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WO2025160262A1 - Systèmes de production d'aav à efficacité accrue en matière d'encapsulation - Google Patents

Systèmes de production d'aav à efficacité accrue en matière d'encapsulation

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Publication number
WO2025160262A1
WO2025160262A1 PCT/US2025/012729 US2025012729W WO2025160262A1 WO 2025160262 A1 WO2025160262 A1 WO 2025160262A1 US 2025012729 W US2025012729 W US 2025012729W WO 2025160262 A1 WO2025160262 A1 WO 2025160262A1
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Prior art keywords
amino acid
acid sequence
production system
seq
aav production
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English (en)
Inventor
Jeremy J. GAM
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Asimov Inc
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Asimov Inc
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Publication of WO2025160262A1 publication Critical patent/WO2025160262A1/fr
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Definitions

  • AAV Adeno- Associated Virus
  • 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.
  • 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.
  • engineered cells and kits comprising an AAV production system and methods of using the same for AAV production.
  • 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.
  • AAV Adeno-Associated Virus
  • the one or more polynucleotides of the AAV production system collectively further comprise a nucleotide sequence encoding for E40rf6 and/or VARNA.
  • the additional viral protein is not an AAV protein or an adenoviral El protein.
  • the additional viral protein is capable of generating conditions for liquid-liquid phase separation when expressed in a cell.
  • the additional viral protein comprises one or more intrinsically disordered region(s).
  • 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.
  • 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.
  • the additional viral protein comprises at least one mutation relative to its wildtype amino acid sequence.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a LI 52K protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a IV a2 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a LI pllla protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a L4 100K protein.
  • 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.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an E1A protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an E1B 19K protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding an E1B 55K protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding an E2B protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an E2B pTP protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding an ORF59 protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding a LANA protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding an EBNA2 protein.
  • 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.
  • 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:
  • the AAV production system comprises a polynucleotide encoding an EBNALP protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a ULI 12-UL113 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a UL57 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an ICP4 protein.
  • 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.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an ICP8 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a ULI 1 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an El protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an E2 protein.
  • 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.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an mSK_082 E2 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a UL12 protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an UL12.5 protein.
  • 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.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an NS2 protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleotide sequence encoding NS2 has been modified to remove potential splice sites.
  • 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).
  • 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).
  • the AAV production system comprises a polynucleotide encoding an E6 protein.
  • 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.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an LI 13.6K protein.
  • 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.
  • the AAV production system comprises a polynucleotide encoding a ORF6 protein.
  • 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.
  • the AAV production system comprises a polynucleotide encoding an NP1 protein.
  • 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.
  • 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.
  • 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.
  • the nucleotide sequence encoding NP1 has been modified to remove potential splice sites.
  • 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).
  • 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) .
  • 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.
  • 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.
  • the AAV production system is capable of producing helper virus-free AAV.
  • the one or more polynucleotides of the AAV production system do not encode a viral capsid protein other than VP1, VP2, and VP3.
  • 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.
  • the AAV production system comprises a second polynucleotide comprising a nucleotide sequence encoding for: E2A; E40rf6; and VARNA.
  • the AAV production system comprises a third polynucleotide comprising a nucleotide sequence encoding the additional viral protein.
  • the AAV production system comprises a transfer plasmid.
  • 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.
  • the one or more polynucleotides of the AAV production system comprising one or more
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV production system does not encode Rep68.
  • the disclosure relates to engineered cells for Adeno-Associated Virus (AAV) production, where the engineered cells comprise an AAV production system described herein.
  • AAV Adeno-Associated Virus
  • one or more polynucleotides of the AAV production system are stably integrated into the genome of the engineered cell.
  • 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.
  • 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.
  • kits comprising an Adeno-Associated Virus (AAV) production system or an engineered cell described herein.
  • AAV Adeno-Associated Virus
  • 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.
  • 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.
  • a method comprises culturing the engineered cell such that it expresses: Rep52; Rep40; Rep78; Rep68; E2A; VP1; VP2; VP3; and the additional viral protein.
  • the disclosure relates to methods for generating an engineered cell for Adeno-Associated Virus (AAV) production.
  • a method comprises introducing an AAV production system described herein into the cell.
  • a method comprises integrating a polynucleotide of the AAV production system into the genome of the cell.
  • 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.
  • Ad5 LI 52K Ad5 LI 52K
  • SUPT5H a control
  • FIG. 2 shows that co-expression of AAV production genes with Ad5 LI 52K boosts AAV titers.
  • FIG. 3 shows results from titration experiments using Ad5 LI 52K.
  • Ad5 LI 52K boosts AAV titers in a concentration dependent manner.
  • 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.
  • 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.
  • 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.
  • 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.
  • LPS liquid-liquid phase separation
  • 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.
  • IDRs intrinsically disordered regions
  • 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).
  • 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.
  • the disclosure relates to adeno-associated virus (AAV) production systems.
  • 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.
  • An AAV production system 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.
  • an AAV production system comprises and additional viral protein component.
  • an AAV production system comprises a viral non-coding RNA component.
  • an AAV production system comprises an additional viral protein component and a viral non-coding RNA component.
  • 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).
  • AAV production components 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 include Rep52, Rep40, Rep78, Rep68, E2A, E4ORF6, VARNA, Cap (VP1, VP2, VP3), AAP, L4 100K, and MAAP.
  • 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.
  • an AAV production system encodes Rep78, but not Rep68.
  • an AAV production system encodes Rep40, but not Rep52.
  • 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.
  • 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.
  • a nucleotide sequence encoding an AAV gene product is operably linked to a promoter.
  • 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).
  • constitutive promoters 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.
  • CMV cytomegalovirus
  • EFla elongation factor 1 a
  • SV40 simian vacuolating virus 40
  • UTC ubiquitin-C
  • U6 promoters U6 promoters
  • p5 promoters p5 promoters
  • pl9 promoters p40 promoters
  • E2A promoters E2A promoters
  • E4 promoters phosphoglycerate kinase
  • 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.
