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TW202309066A - Porcine-derived adeno-associated virus capsids and uses thereof - Google Patents

Porcine-derived adeno-associated virus capsids and uses thereof Download PDF

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TW202309066A
TW202309066A TW111116002A TW111116002A TW202309066A TW 202309066 A TW202309066 A TW 202309066A TW 111116002 A TW111116002 A TW 111116002A TW 111116002 A TW111116002 A TW 111116002A TW 202309066 A TW202309066 A TW 202309066A
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王強
詹姆士Ｍ 威爾森
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賓州大學委員會
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Abstract

Novel porcine-derived adeno-associated virus (AAV) capsids and recombinant AAV vectors comprising the same are provided. Also, provide are methods for delivery of a transgene using the recombinant AAV vectors described herein.

Description

衍生自豬的腺相關病毒衣殼及其用途Adeno-associated virus capsids derived from porcine and uses thereof

本發明係關於衍生自豬的腺相關病毒衣殼及其用途。The present invention relates to porcine-derived adeno-associated virus capsids and uses thereof.

腺相關病毒(AAV)係由於其低免疫原性、非致病性及建立長期表現的能力，而為最有效之用於基因治療的載體候選物。已有報告數十種天然存在的AAV衣殼；它們獨特的衣殼結構使它們能夠辨識及轉導不同的細胞類型及器官。然而，儘管能夠有效的基因轉移，但目前在臨床中使用的AAV載體可因預先存在的對病毒的免疫力及受限制的組織趨性而受到阻礙。Adeno-associated virus (AAV) is the most effective vector candidate for gene therapy due to its low immunogenicity, non-pathogenicity, and ability to establish long-term expression. Dozens of naturally occurring AAV capsids have been reported; their unique capsid structure enables them to recognize and transduce different cell types and organs. However, despite enabling efficient gene transfer, current use of AAV vectors in the clinic can be hampered by pre-existing immunity to the virus and limited tissue tropism.

如此，本領域所需者為用於基因治療的新穎及有效的AAV載體。Thus, what is needed in the art are novel and efficient AAV vectors for gene therapy.

[發明概要][Outline of Invention]

於一態樣，本文提供一種重組腺相關病毒(recombinant adeno-associated virus，rAAV)，其具有包含衣殼蛋白的衣殼並具有包裝於該衣殼中的載體基因體，其中該衣殼蛋白具有下述序列，且該載體基因體包含非AAV核酸序列：AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。In one aspect, this paper provides a recombinant adeno-associated virus (rAAV), which has a capsid comprising a capsid protein and has a vector gene body packaged in the capsid, wherein the capsid protein has The following sequences, and the vector gene body comprises non-AAV nucleic acid sequences: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8) , AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO : 42), AAVpoG024 (SEQ ID NO: 44), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 46); or any one of SEQ ID NO: 2, 4, 6, 8 A sequence sharing at least 98% or at least 99% identity; or sharing at least 96%, at least 97% with any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 , a sequence of at least 98% or at least 99% identity; or at least 90%, at least 95%, at least 96 in common with any of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 %, at least 97%, at least 98%, or at least 99% identical to the sequence.

於一態樣，本文提供一種rAAV，其具有包含衣殼蛋白的衣殼並具有包裝於該衣殼中的載體基因體，其中該衣殼蛋白係由下述序列所編碼，且該載體基因體包含非AAV核酸序列：AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45共有至少70%同一性的序列。於另一態樣，該序列編碼：為AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之序列的vp1、vp2、及/或vp3；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。於再另一態樣，該rAAV具有衣殼蛋白，該衣殼蛋白係由示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的核苷酸序列所編碼。In one aspect, provided herein is a rAAV having a capsid comprising a capsid protein and having a vector gene body packaged in the capsid, wherein the capsid protein is encoded by the following sequence, and the vector gene body Contains non-AAV nucleic acid sequences: AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9) , AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO : 43), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 45); or with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 are sequences that share at least 70% identity. In another aspect, the sequence encoding: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 8), ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20) , AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), vp1, vp2, and/or vp3 of the sequence of AAVpoG024 (SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46); or share at least 98% with any of SEQ ID NO: 2, 4, 6, 8 % or at least 99% identical sequence; or any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 shares at least 96%, at least 97%, at least 98 % or at least 99% identical sequence; or any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 shares at least 90%, at least 95%, at least 96%, at least A sequence that is 97%, at least 98%, or at least 99% identical. In yet another aspect, the rAAV has a capsid protein represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 are encoded by the nucleotide sequence.

於一態樣，提供含有本文所述之rAAV的宿主細胞。亦提供包含rAAV以及生理學上可接受的載劑、緩衝劑、佐劑、及/或稀釋劑的醫藥組成物。In one aspect, a host cell comprising an rAAV described herein is provided. Pharmaceutical compositions comprising rAAV and a physiologically acceptable carrier, buffer, adjuvant, and/or diluent are also provided.

於一態樣，提供一種遞送轉基因至細胞之方法，於該方法包含將該細胞與本文所述rAAV接觸的步驟，且該rAAV包含該轉基因。In one aspect, there is provided a method of delivering a transgene to a cell, the method comprising the step of contacting the cell with an rAAV described herein, the rAAV comprising the transgene.

於一態樣，提供一種生產包含AAV衣殼的rAAV之方法，其中該方法包含培養宿主細胞，該宿主細胞含有：(a)核酸，包含AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之AAV vp1、vp2、及/或vp3序列、或者與示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的vp1、vp2、及/或vp3核苷酸序列共有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列；(b)功能性rep基因；(c)包含AAV反向末端重複序列(ITR)及轉基因的袖珍基因；及(d)充足的輔助功能以允許將該袖珍基因包裝至該AAV衣殼中。於另一態樣，該vp1、vp2、及/或vp3序列編碼：AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。在某些具體實施例中，提供一種組成物，其包含依據此種方法所生產的儲料(stock)。In one aspect, there is provided a method of producing rAAV comprising an AAV capsid, wherein the method comprises culturing a host cell comprising: (a) a nucleic acid comprising AAVpoG001 (SEQ ID NO: 1 ), AAVpoG002 (SEQ ID NO : 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 ( AAV vp1, vp2, and /or the vp3 sequence, or the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, 43, or 45 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of the vp1, vp2, and/or vp3 nucleotide sequences , a sequence of at least 97%, at least 98%, or at least 99% identity; (b) a functional rep gene; (c) a pocket gene comprising an AAV inverted terminal repeat (ITR) and a transgene; and (d) sufficient helper function to allow packaging of the pocket gene into the AAV capsid. In another aspect, the vp1, vp2, and/or vp3 sequence codes: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO : 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 ( The sequence of SEQ ID NO: 42), AAVpoG024 (SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46); or at least 98% in common with any of SEQ ID NO: 2, 4, 6, 8 or A sequence of at least 99% identity; or at least 96%, at least 97%, at least 98% in common with any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or A sequence that is at least 99% identical; or at least 90%, at least 95%, at least 96%, at least 97% in common with any of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 , at least 98%, or at least 99% identical sequences. In certain embodiments, there is provided a composition comprising a stock produced according to such a method.

於一態樣，本文提供一種質體，其包含AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3核苷酸序列；或與示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的vp1、vp2、及/或vp3核苷酸序列共有至少70%同一性的序列。於另一態樣，核苷酸序列編碼：AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。亦提供一種培養的宿主細胞，其含有本文所述的質體。In one aspect, provided herein is a plastid comprising AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 ( SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41 ), AAVpoG024 (SEQ ID NO: 43), or the vp1, vp2, and/or vp3 nucleotide sequence of AAVpoG025 (SEQ ID NO: 45); or shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 vp1, vp2, and/or vp3 nucleotides Sequences that share at least 70% identity. In another aspect, the nucleotide sequence codes: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 ( SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20 ), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42) , AAVpoG024 (SEQ ID NO: 44), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 46); or share at least 98% with any of SEQ ID NO: 2, 4, 6, 8 A sequence of % or at least 99% identity; or at least 96%, at least 97%, at least 98% in common with any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 % or at least 99% identical sequence; or any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 shares at least 90%, at least 95%, at least 96%, at least A sequence that is 97%, at least 98%, or at least 99% identical. Also provided is a cultured host cell comprising a plastid described herein.

由下列本發明之詳細說明將容易明白本發明之其它態樣及優點。 【圖式簡單説明】 Other aspects and advantages of the present invention will be readily apparent from the following detailed description of the present invention. [Simple description of the diagram]

圖1A-圖1G顯示AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、及AAVpoG025 (SEQ ID NO: 45)衣殼蛋白之核苷酸序列的比對。 圖2A-圖2M顯示AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、及AAVpoG025 (SEQ ID NO: 46)衣殼蛋白之胺基酸序列的比對。 圖3顯示從各種豬組織獲得的AAV單離株的頻率。 圖4顯示包括本文所述的新穎豬單離株的系統樹。AAV1-9及AAVpo1、po2.1、po4及po5先前被單離並顯示作為參考。 圖5A及圖5B顯示相對於AAV9而言，具有來自新穎豬單離株的衣殼的病毒顆粒的Huh7之轉導水準。類AAVpo1單離株：001-005；類AAVpo2.1單離株：006、009、012、013、及014；類AAVpo4單離株：015-017；及類AAVpo5單離株：018-025。 圖6顯示相對於AAV8及AAV9而言，AAVpoG013及AAVpoG015載體的生產產率。 圖7顯示IV遞送至小鼠後，具AAVpoG013及AAVpoG015衣殼的載體的轉導效率。 圖8A及圖8B顯示在遞送具AAVpoG015衣殼的重組AAV載體後，載體在非人類靈長類動物中的生物分布。恆河獼猴(rhesus macaque)以5 x 10 13GC/kg之劑量經由靜脈內(IV)注射而接受AAVpoG015.CB7.CI.eGFP.WPRE.RBG。10日後，犧牲動物，並藉由qPCR及RT-qPCR測定組織中的載體基因體拷貝(GC)。結果顯示肝臟、心臟及肌肉中的載體基因體拷貝較高。肝臟L、R、M、C：分別為左、右、中、尾狀葉。對於每個樣品，左欄：GC/µg gDNA，右欄：GC/µg RNA。gDNA：基因體DNA。 圖9顯示在遞送具AAVpoG015衣殼的重組AAV載體後，恆河獼猴中的天冬胺酸轉移酶(AST)及丙胺酸胺基轉移酶(ALT)水準。 圖10A-圖10C顯示在遞送具AAVpoG015衣殼的重組AAV載體後，載體在非人類靈長類動物的生物分布。恆河獼猴以5x10 13GC之劑量經由腦大池內(intra-cisterna magna，ICM)注射而接受AAVpoG015.CB7.CI.eGFP.WPRE.RBG。於15日，犧牲動物，並藉由qPCR及RT-qPCR測定組織中的載體基因體拷貝(GC)。對於每個樣品，左欄：GC/µg gDNA，右欄：GC/µg RNA。gDNA：基因體DNA。 Figures 1A-1G show AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9) , AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO : 43), and the alignment of the nucleotide sequences of AAVpoG025 (SEQ ID NO: 45) capsid protein. Figures 2A-2M show AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10) , AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), AAVpoG024 (SEQ ID NO : 44), and the alignment of the amino acid sequences of the capsid protein of AAVpoG025 (SEQ ID NO: 46). Figure 3 shows the frequency of AAV single isolates obtained from various porcine tissues. Figure 4 shows a phylogenetic tree including the novel porcine isolates described herein. AAV1-9 and AAVpo1, po2.1, po4 and po5 were previously isolated and shown as reference. Figures 5A and 5B show the level of transduction of Huh7 relative to AAV9 with viral particles from capsids of novel porcine isolates. AAVpo1-like isolates: 001-005; AAVpo2.1-like isolates: 006, 009, 012, 013, and 014; AAVpo4-like isolates: 015-017; and AAVpo5-like isolates: 018-025. Figure 6 shows the production yields of AAVpoG013 and AAVpoG015 vectors relative to AAV8 and AAV9. Figure 7 shows the transduction efficiency of vectors with AAVpoG013 and AAVpoG015 capsids after IV delivery to mice. Figures 8A and 8B show the biodistribution of vectors in non-human primates following delivery of recombinant AAV vectors with AAVpoG015 capsids. Rhesus macaques received AAVpoG015.CB7.CI.eGFP.WPRE.RBG via intravenous (IV) injection at a dose of 5 x 1013 GC/kg. Ten days later, animals were sacrificed, and vector gene body copies (GC) in tissues were determined by qPCR and RT-qPCR. The results showed higher copies of the vector gene body in liver, heart and muscle. Liver L, R, M, C: left, right, middle, and caudate lobes, respectively. For each sample, left column: GC/µg gDNA, right column: GC/µg RNA. gDNA: genomic DNA. Figure 9 shows aspartate transferase (AST) and alanine aminotransferase (ALT) levels in rhesus macaques after delivery of recombinant AAV vectors with AAVpoG015 capsids. Figures 10A-10C show vector biodistribution in non-human primates following delivery of recombinant AAV vectors with AAVpoG015 capsids. Rhesus macaques received AAVpoG015.CB7.CI.eGFP.WPRE.RBG via intra-cisterna magna (ICM) injection at a dose of 5x1013 GC. On day 15, animals were sacrificed, and vector gene body copies (GC) in tissues were determined by qPCR and RT-qPCR. For each sample, left column: GC/µg gDNA, right column: GC/µg RNA. gDNA: genomic DNA.

隨著腺相關病毒(AAV)媒介的基因治療擴展而涵蓋更多疾病，對可最適化基因遞送的新穎的AAV衣殼的需求正在上升。有三種獲得新穎的AAV衣殼的主要策略：開發AAV的天然多樣性、定向演化、合理設計，或者此等的組合。本文提供從豬組織中單離的AAV衣殼及包含此等衣殼的rAAV載體。該rAAV有用於遞送基因治療產品，以用於基因編輯、作為疫苗、以及其它適合的用途。As adeno-associated virus (AAV)-mediated gene therapy expands to cover more diseases, the demand for novel AAV capsids that can optimize gene delivery is rising. There are three main strategies for obtaining novel AAV capsids: exploiting the natural diversity of AAV, directed evolution, rational design, or a combination of these. Provided herein are AAV capsids isolated from porcine tissues and rAAV vectors comprising such capsids. The rAAV is useful for the delivery of gene therapy products for gene editing, as a vaccine, and other suitable uses.

除非另有定義，否則本文所用的技術及科學術語具有與本發明所屬領域中具有通常知識者及參照公開文本所通常理解的相同含義，公開文本為本領域中具有通常知識者提供了本申請案中所使用之許多術語的一般指引。提供下列定義僅為了清楚起見，並非意圖限制所請求的發明。Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to this publication which provides the basis for this application to one of ordinary skill in the art. A general guide to many of the terms used in . The following definitions are provided for clarity only and are not intended to limit the claimed invention.

如本文所使用，術語「一」(a或an)係指一或以上，例如，「一質體」應理解為代表一或多個質體。如此，術語「一」(a或an)、「一或以上」及「至少一個(種)」於本文中可互換使用。As used herein, the term "a" (a or an) refers to one or more, for example, "a plastid" should be understood to represent one or more plastids. As such, the terms "a" (a or an), "one or more" and "at least one" are used interchangeably herein.

如本文所使用，術語「約」意指與給定參考值有10%的變異性，除非另有說明。As used herein, the term "about" means a variation of 10% from a given reference value, unless otherwise stated.

儘管說明書中的多個具體實施例使用「包含」語句來呈現，但在其它情況下，相關具體實施例亦意圖使用「由…組成」或「實質上由…組成」語句來解釋及描述。Although various embodiments in the specification are presented using the phrase "comprising", in other cases, related embodiments are also intended to be explained and described using the phrase "consisting of" or "consisting essentially of".

關於下列說明，於另一個具體實施例中，意圖指本文描述的每一組成物可用於本發明的方法。此外，於另一個具體實施例中，亦意圖指於此方法中為有用的本文描述的每一組成物本身為本發明的具體實施例。 A. AAV衣殼 With respect to the following description, in another embodiment, it is intended that each composition described herein can be used in the method of the present invention. Furthermore, in another embodiment, it is also intended that each composition described herein that is useful in this method is itself an embodiment of the invention. A. AAV capsid

編碼AAV衣殼的核酸包括三種重疊的編碼序列，由於替代起始密碼子的使用，其長度會變化。轉譯的蛋白質被稱為VP1、VP2及VP3，其中VP1最長且VP3最短。AAV顆粒係由所有三種衣殼蛋白所構成，比率為~1:1:10 (VP1:VP2:VP3)。VP3被包含於VP1及VP2，位在N端，為構建顆粒的主要結構組件。可使用數種不同的編號系統來指稱衣殼蛋白。為了方便起見，如本文所用，AAV序列係使用VP1編號來指稱，VP1編號係以VP1的第一個殘基之aa 1開頭。然而，本文所述的衣殼蛋白包括VP1、VP2及VP3(在此與vp1、vp2及vp3互換使用)。The nucleic acid encoding the AAV capsid comprises three overlapping coding sequences that vary in length due to the use of alternative start codons. The translated proteins are called VP1, VP2 and VP3, with VP1 being the longest and VP3 being the shortest. AAV particles are composed of all three capsid proteins in a ratio of ~1:1:10 (VP1:VP2:VP3). VP3 is included in VP1 and VP2, located at the N-terminus, and is the main structural component for building particles. Several different numbering systems can be used to refer to capsid proteins. For convenience, as used herein, AAV sequences are referred to using the VP1 numbering, which begins with aa 1 of the first residue of VP1. However, the capsid proteins described herein include VP1, VP2 and VP3 (used interchangeably herein with vp1, vp2 and vp3).

本文提供新穎的AAV衣殼蛋白，由示於序列表的序列所編碼。對應vp1、vp2及vp3的核苷酸及胺基酸的編號如下： 核苷酸 (nt)AAVpoG001：SEQ ID NO: 1之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG002：SEQ ID NO: 3之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG003：SEQ ID NO: 5之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG004：SEQ ID NO: 7之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG005：SEQ ID NO: 9之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG006：SEQ ID NO: 11之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG007：SEQ ID NO: 13之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG008：SEQ ID NO: 15之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG009：SEQ ID NO: 17之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG012：SEQ ID NO: 19之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG013：SEQ ID NO: 21之vp1- nt 1至2184；vp2- nt 409至2184；vp3- nt 604至2184； AAVpoG014：SEQ ID NO: 23之vp1- nt 1至2181；vp2- nt 409至2181；vp3- nt 604至2181； AAVpoG015：SEQ ID NO: 25之vp1- nt 1至2181；vp2- nt 409至2181；vp3- nt 604至2181； AAVpoG016：SEQ ID NO: 27之vp1- nt 1至2181；vp2- nt 409至2181；vp3- nt 604至2181； AAVpoG017：SEQ ID NO: 29之vp1- nt 1至2181；vp2- nt 409至2181；vp3- nt 604至2181； AAVpoG018：SEQ ID NO: 31之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG019：SEQ ID NO: 33之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG020：SEQ ID NO: 35之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG021：SEQ ID NO: 37之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG022：SEQ ID NO: 39之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG023：SEQ ID NO: 41之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG024：SEQ ID NO: 43之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148； AAVpoG025：SEQ ID NO: 45之vp1- nt 1至2148；vp2- nt 409至2148；vp3- nt 550至2148。 胺基酸 (aa)AAVpoG001：SEQ ID NO: 2之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG002：SEQ ID NO: 4之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG003：SEQ ID NO: 6之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG004：SEQ ID NO: 8之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG005：SEQ ID NO: 10之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG006：SEQ ID NO: 12之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG007：SEQ ID NO: 14之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG008：SEQ ID NO: 16之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG009：SEQ ID NO: 18之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG012：SEQ ID NO: 20之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG013：SEQ ID NO: 22之aa vp1–1至728；vp2–aa 137至728；vp3–aa 202至728； AAVpoG014：SEQ ID NO: 24之aa vp1–1至727；vp2–aa 137至727；vp3–aa 202至727； AAVpoG015：SEQ ID NO: 26之aa vp1–1至727；vp2–aa 137至727；vp3–aa 202至727； AAVpoG016：SEQ ID NO: 28之aa vp1–1至727；vp2–aa 137至727；vp3–aa 202至727； AAVpoG017：SEQ ID NO: 30之aa vp1–1至727；vp2–aa 137至727；vp3–aa 202至727； AAVpoG018：SEQ ID NO: 32之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG019：SEQ ID NO: 34之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG020：SEQ ID NO: 36之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG021：SEQ ID NO: 38之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG022：SEQ ID NO: 40之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG023：SEQ ID NO: 42之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG024：SEQ ID NO: 44之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716； AAVpoG025：SEQ ID NO: 46之aa vp1–1至716；vp2–aa 137至716；vp3–aa 184至716。 Provided herein are novel AAV capsid proteins encoded by the sequences shown in the Sequence Listing. The numbers of nucleotides and amino acids corresponding to vp1, vp2 and vp3 are as follows: Nucleotide (nt) AAVpoG001: vp1-nt 1 to 2148 of SEQ ID NO: 1; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG002: vp1-nt 1 to 2148 of SEQ ID NO: 3; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG003: vp1-nt 1 to 2148 of SEQ ID NO: 5; 409 to 2148; vp3-nt 550 to 2148; AAVpoG004: vp1-nt 1 to 2148 of SEQ ID NO: 7; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG005: vp1-nt of SEQ ID NO: 9 nt 1 to 2148; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG006: vp1-nt 1 to 2184 of SEQ ID NO: 11; vp2-nt 409 to 2184; vp3-nt 604 to 2184; vp1-nt 1 to 2184 of SEQ ID NO: 13; vp2-nt 409 to 2184; vp3-nt 604 to 2184; AAVpoG008: vp1-nt 1 to 2184 of SEQ ID NO: 15; - nt 604 to 2184; AAVpoG009: vp1-nt 1 to 2184 of SEQ ID NO: 17; vp2-nt 409 to 2184; vp3-nt 604 to 2184; AAVpoG012: vp1-nt 1 to 2184 of SEQ ID NO: 19; vp2-nt 409 to 2184; vp3-nt 604 to 2184; AAVpoG013: vp1-nt 1 to 2184 of SEQ ID NO: 21; vp2-nt 409 to 2184; vp3-nt 604 to 2184; AAVpoG014: SEQ ID NO: 23 vp1-nt 1 to 2181; vp2-nt 409 to 2181; vp3-nt 604 to 2181; AAVpoG015: vp1-nt 1 to 2181 of SEQ ID NO: 25; vp2-nt 409 to 2181; vp3-nt 604 to 2181 ; AAVpoG016: vp1-nt 1 to 2181 of SEQ ID NO: 27; vp2-nt 409 to 2181; vp3-nt 604 to 2181; AAVpoG017: vp1-nt 1 to 2181 of SEQ ID NO: 29; 2181; vp3-nt 604 to 2181; AAVpoG018: vp1-nt 1 to 2148 of SEQ ID NO: 31; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG019: vp1-nt 1 of SEQ ID NO: 33 vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG020: vp1-nt 1 to 2148 of SEQ ID NO: 35; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG021: SEQ ID NO: vp1-nt 1 to 2148 of 37; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG022: SEQ ID NO: vp1-nt 1 to 2148 of 39; vp2-nt 409 to 2148; 550 to 2148; AAVpoG023: vp1-nt 1 to 2148 of SEQ ID NO: 41; vp2-nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG024: vp1-nt 1 to 2148 of SEQ ID NO: 43; nt 409 to 2148; vp3-nt 550 to 2148; AAVpoG025: vp1-nt 1 to 2148 of SEQ ID NO: 45; vp2-nt 409 to 2148; vp3-nt 550 to 2148. Amino acid (aa) AAVpoG001: aa vp1-1 to 716 of SEQ ID NO: 2; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG002: aa vp1-1 to 716 of SEQ ID NO: 4; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG003: aa of SEQ ID NO: 6 vp1-1 to 716; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG004: SEQ ID NO: 8 vp1 - 1 to 716 of aa of aa; vp1 - 1 to 716 of aa of SEQ ID NO: 10; vp1 - 1 to 716 of aa of vp2 - aa 137 to 716; ; AAVpoG006: aa vp1-1 to 728 of SEQ ID NO: 12; vp2-aa 137 to 728; vp3-aa 202 to 728; AAVpoG007: aa vp1-1 to 728 of SEQ ID NO: 14; vp2-aa 137 to 728; 728; vp3-aa 202 to 728; AAVpoG008: aa vp1-1 to 728 of SEQ ID NO: 16; vp2-aa 137 to 728; vp3-aa 202 to 728; AAVpoG009: aa vp1-1 of SEQ ID NO: 18 vp2-aa 137 to 728; vp3-aa 202 to 728; AAVpoG012: aa of SEQ ID NO: 20 vp1-1 to 728; vp2-aa 137 to 728; vp3-aa 202 to 728; AAVpoG013: SEQ ID NO: aa vp1-1 to 728 of 22; vp2-aa 137 to 728; vp3-aa 202 to 728; AAVpoG014: SEQ ID NO: aa vp1-1 to 727 of 24; vp2-aa 137 to 727; 202 to 727; AAVpoG015: aa vp1-1 to 727 of SEQ ID NO: 26; vp2-aa 137 to 727; vp3-aa 202 to 727; AAVpoG016: aa vp1-1 to 727 of SEQ ID NO: 28; aa 137 to 727; vp3 - aa 202 to 727; AAVpoG017: aa of SEQ ID NO: 30 vp1 - 1 to 727; vp2 - aa 137 to 727; vp3 - aa 202 to 727; AAVpoG018: aa of SEQ ID NO: 32 vp1-1 to 716; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG019: aa of SEQ ID NO: 34 vp1-1 to 716; vp2-aa 137 to 716; vp3-aa 184 to 716; : aa vp1-1 to 716 of SEQ ID NO: 36; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG021: aa vp1-1 to 716 of SEQ ID NO: 38; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG022: aa vp1-1 to 716 of SEQ ID NO:40; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG023: aa vp1-1 to 716 of SEQ ID NO:42 vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG024: aa of SEQ ID NO: 44 vp1-1 to 716; vp2-aa 137 to 716; vp3-aa 184 to 716; AAVpoG025: SEQ ID NO: 46 aa vp1–1 to 716; vp2–aa 137 to 716; vp3–aa 184 to 716.

在某些具體實施例中，本文提供rAAV，其包含AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之任一者的vp1、vp2及vp3中的至少一者。在某些具體實施例中，提供一種具有衣殼蛋白的rAAV，該衣殼蛋白包含與下列序列至少90%、至少95%、至少96%、至少97%、至少98%或至少99%相同的vp1、vp2及/或vp3序列：AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)。在某些具體實施例中，相對於下列序列之vp1、vp2、及/或vp3，vp1、vp2、及/或vp3具有至多1、至多2、至多3、至多4、至多5、至多6、至多7、至多8、至多9、或至多10個胺基酸的差異：AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)。In certain embodiments, provided herein is an rAAV comprising AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), at least one of vp1, vp2, and vp3 of any of AAVpoG024 (SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46). In certain embodiments, there is provided an rAAV having a capsid protein comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to vp1, vp2 and/or vp3 sequences: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO : 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), AAVpoG024 ( SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46). In certain embodiments, relative to vp1, vp2, and/or vp3 of the following sequences, vp1, vp2, and/or vp3 have at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7. A difference of at most 8, at most 9, or at most 10 amino acids: AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO : 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 ( SEQ ID NO: 42), AAVpoG024 (SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46).

本文亦提供一種包含AAV衣殼蛋白的rAAV，該AAV衣殼蛋白係由下述序列所編碼：AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3序列的至少一者；或與SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同的序列。在某些具體實施例中，該序列編碼AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之全長vp1、vp2及/或vp3。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。Also provided herein is an rAAV comprising an AAV capsid protein encoded by the following sequences: AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5 ), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27) , AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO: 43), or at least one of the vp1, vp2, and/or vp3 sequences of AAVpoG025 (SEQ ID NO: 45); or with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 at least A sequence that is 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In certain embodiments, the sequence encodes AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19) , AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), Full length vp1, vp2 and/or vp3 of AAVpoG024 (SEQ ID NO: 43), or AAVpoG025 (SEQ ID NO: 45). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

「重組AAV」或「rAAV」為一種DNA酶抗性的病毒顆粒，含有AAV衣殼及載體基因體兩個元件，該載體基因體至少含有包裝在AAV衣殼內的非AAV編碼序列。除非另有說明，否則此術語可與短語「rAAV載體」互換使用。rAAV為一種「複製缺陷型病毒」或「病毒載體」，因為其缺少任何功能性AAV rep基因或功能性AAV cap基因且不能產生子代。於某些具體實施例，唯一的AAV序列為AAV反向末端重複序列(ITR)，通常位於載體基因體的5’及3’末端，以允許位於ITR之間的基因及調節序列包裝在AAV衣殼內。"Recombinant AAV" or "rAAV" is a DNase-resistant virus particle that contains two elements of an AAV capsid and a vector genome that contains at least non-AAV coding sequences packaged in the AAV capsid. Unless otherwise stated, this term is used interchangeably with the phrase "rAAV vector". rAAV is a "replication defective virus" or "viral vector" because it lacks any functional AAV rep genes or functional AAV cap genes and is unable to produce progeny. In certain embodiments, the only AAV sequence is the AAV inverted terminal repeat (ITR), usually located at the 5' and 3' ends of the vector gene body to allow packaging of genes and regulatory sequences located between the ITRs within the AAV coat inside the shell.

如本文所使用，「載體基因體」係指包裝在形成病毒顆粒的rAAV衣殼內部的核酸序列。此種核酸序列含有AAV反向末端重複序列(ITR)。於本文之例中，載體基因體由5’至3’至少含有AAV 5’ ITR、編碼序列(一或多個)及AAV 3’ ITR。可選擇來自AAV2(不同於衣殼之來源AAV)的ITR或除全長ITRs以外的ITR。在某些具體實施例中，ITR係來自與生產過程中提供rep功能的AAV相同的AAV來源或反式互補AAV。再者，可使用其它ITR。此外，載體基因體含有指導基因產物表現的調節序列。於本文中更詳細地討論載體基因體的適當組件。載體基因體在本文中有時稱為「袖珍基因(minigene)」。As used herein, "vector genome" refers to the nucleic acid sequence packaged inside the rAAV capsid that forms the virus particle. Such nucleic acid sequences contain AAV inverted terminal repeats (ITRs). In the example herein, the vector gene body contains at least AAV 5' ITR, coding sequence (one or more) and AAV 3' ITR from 5' to 3'. ITRs from AAV2 (other than capsid-derived AAV) or ITRs other than full-length ITRs can be selected. In certain embodiments, the ITRs are derived from the same AAV source or trans-complemented AAV as the AAV providing the rep function during production. Again, other ITRs may be used. In addition, the vector gene body contains regulatory sequences that direct the expression of the gene product. Appropriate components of vector gene bodies are discussed in more detail herein. A vector gene body is sometimes referred to herein as a "minigene."

如本文所使用，「表現匣」係指核酸分子，其包含生物學上有用的核酸序列(例如，編碼蛋白質、酵素或其它有用基因產物的基因cDNA、mRNA等)及與其可操作地連接的調節序列，該調節序列指導或調節核酸序列及其基因產物的轉錄、轉譯、及/或表現。As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, an enzyme, or other useful gene product) and regulatory genes operably linked thereto. Sequences that direct or regulate the transcription, translation, and/or expression of nucleic acid sequences and their gene products.

如本文所使用，「可操作地連接的」序列包括與核酸序列鄰接的調節序列及以反式或隔一距離而作用來控制序列的調節序列兩者。此種調節序列通常包括例如：啟動子、強化子、內含子、科札克(Kozak)序列、多腺苷酸化序列及TATA訊息中的一種或多種。表現匣可含有：基因序列上游(相對於基因序列而言為5’)的調節序列，例如，啟動子、強化子、內含子等中的一種或多種；以及基因序列下游(相對於基因序列而言為3’)的強化子、或調節序列的一種或多種，例如，包含多腺苷酸化位的3’非轉譯區；以及其它元件。As used herein, "operably linked" sequences include both regulatory sequences contiguous to a nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences generally include, for example, one or more of promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA messages. An expression cassette may contain: regulatory sequences upstream (5' relative to the gene sequence), e.g., one or more of a promoter, enhancer, intron, etc.; and downstream (relative to the gene sequence) 3'), or one or more of regulatory sequences, eg, a 3' untranslated region comprising a polyadenylation site; and other elements.

rAAV係由AAV衣殼及載體基因體所構成。AAV衣殼為vp1蛋白質之異質族群、vp2蛋白質之異質族群、及vp3蛋白質之異質族群的組裝體。如本文所使用，當用於指vp衣殼蛋白，術語「異質」或其任何語法的變化，係指由不同元件組成的族群，例如，具有具不同的經修飾的胺基酸序列之vp1、vp2或vp3單體(蛋白質)。rAAV is composed of AAV capsid and vector gene body. The AAV capsid is an assembly of a heterogeneous population of vpl proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, the term "heterogeneous" or any grammatical variation thereof, when used in reference to vp capsid proteins, refers to a population consisting of distinct elements, for example, vp1, vp1, vp2 or vp3 monomer (protein).

如本文所使用，與vp1、vp2及vp3蛋白質(亦稱為同功型)組合使用的術語「異質族群」係指衣殼內vp1、vp2及vp3蛋白質的胺基酸序列的差異。AAV衣殼含有具有來自預測的胺基酸殘基的修飾之vp1蛋白質內、vp2蛋白質內及vp3蛋白質內的亞群(subpopulation)。此等亞群至少包括某些脫醯胺化的天冬醯胺酸(N或Asn)殘基。例如，某些亞群包含天冬醯胺酸-甘胺酸對中的至少一、二、三或四個高度脫醯胺化的天冬醯胺酸(N)位置及可選擇地進一步包含其它脫醯胺化的胺基酸，其中該脫醯胺化造成胺基酸改變及其它可選擇的修飾。As used herein, the term "heterogeneous population" used in combination with vp1 , vp2 and vp3 proteins (also known as isoforms) refers to differences in the amino acid sequences of vp1 , vp2 and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein with modifications from predicted amino acid residues. Such subgroups include at least some deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagine (N) positions in the asparagine-glycine pair and optionally further comprise other Deamidated amino acids, wherein the deamidation results in amino acid changes and other optional modifications.

如本文所使用，vp蛋白質之「亞群」係指一組vp蛋白質，其具有至少一個共同的定義特徵，且其係由參考組的至少一組成員到少於所有成員所組成，除非另有指明。例如，vp1蛋白質之「亞群」可為組裝的AAV衣殼中的至少一(1)個vp1蛋白質且少於所有vp1蛋白質，除非另有指明。vp3蛋白質的「亞群」可為組裝的AAV衣殼中的一(1)個vp3蛋白質到少於所有vp3蛋白質，除非另有指明。例如，於組裝的AAV衣殼中，vp1蛋白質可為vp蛋白質之亞群；vp2蛋白質可為vp蛋白質之分開的亞群，且vp3為vp蛋白質之又另一亞群。於另一例中，vp1、vp2及vp3蛋白質可含有具有不同的修飾的亞群，例如，至少一、二、三或四個高度脫醯胺化的天冬醯胺酸，例如，於天冬醯胺酸-甘胺酸對中。As used herein, a "subgroup" of vp proteins refers to a group of vp proteins that have at least one defining characteristic in common and that consist of at least one group to less than all members of a reference group, unless otherwise stated specified. For example, a "subpopulation" of vp1 proteins can be at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of vp3 proteins can range from one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, in an assembled AAV capsid, the vp1 protein can be a subgroup of vp proteins; the vp2 protein can be a separate subgroup of vp proteins, and vp3 is yet another subgroup of vp proteins. In another example, the vp1, vp2 and vp3 proteins may contain subpopulations with different modifications, e.g., at least one, two, three or four highly deamidated asparagine, e.g., in asparagine Amino acid-glycine pair.

除非另有指出，高度脫醯胺化的係指當與於參考胺基酸位置的預測的胺基酸序列比較，於參考的胺基酸位置上有至少45%被脫醯胺化、至少50%被脫醯胺化、至少60%被脫醯胺化、至少65%被脫醯胺化、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少97%、至少99%、或至多約100%被脫醯胺化。此種百分比可使用2D膠體、質譜技術或其它適合的技術來確定。Unless otherwise indicated, highly deamidated means at least 45% deamidated, at least 50% deamidated at a reference amino acid position when compared to the predicted amino acid sequence at a reference amino acid position. % deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at most about 100% deamidated. Such percentages can be determined using 2D colloids, mass spectrometry, or other suitable techniques.

不希望受理論束縛，咸信AAV衣殼中的vp蛋白質中至少高度脫醯胺化的殘基之脫醯胺化本質上主要為非酵素性的，由衣殼蛋白內的官能基所引起，該官能基將所選擇的天冬醯胺酸脫醯胺化，且在較小程度上將麩醯胺酸殘基脫醯胺化。大多數脫醯胺化vp1蛋白質的有效衣殼組裝指出此等事件發生於衣殼組裝後，或者個別單體(vp1、vp2或vp3)中的脫醯胺化在結構上具有良好的耐受性，並且在很大程度上不會影響組裝動力。VP1獨特(VP1-u)區域(~aa 1-137)中的廣泛脫醯胺化通常被認為在細胞進入之前係位於內部，暗示VP脫醯胺化可能發生在衣殼組裝之前。Without wishing to be bound by theory, it is believed that the deamidation of at least highly deamidated residues in the vp protein in the AAV capsid is primarily non-enzymatic in nature, caused by functional groups within the capsid protein that The functional group deamidates selected asparagine and, to a lesser extent, glutamate residues. Efficient capsid assembly of most deamidated vp1 proteins suggests that either these events occur after capsid assembly or that deamidation in individual monomers (vp1, vp2, or vp3) is structurally well tolerated , and does not affect assembly dynamics to a large extent. Extensive deamidation in the VP1 unique (VP1-u) region (~aa 1-137) is generally thought to be internal before cell entry, suggesting that VP deamidation may occur prior to capsid assembly.

不希望受理論束縛，N的脫醯胺化可通過其C端殘基的骨架氮原子對Asn側鏈醯胺基碳原子進行親核攻擊而發生。咸信會形成一個中間體閉環的琥珀醯亞胺殘基。然後此琥珀醯亞胺殘基進行快速水解以產生最終產物天冬胺酸(Asp)或異天冬胺酸(IsoAsp)。因此，於某些具體實施例，天冬醯胺酸(N或Asn)的脫醯胺化會產生Asp或IsoAsp，其可通過琥珀醯亞胺中間體相互轉化，例如，如下所圖示。

Figure 02_image001
Without wishing to be bound by theory, deamidation of N may occur via nucleophilic attack on the amide group carbon atom of the Asn side chain by the backbone nitrogen atom of its C-terminal residue. An intermediate ring-closing succinimide residue is believed to form. This succinimide residue then undergoes rapid hydrolysis to yield the final product aspartic acid (Asp) or isoaspartic acid (IsoAsp). Thus, in certain embodiments, deamidation of asparagine (N or Asn) yields Asp or IsoAsp, which can be interconverted via a succinimide intermediate, eg, as shown below.
Figure 02_image001

如本文所提供，vp1、vp2、或vp3中各脫醯胺化的N可獨立地為天冬胺酸(Asp)、異天冬胺酸(isoAsp)、天冬胺酸鹽、及/或Asp及isoAsp之相互轉化的摻合物、或此等的組合。可存在α-及異天冬胺酸之任何適合的比率。例如，於某些具體實施例，該比率可為由10:1至1:10之天冬胺酸對異天冬胺酸、約50:50之天冬胺酸:異天冬胺酸、或約1:3之天冬胺酸:異天冬胺酸、或其它選擇的比率。As provided herein, each deamidated N in vp1, vp2, or vp3 may independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate, and/or Asp and isoAsp interconvertible blends, or combinations thereof. Any suitable ratio of alpha- and isoaspartic acid may be present. For example, in certain embodiments, the ratio can be from 10:1 to 1:10 aspartic acid to isoaspartic acid, about 50:50 aspartic acid:isoaspartic acid, or About 1:3 aspartic acid:isoaspartic acid, or other selected ratios.

