the two sequences, calculating the number of identical nucleotides, and dividing by the length of the shorter nucleic acid sequence.

[0246] II. Engineered cells

[0247] In some aspects, the disclosure relates to engineered cells for AAV production. An engineered cell may comprise any part (and any combination of parts) of an AAV production system described herein.

[0248] For example, an engineered cell may comprise at least a portion of the AAV production component. For example, and as described above, an AAV production component may comprise multiple polynucleic acids. In such embodiments, an engineered cell comprises one or more of said multiple polynucleic acids - each of which may be located extra-chromosomally or stably integrated into the genome of the engineered cell. In some embodiments, an engineered cell comprises the entire AAV production component.

[0249] Alternatively, or in addition, an engineered cell may comprise an AAV production component or an additional viral protein component.

[0250] In some embodiments, an engineered cell comprises: (i) a first plasmid comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; (ii) a second plasmid comprising a second polynucleotide comprising a nucleotide sequence encoding for E2A, optionally further encoding for E40rf6 and / or VARNA; or (iii) a third plasmid comprising a nucleotide sequence encoding the additional viral protein. In some embodiments, an engineered cell comprises at least two of: (i) a first plasmid comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; (ii) a second plasmid comprising a second polynucleotide comprising a nucleotide sequence encoding for E2A, optionally further encoding for E40rf6 and / or VARNA; and (iii) a third plasmid comprising a nucleotide sequence encoding the additional viral protein. In some embodiments, an engineered cell comprises: (i) a first plasmid comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; (ii) a second plasmid comprising a second polynucleotide comprising a nucleotide sequence encoding for: E2A, optionally further encoding for E40rf6 and / or VARNA; and (iii) a third plasmid comprising a nucleotide sequence encoding the additional viral protein.

[0251] In some embodiments, an engineered cell comprises: a genomic integration of a first nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; optionally wherein the first nucleotide sequence encodes for Rep52, Rep40, Rep78, Rep68, VP1, VP2, and VP3; a genomic integration of a second nucleotide sequence encoding for: E2A; E40rf6; and VARNA; or a genomic integration of a third nucleotide sequence encoding for the additional viral protein. In some embodiments, an engineered cell comprises at least two of: a genomic integration of a first nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; optionally wherein the first nucleotide sequence encodes for Rep52, Rep40, Rep78, Rep68, VP1, VP2, and VP3; a genomic integration of a second nucleotide sequence encoding for: E2A; E40rf6; and VARNA; and a genomic integration of a third nucleotide sequence encoding for the additional viral protein. In some embodiments, an engineered cell comprises: a genomic integration of a first nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; optionally wherein the first nucleotide sequence encodes for Rep52, Rep40, Rep78, Rep68, VP1, VP2, and VP3; a genomic integration of a second nucleotide sequence encoding for: E2A; E40rf6; and VARNA; and a genomic integration of a third nucleotide sequence encoding for the additional viral protein.

[0252] In some embodiments, the engineered cells are derived from known or existing cell lines. In some embodiments, the engineered cells are derived from the group consisting of HEK293 cells, HeLa cells, BHK cells, and Sf9 cells. In some embodiments, the engineered cells comprise nucleotide sequences encoding genes required for AAV production and systems for regulating expression of said genes, as described herein. In some embodiments, the engineered cell comprises genomic sites for stable integration of one or more nucleic acid molecules (e.g., 1, 2, 3, 4, 5, or 6 nucleic acid molecules). These genomics sites for stable integration of nucleic acid molecules are well known to those of ordinary skill in the art. Exemplary sites for stable integration include but are not limited to AAVS1, ROSA26, CCR5, Hl l, and LiPS-A3S. In some embodiments, the stably integrated nucleic acid molecule is randomly integrated into the Engineered cell genome.

[0253] An engineered cell described herein may further comprise a landing pad. As used herein, the term “landing pad” refers to a heterologous polynucleotide sequence that facilitates the targeted insertion of a “payload” sequence into a specific locus (or multiple loci) of the cell’s genome. Accordingly, the landing pad is integrated into the genome of the cell. A fixed integration site is desirable to reduce the variability between experiments that may be caused by positional epigenetic effects or proximal regulatory elements. The ability to control pay load copy number is also desirable to modulate expression levels of the pay load without changing any genetic components.