  • inducible promoters 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.
  • an AAV production component comprises a nucleotide sequence encoding a Rep52 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a Rep40 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a Rep78 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a Rep68 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding an E2A protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding an E4ORF6 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a VARNA.
  • 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.
  • an AAV production component comprises a nucleotide sequence encoding a VP1 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a VP2 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a VP3 protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding an AAP protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a L4 100K protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component comprises a nucleotide sequence encoding a MAAP protein.
  • 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.
  • 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.
  • 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).
  • an AAV production component is (i.e., the gene products of the AAV component are) encoded on a single polynucleotide.
  • 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).
  • 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.
  • an AAV production component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 polynucleotides.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV production component comprises a polynucleotide encoding L4 100K.
  • the one or more polynucleotides of the AAV production system do not encode a viral capsid protein other than VP1, VP2, and VP3.
  • 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.
  • an AAV production system described herein further comprises a transfer polynucleic acid molecule comprising a recombinant AAV genome.
  • the AAV production systems described herein may comprise an additional viral protein component.
  • 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).
  • sequence encoding the additional viral protein may be operably linked to a promoter (as described herein).
  • 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).
  • 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.
  • 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.
  • the additional viral protein is capable of generating conditions for liquid-liquid phase separation (e.g., when expressed in sufficient amounts in a cell).
  • 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.
  • 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.
  • the additional viral protein comprises one or more intrinsically disordered region(s).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the additional viral protein comprises at least one mutation relative to its wildtype amino acid sequence.
  • 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.
  • the additional viral protein is an adenoviral protein.
  • adenoviral protein is an Ad2 protein, and Ad4 protein, or an Ad5 protein.
  • the additional viral protein is a Kaposi's sarcoma-associated herpesvirus (KSHV) protein.
  • KSHV Kaposi's sarcoma-associated herpesvirus
  • the additional viral protein is an Epstein-Barr virus (EBV) protein.
  • EBV Epstein-Barr virus
  • the additional viral protein is a human cytomegalovirus (MCMV) protein.
  • MCMV human cytomegalovirus
  • the additional viral protein is a herpes simplex virus- 1 (HSV- 1) protein.
  • HSV- 1 herpes simplex virus- 1
  • the additional viral protein is a human papillomavirus (HPV) protein.
  • HPV human papillomavirus
  • the additional viral protein is a LI 52K protein.
  • 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.
  • 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.
  • the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 30.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 1.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 2.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 31.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 3.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 4.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 32.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 5.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 6.
  • 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.
  • 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.
  • 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.
  • the LI 52K protein comprises the amino acid sequence of SEQ ID NO: 7.
  • 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.
  • the additional viral protein is a IVa2 protein.
  • 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.
  • 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.
  • the IVa2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 33.
  • 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.
  • 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.
  • the IVa2 protein comprises the amino acid sequence of SEQ ID NO: 8.
  • 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.
  • the additional viral protein is a LI pllla protein.
  • 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.
  • 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.
  • the LI pllla protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 34.
  • 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.
  • 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.
  • the LI pllla protein comprises the amino acid sequence of SEQ ID NO: 9.
  • 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.
  • the additional viral protein is a L4 100K protein.
  • 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.
  • 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.
  • the L4 100K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the L4 100K protein comprises the amino acid sequence of SEQ ID NO: 10.
  • 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.
  • the additional viral protein is an E1A protein.
  • 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.
  • 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.
  • the El A protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 37.
  • 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.
  • 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.
  • the El A protein comprises the amino acid sequence of SEQ ID NO: 11.
  • 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.
  • the additional viral protein is an E1B 19K protein.
  • 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.
  • 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.
  • the E1B 19K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 38.
  • 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.
  • 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.
  • the E1B 19K protein comprises the amino acid sequence of SEQ ID NO: 12.
  • 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.
  • the additional viral protein is an E1B 55K protein.
  • 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.
  • 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.
  • the E1B 55K protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 39.
  • 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.
  • 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.
  • the E1B 55K protein comprises the amino acid sequence of SEQ ID NO: 13.
  • 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.
  • the additional viral protein is an E2B protein.
  • 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.
  • 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.
  • the E2B protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 40.
  • 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.
  • 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.
  • the E2B protein comprises the amino acid sequence of SEQ ID NO: 14.
  • 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.
  • the additional viral protein is an E2B pTP protein.
  • 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.
  • 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.
  • the E2B pTP protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 41.
  • 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.
  • 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.
  • the E2B pTP protein comprises the amino acid sequence of SEQ ID NO: 15.
  • 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.
  • the additional viral protein is a ORF59 protein.
  • 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.
  • 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.
  • the ORF59 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 42.
  • 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.
  • 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.
  • the additional viral protein is a LANA protein.
  • 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.
  • 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.
  • the LANA protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 43.
  • 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.
  • 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.
  • the LANA protein comprises the amino acid sequence of SEQ ID NO: 17.
  • 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.
  • the additional viral protein is an EBNA2 protein.
  • 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.
  • 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.
  • the EBNA2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 44.
  • 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.
  • 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.