在某些具體實施例中，一或多個麩醯胺酸(Q)可脫醯胺化為麩胺酸(Glu)，即，α-麩胺酸、γ-麩胺酸(Glu)、或α-及γ-麩胺酸之摻合物，其可通過共同的戊二醯亞胺(glutarimide)中間體相互轉化。可存在α-及γ-麩胺酸之任何適合的比率。例如，於某些具體實施例，該比率可為由10:1至1:10之α對γ、約50:50之α:γ、或約1:3之α:γ、或其它選擇的比率。

Figure 02_image003
In certain embodiments, one or more glutamic acids (Q) can be deamidated to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or Blends of α- and γ-glutamic acids, which are interconvertible via a common glutarimide intermediate. Any suitable ratio of alpha- and gamma-glutamine may be present. For example, in some embodiments, the ratio can be from 10:1 to 1:10 alpha to gamma, about 50:50 alpha:gamma, or about 1:3 alpha:gamma, or other selected ratios .
Figure 02_image003

如此，rAAV包括具有脫醯胺化的胺基酸的vp1、vp2及/或vp3蛋白質的rAAV衣殼內的亞群，至少包括：包含至少一種高度脫醯胺化的天冬醯胺酸的至少一個亞群。此外，其它修飾可包括異構化，特別於選擇的天冬胺酸(D或Asp)殘基位置上。在另外其它具體實施例中，修飾可包括在Asp位置上的醯胺化。Thus, rAAV includes subpopulations within rAAV capsids of vp1, vp2 and/or vp3 proteins having deamidated amino acids, including at least one protein comprising at least one highly deamidated asparagine. a subgroup. In addition, other modifications may include isomerization, particularly at selected aspartic acid (D or Asp) residue positions. In yet other embodiments, the modification can include amidation at the Asp position.

在某些具體實施例中，AAV衣殼含有具有至少1個、至少2個、至少3個、至少4個、至少5個到至少約25個脫醯胺化的胺基酸殘基位置的vp1、vp2及vp3之亞群，其中當與vp蛋白質的經編碼的胺基酸序列相比時，至少1至10%、至少10至25%、至少25至50%、至少50至70%、至少70至100%、至少75至100%、至少80-100%、或至少90-100%被脫醯胺化。此等中的大部分可為N殘基。然而，Q殘基亦可被脫醯胺化。In certain embodiments, the AAV capsid comprises a vp1 having at least 1, at least 2, at least 3, at least 4, at least 5 to at least about 25 amino acid residue positions for deamidation , a subpopulation of vp2 and vp3, wherein when compared to the encoded amino acid sequence of the vp protein, at least 1 to 10%, at least 10 to 25%, at least 25 to 50%, at least 50 to 70%, at least 70 to 100%, at least 75 to 100%, at least 80-100%, or at least 90-100% is deamidated. Most of these can be N residues. However, the Q residue can also be deamidated.

如本文所使用，「經編碼的胺基酸序列」係指基於轉譯為胺基酸的參考核酸序列之已知DNA密碼子的轉譯而預測的胺基酸。As used herein, "encoded amino acid sequence" refers to an amino acid that is predicted based on the translation of known DNA codons of a reference nucleic acid sequence that translates to the amino acid.

在某些具體實施例中，rAAV具有AAV衣殼，該AAV衣殼具有vp1、vp2及vp3蛋白質，該vp1、vp2及vp3蛋白質具有包含示於本文所提供的表中位置的二、三、四、五個或以上的脫醯胺化的殘基之組合的亞群，且藉由引用併入本文。In certain embodiments, the rAAV has an AAV capsid with vp1, vp2, and vp3 proteins having two, three, and four proteins comprising the positions shown in the tables provided herein. , a subgroup of combinations of five or more deamidated residues, and are incorporated herein by reference.

於rAAV中脫醯胺化可使用2D膠體電泳、及/或質譜分析、及/或蛋白質模擬(protein modelling)技術確定。線上層析可用Acclaim PepMap管柱及Thermo UltiMate 3000 RSLC系統(Thermo Fisher Scientific)連接至具NanoFlex源的Q Exactive HF (Thermo Fisher Scientific)而進行。MS數據係使用Q Exactive HF的依靠數據的top-20方法所獲取，從勘測掃描(200-2000 m/z)中動態選擇最豐富的尚未定序的前驅物離子。經由較高能量的碰撞解離片段化(collisional dissociation fragmentation)進行定序，並以預測性自動增益控制(predictive automatic gain control)確定目標值1e5離子，以4 m/z的區間進行前驅物單離。以m/z 200時的解析度為120,000獲取勘測掃描。在m/z200時，HCD光譜的解析度可設定為30,000，最大離子注射時間為50 ms，歸一化碰撞能量為30。S-lens RF水準可設定為50，以提供來自消化物的肽佔據的m/z區域之最佳透射率。可從片段化選擇中以單個、未賦值或六個或更高電荷狀態來排除前驅物離子。BioPharma Finder 1.0軟體(Thermo Fischer Scientific)可用於分析所獲取的數據。對於肽定位(peptide mapping)，使用單輸入蛋白質FASTA數據庫進行搜索，其中胺甲醯胺基甲基化設定為固定修飾；將氧化、脫醯胺化及磷酸化設定為可變修飾，質量精度為10ppm，高蛋白酶特異性，MS/MS光譜的信賴水準為0.8。適合的蛋白酶之例可包括例如胰蛋白酶或胰凝乳蛋白酶。脫醯胺化的肽的質譜鑑定相對簡單，因脫醯胺化增加完整分子的質量+0.984 Da (-OH及–NH 2基團之間的質量差)。特定肽的脫醯胺化百分比係由脫醯胺化的肽的質量面積除以脫醯胺化與天然的肽的面積之和而確定。考慮到可能的脫醯胺化位的數目，在不同部位脫醯胺化的同量異位物種(isobaric species)可能在一個峰中共遷移。因此，源自具有多個潛在脫醯胺化位的肽的片段離子可用於定位或區分多個脫醯胺化位。於此等情形，觀察到的同位素樣式內的相對強度可用於特異性地確定不同的脫醯胺化的肽異構物的相對豐度。此方法假定所有同量異位物種的片段化效率相同，且在脫醯胺化位上是獨立的。本項技術領域中具通常知識者應理解，可使用此等說明性方法的多種變化。例如，適合的質譜儀可包括例如四極飛行時間質譜儀(QTOF)，諸如Waters Xevo或Agilent 6530或軌道阱(orbitrap)儀器，諸如Orbitrap Fusion或Orbitrap Velos (Thermo Fisher)。適合的液相層析系統包括例如：來自Waters之Acquity UPLC系統、或者Agilent系統(1100或1200系列)。適合的資料分析軟體可包括例如：MassLynx (Waters)、Pinpoint及Pepfinder (Thermo Fischer Scientific)、Mascot (Matrix Science)、Peaks DB (Bioinformatics Solutions)。亦可描述其它技術，例如，描述於X. Jin et al, Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255-267，2017年6月16日線上公開。 Deamidation in rAAV can be determined using 2D gel electrophoresis, and/or mass spectrometry, and/or protein modeling techniques. On-line chromatography was performed with an Acclaim PepMap column and a Thermo UltiMate 3000 RSLC system (Thermo Fisher Scientific) connected to a Q Exactive HF with a NanoFlex source (Thermo Fisher Scientific). MS data were acquired using the Q Exactive HF's data-dependent top-20 method, dynamically selecting the most abundant as yet unsequenced precursor ions from survey scans (200-2000 m/z). Sequencing was performed via higher-energy collisional dissociation fragmentation, and a target value of 1e5 ions was determined with predictive automatic gain control, and precursor isolation was performed in a 4 m/z interval. Survey scans were acquired at a resolution of 120,000 at m/z 200. At m/z 200, the resolution of the HCD spectrum can be set to 30,000, the maximum ion injection time is 50 ms, and the normalized collision energy is 30. The S-lens RF level can be set to 50 to provide optimum transmission in the m/z region occupied by peptides from the digest. Precursor ions can be excluded from fragmentation selection with single, unassigned, or six or higher charge states. BioPharma Finder 1.0 software (Thermo Fischer Scientific) was used to analyze the acquired data. For peptide mapping, a search was performed using the single-input protein FASTA database, where carbamidomethylation was set as a fixed modification; oxidation, deamidation, and phosphorylation were set as variable modifications, with a mass accuracy of 10ppm, high protease specificity, confidence level of 0.8 for MS/MS spectra. Examples of suitable proteases may include, for example, trypsin or chymotrypsin. Mass spectrometric identification of deamidated peptides is relatively straightforward, as deamidation increases the mass of the intact molecule by +0.984 Da (difference in mass between -OH and -NH2 groups). The percent deamidation of a particular peptide was determined by dividing the mass area of the deamidated peptide by the sum of the areas of the deamidated and native peptide. Given the number of possible deamidation sites, isobaric species deamidated at different sites may co-migrate in one peak. Thus, fragment ions derived from peptides with multiple potential deamidation sites can be used to locate or distinguish multiple deamidation sites. In such cases, the relative intensities within the observed isotopic patterns can be used to specifically determine the relative abundance of different deamidated peptide isoforms. This method assumes that all isobaric species fragment with equal efficiency and are independent at the deamidation position. Those of ordinary skill in the art will appreciate that many variations of these illustrative methods can be used. For example, suitable mass spectrometers may include, for example, quadrupole time-of-flight mass spectrometers (QTOF), such as Waters Xevo or Agilent 6530, or orbitrap instruments, such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems include, for example, the Acquity UPLC system from Waters, or the Agilent system (1100 or 1200 series). Suitable data analysis software may include, for example: MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fischer Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions). Other techniques may also be described, for example, as described in X. Jin et al, Hu Gene Therapy Methods, Vol. 28, No. 5, pp. 255-267, published online June 16, 2017.

除了脫醯胺化之外，可發生其它修飾而不會導致一個胺基酸轉換為不同的胺基酸殘基。此種修飾可包括乙醯化殘基、異構化、磷酸化或氧化。In addition to deamidation, other modifications can occur without resulting in the conversion of one amino acid to a different amino acid residue. Such modifications may include acetylation of residues, isomerization, phosphorylation or oxidation.

脫醯胺化的調節：於某些具體實施例，修飾AAV以改變天冬醯胺酸-甘胺酸對中的甘胺酸，以減少脫醯胺化。於其它具體實施例，將天冬醯胺酸改變為不同的胺基酸，例如以較慢的速度脫醯胺化的麩醯胺酸；或改變為缺少醯胺基的胺基酸(例如，麩醯胺酸及天冬醯胺酸含有醯胺基)；及/或改變為缺少胺基的胺基酸(例如，離胺酸、精胺酸及組胺酸含有胺基)。如本文所使用，如本文所使用，缺少醯胺或胺側鏈基團的胺基酸係指例如：甘胺酸、丙胺酸、纈胺酸、白胺酸、異白胺酸、絲胺酸、蘇胺酸、胱胺酸、苯丙胺酸、酪胺酸、或色胺酸、及/或脯胺酸。諸如所述的修飾可位於所編碼的AAV胺基酸序列中發現的一、二或三個天冬醯胺酸-甘胺酸對中。於某些具體實施例，在所有四個天冬醯胺酸-甘胺酸對中沒有進行此種修飾。如此，為一種用於減少AAV及/或經工程化的AAV變異體之脫醯胺化而具有較低脫醯胺化率的方法。另外地或替代地，可將一個或多個其它醯胺胺基酸改變為非醯胺胺基酸以減少AAV的脫醯胺化。於某些具體實施例，本文所述的突變AAV衣殼含有天冬醯胺酸-甘胺酸對中的突變，使得甘胺酸改變為丙胺酸或絲胺酸。突變AAV衣殼可含有一個、兩個或三個突變體，其中參考AAV天然地含有四個NG對。於某些具體實施例，AAV衣殼可含有一個、兩個、三個或四個此種突變體，其中參考AAV天然地含有五個NG對。在某些具體實施例中，突變AAV衣殼僅含有在一NG對中的單個突變。在某些具體實施例中，突變AAV衣殼含有兩個不同NG對中的突變。於某些具體實施例，突變AAV衣殼含有兩個不同的NG對的突變，其位於AAV衣殼中結構上分開的位置。於某些具體實施例，該突變並非位於VP1獨特區域。於某些具體實施例，突變之一者位於VP1獨特區域。可選擇地，突變AAV衣殼不含有在NG對中的修飾，但含有突變以最小化或消除位於NG對之外的一個或多個天冬醯胺酸或麩醯胺酸中的脫醯胺化。Modulation of deamidation: In certain embodiments, AAV is modified to alter the glycine in the asparagine-glycine pair to reduce deamidation. In other embodiments, asparagine is changed to a different amino acid, such as glutamine, which deamidates at a slower rate; or to an amino acid lacking an amido group (e.g., Glutamine and asparagine contain amide groups); and/or are altered to amino acids lacking amine groups (eg, lysine, arginine, and histidine contain amine groups). As used herein, an amino acid lacking an amide or amine side chain group refers to, for example, glycine, alanine, valine, leucine, isoleucine, serine , threonine, cystine, phenylalanine, tyrosine, or tryptophan, and/or proline. Modifications such as those described may be located in one, two or three asparagine-glycine pairs found in the encoded AAV amino acid sequence. In certain embodiments, no such modification is made in all four asparagine-glycine pairs. Thus, there is a method for reducing deamidation of AAV and/or engineered AAV variants with lower deamidation rates. Additionally or alternatively, one or more other amido amino acids can be changed to a non-amid amino acid to reduce deamidation of AAV. In certain embodiments, the mutant AAV capsids described herein contain mutations in the asparagine-glycine pair such that glycine is changed to alanine or serine. Mutant AAV capsids may contain one, two or three mutants, where the reference AAV naturally contains four NG pairs. In certain embodiments, the AAV capsid may contain one, two, three or four such mutants, where the reference AAV naturally contains five NG pairs. In certain embodiments, the mutant AAV capsid contains only a single mutation in an NG pair. In certain embodiments, the mutant AAV capsid contains mutations in two different NG pairs. In certain embodiments, the mutant AAV capsid contains mutations in two different NG pairs located at structurally separate positions in the AAV capsid. In some embodiments, the mutation is not located in a unique region of VP1. In certain embodiments, one of the mutations is in a unique region of VP1. Alternatively, the mutant AAV capsid does not contain a modification in the NG pair, but contains a mutation to minimize or eliminate deamidation at one or more asparagine or glutamine located outside the NG pair change.

在某些具體實施例中，提供一種增加rAAV載體效力的方法，該方法包含將AAV衣殼工程化，其消除了野生型AAV衣殼中的一個或多個NG。在某些具體實施例中，「NG」之「G」的編碼序列被工程化成編碼另一胺基酸。於下列某些例子，「S」或「A」被取代。然而，可選擇其它適合的胺基酸編碼序列。In certain embodiments, a method of increasing potency of an rAAV vector is provided, the method comprising engineering an AAV capsid that eliminates one or more NGs in a wild-type AAV capsid. In certain embodiments, the coding sequence for the "G" of "NG" is engineered to encode another amino acid. In some of the examples below, "S" or "A" are substituted. However, other suitable amino acid coding sequences may be selected.

胺基酸修飾可藉由常規遺傳工程技術進行。例如，可產生含有經修飾的AAV vp密碼子的核酸序列，其中編碼天冬醯胺酸-甘胺酸對中甘胺酸的一至三個密碼子被修飾以編碼甘胺酸以外的胺基酸。於某些具體實施例，含有經修飾的天冬醯胺酸密碼子的核酸序列可在天冬醯胺酸-甘胺酸對中的一至三處工程化，使得經修飾的密碼子編碼天冬醯胺酸以外的胺基酸。每個經修飾的密碼子可編碼不同的胺基酸。或者，一或多個經改變的密碼子可編碼相同的胺基酸。在某些具體實施例中，此等經修飾的核酸序列可用於產生具有比天然AAV3B變異體衣殼更低脫醯胺化衣殼的突變rAAV。此種突變rAAV可具有降低的免疫原性及/或提高儲存穩定性，特別是以懸浮形式儲存。Amino acid modifications can be performed by conventional genetic engineering techniques. For example, nucleic acid sequences containing modified AAV vp codons can be generated in which one to three codons encoding glycine in the asparagine-glycine pair are modified to encode amino acids other than glycine . In certain embodiments, nucleic acid sequences containing modified asparagine codons can be engineered at one to three of the asparagine-glycine pairs such that the modified codons encode asparagine Amino acids other than amide. Each modified codon can encode a different amino acid. Alternatively, one or more altered codons may encode the same amino acid. In certain embodiments, these modified nucleic acid sequences can be used to generate mutant rAAVs with capsids that are less deamidated than native AAV3B variant capsids. Such mutant rAAV may have reduced immunogenicity and/or increased storage stability, particularly in suspension.

本文亦提供編碼具有減少的脫醯胺化之AAV衣殼的核酸序列。設計編碼此AAV衣殼的核酸序列係於本領域技術範圍內，包括DNA(基因體或cDNA)或RNA(例如mRNA)。此種核酸序列可為了在選擇的系統(即，細胞類型)中的表現進行密碼子最適化並且可藉由各種方法設計。可使用可於線上取得的方法(例如，GeneArt)、公開的方法或提供密碼子最適化服務的公司(例如，DNA2.0 (Menlo Park, CA))而進行此最適化。例如，於國際專利公開號WO 2015/012924描述一種密碼子最適化方法，其藉由引用以其整體併入本文。 亦參見例如：US專利公開號2014/0032186及US專利公開號2006/0136184。適合地，產物的開讀框(ORF)的整個長度被修飾。然而，於一些具體實施例，可改變ORF之僅一片段。藉由使用此等方法之一者，可將頻率應用於任何給定的多肽序列，並產生編碼該多肽的經密碼子最適化的編碼區域的核酸片段。許多選項可用於進行對密碼子的實際更改或用於合成如本文所述設計的經密碼子最適化的編碼區域。可使用所屬技術領域中具通常知識者眾所周知的標準及常規分子生物學操作來進行此類修飾或合成。於一方式，藉由標準方法合成各自的長度為80-90個核苷酸並跨越所欲序列的長度之一系列互補的寡核苷酸對。合成此等寡核苷酸對，經過黏合(anneal)，它們形成80-90個鹼基對的雙股片段，其含有黏性末端，例如，對中的每個寡核苷酸被合成以延伸3、4、5、6、7、8、9、10個或更多個鹼基，該鹼基超出與該對中另一個寡核苷酸互補的區域。每對寡核苷酸的單股末端被設計為與另一對寡核苷酸的單股末端黏合。使寡核苷酸對黏合，然後使此等雙股片段中的大約五至六個經由黏性的單股末端一起黏合，然後它們連接在一起並被選殖至標準細菌選殖載體，例如，可獲自Invitrogen Corporation, Carlsbad, Calif的TOPO®載體。然後藉由標準方法定序此構築體。製備數個此等構築體，此等構築體由連接在一起的80至90個鹼基對片段的5至6個片段所組成，即由約500個鹼基對的片段所組成，如此使得整個所欲序列被表示在一系列的質體構築體中。然後將此等質體的插入物以適當的限制酶切開，並連接在一起以形成最終構築體。然後將最終構築體選殖至標準細菌選殖載體，並定序。其它的方法對於所屬技術領域中具通常知識者為顯而易見的。此外，基因合成係商業上容易取得的。 Also provided herein are nucleic acid sequences encoding AAV capsids with reduced deamidation. It is within the skill of the art to design nucleic acid sequences encoding such AAV capsids, including DNA (genome or cDNA) or RNA (eg, mRNA). Such nucleic acid sequences can be codon-optimized for expression in the system of choice (ie, cell type) and can be designed by various methods. This optimization can be performed using methods available online (eg, GeneArt), published methods, or companies that provide codon optimization services (eg, DNA2.0 (Menlo Park, CA)). For example, a codon optimization method is described in International Patent Publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, eg , US Patent Publication No. 2014/0032186 and US Patent Publication No. 2006/0136184. Suitably, the entire length of the open reading frame (ORF) of the product is modified. However, in some embodiments, only one segment of the ORF may be altered. By using one of these methods, frequencies can be applied to any given polypeptide sequence and nucleic acid fragments are generated that encode the codon-optimized coding region of the polypeptide. Many options are available for making actual changes to codons or for synthesizing codon-optimized coding regions designed as described herein. Such modifications or syntheses can be carried out using standard and routine molecular biology procedures well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs each 80-90 nucleotides in length and spanning the length of the desired sequence are synthesized by standard methods. These pairs of oligonucleotides are synthesized, and after annealing, they form double-stranded fragments of 80-90 base pairs containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides are designed to bind to the single-stranded ends of the other pair of oligonucleotides. Oligonucleotide pairs are ligated, and about five to six of these double-stranded fragments are then ligated together via sticky single-stranded ends, which are then ligated together and colonized into standard bacterial colonization vectors, e.g., TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. This construct was then sequenced by standard methods. Several of these constructs were prepared, consisting of 5 to 6 segments of 80 to 90 base pair segments linked together, i.e., of approximately 500 base pair segments, such that the entire Desired sequences are represented in a series of plastid constructs. The inserts of these plastids were then cut with appropriate restriction enzymes and ligated together to form the final construct. The final constructs are then cloned into standard bacterial colonization vectors and sequenced. Other methods will be apparent to those of ordinary skill in the art. Furthermore, gene synthesis is readily available commercially.

在某些具體實施例中，提供的AAV衣殼具有含有多個高度脫醯胺化的「NG」位置之AAV衣殼同功型(即，VP1、VP2、VP3)的異質族群。在某些具體實施例中，參考預測的全長VP1胺基酸序列，高度脫醯胺化的位置位於以下確定的位置。於其它具體實施例，衣殼基因被修飾，使得參考的「NG」被切除，並將突變體「NG」工程化至另一位置。In certain embodiments, AAV capsids are provided that have a heterogeneous population of AAV capsid isoforms (ie, VP1, VP2, VP3) that contain multiple highly deamidated "NG" positions. In certain embodiments, with reference to the predicted full-length VP1 amino acid sequence, the highly deamidated position is located at the position determined below. In other embodiments, the capsid gene is modified such that the reference "NG" is excised and the mutant "NG" is engineered to another position.

本文提供的許多豬AAV衣殼係在小腸中被鑑定的，小腸為人類中不常見的AAV來源。如此，如本文提供的具有豬衣殼(「rAAVpo」)的rAAV可能特別適合靶向腸道中的細胞及組織，包括但不限於小腸。此種rAAVpo載體可用於基因遞送或基因編輯。在某些具體實施例中，此種rAAV載體可用於靶向病毒儲積處(例如，B型肝炎病毒(HBV)、C型肝炎病毒(HCV)、單純疱疹病毒(HSV)、水痘帶狀疱疹病毒(VZV)及人類免疫不全病毒(HIV))或腸道中不希望的細胞(例如細菌)群體。 AAVpoG001 Many of the porcine AAV capsid lines presented herein were identified in the small intestine, an uncommon source of AAV in humans. As such, rAAV with a porcine capsid ("rAAVpo") as provided herein may be particularly suitable for targeting cells and tissues in the intestinal tract, including but not limited to the small intestine. Such rAAVpo vectors can be used for gene delivery or gene editing. In certain embodiments, such rAAV vectors can be used to target viral reservoirs (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), varicella zoster virus (VZV) and human immunodeficiency virus (HIV)) or unwanted populations of cells (such as bacteria) in the gut. AAVpoG001

在某些具體實施例中，提供一種新穎的單離的AAVpoG001衣殼。SEQ ID NO: 1提供編碼AAVpoG001衣殼的核酸序列且SEQ ID NO: 2提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG001 (SEQ ID NO: 2)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG001 (SEQ ID NO: 1)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG001 (SEQ ID NO: 2)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG001 capsid is provided. SEQ ID NO: 1 provides the nucleic acid sequence encoding the AAVpoG001 capsid and SEQ ID NO: 2 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG001 (SEQ ID NO: 2). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG001 (SEQ ID NO: 1). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG001 (SEQ ID NO: 2). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG001衣殼，包含下列一者或多者：(1) AAVpoG001衣殼蛋白，包含：AAVpoG001 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 2的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 1生產的vp1蛋白質、或由與SEQ ID NO: 1至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 2的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG001 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 2的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 1之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 1之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 2的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG001 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 2的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 1之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 1之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 1的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 2之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 2之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 2之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 2中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG001衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG002 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG001 capsid, comprising one or more of the following: (1) AAVpoG001 capsid protein, comprising: AAVpoG001 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 2, the vp1 protein produced by SEQ ID NO: 1, or the vp1 protein produced by combining with SEQ ID NO: 1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 2; a heterogeneous group of AAVpoG001 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 2 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 1, or the vp2 protein produced by the sequence with SEQ ID NO: 1 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 2; a heterogeneous population of AAVpoG001 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 2 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 1, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 1 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 1; and/or (2) a heterogeneous population of vp1 protein , which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2; a heterogeneous population of vp2 proteins, which is a nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 2 The product of sequence; And the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:2 at least aminoacid 184 to 716, wherein: vp1, vp2 and vp3 protein contain subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 2, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG001 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG002

在某些具體實施例中，提供一種新穎的單離的AAVpoG002衣殼。SEQ ID NO: 3提供編碼AAVpoG002衣殼的核酸序列且SEQ ID NO: 4提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG002 (SEQ ID NO: 4)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG002 (SEQ ID NO: 3)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG002 (SEQ ID NO: 4)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG002 capsid is provided. SEQ ID NO: 3 provides the nucleic acid sequence encoding the AAVpoG002 capsid and SEQ ID NO: 4 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG002 (SEQ ID NO: 4). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG002 (SEQ ID NO: 3). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG002 (SEQ ID NO: 4). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG002衣殼，包含下列一者或多者：(1) AAVpoG002衣殼蛋白，包含：AAVpoG002 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 4的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 3生產的vp1蛋白質、或由與SEQ ID NO: 3至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 4的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG002 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 4的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 3之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 3之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 4的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG002 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 4的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 3之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 4之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 4的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 4之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 4之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 4之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 4中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG002衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG003 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG002 capsid, comprising one or more of the following: (1) AAVpoG002 capsid protein, comprising: AAVpoG002 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 4, the vp1 protein produced by SEQ ID NO: 3, or the vp1 protein produced with SEQ ID NO: 3 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 4; a heterogeneous group of AAVpoG002 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 4 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 3, or the vp2 protein produced by the sequence with SEQ ID NO: 3 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 4; a heterogeneous population of AAVpoG002 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 4 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 184 to 716, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 3, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 4 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 4; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 4 The product of sequence; And the heterogeneous group of vp3 protein, it is the product of the nucleotide sequence of coding SEQ ID NO:4 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 4, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG002 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG003

在某些具體實施例中，提供一種新穎的單離的AAVpoG003衣殼。SEQ ID NO: 5提供編碼AAVpoG003衣殼的核酸序列且SEQ ID NO: 6提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG003 (SEQ ID NO: 6)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG003 (SEQ ID NO: 5)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG003 (SEQ ID NO: 6)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG003 capsid is provided. SEQ ID NO: 5 provides the nucleic acid sequence encoding the AAVpoG003 capsid and SEQ ID NO: 6 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG003 (SEQ ID NO: 6). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG003 (SEQ ID NO: 5). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG003 (SEQ ID NO: 6). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG003衣殼，包含下列一者或多者：(1) AAVpoG003衣殼蛋白，包含：AAVpoG003 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 6的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 5生產的vp1蛋白質、或由與SEQ ID NO: 5至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 6的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG003 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 6的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 5之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 5之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 2的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG003 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 6的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 5之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 5之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 5的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 6之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 6之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 6之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 6中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG003衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG004 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG003 capsid, comprising one or more of the following: (1) AAVpoG003 capsid protein, comprising: AAVpoG003 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 6, the vp1 protein produced by SEQ ID NO: 5, or the vp1 protein produced with SEQ ID NO: 5 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 6; a heterogeneous group of AAVpoG003 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 6 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by a sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 5, or produced by a sequence with SEQ ID NO: 5 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 2; a heterogeneous population of AAVpoG003 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 6 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 184 to 716, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 5, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 550 to 2148 of SEQ ID NO: 5 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 5; and/or (2) a heterogeneous population of vp1 protein , which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6; a heterogeneous population of vp2 proteins, which is a nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 6 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:6 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 6, and optionally further comprises other desamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG003 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG004

在某些具體實施例中，提供一種新穎的單離的AAVpoG001衣殼。SEQ ID NO: 7提供編碼AAVpoG001衣殼的核酸序列且SEQ ID NO: 8提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG001 (SEQ ID NO: 8)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG001 (SEQ ID NO: 7)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG001 (SEQ ID NO: 8) 之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG001 capsid is provided. SEQ ID NO: 7 provides the nucleic acid sequence encoding the AAVpoG001 capsid and SEQ ID NO: 8 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG001 (SEQ ID NO: 8). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG001 (SEQ ID NO: 7). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG001 (SEQ ID NO: 8). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG001衣殼，包含下列一者或多者：(1) AAVpoG001衣殼蛋白，包含：AAVpoG001 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 8的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 7生產的vp1蛋白質、或由與SEQ ID NO: 7至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 8的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG001 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 8的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 7之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 7之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 8的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG001 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 8的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 7之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 7之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 7的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質，；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 8之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 8之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 8之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 8中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG001衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG005 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG001 capsid, comprising one or more of the following: (1) AAVpoG001 capsid protein, comprising: AAVpoG001 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 8, the vp1 protein produced by SEQ ID NO: 7, or the vp1 protein produced with SEQ ID NO: 7 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 8; a heterogeneous group of AAVpoG001 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 8 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by a sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 7, or produced by a sequence with SEQ ID NO: 7 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 8; a heterogeneous population of AAVpoG001 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 8 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 7, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 7 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 7; and/or (2) the heterogeneity of the vp1 protein Group, it is the product of the nucleotide sequence of the amino acid sequence of encoding SEQ ID NO:8; The heterogeneous group of vp2 protein, it is the aminoacid sequence of at least about amino acid 137 to 716 of encoding SEQ ID NO:8 The product of nucleic acid sequence; And the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:8 at least aminoacid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subunit with amino acid modification Group, the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 8, and optionally further comprising other a subpopulation of deamidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG001 capsid comprising a nucleic acid molecule comprising The AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG005

在某些具體實施例中，提供一種新穎的單離的AAVpoG005衣殼。SEQ ID NO: 9提供編碼AAVpoG005衣殼的核酸序列且SEQ ID NO: 10提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG005 (SEQ ID NO: 10)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG005 (SEQ ID NO: 9)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG005 (SEQ ID NO: 10)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG005 capsid is provided. SEQ ID NO: 9 provides the nucleic acid sequence encoding the AAVpoG005 capsid and SEQ ID NO: 10 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG005 (SEQ ID NO: 10). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG005 (SEQ ID NO: 9). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG005 (SEQ ID NO: 10). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG005衣殼，包含下列一者或多者：(1) AAVpoG005衣殼蛋白，包含：AAVpoG005 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 10的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 9生產的vp1蛋白質、或由與SEQ ID NO: 9至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 10的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG005 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 10的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 9之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 9之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 10的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG005 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 10的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 9之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 9之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 9的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 10之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 10之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 10之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 10中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG005衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG006 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG005 capsid, comprising one or more of the following: (1) AAVpoG005 capsid protein, comprising: AAVpoG005 vp1 protein A heterogeneous population selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence from 1 to 716 of SEQ ID NO: 10, the vp1 protein produced by SEQ ID NO: 9, or produced by combining with SEQ ID NO: 9 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 10; a heterogeneous group of AAVpoG005 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 10 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by a sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 9, or produced by a sequence with SEQ ID NO: 9 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 10; a heterogeneous population of AAVpoG005 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 10 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 9, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 9 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 9; and/or (2) a heterogeneous group of vp1 proteins , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 10 The product of the sequence; and a heterogeneous group of vp3 proteins, which is the product of the nucleic acid sequence encoding at least amino acids 184 to 716 of SEQ ID NO: 10, wherein: the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 10, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG005 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG006

在某些具體實施例中，提供一種新穎的單離的AAVpoG006衣殼。SEQ ID NO: 11提供編碼AAVpoG006衣殼的核酸序列且SEQ ID NO: 12提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG006 (SEQ ID NO: 12)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG006 (SEQ ID NO: 11)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG006 (SEQ ID NO: 12)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG006 capsid is provided. SEQ ID NO: 11 provides the nucleic acid sequence encoding the AAVpoG006 capsid and SEQ ID NO: 12 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG006 (SEQ ID NO: 12). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG006 (SEQ ID NO: 11). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG006 (SEQ ID NO: 12). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG006衣殼，包含下列一者或多者：(1) AAVpoG006衣殼蛋白，包含：AAVpoG006 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 12的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 11生產的vp1蛋白質、或由與SEQ ID NO: 11至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 12的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG006 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 12的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 11之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 11之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 12的至少約胺基酸137至728之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG006 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 12的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 11之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 11之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 11的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 12之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 12之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 12之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 12中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG006衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG007 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG006 capsid, comprising one or more of the following: (1) AAVpoG006 capsid protein, comprising: AAVpoG006 vp1 protein A heterogeneous population selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 728 of SEQ ID NO: 12, the vp1 protein produced by SEQ ID NO: 11, or produced with SEQ ID NO: 11 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 12; a heterogeneous group of AAVpoG006 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 12 The nucleotide sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 11, or the vp2 protein produced by the sequence with SEQ ID NO: 11 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 12; a heterogeneous population of AAVpoG006 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 12 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 202 to 728, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 11, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 11 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 11; and/or (2) a heterogeneous population of vp1 protein , which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12; a heterogeneous population of vp2 proteins, which is a nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 12 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO: 12 at least amino acid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 12, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG006 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG007

在某些具體實施例中，提供一種新穎的單離的AAVpoG007衣殼。SEQ ID NO: 13提供編碼AAVpoG007衣殼的核酸序列且SEQ ID NO: 14提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG007 (SEQ ID NO: 14)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG007 (SEQ ID NO: 13)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG007 (SEQ ID NO: 14)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG007 capsid is provided. SEQ ID NO: 13 provides the nucleic acid sequence encoding the AAVpoG007 capsid and SEQ ID NO: 14 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG007 (SEQ ID NO: 14). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG007 (SEQ ID NO: 13). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG007 (SEQ ID NO: 14). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG007衣殼，包含下列一者或多者：(1) AAVpoG007衣殼蛋白，包含：AAVpoG007 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 14的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 13生產的vp1蛋白質、或由與SEQ ID NO: 13至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 14的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG007 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 14的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 13之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 13之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 14的至少約胺基酸137至728之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG007 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 14的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 13之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 13之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 13的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 14之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 14之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 14之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 14中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG007衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG008 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG007 capsid, comprising one or more of the following: (1) AAVpoG007 capsid protein, comprising: AAVpoG007 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 728 of the predicted amino acid sequence encoding SEQ ID NO: 14, the vp1 protein produced by SEQ ID NO: 13, or the vp1 protein produced with SEQ ID NO: 13 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 14; a heterogeneous group of AAVpoG007 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 14 The nucleic acid sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 13, or the vp2 protein produced by the sequence with SEQ ID NO: 13 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 14; a heterogeneous population of AAVpoG007 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 14 A vp3 protein produced by a nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 13, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 13 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 13; and/or (2) a heterogeneous population of vp1 protein , which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14; a heterogeneous population of vp2 proteins, which is a nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 14 The product of the sequence; and the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO: 14 at least aminoacid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 14, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG007 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG008

在某些具體實施例中，提供一種新穎的單離的AAVpoG008衣殼。SEQ ID NO: 15提供編碼AAVpoG008衣殼的核酸序列且SEQ ID NO: 16提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG008 (SEQ ID NO: 16)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG008 (SEQ ID NO: 15)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG008 (SEQ ID NO: 16)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG008 capsid is provided. SEQ ID NO: 15 provides the nucleic acid sequence encoding the AAVpoG008 capsid and SEQ ID NO: 16 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG008 (SEQ ID NO: 16). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG008 (SEQ ID NO: 15). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG008 (SEQ ID NO: 16). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG008衣殼，包含下列一者或多者：(1) AAVpoG008衣殼蛋白，包含：AAVpoG008 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 16的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 15生產的vp1蛋白質、或由與SEQ ID NO: 15至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 16的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG008 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 16的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 15之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 15之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 16的至少約胺基酸137至728的預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG008 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 16的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 15之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 15之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 15的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 16之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 16之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 16之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 16中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG008衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG009 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG008 capsid, comprising one or more of the following: (1) AAVpoG008 capsid protein, comprising: AAVpoG008 vp1 protein A heterogeneous population selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 728 of SEQ ID NO: 16, the vp1 protein produced by SEQ ID NO: 15, or produced with SEQ ID NO: 15 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 16; a heterogeneous group of AAVpoG008 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 16 The nucleotide sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 15, or the vp2 protein produced by the sequence with SEQ ID NO: 15 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 16; a heterogeneous population of AAVpoG008 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 16 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 202 to 728, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 15, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 15 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 15; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 16; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 728 of SEQ ID NO: 16 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO: 16 at least aminoacid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 16, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG008 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG009

在某些具體實施例中，提供一種新穎的單離的AAVpoG009衣殼。SEQ ID NO: 17提供編碼AAVpoG009衣殼的核酸序列且SEQ ID NO: 18提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG009 (SEQ ID NO: 18)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG009 (SEQ ID NO: 17)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG009 (SEQ ID NO: 18)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG009 capsid is provided. SEQ ID NO: 17 provides the nucleic acid sequence encoding the AAVpoG009 capsid and SEQ ID NO: 18 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG009 (SEQ ID NO: 18). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG009 (SEQ ID NO: 17). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG009 (SEQ ID NO: 18). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG009衣殼，包含下列一者或多者：(1) AAVpoG009衣殼蛋白，包含：AAVpoG009 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 18的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 17生產的vp1蛋白質、或由與SEQ ID NO: 17至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 18的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG009 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 18的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 17之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 17之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 18的至少約胺基酸137至728之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG009 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 18的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 17之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 17之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 17的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 18之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 18之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 18之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 18中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG009衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG012 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG009 capsid, comprising one or more of the following: (1) AAVpoG009 capsid protein, comprising: AAVpoG009 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 728 of the predicted amino acid sequence encoding SEQ ID NO: 18, the vp1 protein produced by SEQ ID NO: 17, or the vp1 protein produced with SEQ ID NO: 17 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 18; a heterogeneous group of AAVpoG009 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 18 The nucleic acid sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 17, or the vp2 protein produced by the sequence with SEQ ID NO: 17 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 18; a heterogeneous population of AAVpoG009 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 18 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 17, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 17 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 17; and/or (2) a heterogeneous population of vp1 protein , which is the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18; a heterogeneous population of vp2 proteins, which is a nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 18 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleotide sequence of coding SEQ ID NO: 18 at least aminoacid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 18, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG009 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG012

在某些具體實施例中，提供一種新穎的單離的AAVpoG012衣殼。SEQ ID NO: 19提供編碼AAVpoG012衣殼的核酸序列且SEQ ID NO: 20提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG012 (SEQ ID NO: 20)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG012 (SEQ ID NO: 19)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG012 (SEQ ID NO: 20)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG012 capsid is provided. SEQ ID NO: 19 provides the nucleic acid sequence encoding the AAVpoG012 capsid and SEQ ID NO: 20 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG012 (SEQ ID NO: 20). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG012 (SEQ ID NO: 19). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG012 (SEQ ID NO: 20). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG012衣殼，包含下列一者或多者：(1) AAVpoG012衣殼蛋白，包含：AAVpoG012 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 20的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 19生產的vp1蛋白質、或由與SEQ ID NO: 19至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 20的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG012 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 20的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 19之至少核苷酸409至2187的序列所生產的vp2蛋白質、或由與SEQ ID NO: 19之至少核苷酸409至2187為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 20的至少約胺基酸137至728之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG012 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 20的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 19之至少核苷酸604至2187的序列所生產的vp3蛋白質、或由與SEQ ID NO: 19之至少核苷酸604至2187為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 19的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 20之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 20之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 20之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 20中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG012衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導基因產物在宿主細胞中表現的序列。 AAVpoG013 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG012 capsid, comprising one or more of the following: (1) AAVpoG012 capsid protein, comprising: AAVpoG012 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 728 of the predicted amino acid sequence encoding SEQ ID NO: 20, the vp1 protein produced by SEQ ID NO: 19, or the vp1 protein produced with SEQ ID NO: 19 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 20; a heterogeneous group of AAVpoG012 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 20 The nucleotide sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2187 of SEQ ID NO: 19, or the vp2 protein produced by the sequence with SEQ ID NO: 19 at least nucleotides 409 to 2187 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 20; a heterogeneous population of AAVpoG012 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 20 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2187 of SEQ ID NO: 19, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2187 of SEQ ID NO: 19 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 19; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 728 of SEQ ID NO: 20 The product of the sequence; and the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:20 at least aminoacid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 20, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG012 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to a sequence directing expression of the gene product in a host cell. AAVpoG013

在某些具體實施例中，提供一種新穎的單離的AAVpoG013衣殼。SEQ ID NO: 21提供編碼AAVpoG013衣殼的核酸序列且SEQ ID NO: 22提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG013 (SEQ ID NO: 22)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG013 (SEQ ID NO: 21)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG013 (SEQ ID NO: 22)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG013 capsid is provided. SEQ ID NO: 21 provides the nucleic acid sequence encoding the AAVpoG013 capsid and SEQ ID NO: 22 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG013 (SEQ ID NO: 22). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG013 (SEQ ID NO: 21). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG013 (SEQ ID NO: 22). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG013衣殼，包含下列一者或多者：(1) AAVpoG013衣殼蛋白，包含：AAVpoG013 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 22的1至728之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 21生產的vp1蛋白質、或由與SEQ ID NO: 21至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 22的1至728之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG013 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 22的至少約胺基酸137至728的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 21之至少核苷酸409至2187的序列所生產的vp2蛋白質、或由與SEQ ID NO: 21之至少核苷酸409至2187為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 22的至少約胺基酸137至728之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG013 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 22的至少約胺基酸202至728之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 21之至少核苷酸604至2187的序列所生產的vp3蛋白質、或由與SEQ ID NO: 21之至少核苷酸604至2187為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 21的至少約胺基酸202至728之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 22之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 22之至少約胺基酸137至728之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 22之至少胺基酸202至728的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 22中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG013衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG013 capsid, comprising one or more of the following: (1) AAVpoG013 capsid protein, comprising: AAVpoG013 vp1 protein The heterogeneous population is selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 728 of SEQ ID NO: 22, the vp1 protein produced by SEQ ID NO: 21, or produced with SEQ ID NO: 21 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 728 of NO: 22; a heterogeneous group of AAVpoG013 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 22 The nucleic acid sequence of the predicted amino acid sequence of 728 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2187 of SEQ ID NO: 21, or the vp2 protein produced by the sequence with SEQ ID NO: 21 at least nucleotides 409 to 2187 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 22; a heterogeneous population of AAVpoG013 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 22 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 202 to 728, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2187 of SEQ ID NO: 21, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2187 of SEQ ID NO: 21 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 728 of SEQ ID NO: 21; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 728 of SEQ ID NO: 22 The product of the sequence; and the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:22 at least amino acid 202 to 728, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 22, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG013 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell.