[0254] In some embodiments, the landing pad is located at a safe harbor site in the genome of the engineered cell. As used herein, the term “safe harbor site” refers to a location in the genome where genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes and / or adjacent genomic elements do not disrupt expression or regulation of the introduced genes or genetic elements. Examples of safe harbor sites are known to those having skill in the art and include, but are not limited to, AAVS1, ROSA26, COSMIC, Hl l, CCR5, and LiPS-A3S. See e.g., Gaidukov et al., Nucleic Acids Res. 2018 May 4; 46(8): 4072-4086; Patent No.: US 8,980,579 B2; Patent No.: US 10,017,786 B2; Patent No.: US 9,932,607 B2; Pub. No.: US 2013 / 280222 A; Pub. No.: WO 2017 / 180669 Al - the entireties of which are incorporated by reference herein, particularly for the disclosure relating to safe harbor sites. In some embodiments, the safe harbor site is a known site. In other embodiments, the safe harbor site is a previously undisclosed site. See “Methods of Identifying High-Expressing Genomic Loci and Uses Thereof’ herein. In some embodiments, an engineered cell described herein comprises a landing pad that is integrated at a safe harbor locus selected from the group consisting of AAVS1, ROSA26, COSMIC, H11, CCR5, and LiPS-A3S.

[0255] In some embodiments, the engineered cell is derived from a HEK293 cell. In some embodiments, the engineered HEK293 cell comprises a landing pad that is integrated at a safe harbor locus selected from the group consisting of AAVS1, ROSA26, CCR5, and LiPS- A3S.

[0256] In some embodiments, the engineered cell is derived from a CHO cell. In some embodiments, the engineered CHO cell comprises a landing pad that is integrated at a safe harbor locus selected from the group consisting of ROSA26, COSMIC, and Hl l.

[0257] Each of the landing pads described herein comprises at least one recombination site. Recombination sites for various integrases have been identified previously. For example, a landing pad may comprise recombination sites corresponding to a Bxbl integrase, lambdaintegrase, Cre recombinase, Flp recombinase, gamma-delta resolvase, Tn3 resolvase, cpC31 integrase, or R4 integrase. Exemplary recombination site sequences are known in the art (e.g., attP, attB, attR, attL, Lox, and Frt).

[0258] III. Kits

[0259] In some aspects, the disclosure relates to kits for AAV production. In some embodiments, a kit comprises one or more polynucleic acids collectively comprising an AAV production system described herein. In some embodiments, a kit comprises an engineered cell described herein.

[0260] In some embodiments, a kit comprises a polynucleotide comprising, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted terminal repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted terminal repeat. In some embodiments, the polynucleotide is a plasmid or a vector.

[0261] The central nucleic acid of a transfer polynucleic acid may comprise a nucleotide sequence of a multiple cloning site. Exemplary multiple cloning sites are known to those having ordinary skill in the art. A multiple cloning site can be used for cloning a payload molecule (or gene of interest) - or an expression cassette encoding a payload molecule - into the transfer polynucleic acid prior to the generation of viral vectors in a host cell.

[0262] IV. Methods of AAV production

[0263] In some aspects, the disclosure provides methods of producing AAVs using an engineered cell or kit described herein.

[0264] In some embodiments, the method comprises culturing an engineered cell described herein such that it expresses: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and the additional viral protein (and optionally E40rf6 and / or VARNA). In some embodiments, the method comprises culturing the engineered cell such that it expresses: Rep52; Rep40; Rep78; Rep68; E2A; VP1; VP2; VP3; and the additional viral protein (and optionally E40rf6 and / or VARNA).

[0265] In some embodiments, the method comprises growing the engineered cell to a confluency that is optimal for AAV production. An optimal confluency will be dependent on the type of cell the engineered cell is derived from. The skilled person will know or be able to determine the optimal confluency for AAV production.

