Stuffer sequences

Abstract

The technology described herein relates generally to recombinant AAV preparation and production.

Description

The techniques described in this article typically involve the preparation and production of recombinant AAV. Abstract

Claims

CLAIMS What is claimed is:

1. A nucleic acid comprising a short stuffer sequence, wherein the short stuffer sequence comprises a nucleotide sequence having at least 85% identity to: (i) a nucleotide sequence of any one of SEQ ID NOs: 1-6 or a nucleotide sequence complementary to any one of SEQ ID NO: 1-6; or (ii) a nucleotide sequence of a contiguous fragment of from about 250 nucleotides to about 1500 nucleotides of any one of SEQ ID NO: 7-9, 81 or a nucleotide sequence complementary to a contiguous fragment of from about 250 nucleotides to about 1500 nucleotides of any one of SEQ ID NO: 7-9, 81.

2. The nucleic acid of claim 1, wherein the nucleic acid is linear DNA.

3. The nucleic acid of claim 1 or 2, wherein the nucleic acid is a closed ended linear duplexed DNA (clDNA).

4. The nucleic acid of any one of claims 1-3, wherein the nucleic acid further comprises at least one protelomerase binding site.

5. The nucleic acid of claim 4, wherein the short stuffer is located downstream of the protelomerase binding site.

6. The nucleic acid of claim 4, wherein the short stuffer is located upstream of the protelomerase binding site.

7. The nucleic acid of claim 4, wherein the nucleic acid comprises two protelomerase binding sites and the short stuffer is located between the two protelomerase binding sites.

8. The nucleic acid of any one of claims 1-7, wherein the nucleic acid further comprises a heterologous transgene operably linked to one or more regulatory elements.

9. The nucleic acid of claim 8, wherein the short stuffer is located upstream of the heterologous transgene.

10. The nucleic acid of claim 9, wherein the short stuffer is located downstream of the heterologous transgene.

11. The nucleic acid of any one of claims 8-10 wherein the nucleic acid further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

12. The nucleic acid of claim 11, wherein the short stuffer is upstream of the at least one ITR sequence.

13. The nucleic acid of claim 11, wherein the short stuffer is downstream of the at least one ITR sequence.

14. The nucleic acid of any one of claims 11-13, wherein the at least one ITR sequence is located between the short stuffer and the heterologous transgene.

15. The nucleic acid of any one of claims 10-13, wherein the nucleic acid further comprises at least one protelomerase binding site and the short stuffer is located between the at least one protelomerase binding site and the at least ITR.

16. The nucleic acid of any one of claims 11-15, wherein the nucleic acid comprises at least two ITRs and the short stuffer located outside the two ITRs.

17. The nucleic acid of claim 16, wherein the heterologous transgene is located between the two ITRs.

18. The nucleic acid of claims 16, wherein the heterologous transgene is located between the two ITRs, and one of the ITRs is located between the short stuffer and the heterologous transgene.

19. The nucleic acid of claim 16, wherein the nucleic acid comprises a first ITR (e.g., left ITR) sequence and a second ITR (e.g., right ITR) sequence, wherein the heterologous transgene sequence is located between the first and second ITR sequences, and wherein the short stuffer is upstream of the first ITR sequence.

20. The nucleic acid of claim 16, wherein the nucleic acid comprises a first ITR (e.g., left ITR) sequence and a second ITR (e.g., right ITR) sequence, wherein the heterologous polynucleotide sequence is located between first and second ITR sequences, wherein the short stuffer is upstream of the first ITR sequence, and wherein the nucleic acid does not comprise a short stuffer downstream of the second ITR sequence.

21. The nucleic acid of claim 16, wherein the nucleic acid comprises a first ITR (e.g., left ITR) sequence and a second ITR (e.g., right ITR) sequence, wherein the short stuffer is located upstream of the 5’-end of the first and second ITRs.

22. The nucleic acid of any one of claims 7-21, wherein each ITR sequence is selected independently from an ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

23. The nucleic acid of claim 22, wherein the ITR sequences are from the same AAV serotype.

24. The nucleic acid of claim 22, wherein the ITR sequences are from the different AAV serotype.

25. The nucleic acid of any one of claims 1-7, wherein the nucleic acid comprises a nucleic acid sequence encoding one or more helper proteins that assist AAV replication in rAAV production.