在某些具體實施例中，具有如本文所述的poG013衣殼的rAAV係非常適合用於將基因遞送至肝臟。在某些具體實施例中，rAAVpoG013載體係非常適合用於基因編輯在肝臟中的目標。在其它具體實施例中，可選擇rAAVpoG013以及利用其之組成物及方案以用於靶向其它組織或細胞。 AAVpoG014 In certain embodiments, rAAV lines with poG013 capsids as described herein are well suited for gene delivery to the liver. In certain embodiments, the rAAVpoG013 vector is well suited for gene editing targeting in the liver. In other embodiments, rAAVpoG013 and compositions and protocols utilizing it can be selected for targeting other tissues or cells. AAVpoG014

在某些具體實施例中，提供一種新穎的單離的AAVpoG014衣殼。SEQ ID NO: 23提供編碼AAVpoG014衣殼的核酸序列且SEQ ID NO: 24提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG014 (SEQ ID NO: 24)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG014 (SEQ ID NO: 23)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG014 (SEQ ID NO: 24)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG014 capsid is provided. SEQ ID NO: 23 provides the nucleic acid sequence encoding the AAVpoG014 capsid and SEQ ID NO: 24 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG014 (SEQ ID NO: 24). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG014 (SEQ ID NO: 23). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG014 (SEQ ID NO: 24). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG014衣殼，包含下列一者或多者：(1) AAVpoG014衣殼蛋白，包含：AAVpoG014 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 24的1至727之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 23生產的vp1蛋白質、或由與SEQ ID NO: 23至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 24的1至727之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG014 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 24的至少約胺基酸137至727的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 23之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 23之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 24的至少約胺基酸137至727之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG014 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 24的至少約胺基酸202至727之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 23之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 23之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 23的至少約胺基酸202至727之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 24之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 24之至少約胺基酸137至727之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 24之至少胺基酸202至727的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 24中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG014衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG015 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG014 capsid, comprising one or more of the following: (1) AAVpoG014 capsid protein, comprising: AAVpoG014 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 727 of the predicted amino acid sequence encoding SEQ ID NO: 24, the vp1 protein produced by SEQ ID NO: 23, or the vp1 protein produced with SEQ ID NO: 23 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 727 of NO: 24; a heterogeneous group of AAVpoG014 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 24 The nucleic acid sequence of the predicted amino acid sequence of 727 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 23, or the vp2 protein produced by the sequence with SEQ ID NO: 23 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 727 of SEQ ID NO: 24; a heterogeneous population of AAVpoG014 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 24 A vp3 protein produced by a nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 23, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 23 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727 of SEQ ID NO: 23; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 727 of SEQ ID NO: 24 The product of sequence; And the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:24 at least aminoacid 202 to 727, wherein: vp1, vp2 and vp3 protein contain subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 24, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG014 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG015

在某些具體實施例中，提供一種新穎的單離的AAVpoG015衣殼。SEQ ID NO: 25提供編碼AAVpoG015衣殼的核酸序列且SEQ ID NO: 26提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG015 (SEQ ID NO: 26)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG015 (SEQ ID NO: 25)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG015 (SEQ ID NO: 26)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG015 capsid is provided. SEQ ID NO: 25 provides the nucleic acid sequence encoding the AAVpoG015 capsid and SEQ ID NO: 26 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG015 (SEQ ID NO: 26). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG015 (SEQ ID NO: 25). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG015 (SEQ ID NO: 26). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG015衣殼，包含下列一者或多者：(1) AAVpoG015衣殼蛋白，包含：AAVpoG015 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 26的1至727之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 25生產的vp1蛋白質、或由與SEQ ID NO: 25至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 26的1至727之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG015 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 26的至少約胺基酸137至727的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 25之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 25之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 26的至少約胺基酸137至727之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG015 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 26的至少約胺基酸202至727之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 25之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 25之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 25的至少約胺基酸202至727之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 26之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 26之至少約胺基酸137至727之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 26之至少胺基酸202至727的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 26中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG015衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG015 capsid, comprising one or more of the following: (1) AAVpoG015 capsid protein, comprising: AAVpoG015 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 727 of the predicted amino acid sequence encoding SEQ ID NO: 26, the vp1 protein produced by SEQ ID NO: 25, or the vp1 protein produced with SEQ ID NO: 25 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 727 of NO: 26; a heterogeneous group of AAVpoG015 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 26 The nucleic acid sequence of the predicted amino acid sequence of 727 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 25, or the vp2 protein produced by the sequence with SEQ ID NO: 25 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 727 of SEQ ID NO: 26; a heterogeneous population of AAVpoG015 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 26 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 202 to 727, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 25, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 604 to 2184 of SEQ ID NO: 25 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727 of SEQ ID NO: 25; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acids 137 to 727 of SEQ ID NO: 26 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:26 at least amino acid 202 to 727, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 26, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG015 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell.

在某些具體實施例中，具有如本文所述的poG015衣殼的rAAV載體係非常適合用於將基因遞送至肝臟。在某些具體實施例中，具有poG015衣殼的rAAV載體係非常適合用於遞送基因編輯核酸酶及/或供體構築體至肝臟的。在其它具體實施例中，可選擇具有poG015衣殼的rAAV載體以及利用其之組成物及方案以用於靶向其它組織或細胞。In certain embodiments, rAAV vectors with poG015 capsids as described herein are well suited for gene delivery to the liver. In certain embodiments, rAAV vectors with poG015 capsids are well suited for delivering gene editing nucleases and/or donor constructs to the liver. In other embodiments, rAAV vectors with poG015 capsids and compositions and protocols utilizing them can be selected for targeting other tissues or cells.

在某些具體實施例中，具有如本文所述的poG015衣殼的rAAV載體係非常適合用於將基因遞送至心臟。在某些具體實施例中，具有poG015衣殼的rAAV載體係非常適合用於遞送基因編輯核酸酶及/或供體構築體至心臟。In certain embodiments, rAAV vectors with poG015 capsids as described herein are well suited for gene delivery to the heart. In certain embodiments, rAAV vectors with poG015 capsids are well suited for delivering gene editing nucleases and/or donor constructs to the heart.

在某些具體實施例中，具有如本文所述的poG015衣殼的rAAV載體係非常適合用於將基因遞送至肌肉。在某些具體實施例中，具有poG015衣殼的rAAV載體係非常適合用於遞送基因編輯核酸酶及/或供體構築體至肌肉。 AAVpoG016 In certain embodiments, rAAV vectors with poG015 capsids as described herein are well suited for gene delivery to muscle. In certain embodiments, rAAV vectors with poG015 capsids are well suited for delivering gene editing nucleases and/or donor constructs to muscle. AAVpoG016

在某些具體實施例中，提供一種新穎的單離的AAVpoG016衣殼。SEQ ID NO: 27提供編碼AAVpoG016衣殼的核酸序列且SEQ ID NO: 28提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG016 (SEQ ID NO: 28)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG016 (SEQ ID NO: 27)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG016 (SEQ ID NO: 28)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG016 capsid is provided. SEQ ID NO: 27 provides the nucleic acid sequence encoding the AAVpoG016 capsid and SEQ ID NO: 28 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG016 (SEQ ID NO: 28). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG016 (SEQ ID NO: 27). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG016 (SEQ ID NO: 28). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG016衣殼，包含下列一者或多者：(1) AAVpoG016衣殼蛋白，包含：AAVpoG016 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 28的1至727之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 27生產的vp1蛋白質、或由與SEQ ID NO: 27至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 28的1至727之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG016 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 28的至少約胺基酸137至727的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 27之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 27之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 28的至少約胺基酸137至727之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG016 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 28的至少約胺基酸202至727之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 27之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 27之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 27的至少約胺基酸202至727之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 28之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 28之至少約胺基酸137至727之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 28之至少胺基酸202至727的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 28中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG016衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG017 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG016 capsid, comprising one or more of the following: (1) AAVpoG016 capsid protein, comprising: AAVpoG016 vp1 protein A heterogeneous population selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 727 of SEQ ID NO: 28, the vp1 protein produced by SEQ ID NO: 27, or produced with SEQ ID NO: 27 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 727 of NO: 28; a heterogeneous group of AAVpoG016 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 28 The nucleic acid sequence of the predicted amino acid sequence of 727 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 27, or the vp2 protein produced by the sequence with SEQ ID NO: 27 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 727 of SEQ ID NO: 28; a heterogeneous population of AAVpoG016 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 28 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 27, or At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 604 to 2184 of SEQ ID NO: 27 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727 of SEQ ID NO: 27; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 727 of SEQ ID NO: 28 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:28 at least aminoacid 202 to 727, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 28, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG016 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG017

在某些具體實施例中，提供一種新穎的單離的AAVpoG017衣殼。SEQ ID NO: 29提供編碼AAVpoG017衣殼的核酸序列且SEQ ID NO: 30提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG017 (SEQ ID NO: 30)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG017 (SEQ ID NO: 29)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG017 (SEQ ID NO: 30)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG017 capsid is provided. SEQ ID NO: 29 provides the nucleic acid sequence encoding the AAVpoG017 capsid and SEQ ID NO: 30 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG017 (SEQ ID NO: 30). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG017 (SEQ ID NO: 29). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG017 (SEQ ID NO: 30). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG017衣殼，包含下列一者或多者：(1) AAVpoG017衣殼蛋白，包含：AAVpoG017 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 30的1至727之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 29生產的vp1蛋白質、或由與SEQ ID NO: 29至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 30的1至727之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG017 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 30的至少約胺基酸137至727的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 29之至少核苷酸409至2184的序列所生產的vp2蛋白質、或由與SEQ ID NO: 29之至少核苷酸409至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 30的至少約胺基酸137至727之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG017 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 30的至少約胺基酸202至727之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 29之至少核苷酸604至2184的序列所生產的vp3蛋白質、或由與SEQ ID NO: 29之至少核苷酸604至2184為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 29的至少約胺基酸202至727之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 30之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 30之至少約胺基酸137至727之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 30之至少胺基酸202至727的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 30中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG017衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG018 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG017 capsid, comprising one or more of the following: (1) AAVpoG017 capsid protein, comprising: AAVpoG017 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 727 of the predicted amino acid sequence encoding SEQ ID NO: 30, the vp1 protein produced by SEQ ID NO: 29, or the vp1 protein produced with SEQ ID NO: 29 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 727 of NO: 30; a heterogeneous group of AAVpoG017 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 30 The nucleic acid sequence of the predicted amino acid sequence of 727 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2184 of SEQ ID NO: 29, or the vp2 protein produced by the sequence with SEQ ID NO: 29 at least nucleotides 409 to 2184 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 727 of SEQ ID NO: 30; a heterogeneous population of AAVpoG017 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 30 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 202 to 727, a vp3 protein produced by a sequence comprising at least nucleotides 604 to 2184 of SEQ ID NO: 29, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 604 to 2184 of SEQ ID NO: 29 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 202 to 727 of SEQ ID NO: 29; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 727 of SEQ ID NO: 30 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:30 at least amino acid 202 to 727, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 30, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in the AAVpoG017 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG018

在某些具體實施例中，提供一種新穎的單離的AAVpoG018衣殼。SEQ ID NO: 31提供編碼AAVpoG018衣殼的核酸序列且SEQ ID NO: 32提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG018 (SEQ ID NO: 32)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG018 (SEQ ID NO: 31)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG018 (SEQ ID NO: 32)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG018 capsid is provided. SEQ ID NO: 31 provides the nucleic acid sequence encoding the AAVpoG018 capsid and SEQ ID NO: 32 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG018 (SEQ ID NO: 32). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG018 (SEQ ID NO: 31). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG018 (SEQ ID NO: 32). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG018衣殼，包含下列一者或多者：(1) AAVpoG018衣殼蛋白，包含：AAVpoG018 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 32的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 31生產的vp1蛋白質、或由與SEQ ID NO: 31至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 32的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG018 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 32的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 31之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 31之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 32的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG018 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 32的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 31之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 31之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 31的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 32之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 32之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 32之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 32中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG018衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG019 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG018 capsid, comprising one or more of the following: (1) AAVpoG018 capsid protein, comprising: AAVpoG018 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 32, the vp1 protein produced by SEQ ID NO: 31, or the vp1 protein produced with SEQ ID NO: 31 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 32; a heterogeneous group of AAVpoG018 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 32 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 31, or the vp2 protein produced by the sequence with SEQ ID NO: 31 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 32; a heterogeneous population of AAVpoG018 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 32 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 31, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 31 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 31; and/or (2) a heterogeneous population of vp1 proteins , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 32 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO: 32 at least aminoacid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 32, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG018 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG019

在某些具體實施例中，提供一種新穎的單離的AAVpoG019衣殼。SEQ ID NO: 33提供編碼AAVpoG019衣殼的核酸序列且SEQ ID NO: 34提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG019 (SEQ ID NO: 34)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG019 (SEQ ID NO: 33)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG019 (SEQ ID NO: 34)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG019 capsid is provided. SEQ ID NO: 33 provides the nucleic acid sequence encoding the AAVpoG019 capsid and SEQ ID NO: 34 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG019 (SEQ ID NO: 34). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG019 (SEQ ID NO: 33). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG019 (SEQ ID NO: 34). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG019衣殼，包含下列一者或多者：(1) AAVpoG019衣殼蛋白，包含：AAVpoG019 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 34的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 33生產的vp1蛋白質、或由與SEQ ID NO: 33至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 34的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG019 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 34的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 33之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 33之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 34的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG019 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 34的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 33之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 33之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 33的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 34之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 34之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 34之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 34中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG019衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG020 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG019 capsid, comprising one or more of the following: (1) AAVpoG019 capsid protein, comprising: AAVpoG019 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 34, the vp1 protein produced by SEQ ID NO: 33, or the vp1 protein produced with SEQ ID NO: 33 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 34; a heterogeneous group of AAVpoG019 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 34 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 33, or the vp2 protein produced by the sequence with SEQ ID NO: 33 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 34; a heterogeneous population of AAVpoG019 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 34 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 33, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 33 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 33; and/or (2) a heterogeneous population of vp1 proteins , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 34 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO: 34 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 34, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in the AAVpoG019 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG020

在某些具體實施例中，提供一種新穎的單離的AAVpoG020衣殼。SEQ ID NO: 35提供編碼AAVpoG020衣殼的核酸序列且SEQ ID NO: 36提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG020 (SEQ ID NO: 36)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG020 (SEQ ID NO: 35)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG020 (SEQ ID NO: 36)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG020 capsid is provided. SEQ ID NO: 35 provides the nucleic acid sequence encoding the AAVpoG020 capsid and SEQ ID NO: 36 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG020 (SEQ ID NO: 36). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG020 (SEQ ID NO: 35). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG020 (SEQ ID NO: 36). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG020衣殼，包含下列一者或多者：(1) AAVpoG020衣殼蛋白，包含：AAVpoG020 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 36的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 35生產的vp1蛋白質、或由與SEQ ID NO: 35至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 36的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG020 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 36的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 35之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 35之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 36的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG020 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 36的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 35之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 35之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 35的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 36之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 36之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 36之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 36中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG020衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG021 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG020 capsid, comprising one or more of the following: (1) AAVpoG020 capsid protein, comprising: AAVpoG020 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 36, the vp1 protein produced by SEQ ID NO: 35, or the vp1 protein produced with SEQ ID NO: 35 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 36; a heterogeneous group of AAVpoG020 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 36 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 35, or the vp2 protein produced by the sequence with SEQ ID NO: 35 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 36; a heterogeneous population of AAVpoG020 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 36 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 184 to 716, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 35, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 550 to 2148 of SEQ ID NO: 35 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 35; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 36 The product of the sequence; and a heterogeneous population of vp3 proteins, which is the product of the nucleic acid sequence encoding at least amino acids 184 to 716 of SEQ ID NO: 36, wherein: the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 36, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG020 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG021

在某些具體實施例中，提供一種新穎的單離的AAVpoG021衣殼。SEQ ID NO: 37提供編碼AAVpoG021衣殼的核酸序列且SEQ ID NO: 38提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG021 (SEQ ID NO: 38)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG021 (SEQ ID NO: 37)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG021 (SEQ ID NO: 38)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG021 capsid is provided. SEQ ID NO: 37 provides the nucleic acid sequence encoding the AAVpoG021 capsid and SEQ ID NO: 38 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG021 (SEQ ID NO: 38). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG021 (SEQ ID NO: 37). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG021 (SEQ ID NO: 38). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG021衣殼，包含下列一者或多者：(1) AAVpoG021衣殼蛋白，包含：AAVpoG021 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 38的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 37生產的vp1蛋白質、或由與SEQ ID NO: 37至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 38的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG021 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 38的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 37之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 37之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 38的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG021 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 38的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 37之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 37之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 37的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 38之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 38之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 38之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 38中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG021衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG022 In another specific embodiment, there is provided a recombinant adeno-associated virus (rAAV), which comprises: (A) AAVpoG021 capsid, comprising one or more of the following: (1) AAVpoG021 capsid protein, comprising: AAVpoG021 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 38, the vp1 protein produced by SEQ ID NO: 37, or the vp1 protein produced with SEQ ID NO: 37 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 38; a heterogeneous group of AAVpoG021 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 38 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 37, or the vp2 protein produced by the sequence with SEQ ID NO: 37 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 38; a heterogeneous population of AAVpoG021 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 38 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 37, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 550 to 2148 of SEQ ID NO: 37 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 37; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 38 The product of sequence; And the heterogeneous group of vp3 protein, it is the product of the nucleotide sequence of coding SEQ ID NO:38 at least aminoacid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 38, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG021 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG022

在某些具體實施例中，提供一種新穎的單離的AAVpoG022衣殼。SEQ ID NO: 39提供編碼AAVpoG022衣殼的核酸序列且SEQ ID NO: 40提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG022 (SEQ ID NO: 40)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG022 (SEQ ID NO: 39)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG022 (SEQ ID NO: 40)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG022 capsid is provided. SEQ ID NO: 39 provides the nucleic acid sequence encoding the AAVpoG022 capsid and SEQ ID NO: 40 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG022 (SEQ ID NO: 40). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG022 (SEQ ID NO: 39). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG022 (SEQ ID NO: 40). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG022衣殼，包含下列一者或多者：(1) AAVpoG022衣殼蛋白，包含：AAVpoG022 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 40的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 39生產的vp1蛋白質、或由與SEQ ID NO: 39至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 40的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG022 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 40的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 39之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 39之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 40的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG022 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 40的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 39之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 39之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 39的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 40之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 40之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 40之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 40中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG022衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG023 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG022 capsid, comprising one or more of the following: (1) AAVpoG022 capsid protein, comprising: AAVpoG022 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 40, the vp1 protein produced by SEQ ID NO: 39, or the vp1 protein produced with SEQ ID NO: 39 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 40; a heterogeneous group of AAVpoG022 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 40 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 39, or the vp2 protein produced by the sequence with SEQ ID NO: 39 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 40; a heterogeneous population of AAVpoG022 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 40 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 39, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 550 to 2148 of SEQ ID NO: 39 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 39; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 40 The product of sequence; And the heterogeneous group of vp3 protein, it is the product of the nucleic acid sequence of coding SEQ ID NO:40 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 40, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG022 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG023

在某些具體實施例中，提供一種新穎的單離的AAVpoG023衣殼。SEQ ID NO: 41提供編碼AAVpoG023衣殼的核酸序列且SEQ ID NO: 42提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG023 (SEQ ID NO: 42)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG023 (SEQ ID NO: 41)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG023 (SEQ ID NO: 42)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG023 capsid is provided. SEQ ID NO: 41 provides the nucleic acid sequence encoding the AAVpoG023 capsid and SEQ ID NO: 42 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG023 (SEQ ID NO: 42). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG023 (SEQ ID NO: 41). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG023 (SEQ ID NO: 42). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG023衣殼，包含下列一者或多者：(1) AAVpoG023衣殼蛋白，包含：AAVpoG023 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 42的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 41生產的vp1蛋白質、或由與SEQ ID NO: 41至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 42的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG023 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 42的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 41之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 41之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 42的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG023 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 42的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 41之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 41之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 41的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 42之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 42之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 42之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 42中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG023衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG024 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG023 capsid, comprising one or more of the following: (1) AAVpoG023 capsid protein, comprising: AAVpoG023 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 42, the vp1 protein produced by SEQ ID NO: 41, or the vp1 protein produced with SEQ ID NO: 41 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 42; a heterogeneous group of AAVpoG023 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 42 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 41, or the vp2 protein produced by the sequence with SEQ ID NO: 41 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 42; a heterogeneous population of AAVpoG023 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 42 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 41, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 41 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 41; and/or (2) a heterogeneous population of vp1 proteins , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42; a heterogeneous group of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 42 The product of the sequence; and the heterogeneous population of vp3 protein, it is the product of the nucleotide sequence of coding SEQ ID NO:42 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 42, and optionally further comprises other deamidated a subpopulation of amidated amino acids, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG023 capsid comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG024

在某些具體實施例中，提供一種新穎的單離的AAVpoG024衣殼。SEQ ID NO: 43提供編碼AAVpoG024衣殼的核酸序列且SEQ ID NO: 44提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG024 (SEQ ID NO: 44)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG024 (SEQ ID NO: 43)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG024 (SEQ ID NO: 44)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG024 capsid is provided. SEQ ID NO: 43 provides the nucleic acid sequence encoding the AAVpoG024 capsid and SEQ ID NO: 44 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG024 (SEQ ID NO: 44). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG024 (SEQ ID NO: 43). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG024 (SEQ ID NO: 44). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG024衣殼，包含下列一者或多者：(1) AAVpoG024衣殼蛋白，包含：AAVpoG024 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 44的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 43生產的vp1蛋白質、或由與SEQ ID NO: 43至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 44的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG024 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 44的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 43之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 43之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 44的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG024 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 44的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 43之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 43之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 43的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 44之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 44之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 44之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 44中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG024衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 AAVpoG025 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, comprising: (A) AAVpoG024 capsid, comprising one or more of the following: (1) AAVpoG024 capsid protein, comprising: AAVpoG024 vp1 protein A heterogeneous population selected from the group consisting of vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 716 of SEQ ID NO: 44, the vp1 protein produced by SEQ ID NO: 43, or produced with SEQ ID NO: 43 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 44; a heterogeneous group of AAVpoG024 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 44 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 43, or the vp2 protein produced by the sequence with SEQ ID NO: 43 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 44; a heterogeneous population of AAVpoG024 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 44 A nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 represents a vp3 protein produced, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 43, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of at least nucleotides 550 to 2148 of SEQ ID NO: 43 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 43; and/or (2) a heterogeneous population of vp1 protein , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 44 The product of sequence; And the heterogeneous population of vp3 protein, it is the product of the nucleotide sequence of coding SEQ ID NO:44 at least amino acid 184 to 716, wherein: vp1, vp2 and vp3 protein contain the subpopulation with amino acid modification , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 44, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG024 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. AAVpoG025

在某些具體實施例中，提供一種新穎的單離的AAVpoG025衣殼。SEQ ID NO: 45提供編碼AAVpoG025衣殼的核酸序列且SEQ ID NO: 46提供所編碼的胺基酸序列。本文提供一種rAAV，包含AAVpoG025 (SEQ ID NO: 46)的vp1、vp2及vp3之至少一者。本文亦提供一種rAAV，包含由AAVpoG025 (SEQ ID NO: 45)的vp1、vp2及vp3之至少一者所編碼的AAV衣殼。在某些具體實施例中，vp1、vp2及/或vp3為AAVpoG025 (SEQ ID NO: 46)之全長衣殼蛋白。在其它具體實施例中，vp1、vp2及/或vp3具有N端及/或C端截短(例如，約1至約10個胺基酸的截短)。In certain embodiments, a novel isolated AAVpoG025 capsid is provided. SEQ ID NO: 45 provides the nucleic acid sequence encoding the AAVpoG025 capsid and SEQ ID NO: 46 provides the encoded amino acid sequence. Provided herein is an rAAV comprising at least one of vp1, vp2, and vp3 of AAVpoG025 (SEQ ID NO: 46). Also provided herein is an rAAV comprising an AAV capsid encoded by at least one of vp1, vp2, and vp3 of AAVpoG025 (SEQ ID NO: 45). In certain embodiments, vp1, vp2 and/or vp3 is the full-length capsid protein of AAVpoG025 (SEQ ID NO: 46). In other embodiments, vp1, vp2, and/or vp3 have N-terminal and/or C-terminal truncations (eg, truncations of about 1 to about 10 amino acids).

在另一具體實施例中，提供一種重組腺相關病毒(rAAV)，其包含：(A) AAVpoG025衣殼，包含下列一者或多者：(1) AAVpoG025衣殼蛋白，包含：AAVpoG025 vp1蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 46的1至716之預測的胺基酸序列的核酸序列表現所生產的vp1蛋白質、由SEQ ID NO: 45生產的vp1蛋白質、或由與SEQ ID NO: 45至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 46的1至716之預測的胺基酸序列的核酸序列所生產的vp1蛋白質；AAVpoG025 vp2蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 46的至少約胺基酸137至716的預測的胺基酸序列的核酸序列表現所生產的vp2蛋白質、由包含SEQ ID NO: 45之至少核苷酸409至2148的序列所生產的vp2蛋白質、或由與SEQ ID NO: 45之至少核苷酸409至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 46的至少約胺基酸137至716之預測的胺基酸序列的核酸序列所生產的vp2蛋白質；AAVpoG025 vp3蛋白質之異質族群，選自：藉由從編碼SEQ ID NO: 46的至少約胺基酸184至716之預測的胺基酸序列的核酸序列表現所生產的vp3蛋白質、由包含SEQ ID NO: 45之至少核苷酸550至2148的序列所生產的vp3蛋白質、或由與SEQ ID NO: 45之至少核苷酸550至2148為至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%相同且編碼SEQ ID NO: 45的至少約胺基酸184至716之預測的胺基酸序列的核酸序列所生產的vp3蛋白質；及/或(2) vp1蛋白質之異質族群，其為編碼SEQ ID NO: 46之胺基酸序列的核酸序列的產物；vp2蛋白質之異質族群，其為編碼SEQ ID NO: 46之至少約胺基酸137至716之胺基酸序列的核酸序列的產物；及vp3蛋白質之異質族群，其為編碼SEQ ID NO: 46之至少胺基酸184至716的核酸序列的產物，其中：vp1、vp2及vp3蛋白質含有具有胺基酸修飾的亞群，該修飾包含在SEQ ID NO: 46中的天冬醯胺酸-甘胺酸對中至少兩個高度脫醯胺化的天冬醯胺酸(N)，且可選擇地進一步包含含有其它脫醯胺化的胺基酸的亞群，其中該脫醯胺化造成胺基酸改變；及(B) 在AAVpoG025衣殼中的載體基因體，該載體基因體包含核酸分子，該核酸分子包含AAV反向末端重複序列及編碼基因產物的非AAV核酸序列，該核酸序列可操作地連接至指導該產物在宿主細胞中表現的序列。 B. rAAV載體及組成物 In another specific embodiment, a recombinant adeno-associated virus (rAAV) is provided, which comprises: (A) AAVpoG025 capsid, comprising one or more of the following: (1) AAVpoG025 capsid protein, comprising: AAVpoG025 vp1 protein The heterogeneous population is selected from: the vp1 protein produced by expression of the nucleic acid sequence from 1 to 716 of the predicted amino acid sequence encoding SEQ ID NO: 46, the vp1 protein produced by SEQ ID NO: 45, or the vp1 protein produced with SEQ ID NO: 45 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and encodes a SEQ ID The vp1 protein produced by the nucleic acid sequence of the predicted amino acid sequence of 1 to 716 of NO: 46; a heterogeneous group of AAVpoG025 vp2 proteins selected from the group consisting of at least about amino acids 137 to 137 of encoding SEQ ID NO: 46 The nucleic acid sequence of the predicted amino acid sequence of 716 represents the vp2 protein produced, the vp2 protein produced by the sequence comprising at least nucleotides 409 to 2148 of SEQ ID NO: 45, or the vp2 protein produced by the sequence with SEQ ID NO: 45 at least nucleotides 409 to 2148 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and A vp2 protein produced by a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 137 to 716 of SEQ ID NO: 46; a heterogeneous population of AAVpoG025 vp3 proteins selected from the group consisting of encoding SEQ ID NO: 46 A vp3 protein produced by a nucleic acid sequence representing a predicted amino acid sequence of at least about amino acids 184 to 716, a vp3 protein produced by a sequence comprising at least nucleotides 550 to 2148 of SEQ ID NO: 45, or From at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% from at least nucleotides 550 to 2148 of SEQ ID NO: 45 %, or at least 99% identical and encoding the vp3 protein produced by the nucleic acid sequence of at least about the predicted amino acid sequence of amino acids 184 to 716 of SEQ ID NO: 45; and/or (2) a heterogeneous population of vp1 proteins , which is the product of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46; a heterogeneous population of vp2 proteins, which is the nucleic acid encoding the amino acid sequence of at least about amino acid 137 to 716 of SEQ ID NO: 46 The product of the sequence; and a heterogeneous group of vp3 proteins, which is the product of the nucleic acid sequence encoding at least amino acids 184 to 716 of SEQ ID NO: 46, wherein: the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications , the modification comprises at least two highly deamidated asparagine (N) in the asparagine-glycine pair in SEQ ID NO: 46, and optionally further comprises other deamidated A subset of amino acids that are amidated, wherein the deamidation results in an amino acid change; and (B) a vector gene body in an AAVpoG025 capsid, the vector gene body comprising a nucleic acid molecule comprising an AAV Inverted terminal repeats and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences directing expression of the product in a host cell. B. rAAV vectors and components

於一態樣，本文提供具有本文所述AAV衣殼序列的核酸，包括其片段，用於生產有用於遞送異源基因或其它核酸序列至目標細胞的病毒載體。在某些具體實施例中，提供的rAAV具有本文所述衣殼以及已包裝於該衣殼中的載體基因體，該載體基因體包含非AAV核酸序列。在某些具體實施例中，有用於本文所述組成物及方法中的該載體至少含有AAV衣殼vp1、vp2、及/或vp3、或其片段，此等係由本文提供的序列所編碼。在某些具體實施例中，有用的載體至少含有編碼所選擇的AAV血清型rep蛋白質的序列、或其片段。可選擇地，此種載體可含有AAV cap及rep蛋白質兩者。在提供有AAV rep及 cap兩者的載體中，該AAV rep及AAV cap序列可兩者皆為一種血清型來源者，例如，皆為AAVpoG001、AAVpoG002、AAVpoG003、AAVpoG004、AAVpoG005、AAVpoG006、AAVpoG007、AAVpoG008、AAVpoG009、AAVpoG012、AAVpoG013、AAVpoG014、AAVpoG015、AAVpoG016、AAVpoG017、AAVpoG018、AAVpoG019、AAVpoG020、AAVpoG021、AAVpoG022、AAVpoG023、AAVpoG024、或AAVpoG025來源。或者，可使用的載體中， rep序列來自與提供 cap序列的野生型AAV不同的AAV，例如，來自提供ITR及 rep的相同AAV。 In one aspect, provided herein are nucleic acids having the AAV capsid sequences described herein, including fragments thereof, for use in the production of viral vectors useful for delivering heterologous genes or other nucleic acid sequences to target cells. In certain embodiments, rAAV is provided having a capsid as described herein and a vector gene body packaged in the capsid, the vector gene body comprising a non-AAV nucleic acid sequence. In certain embodiments, the vectors useful in the compositions and methods described herein comprise at least AAV capsid vp1, vp2, and/or vp3, or fragments thereof, encoded by the sequences provided herein. In certain embodiments, useful vectors contain at least a sequence encoding the rep protein of a selected AAV serotype, or a fragment thereof. Alternatively, such vectors may contain both AAV cap and rep proteins. In vectors providing both AAV rep and cap , the AAV rep and AAV cap sequences may both be of one serotype origin, for example, both AAVpoG001, AAVpoG002, AAVpoG003, AAVpoG004, AAVpoG005, AAVpoG006, AAVpoG007, AAVpoG008 , AAVpoG009, AAVpoG012, AAVpoG013, AAVpoG014, AAVpoG015, AAVpoG016, AAVpoG017, AAVpoG018, AAVpoG019, AAVpoG020, AAVpoG021, AAVpoG022, AAVpoG023, AAVpoG024, or AAVpoG025 origin. Alternatively, vectors may be used in which the rep sequence is from an AAV different from the wild-type AAV providing the cap sequence, eg, from the same AAV providing the ITR and rep .

在一具體實施例中， rep及 cap序列由各别的來源(例如，各别的載體、或宿主細胞及載體)表現。在另一具體實施例中，此等 rep序列在框內與不同AAV血清型的 cap序列融合以形成嵌合AAV載體，如美國專利號7,282,199所述的AAV2/8，其藉由引用併入本文。可選擇地，載體進一步含有袖珍基因，該袖珍基因包含所選擇的轉基因，該轉基因兩側為AAV 5’ ITR及AAV 3’ ITR。在另一具體實施例中，AAV為自互補AAV (sc-AAV)(參見US 2012/0141422，其藉由引用併入本文)。自互補載體包裝一反向重複基因體，其可折疊成dsDNA，而無需DNA合成或多個載體基因體之間的鹼基配對。因為scAAV不需於表現之前將單股DNA (ssDNA)基因體轉化成為雙股DNA (dsDNA)，scAAV為更有效率的載體。然而，此效率的代價係載體之一半編碼能力的喪失，scAAV有用於小蛋白質編碼基因(至多~55 kd)及任何目前可使用之基於RNA的治療。 In one embodiment, the rep and cap sequences are expressed from separate sources (eg, separate vectors, or host cells and vectors). In another embodiment, these rep sequences are fused in frame with the cap sequences of different AAV serotypes to form chimeric AAV vectors, such as AAV2/8 as described in U.S. Patent No. 7,282,199, which is incorporated herein by reference . Optionally, the vector further contains a minigene comprising a selected transgene flanked by AAV 5'ITR and AAV 3'ITR. In another specific embodiment, the AAV is a self-complementary AAV (sc-AAV) (see US 2012/0141422, which is incorporated herein by reference). Self-complementary vectors package an inverted repeat gene body that can be folded into dsDNA without DNA synthesis or base pairing between multiple vector gene bodies. Because scAAV does not require conversion of single-stranded DNA (ssDNA) gene bodies to double-stranded DNA (dsDNA) prior to expression, scAAV is a more efficient vector. However, this efficiency comes at the cost of the loss of half of the vector's coding capacity, and scAAV is useful for small protein-coding genes (up to ~55 kd) and any RNA-based therapy currently available.

一種AAV之衣殼以異源衣殼蛋白質替代的假型載體於此處為有用的。例如，利用如本文所述AAVpoG001、AAVpoG002、AAVpoG003、AAVpoG004、AAVpoG005、AAVpoG006、AAVpoG007、AAVpoG008、AAVpoG009、AAVpoG012、AAVpoG013、AAVpoG014、AAVpoG015、AAVpoG016、AAVpoG017、AAVpoG018、AAVpoG019、AAVpoG020、AAVpoG021、AAVpoG022、AAVpoG023、AAVpoG024、或AAVpoG025衣殼的AAV載體，具有AAV2 ITR。參見Mussolini等人。除非另有指出，本文所述AAV ITR及其它所選擇的AAV組件，可各別選自任何AAV血清型，包括但不限於AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9或其它已知及未知的AAV血清型。於一理想的具體實施例中，使用AAV血清型2之ITR。然而，可選擇來自其它適合血清型的ITR。此等ITR或其它AAV組件可使用所屬技術領域中具通常知識者可用的技術自一AAV血清型容易地單離。此種AAV可自學術、商業或公共資源(例如，美國典型培養物保藏中心(the American Type Culture Collection, Manassas, VA))單離或獲得。或者，AAV序列可通過合成或其它適合的手段藉由參考公開的序列而獲得，該公開的序列例如可在文獻或資料庫中獲得，諸如例如GenBank、PubMed等。A pseudotyped vector in which the capsid of AAV is replaced with a heterologous capsid protein is useful herein.例如，利用如本文所述AAVpoG001、AAVpoG002、AAVpoG003、AAVpoG004、AAVpoG005、AAVpoG006、AAVpoG007、AAVpoG008、AAVpoG009、AAVpoG012、AAVpoG013、AAVpoG014、AAVpoG015、AAVpoG016、AAVpoG017、AAVpoG018、AAVpoG019、AAVpoG020、AAVpoG021、AAVpoG022、AAVpoG023、AAVpoG024 , or AAVpoG025 capsid AAV vectors with AAV2 ITRs. See Mussolini et al. Unless otherwise indicated, the AAV ITRs described herein, and other selected AAV components, may each be selected from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or Other known and unknown AAV serotypes. In a desirable embodiment, the ITR of AAV serotype 2 is used. However, ITRs from other suitable serotypes can be selected. These ITRs or other AAV components can be readily isolated from an AAV serotype using techniques available to those of ordinary skill in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (eg, the American Type Culture Collection (Manassas, VA)). Alternatively, AAV sequences may be obtained synthetically or by other suitable means by reference to published sequences, eg, available in the literature or in databases, such as, eg, GenBank, PubMed, and the like.

本文提供的rAAV包含載體基因體。該載體基因體至少由下列組成：非AAV或異源核酸序列(轉基因)(如下文所述)、調節序列、以及5’及3’AAV反向末端重複序列(ITR)。正是這種袖珍基因被包裝於衣殼蛋白中並被遞送到所選擇的目標細胞或目標組織。The rAAV provided herein comprise vector gene bodies. The vector gene body consists of at least the following: non-AAV or heterologous nucleic acid sequence (transgene) (as described below), regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this pocket gene that is packaged in the capsid protein and delivered to the target cell or tissue of choice.

轉基因為與轉基因兩側的載體序列異源的核酸序列，該轉基因編碼感興趣的多肽、蛋白質或其它產物。該核酸編碼序列係以在目標細胞中允許轉基因轉錄、轉譯及/或表現的方式而可操作地連接至調節組件。異源核酸序列(轉基因)可衍生自任何生物體。AAV可包含一或多個轉基因。A transgene is a nucleic acid sequence that is heterologous to the vector sequences flanking the transgene encoding a polypeptide, protein or other product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that permits transcription, translation and/or expression of the transgene in the target cell. A heterologous nucleic acid sequence (transgene) can be derived from any organism. AAV can contain one or more transgenes.