[0266] In some embodiments, the method comprises harvesting the AAV produced from the culture of engineered cells using methods that are well known to those of skill in the art.

[0267] V. Methods of Generating an Engineered Cell for AAV Production

[0268] In some aspects, the disclosure relates to a method of generating an engineered cell for AAV production. In some embodiments, the method comprises introducing an AAV production system described herein into the cell. Methods of introducing polynucleotides into a cell are known to those having ordinary skill in the art.

[0269] In some embodiments, the method comprising integrating a polynucleotide of the AAV production system into the genome of the cell.

[0270] EXAMPLES

[0271] Example 1. Ad5 LI 52K Expression Boosts AAV Titers

[0272] Description of Approach

[0273] Adenovirus LI 52K was recently identified as a phase separating protein that is important for scaffolding other viral structural proteins. See Charman et al., Nature. 2023 Apr;616(7956):332-338. Previously, adenovirus DNA binding protein (DBP or E2A) was also implicated in liquid-liquid phase separation (LLPS) and interestingly is one of the proteins essential for AAV packaging. See Hildago et la., Viruses. 2021 Sep 6; 13(9): 1778.

[0274] Since AAV uses adenovirus to provide helper function to replicate and package its own genome, it was hypothesized that AAV may also use adenovirus LLPS to better coordinate with adenoviral helper factors. Also, since other viruses also utilize LLPS, it was hypothesized that other viral proteins (such as viral proteins with intrinsically disordered regions, or IDRs (see TABLE 1) or other proteins of interest identified by the inventors (see TABLE 2)) would provide a similar helper function to AAV to enhance the formation of viral biomolecular condensates. In this way a greater percentage of filled capsid compared to empty capsid, could be achieved.

[0275] Data and Experiment Description:

[0276] In several experiments, AAV titer was quantified when using the traditional 3-plasmid transfection system (pHelper plasmid, pRepCap plasmid, pAAV transfer plasmid) supplemented with a fourth plasmid expressing LI 52K (EIG. 1).

[0277] In an initial experiment, 125 ng of Ad5 LI 52K encoding DNA was supplemented into the AAV transfection mixture and compared to a wt production sample lacking any supplementation with exogenous genes. Human host genes CELF2, CEP128, and SUPT5H were tested but showed minimal effect compared to the viral LI 52K gene. Ad5 LI 52K showed a 3.2-fold boost in infectious titers using an EGFP AAV pay load (FIG. 2).

[0278] Additional titrating experiments were performed (FIG. 3). Interestingly, when 12.5 or 25 ng of LI 52K was supplemented into the AAV transfection mixture, AAV infectious titers were moderately decreased. However, AAV infectious titers were boosted when added at 100 ng or 200 ng DNA amounts.

[0279] Example 2. Screening for Additional Viral Proteins that Boost AAV Titers

[0280] Additional viral were transfected at 150 ng DNA amount while the other AAV genes (pHelper, pRepCap, pAAV) were transfected at a total amount of 600 ng. AAV infectious titers were measured by flow cytometry of an EGFP payload. Genes that were codon optimized (CO) are indicated. See FIG. 4; TABLES 1-2. Genes providing the greatest boost to titers included Adenovirus 5 LI 52K, HPV E2 (all tested types), Adenovirus 5 E1B 55K, Adenovirus 5 E2B, Adenovirus 5 E2B pTP, EBV EBNA2, HPV5 E6.

[0281] Example 3. Screening for HSV and Bocavirus Viral Proteins that Boost AAV Titers

[0282] HSV1 UL12, HSV1 UL12.5, and Bocavirus 1 NS2 were tested alone and in combination for ability to boost AAV infectious titers. Two codon optimized variant sequences (CO_1 and CO_2) were tested for each. KSHV LANA and EBV EBNALP were also tested. See FIG. 5; TABLES 1-2. Generally, UL12, UL12.5, Bocavirus NS2, and KSHV LANA show boosted AAV titers.