26. The nucleic acid of claim 25, wherein the nucleic acid comprises at least one protelomerase binding site and wherein the short stuffer is located between the protelomerase binding site and the nucleic acid sequence encoding one or more helper proteins.

27. The nucleic acid of claim 26, wherein the short stuffer is upstream of the 5’-end of the nucleic acid sequence encoding one or more helper proteins.

28. The nucleic acid of claim 26, wherein the short stuffer is downstream of the 3’-end of the nucleic acid sequence encoding one or more helper proteins.

29. The nucleic acid of any one of claims 25-28, wherein the helper protein sufficient for AAV replication comprises one or more of an E2A region, an E4 region, and a virus associated (VA) RNA region, and optionally, an E1 region, an E3 region and / or a Major Late Promoter (MLP) region.

30. The nucleic acid of any one of claims 25-29, wherein the nucleic acid sequence encoding one or more helper proteins comprises the nucleotide sequence of any one of SEQ ID NOs: 67-70.

31. The nucleic acid of any one of claims 1-7, wherein the nucleic acid further comprises nucleic acid sequence encoding a AAV rep protein and a AAV cap protein.

32. The nucleic acid of claim 31, wherein the nucleic acid comprises at least one protelomerase binding site and wherein the short stuffer is located between the protelomerase binding site and the nucleic acid sequence encoding the AAV rep and AAV cap proteins.

33. The nucleic acid of claim 31, wherein the short stuffer is upstream of the 5’-end of the nucleic acid sequence encoding the AAV rep and AAV cap proteins, and the short stuffer is located between the protelomerase binding site and the nucleic acid sequence encoding the AAV rep and AAV cap proteins.

34. The nucleic acid of claim 31, wherein the short stuffer is downstream of the 3’-end of the nucleic acid sequence encoding the AAV rep and AAV cap proteins, and the short stuffer is located between the protelomerase binding site and the nucleic acid sequence encoding the AAV rep and AAV cap proteins.

35. The nucleic acid of any one of claims 31-34, wherein AAV Rep protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAVAnc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

36. The nucleic acid of any one of claims 31-35, wherein AAV Cap protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

37. The nucleic acid of any one of claims 31-36, wherein AAV Rep protein and the AAV Cap protein are from the same AAV serotype.

38. The nucleic acid of any one of claims 31-36, wherein AAV Rep protein and the AAV Cap protein are from different AAV serotypes.

39. The nucleic acid of any one of claims 1-6, wherein the nucleic acid comprises an ITR sequence and a protelomerase binding upstream of the ITR sequence, wherein the nucleic acid comprises the sequence of SEQ ID NO: 1 or 4 between the ITR and the protelomerase binding site.

40. The nucleic acid of any one of claims 1-6, wherein the nucleic acid comprises an ITR sequence and a protelomerase binding downstream of the ITR sequence, wherein the nucleic acid comprises the sequence of SEQ ID NO: 1 or 4 between the ITR and the protelomerase binding site.

41. The nucleic acid of any one of claims 1-40, wherein the nucleic acid further comprises a stop codon (e.g., TAA, TAG or TGA) upstream of the short stuffer.

42. The nucleic acid of any one of claims 1-40, wherein the nucleic acid further comprises a stop codon (e.g., TAA, TAG or TGA) downstream of the short stuffer.

43. The nucleic acid of any one of claims 1-42, wherein the nucleic acid is a vector.

44. The nucleic acid of any one of claims 1-43, wherein the nucleic acid is a plasmid.

45. A host cell comprising the nucleic acid of any one of claims 1-44.

46. The host cell of claim 45, wherein the host cell is an insect cell or a mammalian cell.

47. The host cell of claim 46, wherein the mammalian cell is a HEK293 cell or a HeLa cell.

48. Use of a nucleic acid of any one of claims 1-44 in a method of producing a plurality of viral particles.

49. Use of claim 48, wherein the viral particles are recombinant AAV (rAAV) particles.

50. A method for producing a plurality of viral particles, the method comprising culturing a host cell of claim 46 or 47 in a culture medium under conditions in which viral particles are produced.

51. A method for producing a plurality of viral particles, the method comprises culturing a host cell comprising a nucleic acid of any one of claims 1-44 in a culture medium under conditions in which viral particles are produced.

52. The method of claim 50 or 51, wherein the host cell comprises at least one nucleic acid encoding one or more helper proteins sufficient for AAV replication, at least one nucleic acid encoding AAV rep and AAV cap proteins, and at least one nucleic acid encoding a transgene of interest.