如本文所使用，術語「目標細胞」及「目標組織」可指意圖藉由測試AAV轉導的任何細胞或組織。該術語可指肌肉、肝臟、肺臟、呼吸道上皮、中樞神經系統、神經元、眼睛(眼細胞)或心臟之任何一種或多種。在一具體實施例中，目標組織為肝臟。在另一具體實施例中，目標組織為心臟。在另一具體實施例中，目標組織為腦。在另一具體實施例中，目標組織為肌肉。在另一具體實施例中，目標組織為視網膜。As used herein, the terms "target cell" and "target tissue" may refer to any cell or tissue intended to be transduced by a test AAV. The term may refer to any one or more of muscle, liver, lung, respiratory epithelium, central nervous system, neurons, eye (ocular cells), or heart. In a specific embodiment, the target tissue is the liver. In another specific embodiment, the target tissue is the heart. In another specific embodiment, the target tissue is the brain. In another specific embodiment, the target tissue is muscle. In another specific embodiment, the target tissue is the retina.

如本文所使用，術語「哺乳動物受試者」或「受試者」包括需要本文所述治療或預防方法的任何哺乳動物，特別是包括人類。其它需要此種治療或預防的哺乳動物包括狗、貓、或其它馴養動物、馬、家畜、包括非人類靈長類的實驗動物等。受試者可為雄性或雌性。As used herein, the term "mammalian subject" or "subject" includes any mammal in need of the methods of treatment or prevention described herein, including specifically humans. Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, experimental animals including non-human primates, and the like. A subject can be male or female.

如本文所使用，術語「宿主細胞」可指包裝細胞株(packaging cell line)，其中rAAV係由質體產生。或者，術語「宿主細胞」可指期望轉基因表現之目標細胞。As used herein, the term "host cell" may refer to a packaging cell line in which rAAV is produced from plastids. Alternatively, the term "host cell" may refer to a target cell in which expression of a transgene is desired.

如本文所使用，rAAV之「儲料(stock)」係指一群rAAV。儘管由於脫醯胺化，其衣殼蛋白質具有異質性，但是儲料中的rAAV被預期共有相同的載體基因體。儲料可包括具有衣殼之rAAV載體，該衣殼具有例如所選擇AAV衣殼蛋白質及所選擇生產系統的特徵性的異質脫醯胺化樣式。儲料可從單個生產系統生產，或從生產系統的多個運行中儲集。可選擇各種生產系統，包括但不限於本文所述彼等。 治療性轉基因 As used herein, a "stock" of rAAV refers to a population of rAAV. Despite the heterogeneity of their capsid proteins due to deamidation, the rAAV in the stock were expected to share the same vector gene body. The stock may comprise rAAV vectors with capsids having, for example, the selected AAV capsid protein and the heterogeneous deamidation pattern characteristic of the selected production system. Stock can be produced from a single production system, or can be pooled from multiple runs of a production system. Various production systems can be chosen, including but not limited to those described herein. therapeutic transgene

由轉基因編碼的有用產物包括多種基因產物，其替代缺陷或缺乏的基因，或者不活化或「剔除(knock-out)」、或「減弱(knock-down)」或降低以不希望的高水準表現的基因表現，或者遞送具有所欲治療效果的基因產物。在大部份具體實施例中，治療將為「體細胞基因治療(somatic gene therapy)」，即，將基因轉移到不產生精子或卵子的身體的細胞中。在某些具體實施例中，轉基因表現具有天然人類序列的序列之蛋白質。然而，在其它具體實施例中，表現合成蛋白質。此類蛋白質可意圖用於治療人類，或在其它具體實施例中，設計用於治療動物，包括諸如犬科或貓科族群的伴侶動物，或者用於治療與人群接觸的家畜或其它動物。Useful products encoded by transgenes include various gene products that replace defective or absent genes, or are either inactivated or "knock-out", or "knock-down" or reduced to undesirably high levels of expression gene expression, or delivery of a gene product with a desired therapeutic effect. In most embodiments, the treatment will be "somatic gene therapy," which involves transferring genes into cells of the body that do not produce sperm or eggs. In certain embodiments, the transgene expresses a protein having a sequence of a native human sequence. However, in other embodiments, synthetic proteins are represented. Such proteins may be intended for use in the treatment of humans, or in other embodiments, designed for use in the treatment of animals, including companion animals such as canine or feline populations, or in the treatment of livestock or other animals in contact with humans.

適合的基因產物之例可包括彼等與家族性高膽固醇血症、肌肉營養不良、囊腫纖維化及罕見疾病或孤兒病有關者。此種罕見疾病之例可包括脊髓性肌肉萎縮症(SMA)、杭丁頓氏舞蹈症(Huntingdon’s Disease)、雷特氏症候群(Rett Syndrome)(例如，甲基-CpG-結合蛋白2(MeCP2)；UniProtKB-P51608)、肌肉萎縮性脊髓側索硬化症(ALS)、裘馨氏型肌肉萎縮症(Duchenne Type Muscular dystrophy)、弗裏德賴希共濟失調(Friedrichs Ataxia)(例如，共濟失調蛋白(frataxin))、與脊髓小腦性共濟失調症第2型(spinocerebellar ataxia type 2，SCA2)/ALS有關的ATXN2；與ALS有關的TDP-43、顆粒蛋白前體(progranulin，PRGN)(與非阿茲海默氏症的腦退化症有關，包括額顳葉失智症(FTD)、進行性非流暢性失語症(progressive non-fluent aphasia，PNFA)及語意型失智症(semantic dementia)等。參見例如www.orpha.net/consor/cgi-bin/Disease_Search_List.php；rarediseases.info.nih.gov/diseases。在一具體實施例中，轉基因並非人類低密度脂蛋白受體(hLDLR)。在另一具體實施例中，轉基因並非經工程化的人類低密度脂蛋白受體(hLDLR)變異體，諸如彼等WO 2015/164778中所述者。Examples of suitable gene products may include those associated with familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare diseases may include spinal muscular atrophy (SMA), Huntingdon's Disease, Rett Syndrome (e.g., methyl-CpG-binding protein 2 (MeCP2) ; UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedrichs Ataxia (eg, ataxia ATXN2 related to spinocerebellar ataxia type 2 (SCA2)/ALS; TDP-43 related to ALS, progranulin (PRGN) (related to Non-Alzheimer's disease dementia, including frontotemporal dementia (FTD), progressive non-fluent aphasia (progressive non-fluent aphasia, PNFA) and semantic dementia (semantic dementia) See eg www.orpha.net/consor/cgi-bin/Disease_Search_List.php; rarediseases.info.nih.gov/diseases. In a specific embodiment, the transgene is not human low density lipoprotein receptor (hLDLR). In In another embodiment, the transgene is not an engineered variant of the human low density lipoprotein receptor (hLDLR), such as those described in their WO 2015/164778.

適合的基因之例可包括例如荷爾蒙以及生長及分化因子，包括但不限於胰島素、升糖素、類升糖素肽-1 (GLP1)、生長激素(GH)、副甲狀腺素(PTH)、生長激素釋放因子(GRF)、促濾泡素(FSH)、黃體激素(LH)、人類絨毛膜促性腺激素(hCG)、血管內皮生長因子(VEGF)、血管生成素(angiopoietin)、血管抑制素、顆粒球群落刺激因子(GCSF)、紅血球生成素(EPO)(包括例如人類、犬或貓epo)、結締組織生長因子(CTGF)、神經營養因子，包括例如鹼性纖維母細胞生長因子(bFGF)、酸性纖維母細胞生長因子(aFGF)、表皮生長因子(EGF)、血小板衍生生長因子(PDGF)、胰島素生長因子I及II (IGF-I及IGF-II)、轉形生長因子α超家族之任一者，包括TGFα、活化素(activin)、抑制素(inhibin)、或骨形態發生蛋白(BMP) BMPs 1-15之任一者、生長因子之調蛋白(heregluin)/神經調節蛋(neuregulin)/ARIA/neu分化因子(NDF)家族之任一者、神經生長因子(NGF)、腦衍生的神經營養因子(BDNF)、神經滋養蛋白(neurotrophin) NT-3及NT-4/5、睫狀神經營養因子(ciliary neurotrophic factor，CNTF)、神經膠細胞株衍生的神經營養因子(GDNF)、神經營養素(neurturin)、集聚蛋白(agrin)、腦訊息蛋白(semaphorin)/折疊蛋白(collapsin)家族之任一者、神經軸突導向分子-1 (netrin-1)及神經軸突導向分子-2 (netrin-2)、肝細胞生長因子(HGF)、肝配蛋白(ephrin)、頭蛋白(noggin)、音猬因子(sonic hedgehog)及酪胺酸羥化酶。Examples of suitable genes may include, for example, hormones and growth and differentiation factors including, but not limited to, insulin, glucagon, glucagon-like peptide-1 (GLP1), growth hormone (GH), parathyroid hormone (PTH), growth Hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, Granulocyte colony stimulating factor (GCSF), erythropoietin (EPO) (including e.g. human, canine or feline epo), connective tissue growth factor (CTGF), neurotrophic factors including e.g. basic fibroblast growth factor (bFGF) , acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), transforming growth factor α superfamily Any, including TGFα, activin (activin), inhibitor (inhibin), or bone morphogenetic protein (BMP) any one of BMPs 1-15, growth factor heregluin/neuregulin )/ARIA/neu differentiation factor (NDF) family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (neurotrophin) NT-3 and NT-4/5, ciliary Ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin (neurturin), agrin, semaphorin/collapsin family Any of them, axon guidance molecule-1 (netrin-1) and axon guidance molecule-2 (netrin-2), hepatocyte growth factor (HGF), ephrin (ephrin), noggin (noggin ), sonic hedgehog and tyrosine hydroxylase.

其它有用的轉基因產物包括調節免疫系統的蛋白質，包括但不限於細胞激素及淋巴激素，如促血小板生成素(thrombopoietin，TPO)、介白素(IL) IL-1至IL-36(包括例如人類介白素IL-1、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-6、IL-8、IL-12、IL-11、IL-12、IL-13、IL-18、IL-31、IL-35)、單核球趨化蛋白(monocyte chemoattractant protein)、白血病抑制性因子、顆粒球-巨噬細胞群落刺激因子、Fas配體、腫瘤壞死因子α及β、干擾素α、β及γ、幹細胞因子、flk-2/flt3配體。免疫系統所產生的基因產物亦有用於本發明。此等包括但不限於免疫球蛋白IgG、IgM、IgA、IgD及IgE、嵌合免疫球蛋白、人源化抗體、單鏈抗體、T細胞受體、嵌合T細胞受體、單鏈T細胞受體、第I及II類MHC分子、以及經工程化的免疫球蛋白及MHC分子。例如，在某些具體實施例中，rAAV抗體可被設計以遞送犬或貓抗體，例如，如抗IgE、抗IL31、抗IL33、抗CD20、抗NGF、抗GnRH。有用的基因產物亦包括補體調節蛋白，如補體調節蛋白、膜輔因子蛋白質(membrane cofactor protein，MCP)、衰退加速因子(decay accelerating factor，DAF)、CR1、CF2、CD59、及C1酯酶抑制劑(C1-INH)。Other useful transgene products include proteins that regulate the immune system, including but not limited to cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-36 (including, for example, human Interleukins IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-12, IL-11, IL-12, IL-13 , IL-18, IL-31, IL-35), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and γ, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These include but are not limited to immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell Receptors, MHC class I and II molecules, and engineered immunoglobulin and MHC molecules. For example, in certain embodiments, rAAV antibodies can be designed to deliver canine or feline antibodies, such as, for example, anti-IgE, anti-IL31, anti-IL33, anti-CD20, anti-NGF, anti-GnRH. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, CD59, and C1 esterase inhibitors (C1-INH).

再其它有用的基因產物包括對於荷爾蒙、生長激素、細胞激素、淋巴激素、調節性蛋白質及免疫系統蛋白質之受體的任一者。本發明涵蓋膽固醇調節及/或脂質調節之受體，包括低密度脂蛋白(LDL)受體、高密度脂蛋白(HDL)受體、極低密度脂蛋白(VLDL)受體、及清道夫受體(scavenger receptor)。本發明亦涵蓋基因產物，如類固醇激素受體超家族成員，包括醣皮質素受體及雌激素受體、維生素D受體及其它核受體。此外，有用的基因產物包括轉錄因子，如 jun、 fos、max、mad、血清反應因子(serum response factor，SRF)、AP-1、AP2、 myb、MyoD及肌細胞生成素(myogenin)、含ETS匣的蛋白、TFE3、E2F、ATF1、ATF2、ATF3、ATF4、ZF5、NFAT、CREB、HNF-4、C/EBP、SP1、CCAAT匣結合蛋白、干擾素調節因子(IRF-1)、威爾姆斯瘤蛋白(Wilms tumor protein)、ETS-結合蛋白、STAT、GATA匣結合蛋白，例如GATA-3、及有翼螺旋(winged helix)蛋白的叉頭(forkhead)家族。 Still other useful gene products include receptors for any of hormones, growth hormones, cytokines, lymphokines, regulatory proteins, and immune system proteins. The invention encompasses cholesterol modulating and/or lipid modulating receptors, including low density lipoprotein (LDL) receptors, high density lipoprotein (HDL) receptors, very low density lipoprotein (VLDL) receptors, and scavenger receptors Body (scavenger receptor). The invention also encompasses gene products, such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. In addition, useful gene products include transcription factors, such as jun , fos , max, mad, serum response factor (serum response factor, SRF), AP-1, AP2, myb , MyoD and myogenin (myogenin), containing ETS Cassette proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT cassette binding protein, interferon regulatory factor (IRF-1), Wilm Wilms tumor proteins, ETS-binding proteins, STATs, GATA box-binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

其它有用的基因產物包括羥甲基膽汁烷合成酶(hydroxymethylbilane synthase，HMBS)、胺甲醯基合成酶I(carbamoyl synthetase I)、鳥胺酸胺甲醯基轉移酶(ornithine transcarbamylase，OTC)、精胺琥珀酸合成酶(arginosuccinate synthetase)、治療精胺琥珀酸裂解酶缺乏症之精胺琥珀酸裂解酶(arginosuccinate lyase，ASL)、精胺酸酶(arginase)、延胡索醯乙酸水解酶(fumarylacetate hydrolase)、苯丙胺酸羥化酶、α-1抗胰蛋白酶、恆河猴α胎兒蛋白(alpha- fetoprotein，AFP)、絨毛膜促性腺激素(CG)、葡萄糖-6-磷酸酶(glucose-6-phosphatase)、膽色素原脫胺酶(porphobilinogen deaminase)、胱硫醚β合成酶(cystathione beta-synthase)、支鏈酮酸脫羧酶(branched chain ketoacid decarboxylase)、白蛋白、異戊醯輔酶A脫氫酶(isovaleryl-coA dehydrogenase)、丙醯輔酶A羧化酶(propionyl CoA carboxylase)、甲基丙二醯輔酶A變位酶(methyl malonyl CoA mutase)、戊二醯輔酶A脫氫酶(glutaryl CoA dehydrogenase)、胰島素、β-葡萄糖苷酶(beta-glucosidase)、丙酮酸羧化酶(pyruvate carboxylate)、肝磷酸化酶(hepatic phosphorylase)、磷酸化酶激酶(phosphorylase kinase)、甘胺酸脫羧酶、H-蛋白、T-蛋白、囊腫纖維化跨膜調節子(cystic fibrosis transmembrane regulator，CFTR)序列、及肌肉萎縮蛋白(dystrophin)基因產物[例如，迷你-或微小-肌肉萎縮蛋白]。又其它有用的基因產物包括酵素，如可有用於酵素替代療法(enzyme replacement therapy)，其有用於各種由於酵素活性不足而引起的病況。例如，含有甘露糖-6-磷酸的酵素可用於溶酶體儲積症(lysosomal storage diseases)之治療(例如，包括編碼β-葡萄醣醛酸酶(β-glucuronidase，GUSB)之適合的基因)。於另一例中，基因產物為泛素蛋白連接酶E3A (ubiquitin protein ligase E3A，UBE3A)。另有用的基因產物包括UDP葡萄糖醛酸基轉移酶家族1成員A1 (UDP Glucuronosyltransferase Family 1 Member A1，UGT1A1)。Other useful gene products include hydroxymethylbilane synthase (HMBS), carbamoyl synthetase I, ornithine transcarbamylase (OTC), sperm Arginine succinate synthetase (arginosuccinate synthetase), arginine succinate lyase (ASL) for the treatment of arginine succinate lyase deficiency, arginase, fumarylacetate hydrolase , phenylalanine hydroxylase, α-1 antitrypsin, rhesus monkey α-fetoprotein (alpha-fetoprotein, AFP), chorionic gonadotropin (CG), glucose-6-phosphatase (glucose-6-phosphatase) , porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isopentyl-CoA dehydrogenase ( isovaleryl-coA dehydrogenase), propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, Insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein , T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin gene product (eg, mini- or micro-dystrophin). Still other useful gene products include enzymes, such as enzyme replacement therapy (enzyme replacement therapy), which is useful for various conditions caused by insufficient enzyme activity. For example, enzymes containing mannose-6-phosphate can be used in the treatment of lysosomal storage diseases (eg, including a suitable gene encoding β-glucuronidase (GUSB)). In another example, the gene product is ubiquitin protein ligase E3A (UBE3A). Another useful gene product includes UDP Glucuronosyltransferase Family 1 Member A1 (UDP Glucuronosyltransferase Family 1 Member A1, UGT1A1).

在某些具體實施例中，rAAV可用於基因編輯系統中，該系統可涉及一種rAAV或多種rAAV儲料的共同投予。例如，rAAV可經工程化以遞送SpCas9、SaCas9、ARCUS、Cpf1(亦已知為Cas12a)、CjCas9、及其它適合的基因編輯構築體。In certain embodiments, rAAV can be used in gene editing systems, which can involve the co-administration of one rAAV or multiple rAAV stocks. For example, rAAV can be engineered to deliver SpCas9, SaCas9, ARCUS, Cpf1 (also known as Cas12a), CjCas9, and other suitable gene editing constructs.

又其它有用的基因產物包括用於治療血友病的基因產物，包括血友病B(包括因子IX)及血友病A(包括因子VIII及其變異體，例如異二聚體的輕鏈及重鏈以及B缺失的域；美國專利號6,200,560及美國專利號6,221,349)。在一些具體實施例中，袖珍基因包含因子VIII重鏈的最初57個鹼基對，其編碼10個胺基酸訊息序列；以及人類生長激素(hGH)多腺苷酸化序列。於另外的具體實施例中，袖珍基因另包含A1及A2域；以及5個來自B域之N端的胺基酸、及/或B域之C端之85個胺基酸；以及A3、C1及C2域。於再其它具體實施例，於單一袖珍基因中提供編碼因子VIII重鏈及輕鏈之核酸，其被編碼B域之14個胺基酸的42個核酸分隔開[美國專利號6,200,560]。Still other useful gene products include gene products for the treatment of hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and variants thereof, such as the light chain of a heterodimer and Heavy chain and B deleted domain; US Patent No. 6,200,560 and US Patent No. 6,221,349). In some embodiments, the minigene comprises the first 57 base pairs of the Factor VIII heavy chain, which encodes a 10 amino acid message sequence; and a human growth hormone (hGH) polyadenylation sequence. In another specific embodiment, the pocket gene further comprises A1 and A2 domains; and 5 amino acids from the N-terminal of the B domain, and/or 85 amino acids from the C-terminal of the B domain; and A3, C1 and C2 domain. In still other embodiments, the nucleic acids encoding the heavy and light chains of Factor VIII are provided in a single pocket gene separated by 42 nucleic acids encoding the 14 amino acids of the B domain [US Pat. No. 6,200,560].

其它有用的基因產物包括非天然存在的多肽，諸如含有插入、缺失或胺基酸取代之具有非天然存在的胺基酸序列嵌合或雜合多肽。例如，於某些免疫功能不全的患者中，單鏈經工程化的免疫球蛋白可為有用的。非天然存在的基因序列之其它類型包括反義分子及催化性核酸，諸如，核糖核酸酵素(ribozyme)，其可被用來減少目標的過表現。Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, in certain immunocompromised patients, single-chain engineered immunoglobulins may be useful. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce target overexpression.

基因表現的減少及/或調節對於治療以細胞過度增生為特徵的過度增生性病況特別理想，如癌症及牛皮癬。目標多肽包括彼等與正常細胞相比在過度增生的細胞中排他地產生或以更高水準產生的多肽。目標抗原包括由致癌基因所編碼的多肽，該致癌基因例如為myb、myc、fyn、以及轉位基因bcr/abl、ras、src、P53、neu、trk及EGRF。除了致癌基因產物作為目標抗原外，用於抗癌治療及保護性方案的目標多肽包括由B細胞淋巴瘤產生的抗體的可變區及T細胞淋巴瘤的T細胞受體的可變區，於一些具體實施例，其亦使用作為自體免疫疾病的目標抗原。其它腫瘤相關多肽可用於作為目標多肽，諸如在腫瘤細胞中發現有較高水準的多肽，包括被單株抗體17-1A辨識的多肽及葉酸結合多肽。Reduction and/or modulation of gene expression is particularly desirable for the treatment of hyperproliferative conditions characterized by cellular hyperplasia, such as cancer and psoriasis. Polypeptides of interest include those that are produced exclusively or at higher levels in hyperproliferative cells as compared to normal cells. Antigens of interest include polypeptides encoded by oncogenes such as myb, myc, fyn, and the transposons bcr/abl, ras, src, P53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides for anticancer therapy and protective regimens include the variable regions of antibodies produced by B-cell lymphomas and the variable regions of T-cell receptors for T-cell lymphomas, in In some embodiments, it is also used as a target antigen for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides found at higher levels in tumor cells, including polypeptides recognized by monoclonal antibody 17-1A and folate-binding polypeptides.

其它適合的治療性多肽及蛋白質包括藉由賦予針對與自體免疫有關目標之廣泛基礎的保護性免疫反應而可用於治療罹患自體免疫疾病及失調的個體的多肽及蛋白質，該目標包括細胞受體及產生「自」導向抗體的細胞。T細胞媒介的自體免疫疾病包括類風濕性關節炎(Rheumatoid arthritis，RA)、多發性硬化症(multiple sclerosis，MS)、休格倫氏症候群(Sjögren’s syndrome)、類肉瘤病(sarcoidosis)、胰島素依賴型糖尿病(IDDM)、自體免疫甲狀腺炎(autoimmune thyroiditis)、反應性關節炎(reactive arthritis)、關節黏連性脊椎炎(ankylosing spondylitis)、硬皮病(scleroderma)、多發性肌炎(polymyositis)、皮肌炎(dermatomyositis)、牛皮癬、血管炎、華格納氏肉芽病(Wegener’s granulomatosis)、克隆氏病(Crohn’s disease)及潰瘍性結腸炎。此等疾病中的每一種皆以與內源性抗原結合並引發和自體免疫疾病有關的發炎級聯反應的T細胞受體(TCR)為特徵。Other suitable therapeutic polypeptides and proteins include those useful in the treatment of individuals suffering from autoimmune diseases and disorders by conferring a broad-based protective immune response against targets associated with autoimmunity, including cellular immune body and cells that produce self-directing antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin Dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis ), dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

可經由本文提供的rAAV遞送之另外說明性的基因用於治療例如肝臟適應症，該基因包括但不限於：葡萄糖-6-磷酸酶，與肝醣儲積症或缺乏症1A型(GSD1)有關；磷酸烯醇丙酮酸羧激酶(PEPCK)，與PEPCK缺乏症有關；類周期蛋白依賴型激酶5(cyclin-dependent kinase-like 5，CDKL5)，亦已知為絲胺酸/蘇胺酸激酶9(STK9)，與癲癇發作及嚴重的神經發展障礙有關；半乳糖-1-磷酸尿苷醯基轉移酶(galactose-1 phosphate uridyl transferase)，與半乳糖血症(galactosemia)有關；苯丙胺酸羥化酶(PAH)，與苯丙酮尿症(phenylketonuria，PKU)有關；與原發性高草酸鹽尿症第1型(Primary Hyperoxaluria Type 1)有關的基因產物，包括羥基酸氧化酶1(Hydroxyacid Oxidase 1，GO/HAO1)及AGXT；支鏈α-酮酸脫氫酶(branched chain alpha-ketoacid dehydrogenase)，包括BCKDH、BCKDH-E2、BAKDH-E1a及BAKDH-E1b，與楓糖尿症(Maple syrup urine disease)有關；延胡索醯乙醯乙酸水解酶(fumarylacetoacetate hydrolase)，與酪胺酸血症第1型有關；甲基丙二醯輔酶A變位酶，與甲基丙二酸血症(methylmalonic acidemia)有關；中鏈乙醯輔酶A脫氫酶(medium chain acyl CoA dehydrogenase)，與中鏈乙醯輔酶A缺乏症有關；鳥胺酸胺甲醯基轉移酶(OTC)，與鳥胺酸胺甲醯基轉移酶缺乏症有關；精胺琥珀酸合成酶(argininosuccsinate synthase，ASS1)，與瓜胺酸血症(citrullinemia)有關；卵磷脂-膽固醇醯基轉移酶(lecithin-cholesterol acyltransferase，LCAT)缺乏症；甲基丙二酸血症(MMA)；與Cl型尼曼匹克症(Niemann-Pick disease, type C1)有關的NPC1；丙酸血症(propionic academia，PA)；與轉甲狀腺素蛋白相關遺傳性類澱粉變性症(Transthyretin (TTR)-related Hereditary Amyloidosis)有關的TTR；低密度脂蛋白受體(LDLR)蛋白，與家族性高膽固醇血症(FH)有關；LDLR變異體，諸如彼等描述於WO 2015/164778者；PCSK9；ApoE及ApoC蛋白，與失智症有關；UDP葡萄糖醛酸基轉移酶(UDP-glucouronosyltransferase)，與克-納二氏症(Crigler-Najjar disease)有關；腺苷脫胺酶(adenosine deaminase)，與嚴重合併性免疫不全病(severe combined immunodeficiency disease)有關；次黃嘌呤-鳥嘌呤磷酸核糖轉移酶(hypoxanthine guanine phosphoribosyl transferase)，與痛風及勒-奈二氏症候群(Lesch-Nyan syndrome)有關；生物素酶(biotimidase)，與生物素酶缺乏症有關；與法布瑞氏症(Fabry disease)有關的α-半乳糖苷酶(a-Gal A)；與GM1神經節苷脂沉積症(GM1 gangliosidosis)有關的β-半乳糖苷酶(GLB1)；與威爾森氏症(Wilson’s Disease)有關的ATP7B；β-葡萄糖腦苷脂酶(beta-glucocerebrosidase)，與高歇氏病(Gaucher disease)第2及3型有關；過氧化體膜蛋白(peroxisome membrane protein) 70 kDa，與齊威格氏症候群(Zellweger syndrome)有關；與異染性白質失養症(metachromatic leukodystrophy)有關的芳基硫酸酯酶A(ARSA)；與克拉培氏病(Krabbe disease)有關的半乳糖腦苷脂酶(galactocerebrosidase，GALC)酵素；與龐貝氏症(Pompe disease)有關的α-葡萄糖苷酶(GAA)；神經髓磷脂酶(sphingomyelinase，SMPD1)基因，與A型尼曼匹克氏症(Nieman Pick disease type A)有關；與成年發病第II型瓜胺酸血症(CTLN2)有關的精胺琥珀酸合成酶；與尿素循環失調有關的胺甲醯基-磷酸合成酶1(CPS1)；運動神經元存活(survival motor neuron，SMN)蛋白，與脊髓性肌肉萎縮症有關；與法伯脂肉芽腫症(Farber lipogranulomatosis)有關的神經醯胺酶(ceramidase)；與GM2神經節苷脂沉積症以及泰-薩二氏(Tay-Sachs)及山多夫氏(Sandhoff)病有關的β-己醣胺酶(b-hexosaminidase)；與天冬胺醯基葡萄糖胺尿(aspartyl-glucosaminuria)有關的天冬胺醯基葡萄胺糖苷酶(aspartylglucosaminidase)；與岩藻糖沉積症(fucosidosis)有關的α-岩藻糖苷酶(α-fucosidase)；與α-甘露糖沉積症(alpha-mannosidosis)有關的α-甘露糖苷酶(α-mannosidase)；膽色素原脫胺酶，與急性間歇性紫質症(acute intermittent porphyria，AIP)有關；用於治療α-1抗胰蛋白酶缺乏症(肺氣腫(emphysema))之α-1抗胰蛋白酶；用於治療因地中海貧血或腎衰竭引起的貧血之紅血球生成素；用於治療缺血性疾病(ischemic diseases)之血管內皮生長因子、血管生成素-1 (angiopoietin-1)及纖維母細胞生長因子；用於治療於下列所見的血管阻塞之凝血酶調節素(thrombomodulin)及組織因子路徑抑制劑(tissue factor pathway inhibitor)，例如動脈粥樣硬化、血栓症或栓塞；用於治療帕金森氏症之芳香族胺基酸脫羧酶(AADC)及酪胺酸羥化酶(TH)；用於治療充血性心臟衰竭之β腎上腺素受體、受磷蛋白(phospholamban)之反義或突變型、肌(內)質網腺苷三磷酸酶-2 (sarco(endo)plasmic reticulum adenosine triphosphatase-2，SERCA2)及心臟腺苷酸環化酶(cardiac adenylyl cyclase)；用於治療各種癌症之腫瘤抑制基因，諸如p53；細胞激素，諸如各種介白素之一者，用於治療發炎及免疫失調及癌症；用於治療肌肉營養不良之肌肉萎縮蛋白(dystrophin)或迷你肌肉萎縮蛋白(minidystrophin)及肌營養相關蛋白(utrophin)或迷你肌營養相關蛋白(miniutrophin)；以及用於治療糖尿病之胰島素或GLP-1。Additional illustrative genes that can be delivered via the rAAV provided herein for the treatment of, for example, liver indications include, but are not limited to: glucose-6-phosphatase, associated with glycogen storage disease or deficiency type 1A (GSD1); Phosphoenolpyruvate carboxykinase (PEPCK), associated with PEPCK deficiency; cyclin-dependent kinase-like 5 (CDKL5), also known as serine/threonine kinase 9 ( STK9), associated with seizures and severe neurodevelopmental disorders; galactose-1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase (PAH), associated with phenylketonuria (PKU); gene products associated with Primary Hyperoxaluria Type 1, including Hydroxyacid Oxidase 1 , GO/HAO1) and AGXT; branched chain alpha-ketoacid dehydrogenase (branched chain alpha-ketoacid dehydrogenase), including BCKDH, BCKDH-E2, BAKDH-E1a and BAKDH-E1b, and maple syrup urine disease (Maple syrup urine disease ), fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia ; medium chain acetyl-CoA dehydrogenase (medium chain acyl-CoA dehydrogenase), associated with medium-chain acetyl-CoA deficiency; ornithine formyltransferase (OTC), associated with ornithine formyl Argininosuccsinate synthase (ASS1), which is related to citrullinemia; lecithin-cholesterol acyltransferase (LCAT) deficiency; MMA; NPC1 associated with Niemann-Pick disease (type C1); propionic academia (PA); genetics associated with transthyretin Transthyretin (TTR)-related Hereditary Amyloidosis-related TTR; low-density lipoprotein receptor (LDLR) protein, associated with familial hypercholesterolemia (FH); LDLR variants, such as those described in WO 2015/164778; PCSK9; ApoE and ApoC proteins, related to dementia; UDP-glucuronosyltransferase (UDP-glucouronosyltransferase), related to Crigler-Najjar disease; adenosine deamination Enzyme (adenosine deaminase), which is related to severe combined immunodeficiency disease; hypoxanthine-guanine phosphoribosyl transferase (hypoxanthine guanine phosphoribosyl transferase), which is related to gout and Lesch-Nay syndrome Nyan syndrome; biotinidase, associated with biotinidase deficiency; alpha-galactosidase (a-Gal A), associated with Fabry disease; associated with GM1 gangliosides β-galactosidase (GLB1) associated with GM1 gangliosidosis; ATP7B associated with Wilson's Disease; beta-glucocerebrosidase, associated with Gaucher's associated with Gaucher disease types 2 and 3; peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; associated with metachromatic leukodystrophy Arylsulfatase A (ARSA); galactocerebrosidase (GALC) enzyme associated with Krabbe disease; α-glucoside associated with Pompe disease enzyme (GAA); neuromyelinase (sphingomyelinase, SMPD1) gene, associated with Nieman Pick disease type A (Nieman Pick disease type A); Aminosuccinate synthase; carbamoyl-phosphate synthase 1 (CPS1), implicated in urea cycle disorders; survival motor neuron (SMN) protein, implicated in spinal muscular atrophy; Ceramidase associated with Farber lipogranulomatosis; β-hexamic acid associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff diseases glucosaminidase (b-hexosaminidase); aspartylglucosaminidase (aspartyl-glucosaminidase) related to aspartyl-glucosaminuria (aspartyl-glucosaminuria); -Fucosidase (alpha-fucosidase); alpha-mannosidase (alpha-mannosidase) associated with alpha-mannosidosis; porphobilinogen deaminase, associated with acute intermittent porphyria (acute intermittent porphyria, AIP); alpha-1 antitrypsin used to treat alpha-1 antitrypsin deficiency (emphysema); red blood cells used to treat anemia caused by thalassemia or renal failure Propoietin; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; regulation of thrombin for the treatment of vascular occlusion seen in Thrombomodulin and tissue factor pathway inhibitors, such as atherosclerosis, thrombosis or embolism; aromatic amino acid decarboxylase (AADC) and tyrosine for the treatment of Parkinson's disease Hydroxylase (TH); β-adrenoceptor for the treatment of congestive heart failure, antisense or mutant of phospholamban, sarcoplasmic reticulum adenosine triphosphatase-2 (sarco( endo) plasma reticulum adenosine triphosphatase-2, SERCA2) and cardiac adenylyl cyclase (cardiac adenylyl cyclase); tumor suppressor genes for the treatment of various cancers, such as p53; cytokines, such as one of various interleukins, for Treatment of inflammation and immune disorders and cancer; dystrophin or mini-dystrophin and utrophin or miniutrophin for the treatment of muscular dystrophy; and Insulin or GLP-1 for diabetes.

另外感興趣的基因及疾病包括例如與疾病有關的肌張力異常蛋白(dystonin)基因，如第IV型遺傳性感覺及自主神經病變(Hereditary Sensory and Autonomic Neuropathy Type VI) (DST基因編碼肌張力異常蛋白；由於蛋白質的大小(~7570 aa)而可能需要雙重AAV載體；SCN9A相關疾病，其中功能突變體的喪失導致無法感覺疼痛，而功能突變體的獲得引起疼痛狀況，如肢端紅痛症(erythromelagia)。另一病況係由於NEFL基因(神經纖維絲輕鏈)發生突變而導致的1F及2E型夏馬杜三氏病(Charcot-Marie-Tooth，CMT)，其特徵為進行性周圍運動及感覺神經病變，具有可變的臨床及電生理表現。與CMT有關的其它基因產物包括粒線體融合蛋白2(mitofusin 2，MFN2)。Additional genes and diseases of interest include, for example, the dystonia gene associated with diseases such as Hereditary Sensory and Autonomic Neuropathy Type VI (DST gene encodes dystonia ; dual AAV vectors may be required due to protein size (~7570 aa); SCN9A-associated diseases in which loss-of-function mutants result in inability to feel pain and gain-of-function mutants cause painful conditions such as erythromelagia ). Another condition is Charcot-Marie-Tooth (CMT) type 1F and 2E caused by mutations in the NEFL gene (neurofilament light chain), which is characterized by progressive peripheral motor and sensory Neuropathy with variable clinical and electrophysiological manifestations. Other gene products associated with CMT include mitochondrial fusion protein 2 (mitofusin 2, MFN2).

在某些具體實施例中，本文所述rAAV可用於治療黏多醣病(MPS)。此種rAAV可含有攜帶：編碼治療MPS I(赫勒氏(Hurler)、赫勒-施艾氏(Hurler-Scheie)及施艾氏(Scheie)症候群)之α-L-艾杜糖醛酸酶(α-L-iduronidase，IDUA)之核酸序列；編碼治療MPS II(韓特氏症候群(Hunter syndrome))之艾杜糖醛酸-2-硫酸脂酶(iduronate-2-sulfatase，IDS)之核酸序列；編碼治療MPSIII A、B、C、及D(聖菲利柏氏症候群(Sanfilippo syndrome))之磺醯胺酶(sulfamidase，SGSH)之核酸序列；編碼治療MPS IV A及B(莫奎歐氏症候群(Morquio syndrome))之N-乙醯半乳胺糖-6-硫酸硫酸酯酶(N-acetylgalactosamine-6-sulfate sulfatase，GALNS)之核酸序列；編碼治療MPS VI(馬-拉二氏症候群(Maroteaux-Lamy syndrome))之芳基硫酸酯酶B(ARSB)之核酸序列；編碼治療MPSI IX(玻尿酸酶缺乏症)之玻尿酸酶之核酸序列及編碼治療MPS VII(史萊氏症候群(Sly syndrome))之β-葡萄醣醛酸酶之核酸序列。In certain embodiments, the rAAV described herein can be used to treat mucopolysaccharidosis (MPS). Such rAAV may contain α-L-iduronidase encoding the treatment of MPS I (Hurler, Hurler-Scheie and Scheie syndromes) Nucleic acid sequence of (α-L-iduronidase, IDUA); nucleic acid encoding iduronate-2-sulfatase (iduronate-2-sulfatase, IDS) for the treatment of MPS II (Hunter syndrome) Sequence; Nucleic acid sequence encoding the sulfamidase (sulfamidase, SGSH) for the treatment of MPSIII A, B, C, and D (Sanfilippo syndrome); encoding for the treatment of MPS IV A and B (Morquio's Syndrome (Morquio syndrome)) N-acetylgalactosamine-6-sulfate sulfatase (N-acetylgalactosamine-6-sulfate sulfatase, GALNS) nucleic acid sequence; coding treatment MPS VI (Marine-Laurent syndrome ( Nucleic acid sequence of aryl sulfatase B (ARSB) of Maroteaux-Lamy syndrome); Nucleic acid sequence of hyaluronidase encoding treatment of MPSI IX (hyaluronidase deficiency) and encoding treatment of MPS VII (Sly syndrome (Sly syndrome) ) nucleic acid sequence of β-glucuronidase.