[0283] Example 4. Non-Coding RNA BocaSR Boosts AAV Titers

[0284] Experiments were performed to assess whether the co-expression of Bocavirus noncoding RNA BocaSR and Bocavirus proteins NP1 and NS2 with AAV gene products is capable of boosting AAV titers.

[0285] The following genetic architecture was prepared:

[0286] • a first polynucleotide encoding large Rep (Rep78 and / or Rep68) and Cap (VP1 + VP2 + VP3), each operably linked to an inducible TRE promoter;

[0287] • a second polynucleotide encoding small Rep (Rep52 and / or Rep40) and Cap (VP1 + VP2 + VP3), each operably linked to an inducible TRE promoter;

[0288] • a third polynucleotide encoding E2A, E4orf6, and VARNA, each operably linked to an inducible TRE promoter;

[0289] • a fourth polynucleotide encoding BocaSR (see TABLE 4), Bocavirus NP1 (SEQ ID NO: 121) and Bocavirus NS2 (SEQ ID NO: 119), each operably linked to a hEFla promoter or an inducible TRE promoter;

[0290] • a fifth polynucleotide encoding a transactivator (TetOn) operably linked to a hEFla promoter; and

[0291] • a sixth polynucleotide encoding a pAAV-EGFP transgene plasmid for measuring AAV production.

[0292] HEK293T cells were transiently transfected with the AAV architecture (plus or minus the Bocavirus polynucleotide (i.e., the fourth polynucleotide)) and AAV production was measured. Specifically, adherent HEK293T cells were transiently transfected with: 22 ng of the first polynucleotide; 180 ng of the second polynucleotide; 80 ng of the third polynucleotide; 80 ng of the fourth polynucleotide (if included); 80 ng of the fifth polynucleotide; and 140 ng of the sixth polynucleotide.

[0293] Cells were plated at 400,000 cells / well in 0.5 mL of DMEM supplemented with 10% FBS, in TC-treated 24-well plates immediately prior to transfection. The transfection mix for each well was made by adding 600 ng of DNA to a microcentrifuge tube, adding Opti-MEM to a final volume of 50 uL, adding 1 uL of P3000 reagent and mixing, then adding 1 uL of lipofectamine 3000 and mixing. Transfection mixes were incubated for 15-30 minutes at room temperature prior to adding to cells. After transfection, doxycycline was added to select samples at 200 nM concentration.

[0294] Three days after transfection, cells and supernatant were harvested by pipetting and transferred to a microcentrifuge tube. Virus solutions produced this way were lysed by freeze-thaw cycling between a dry ice isopropanol bath and a 37°C bead bath. Three freezethaw cycles were used and cellular debris was removed by centrifugation at 4000 ref. Infectious titers were measured by transducing HEK293T target cells within a 96-well plate. Briefly, virus was serially diluted lOx in growth medium, then 0.1 uL, 1 uL, or 10 uL of virus was added to plated cells. Cells were plated at 50,000 cells per well in 100 uL of supplemented DMEM. After three days incubation at 37°C and 5% CO2, transduced cells were trypsinized and resuspended in PBS before % EGFP positive was measured using flow cytometry.

[0295] Results are shown in FIG. 6. These results demonstrate that the co-expression of Bocavirus non-coding RNA BocaSR and Bocavirus proteins NP1 and NS2 with AAV gene products is capable of boosting AAV titers. Moreover, embodiments utilizing codon- optimized NP1 and codon-optimized NS1 (to remove potential splice sites) was found superior to utilizing the wt coding sequences for NP1 and NS1 (data not shown).

[0296] TABLE 1: Exemplary IDR-containing proteins. Amino acid and nucleotide sequences are provided. Underling labels known or predicted intrinsically disordered regions (IDRs) in the amino acid sequences. TABLE 2: Additional exemplary proteins for promoting LLPS conditions. Amino acid and nucleotide sequences are provided.

[0297] TABLE 3: Exemplary amino acid sequence for AAV gene products.

[0298] TABLE 4: Exemplary non-coding RNAs for promoting AAV production.