  • the EBNA2 protein comprises the amino acid sequence of SEQ ID NO: 18.
  • 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.
  • the additional viral protein is an EBNALP protein.
  • 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.
  • 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.
  • the EBNALP protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 45.
  • 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.
  • 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.
  • the EBNALP protein comprises the amino acid sequence of SEQ ID NO: 19.
  • 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.
  • the additional viral protein is a ULI 12-UL113 protein.
  • 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.
  • 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.
  • the ULI 12-UL113 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 46.
  • 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.
  • 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.
  • the ULI 12- UL113 protein comprises the amino acid sequence of SEQ ID NO: 20.
  • 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.
  • the additional viral protein is a UL57 protein.
  • 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.
  • 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.
  • the UL57 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 47.
  • 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.
  • 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.
  • the UL57 protein comprises the amino acid sequence of SEQ ID NO: 21.
  • 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.
  • the additional viral protein is an ICP4 protein.
  • 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.
  • 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.
  • the ICP4 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ICP4 protein comprises the amino acid sequence of SEQ ID NO: 22.
  • 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.
  • the additional viral protein is an ICP8 protein.
  • 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.
  • 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.
  • the ICP8 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 50.
  • 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.
  • 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.
  • the ICP8 protein comprises the amino acid sequence of SEQ ID NO: 23.
  • 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.
  • 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.
  • 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.
  • the ULI 1 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 51.
  • the additional viral protein is an El protein.
  • 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.
  • 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.
  • the El protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 52.
  • 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.
  • 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.
  • the El protein comprises the amino acid sequence of SEQ ID NO: 25.
  • 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.
  • the additional viral protein is an E2 protein.
  • 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.
  • 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.
  • the E2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 53.
  • 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.
  • 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.
  • the E2 protein comprises the amino acid sequence of SEQ ID NO: 26.
  • 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.
  • 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.
  • 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.
  • 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.
  • the E2 protein comprises the amino acid sequence of SEQ ID NO: 96.
  • 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.
  • the additional viral protein is an mSK_082 E2 protein.
  • 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.
  • 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.
  • the mSK_082 E2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 106.
  • 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.
  • 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.
  • the mSK_082 E2 protein comprises the amino acid sequence of SEQ ID NO: 97.
  • 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.
  • the additional viral protein is a UL12 protein.
  • 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.
  • 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.
  • the UL12 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108.
  • 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.
  • 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: 108.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the UL12 protein comprises the amino acid sequence of SEQ ID NO: 98.
  • 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.
  • 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.
  • 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.
  • the UL12 protein comprises the amino acid sequence of SEQ ID NO: 99.
  • 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.
  • the additional viral protein is a UL12.5 protein.
  • 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.
  • 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.
  • the UL12.5 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 108.
  • 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.
  • 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.
  • the ULI 2.5 protein comprises the amino acid sequence of SEQ ID NO: 100.
  • 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.
  • 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.
  • 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.
  • the ULI 2.5 protein comprises the amino acid sequence of SEQ ID NO: 101.
  • 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.
  • the additional viral protein is an NS2 protein.
  • 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.
  • 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.
  • the NS2 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 109.
  • 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.
  • 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.
  • the NS2 protein comprises the amino acid sequence of SEQ ID NO: 102.
  • 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.
  • 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.
  • the NS2 protein comprises the amino acid sequence of SEQ ID NO: 103.
  • 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.
  • 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.
  • 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).
  • 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).
  • the additional viral protein is an E6 protein.
  • 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.
  • 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.
  • the E6 protein comprises the amino acid sequence of SEQ ID NO: 27.
  • 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.
  • the nucleotide sequence encoding the E6 protein comprises the sequence of SEQ ID NO: 80.
  • 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.
  • 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.
  • the E6 protein comprises the amino acid sequence of SEQ ID NO: 104.
  • 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.
  • the additional viral protein is a LI 13.6K protein.
  • 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.
  • 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.
  • the LI 13.6K protein comprises the amino acid sequence of SEQ ID NO: 28.
  • 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.
  • the additional viral protein is an ORF6 protein.
  • 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.
  • 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.
  • the ORF6 protein comprises the amino acid sequence of SEQ ID NO: 29.
  • 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.
  • the additional viral protein is an NP1 protein.
  • 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.
  • 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.
  • the NP1 protein comprises an intrinsically disordered region comprising the amino acid sequence of SEQ ID NO: 123.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits for AAV production 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • the disclosure relates to a method of generating an engineered cell for AAV production.
  • 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.
  • the method comprising integrating a polynucleotide of the AAV production system into the genome of the cell.
  • 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.
  • AAV uses adenovirus to provide helper function to replicate and package its own genome
  • AAV may also use adenovirus LLPS to better coordinate with adenoviral helper factors.
  • LLPS 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.
  • 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).
  • Ad5 LI 52K 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).
  • 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.
  • 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.
  • UL12, UL12.5, Bocavirus NS2, and KSHV LANA show boosted AAV titers.
  • BocaSR • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • IDR-containing proteins 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.
  • inventive 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.
  • 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.
  • 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.
  • “at least one of A and 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.