在一些具體實施例中，可使用包含編碼與癌症有關的基因產物(例如，腫瘤抑制因子)之核酸的rAAV載體，藉由投予攜帶該rAAV載體之rAAV至具有癌症的受試者，以治療癌症。在一些具體實施例中，可使用包含編碼抑制與癌症有關的基因產物的表現(例如，致癌基因)之短小干擾核酸(例如，shRNA、miRNA)之核酸的rAAV載體，藉由投予攜帶該rAAV載體之rAAV至具有癌症的受試者，以治療癌症。在一些具體實施例中，可使用包含編碼與癌症有關的基因產物的核酸(或抑制與癌症有關的基因表現的功能性RNA)的rAAV載體於研究目的，例如，研究癌症或確定治療癌症的療法。下列為已知與癌症發展有關的示例性基因的非限制列表(例如，致癌基因及腫瘤抑制因子)：AARS、ABCB1、ABCC4、ABI2、ABL1、ABL2、ACK1、ACP2、ACY1、ADSL、AK1、AKR1C2、AKT1、ALB、ANPEP、ANXA5、ANXA7、AP2M1、APC、ARHGAP5、ARHGEF5、ARID4A、ASNS、ATF4、ATM、ATP5B、ATP5O、AXL、BARD1、BAX、BCL2、BHLHB2、BLMH、BRAF、BRCA1、BRCA2、BTK、CANX、CAP1、CAPN1、CAPNS1、CAV1、CBFB、CBLB、CCL2、CCND1、CCND2、CCND3、CCNE1、CCT5、CCYR61、CD24、CD44、CD59、CDC20、CDC25、CDC25A、CDC25B、CDC2L5、CDK10、CDK4、CDK5、CDK9、CDKL1、CDKN1A、CDKN1B、CDKN1C、CDKN2A、CDKN2B、CDKN2D、CEBPG、CENPC1、CGRRF1、CHAF1A、CIB1、CKMT1、CLK1、CLK2、CLK3、CLNS1A、CLTC、COL1A1、COL6A3、COX6C、COX7A2、CRAT、CRHR1、CSF1R、CSK、CSNK1G2、CTNNA1、CTNNB1、CTPS、CTSC、CTSD、CUL1、CYR61、DCC、DCN、DDX10、DEK、DHCR7、DHRS2、DHX8、DLG3、DVL1、DVL3、E2F1、E2F3、E2F5、EGFR、EGR1、EIF5、EPHA2、ERBB2、ERBB3、ERBB4、ERCC3、ETV1、ETV3、ETV6、F2R、FASTK、FBN1、FBN2、FES、FGFR1、FGR、FKBP8、FN1、FOS、FOSL1、FOSL2、FOXG1A、FOXO1A、FRAP1、FRZB、FTL、FZD2、FZD5、FZD9、G22P1、GAS6、GCN5L2、GDF15、GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3A、GSPT1、GTF2I、HDAC1、HDGF、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB1、HSPH1、HYAL1、HYOU1、ICAM1、ID1、ID2、IDUA、IER3、IFITM1、IGF1R、IGF2R、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、IRF3、ITGA3、ITGA6、ITGB4、JAK1、JARID1A、JUN、JUNB、JUND、K-ALPHA-1、KIT、KITLG、KLK10、KPNA2、KRAS2、KRT18、KRT2A、KRT9、LAMB1、LAMP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MAP3K8、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MAS1、MCC、MCM2、MCM4、MDM2、MDM4、MET、MGST1、MICB、MLLT3、MME、MMP1、MMP14、MMP17、MMP2、MNDA、MSH2、MSH6、MT3、MYB、MYBL1、MYBL2、MYC、MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NMBR、NME1、NME2、NME3、NOTCH1、NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、PA2G4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT1、PLK2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP、PRSS15、PSMA1、PTCH、PTEN、PTGS1、PTMA、PTN、PTPRN、RAB5A、RAC1、RAD50、RAF1、RALBP1、RAP1A、RARA、RARB、RASGRF1、RB1、RBBP4、RBL2、REA、REL、RELA、RELB、RET、RFC2、RGS19、RHOA、RHOB、RHOC、RHOD、RIPK1、RPN2、RPS6 KB1、RRM1、SARS、SELENBP1、SEMA3C、SEMA4D、SEPP1、SERPINH1、SFN、SFPQ、SFRS7、SHB、SHH、SIAH2、SIVA、SIVA TP53、SKI、SKIL、SLC16A1、SLC1A4、SLC20A1、SMO、神經髓磷脂磷酸二脂酶1(sphingomyelin phosphodiesterase 1，SMPD1)、SNAI2、SND1、SNRPB2、SOCS1、SOCS3、SOD1、SORT1、SPINT2、SPRY2、SRC、SRPX、STAT1、STAT2、STAT3、STAT5B、STC1、TAF1、TBL3、TBRG4、TCF1、TCF7L2、TFAP2C、TFDP1、TFDP2、TGFA、TGFB1、TGFBI、TGFBR2、TGFBR3、THBS1、TIE、TIMP1、TIMP3、TJP1、TK1、TLE1、TNF、TNFRSF10A、TNFRSF10B、TNFRSF1A、TNFRSF1B、TNFRSF6、TNFSF7、TNK1、TOB1、TP53、TP53BP2、TP5313、TP73、TPBG、TPT1、TRADD、TRAM1、TRRAP、TSG101、TUFM、TXNRD1、TYRO3、UBC、UBE2L6、UCHL1、USP7、VDAC1、VEGF、VHL、VIL2、WEE1、WNT1、WNT2、WNT2B、WNT3、WNT5A、WT1、XRCC1、YES1、YWHAB、YWHAZ、ZAP70、及ZNF9。In some embodiments, an rAAV vector comprising a nucleic acid encoding a cancer-related gene product (e.g., a tumor suppressor) can be used to treat a subject with cancer by administering rAAV carrying the rAAV vector to a subject with cancer. cancer. In some embodiments, an rAAV vector comprising a nucleic acid encoding a short interfering nucleic acid (e.g., shRNA, miRNA) that inhibits the expression of a gene product associated with cancer (e.g., an oncogene) can be used by administering an rAAV vector carrying the rAAV Vectoring rAAV to a subject with cancer to treat the cancer. In some embodiments, rAAV vectors comprising a nucleic acid encoding a cancer-related gene product (or a functional RNA that inhibits the expression of a cancer-related gene) can be used for research purposes, e.g., to study cancer or to determine a therapy for treating cancer . The following is a non-limiting list of exemplary genes known to be involved in cancer development (e.g., oncogenes and tumor suppressors): AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2 , AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK , CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5 , CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1 , CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1 , EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB , FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2, GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB, HSPA4, HSPA5, HSPA8 , HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA, IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3, ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN , JUNB, JUND, K-ALPHA-1, KIT, KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2, LEP, LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2 , MAP3K8, MAPK12, MAPK13, MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6, MT3 , MYB, MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK, NEO1, NF1, NF2, NFKB1, NFKB2, NFSF7, NID, NINE, NMBR, NME1, NME2, NME3, NOTCH1, NOTCH2, NOTCH4, NPM1 , NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1, NSEP1, OSM, PA2G4, PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB, PDGFRA, PDPK1, PEA15, PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB , PIK3CG, PIM1, PKM2, PKMYT1, PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2, PRDX4, PRKAR1A, PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN, RAB5A, RAC1 , RAD50, RAF1, RALBP1, RAP1A, RARA, RARB, RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6 KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, myelin phosphodiesterase 1 (sphingomyelin phosphodiesterase 1, SMPD1), SNAI2, SND1, SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2 , TFAP2C, TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1, TOB1, TP53 , TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3 , WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

rAAV載體可包含編碼調節細胞凋亡之蛋白質或功能性RNA的轉基因、核酸。下列為與細胞凋亡有關的基因以及編碼此等基因及其同源物的產物的核酸、以及編碼抑制此等基因及其同源物表現的短小干擾核酸(例如，shRNA、miRNA)的非限制列表，於本發明之某些具體實施例，有用於作為轉基因：RPS27A、ABL1、AKT1、APAF1、BAD、BAG1、BAG3、BAG4、BAK1、BAX、BCL10、BCL2、BCL2A1、BCL2L1、BCL2L10、BCL2L11、BCL2L12、BCL2L13、BCL2L2、BCLAF1、BFAR、BID、BIK、NAIP、BIRC2、BIRC3、XIAP、BIRC5、BIRC6、BIRC7、BIRC8、BNIP1、BNIP2、BNIP3、BNIP3L、BOK、BRAF、CARD10、CARD11、NLRC4、CARD14、NOD2、NOD1、CARD6、CARDS、CARDS、CASP1、CASP10、CASP14、CASP2、CASP3、CASP4、CASP5、CASP6、CASP7、CASP8、CASP9、CFLAR、CIDEA、CIDEB、CRADD、DAPK1、DAPK2、DFFA、DFFB、FADD、GADD45A、GDNF、HRK、IGF1R、LTA、LTBR、MCL1、NOL3、PYCARD、RIPK1、RIPK2、TNF、TNFRSF10A、TNFRSF10B、TNFRSF10C、TNFRSF10D、TNFRSF11B、TNFRSF12A、TNFRSF14、TNFRSF19、TNFRSF1A、TNFRSF1B、TNFRSF21、TNFRSF25、CD40、FAS、TNFRSF6B、CD27、TNFRSF9、TNFSF10、TNFSF14、TNFSF18、CD40LG、FASLG、CD70、TNFSF8、TNFSF9、TP53、TP53BP2、TP73、TP63、TRADD、TRAF1、TRAF2、TRAF3、TRAF4、及TRAF5。rAAV vectors may contain transgenes, nucleic acids encoding proteins or functional RNAs that regulate apoptosis. The following are non-limiting examples of genes associated with apoptosis and nucleic acids encoding the products of these genes and their homologues, as well as encoding short interfering nucleic acids (e.g., shRNA, miRNA) that inhibit the expression of these genes and their homologues List, in certain embodiments of the invention, useful as transgenes: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12 , BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2 , NOD1, CARD6, CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A , GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3, PYCARD, RIPK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1AS, CDNFRSF1B, TNFRSF4 , TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5.

有用的轉基因產物亦包括miRNA。miRNA及其它短小干擾核酸經由目標RNA轉錄本的切割/降解或目標傳訊RNA (mRNA)的轉譯壓制來調節基因表現。miRNA係天然地表現，通常作為最終19-25個非轉譯的RNA產物。miRNA通過與目標mRNA的3′非轉譯區(UTR)之序列特異性相互作用來展現其活性。此等內源性表現的miRNA形成髮夾前驅物，隨後被加工成雙股miRNA (miRNA duplex)，並進一步加工成「成熟」單股miRNA分子。此成熟miRNA引導多蛋白複合物miRISC，其根據與成熟miRNA的互補性而識別目標miRNA的目標部位，例如，於3′ UTR區。Useful transgene products also include miRNAs. miRNAs and other short interfering nucleic acids regulate gene expression through cleavage/degradation of target RNA transcripts or translational repression of target messenger RNAs (mRNAs). miRNAs are expressed naturally, usually as the final 19-25 non-translated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into double-stranded miRNAs (miRNA duplexes) and further processed into "mature" single-stranded miRNA molecules. This mature miRNA guides the multiprotein complex miRISC, which recognizes the target site of the miRNA of interest, eg, in the 3' UTR region, based on complementarity to the mature miRNA.

下列miRNA基因及其同源物的非限制性列表，在本方法的某些具體實施例中，有用於作為轉基因或作為由轉基因所編碼的短小干擾核酸(例如，miRNA海綿(miRNA sponge)、反義寡核苷酸、TuD RNA)之目標：hsa-let-7a、hsa-let-7a*、hsa-let-7b、hsa-let-7b*、hsa-let-7c、hsa-let-7c*、hsa-let-7d、hsa-let-7d*、hsa-let-7e、hsa-let-7e*、hsa-let-7f、hsa-let-7f-1*、hsa-let-7f-2*、hsa-let-7g、hsa-let-7g*、hsa-let-71、hsa-let-71*、hsa-miR-1、hsa-miR-100、hsa-miR-100*、hsa-miR-101、hsa-miR-101*、hsa-miR-103、hsa-miR-105、hsa-miR-105*、hsa-miR-106a、hsa-miR-106a*、hsa-miR-106b、hsa-miR-106b*、hsa-miR-107、hsa-miR-10a、hsa-miR-10a*、hsa-miR-10b、hsa-miR-10b*、hsa-miR-1178、hsa-miR-1179、hsa-miR-1180、hsa-miR-1181、hsa-miR-1182、hsa-miR-1183、hsa-miR-1184、hsa-miR-1185、hsa-miR-1197、hsa-miR-1200、hsa-miR-1201、hsa-miR-1202、hsa-miR-1203、hsa-miR-1204、hsa-miR-1205、hsa-miR-1206、hsa-miR-1207-3p、hsa-miR-1207-5p、hsa-miR-1208、hsa-miR-122、hsa-miR-122*、hsa-miR-1224-3p、hsa-miR-1224-5p、hsa-miR-1225-3p、hsa-miR-1225-5p、hsa-miR-1226、hsa-miR-1226*、hsa-miR-1227、hsa-miR-1228、hsa-miR-1228*、hsa-miR-1229、hsa-miR-1231、hsa-miR-1233、hsa-miR-1234、hsa-miR-1236、hsa-miR-1237、hsa-miR-1238、hsa-miR-124、hsa-miR-124*、hsa-miR-1243、hsa-miR-1244、hsa-miR-1245、hsa-miR-1246、hsa-miR-1247、hsa-miR-1248、hsa-miR-1249、hsa-miR-1250、hsa-miR-1251、hsa-miR-1252、hsa-miR-1253、hsa-miR-1254、hsa-miR-1255a、hsa-miR-1255b、hsa-miR-1256、hsa-miR-1257、hsa-miR-1258、hsa-miR-1259、hsa-miR-125a-3p、hsa-miR-125a-5p、hsa-miR-125b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-miR-126、hsa-miR-126*、hsa-miR-1260、hsa-miR-1261、hsa-miR-1262、hsa-miR-1263、hsa-miR-1264、hsa-miR-1265、hsa-miR-1266、hsa-miR-1267、hsa-miR-1268、hsa-miR-1269、hsa-miR-1270、hsa-miR-1271、hsa-miR-1272、hsa-miR-1273、hsa-miR-127-3p、hsa-miR-1274a、hsa-miR-1274b、hsa-miR-1275、hsa-miR-127-5p、hsa-miR-1276、hsa-miR-1277、hsa-miR-1278、hsa-miR-1279、hsa-miR-128、hsa-miR-1280、hsa-miR-1281、hsa-miR-1282、hsa-miR-1283、hsa-miR-1284、hsa-miR-1285、hsa-miR-1286、hsa-miR-1287、hsa-miR-1288、hsa-miR-1289、hsa-miR-129*、hsa-miR-1290、hsa-miR-1291、hsa-miR-1292、hsa-miR-1293、hsa-miR-129-3p、hsa-miR-1294、hsa-miR-1295、hsa-miR-129-5p、hsa-miR-1296、hsa-miR-1297、hsa-miR-1298、hsa-miR-1299、hsa-miR-1300、hsa-miR-1301、hsa-miR-1302、hsa-miR-1303、hsa-miR-1304、hsa-miR-1305、hsa-miR-1306、hsa-miR-1307、hsa-miR-1308、hsa-miR-130a、hsa-miR-130a*、hsa-miR-130b、hsa-miR-130b*、hsa-miR-132、hsa-miR-132*、hsa-miR-1321、hsa-miR-1322、hsa-miR-1323、hsa-miR-1324、hsa-miR-133a、hsa-miR-133b、hsa-miR-134、hsa-miR-135a、hsa-miR-135a*、hsa-miR-135b、hsa-miR-135b*、hsa-miR-136、hsa-miR-136*、hsa-miR-137、hsa-miR-138、hsa-miR-138-1*、hsa-miR-138-2*、hsa-miR-139-3p、hsa-miR-139-5p、hsa-miR-140-3p、hsa-miR-140-5p、hsa-miR-141、hsa-miR-141*、hsa-miR-142-3p、hsa-miR-142-5p、hsa-miR-143、hsa-miR-143*、hsa-miR-144、hsa-miR-144*、hsa-miR-145、hsa-miR-145*、hsa-miR-146a、hsa-miR-146a*、hsa-miR-146b-3p、hsa-miR-146b-5p、hsa-miR-147、hsa-miR-147b、hsa-miR-148a、hsa-miR-148a*、hsa-miR-148b、hsa-miR-148b*、hsa-miR-149、hsa-miR-149*、hsa-miR-150、hsa-miR-150*、hsa-miR-151-3p、hsa-miR-151-5p、hsa-miR-152、hsa-miR-153、hsa-miR-154、hsa-miR-154*、hsa-miR-155、hsa-miR-155*、hsa-miR-15a、hsa-miR-15a*、hsa-miR-15b、hsa-miR-15b*、hsa-miR-16、hsa-miR-16-1*、hsa-miR-16-2*、hsa-miR-17、hsa-miR-17*、hsa-miR-181a、hsa-miR-181a*、hsa-miR-181a-2*、hsa-miR-181b、hsa-miR-181c、hsa-miR-181c*、hsa-miR-181d、hsa-miR-182、hsa-miR-182*、hsa-miR-1825、hsa-miR-1826、hsa-miR-1827、hsa-miR-183、hsa-miR-183*、hsa-miR-184、hsa-miR-185、hsa-miR-185*、hsa-miR-186、hsa-miR-186*、hsa-miR-187、hsa-miR-187*、hsa-miR-188-3p、hsa-miR-188-5p、hsa-miR-18a、hsa-miR-18a*、hsa-miR-18b、hsa-miR-18b*、hsa-miR-190、hsa-miR-190b、hsa-miR-191、hsa-miR-191*、hsa-miR-192、hsa-miR-192*、hsa-miR-193a-3p、hsa-miR-193a-5p、hsa-miR-193b、hsa-miR-193b*、hsa-miR-194、hsa-miR-194*、hsa-miR-195、hsa-miR-195*、hsa-miR-196a、hsa-miR-196a*、hsa-miR-196b、hsa-miR-197、hsa-miR-198、hsa-miR-199a-3p、hsa-miR-199a-5p、hsa-miR-199b-5p、hsa-miR-19a、hsa-miR-19a*、hsa-miR-19b、hsa-miR-19b-1*、hsa-miR-19b-2*、hsa-miR-200a、hsa-miR-200a*、hsa-miR-200b、hsa-miR-200b*、hsa-miR-200c、hsa-miR-200c*、hsa-miR-202、hsa-miR-202*、hsa-miR-203、hsa-miR-204、hsa-miR-205、hsa-miR-206、hsa-miR-208a、hsa-miR-208b、hsa-miR-20a、hsa-miR-20a*、hsa-miR-20b、hsa-miR-20b*、hsa-miR-21、hsa-miR-21*、hsa-miR-210、hsa-miR-211、hsa-miR-212、hsa-miR-214、hsa-miR-214*、hsa-miR-215、hsa-miR-216a、hsa-miR-216b、hsa-miR-217、hsa-miR-218、hsa-miR-218-1*、hsa-miR-218-2*、hsa-miR-219-1-3p、hsa-miR-219-2-3p、hsa-miR-219-5p、hsa-miR-22、hsa-miR-22*、hsa-miR-220a、hsa-miR-220b、hsa-miR-220c、hsa-miR-221、hsa-miR-221*、hsa-miR-222、hsa-miR-222*、hsa-miR-223、hsa-miR-223*、hsa-miR-224、hsa-miR-23a、hsa-miR-23a*、hsa-miR-23b、hsa-miR-23b*、hsa-miR-24、hsa-miR-24-1*、hsa-miR-24-2*、hsa-miR-25、hsa-miR-25*、hsa-miR-26a、hsa-miR-26a-1*、hsa-miR-26a-2*、hsa-miR-26b、hsa-miR-26b*、hsa-miR-27a、hsa-miR-27a*、hsa-miR-27b、hsa-miR-27b*、hsa-miR-28-3p、hsa-miR-28-5p、hsa-miR-296-3p、hsa-miR-296-5p、hsa-miR-297、hsa-miR-298、hsa-miR-299-3p、hsa-miR-299-5p、hsa-miR-29a、hsa-miR-29a*、hsa-miR-29b、hsa-miR-296-1*、hsa-miR-296-2*、hsa-miR-29c、hsa-miR-29c*、hsa-miR-300、hsa-miR-301a、hsa-miR-301b、hsa-miR-302a、hsa-miR-302a*、hsa-miR-302b、hsa-miR-302b*、hsa-miR-302c、hsa-miR-302c*、hsa-miR-302d、hsa-miR-302d*、hsa-miR-302e、hsa-miR-302f、hsa-miR-30a、hsa-miR-30a*、hsa-miR-30b、hsa-miR-30b*、hsa-miR-30c、hsa-miR-30c-1*、hsa-miR-30c-2*、hsa-miR-30d、hsa-miR-30d*、hsa-miR-30e、hsa-miR-30e*、hsa-miR-31、hsa-miR-31*、hsa-miR-32、hsa-miR-32*、hsa-miR-320a、hsa-miR-320b、hsa-miR-320c、hsa-miR-320d、hsa-miR-323-3p、hsa-miR-323-5p、hsa-miR-324-3p、hsa-miR-324-5p、hsa-miR-325、hsa-miR-326、hsa-miR-328、hsa-miR-329、hsa-miR-330-3p、hsa-miR-330-5p、hsa-miR-331-3p、hsa-miR-331-5p、hsa-miR-335、hsa-miR-335*、hsa-miR-337-3p、hsa-miR-337-5p、hsa-miR-338-3p、hsa-miR-338-5p、hsa-miR-339-3p、hsa-miR-339-5p、hsa-miR-33a、hsa-miR-33a*、hsa-miR-33b、hsa-miR-33b*、hsa-miR-340、hsa-miR-340*、hsa-miR-342-3p、hsa-miR-342-5p、hsa-miR-345、hsa-miR-346、hsa-miR-34a、hsa-miR-34a*、hsa-miR-34b、hsa-miR-34b*、hsa-miR-34c-3p、hsa-miR-34c-5p、hsa-miR-361-3p、hsa-miR-361-5p、hsa-miR-362-3p、hsa-miR-362-5p、hsa-miR-363、hsa-miR-363*、hsa-miR-365、hsa-miR-367、hsa-miR-367*、hsa-miR-369-3p、hsa-miR-369-5p、hsa-miR-370、hsa-miR-371-3p、hsa-miR-371-5p、hsa-miR-372、hsa-miR-373、hsa-miR-373*、hsa-miR-374a、hsa-miR-374a*、hsa-miR-374b、hsa-miR-374b*、hsa-miR-375、hsa-miR-376a、hsa-miR-376a*、hsa-miR-376b、hsa-miR-376c、hsa-miR-377、hsa-miR-377*、hsa-miR-378、hsa-miR-378*、hsa-miR-379、hsa-miR-379*、hsa-miR-380、hsa-miR-380*、hsa-miR-381、hsa-miR-382、hsa-miR-383、hsa-miR-384、hsa-miR-409-3p、hsa-miR-409-5p、hsa-miR-410、hsa-miR-411、hsa-miR-411*、hsa-miR-412、hsa-miR-421、hsa-miR-422a、hsa-miR-423-3p、hsa-miR-423-5p、hsa-miR-424、hsa-miR-424*、hsa-miR-425、hsa-miR-425*、hsa-miR-429、hsa-miR-431、hsa-miR-431*、hsa-miR-432、hsa-miR-432*、hsa-miR-433、hsa-miR-448、hsa-miR-449a、hsa-miR-449b、hsa-miR-450a、hsa-miR-450b-3p、hsa-miR-450b-5p、hsa-miR-451、hsa-miR-452、hsa-miR-452*、hsa-miR-453、hsa-miR-454、hsa-miR-454*、hsa-miR-455-3p、hsa-miR-455-5p、hsa-miR-483-3p、hsa-miR-483-5p、hsa-miR-484、hsa-miR-485-3p、hsa-miR-485-5p、hsa-miR-486-3p、hsa-miR-486-5p、hsa-miR-487a、hsa-miR-487b、hsa-miR-488、hsa-miR-488*、hsa-miR-489、hsa-miR-490-3p、hsa-miR-490-5p、hsa-miR-491-3p、hsa-miR-491-5p、hsa-miR-492、hsa-miR-493、hsa-miR-493*、hsa-miR-494、hsa-miR-495、hsa-miR-496、hsa-miR-497、hsa-miR-497*、hsa-miR-498、hsa-miR-499-3p、hsa-miR-499-5p、hsa-miR-500、hsa-miR-500*、hsa-miR-501-3p、hsa-miR-501-5p、hsa-miR-502-3p、hsa-miR-502-5p、hsa-miR-503、hsa-miR-504、hsa-miR-505、hsa-miR-505*、hsa-miR-506、hsa-miR-507、hsa-miR-508-3p、hsa-miR-508-5p、hsa-miR-509-3-5p、hsa-miR-509-3p、hsa-miR-509-5p、hsa-miR-510、hsa-miR-511、hsa-miR-512-3p、hsa-miR-512-5p、hsa-miR-513a-3p、hsa-miR-513a-5p、hsa-miR-513b、hsa-miR-513c、hsa-miR-514、hsa-miR-515-3p、hsa-miR-515-5p、hsa-miR-516a-3p、hsa-miR-516a-5p、hsa-miR-516b、hsa-miR-517*、hsa-miR-517a、hsa-miR-517b、hsa-miR-517c、hsa-miR-518a-3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、hsa-miR-518d-3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa-miR-518f、hsa-miR-518f*、hsa-miR-519a、hsa-miR-519b-3p、hsa-miR-519c-3p、hsa-miR-519d、hsa-miR-519e、hsa-miR-519e*、hsa-miR-520a-3p、hsa-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、hsa-miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR-520h、hsa-miR-521、hsa-miR-522、hsa-miR-523、hsa-miR-524-3p、hsa-miR-524-5p、hsa-miR-525-3p、hsa-miR-525-5p、hsa-miR-526b、hsa-miR-526b*、hsa-miR-532-3p、hsa-miR-532-5p、hsa-miR-539、hsa-miR-541、hsa-miR-541*、hsa-miR-542-3p、hsa-miR-542-5p、hsa-miR-543、hsa-miR-544、hsa-miR-545、hsa-miR-545*、hsa-miR-548a-3p、hsa-miR-548a-5p、hsa-miR-548b-3p、hsa-miR-5486-5p、hsa-miR-548c-3p、hsa-miR-548c-5p、hsa-miR-548d-3p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-miR-548g、hsa-miR-548h、hsa-miR-548i、hsa-miR-548j、hsa-miR-548k、hsa-miR-5481、hsa-miR-548m、hsa-miR-548n、hsa-miR-548o、hsa-miR-548p、hsa-miR-549、hsa-miR-550、hsa-miR-550*、hsa-miR-551a、hsa-miR-551b、hsa-miR-551b*、hsa-miR-552、hsa-miR-553、hsa-miR-554、hsa-miR-555、hsa-miR-556-3p、hsa-miR-556-5p、hsa-miR-557、hsa-miR-558、hsa-miR-559、hsa-miR-561、hsa-miR-562、hsa-miR-563、hsa-miR-564、hsa-miR-566、hsa-miR-567、hsa-miR-568、hsa-miR-569、hsa-miR-570、hsa-miR-571、hsa-miR-572、hsa-miR-573、hsa-miR-574-3p、hsa-miR-574-5p、hsa-miR-575、hsa-miR-576-3p、hsa-miR-576-5p、hsa-miR-577、hsa-miR-578、hsa-miR-579、hsa-miR-580、hsa-miR-581、hsa-miR-582-3p、hsa-miR-582-5p、hsa-miR-583、hsa-miR-584、hsa-miR-585、hsa-miR-586、hsa-miR-587、hsa-miR-588、hsa-miR-589、hsa-miR-589*、hsa-miR-590-3p、hsa-miR-590-5p、hsa-miR-591、hsa-miR-592、hsa-miR-593、hsa-miR-593*、hsa-miR-595、hsa-miR-596、hsa-miR-597、hsa-miR-598、hsa-miR-599、hsa-miR-600、hsa-miR-601、hsa-miR-602、hsa-miR-603、hsa-miR-604、hsa-miR-605、hsa-miR-606、hsa-miR-607、hsa-miR-608、hsa-miR-609、hsa-miR-610、hsa-miR-611、hsa-miR-612、hsa-miR-613、hsa-miR-614、hsa-miR-615-3p、hsa-miR-615-5p、hsa-miR-616、hsa-miR-616*、hsa-miR-617、hsa-miR-618、hsa-miR-619、hsa-miR-620、hsa-miR-621、hsa-miR-622、hsa-miR-623、hsa-miR-624、hsa-miR-624*、hsa-miR-625、hsa-miR-625*、hsa-miR-626、hsa-miR-627、hsa-miR-628-3p、hsa-miR-628-5p、hsa-miR-629、hsa-miR-629*、hsa-miR-630、hsa-miR-631、hsa-miR-632、hsa-miR-633、hsa-miR-634、hsa-miR-635、hsa-miR-636、hsa-miR-637、hsa-miR-638、hsa-miR-639、hsa-miR-640、hsa-miR-641、hsa-miR-642、hsa-miR-643、hsa-miR-644、hsa-miR-645、hsa-miR-646、hsa-miR-647、hsa-miR-648、hsa-miR-649、hsa-miR-650、hsa-miR-651、hsa-miR-652、hsa-miR-653、hsa-miR-654-3p、hsa-miR-654-5p、hsa-miR-655、hsa-miR-656、hsa-miR-657、hsa-miR-658、hsa-miR-659、hsa-miR-660、hsa-miR-661、hsa-miR-662、hsa-miR-663、hsa-miR-663b、hsa-miR-664、hsa-miR-664*、hsa-miR-665、hsa-miR-668、hsa-miR-671-3p、hsa-miR-671-5p、hsa-miR-675、hsa-miR-7、hsa-miR-708、hsa-miR-708*、hsa-miR-7-1*、hsa-miR-7-2*、hsa-miR-720、hsa-miR-744、hsa-miR-744*、hsa-miR-758、hsa-miR-760、hsa-miR-765、hsa-miR-766、hsa-miR-767-3p、hsa-miR-767-5p、hsa-miR-768-3p、hsa-miR-768-5p、hsa-miR-769-3p、hsa-miR-769-5p、hsa-miR-770-5p、hsa-miR-802、hsa-miR-873、hsa-miR-874、hsa-miR-875-3p、hsa-miR-875-5p、hsa-miR-876-3p、hsa-miR-876-5p、hsa-miR-877、hsa-miR-877*、hsa-miR-885-3p、hsa-miR-885-5p、hsa-miR-886-3p、hsa-miR-886-5p、hsa-miR-887、hsa-miR-888、hsa-miR-888*、hsa-miR-889、hsa-miR-890、hsa-miR-891a、hsa-miR-891b、hsa-miR-892a、hsa-miR-892b、hsa-miR-9、hsa-miR-9*、hsa-miR-920、hsa-miR-921、hsa-miR-922、hsa-miR-923、hsa-miR-924、hsa-miR-92a、hsa-miR-92a-1*、hsa-miR-92a-2*、hsa-miR-92b、hsa-miR-92b*、hsa-miR-93、hsa-miR-93*、hsa-miR-933、hsa-miR-934、hsa-miR-935、hsa-miR-936、hsa-miR-937、hsa-miR-938、hsa-miR-939、hsa-miR-940、hsa-miR-941、hsa-miR-942、hsa-miR-943、hsa-miR-944、hsa-miR-95、hsa-miR-96、hsa-miR-96*、hsa-miR-98、hsa-miR-99a、hsa-miR-99a*、hsa-miR-99b、及hsa-miR-99b*。例如，可能感興趣的是靶向表現與肌肉萎縮性脊髓側索硬化症(ALS)有關的超氧化物歧化酶(superoxide dismutase，SOD1)之8號染色體開讀框72 (C9orf72)的miRNA。The following non-limiting list of miRNA genes and their homologues are useful in certain embodiments of the present methods as transgenes or as short interfering nucleic acids encoded by transgenes (e.g., miRNA sponges, anti- Sense oligonucleotides, TuD RNA) targets: hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c* , hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2* , hsa-let-7g, hsa-let-7g*, hsa-let-71, hsa-let-71*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR- 101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR -106b*, hsa-miR-107, hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178, hsa-miR-1179, hsa- miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR- 1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-1207-3p, hsa-miR-1207-5p, hsa- miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa -miR-1226, hsa-miR-1226*, hsa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233, hsa -miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124, hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa- miR-1245, hsa-miR-1246, hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR- 1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b, hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a- 3p, hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*, hsa-miR-126, hsa-miR-126*, hsa-miR -1260, hsa-miR-1261, hsa-miR-1262, hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267, hsa-miR-1268 , hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b , hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278, hsa-miR-1279, hsa-miR-128, hsa-miR-1280 , hsa-miR-1281, hsa-miR-1282, hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287, hsa-miR-1288, hsa -miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299, hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a, hsa- miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*, hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa -miR-1324, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b, hsa-miR-135b*, hsa -miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138, hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*, hsa-miR-142-3p, hsa-miR-142 -5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa- miR-146a*, hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b, hsa-miR-148a, hsa-miR-148a*, hsa-miR- 148b, hsa-miR-148b*, hsa-miR-149, hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p , hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155, hsa-miR-155*, hsa-miR-15a, hsa-miR-15a *, hsa-miR-15b, hsa-miR-15b*, hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17 *, hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa -miR-182, hsa-miR-182*, hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*, hsa-miR-184, hsa -miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*, hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR- 188-5p, hsa-miR-18a, hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b, hsa-miR-191, hsa- miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR -194, hsa-miR-194*, hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b, hsa-miR-197, hsa- miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR -19b-1*, hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b, hsa-miR-200b*, hsa-miR-200c, hsa-miR -200c*, hsa-miR-202, hsa-miR-202*, hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a, hsa-miR -208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*, hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa- miR-211, hsa-miR-212, hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR -218, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa -miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221, hsa-miR-221*, hsa-miR-222, hsa -miR-222*, hsa-miR-223, hsa-miR-223*, hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b* , hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*, hsa-miR-26a, hsa-miR-26a- 1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b* , hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298, hsa-miR-299 -3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b, hsa-miR-296-1*, hsa-miR-296-2*, hsa- miR-29c, hsa-miR-29c*, hsa-miR-300, hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b, hsa- miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*, hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa -miR-30a*, hsa-miR-30b, hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d, hsa -miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*, hsa-miR-32, hsa-miR-32*, hsa-miR-320a , hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p, hsa-miR-324-5p , hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329, hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa -miR-331-5p, hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p, hsa-miR-338- 5p, hsa-miR-339-3p, hsa-miR-339-5p, hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, hsa-miR-34a, hsa-miR-34a*, hsa- miR-34b, hsa-miR-34b*, hsa-miR-34c-3p, hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p , hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-369-3p, hsa -miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*, hsa- miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375, hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa -miR-376c, hsa-miR-377, hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR -410, hsa-miR-411, hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432 , hsa-miR-432*, hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, hsa-miR-450b-3p, hsa-miR- 450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR- 486-3p, hsa-miR-486-5p, hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489, hsa-miR-490-3p , hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495, hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p, hsa-miR -500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503, hsa- miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509 -3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa - miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b, hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p, hsa -miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*, hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR- 518a-3p, hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518c*, hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR -518e, hsa-miR-518e*, hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p, hsa-miR-519c-3p, hsa-miR-519d , hsa-miR-519e, hsa-miR-519e*, hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p, hsa-miR-520d- 3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f, hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR- 523, hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-526b, hsa-miR-526b*, hsa-miR -532-3p, hsa-miR-532-5p, hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p, hsa- miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p, hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR -5486-5p, hsa-miR-548c-3p, hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e, hsa-miR-548f, hsa - miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j, hsa-miR-548k, hsa-miR-5481, hsa-miR-548m, hsa-miR-548n, hsa-miR -548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a, hsa-miR-551b, hsa-miR-551b*, hsa-miR -552, hsa-miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa - miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR -569, hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-575, hsa - miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582 -3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR-588, hsa-miR -589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593* , hsa-miR-595, hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa - miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610, hsa-miR -611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p, hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa- miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR- 629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636 , hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa - miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR -653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa -miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa- miR-668, hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7 -1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744, hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766, hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p, hsa-miR-769-3p, hsa-miR- 769-5p, hsa-miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR- 876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*, hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa -miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923, hsa -miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa- miR-93*, hsa-miR-933, hsa-miR-934, hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939, hsa-miR -940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944, hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR- 98. hsa-miR-99a, hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*. For example, miRNAs targeting chromosome 8 open reading frame 72 (C9orf72) expressing superoxide dismutase (SOD1) associated with amyotrophic lateral sclerosis (ALS) may be of interest.

miRNA抑制了其靶向的mRNA的功能，結果抑制由該mRNA所編碼的多肽的表現。如此，阻斷(部分或全部)miRNA的活性(例如使miRNA緘默化)可有效地誘導或恢復其表現被抑制的多肽的表現(將該多肽去壓制)。在一具體實施例中，藉由於細胞中通過許多方法中之任一種來抑制miRNA活性而達成由miRNA之mRNA目標所編碼的多肽之去壓制作用。例如，藉由與短小干擾核酸(例如，反義寡核苷酸、miRNA海綿、TuD RNA)雜交可達成miRNA的活性的阻斷，該短小干擾核酸與miRNA互補或實質上互補，因而阻斷miRNA與其目標mRNA的相互作用。如本文所使用，與miRNA實質上互補的短小干擾核酸為能夠與miRNA雜交且阻斷該miRNA的活性者。在一些具體實施例中，與miRNA實質上互補的短小干擾核酸為除了1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18個鹼基外，皆與miRNA互補的短小干擾核酸。「miRNA抑制劑」為阻斷miRNA功能、表現及/或加工的試劑。例如，此等分子包括但不限於抑制miRNA與Drosha複合物相互作用的微小RNA特異性反義、微小RNA海綿、強誘餌(tough decoy) RNA (TuD RNA)及微小RNA寡核苷酸(雙股、髮夾、短寡核苷酸)。miRNAs inhibit the function of the mRNA they target and, as a result, inhibit the expression of the polypeptide encoded by the mRNA. Thus, blocking (partial or complete) the activity of a miRNA (eg, silencing the miRNA) can effectively induce or restore the expression of a polypeptide whose expression is inhibited (derepress the polypeptide). In one embodiment, derepression of the polypeptide encoded by the mRNA target of the miRNA is achieved by inhibiting miRNA activity in the cell by any of a number of methods. For example, blocking of the activity of miRNAs can be achieved by hybridization to short interfering nucleic acids (e.g., antisense oligonucleotides, miRNA sponges, TuD RNAs) that are complementary or substantially complementary to the miRNA, thereby blocking the miRNA Interaction with its target mRNA. As used herein, a short interfering nucleic acid that is substantially complementary to a miRNA is one that is capable of hybridizing to and blocking the activity of the miRNA. In some embodiments, the short interfering nucleic acid that is substantially complementary to the miRNA is other than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Outside 17 or 18 bases, all short interfering nucleic acids are complementary to miRNA. "miRNA inhibitors" are agents that block the function, expression and/or processing of miRNAs. For example, such molecules include, but are not limited to, microRNA-specific antisenses, microRNA sponges, tough decoy RNA (TuD RNA), and microRNA oligonucleotides (double-stranded , hairpins, short oligonucleotides).

又其它有用的轉基因可包括彼等編碼免疫球蛋白的基因，該免疫球蛋白賦予對病原的被動免疫力。「免疫球蛋白分子」為含有共價連接在一起並且能夠與抗原特異性結合的免疫球蛋白重鏈及免疫球蛋白輕鏈之免疫活性部分的蛋白質。免疫球蛋白分子為任一類型(例如，IgG、IgE、IgM、IgD、IgA及IgY)、類別(例如，IgG1、IgG2、IgG3、IgG4、IgA1及IgA2)或子類。於本文中術語「抗體」及「免疫球蛋白」可互換使用。Still other useful transgenes may include those encoding immunoglobulins that confer passive immunity to pathogens. An "immunoglobulin molecule" is a protein comprising the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently linked together and capable of specifically binding an antigen. Immunoglobulin molecules are of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass. The terms "antibody" and "immunoglobulin" are used interchangeably herein.

「免疫球蛋白重鏈」為一含有免疫球蛋白之抗原結合域(antigen binding domain)之至少一部分及免疫球蛋白重鏈可變區之至少一部分或免疫球蛋白重鏈恆定區之至少一部分之多肽。如此，免疫球蛋白衍生的重鏈與免疫球蛋白基因超家族的成員具有顯著的胺基酸序列同源性區域。例如，Fab片段中的重鏈為免疫球蛋白衍生的重鏈。"Immunoglobulin heavy chain" is a polypeptide comprising at least a portion of an immunoglobulin antigen binding domain and at least a portion of an immunoglobulin heavy chain variable region or at least a portion of an immunoglobulin heavy chain constant region . Thus, immunoglobulin-derived heavy chains have significant regions of amino acid sequence homology to members of the immunoglobulin gene superfamily. For example, the heavy chains in the Fab fragments are immunoglobulin derived heavy chains.

「免疫球蛋白輕鏈」為一含有免疫球蛋白之抗原結合域之至少一部分及免疫球蛋白輕鏈可變區之至少一部分或免疫球蛋白輕鏈恆定區之至少一部分之多肽。如此，免疫球蛋白衍生的輕鏈與免疫球蛋白基因超家族的成員具有顯著的胺基酸序列同源性區域。An "immunoglobulin light chain" is a polypeptide comprising at least a portion of the antigen binding domain of an immunoglobulin and at least a portion of the variable region of an immunoglobulin light chain or at least a portion of the constant region of an immunoglobulin light chain. Thus, immunoglobulin-derived light chains share significant regions of amino acid sequence homology with members of the immunoglobulin gene superfamily.

「免疫黏附素(immunoadhesin)」為一嵌合、類抗體的分子，其將結合蛋白質(通常為受體、配體、或細胞黏附分子)的功能域與免疫球蛋白恆定域(通常包括鉸鏈區域及Fc區域)予以組合。"Immunoadhesin" is a chimeric, antibody-like molecule that binds a functional domain of a protein (usually a receptor, ligand, or cell adhesion molecule) to an immunoglobulin constant domain (usually including the hinge region). and Fc region) to be combined.

「片段抗原結合(fragment antigen-binding)」(Fab)片段」係與抗原結合的抗體上的區域。其係由重鏈及輕鏈之每一者的一個恆定域與一個可變域所組成。A "fragment antigen-binding" (Fab fragment) is a region on an antibody that binds to an antigen. It consists of one constant domain and one variable domain of each of the heavy and light chains.