[0299] OTHER EMBODIMENTS

[0300] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0301] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0302] EQUIVALENTS

[0303] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0304] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0305] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0306] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0307] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0308] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0309] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0310] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims

CLAIMSWhat is claimed is:

1. An Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and an additional viral protein, wherein the additional viral protein is not an AAV protein or an adenoviral El protein, and wherein the additional viral protein is capable of generating conditions for liquid- liquid phase separation when expressed in a cell.

2. An Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and an additional viral protein, wherein the additional viral protein is not an AAV protein or an adenoviral El protein, and wherein the additional viral protein comprises one or more intrinsically disordered region(s).

3. The AAV production system of claim 2, wherein the additional viral protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 30-53, 105-109, or 123.

4. An Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and an additional viral protein, wherein the additional viral protein comprises an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 1-29, 96-104, or 120, wherein the additional viral protein does not comprise the amino acid sequence of any one of SEQ ID NOs: 11-13.

5. The AAV production system of any one of claims 1-4, wherein the one or more polynucleotides of the AAV production system collectively further comprise a nucleotide sequence encoding for E40rf6 and / or VARNA.

6. The AAV production system of any one of claims 1-5, wherein the additional viral protein comprises at least one mutation relative to its wildtype amino acid sequence.

7. The AAV production system of any one of claims 1-6, wherein the nucleotide sequence encoding the additional viral protein comprises at least one codon that has been optimized, relative to a wild type nucleotide sequence encoding the additional viral protein, for expression in a human cell.

8. The AAV production system of any one of claims 1-7, wherein the amino acid sequence of Rep52, the amino acid sequence of Rep40, the amino acid sequence of Rep78, the amino acid sequence of Rep68, the amino acid sequence of E2A, the amino acid sequence of VP1, the amino acid sequence of VP2, and / or the amino acid sequence of VP3 comprises one or more mutations relative to its corresponding wild type amino acid sequence.

9. The AAV production system of any one of claims 1-8, wherein the AAV production system comprises a polynucleotide encoding a LI 52K protein.

10. The AAV production system of claim 9, wherein the LI 52K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 30-32.

11. The AAV production system of claim 9 or claim 10, wherein the LI 52K protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-7.

12. The AAV production system of any one of claims 1-11, wherein the AAV production system comprises a polynucleotide encoding a IV a2 protein.

13. The AAV production system of claim 12, wherein the IVa2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 33.

14. The AAV production system of claim 12 or claim 13, wherein the IVa2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 8.

15. The AAV production system of any one of claims 1-14, wherein the AAV production system comprises a polynucleotide encoding a LI pllla protein.

16. The AAV production system of claim 15, wherein the LI pllla protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 34.

17. The AAV production system of claim 15 or claim 16, wherein the LI pllla protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 9.

18. The AAV production system of any one of claims 1-17, wherein the AAV production system comprises a polynucleotide encoding a L4 100K protein.

19. The AAV production system of claim 18, wherein the L4 100K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, optionally wherein the L4 100K protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 35 and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 36.

20. The AAV production system of claim 18 or claim 19, wherein the L4 100K protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10.

21. The AAV production system of any one of claims 1-20, wherein the AAV production system comprises a polynucleotide encoding an E1A protein.

22. The AAV production system of claim 21, wherein the E1A protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 37.

23. The AAV production system of claim 21 or claim 22, wherein the E1A protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 11.

24. The AAV production system of any one of claims 1-23, wherein the AAV production system comprises a polynucleotide encoding an E1B 19K protein.

25. The AAV production system of claim 24, wherein the E1B 19K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 38.

26. The AAV production system of claim 24 or claim 25, wherein the E1B 19K protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 12.

27. The AAV production system of any one of claims 1-26, wherein the AAV production system comprises a polynucleotide encoding an E1B 55K protein.

28. The AAV production system of claim 27, wherein the E1B 55K protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 39.

29. The AAV production system of claim 27 or claim 28, wherein the E1B 55K protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 13.

30. The AAV production system of any one of claims 1-29, wherein the AAV production system comprises a polynucleotide encoding an E2B protein.