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Abstract

La présente invention porte sur des cellules génétiquement ingénierisées pour la production d'AAV. La cellule génétiquement ingénierisée comporte des systèmes moléculaires pour la régulation temporelle de l'expression des gènes nécessaires à la production d'AAV. La présente invention porte également sur des procédés d'utilisation de cellules génétiquement ingénierées pour la production d'AAV.
PCT/US2025/012729 2024-01-23 2025-01-23 Systèmes de production d'aav à efficacité accrue en matière d'encapsulation Pending WO2025160262A1 (fr)

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WO2023086822A2 (fr) * 2021-11-09 2023-05-19 Asimov Inc. Systèmes de production stables pour la production de vecteurs de vaa
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US20220154213A1 (en) * 2017-01-13 2022-05-19 University Of Iowa Research Foundation Bocaparvovirus small noncoding rna and uses thereof
WO2020023612A1 (fr) * 2018-07-24 2020-01-30 Voyager Therapeutics, Inc. Systèmes et méthodes de production de formulations de thérapie génique
WO2023086822A2 (fr) * 2021-11-09 2023-05-19 Asimov Inc. Systèmes de production stables pour la production de vecteurs de vaa
WO2023250416A2 (fr) * 2022-06-22 2023-12-28 Asklepios Biopharmaceutical, Inc. Acides nucléiques à base d'adénovirus et procédés associés

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