53. The method of any one of claims 50-52, wherein the host cell comprises at least one nucleic acid of any one of claims 7-24.

54. The method of any one of claims 50-53, wherein the host cell comprises at least one nucleic acid of any one of claims 25-30.

55. The method of any one of claim 50-54, wherein the host cell comprises at least one nucleic acid of any one of claims 31-38.

56. The method of any one of claims 50-55, wherein the host cell comprises at least one nucleic acid of any one of claims 7-24, at least one nucleic acid of any one of claims 25- 30, and at least one nucleic acid of any one of claims 31-38.

57. The method of any one of claims 50-56, wherein the plurality of viral particles comprises rAAV particles.

58. A nucleic acid comprising a large stuffer, wherein the large stuffer comprises a nucleotide sequence having at least 85% identity to a nucleotide sequence of any one of SEQ ID NOs: 7-9 or 81.

59. The nucleic acid of claim 58, wherein the nucleic acid further comprises a nucleic acid sequence encoding an AAV Rep protein operably linked to a promoter.

60. The nucleic acid of claim 59, wherein the large stuffer is located in the nucleic acid sequence encoding the AAV Rep protein.

61. The nucleic acid of claim 60, wherein the large stuffer is located in an intron in the nucleic acid encoding the AAV Rep protein.

62. The nucleic acid of any one of claims 59-61, wherein the large stuffer is located upstream of the promoter.

63. The nucleic acid of any one of claims 59-61, wherein the large stuffer is located downstream of the promoter.

64. The nucleic acid of any one of claims 59-63, wherein the promoter is a p19 promoter.

65. The nucleic acid of any one of claims 59-64, wherein the AAV Rep is large Rep.

66. The nucleic acid of any one of claims 59-65, wherein the AAV Rep is Rep68 or Rep78.

67. The nucleic acid of any one of claims 59-66, wherein the AAV Rep is Rep68.

68. The nucleic acid of any one of claims 59-67, wherein the AAV Rep protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

69. The nucleic acid of any one of claims 59-68, wherein the AAV Rep protein is a modified Rep protein.

70. The nucleic acid of any one of claims 59-69, wherein the nucleic acid further comprises a nucleic acid sequence encoding an AAV Cap protein.

71. The nucleic acid of claim 70, wherein the nucleic acid sequence encoding the AAV Cap protein is downstream of the nucleic acid sequence encoding the AAV Rep protein.

72. The nucleic acid of claim 70, wherein the nucleic acid sequence encoding the AAV Cap protein is upstream of the nucleic acid sequence encoding the AAV Rep protein.

73. The nucleic acid of any one of claims 70-72, wherein the nucleic acid sequence encoding the AAV Cap protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9- PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4A and / or any chimeras thereof.

74. The nucleic acid of any one of claims 70-73, wherein AAV Rep protein and the AAV Cap protein are from the same AAV serotype.

75. The nucleic acid of any one of claims 70-73, wherein AAV Rep protein and the AAV Cap protein are from different AAV serotypes.

76. The nucleic acid of any one of claims 70-75, wherein the nucleic acid sequence encoding the AAV Rep protein is upstream of the nucleic acid sequence encoding the AAV Cap protein.

77. The nucleic acid of any one of claims 70-75, wherein the nucleic acid sequence encoding the AAV Rep protein is downstream of the nucleic acid sequence encoding the AAV Cap protein.

78. The nucleic acid of any one of 58-77, wherein the large stuffer does not comprise a nucleotide sequence of mammalian origin.

79. The nucleic acid of any one of 58-78, wherein the large stuffer comprises a nucleotide sequence of non-mammalian origin.

80. The nucleic acid of any one of 58-79, wherein the large stuffer is synthetic.

81. The nucleic acid of any one of 58-80, wherein the large stuffer does not comprise more than one of the following: a. a transcription factor binding site; b. a regulatory element; c. an AAV Rep binding site; d. a donor or acceptor splicing site; e. an endonuclease cleavage site, optionally where the endonuclease is ApaLI, BamHI, ClaI, DrdI, FspI, RsrII, XbaI, NcoI, SacII, CsiI, AflII, or PacI; f. a repetitive or palindrome sequence longer than 5 nucleotides; g. a strong secondary structure; and / or h. a repetitive or palindrome sequence, optionally a repetitive or palindrome sequence longer than 5 nucleotides.