31. The AAV production system of claim 30, wherein the E2B protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 40.

32. The AAV production system of claim 30 or claim 31, wherein the E2B protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14.

33. The AAV production system of any one of claims 1-32, wherein the AAV production system comprises a polynucleotide encoding an E2B pTP protein.

34. The AAV production system of claim 33, wherein the E2B pTP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 41.

35. The AAV production system of claim 33 or claim 34, wherein the E2B pTP protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 15.

36. The AAV production system of any one of claims 1-35, wherein the AAV production system comprises a polynucleotide encoding an ORF59 protein.

37. The AAV production system of claim 36, wherein the ORF59 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 42.

38. The AAV production system of claim 36 or claim 37, wherein the ORF59 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16.

39. The AAV production system of any one of claims 1-38, wherein the AAV production system comprises a polynucleotide encoding a LANA protein.

40. The AAV production system of claim 39, wherein the LANA protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 43.

41. The AAV production system of claim 39 or claim 40, wherein the LANA protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 17.

42. The AAV production system of any one of claims 1-41, wherein the AAV production system comprises a polynucleotide encoding an EBNA2 protein.

43. The AAV production system of claim 42, wherein the EBNA2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 44.

44. The AAV production system of claim 42 or claim 43, wherein the EBNA2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 18.

45. The AAV production system of any one of claims 1-44, wherein the AAV production system comprises a polynucleotide encoding an EBNALP protein.

46. The AAV production system of claim 45, wherein the EBNALP protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 45.

47. The AAV production system of claim 45 or claim 46, wherein the EBNALP protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 19.

48. The AAV production system of any one of claims 1-47, wherein the AAV production system comprises a polynucleotide encoding a ULI 12-UL113 protein.

49. The AAV production system of claim 48, wherein the ULI 12-UL113 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 46.

50. The AAV production system of claim 48 or claim 49, wherein the ULI 12-UL113 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 20.

51. The AAV production system of any one of claims 1-50, wherein the AAV production system comprises a polynucleotide encoding a UL57 protein.

52. The AAV production system of claim 51, wherein the UL57 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 47.

53. The AAV production system of claim 51 or claim 52, wherein the UL57 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 21.

54. The AAV production system of any one of claims 1-53, wherein the AAV production system comprises a polynucleotide encoding an ICP4 protein.

55. The AAV production system of claim 54, wherein the ICP4 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49, optionally wherein the ICP4 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 48 and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 49.

56. The AAV production system of claim 54 or claim 55, wherein the ICP4 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 22.

57. The AAV production system of any one of claims 1-56, wherein the AAV production system comprises a polynucleotide encoding an ICP8 protein.

58. The AAV production system of claim 57, wherein the ICP8 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 50.

59. The AAV production system of claim 57 or claim 58, wherein the ICP8 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 23.

60. The AAV production system of any one of claims 1-59, wherein the AAV production system comprises a polynucleotide encoding a ULI 1 protein.

61. The AAV production system of claim 60, wherein the ULI 1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 51.

62. The AAV production system of claim 60 or claim 61, wherein the ULI 1 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 24.

63. The AAV production system of any one of claims 1-62, wherein the AAV production system comprises a polynucleotide encoding an El protein.

64. The AAV production system of claim 63, wherein the El protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 52.

65. The AAV production system of claim 63 or claim 64, wherein the El protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 25.

66. The AAV production system of any one of claims 1-65, wherein the AAV production system comprises a polynucleotide encoding an E2 protein.

67. The AAV production system of claim 66, wherein the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 53.

68. The AAV production system of claim 66 or claim 67, wherein the E2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 26.

69. The AAV production system of claim 66, wherein the E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 105.

70. The AAV production system of claim 66 or claim 69, wherein the E2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 96.

71. The AAV production system of any one of claims 1-70, wherein the AAV production system comprises a polynucleotide encoding an mSK_082 E2 protein.

72. The AAV production system of claim 71, wherein the mSK_082 E2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 106.

73. The AAV production system of claim 71 or claim 72, wherein the mSK_082 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 97.