基於尋求針對其進行保護的疾病的病因(病原)而選擇抗病原構築體。此等病原可為病毒、細菌或真菌來源，並且可用於預防人類感染人類疾病，或者用於非人類哺乳動物或其它動物以預防獸醫學疾病。Antipathogenic constructs are selected based on the etiology (pathogen) of the disease against which protection is sought. These pathogens may be of viral, bacterial or fungal origin and may be used to prevent human disease in humans, or in non-human mammals or other animals to prevent veterinary disease.

rAAV可包括編碼抗體的基因，特別是針對病毒性病原的中和抗體。此類抗病毒抗體可包括針對A型流感、B型流感及C型流感中的一種或多種的抗流感抗體。A型病毒為最具毒性的人類病原。已與大流行有關的A型流感的血清型包括：H1N1，導致了1918年的西班牙流感及2009年的豬流感；H2N2，導致了1957年的亞洲流感；H3N2，導致了1968年的香港流感；H5N1，導致了2004年的禽流感；H7N7；H1N2；H9N2；H7N2；H7N3；及H10N7。其它目標病原性病毒包括：沙狀病毒(arenavirus)(包括胡寧病毒(funin virus)、馬丘波病毒(Machupo virus)及賴薩病毒(Lassa virus))、絲狀病毒(filovirus)(包括馬堡病毒(Marburg virus)及伊波拉病毒(Ebola virus))、漢他病毒(hantavirus)、小核糖核酸病毒科(picornoviridae)(包括鼻病毒(rhinovirus)、ECHO病毒(echovirus))、冠狀病毒(coronavirus)、副黏液病毒(paramyxovirus)、麻疹病毒(morbillivirus)、呼吸道融合病毒(respiratory synctial virus)、披衣病毒(togavirus)、柯沙奇病毒(coxsackievirus)、JC病毒、小病毒(parvovirus) B19、副流感病毒、腺病毒、里奧病毒(reovirus)、天花(主天花病毒(Variola major (Smallpox))及來自痘病毒科的牛痘 (Vaccinia (Cowpox))、及水痘帶狀疱疹(varicella-zoster)(假性狂犬病(pseudorabies))。由沙狀病毒科的成員(賴薩熱)(該科亦與淋巴球性脈絡叢腦膜炎(Lymphocytic choriomeningitis，LCM)有關)、絲狀病毒(伊波拉病毒)、及漢他病毒(普馬拉病毒(puremala virus))所引起的病毒性出血熱。小核糖核酸病毒(picornavirus)的成員(鼻病毒之亞科)與人類普通感冒有關。冠狀病毒科，包括許多非人類病毒，如傳染性支氣管炎病毒(家禽)、豬傳染性胃腸病毒(豬)、豬血球凝集性腦脊髓炎病毒(豬)、貓傳染性腹膜炎病毒(貓)、貓腸冠狀病毒(貓)、犬冠狀病毒(狗)。已推定人類呼吸道冠狀病毒與普通感冒、非A、B或C型肝炎及突發性急性呼吸道症候群(SARS)有關。副黏液病毒科包括第1型副流感病毒、第3型副流感病毒、第3型牛副流感病毒、腮腺炎病毒(rubulavirus (mumps virus))、第2型副流感病毒、第4型副流感病毒、新城雞瘟病毒(Newcastle disease virus)(雞)、牛瘟、麻疹病毒(其包括麻疹及犬瘟熱)、及肺炎病毒(pneumovirus)(其包括呼吸道融合病毒(RSV))。小病毒科包括貓小病毒(貓腸炎)、貓泛白血球減少症病毒、犬小病毒、及豬小病毒。腺病毒科包括病毒(EX、AD7、ARD、O.B.)，其引起呼吸道疾病。如此，在某些具體實施例中，如本文所述之rAAV載體可經工程化以表現抗伊波拉抗體，例如2G4、4G7、13C6、抗流感抗體，例如FI6、CR8033、及抗RSV抗體，例如帕利珠單抗(palivizumab)、莫維珠單抗(motavizumab)。亦可選擇針對細菌性病原之中和抗體構築體用於本發明。在一具體實施例中，中和抗體構築體係針對細菌本身。在另一具體實施例中，中和抗體構築體係針對由細菌所產生的毒素。空氣傳播的細菌性病原之例包括例如腦膜炎雙球菌( Neisseria meningitidis)(腦膜炎)、克雷伯氏肺炎菌( Klebsiella pneumonia)(肺炎)、銅綠假單胞菌( Pseudomonas aeruginosa)(肺炎)、類寄疽假單胞菌( Pseudomonas pseudomallei)(肺炎)、鼻疽假單胞菌( Pseudomonas mallei)(肺炎)、不動桿菌(Acinetobacter)(肺炎)、卡他莫拉菌( Moraxella catarrhalis)、腔隙莫拉菌( Moraxella lacunata)、產鹼桿菌屬( Alkaligenes)、心桿菌屬( Cardiobacterium)、流感嗜血桿菌( Haemophilus influenzae)(流感)、副流感嗜血桿菌( Haemophilus parainfluenzae)、百日咳博德氏桿菌( Bordetella pertussis)(百日咳)、土倫病法蘭西斯桿菌( Francisella tularensis)(肺炎/熱病)、退伍軍人菌( Legionella pneumonia)(退伍軍人病)、鸚鵡熱披衣菌( Chlamydia psittaci)(肺炎)、肺炎披衣菌( Chlamydia pneumoniae)(肺炎)、結核分枝桿菌( Mycobacterium tuberculosis)(結核病(TB))、堪塞斯分枝桿菌( Mycobacterium kansasii)(TB)、鳥分枝桿菌( Mycobacterium avium)(肺炎)、星形土壤絲菌( Nocardia asteroides)(肺炎)、炭疽桿菌( Bacillus anthracis)(炭疽)、金黃色葡萄球菌( Staphylococcus aureus)(肺炎)、釀膿鏈球菌( Streptococcus pyogenes)(猩紅熱)、肺炎鏈球菌( Streptococcus pneumoniae)(肺炎)、白喉桿菌( Corynebacteria diphtheria)(白喉)、肺炎黴漿菌( Mycoplasma pneumoniae)(肺炎)。 rAAV may include genes encoding antibodies, particularly neutralizing antibodies against viral pathogens. Such antiviral antibodies may include anti-influenza antibodies against one or more of influenza A, influenza B, and influenza C. Type A viruses are the most virulent human pathogens. Influenza A serotypes that have been linked to pandemics include: H1N1, which caused the Spanish flu in 1918 and swine flu in 2009; H2N2, which caused the Asian flu in 1957; H3N2, which caused the Hong Kong flu in 1968; H5N1, which caused the bird flu in 2004; H7N7; H1N2; H9N2; H7N2; H7N3; and H10N7. Other pathogenic viruses of interest include: arenaviruses (including funin virus, Machupo virus, and Lassa virus), filoviruses (including horse Marburg virus and Ebola virus), hantavirus, picornoviridae (including rhinovirus, ECHO virus), coronavirus ), paramyxovirus, morbillivirus, respiratory synctial virus, togavirus, coxsackievirus, JC virus, parvovirus B19, paravirus Influenza virus, adenovirus, reovirus, smallpox (Variola major (Smallpox)) and Vaccinia (Cowpox) from the family Poxviridae, and varicella-zoster ( Pseudorabies). Consisting of members of the arenavirus family (Lyssa fever) (which is also associated with lymphocytic choriomeningitis (LCM)), filoviruses (Ebola virus), Viral hemorrhagic fever caused by Hantavirus (puremala virus). Members of picornaviruses (subfamily of rhinoviruses) are associated with the common cold in humans. Coronaviridae, including many Nonhuman viruses such as infectious bronchitis virus (poultry), porcine transmissible gastrointestinal virus (pig), porcine hemagglutinating encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enterocoronavirus (feline ), canine coronaviruses (dogs). Human respiratory coronaviruses have been presumed to be associated with the common cold, hepatitis other than A, B or C, and sudden acute respiratory syndrome (SARS). Paramyxoviridae includes parainfluenza virus type 1 , type 3 parainfluenza virus, type 3 bovine parainfluenza virus, rubulavirus (mumps virus)), type 2 parainfluenza virus, type 4 parainfluenza virus, Newcastle disease virus (Newcastle disease virus) ( chicken), rinderpest, measles virus (which includes measles and canine distemper), and pneumovirus (which includes respiratory fusion virus (RSV)). Parvoviridae includes feline parvovirus (feline enteritis), feline panleukocyte Attenuation virus, canine parvovirus, and porcine parvovirus. The Adenoviridae family includes viruses (EX, AD7, ARD, OB) that cause respiratory disease. Thus, in certain embodiments, rAAV vectors as described herein can be engineered to express anti-Ebola antibodies, such as 2G4, 4G7, 13C6, anti-influenza antibodies, such as FI6, CR8033, and anti-RSV antibodies, such as Palivizumab, Motavizumab. Neutralizing antibody constructs against bacterial pathogens may also be selected for use in the present invention. In one embodiment, the neutralizing antibody construct is directed against the bacterium itself. In another embodiment, the neutralizing antibody construct is directed against a toxin produced by bacteria. Examples of airborne bacterial pathogens include, for example, Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis , lacunae Moraxella lacunata , Alkaligenes , Cardiobacterium , Haemophilus influenzae (influenzae), Haemophilus parainfluenzae , Bordetella pertussis ( Bordetella pertussis ) (pertussis), Francisella tularensis (pneumonia/fever), Legionella pneumonia (Legion's disease), Chlamydia psittaci (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis ( tuberculosis (TB)), Mycobacterium kansasii (TB), Mycobacterium avium ( Pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria (diphtheria), Mycoplasma pneumoniae (pneumonia).

rAAV可包括編碼抗體的基因，特別是針對如炭疽之病因(一種由炭疽桿菌( Bacillius anthracis)所產生的毒素)的細菌性病原的中和抗體。已描述針對保護物質(PA)(形成類毒素之三種肽之一)的中和抗體。另外兩種多肽係由致死因子(lethal factor，LF)及水腫因子(edema factor，EF)所組成。抗PA中和抗體已被描述於針對炭疽的被動免疫中有效。參見例如：US專利號7,442,373；R. Sawada-Hirai et al, J Immune Based Ther Vaccines. 2004; 2: 5. (2004年5月12日上線)。其它抗炭疽毒素中和抗體已被描述及/或可被生成。相似地，可使用針對其它細菌及/或細菌毒素的中和抗體來產生如本文所述的AAV遞送的抗病原構築體。 rAAV may include genes encoding antibodies, particularly neutralizing antibodies against bacterial pathogens such as the cause of anthrax, a toxin produced by Bacillius anthracis . Neutralizing antibodies have been described against protective substance (PA), one of the three peptides forming toxoids. The other two polypeptides are composed of lethal factor (LF) and edema factor (EF). Anti-PA neutralizing antibodies have been described to be effective in passive immunization against anthrax. See eg: US Patent No. 7,442,373; R. Sawada-Hirai et al, J Immune Based Ther Vaccines. 2004; 2: 5. (accessed May 12, 2004). Other neutralizing antibodies against anthrax toxin have been described and/or can be generated. Similarly, neutralizing antibodies against other bacteria and/or bacterial toxins can be used to generate AAV-delivered antipathogenic constructs as described herein.

抗傳染病的抗體可能由寄生蟲或真菌引起，包括例如麴菌屬物種( Aspergillusspecies)、繖狀犁頭黴( Absidia corymbifera)、匍枝根黴( Rhixpus stolonifer)、毛黴菌( Mucor plumbeaus)、新型隱球菌( Cryptococcus neoformans)、莢膜組織孢漿菌( Histoplasm capsulatum)、皮炎芽生菌( Blastomyces dermatitidis)、粗球黴菌( Coccidioides immitis)、青黴菌屬物種( Penicilliumspecies)、乾草小多孢菌( Micropolyspora faeni)、普通耐熱放線菌( Thermoactinomyces vulgaris)、互生鏈隔孢菌( Alternaria alternate)、分枝孢子菌屬物種( Cladosporiumspecies)、長蠕孢黴屬( Helminthosporium)、及葡萄穗黴屬物種( Stachybotrysspecies)。 Antibodies against infectious diseases may be caused by parasites or fungi, including, for example, Aspergillus species, Absidia corymbifera , Rhixpus stolonifer , Mucor plumbeaus , Cryptococcus neoformans , Histoplasm capsulatum, Blastomyces dermatitidis , Coccidioides immitis , Penicillium species, Micropolyspora hay Micropolyspora faeni ), Thermoactinomyces vulgaris , Alternaria alternate , Cladosporium species, Helminthosporium , and Staphylocera species ( Stachybotrys species).

rAAV可包括編碼抗體的基因，特別是針對疾病的病原因子之中和抗體，該疾病如阿茲海默氏症(AD)、帕金森氏症(PD)、GBA相關帕金森氏症(GBA-PD)、類風濕性關節炎(RA)、腸躁症候群(Irritable bowel syndrome，IBS)、慢性阻塞性肺病(COPD)、癌症、腫瘤、全身性硬化症、氣喘及其它疾病。此種抗體可為但不限於例如α-突觸核蛋白(α-synuclein)、抗血管內皮生長因子(VEGF)(抗VEGF)、抗VEGFA、抗PD-1、抗PDL1、抗CTLA-4、抗TNF-alpha、抗IL-17、抗IL-23、抗IL-21、抗IL-6、抗IL-6受體、抗IL-5、抗IL-7、抗因子XII、抗IL-2、抗HIV、抗IgE、抗腫瘤壞死因子受體-1 (TNFR1)、抗刻痕蛋白(notch) 2/3、抗刻痕蛋白1、抗OX40、抗erb-b2受體酪胺酸激酶3(ErbB3)、抗ErbB2、抗β細胞成熟抗原、抗B淋巴球刺激因子、抗CD20、抗HER2、抗顆粒球巨噬細胞群落刺激因子、抗抑癌蛋白(oncostatin)M (OSM)、抗淋巴球活化基因3(LAG3)蛋白、抗CCL20、抗血清類澱粉P成分(SAP)、抗脯胺醯基羥化酶抑制劑、抗CD38、抗醣蛋白IIb/IIIa、抗CD52、抗CD30、抗IL-1beta、抗表皮生長因子受體、抗CD25、抗RANK配體、抗補體系統蛋白C5、抗CD11a、抗CD3受體、抗alpha-4 (α4)整合素、抗RSV F蛋白、及抗整合素α 4β 7。對於所屬技術領域中具通常知識者而言，其它病原及疾病將為顯而易見的。其它適合的抗體可包括彼等有用於治療阿茲海默氏症者，諸如例如抗β類澱粉(例如，克雷內治單抗(crenezumab)、索拉珠單抗(solanezumab)、阿杜卡單抗(aducanumab))、抗β類澱粉纖絲、抗β類澱粉斑、抗tau、巴皮紐阻單抗(bapineuzamab)等。治療多種適應症之其它適合的抗體包括彼等描述於例如PCT/US2016/058968者，其於2016年10月27日申請，公開為WO 2017/075119A1。 rAAV may include genes encoding antibodies, particularly neutralizing antibodies against pathogenic factors of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), GBA-associated Parkinson's disease (GBA- PD), rheumatoid arthritis (RA), irritable bowel syndrome (Irritable bowel syndrome, IBS), chronic obstructive pulmonary disease (COPD), cancer, tumor, systemic sclerosis, asthma and other diseases. Such antibodies can be, but are not limited to, for example, α-synuclein (α-synuclein), anti-vascular endothelial growth factor (VEGF) (anti-VEGF), anti-VEGFA, anti-PD-1, anti-PDL1, anti-CTLA-4, Anti-TNF-alpha, anti-IL-17, anti-IL-23, anti-IL-21, anti-IL-6, anti-IL-6 receptor, anti-IL-5, anti-IL-7, anti-factor XII, anti-IL-2 , Anti-HIV, Anti-IgE, Anti-Tumor Necrosis Factor Receptor-1 (TNFR1), Anti-Notch 2/3, Anti-Notch 1, Anti-OX40, Anti-ERB-B2 Receptor Tyrosine Kinase 3 (ErbB3), anti-ErbB2, anti-β-cell maturation antigen, anti-B lymphocyte-stimulating factor, anti-CD20, anti-HER2, anti-granular macrophage colony-stimulating factor, anti-oncostatin M (OSM), anti-lymphocyte Globe-activating gene 3 (LAG3) protein, anti-CCL20, anti-serum amyloid P component (SAP), anti-prolyl hydroxylase inhibitor, anti-CD38, anti-glycoprotein IIb/IIIa, anti-CD52, anti-CD30, anti- IL-1beta, anti-epidermal growth factor receptor, anti-CD25, anti-RANK ligand, anti-complement system protein C5, anti-CD11a, anti-CD3 receptor, anti-alpha-4 (α4) integrin, anti-RSV F protein, and anti- Integrin α 4 β 7 . Other pathogens and diseases will be apparent to those of ordinary skill in the art. Other suitable antibodies may include those useful in the treatment of Alzheimer's disease, such as, for example, anti-beta-amyloids (e.g., crenezumab, solanezumab, aducan Monoclonal antibody (aducanumab)), anti-β-amyloid fibrils, anti-β-amyloid plaques, anti-tau, bapineuzamab, etc. Other suitable antibodies for the treatment of various indications include those described, eg, in PCT/US2016/058968, which was filed on October 27, 2016 and published as WO 2017/075119A1.

減少及/或調節基因表現對於治療以細胞過度增生為特徵的過度增生性病況特別理想，如癌症及牛皮癬。目標多肽包括彼等與正常細胞相比在過度增生的細胞中排他地產生或以更高水準產生的多肽。目標抗原包括由致癌基因所編碼的多肽，該致癌基因例如為myb、myc、fyn、以及轉位基因 bcr/abl、ras、src、P53、neu、trk及EGRF。除了致癌基因產物作為目標抗原外，用於抗癌治療及保護性方案之目標多肽包括由B細胞淋巴瘤產生的抗體的可變區及T細胞淋巴瘤的T細胞受體的可變區，於一些具體實施例，其亦使用作為自體免疫疾病的目標抗原。其它腫瘤相關多肽可用於作為目標多肽，諸如在腫瘤細胞中發現有較高水準的多肽，包括被單株抗體17-1A辨識的多肽及葉酸結合多肽。Reducing and/or modulating gene expression is particularly desirable for the treatment of hyperproliferative conditions characterized by cellular hyperplasia, such as cancer and psoriasis. Polypeptides of interest include those that are produced exclusively or at higher levels in hyperproliferative cells as compared to normal cells. Antigens of interest include polypeptides encoded by oncogenes such as myb, myc, fyn, and the transposons bcr/abl, ras, src, P53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides for anticancer therapy and protective regimens include the variable regions of antibodies produced by B-cell lymphomas and the variable regions of T-cell receptors for T-cell lymphomas, in In some embodiments, it is also used as a target antigen for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides found at higher levels in tumor cells, including polypeptides recognized by monoclonal antibody 17-1A and folate-binding polypeptides.

其它適合的治療性多肽及蛋白質包括彼等藉由賦予針對與自體免疫有關目標之廣泛基礎的保護性免疫反應而可用於治療罹患自體免疫疾病及失調的個體的多肽及蛋白質，該目標包括細胞受體及產生「自」導向抗體的細胞。T細胞媒介的自體免疫疾病包括類風濕性關節炎(RA)、多發性硬化症(MS)、休格倫氏症候群、類肉瘤病、胰島素依賴型糖尿病(IDDM)、自體免疫甲狀腺炎、反應性關節炎、關節黏連性脊椎炎、硬皮病、多發性肌炎、皮肌炎、牛皮癬、血管炎、華格納氏肉芽病、克隆氏病及潰瘍性結腸炎。此等疾病中的每一種皆以與內源性抗原結合並引發和自體免疫疾病有關的發炎級聯反應的T細胞受體(TCR)為特徵。Other suitable therapeutic polypeptides and proteins include those useful in the treatment of individuals suffering from autoimmune diseases and disorders by conferring a broad-based protective immune response against targets associated with autoimmunity, including Cell receptors and cells that produce "self" directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Shoegren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, Reactive arthritis, adhesive spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wagner's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

替代地或附加地，載體可含有本發明的AAV序列及編碼誘導對所選免疫原的免疫反應的肽、多肽或蛋白質的轉基因。例如，免疫原可選自多種病毒科。期望針對其進行免疫反應的理想病毒科之例包括小核糖核酸病毒科，其包括：鼻病毒屬，造成約50%的普通感冒病例；腸病毒屬，其包括脊髓灰白質炎病毒(poliovirus)、柯沙奇病毒、ECHO病毒、及人類腸病毒如A型肝炎病毒；及口蹄疫病毒屬(apthovirus)，其造成口蹄疫，主要於非人類動物。小核糖核酸病毒科之病毒中，目標抗原包括VP1、VP2、VP3、VP4及VPG。另外的病毒科包括杯狀病毒科(calcivirus family)，其涵蓋諾瓦克病毒群(Norwalk group of viruses)，其為流行性胃腸炎的重要病因。期望用於靶向抗原以誘導於人類及非人類動物中的免疫反應的另一種病毒科為披衣病毒科(togavirus family)，該病毒科包括：α病毒屬(alphavirus)，包括辛得比斯病病毒(Sindbis virus)、羅氏河病毒(RossRiver virus)、以及委內瑞拉、東方及西方馬腦炎；以及風疹病毒屬(rubivirus)，包括風疹病毒。黃病毒科(flaviviridae family)包括登革熱、黃熱病、日本腦炎、聖路易腦炎(St. Louis encephalitis)、以及蜱傳染的腦炎之病毒。其它目標抗原可由C型肝炎或冠狀病毒科產生，其包括許多非人類病毒，如傳染性支氣管炎病毒(家禽)、豬傳染性胃腸病毒(豬)、豬血球凝集性腦脊髓炎病毒(豬)、貓傳染性腹膜炎病毒(貓)、貓腸冠狀病毒(貓)、犬冠狀病毒(狗)以及人類呼吸道冠狀病毒，此等病毒可引起感冒及/或非A型、B型或C型肝炎。於冠狀病毒科，目標抗原包括E1(亦稱為M或基質蛋白)、E2(亦稱為S或棘蛋白(Spike protein))、E3(亦稱為HE或血球凝集素酯酶)醣蛋白(不存在於所有冠狀病毒中)、或者N(核酸蛋白殼)。再其它抗原可為針對棒狀病毒科(rhabdovirus family)所靶向的，棒狀病毒科包括水泡病毒屬(vesiculovirus)(例如，水泡性口炎病毒)及麗沙病毒屬(lyssavirus)(例如，狂犬病)。於棒狀病毒科中，適合的抗原可衍生自G蛋白或N蛋白。絲狀病毒科(filoviridae)可為一種適合的抗原來源，其包括出血熱病毒如馬堡病毒及伊波拉病毒。副黏病毒科包括第1型副流感病毒、第3型副流感病毒、第3型牛副流感病毒、腮腺炎病毒(rubulavirus (mumps virus))、第2型副流感病毒、第4型副流感病毒、新城雞瘟病毒(雞)、牛瘟、麻疹病毒(包括麻疹及犬瘟熱病毒)、以及肺炎病毒(包括呼吸道融合病毒)。流感病毒被分類於正黏液病毒科，為一種適合的抗原來源(例如，HA蛋白、N1蛋白)。崩芽病毒科(bunyavirus family)包括崩芽病毒屬(加州腦炎、拉克羅斯(La Crosse)腦炎)、沙蠅病毒屬(phlebovirus)(裂谷熱(Rift Valley Fever))、漢他病毒屬(普馬拉病毒(puremala)為一種出血熱病毒)、內羅畢病毒屬(nairovirus)(奈洛比綿羊病)以及各種未命名的崩芽病毒。沙狀病毒科提供針對LCM及賴薩熱病毒的抗原來源。里奧病毒科包括里奧病毒屬、輪狀病毒屬(rotavirus)(其引起而兒童急性胃腸炎)、環狀病毒屬(orbivirus)、及科羅拉多壁蝨熱病毒屬(cultivirus)(科羅拉多壁蝨熱、勒邦博病(Lebombo)(人類)、馬器質性腦病、藍舌病)。Alternatively or additionally, the vector may contain the AAV sequences of the invention and a transgene encoding a peptide, polypeptide or protein that induces an immune response to the immunogen of choice. For example, the immunogen can be selected from a variety of viral families. Examples of ideal viral families against which an immune response is desired include the picornaviridae, which includes: rhinoviruses, which cause about 50% of common cold cases; enteroviruses, which include polioviruses, Coxsackieviruses, ECHOviruses, and human enteroviruses such as hepatitis A virus; and apthoviruses, which cause foot-and-mouth disease, mainly in non-human animals. Among viruses of the Picornaviridae family, target antigens include VP1, VP2, VP3, VP4, and VPG. Additional viral families include the calcivirus family, which encompasses the Norwalk group of viruses, an important cause of epidemic gastroenteritis. Another family of viruses that is expected to be used to target antigens to induce immune responses in humans and non-human animals is the togavirus family, which includes: alphaviruses, including Sindbis Sindbis virus, Ross River virus, and Venezuelan, Eastern, and Western equine encephalitis; and rubiviruses, including rubella virus. The flaviviridae family includes viruses of dengue fever, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis. Other target antigens can be produced by Hepatitis C or Coronaviridae, which includes many non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenterovirus (pig), porcine hemagglutinating encephalomyelitis virus (pig) , feline infectious peritonitis virus (cats), feline enterocoronavirus (cats), canine coronavirus (dogs), and human respiratory coronavirus, which can cause colds and/or non-A, B, or C hepatitis. In Coronaviridae, target antigens include E1 (also known as M or matrix protein), E2 (also known as S or Spike protein), E3 (also known as HE or hemagglutinin esterase) glycoproteins ( Not present in all coronaviruses), or N (nucleic acid protein shell). Still other antigens may be targeted against the rhabdovirus family, which includes vesiculoviruses (e.g., vesicular stomatitis virus) and lyssaviruses (e.g., rabies). In the Rhabdoviridae, suitable antigens may be derived from the G protein or the N protein. Filoviridae may be a suitable source of antigens and include hemorrhagic fever viruses such as Marburg virus and Ebola virus. Paramyxoviridae include parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubulavirus (mumps virus), parainfluenza virus type 2, parainfluenza type 4 virus, Newcastle disease virus (chicken), rinderpest, measles virus (including measles and canine distemper virus), and pneumovirus (including respiratory fusion virus). Influenza viruses are classified in the Orthomyxoviridae family and are a suitable source of antigens (eg, HA protein, N1 protein). The bunyavirus family includes bunyaviruses (California encephalitis, La Crosse encephalitis), phleboviruses (Rift Valley Fever), hantaviruses (puremala, a hemorrhagic fever virus), nairovirus (Nairobi sheep disease), and various unnamed collapse bud viruses. The Arenaviridae provide a source of antigens against LCM and Lyssa virus. The Rioviridae family includes the genera Riovirus, rotavirus (which causes acute gastroenteritis in children), orbivirus, and cultivirus (Colorado tick fever, Le Lebombo (human), equine organic encephalopathy, bluetongue).

反轉錄病毒科(retrovirus family)包括：致癌病毒(oncorivirinal)亞科，其涵蓋此類人類及獸醫學疾病，如猫白血病病毒、HTLVI及HTLVII；慢病毒(lentivirinal)亞科(包括人類免疫不全病毒(HIV)、猿猴免疫不全病毒(SIV)、猫免疫不全病毒(FIV)、馬傳染性貧血病毒、以及泡沫病毒(spumavirinal))。於HIV及SIV之間，許多適合的抗原已被描述且可容易地選擇。適合的HIV及SIV抗原之例包括但不限於gag、pol、Vif、Vpx、VPR、Env、Tat及Rev蛋白、以及此等各種片段。此外，已描述對此等抗原的多種修飾。為了此目的之適合的抗原為所屬技術領域中具通常知識者已知的。例如，可選擇編碼gag、pol、Vif、及Vpr、Env、Tat及Rev以及其它蛋白質等的序列。參見例如：經修飾的gag蛋白描述於美國專利5,972,596。亦參見描述於D.H. Barouch et al, J. Virol., 75(5):2462-2467 (2001年3月)、及R.R. Amara, et al, Science, 292:69-74 (2001年4月6日)之HIV及SIV蛋白質。此等蛋白質或其次單元可被單獨遞送，或者經由各別載體或形成單一載體而組合遞送。The retrovirus family includes: the oncorivirinal subfamily, which covers such human and veterinary diseases, such as feline leukemia virus, HTLVI and HTLVII; the lentivirinal subfamily (including human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, and spumavirinal). Between HIV and SIV, many suitable antigens have been described and can be easily selected. Examples of suitable HIV and SIV antigens include, but are not limited to, the gag, pol, Vif, Vpx, VPR, Env, Tat and Rev proteins, and various fragments of these. Furthermore, various modifications of these antigens have been described. Suitable antigens for this purpose are known to those of ordinary skill in the art. For example, sequences encoding gag, pol, Vif, and Vpr, Env, Tat, and Rev, among other proteins, can be selected. See eg: Modified gag proteins are described in US Patent 5,972,596. See also described in D.H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R.R. Amara, et al, Science, 292:69-74 (April 6, 2001 ) HIV and SIV proteins. These proteins or subunits may be delivered individually or in combination via separate carriers or forming a single carrier.

乳多泡病毒科(papovavirus family)包括多瘤病毒(polyomavirus)亞科(BKU及JCU病毒)及乳頭瘤病毒(papillomavirus)亞科(與癌症或乳頭狀瘤的惡性進展有關)。腺病毒科包括引起呼吸道疾病及/或腸炎的病毒(EX、AD7、ARD、O.B.)。小病毒科包括貓小病毒(貓腸炎)、貓泛白血球減少症病毒、犬小病毒、以及豬小病毒。疱疹病毒科包括：α疱疹病毒亞科，其涵蓋單純疱疹病毒屬(simplexvirus) (HSVI、HSVII)、水痘疱疹病毒屬(varicellovirus)(假性狂犬病、水痘帶狀疱疹)；以及β疱疹病毒亞科，其包括巨細胞病毒屬(HCMV、鼠巨細胞病毒)；以及γ疱疹病毒亞科，其包括淋巴潛隱病毒屬(lymphocryptovirus)、EBV(伯奇氏淋巴瘤(Burkitts lymphoma))、傳染性鼻氣管炎、馬立克氏病病毒(Marek’s disease virus)、以及蛛猴病毒屬(rhadinovirus)。痘病毒科包括痘病毒脊索亞科(subfamily chordopoxvirinae)，其涵蓋正痘病毒屬(orthopoxvirus)(天花(Variola (Smallpox))及牛痘(Vaccinia (Cowpox)))、副痘病毒屬(parapoxvirus)、禽痘病毒屬(avipoxvirus)、羊痘病毒屬(capripoxvirus)、兔痘病毒屬(leporipoxvirus)、豬痘病毒屬(suipoxvirus)；以及痘病毒昆蟲亞科(subfamily entomopoxvirinae)。肝DNA病毒科(hepadnavirus family)包括B型肝炎病毒。一種可為適合的抗原來源的未分類病毒是D型肝炎病毒。再其它病毒來源可包括禽類傳染性華氏囊病病毒及豬生殖與呼吸綜合症病毒。α病毒科包括馬動脈炎病毒及各種腦炎病毒。The papovavirus family includes the polyomavirus subfamily (BKU and JCU viruses) and the papillomavirus subfamily (associated with malignant progression of cancer or papilloma). The Adenoviridae family includes viruses (EX, AD7, ARD, O.B.) that cause respiratory disease and/or enteritis. The parvovirus family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The Herpesviridae family includes: Alphaherpesvirinae, which covers the genera simplexvirus (HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster); and Betaherpesvirinae , which includes the genus cytomegalovirus (HCMV, murine cytomegalovirus); Tracheitis, Marek's disease virus, and rhadinovirus. The Poxviridae includes the subfamily chordopoxvirinae, which covers the genera orthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus, avian avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus; and subfamily entomopoxvirinae. The hepadnavirus family includes hepatitis B viruses. One unclassified virus that may be a suitable source of antigen is hepatitis D virus. Still other viral sources may include avian infectious bursal disease virus and porcine reproductive and respiratory syndrome virus. The Alphaviridae family includes equine arteritis viruses and various encephalitis viruses.

rAAV亦可遞送編碼免疫原的序列，該序列有用於免疫人類或非人類動物以對抗其它病原，該病原包括感染人類及非人類脊椎動物之細菌、真菌、寄生性微生物或多細胞寄生蟲，或者是來自癌細胞或腫瘤細胞。細菌性病原之例包括病原性革蘭氏陽性球菌，包括肺炎雙球菌(pneumococci)；葡萄球菌(staphylococci)；以及鏈球菌。病原性革蘭氏陰性球菌包括腦膜炎雙球菌；淋球菌。病原性腸道革蘭氏陰性桿菌包括腸桿菌科；假單胞菌屬、不動桿菌屬(acinetobacteria)及艾肯菌屬(eikenella)；類鼻疽(melioidosis)；沙門氏桿菌屬；志賀桿菌屬；嗜血桿菌屬；莫拉氏菌屬(moraxella)；杜克氏嗜血桿菌( H. ducreyi)(引起軟下疳(chancroid))；布氏桿菌屬(Brucella)；土倫病法蘭西斯桿菌( Franisella tularensis)(引起兔熱病)；耶氏桿菌(yersinia)(巴斯德桿菌屬)；念珠狀鏈桿菌(streptobacillus moniliformis)及螺菌屬(spirillum)；革蘭氏陽性桿菌包括李斯特單胞菌(listeria monocytogenes)；紅斑丹毒絲菌(erysipelothrix rhusiopathiae)；白喉棒狀桿菌( Corynebacterium diphtheria)(白喉)；霍亂(cholera)；炭疽桿菌(炭疽)；杜諾凡病(donovanosis)(腹股溝肉芽腫(granuloma inguinale))；以及巴東體症(bartonellosis)。由病原性厭氧菌引起的疾病包括破傷風；肉毒桿菌中毒(botulism)；其它梭狀芽孢桿菌；結核病；麻風；以及其它分枝桿菌。病原性螺旋體病包括梅毒；密螺旋體病(treponematoses)：莓疹病(yaws)、品他病(pinta)及地方性梅毒；以及鉤端螺旋體病(leptospirosis)。由高等病原性細菌及病原性真菌所引起的其它感染包括放線菌病；土壤絲菌病；隱球菌病、芽生菌病(blastomycosis)、組織漿菌病以及球黴菌病；念珠菌病、麴菌病及白黴菌症(mucormycosis)；孢子絲菌病(sporotrichosis)；副球黴菌病(paracoccidiodomycosis)、囊子菌病(petriellidiosis)、球擬酵母菌病(torulopsosis)、足菌腫(mycetoma)及產色黴菌病(chromomycosis)；以及皮癬菌病。立克次體感染包括斑疹傷寒熱、落磯山斑疹熱、Q熱(Q fever)、及立克次體痘(Rickettsialpox)。黴漿菌及披衣菌感染之例包括：肺炎黴漿菌；性病性淋巴肉芽腫(lymphogranuloma venereum)；鸚鵡熱；及週產期披衣菌感染(perinatal chlamydial infection)。病原性真核生物涵蓋病原性原生動物及蠕蟲且由其所產生的感染包括：阿米巴症；瘧疾；利什曼病；錐蟲病；弓蟲病；卡氏肺囊蟲( Pneumocystis carinii)；Trichans；剛第弓蟲( Toxoplasma gondii)；焦蟲病；梨形鞭毛蟲症；旋毛蟲病；絲蟲病；血吸蟲病；線蟲(nematode)；吸蟲(trematode或fluke)；及絛蟲(cestode (tapeworm))感染。 rAAV can also deliver sequences encoding immunogens useful for immunizing humans or non-human animals against other pathogens, including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates, or from cancer cells or tumor cells. Examples of bacterial pathogens include pathogenic Gram-positive cocci, including pneumococci; staphylococci; and streptococci. Pathogenic Gram-negative cocci include Neisseria meningitidis; Neisseria gonorrhoeae. Pathogenic enteric gram-negative bacilli include Enterobacteriaceae; Pseudomonas, acinetobacteria, and eikenella; melioidosis; Salmonella; Shigella; Haemophilus; Moraxella; H. ducreyi (causes chancroid); Brucella; Franisella tularensis ) (causes tularemia); yersinia (pasteurella genus); streptobacillus moniliformis and spirillum; gram-positive bacilli including listeria monocytogenes); erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; Bacillus anthracis (anthrax); donovanosis (granuloma inguinale ); and bartonellosis. Diseases caused by pathogenic anaerobes include tetanus; botulism; other Clostridia; tuberculosis; leprosy; Pathogenic treponematosis includes syphilis; treponematoses: yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by higher pathogenic bacteria and pathogenic fungi include actinomycosis; agrothrichosis; cryptococcosis, blastomycosis, histoplasmosis, and coccidiomycosis; candidiasis, aspergillus fungal disease and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include typhus fever, Rocky Mountain spotted fever, Q fever, and Rickettsial pox. Examples of mycoplasma and chlamydial infections include: Mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infection. Pathogenic eukaryotes include pathogenic protozoa and helminths and infections resulting from them include: amoebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii ); Trichans; Toxoplasma gondii ; Pyrosomiasis; Piropiasis; Trichinellosis; Filariasis; Schistosomiasis; cestode (tapeworm)) infection.

許多此等生物體及/或由其所產生的毒素已被疾病管制中心[(CDC)，美國衛生與人群服務部]認定為具有用於生物攻擊的潛力的物質。例如，一些此類生物物質包括炭疽桿菌(炭疽)、肉毒桿菌及其毒素(肉毒桿菌中毒)、鼠疫耶氏桿菌( Yersinia pestis)(鼠疫)、主天花病毒(Variola major (Smallpox))、土倫病法蘭西斯桿菌(兔熱病)及病毒性出血熱，其所有目前皆被分類為A類物質；伯納特氏柯克斯氏體( Coxiella burnetti)(Q熱)、布氏桿菌屬物種(布氏桿菌病)、鼻疽伯克霍爾德氏菌( Burkholderia mallei)(鼻疽)、蓖麻( Ricinus communis)及其毒素(蓖麻毒蛋白毒素)、產氣莢膜梭狀芽孢桿菌( Clostridium perfringens)及其毒素(ε毒素)、葡萄球菌屬物種及其毒素(腸毒素B)，其所有目前皆被分類為B類物質；以及立百病毒(Nipan virus)及漢他病毒，其所有目前皆被分類為C類物質。此外，其它如此分類或不同分類的生物體可於將來被認定及/或用於這種目的。容易理解的是，本文所述的病毒載體及其它構築體有用於遞送來自此等生物體、病毒、其毒素或其它副產物的抗原，其將預防及/或治療此等生物物質引起的感染或其它不良反應。 Many of these organisms and/or toxins produced by them have been identified by the Centers for Disease Control [(CDC), US Department of Health and Human Services] as substances with potential for use in biological attacks. Some such biological agents include, for example, Bacillus anthracis (anthrax), Bacillus botulinum and its toxin (botulism), Yersinia pestis (plague), Variola major (Smallpox), Francisella tularensis (tularemia) and viral hemorrhagic fever, all of which are currently classified as category A substances; Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (mallei), castor plant ( Ricinus communis ) and its toxin (ricin toxin), Clostridium perfringens ( Clostridium perfringens ) and its toxin (ε toxin), Staphylococcus species and its toxin (enterotoxin B), all of which are currently classified as Category B substances; and Nipah virus (Nipan virus) and Hantavirus, which All are currently classified as Category C substances. Furthermore, other organisms classified as such or differently may be identified and/or used for such purposes in the future. It will be readily understood that the viral vectors and other constructs described herein are useful for delivering antigens from such organisms, viruses, their toxins or other by-products that will prevent and/or treat infections or Other adverse reactions.