82. The nucleic acid of any one of claims 58-81, wherein the large stuffer comprises a GC content of less than about 50%, e.g., less than about 45%, or less than about 40%.

83. The nucleic acid of any one of claim 58-82, wherein the nucleic acid is larger than 5.5kb.

84. The nucleic acid of any one of claims 58-83, wherein the nucleic acid comprises the sequence MAG, where M is A or C, upstream of the large stuffer.

85. The nucleic acid of any one of claims 58-84, wherein the nucleic acid is a vector.

86. The nucleic acid of any one of claims 58-85, wherein the nucleic acid is a plasmid.

87. The nucleic acid of any one of claims 58-86, wherein the nucleic acid is linear DNA.

88. The nucleic acid of any one of claims 58-87, wherein the nucleic acid is closed ended linear duplexed DNA (clDNA).

89. A host cell comprising a nucleic acid of any one of claims 58-88.

90. The host cell of claim 89, wherein the host cell is an insect cell or a mammalian cell, optionally the mammalian cell is a HEK293 cell or a HeLa cell.

91. Use of a nucleic acid of any one of claims 59-88 or a host cell of claim 89 or 90 in a method of preventing manufacturing of a replication competent rAAV.

92. A nucleic acid comprising a nucleotide sequence having a having at least 85% identity to SEQ ID NO: 10 and a large stuffer of a size at least 2 kb.

93. The nucleic acid of claim 92, wherein the nucleic acid comprises one or more nucleotides between positions 44 and 45 of the nucleotide sequence having a having at least 85% identity to SEQ ID NO:

10.

94. The nucleic acid of claim 93, wherein the nucleic acid comprises from about 10 to about 10,000 nucleotides between positions 44 and 45 of the nucleotide sequence having a having at least 85% identity to SEQ ID NO:

10.

95. The nucleic acid of claim 94, wherein the nucleic acid comprises from about 2,000 to about 5,000 nucleotides between positions 44 and 45 of the nucleotide sequence having a having at least 85% identity to SEQ ID NO:

10.

96. The nucleic acid of any one of claims 92-95, wherein the large stuffer is located between positions 44 and 45 of the nucleotide sequence having a having at least 85% identity to SEQ ID NO:

10.

97. The nucleic acid of any one of claims 92-96, wherein the large stuffer comprises a nucleotide sequence having at least 85% identity to a nucleotide sequence of any one of SEQ ID NO: 7-9 or 81.

98. The nucleic acid of any one of claims 92-97, wherein the nucleic acid further comprises a nucleic acid sequence encoding an AAV Rep protein operably linked to a promoter.

99. The nucleic acid of claim 98, wherein the nucleotide sequence having a having at least 85% identity to SEQ ID NO: 10 is located in the nucleic acid sequence encoding the AAV Rep protein.

100. The nucleic acid of claim 98 or 99, wherein the nucleotide sequence having at least 85% identity to SEQ ID NO: 10 is located in an intron in the nucleic acid sequence encoding an AAV Rep protein.

101. The nucleic acid of any one of claims 98-100, wherein the nucleotide sequence having at least 85% identity to SEQ ID NO: 10 is upstream of the promoter.

102. The nucleic acid of any one of claims 98-100, wherein the nucleotide sequence having at least 85% identity to SEQ ID NO: 10 is downstream of the promoter.

103. The nucleic acid of any one of claims 98-102, wherein the promoter is a p19 promoter.

104. The nucleic acid of any one of claims 98-103, wherein the AAV Rep is large Rep (Rep68).

105. The nucleic acid of any one of claims 98-104, wherein the AAV Rep protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

106. The nucleic acid of any one of claims 98-105, wherein the AAV Rep protein is a modified Rep protein.

107. The nucleic acid of any one of claims 98-106, wherein the nucleic acid further comprises a nucleic acid sequence encoding an AAV Cap protein.

108. The nucleic acid of claim 107, wherein the nucleic acid sequence encoding the AAV Cap protein is downstream of the nucleic acid sequence encoding the AAV Rep protein.

109. The nucleic acid of claim 107, wherein the nucleic acid sequence encoding the AAV Cap protein is upstream of the nucleic acid sequence encoding the AAV Rep protein.