74. The AAV production system of any one of claims 1-73, wherein the AAV production system comprises a polynucleotide encoding a UL12 protein.

75. The AAV production system of claim 74, wherein the UL12 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108, optionally wherein the ULI 2 protein comprises a first intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 107 and a second intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 108.

76. The AAV production system of claim 74 or claim 75, wherein the UL12 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 98.

77. The AAV production system of claim 74 or claim 75, wherein the UL12 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 99.

78. The AAV production system of any one of claims 1-77, wherein the AAV production system comprises a polynucleotide encoding an UL12.5 protein.

79. The AAV production system of claim 78, wherein the UL12.5 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 108.

80. The AAV production system of claim 78 or claim 79, wherein the UL12.5 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 100.

81. The AAV production system of claim 78 or claim 79, wherein the UL12.5 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 101.

82. The AAV production system of any one of claims 1-81, wherein the AAV production system comprises a polynucleotide encoding an NS2 protein.

83. The AAV production system of claim 82, wherein the NS2 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 109.

84. The AAV production system of claim 82 or claim 83, wherein the NS2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 102.

85. The AAV production system of claim 82 or claim 83, wherein the NS2 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 103.

86. The AAV production system of any one of claims 1-85, wherein the AAV production system comprises a polynucleotide encoding an E6 protein.

87. The AAV production system of claim 86, wherein the E6 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 27.

88. The AAV production system of claim 86, wherein the E6 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 104.

89. The AAV production system of any one of claims 1-88, wherein the AAV production system comprises a polynucleotide encoding an LI 13.6K protein.

90. The AAV production system of claim 89, wherein the LI 13.6K protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:28.

91. The AAV production system of any one of claims 1-90, wherein the AAV production system comprises a polynucleotide encoding a ORF6 protein.

92. The AAV production system of claim 91, wherein the ORF6 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:29.

93. The AAV production system of any one of claims 1-92, wherein the AAV production system comprises a polynucleotide encoding an NP1 protein.

94. The AAV production system of claim 93, wherein the NP1 protein comprises an intrinsically disordered region comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 123.

95. The AAV production system of claim 93 or claim 94, wherein the NP1 protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120.

96. The AAV production system of any one of claims 1-95, wherein the one or more polynucleotides of the AAV production system further comprise a nucleotide sequence encoding for a non-coding RNA comprising a sequence having at least 80% identity to the sequence of SEQ ID NO: 122.

97. The AAV production system of any one of claims 1-96, wherein the AAV production system comprises a polynucleotide comprising a sequence having at least 80% identity with the nucleotide sequence of one or more of SEQ ID NOs: 54-82, 110-119, or 121.

98. The AAV production system of any one of claims 1-97, wherein the AAV production system is capable of producing helper virus-free AAV.

99. The AAV production system of any one of claims 1-98, wherein the one or more polynucleotides of the AAV production system do not encode a viral capsid protein other than VP1, VP2, and VP3.

100. The AAV production system of any one of claims 1-99, comprising a first polynucleotide comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3.

101. The AAV production system of any one of claims 1-100, comprising a first polynucleotide comprising a nucleotide sequence encoding for: Rep52; Rep40; Rep78; Rep68; VP1; VP2; and VP3.

102. The AAV production system of any one of claims 1-101, comprising a second polynucleotide comprising a nucleotide sequence encoding for: E2A; E40rf6; and VARNA.

103. The AAV production system of any one of claims 1-102, comprising a third polynucleotide comprising a nucleotide sequence encoding the additional viral protein.

104. The AAV production system of any one of claims 1-103, comprising a transfer plasmid.

105. An Adeno-Associated Virus (AAV) production system comprising one or more polynucleotides collectively comprising a nucleotide sequence encoding for each of: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; a viral noncoding RNA comprising at least 80% identity to the sequence of SEQ ID NO: 122; a first additional viral protein; and a second additional viral protein; wherein the AAV production system comprises a polynucleotide comprising a sequence encoding a polycistronic RNA, wherein the polycistronic RNA comprises the sequence encoding for the viral noncoding RNA, the sequence encoding for the first additional viral protein, and the sequence encoding for the second additional viral protein.