投予本發明之載體以遞送免疫原，該免疫原針對T細胞的可變區誘發免疫反應(包括CTL)以消除彼等T細胞。於類風濕性關節炎(RA)中，涉及此疾病的T細胞受體(TCR)的數種特異性可變區已被特性分析。此等TCR包括V-3、V-14、V-17及Vα-17。如此，遞送編碼此等多肽的至少一者的核酸序列將誘發免疫反應，該免疫反應將靶向涉及RA的T細胞。於多發性硬化症(MS)，涉及此疾病的TCR的數種特異性可變區已被特性分析。此等TCR包括V-7及Vα-10。如此，遞送編碼此等多肽的至少一者的核酸序列將誘發免疫反應，該免疫反應將靶向涉及MS的T細胞。於硬皮病，涉及此疾病的TCR的數種特異性可變區已被特性分析。此等TCR包括V-6、V-8、V-14及Vα-16、Vα-3C、Vα-7、Vα-14、Vα-15、Vα-16、Vα-28及Vα-12。如此，遞送編碼此等多肽的至少一者的核酸分子將誘發免疫反應，該免疫反應將靶向涉及硬皮病的T細胞。The vectors of the invention are administered to deliver an immunogen that elicits an immune response (including CTL) against the variable regions of T cells to eliminate those T cells. In rheumatoid arthritis (RA), several specific variable regions of the T cell receptor (TCR) involved in this disease have been characterized. Such TCRs include V-3, V-14, V-17 and Va-17. As such, delivery of a nucleic acid sequence encoding at least one of these polypeptides will induce an immune response that will target T cells involved in RA. In multiple sclerosis (MS), several specific variable regions of TCRs involved in this disease have been characterized. Such TCRs include V-7 and Va-10. As such, delivery of a nucleic acid sequence encoding at least one of these polypeptides will induce an immune response that will target T cells involved in MS. In scleroderma, several specific variable regions of TCRs involved in this disease have been characterized. Such TCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. As such, delivery of a nucleic acid molecule encoding at least one of these polypeptides will induce an immune response that will target T cells involved in scleroderma.

在一具體實施例中，選擇轉基因以提供光遺傳學治療。在光遺傳學治療中，人造光受體係藉由將光活化通道或泵基因遞送至剩餘的視網膜迴路中存活的細胞類型而構築。此對於失去大量光受體功能，但神經節細胞及視神經的雙極細胞迴路仍然完整的患者特別有用。在一具體實施例中，異源性核酸序列(轉基因)為一種視蛋白(opsin)。視蛋白序列可衍生自任何適合的單或多細胞生物體，包括人類、藻類及細菌。在一具體實施例中，該視蛋白為視紫紅質(rhodopsin)、光蛋白(photopsin)、L/M波長(紅/綠)-視蛋白、或短波長(S)視蛋白(藍)。在另一具體實施例中，該視蛋白為光敏通道蛋白(channelrhodopsin)或嗜鹽視紫紅質(halorhodopsin)。In a specific embodiment, the transgene is selected to provide optogenetic therapy. In optogenetic therapy, artificial photoreceptors are constructed by delivering light-activated channel or pump genes to surviving cell types in the remaining retinal circuits. This is especially useful in patients who have lost a significant amount of photoreceptor function but have intact ganglion cells and bipolar cell circuits in the optic nerve. In a specific embodiment, the heterologous nucleic acid sequence (transgene) is an opsin. Opsin sequences may be derived from any suitable uni- or multicellular organism, including humans, algae, and bacteria. In a specific embodiment, the opsin is rhodopsin, photopsin, L/M wavelength (red/green)-opsin, or short wavelength (S) opsin (blue). In another specific embodiment, the opsin is channelrhodopsin or halorhodopsin.

在另一具體實施例中，選擇轉基因以用於基因增強治療(gene augmentation therapy)，即，提供缺失或缺陷的基因的替代副本。於此具體實施例中，所屬技術領域中具通常知識者可容易地選擇轉基因以提供必要的替代基因。在一具體實施例中，此缺失/缺陷的基因係關於眼部病症。在另一具體實施例中，轉基因為NYX、GRM6、TRPM1L或GPR179且該眼部病症為先天性停滯型夜盲症(Congenital Stationary Night Blindness)。參見例如：Zeitz et al, Am J Hum Genet. 2013 Jan 10;92(1):67-75. Epub 2012年12月13日，其藉由引用併入本文。於另一具體實施例，轉基因為RPGR。於另一具體實施例，該基因為由CHM所編碼的Rab護衛蛋白(Rab escort protein 1，REP-1)，與無脈絡膜有關。In another specific embodiment, a transgene is selected for gene augmentation therapy, ie, to provide a replacement copy of a missing or defective gene. In this embodiment, one of ordinary skill in the art can readily select a transgene to provide the necessary replacement gene. In one embodiment, the deleted/defective gene is related to an eye disorder. In another specific embodiment, the transgene is NYX, GRM6, TRPM1L or GPR179 and the ocular disorder is Congenital Stationary Night Blindness. See eg: Zeitz et al, Am J Hum Genet. 2013 Jan 10;92(1):67-75. Epub 13 Dec. 2012, which is incorporated herein by reference. In another embodiment, the transgene is RPGR. In another embodiment, the gene is Rab escort protein 1 (REP-1) encoded by CHM, which is related to choroideremia.

在另一具體實施例中，選擇轉基因以用於基因抑制治療(gene suppression therapy)，即，一種以上天然基因的表現於轉錄或轉譯層級被中斷或抑制。此可使用短髮夾RNA (shRNA)或本領域眾所周知的其它技術來完成。參見例如：Sun et al, Int J Cancer. 2010 Feb 1;126(3):764-74及O'Reilly M, et al. Am J Hum Genet. 2007 Jul;81(1):127-35，此等藉由引用而併入本文。在此具體實施例中，所屬技術領域中具通常知識者基於所欲緘默化的基因可容易地選擇轉基因。In another embodiment, a transgene is selected for gene suppression therapy, ie, the expression of one or more native genes is disrupted or inhibited at the transcriptional or translational level. This can be done using short hairpin RNA (shRNA) or other techniques well known in the art. See for example: Sun et al, Int J Cancer. 2010 Feb 1;126(3):764-74 and O'Reilly M, et al. Am J Hum Genet. 2007 Jul;81(1):127-35, here et al. are incorporated herein by reference. In this embodiment, one of ordinary skill in the art can readily select a transgene based on the gene to be silenced.

在另一具體實施例中，轉基因包含多於一個轉基因。此可使用帶有二種以上異源性序列的單一載體或使用各自帶有一種以上異源性序列的二種以上之rAAV而完成。在一具體實施例中，該rAAV用於基因抑制(或減弱(knockdown))及基因增強協同治療。於減弱/增強協同治療中，感興趣的基因的缺陷型拷貝被緘默化，且提供未突變的拷貝。在一具體實施例中，此係使用兩種以上共同投予的載體而完成。參見Millington-Ward et al, Molecular Therapy, April 2011, 19(4):642–649，其藉由引用併入本文。基於所欲結果，所屬技術領域中具通常知識者可容易地選擇該轉基因。In another specific embodiment, the transgene comprises more than one transgene. This can be accomplished using a single vector with two or more heterologous sequences or using two or more rAAVs each with more than one heterologous sequence. In a specific embodiment, the rAAV is used for synergistic therapy of gene suppression (or knockdown) and gene enhancement. In attenuation/enhancement combination therapy, the defective copy of the gene of interest is silenced and an unmutated copy is provided. In one embodiment, this is accomplished using two or more co-administered vectors. See Millington-Ward et al, Molecular Therapy, April 2011, 19(4):642-649, which is incorporated herein by reference. The transgene can be readily selected by one of ordinary skill in the art based on the desired outcome.

在另一具體實施例中，選擇轉基因以用於基因矯正治療(gene correction therapy)。其可使用下列而完成，例如，組合鋅指核酸酶(zinc-finger nuclease，ZFN)誘導的DNA雙股斷裂與外源DNA供體基質。參見例如Ellis et al, Gene Therapy (epub January 2012) 20:35-42，其藉由引用併入本文。在一具體實施例中，該轉基因編碼選自巨核酸酶(meganuclease)、鋅指核酸酶、類轉錄活化因子(transcription activator-like，TAL)效應核酸酶(TALEN)及重複短迴文序列簇(clustered, regularly interspaced short palindromic repeat，CRISPR)/核酸內切酶(Cas9、Cpf1等)的核酸酶。適合的巨核酸酶之例描述於例如美國專利8,445,251；US 9,340,777；US 9,434,931；US 9,683,257及WO 2018/195449。其它適合的酵素包括可用核酸編程的方式結合RNA之核酸酶不活化的釀膿鏈球菌(S. pyogenes) CRISPR/Cas9 (Nelles et al, Programmable RNA Tracking in Live Cells with CRISPR/Cas9, Cell, 165(2):P488-96 (2016年4月))、及鹼基編輯器(base editor)(例如，Levy et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses, Nature Biomedical Engineering, 4, 97–110 (2020年1月))。在某些具體實施例中，該核酸酶不為鋅指核酸酶。在某些具體實施例中，該核酸酶不為CRISPR相關核酸酶。在某些具體實施例中，該核酸酶不為TALEN。在一具體實施例中，該核酸酶不為巨核酸酶。在某些具體實施例中，該核酸酶為歸巢核酸內切酶(homing endonuclease)之LAGLIDADG (SEQ ID NO: 47)家族之一員。在某些具體實施例中，該核酸酶為歸巢核酸內切酶之I-CreI家族之一員，其辨識並切割22個鹼基對辨識序列SEQ ID NO: 48 - CAAAACGTCGTGAGACAGTTTG。參見例如：WO 2009/059195。描述合理設計單-LAGLIDADG (SEQ ID NO: 47)歸巢核酸內切酶的方法，該方法能夠全面地重新設計ICreI及其它歸巢核酸內切酶以靶向廣泛不同的DNA部位，包括在哺乳動物、酵母、植物、細菌及病毒基因體中的部位(WO 2007/047859)。In another specific embodiment, the transgene is selected for gene correction therapy. This can be accomplished using, for example, zinc-finger nuclease (ZFN)-induced DNA double-strand breaks in combination with an exogenous DNA donor matrix. See, eg, Ellis et al, Gene Therapy (epub January 2012) 20:35-42, which is incorporated herein by reference. In a specific embodiment, the transgene encoding is selected from meganuclease (meganuclease), zinc finger nuclease, class transcription activator-like (transcription activator-like, TAL) effector nuclease (TALEN) and repeated short palindromic sequence cluster ( clustered, regularly interspaced short palindromic repeat, CRISPR)/endonuclease (Cas9, Cpf1, etc.) nuclease. Examples of suitable meganucleases are described in eg US Patent 8,445,251; US 9,340,777; US 9,434,931; US 9,683,257 and WO 2018/195449. Other suitable enzymes include nuclease-inactivated Streptococcus pyogenes (S. pyogenes) CRISPR/Cas9 (Nelles et al, Programmable RNA Tracking in Live Cells with CRISPR/Cas9, Cell, 165( 2): P488-96 (April 2016)), and base editor (for example, Levy et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses, Nature Biomedical Engineering, 4, 97–110 (January 2020). In certain embodiments, the nuclease is not a zinc finger nuclease. In certain embodiments, the nuclease is not a CRISPR-associated nuclease. In certain embodiments, the nuclease is not a TALEN. In a specific embodiment, the nuclease is not a meganuclease. In certain embodiments, the nuclease is a member of the LAGLIDADG (SEQ ID NO: 47) family of homing endonucleases. In certain embodiments, the nuclease is a member of the I-CreI family of homing endonucleases, which recognizes and cleaves the 22 base pair recognition sequence of SEQ ID NO: 48 - CAAAACGTCGTGAGACAGTTTG. See eg WO 2009/059195. Describes a method for the rational design of a mono-LAGLIDADG (SEQ ID NO: 47) homing endonuclease that enables comprehensive redesign of ICreI and other homing endonucleases to target a wide variety of DNA sites, including in mammals Sites in animal, yeast, plant, bacterial and viral genomes (WO 2007/047859).

適合的基因編輯目標包括例如肝臟表現的基因，例如但不限於前蛋白轉化酶枯草桿菌蛋白酶/kexin 9型(proprotein convertase subtilisin/kexin type 9，PCSK9)(膽固醇相關失調)、轉甲狀腺素蛋白(transthyretin，TTR)(轉甲狀腺素蛋白類澱粉變性症)、HAO、脂蛋白元(apolipoprotein) C-III (APOC3)、因子VIII、因子IX、低密度脂蛋白受體(LDLr)、脂蛋白脂酶(LPL)(脂蛋白脂酶缺乏症)、卵磷脂-膽固醇醯基轉移酶(LCAT)、鳥胺酸胺甲醯基轉移酶(OTC)、肌肽酶(carnosinase，CN1)、神經髓磷脂磷酸二脂酶(SMPD1)(尼曼匹克症)、次黃嘌呤-鳥嘌呤磷酸核糖轉移酶(HGPRT)、支鏈α-酮酸脫氫酶複合物(branched-chain alpha-keto acid dehydrogenase complex，BCKDC)(楓糖尿症)、紅血球生成素(EPO)、胺甲醯基磷酸合成酶(CPS1)、N-乙醯麩胺酸合成酶(N-Acetylglutamate Synthetase，NAGS)、精胺琥珀酸合成酶(瓜胺酸血症)、精胺琥珀酸裂解酶(ASL)(精胺琥珀酸尿症(Argininosuccinic Aciduria))、及精胺酸酶(AG)。Suitable gene editing targets include, for example, liver-expressed genes such as, but not limited to, proprotein convertase subtilisin/kexin type 9 (PCSK9) (cholesterol-related disorders), transthyretin , TTR) (transthyretin-like amylosis), HAO, apolipoprotein C-III (APOC3), factor VIII, factor IX, low-density lipoprotein receptor (LDLr), lipoprotein lipase ( LPL) (lipoprotein lipase deficiency), lecithin-cholesterol acyltransferase (LCAT), ornithine aminotransferase (OTC), carnosinase (CN1), myelin phosphodiesterase enzyme (SMPD1) (Niemann-Pick syndrome), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), branched-chain alpha-keto acid dehydrogenase complex (BCKDC) ( maple syrup urine), erythropoietin (EPO), carbamoyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (N-Acetylglutamate Synthetase, NAGS), arginine succinate synthase (citrulline acidemia), arginine succinate lyase (ASL) (Argininosuccinic Aciduria), and arginine enzyme (AG).

其它基因編輯目標可包括例如羥甲基膽汁烷合成酶(HMBS)、胺甲醯基合成酶I、鳥胺酸胺甲醯基轉移酶(OTC)、精胺琥珀酸合成酶、α-1抗胰蛋白酶(A1AT)、用於治療精胺琥珀酸裂解酶缺乏症之精胺琥珀酸裂解酶(ASL)、精胺酸酶、延胡索醯乙酸水解酶、苯丙胺酸羥化酶、α-1抗胰蛋白酶、恆河猴α胎兒蛋白(AFP)、恆河猴絨毛膜促性腺激素(CG)、葡萄糖-6-磷酸酶、膽色素原脫胺酶、胱硫醚β合成酶、支鏈酮酸脫羧酶、白蛋白、異戊醯輔酶A脫氫酶、丙醯輔酶A羧化酶、甲基丙二醯輔酶A變位酶(MUT)、戊二醯輔酶A脫氫酶、胰島素、β-葡萄糖苷酶、丙酮酸羧化酶、肝磷酸化酶、磷酸化酶激酶、甘胺酸脫羧酶、H-蛋白、T-蛋白、囊腫纖維化跨膜調節子(CFTR)序列、及肌肉萎縮蛋白基因產物[例如，迷你-或微小-肌肉萎縮蛋白]。又其它有用的基因產物包括酵素，如可有用於酵素替代療法，其有用於各種由於酵素活性不足而引起的病況。例如，含有甘露糖-6-磷酸的酵素可用於溶酶體儲積症之治療(例如，包括編碼β-葡萄醣醛酸酶(GUSB)之適合的基因)。於另一例中，基因產物為泛素蛋白連接酶。葡萄糖-6-磷酸酶，與肝醣儲積症或缺乏症1A型(GSD1)有關；磷酸烯醇丙酮酸羧激酶(PEPCK)，與PEPCK缺乏症有關；類周期蛋白依賴型激酶5(CDKL5)，亦已知為絲胺酸/蘇胺酸激酶9(STK9)，與癲癇發作及嚴重的神經發展障礙有關；半乳糖-1-磷酸尿苷醯基轉移酶，與半乳糖血症有關；苯丙胺酸羥化酶(PAH)，與苯丙酮尿症(PKU)有關；與原發性高草酸鹽尿症第1型有關的基因產物，包括羥基酸氧化酶1(GO/HAO1)及AGXT；支鏈α-酮酸脫氫酶，包括BCKDH、BCKDH-E2、BAKDH-E1a、及BAKDH-E1b，與楓糖尿症有關；延胡索醯乙醯乙酸水解酶，與酪胺酸血症第1型有關；甲基丙二醯輔酶A變位酶，與甲基丙二酸血症有關；中鏈乙醯輔酶A脫氫酶，與中鏈乙醯輔酶A缺乏症有關；鳥胺酸胺甲醯基轉移酶(OTC)，與鳥胺酸胺甲醯基轉移酶缺乏症有關；精胺琥珀酸合成酶(ASS1)，與瓜胺酸血症有關；卵磷脂-膽固醇醯基轉移酶(LCAT)缺乏症；甲基丙二酸血症(MMA)；與Cl型尼曼匹克症有關的NPC1；丙酸血症(PA)；與轉甲狀腺素蛋白(TTR)相關遺傳性類澱粉變性症有關的TTR；低密度脂蛋白受體(LDLR)蛋白，與家族性高膽固醇血症(FH)有關；LDLR變異體，諸如彼等描述於WO 2015/164778者；PCSK9；ApoE及ApoC蛋白，與失智症有關；UDP葡萄糖醛酸基轉移酶，與克-納二氏症有關；腺苷脫胺酶，與嚴重合併性免疫不全病有關；次黃嘌呤-鳥嘌呤磷酸核糖轉移酶，與痛風及勒-奈二氏症候群有關；生物素酶，與生物素酶缺乏症有關；與法布瑞氏症有關的α-半乳糖苷酶(a-Gal A)；與GM1神經節苷脂沉積症有關的β-半乳糖苷酶(GLB1)；與威爾森氏症有關的ATP7B；β-葡萄糖腦苷脂酶，與高歇氏病第2及3型有關；過氧化體膜蛋白70 kDa，與齊威格氏症候群有關；與異染性白質失養症有關的芳基硫酸酯酶A(ARSA)；與克拉培氏病有關的半乳糖腦苷脂酶( GALC)酵素；與龐貝氏症有關的α-葡萄糖苷酶(GAA)；神經髓磷脂酶(SMPD1)基因，與A型尼曼匹克氏症有關；與成年發病第II型瓜胺酸血症(CTLN2)有關的精胺琥珀酸合成酶；與尿素循環失調有關的胺甲醯基-磷酸合成酶1(CPS1)；運動神經元存活(SMN)蛋白，與脊髓性肌肉萎縮症有關；與法伯脂肉芽腫症有關的神經醯胺酶；與GM2神經節苷脂沉積症以及泰-薩二氏及山多夫氏病有關的β-己醣胺酶；與天冬胺醯基葡萄糖胺尿有關的天冬胺醯基葡萄胺糖苷酶；與岩藻糖沉積症有關的α-岩藻糖苷酶；與α-甘露糖沉積症有關的α-甘露糖苷酶；膽色素原脫胺酶，與急性間歇性紫質症(AIP)有關；用於治療α-1抗胰蛋白酶缺乏症(肺氣腫)之α-1抗胰蛋白酶；用於治療因地中海貧血或腎衰竭引起的貧血之紅血球生成素；用於治療缺血性疾病之血管內皮生長因子、血管生成素-1及纖維母細胞生長因子；用於治療於下列所見的血管阻塞之凝血酶調節素及組織因子路徑抑制劑，例如動脈粥樣硬化、血栓症或栓塞；用於治療帕金森氏症之芳香族胺基酸脫羧酶(AADC)及酪胺酸羥化酶(TH)；用於治療充血性心臟衰竭之β腎上腺素受體、受磷蛋白之反義或突變型、肌(內)質網腺苷三磷酸酶-2 (SERCA2)及心臟腺苷酸環化酶；用於治療各種癌症之腫瘤抑制基因，諸如p53；細胞激素，諸如各種介白素之一者，用於治療發炎及免疫失調及癌症；用於治療肌肉營養不良之肌肉萎縮蛋白或小肌肉萎縮蛋白及肌營養相關蛋白或小肌營養相關蛋白；以及用於治療糖尿病之胰島素或GLP-1。 Other gene editing targets can include, e.g., hydroxymethylbilane synthase (HMBS), carbamoyl synthetase I, ornithine carboxytransferase (OTC), spermine succinate synthase, alpha-1 antibody Trypsin (A1AT), arginine succinate lyase (ASL) for the treatment of arginine succinate lyase deficiency, argininase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1 antipancreatic Protease, rhesus alpha-fetal protein (AFP), rhesus chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, cystathionine beta synthase, branched-chain ketoacid decarboxylation Enzymes, albumin, isopentyl-CoA dehydrogenase, acyl-CoA carboxylase, methylmalonyl-CoA mutase (MUT), glutaryl-CoA dehydrogenase, insulin, beta-glucose Glycidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin gene product [eg, mini- or micro-dystrophin]. Still other useful gene products include enzymes, such as enzyme replacement therapy, which is useful for various conditions caused by insufficient enzyme activity. For example, mannose-6-phosphate-containing enzymes can be used in the treatment of lysosomal storage disorders (eg, including a suitable gene encoding beta-glucuronidase (GUSB)). In another example, the gene product is a ubiquitin protein ligase. Glucose-6-phosphatase, associated with glycogen storage disease or deficiency type 1A (GSD1); phosphoenolpyruvate carboxykinase (PEPCK), associated with PEPCK deficiency; cyclin-dependent kinase 5 (CDKL5), Also known as serine/threonine kinase 9 (STK9), associated with seizures and severe neurodevelopmental disorders; galactose-1-phosphate uridine acyltransferase, associated with galactosemia; phenylalanine Hydroxylase (PAH), associated with phenylketonuria (PKU); gene products associated with primary hyperoxaluria type 1, including hydroxyacid oxidase 1 (GO/HAO1) and AGXT; Chain alpha-ketoacid dehydrogenases, including BCKDH, BCKDH-E2, BAKDH-E1a, and BAKDH-E1b, are associated with maple syrup urine; fumarylacetate hydrolase, associated with tyrosinemia type 1; Methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium-chain acetyl-CoA dehydrogenase, associated with medium-chain acetyl-CoA deficiency; ornithine formyl transfer Enzymes (OTC), associated with ornithine aminotransferase deficiency; argininesuccinate synthase (ASS1), associated with citrullinemia; lecithin-cholesterol acyltransferase (LCAT) deficiency ; methylmalonic acidemia (MMA); NPC1 associated with Niemann-Pick disease type Cl; propionic acidemia (PA); TTR associated with transthyretin (TTR)-related hereditary amyloidosis; Low-density lipoprotein receptor (LDLR) protein, associated with familial hypercholesterolemia (FH); LDLR variants, such as those described in WO 2015/164778; PCSK9; ApoE and ApoC proteins, associated with dementia ; UDP glucuronosyltransferase, associated with Krona-Sehr syndrome; adenosine deaminase, associated with severe comorbid immunodeficiency disease; hypoxanthine-guanine phosphoribosyltransferase, associated with gout and Leh-Nye Associated with Gemini syndrome; biotinidase, associated with biotinidase deficiency; α-galactosidase (a-Gal A), associated with Fabry's disease; β-galactosidase (a-GalA), associated with GM1 gangliosidosis Galactosidase (GLB1); ATP7B, associated with Wilson's disease; β-glucocerebrosidase, associated with Gaucher disease types 2 and 3; peroxosomal membrane protein 70 kDa, associated with Zellweger syndrome; arylsulfatase A (ARSA) associated with metachromatic leukodystrophy; galactocerebrosidase ( GALC ) enzyme associated with Clapey's disease; alpha - Glucosidase (GAA); neuromyelinase (SMPD1) gene, associated with Niemann-Pick disease type A; argininosuccinate synthase associated with adult-onset citrullinemia type II (CTLN2); Carbamoyl-phosphate synthase 1 (CPS1), associated with urea cycle disorders; Survival motor neuron (SMN) protein, associated with spinal muscular atrophy; Ceramidase, associated with Faber lipogranulomatosis; β-hexosaminidase associated with GM2 gangliosidosis and Tay-Sarger and Sanddorf diseases; aspartaginal glucosaminidase associated with aspartaginal glucosamineuria; α-fucosidase associated with fucosidosis; α-mannosidase associated with α-mannosidosis; porphobilinogen deaminase, associated with acute intermittent porphyria (AIP); α-1 antitrypsin for the treatment of α-1 antitrypsin deficiency (emphysema); erythropoietin for the treatment of anemia caused by thalassemia or renal failure; vascular endothelial for the treatment of ischemic diseases Growth Factors, Angiopoietin-1, and Fibroblast Growth Factor; Thrombomodulin and Tissue Factor Pathway Inhibitors for the treatment of vascular obstruction seen in, for example, atherosclerosis, thrombosis, or embolism; for the treatment of Aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) for Parkinson's disease; β-adrenergic receptors, antisense or mutant forms of phospholamban for the treatment of congestive heart failure, Sarcoplasmic reticulum adenosine triphosphatase-2 (SERCA2) and cardiac adenylate cyclase; tumor suppressor genes, such as p53, for the treatment of various cancers; cytokines, such as one of various interleukins, for Treatment of inflammation and immune disorders and cancer; dystrophin or dystrophin and dystrophin or dystrophin for the treatment of muscular dystrophy; and insulin or GLP-1 for the treatment of diabetes.

在一具體實施例中，本文所述衣殼有用於CRISPR-Cas雙載體系統，其描述於US公開專利申請案2018/0110877，2018年4月26日申請，其藉由引用併入本文。該衣殼亦有用於遞送歸巢核酸內切酶或其它巨核酸酶。In one embodiment, the capsids described herein are useful in a CRISPR-Cas dual vector system, which is described in US Published Patent Application 2018/0110877, filed April 26, 2018, which is incorporated herein by reference. The capsid is also useful for the delivery of homing endonucleases or other meganucleases.

在另一具體實施例中，於本文中有用的轉基因包括報導序列(reporter sequence)，其表現產生可偵測到的訊號。此種報導序列包括但不限於編碼下列的DNA序列：β-內醯胺酶、β-半乳糖苷酶(LacZ)、鹼性磷酸酶、胸苷激酶、綠色螢光蛋白(GFP)、紅色螢光蛋白(RFP)、氯黴素乙醯基轉移酶(chloramphenicol acetyltransferase，CAT)、螢光素酶、膜結合蛋白質，包括例如CD2、CD4、CD8、流感血球凝集素蛋白、及所屬技術領域中其它熟知者，針對其的高親和力抗體係存在或可藉由常規手段產生，且包含膜結合蛋白質的融合蛋白適當融合至抗原標籤域，其中抗原標籤域來自如血球凝集素或Myc。In another embodiment, a transgene useful herein includes a reporter sequence that expresses a detectable signal. Such reporter sequences include, but are not limited to, DNA sequences encoding the following: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein Photoprotein (RFP), chloramphenicol acetyltransferase (chloramphenicol acetyltransferase, CAT), luciferase, membrane-bound proteins including, for example, CD2, CD4, CD8, influenza hemagglutinin protein, and others known in the art It is well known that high affinity antibodies against it exist or can be generated by conventional means, and that fusion proteins comprising membrane-bound proteins are suitably fused to antigen tag domains from eg hemagglutinin or Myc.

在某些具體實施例中，除了轉基因編碼序列，可包括另外的非AAV編碼序列，例如，肽、多肽、蛋白質、功能性RNA分子(例如，miRNA、miRNA抑制劑)或其它感興趣的基因產物。有用的基因產物可包括miRNA。miRNA及其它短小干擾核酸經由目標RNA轉錄本的切割/降解或目標傳訊RNA (mRNA)的轉譯壓制而調節基因表現。miRNA係天然地表現，通常作為最終19-25個非轉譯的RNA產物。miRNA通過與目標mRNAs之3′未轉譯區域(UTR)的序列特異性相互作用而展示其活性。此等內源性表現的miRNA形成髮夾前驅物，隨後被加工成雙股miRNA，並進一步加工成「成熟」單股miRNA分子。此成熟miRNA引導一種多蛋白複合物miRISC，其基於與成熟miRNA的互補性而識別目標mRNAs之目標部位，例如，於3′ UTR區域。In certain embodiments, in addition to the transgene coding sequence, additional non-AAV coding sequences may be included, e.g., peptides, polypeptides, proteins, functional RNA molecules (e.g., miRNA, miRNA inhibitors), or other gene products of interest . Useful gene products can include miRNAs. miRNAs and other short interfering nucleic acids regulate gene expression through cleavage/degradation of target RNA transcripts or translational repression of target messenger RNAs (mRNAs). miRNAs are expressed naturally, usually as final 19-25 non-translated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into double-stranded miRNAs and further processed into "mature" single-stranded miRNA molecules. The mature miRNA guides miRISC, a multiprotein complex that recognizes target sites of target mRNAs based on complementarity to the mature miRNA, for example, in the 3'UTR region.

此等上述編碼序列，當與驅動其表現的調節元件組合時，提供可藉由常規手段檢測到的訊號，包括酵素性的、放射線攝影的、比色的、螢光或其它光譜的檢定、螢光活化細胞分選檢定及免疫檢定，包括酶聯免疫吸附檢定(ELISA)、放射免疫檢定(RIA)及免疫組織化學。例如，於標記序列為LacZ基因的情況下，藉由β-半乳糖苷酶活性檢定來檢測帶有訊號的載體的存在。當轉基因為綠色螢光蛋白或螢光素酶時，可在光度計中藉由產生的顏色或光來目測帶有訊號的載體。These aforementioned coding sequences, when combined with regulatory elements driving their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorometric or other spectroscopic assays, fluorescent Photoactivated cell sorting assays and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by a β-galactosidase activity assay. When the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visualized in a luminometer by the color or light produced.

理想地，轉基因編碼在生物學及醫學上有用的產物，如蛋白質、肽、RNA、酵素或催化性RNA。理想的RNA分子包括shRNA、tRNA、dsRNA、核糖體RNA、催化性RNA及反義RNA。有用的RNA序列之一例為在目標細胞中消除所靶向的核酸序列的表現的序列。Ideally, the transgene encodes a biologically and medically useful product, such as a protein, peptide, RNA, enzyme or catalytic RNA. Ideal RNA molecules include shRNA, tRNA, dsRNA, ribosomal RNA, catalytic RNA, and antisense RNA. An example of a useful RNA sequence is one that eliminates the expression of the targeted nucleic acid sequence in the target cell.

調節序列包括常規控制元件，其以允許轉基因在用載體轉染或用如本文所述產生的病毒感染的細胞中轉錄、轉譯及/或表現的方式，與轉基因可操作地連接。Regulatory sequences include conventional control elements that are operably linked to the transgene in a manner that permits its transcription, translation and/or expression in cells transfected with the vector or infected with viruses produced as described herein.

表現控制序列包括適當的轉錄起始、終止、啟動子及強化子序列；有效的RNA加工訊息如剪接及多腺苷酸化(polyA)訊息；穩定細胞質mRNA的序列；增強轉譯效率的序列(即Kozak共通序列)；增強蛋白質穩定性的序列；及當期望時，增強所編碼的產物分泌的序列。許多表現控制序列(包括啟動子)在本領域中係已知且可被利用。Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing messages such as splicing and polyadenylation (polyA) messages; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (ie, Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product. Many expression control sequences, including promoters, are known and available in the art.

本文提供的構築體中有用的調節序列亦可含有內含子，理想地，位於啟動子/強化子序列與基因之間。一理想的內含子序列衍生自SV-40，為100 bp迷你內含子(mini-intron)剪接供體/剪接受體(splice donor/splice acceptor)，稱為SD-SA。另外適合的序列包括土撥鼠肝炎病毒(woodchuck hepatitis virus)轉錄後元件。(參見例如：L. Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910)。多腺苷酸化訊息可衍生自許多適合的物種，包括但不限於SV-40、人類及牛。Regulatory sequences useful in the constructs provided herein may also contain introns, ideally located between the promoter/enhancer sequence and the gene. An ideal intron sequence derived from SV-40 is a 100 bp mini-intron splice donor/splice acceptor, called SD-SA. Additional suitable sequences include woodchuck hepatitis virus post-transcriptional elements. (See eg: L. Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910). The polyadenylation message can be derived from many suitable species, including but not limited to SV-40, human and bovine.

在本文所述的方法中有用的rAAV的另一調節組件為內部核糖體進入位(internal ribosome entry site，IRES)。可使用IRES序列或其它適合的系統從單個基因轉錄本中產生多於一個的多肽。IRES(或其它適合的序列)用於產生含有多於一條的多肽鏈的蛋白質，或用於從相同細胞或在同一細胞內表現兩種不同的蛋白質。示例性IRES為脊髓灰白質炎病毒內部核糖體進入序列，其支持在光受體、RPE及神經節細胞中的轉基因表現。較佳地，IRES位於rAAV載體中轉基因的3’。Another regulatory component of rAAV useful in the methods described herein is the internal ribosome entry site (IRES). More than one polypeptide can be produced from a single gene transcript using an IRES sequence or other suitable system. IRES (or other suitable sequences) are used to generate proteins containing more than one polypeptide chain, or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosomal entry sequence, which supports transgene expression in photoreceptors, RPE, and ganglion cells. Preferably, the IRES is located 3' of the transgene in the rAAV vector.

在某些具體實施例中，載體基因體包含啟動子(或啟動子之功能性片段)。rAAV中運用的啟動子的選擇可從大量組成型(constitutive)或可誘導型(inducible)啟動子中進行，該等啟動子可在所欲目標細胞中表現所選的轉基因。在一具體實施例中，目標細胞為眼細胞。啟動子可衍生自任何物種，包括人類。理想地，在一具體實施例中，啟動子為「細胞特異性的」。術語「細胞特異性的」係指為了重組載體所選的特定啟動子可指導所選的轉基因在特定細胞組織中的表現。在一具體實施例中，啟動子對於轉基因在肌肉細胞中的表現為特異性的。在另一具體實施例中，啟動子對於在肺中的表現為特異性的。在另一具體實施例中，啟動子對於在肝臟細胞中的表現為特異性的。在另一具體實施例中，啟動子對於在呼吸道上皮中的表現為特異性的。在另一具體實施例中，啟動子對於在神經元中的表現為特異性的。在另一具體實施例中，啟動子對於在心臟中的表現為特異性的。In some embodiments, the vector gene body comprises a promoter (or a functional fragment of a promoter). The choice of promoters for use in rAAV can be made from a large number of constitutive or inducible promoters that can express the selected transgene in the desired target cell. In a specific embodiment, the target cells are eye cells. Promoters can be derived from any species, including humans. Ideally, in one embodiment, the promoter is "cell specific". The term "cell-specific" refers to the fact that the specific promoter selected for the recombinant vector directs the expression of the selected transgene in a specific cell tissue. In a specific embodiment, the promoter is specific for expression of the transgene in muscle cells. In another embodiment, the promoter is specific for expression in the lung. In another embodiment, the promoter is specific for expression in liver cells. In another embodiment, the promoter is specific for expression in respiratory epithelium. In another embodiment, the promoter is specific for expression in neurons. In another specific embodiment, the promoter is specific for expression in the heart.

載體基因體通常含有啟動子序列作為表現控制序列之一部分，例如，位於所選擇的5’ ITR序列及編碼序列之間。在一具體實施例中，期望於肝臟中表現。如此，在一具體實施例中，使用肝臟特異性啟動子。肝臟特異性啟動子之例可包括例如甲狀腺素結合球蛋白(TBG)、白蛋白，Miyatake et al., (1997) J. Virol., 71:5124 32；B型肝炎病毒核心啟動子，Sandig et al., (1996) Gene Ther., 3:1002 9；或者人類α1-抗胰蛋白酶、磷酸烯醇丙酮酸羧激酶(PECK)、或α胎兒蛋白(AFP)，Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503 14)。組織特異性啟動子、組成型啟動子、可調節型啟動子[參見例如WO 2011/126808及WO 2013/04943]、或對生理學提示反應的啟動子可用於本文所述的載體中。在另一具體實施例中，期望於肌肉中表現。如此，在一具體實施例中，使用肌肉特異性啟動子。在一具體實施例中，啟動子為MCK系啟動子，如dMCK (509-bp)或tMCK (720-bp)啟動子(參見例如Wang et al, Gene Ther. 2008 Nov;15(22):1489-99. doi: 10.1038/gt.2008.104. Epub 2008 Jun 19，其藉由引用併入本文)。另外有用的啟動子為SPc5-12啟動子(參見Rasowo et al, European Scientific Journal June 2014 edition vol.10, No.18，其藉由引用併入本文)。在某些具體實施例中，可選擇對眼或其子部件(subpart)(例如，視網膜)為特異性之啟動子。The vector gene body usually contains a promoter sequence as part of the expression control sequence, for example, between the selected 5' ITR sequence and the coding sequence. In one embodiment, expression in the liver is desired. Thus, in a specific embodiment, a liver-specific promoter is used. Examples of liver-specific promoters may include, for example, thyroxine-binding globulin (TBG), albumin, Miyatake et al., (1997) J. Virol., 71:5124 32; hepatitis B virus core promoter, Sandig et al. al., (1996) Gene Ther., 3:1002 9; or human α1-antitrypsin, phosphoenolpyruvate carboxykinase (PECK), or α-fetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503 14). Tissue-specific promoters, constitutive promoters, regulatable promoters [see eg WO 2011/126808 and WO 2013/04943], or promoters responsive to physiological cues can be used in the vectors described herein. In another embodiment, expression in muscle is desired. Thus, in a specific embodiment, a muscle-specific promoter is used. In a specific embodiment, the promoter is an MCK promoter, such as dMCK (509-bp) or tMCK (720-bp) promoter (see, for example, Wang et al, Gene Ther. 2008 Nov; 15(22): 1489 -99. doi: 10.1038/gt.2008.104. Epub 2008 Jun 19, which is incorporated herein by reference). Another useful promoter is the SPc5-12 promoter (see Rasowo et al, European Scientific Journal June 2014 edition vol. 10, No. 18, which is incorporated herein by reference). In certain embodiments, a promoter can be selected that is specific for the eye or a subpart thereof (eg, the retina).

在一具體實施例中，啟動子為CMV啟動子。在另一具體實施例中，啟動子為TBG啟動子。在另一具體實施例中，使用CB7啟動子。CB7為一雞β-肌動蛋白啟動子，具巨細胞病毒強化子元件。或者，可使用其它肝臟特異性啟動子[參見例如：肝臟特異性基因啟動子資料庫，Cold Spring Harbor，rulai.schl.edu/LSPD，α1-抗胰蛋白酶(A1AT)；人類白蛋白，Miyatake et al., J. Virol., 71:5124 32 (1997), humAlb；及B型肝炎病毒核心啟動子，Sandig et al.,Gene Ther., 3:1002 9 (1996)]。TTR最小強化子/啟動子、α-抗胰蛋白酶啟動子、LSP (845 nt) 25(需要無內含子scAAV)。 In a specific embodiment, the promoter is a CMV promoter. In another specific embodiment, the promoter is a TBG promoter. In another specific embodiment, the CB7 promoter is used. CB7 is a chicken β-actin promoter with a cytomegalovirus enhancer element. Alternatively, other liver-specific promoters can be used [see e.g.: Liver-Specific Gene Promoter Database, Cold Spring Harbor, rulai.schl.edu/LSPD, α1-antitrypsin (A1AT); human albumin, Miyatake et al. al., J. Virol., 71:5124 32 (1997), humAlb; and the hepatitis B core promoter, Sandig et al., Gene Ther., 3:1002 9 (1996)]. TTR minimal enhancer/promoter, alpha-antitrypsin promoter, LSP (845 nt) 25 (requires intronless scAAV).

啟動子可選自不同來源，例如，人類巨細胞病毒(CMV)立即早期強化子/啟動子、SV40早期強化子/啟動子、JC多瘤病毒(polymovirus)啟動子、髓磷脂鹼性蛋白(MBP)或神經膠原纖維酸性蛋白(glial fibrillary acidic protein，GFAP)啟動子、單純疱疹病毒(HSV-1)潛伏相關啟動子(LAP)、勞斯氏肉瘤病毒(rouse sarcoma virus，RSV)末端長重複序列(long terminal repeat，LTR)啟動子、神經元特異性啟動子(NSE)、血小板衍生的生長因子(PDGF)啟動子、hSYN、黑色素聚集激素(melanin-concentrating hormone，MCH)啟動子、CBA、基質金屬蛋白(matrix metalloprotein)啟動子(MPP)、及雞β-肌動蛋白啟動子。Promoters can be selected from different sources, for example, human cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus (polymovirus) promoter, myelin basic protein (MBP ) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (rouse sarcoma virus, RSV) terminal long repeat (long terminal repeat, LTR) promoter, neuron-specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix The matrix metalloprotein promoter (MPP), and the chicken β-actin promoter.