110. The nucleic acid of any one of claims 107-109, wherein the nucleic acid sequence encoding the AAV Cap protein is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-ePHP.B, AAV LK03, AAV Anc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV23xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5i82g9, AAV2.5, AAVr10pLDB_L2, AAVr10pLDB_P31, AAV4E, and AAV4Aand / or any chimeras thereof.

111. The nucleic acid of any one of claims 107-110, wherein AAV Rep protein and the AAV Cap protein are from the same AAV serotype.

112. The nucleic acid of any one of claims 107-110, wherein AAV Rep protein and the AAV Cap protein are from different AAV serotypes.

113. The nucleic acid of any one of claims 92-112, which is larger than 5.5 kb.

114. The nucleic acid of any one of 92-113, wherein the large stuffer does not comprise a nucleotide sequence of mammalian origin.

115. The nucleic acid of any one of 92-114 wherein the large stuffer comprises a nucleotide sequence of non-mammalian origin.

116. The nucleic acid of any one of 92-115, wherein the large stuffer is synthetic.

117. The nucleic acid of any one of 92-116, wherein the large stuffer does not comprise more than one of the following: a. a transcription factor binding site; b. a regulatory element; c. an AAV Rep binding site; d. a donor or acceptor splicing site; e. an endonuclease cleavage site, optionally where the endonuclease is ApaLI, BamHI, ClaI, DrdI, FspI, RsrII, XbaI, NcoI, SacII, CsiI, AflII, or PacI; f. a repetitive or palindrome sequence longer than 5 nucleotides; g. a strong secondary structure; and / or h. a repetitive or palindrome sequence, optionally a repetitive or palindrome sequence longer than 5 nucleotides.

118. The nucleic acid of any one of claims 92-117, wherein the large stuffer comprises a GC content of less than about 50%, e.g., less than about 45%, or less than about 40%.

119. The nucleic acid of any one of claim 92-118, wherein the nucleic acid is larger than 5.5kb.

120. The nucleic acid of any one of claims 92-119, wherein the 5’-end of the nucleotide sequence having at least 85% identity to SEQ ID NO: 10 is linked to the sequence MAG, where M is A or C.

121. The nucleic acid of any one of claims 92-120, wherein the 3’-end of the nucleotide sequence having at least 85% identity to SEQ ID NO: 10 is linked to A or G.

122. The nucleic acid of any one of claims 92-121, wherein the large stuffer is located downstream of the sequence MAG, where M is A or C.

123. The nucleic acid of any one of claims 92-122, wherein the nucleic acid comprises a nucleotide sequence having at least 85% identity to a nucleotide sequence of any one of SEQ ID NOs: 11-13.

124. The nucleic acid of claim 123, wherein 5’-end of the nucleotide sequence having at least 85% identity to any of SEQ ID NOs: 11-13 is linked to the sequence MAG, where M is A or C.

125. The nucleic acid of claim 123 or 124, wherein the 3’-end of the nucleotide sequence having at least 85% identity to any of SEQ ID NOs: 11-13 is linked to A or G.

126. The nucleic acid of any one of claims 92-125, wherein the nucleic acid comprises a nucleotide sequence having at least 85% identity to SEQ ID NO:

11.

127. The nucleic acid of any one of claims 92-126, wherein the nucleic acid prevents production of replication competent rAAV vector.

128. The nucleic acid of any one of claims 92-127, wherein the nucleic acid is a vector.

129. The nucleic acid of any one of claims 92-128, wherein the nucleic acid is a plasmid.

130. The nucleic acid of any one of claims 92-129, wherein the nucleic acid is linear DNA.

131. The nucleic acid of any one of claims 92-130, wherein the nucleic acid is closed ended linear duplexed DNA (clDNA).

132. A host cell comprising a nucleic acid of any one of claims 92-131.

133. The host cell of claim 132, wherein the host cell is an insect cell or a mammalian cell, ptionally the mammalian cell s a HEK293 cell or a HeLa cell.

134. Use of a nucleic acid of any one of claims 92-131 or a host cell of claim 132or 133 in a method of preventing manufacturing of a replication competent rAAV.

135. A method of detecting residual DNA in a population of viral particles, the method comprising using at least one oligonucleotide sequence that anneals to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:

6.

136. The method of claim 135, the method comprises using at least two oligonucleotide sequences that anneal to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:

6.

137. The method of claim 136, wherein the oligonucleotide is about 20 nucleotides long.

138. The method of claim136, wherein the oligonucleotide is about 25 nucleotides long.

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