106. The AAV production system of claim 105, wherein the one or more polynucleotides of the AAV production system further comprise a nucleotide sequence encoding for E40rf6.

107. The AAV production system of claim 105 or claim 106, wherein the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the first additional viral protein; and the sequence encoding the second additional viral protein.

108. The AAV production system of claim 107, wherein the polycistronic RNA comprises, 5’ to 3’: the sequence encoding the viral noncoding RNA; the sequence encoding the firstadditional viral protein; a sequence encoding a viral 2a peptide; and the sequence encoding the second additional viral protein.

109. The AAV production system of any one of claims 105-108, wherein the first additional viral protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26, 96-104, or 120.

110. The AAV production system of any one of claims 105-108, wherein the second additional viral protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26, 96-104, or 120.

111. The AAV production system of any one of claims 105-110, wherein the first additional viral protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120.

112. The AAV production system of any one of claims 105-111, wherein the second additional viral protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 103.

113. The AAV production system of any one of claims 105-112, comprising: a first polynucleotide comprising the sequence encoding for the polycistronic RNA; a second polynucleotide comprising a sequence of encoding for: Rep52 or Rep40;VP1; VP2; VP3; E2A; and E40rf6; and a third polynucleotide comprising a sequence encoding for: Rep78 or Rep68; VP1; VP2; VP3; E2A; and E40rf6.

114. The AAV production system of any one of claims 1-113, wherein the AAV production system does not encode Rep68.

115. An engineered cell for Adeno- Associated Virus (AAV) production, comprising an AAV production system of any one of claims 1-114.

116. The engineered cell of claim 115, wherein one or more polynucleotides of the AAV production system are stably integrated into the genome of the engineered cell.

117. The engineered cell of claim 115, wherein the engineered cell comprises:(i) a first plasmid comprising a nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3;(ii) a second plasmid comprising a second polynucleotide comprising a nucleotide sequence encoding for E2A, optionally wherein the second nucleotide further comprises a nucleotide sequence encoding for E40rf6 and / or VARNA; and(iii) a third plasmid comprising a nucleotide sequence encoding the additional viral protein.

118. An engineered cell for Adeno- Associated Virus (AAV) production, comprising:(i) a genomic integration of a first nucleotide sequence encoding for: Rep52 or Rep40; Rep78 or Rep68; VP1; VP2; and VP3; optionally wherein the first nucleotide sequence encodes for Rep52, Rep40, Rep78, Rep68, VP1, VP2, and VP3;(ii) a genomic integration of a second nucleotide sequence encoding for E2A, optionally wherein the second nucleotide sequence further encodes for E40rf6 and / or VARNA; and(iii) a genomic integration of a third nucleotide sequence encoding for an additional viral protein, optionally wherein the additional viral protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-26, 96-104, or 120.

119. A kit comprising the Adeno-Associated Virus (AAV) production system of any one of claims 1-114 or the engineered cell of any one of claims 115-118.

120. The kit of claim 119, comprising a transfer plasmid, wherein the transfer plasmid comprises, from 5’ to 3’: (i) a nucleotide sequence of a 5’ inverted tandem repeat; (ii) a multiple cloning site; and (iii) a nucleotide sequence of a 3’ inverted tandem repeat.

121. A method for Adeno-Associated Virus (AAV) production, comprising culturing the engineered cell of any one of claims 115-118 such that it expresses: Rep52 or Rep40; Rep78 or Rep68; E2A; VP1; VP2; VP3; and the additional viral protein.

122. The method of claim 121, comprising culturing the engineered cell such that it expresses: Rep52; Rep40; Rep78; Rep68; VARNA; VP1; VP2; VP3; and the additional viral protein.

123. A method for generating an engineered cell for Adeno-Associated Virus (AAV) production, comprising introducing the AAV production system of any one of claims 1-116 into the cell.

124. The method of claim 123, where the method comprises integrating a polynucleotide of the AAV production system into the genome of the cell.