載體基因體可含有至少一個強化子，即，CMV強化子。再其它強化子元件可包括例如脂蛋白元強化子、斑馬魚強化子、GFAP強化子元件、及腦特異性強化子(如描述於WO 2013/1555222)、土撥鼠肝炎病毒轉錄後調節元件。另外或可替代地，可選擇其它例如雜合人類巨細胞病毒(HCMV)立即早期(IE)-PDGR啟動子(hybrid human cytomegalovirus (HCMV)-immediate early (IE)-PDGR promoter)啟動子或其它啟動子-強化子元件。於本文中有用的其它強化子序列包括IRBP強化子(Nicoud 2007, J Gene Med. 2007 Dec;9(12):1015-23)、立即早期巨細胞病毒強化子、衍生自免疫球蛋白基因者或SV40強化子、在小鼠近端啟動子鑑定出的順式作用元件(cis-acting element)等。The vector gene body may contain at least one enhancer, ie, a CMV enhancer. Still other enhancer elements may include, for example, lipoprotein enhancers, zebrafish enhancers, GFAP enhancer elements, and brain-specific enhancers (as described in WO 2013/1555222), woodchuck hepatitis virus post-transcriptional regulatory elements. Additionally or alternatively, other promoters such as the hybrid human cytomegalovirus (HCMV)-immediate early (IE)-PDGR promoter or other promoters may be selected. child - reinforces the child element. Other enhancer sequences useful herein include the IRBP enhancer (Nicoud 2007, J Gene Med. 2007 Dec;9(12):1015-23), the immediate early cytomegalovirus enhancer, those derived from immunoglobulin genes or The SV40 enhancer, the cis-acting element identified in the mouse proximal promoter, etc.

除了啟動子，載體基因體可含有其它適當轉錄起始、終止、強化子序列、有效的RNA加工訊息如剪接及多腺苷酸化(polyA)訊息；穩定細胞質mRNA的序列；增強轉譯效率的序列(即Kozak共通序列)；增強蛋白質穩定性的序列；及當期望時，增強所編碼的產物分泌的序列。許多適合的polyA為已知的。於一例中，該polyA為兔β球蛋白，諸如127 bp兔β球蛋白多腺苷酸化訊息(GenBank # V00882.1)。在其它具體實施例中，選擇SV40 polyA訊息。可選擇再其它適合的polyA序列。在某些具體實施例中，包括內含子。一適合的內含子為雞β-肌動蛋白內含子。在一具體實施例中，該內含子為875 bp (GenBank # X00182.1)。在另一具體實施例中，使用可獲自Promega的嵌合內含子。然而，可選擇其它適合的內含子。在一具體實施例中，包括間隔子(spacers)使得載體基因體與天然AAV載體基因體為大約相同大小(例如，4.1至5.2 kb之間)。在一具體實施例中，包括間隔子使得載體基因體為約4.7 kb。參見Wu et al, Effect of Genome Size on AAV Vector Packaging, Mol Ther. 2010 Jan; 18(1): 80–86，其藉由引用併入本文。In addition to the promoter, the vector gene body can contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing information such as splicing and polyadenylation (polyA) information; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency ( ie, the Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product. Many suitable polyAs are known. In one example, the polyA is a rabbit beta globin, such as the 127 bp rabbit beta globin polyadenylation message (GenBank # V00882.1). In other embodiments, SV40 polyA messages are selected. Still other suitable polyA sequences can be selected. In certain embodiments, introns are included. A suitable intron is the chicken β-actin intron. In a specific embodiment, the intron is 875 bp (GenBank # X00182.1). In another specific embodiment, chimeric introns available from Promega are used. However, other suitable introns may be selected. In one embodiment, spacers are included such that the vector gene body is about the same size as the native AAV vector gene body (eg, between 4.1 and 5.2 kb). In a specific embodiment, spacers are included such that the vector gene body is about 4.7 kb. See Wu et al, Effect of Genome Size on AAV Vector Packaging, Mol Ther. 2010 Jan; 18(1): 80-86, which is incorporated herein by reference.

在某些具體實施例中，載體基因體進一步包含可操作地連接至轉基因編碼序列的背根神經節(drg)特異性miRNA脫靶序列(dorsal root ganglion (drg)-specific miRNA detargeting sequence)。在某些具體實施例中，縱排miRNA目標序列為連續的或被1至10個核酸之間隔子分隔開，其中該間隔子不為miRNA目標序列。在某些具體實施例中，有至少二個drg特異性miRNA序列位於功能性轉基因編碼序列的3’。在某些具體實施例中，該至少二個drg特異性miRNA縱排重複序列的第一個起始於距離轉基因編碼序列的3’端之20個核苷酸內。在某些具體實施例中，該至少二個drg特異性miRNA縱排重複序列的第一個起始於距離功能性轉基因編碼序列的3’端之至少100個核苷酸。在某些具體實施例中，miRNA縱排重複序列包含長度200至1200個核苷酸。在某些具體實施例中，有至少有二個drg特異性miRNA目標序列位於功能性轉基因編碼序列的5’。在某些具體實施例中，至少二個drg特異性miRNA目標序列位於功能性轉基因編碼序列的5’及3’兩端。在某些具體實施例中，表現匣mRNA或DNA正股的至少第一及/或至少第二miRNA目標序列的該miRNA目標序列係選自(i) AGTGAATTCTACCAGTGCCATA (miR183，SEQ ID NO: 49)；(ii) AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 50)；(iii) AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 51)；或(iv) AGGGATTCCTGGGAAAACTGGAC (SEQ ID NO: 52)。在某些具體實施例中，表現匣mRNA或DNA正股的至少第一及/或至少第二miRNA目標序列的該miRNA目標序列為AGTGAATTCTACCAGTGCCATA (miR183，SEQ ID NO: 49)。在某些具體實施例中，表現匣mRNA或DNA正股的至少第一及/或至少第二miRNA目標序列的該miRNA目標序列為AGTGAATTCTACCAGTGCCATA (miR182，SEQ ID NO: 49)。在某些具體實施例中，二或多個連續的miRNA目標序列為連續的且未被間隔子分隔開。在某些具體實施例中，二或多個該miRNA目標序列被間隔子分隔開且各間隔子獨立選自(A) GGAT；(B) CACGTG；或(C) GCATGC之一種以上。在某些具體實施例中，位於該miRNA目標序列之間的間隔子可位於第一miRNA目標序列之3’及/或最後的miRNA目標序列之5’。在某些具體實施例中，miRNA目標序列之間的間隔子為相同。參見國際專利申請號WO 2020/132455，2020年12月20日申請；及國際專利申請號WO 2021/081217，2020年10月22日申請，此等藉由引用以其整體併入。In certain embodiments, the vector gene body further comprises a dorsal root ganglion (drg)-specific miRNA detargeting sequence operably linked to the transgene coding sequence. In certain embodiments, the tandem miRNA target sequences are contiguous or separated by 1 to 10 internucleotide spacers, wherein the spacers are not miRNA target sequences. In certain embodiments, at least two drg-specific miRNA sequences are located 3' to the coding sequence of the functional transgene. In certain embodiments, the first of the at least two drg-specific miRNA tandem repeats begins within 20 nucleotides of the 3' end of the coding sequence of the transgene. In certain embodiments, the first of the at least two drg-specific miRNA tandem repeats begins at least 100 nucleotides from the 3' end of the functional transgene coding sequence. In certain embodiments, the miRNA tandem repeat comprises 200 to 1200 nucleotides in length. In certain embodiments, there are at least two drg-specific miRNA target sequences located 5' to the coding sequence of the functional transgene. In certain embodiments, at least two drg-specific miRNA target sequences are located at the 5' and 3' ends of the coding sequence of the functional transgene. In certain embodiments, the miRNA target sequence representing at least a first and/or at least a second miRNA target sequence of a cassette mRNA or DNA strand is selected from (i) AGTGAATTCTACCAGTGCCATA (miR183, SEQ ID NO: 49); (ii) AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 50); (iii) AGTGTGAGTTTCTACCATTGCCAAA (SEQ ID NO: 51); or (iv) AGGGATTCCTGGGAAAACTGGAC (SEQ ID NO: 52). In certain embodiments, the miRNA target sequence representing at least a first and/or at least a second miRNA target sequence of a cassette mRNA or DNA strand is AGTGAATTCTACCAGTGCCATA (miR183, SEQ ID NO: 49). In certain embodiments, the miRNA target sequence representing at least a first and/or at least a second miRNA target sequence of a cassette mRNA or DNA strand is AGTGAATTCTACCAGTGCCATA (miR182, SEQ ID NO: 49). In certain embodiments, two or more contiguous miRNA target sequences are contiguous and not separated by a spacer. In certain embodiments, two or more of the miRNA target sequences are separated by a spacer and each spacer is independently selected from one or more of (A) GGAT; (B) CACGTG; or (C) GCATGC. In certain embodiments, the spacer between the miRNA target sequences can be located 3' to the first miRNA target sequence and/or 5' to the last miRNA target sequence. In certain embodiments, the spacers between miRNA target sequences are identical. See International Patent Application No. WO 2020/132455, filed December 20, 2020; and International Patent Application No. WO 2021/081217, filed October 22, 2020, which are incorporated by reference in their entirety.

此等及其它常見載體及調節元件的選擇為常規的且許多這樣的序列為可取得的。參見例如Sambrook et al及其中引用的參考，例如，第3.18-3.26及16.17-16.27頁、以及Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989。當然，並非所有載體及表現控制序列都將同樣良好地作用以表現本文所述的所有轉基因。然而，所屬技術領域中具通常知識者可於此等以及其它表現控制序列中進行選擇，而不脫離本發明的範疇。The choice of these and other common vectors and regulatory elements is routine and many such sequences are available. See, eg, Sambrook et al and references cited therein, eg, pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989. Of course, not all vectors and expression control sequences will work equally well to express all of the transgenes described herein. However, one of ordinary skill in the art may make selections among these and other presentation control sequences without departing from the scope of the present invention.

在另一具體實施例中，提供一種產生重組腺相關病毒之方法。藉由培養宿主細胞而產生適合的重組腺相關病毒(AAV)，該宿主細胞含有編碼本文所述AAV衣殼蛋白或其片段的核酸序列；功能性rep基因；袖珍基因，至少由AAV反向末端重複序列(ITR)及編碼所欲轉基因的異源性核酸序列所構成；及充足的輔助功能以允許將袖珍基因包裝於AAV衣殼蛋白中。需要在宿主細胞中培養以將AAV袖珍基因包裝在AAV衣殼中的組件可反式地提供給宿主細胞。或者，可藉由穩定的宿主細胞提供所需組件之任一種以上(例如，袖珍基因、 rep序列、 cap序列、及/或輔助功能)，該穩定的宿主細胞已使用所屬技術領域中具通常知識者已知的方法被工程化為含有一種以上之所需組件。 In another embodiment, a method of producing a recombinant adeno-associated virus is provided. A suitable recombinant adeno-associated virus (AAV) is produced by culturing a host cell containing a nucleic acid sequence encoding the AAV capsid protein or fragment thereof as described herein; a functional rep gene; a pocket gene consisting of at least the reverse end of the AAV repeat sequence (ITR) and heterologous nucleic acid sequence encoding the desired transgene; and sufficient accessory functions to allow packaging of the pocket gene in the AAV capsid protein. The components required to be cultured in the host cell to package the AAV pocket gene in the AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., pocket genes, rep sequences, cap sequences, and/or helper functions) can be provided by a stable host cell that has been used with ordinary knowledge in the art Known methods are engineered to contain more than one required component.

本文亦提供用本文所述之AAV轉染的宿主細胞。最適合地，此種穩定的宿主細胞將含有在可誘導型啟動子的控制下的所需組件。然而，所需組件可在組成型啟動子之控制下。本文於下述適合與轉基因一起使用的調節元件的討論中提供適合的可誘導型及組成型啟動子之例。再或者，所選擇的穩定宿主細胞可含有在組成型啟動子控制下的所選擇的組件及在一個或多個可誘導型啟動子控制下的其它所選擇的組件。例如，可產生穩定的宿主細胞，其衍生自293細胞(含有在組成型啟動子的控制下的E1輔助功能)，但含有在可誘導型啟動子的控制下的 rep及/或 cap蛋白。由所屬技術領域中具通常知識者亦可產生其它穩定的宿主細胞。在另一具體實施例中，宿主細胞包含如本文所述的核酸分子(例如，質體)。 Also provided herein are host cells transfected with the AAV described herein. Most suitably, such stable host cells will contain the desired components under the control of inducible promoters. However, the desired component can be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein below in the discussion of regulatory elements suitable for use with transgenes. Still alternatively, the selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells derived from 293 cells (containing El helper functions under the control of constitutive promoters) but containing rep and/or cap proteins under the control of inducible promoters can be produced. Other stable host cells can also be generated by those of ordinary skill in the art. In another specific embodiment, the host cell comprises a nucleic acid molecule (eg, a plastid) as described herein.

產生本文所述的rAAV所需的袖珍基因、 rep序列、 cap序列、及輔助功能，可用任何轉移其上攜帶的序列的遺傳元件的形式遞送至包裝宿主細胞。所選擇的遺傳元件可藉由任何適合的方法遞送，包括本文所述的彼等方法。用於構建本發明任何具體實施例的方法為核酸操作技術領域中具有通常知識者已知，且包括基因工程、重組工程及合成技術。參見例如Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY。相似地，產生rAAV病毒顆粒的方法為眾所周知，且選擇適合的方法並非對本發明的限制。參見例如K. Fisher et al, 1993 J. Virol., 70:520-532及US專利5,478,745等。此等公開文獻藉由引用而併入本文。 The minigenes, rep sequences, cap sequences, and accessory functions required to produce the rAAV described herein can be delivered to the packaging host cell in the form of any genetic element that transfers the sequences carried thereon. The selected genetic elements can be delivered by any suitable method, including those described herein. Methods for constructing any embodiment of the invention are known to those of ordinary skill in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering and synthetic techniques. See, eg, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and selection of a suitable method is not a limitation of the invention. See, eg, K. Fisher et al, 1993 J. Virol. , 70:520-532 and US Patent 5,478,745, among others. These publications are incorporated herein by reference.

本文亦提供用於產生本文所述的載體的質體。此種質體包括編碼AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG015 (SEQ ID NO: 26)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及vp3的至少一者的核酸序列；或與SEQ ID NO: 2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46之vp1、vp2、及/或vp3序列共有至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。在某些具體實施例中，提供一種質體，其具有AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG015 (SEQ ID NO: 25)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45之vp1、vp2、及/或vp3核苷酸序列共有至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。在另外的具體實施例中，該質體包括非AAV序列。亦提供培養的宿主細胞，其含有本文所述的質體。 C.醫藥組成物及投予 Also provided herein are plastids for use in generating the vectors described herein. Such germplasms include genes encoding AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10) , AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG015 (SEQ ID NO: 26), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), AAVpoG024 (SEQ ID NO : 44), or the nucleic acid sequence of at least one of vp1, vp2, and vp3 of AAVpoG025 (SEQ ID NO: 46); or with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, The vp1, vp2, and/or vp3 sequences of 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 share at least 95%, at least 96%, at least 97% %, at least 98%, or at least 99% identical sequences. In certain embodiments, there is provided a plastid having AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7) , AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG015 (SEQ ID NO: 25), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO : 41), AAVpoG024 (SEQ ID NO: 43), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 45); or with SEQ ID NO: 1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 vp1, vp2, and/or vp3 nucleotide sequences share at least A sequence that is 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In other specific embodiments, the plastid comprises non-AAV sequences. Also provided are cultured host cells comprising the plastids described herein. C. Pharmaceutical composition and administration

在一具體實施例中，可選擇地藉由常規方法評估含有所欲轉基因及啟動子的用於上列詳述的目標細胞的重組AAV的污染，然後調配至意圖投予需要其之受試者之醫藥組成物中。此種調配劑涉及使用醫藥上及/或生理學上可接受的媒劑或載劑，如緩衝鹽水或其它緩衝劑，例如HEPES，以維持pH於適當的生理學上的水準，且可選擇地使用其它藥物、醫藥劑、穩定劑、緩衝劑、載劑、佐劑、稀釋劑等。於注射，載體通常為液體。示例性的生理上可接受的載劑包括無菌、無熱原(pyrogen)的水及無菌、無熱原的磷酸鹽緩衝鹽水。於美國專利公開號7,629,322中提供了各式各樣之此種已知載劑，藉由引用被併入本文中。在一具體實施例中，該載劑為等張的氯化鈉溶液。在另一具體實施例中，載劑為經平衡的鹽溶液。在一具體實施例中，該載劑包括吐溫(Tween)。若病毒要長期保存，則可於甘油或Tween20的存在下將其冷凍。在另一具體實施例中，醫藥上可接受的載劑包含界面活性劑，如全氟辛烷(Perfluoron液體)。將該載體調配在適於人類受試者的輸注之緩衝液/載劑中。緩衝液/載劑應包括防止rAAV黏附到輸液管上但不干擾rAAV活體內結合活性的成分。In one embodiment, recombinant AAV containing the desired transgene and promoter for the target cells detailed above can optionally be assessed for contamination by conventional methods and then formulated for intended administration to a subject in need thereof in pharmaceutical compositions. Such formulation involves the use of pharmaceutically and/or physiologically acceptable vehicles or carriers, such as buffered saline or other buffers, such as HEPES, to maintain the pH at an appropriate physiological level, and optionally Other drugs, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. are used. For injection, the carrier is usually a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate-buffered saline. A variety of such known carriers are provided in US Patent Publication No. 7,629,322, incorporated herein by reference. In a specific embodiment, the carrier is isotonic sodium chloride solution. In another specific embodiment, the carrier is a balanced salt solution. In a specific embodiment, the carrier includes Tween. If the virus is to be stored for a long time, it can be frozen in the presence of glycerol or Tween20. In another embodiment, the pharmaceutically acceptable carrier comprises a surfactant, such as perfluorooctane (Perfluoron liquid). The carrier is formulated in a buffer/vehicle suitable for infusion in a human subject. The buffer/carrier should include components that prevent rAAV from adhering to the infusion tubing but do not interfere with rAAV binding activity in vivo.

在本文所述的方法之某些具體實施例中，將上述醫藥組成物肌內(IM)投予至受試者。在其它具體實施例中，醫藥組成物係藉由靜脈內(IV)投予。在其它具體實施例中，醫藥組成物係藉由腦室內(intracerebroventricular，ICV)注射投予。在其它具體實施例中，醫藥組成物係藉由腦大池內(ICM)注射投予。可有用於本文所述方法的投予之其它形式包括但不限於直接遞送至所欲器官(例如，眼睛)，包括視網膜下或玻璃體內遞送、經口、吸入、鼻內、氣管內、靜脈內、肌內、皮下、皮內、及其它非腸胃道路徑之投予。若期望，可組合投予路徑。In certain embodiments of the methods described herein, the pharmaceutical composition described above is administered intramuscularly (IM) to the subject. In other embodiments, the pharmaceutical composition is administered intravenously (IV). In other embodiments, the pharmaceutical composition is administered by intracerebroventricular (ICV) injection. In other embodiments, the pharmaceutical composition is administered by intracisternal (ICM) injection. Other forms of administration that may be useful in the methods described herein include, but are not limited to, direct delivery to the desired organ (e.g., the eye), including subretinal or intravitreal delivery, oral, inhalation, intranasal, intratracheal, intravenous , intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. Routes of administration can be combined if desired.

如本文所使用，術語「鞘內遞送」或「鞘內投予」係指經由注射至椎管的投予途徑，更具體而言為注射至蜘蛛膜下腔以使其到達腦脊髓液(CSF)。鞘內遞送可包括腰椎穿刺、室內(包括腦室內(ICV))、枕骨下/腦池內、及/或C1-2穿刺。例如，可藉由腰椎穿刺的手段導入物質以在整個蜘蛛膜下腔擴散。於另一例，可注射至腦大池。As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to the route of administration via injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF ). Intrathecal delivery may include lumbar puncture, intraventricular (including intraventricular (ICV)), suboccipital/intracisternal, and/or C1-2 puncture. For example, a substance may be introduced by means of a lumbar puncture to spread throughout the subarachnoid space. In another example, it can be injected into the cisterns of the brain.

如本文所使用，術語「腦池內遞送」或「腦池內投予」係指直接至腦大池小腦延髓之腦脊髓液中的投予途徑，更具體而言係經由枕骨下穿刺或藉由直接注射至腦大池，或者經由永久定位的管子。As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to a route of administration directly into the cerebrospinal fluid of the cerebellum and medulla in the large cisterns of the brain, more specifically via a suboccipital puncture or via Injected directly into the cistern, or via a permanently positioned tube.

組成物可用約0.1 μL至約10 mL的體積遞送，包括此範圍內所有數量，取決於欲治療區域的大小、使用的病毒力價、投予途徑、及該方法之所欲效果。在一具體實施例中，體積為約50 µL。在另一具體實施例中，體積為約70 µL。在另一具體實施例中，體積為約100 µL。在另一具體實施例中，體積為約125 µL。在另一具體實施例中，體積為約150 µL。在另一具體實施例中，體積為約175 µL。又一具體實施例中，體積為約200 µL。在另一具體實施例中，體積為約250 µL。在另一具體實施例中，體積為約300 µL。在另一具體實施例中，體積為約450 µL。在另一具體實施例中，體積為約500 µL。在另一具體實施例中，體積為約600 µL。在另一具體實施例中，體積為約750 µL。在另一具體實施例中，體積為約850 µL。在另一具體實施例中，體積為約1000 µL。在另一具體實施例中，體積為約1.5 mL。在另一具體實施例中，體積為約2 mL。在另一具體實施例中，體積為約2.5 mL。在另一具體實施例中，體積為約3 mL。在另一具體實施例中，體積為約3.5 mL。在另一具體實施例中，體積為約4 mL。在另一具體實施例中，體積為約5 mL。在另一具體實施例中，體積為約5.5 mL。在另一具體實施例中，體積為約6 mL。在另一具體實施例中，體積為約6.5 mL。在另一具體實施例中，體積為約7 mL。在另一具體實施例中，體積為約8 mL。在另一具體實施例中，體積為約8.5 mL。在另一具體實施例中，體積為約9 mL。在另一具體實施例中，體積為約9.5 mL。在另一具體實施例中，體積為約10 mL。The composition can be delivered in a volume of about 0.1 μL to about 10 mL, including all amounts within this range, depending on the size of the area to be treated, the titer of virus used, the route of administration, and the desired effect of the method. In a specific embodiment, the volume is about 50 µL. In another specific embodiment, the volume is about 70 µL. In another specific embodiment, the volume is about 100 µL. In another specific embodiment, the volume is about 125 µL. In another specific embodiment, the volume is about 150 µL. In another specific embodiment, the volume is about 175 µL. In yet another specific embodiment, the volume is about 200 µL. In another specific embodiment, the volume is about 250 µL. In another specific embodiment, the volume is about 300 µL. In another specific embodiment, the volume is about 450 µL. In another specific embodiment, the volume is about 500 µL. In another specific embodiment, the volume is about 600 µL. In another specific embodiment, the volume is about 750 µL. In another specific embodiment, the volume is about 850 µL. In another specific embodiment, the volume is about 1000 µL. In another specific embodiment, the volume is about 1.5 mL. In another specific embodiment, the volume is about 2 mL. In another specific embodiment, the volume is about 2.5 mL. In another specific embodiment, the volume is about 3 mL. In another specific embodiment, the volume is about 3.5 mL. In another specific embodiment, the volume is about 4 mL. In another specific embodiment, the volume is about 5 mL. In another specific embodiment, the volume is about 5.5 mL. In another specific embodiment, the volume is about 6 mL. In another specific embodiment, the volume is about 6.5 mL. In another specific embodiment, the volume is about 7 mL. In another specific embodiment, the volume is about 8 mL. In another specific embodiment, the volume is about 8.5 mL. In another specific embodiment, the volume is about 9 mL. In another specific embodiment, the volume is about 9.5 mL. In another specific embodiment, the volume is about 10 mL.

攜帶編碼在調節序列控制下的所欲轉基因的核酸序列的重組腺相關病毒的有效濃度範圍理想為每毫升約10 7至10 14載體基因體(vg/mL)(亦稱為基因體拷貝/mL (GC/mL))。在一具體實施例中，藉由即時PCR測量rAAV載體基因體。在另一具體實施例中，藉由數位PCR測量rAAV載體基因體。參見Lock et al, Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb 14，其藉由引用而併入本文。在另一具體實施例中，測量rAAV感染單位，如S.K. McLaughlin et al, 1988 J. Virol., 62:1963所述，其藉由引用併入本文。 The effective concentration range of the recombinant adeno-associated virus carrying the nucleic acid sequence encoding the desired transgene under the control of the regulatory sequences is ideally about 107 to 1014 vector gene bodies per milliliter (vg/mL) (also known as gene body copies/mL (GC/mL)). In one embodiment, rAAV vector gene bodies are measured by real-time PCR. In another embodiment, rAAV vector gene bodies are measured by digital PCR. See Lock et al, Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi: 10.1089/hgtb. 2013.131. Epub 2014 Feb 14, which is incorporated herein by reference. In another embodiment, rAAV infectious units are measured as described in SK McLaughlin et al, 1988 J. Virol., 62:1963, which is incorporated herein by reference.

較佳地，濃度為約1.5 x 10 9vg/mL至約1.5 x 10 13vg/mL，更佳為約1.5 x 10 9vg/mL至約1.5 x 10 11vg/mL。在一具體實施例中，有效濃度為約1.4 x 10 8vg/mL。在一具體實施例中，有效濃度為約3.5 x 10 10vg/mL。在另一具體實施例中，有效濃度為約5.6 x 10 11vg/mL。在另一具體實施例中，有效濃度為約5.3 x 10 12vg/mL。又一具體實施例中，有效濃度為約1.5 x 10 12vg/mL。在另一具體實施例中，有效濃度為約1.5 x 10 13vg/mL。本文所述的所有範圍均包括端點。 Preferably, the concentration is about 1.5 x 10 9 vg/mL to about 1.5 x 10 13 vg/mL, more preferably about 1.5 x 10 9 vg/mL to about 1.5 x 10 11 vg/mL. In a specific embodiment, the effective concentration is about 1.4 x 10 8 vg/mL. In a specific embodiment, the effective concentration is about 3.5 x 10 10 vg/mL. In another specific embodiment, the effective concentration is about 5.6 x 1011 vg/mL. In another specific embodiment, the effective concentration is about 5.3 x 1012 vg/mL. In yet another specific embodiment, the effective concentration is about 1.5 x 10 12 vg/mL. In another specific embodiment, the effective concentration is about 1.5 x 1013 vg/mL. All ranges stated herein include endpoints.

在一具體實施例中，劑量為約1.5 x 10 9vg/kg體重至約1.5 x 10 13vg/kg，更佳為約1.5 x 10 9vg/kg至約1.5 x 10 11vg/kg。在一具體實施例中，劑量為約1.4 x 10 8vg/kg。在一具體實施例中，劑量為約3.5 x 10 10vg/kg。在另一具體實施例中，劑量為約5.6 x 10 11vg/kg。在另一具體實施例中，劑量為約5.3 x 10 12vg/kg。又一具體實施例中，劑量為約1.5 x 10 12vg/kg。在另一具體實施例中，劑量為約1.5 x 10 13vg/kg。在另一具體實施例中，劑量為約3.0 x 10 13vg/kg。在另一具體實施例中，劑量為約1.0 x 10 14vg/kg。本文所述的所有範圍均包括端點。 In a specific embodiment, the dose is about 1.5 x 10 9 vg/kg body weight to about 1.5 x 10 13 vg/kg, more preferably about 1.5 x 10 9 vg/kg to about 1.5 x 10 11 vg/kg. In a specific embodiment, the dosage is about 1.4 x 108 vg/kg. In a specific embodiment, the dose is about 3.5 x 1010 vg/kg. In another specific embodiment, the dose is about 5.6 x 1011 vg/kg. In another specific embodiment, the dose is about 5.3 x 1012 vg/kg. In yet another specific embodiment, the dose is about 1.5 x 1012 vg/kg. In another specific embodiment, the dose is about 1.5 x 1013 vg/kg. In another specific embodiment, the dose is about 3.0 x 1013 vg/kg. In another specific embodiment, the dose is about 1.0 x 1014 vg/kg. All ranges stated herein include endpoints.

在一具體實施例中，有效劑量(遞送的總基因體拷貝)為約10 7至10 13載體基因體。在一具體實施例中，總劑量為約10 8基因體拷貝。在一具體實施例中，總劑量為約10 9基因體拷貝。在一具體實施例中，總劑量為約10 10基因體拷貝。在一具體實施例中，總劑量為約10 11基因體拷貝。在一具體實施例中，總劑量為約10 12基因體拷貝。在一具體實施例中，總劑量為約10 13基因體拷貝。在一具體實施例中，總劑量為約10 14基因體拷貝。在一具體實施例中，總劑量為約10 15基因體拷貝。 In a specific embodiment, the effective dose (total gene body copies delivered) is about 10 7 to 10 13 vector gene bodies. In a specific embodiment, the total dose is about 108 gene body copies. In a specific embodiment, the total dose is about 109 gene body copies. In a specific embodiment, the total dose is about 1010 gene body copies. In a specific embodiment, the total dose is about 10 11 gene body copies. In a specific embodiment, the total dose is about 1012 gene body copies. In a specific embodiment, the total dose is about 1013 gene body copies. In a specific embodiment, the total dose is about 1014 gene body copies. In a specific embodiment, the total dose is about 1015 gene body copies.

理想為使用最低有效濃度的病毒以便降低不希望的影響(如毒性)的風險。考慮到受治療的受試者(較佳為人類)的身體狀態、受試者的年齡、特定的病症及病症的程度，若已經發展為進行性的，主治醫師可選擇此等範圍內的其它劑量及投予體積。例如，靜脈投予可能需要約1.5 X 10 13vg/kg的劑量。 D.方法 It is desirable to use the lowest effective concentration of virus in order to reduce the risk of undesired effects such as toxicity. Taking into account the physical condition of the subject (preferably a human being) to be treated, the age of the subject, the specific disease and the extent of the disease, if it has developed progressively, the attending physician can choose other medicines within these ranges. Dose and administration volume. For example, intravenous administration may require a dose of about 1.5 X 1013 vg/kg. D. method

於另一態樣，提供一種轉導目標細胞或組織之方法。在一具體實施例中，該方法包括投予如本文所述之rAAV至受試者。在其它具體實施例中，該方法在活體外進行。In another aspect, a method of transducing target cells or tissues is provided. In a specific embodiment, the method comprises administering to a subject an rAAV as described herein. In other specific embodiments, the method is performed in vitro.

病毒載體(例如，rAAV)的劑量主要取決於要治療的病況、患者的年齡、體重及健康狀況等因素，因此可能因患者而異。例如，病毒載體之治療上有效的人類劑量一般範圍為約25至約1000 微升至約100 mL之溶液，該溶液含有濃度為約1 x 10 9至1 x 10 16的載體基因體拷貝。在某些具體實施例中，遞送體積約1 mL至約15 mL、或約2.5 mL至約10 mL、或約5 mL懸浮液。在某些具體實施例中，遞送體積約1、約2、約3、約4、約5、約6、約7、約8、約9、約10、約11、約12、約13、約14、或約15 mL懸浮液。在某些具體實施例中，以此體積投予總共約8.9 x 10 12至2.7 x 10 14GC之劑量。在某些具體實施例中，以此體積投予約1.1 x10 10GC/g腦質量至約3.3 x 10 11GC/g腦質量之劑量。在某些具體實施例中，以此體積投予約3.0 x10 9、約4.0 x10 9、約5.0 x10 9、約6.0 x10 9、約7.0 x10 9、約8.0 x10 9、約9.0 x10 9、約1.0 x10 10、約1.1 x10 10、約1.5 x10 10、約2.0 x10 10、約2.5 x10 10、約3.0 x10 10、約3.3 x10 10、約3.5 x10 10、約4.0 x10 10、約4.5 x10 10、約5.0 x10 10、約5.5 x10 10、約6.0 x10 10、約6.5 x10 10、約7.0 x10 10、約7.5 x10 10、約8.0 x10 10、約8.5 x10 10、約9.0 x10 10、約9.5 x10 10、約1.0 x10 11、約1.1 x10 11、約1.5 x10 11、約2.0 x10 11、約2.5 x10 11、約3.0 x10 11、約3.3 x10 11、約3.5 x10 11、約4.0 x10 11、約4.5 x10 11、約5.0 x10 11、約5.5 x10 11、約6.0 x10 11、約6.5 x10 11、約7.0 x10 11、約7.5 x10 11、約8.0 x10 11、約8.5 x10 11、約9.0 x10 11GC每公克腦質量之劑量。 Dosages of viral vectors (eg, rAAV) depend primarily on the condition to be treated, the age, weight, and health of the patient, and thus may vary from patient to patient. For example, a therapeutically effective human dose of a viral vector generally ranges from about 25 to about 1000 microliters to about 100 mL of a solution containing copies of the vector genome at a concentration of about 1 x 109 to 1 x 1016 . In certain embodiments, the delivery volume is about 1 mL to about 15 mL, or about 2.5 mL to about 10 mL, or about 5 mL of suspension. In certain embodiments, the delivered volume is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 mL of suspension. In certain embodiments, a total of about 8.9 x 1012 to 2.7 x 1014 GC is dosed in this volume. In certain embodiments, a dose of about 1.1 x 1010 GC/g brain mass to about 3.3 x 1011 GC/g brain mass is administered in this volume. In certain embodiments, about 3.0 x 10 9 , about 4.0 x 10 9 , about 5.0 x 10 9 , about 6.0 x 10 9 , about 7.0 x 10 9 , about 8.0 x 10 9 , about 9.0 x 10 9 , about 1.0 x 10 9 are administered in this volume. 10 , about 1.1 x10 10 , about 1.5 x10 10 , about 2.0 x10 10 , about 2.5 x10 10 , about 3.0 x10 10 , about 3.3 x10 10 , about 3.5 x10 10 , about 4.0 x10 10 , about 4.5 x10 10 , about 5.0 x10 10 , about 5.5 x10 10 , about 6.0 x10 10 , about 6.5 x10 10 , about 7.0 x10 10 , about 7.5 x10 10 , about 8.0 x10 10 , about 8.5 x10 10 , about 9.0 x10 10 , about 9.5 x10 10 , about 1.0 x10 11 , about 1.1 x10 11 , about 1.5 x10 11 , about 2.0 x10 11 , about 2.5 x10 11 , about 3.0 x10 11 , about 3.3 x10 11 , about 3.5 x10 11 , about 4.0 x10 11 , about 4.5 x10 11 , about 5.0 x10 11. About 5.5 x10 11 , about 6.0 x10 11 , about 6.5 x10 11 , about 7.0 x10 11 , about 7.5 x10 11 , about 8.0 x10 11 , about 8.5 x10 11 , about 9.0 x10 11 GC per gram of brain mass.

在某些具體實施例中，rAAV之劑量為每劑約1 x 10 9GC至約1 x 10 15基因體拷貝(GC)(治療平均體重70公斤的受試者)，對人類患者較佳為1.0 x 10 12GC至2.0 x 10 15GC。在另一具體實施例中，劑量為小於約1 x 10 14GC/kg受試者體重。在某些具體實施例中，投予患者的劑量為至少約1.0 x 10 9GC/kg、約1.5 x 10 9GC/kg、約2.0 x 10 9GC/g、約2.5 x 10 9GC/kg、約3.0 x 10 9GC/kg、約3.5 x 10 9GC/kg、約4.0 x 10 9GC/kg、約4.5 x 10 9GC/kg、約5.0 x 10 9GC/kg、約5.5 x 10 9GC/kg、約6.0 x 10 9GC/kg、約6.5 x 10 9GC/kg、約7.0 x 10 9GC/kg、約7.5 x 10 9GC/kg、約8.0 x 10 9GC/kg、約8.5 x 10 9GC/kg、約9.0 x 10 9GC/kg、約9.5 x 10 9GC/kg、約1.0 x 10 10GC/kg、約1.5 x 10 10GC/kg、約2.0 x 10 10GC/kg、約2.5 x 10 10GC/kg、約3.0 x 10 10GC/kg、約3.5 x 10 10GC/kg、約4.0 x 10 10GC/kg、約4.5 x 10 10GC/kg、約5.0 x 10 10GC/kg、約5.5 x 10 10GC/kg、約6.0 x 10 10GC/kg、約6.5 x 10 10GC/kg、約7.0 x 10 10GC/kg、約7.5 x 10 10GC/kg、約8.0 x 10 10GC/kg、約8.5 x 10 10GC/kg、約9.0 x 10 10GC/kg、約9.5 x 10 10GC/kg、約1.0 x 10 11GC/kg、約1.5 x 10 11GC/kg、約2.0 x 10 11GC/kg、約2.5 x 10 11GC/kg、約3.0 x 10 11GC/kg、約3.5 x 10 11GC/kg、約4.0 x 10 11GC/kg、約4.5 x 10 11GC/kg、約5.0 x 10 11GC/kg、約5.5 x 10 11GC/kg、約6.0 x 10 11GC/kg、約6.5 x 10 11GC/kg、約7.0 x 10 11GC/kg、約7.5 x 10 11GC/kg、約8.0 x 10 11GC/kg、約8.5 x 10 11GC/kg、約9.0 x 10 11GC/kg、約9.5 x 10 11GC/kg、約1.0 x 10 12GC/kg、約1.5 x 10 12GC/kg、約2.0 x 10 12GC/kg、約2.5 x 10 12GC/kg、約3.0 x 10 12GC/kg、約3.5 x 10 12GC/kg、約4.0 x 10 12GC/kg、約4.5 x 10 12GC/kg、約5.0 x 10 12GC/kg、約5.5 x 10 12GC/kg、約6.0 x 10 12GC/kg、約6.5 x 10 12GC/kg、約7.0 x 10 12GC/kg、約7.5 x 10 12GC/kg、約8.0 x 10 12GC/kg、約8.5 x 10 12GC/kg、約9.0 x 10 12GC/kg、約9.5 x 10 12GC/kg、約1.0 x 10 13GC/kg、約1.5 x 10 13GC/kg、約2.0 x 10 13GC/kg、約2.5 x 10 13GC/kg、約3.0 x 10 13GC/kg、約3.5 x 10 13GC/kg、約4.0 x 10 13GC/kg、約4.5 x 10 13GC/kg、約5.0 x 10 13GC/kg、約5.5 x 10 13GC/kg、約6.0 x 10 13GC/kg、約6.5 x 10 13GC/kg、約7.0 x 10 13GC/kg、約7.5 x 10 13GC/kg、約8.0 x 10 13GC/kg、約8.5 x 10 13GC/kg、約9.0 x 10 13GC/kg、約9.5 x 10 13GC/kg、或約1.0 x 10 14GC/kg受試者體重。 In certain embodiments, the dose of rAAV is about 1 x 10 9 GC to about 1 x 10 15 gene body copies (GC) per dose (for subjects with an average body weight of 70 kg), preferably for human patients 1.0 x 10 12 GC to 2.0 x 10 15 GC. In another specific embodiment, the dose is less than about 1 x 1014 GC/kg body weight of the subject. In certain embodiments, the dose administered to the patient is at least about 1.0 x 10 9 GC/kg, about 1.5 x 10 9 GC/kg, about 2.0 x 10 9 GC/g, about 2.5 x 10 9 GC/kg , about 3.0 x 10 9 GC/kg, about 3.5 x 10 9 GC/kg, about 4.0 x 10 9 GC/kg, about 4.5 x 10 9 GC/kg, about 5.0 x 10 9 GC/kg, about 5.5 x 10 9 GC/kg, about 6.0 x 10 9 GC /kg, about 6.5 x 10 9 GC/kg, about 7.0 x 10 9 GC/kg, about 7.5 x 10 9 GC/kg, about 8.0 x 10 9 GC/kg, About 8.5 x 10 9 GC/kg, about 9.0 x 10 9 GC/kg, about 9.5 x 10 9 GC/kg, about 1.0 x 10 10 GC/kg, about 1.5 x 10 10 GC/kg, about 2.0 x 10 10 GC/kg, about 2.5 x 10 10 GC/kg, about 3.0 x 10 10 GC/kg, about 3.5 x 10 10 GC/kg, about 4.0 x 10 10 GC/kg, about 4.5 x 10 10 GC/kg, about 5.0 x 10 10 GC/kg, about 5.5 x 10 10 GC/kg, about 6.0 x 10 10 GC/kg, about 6.5 x 10 10 GC/kg, about 7.0 x 10 10 GC/kg, about 7.5 x 10 10 GC /kg, about 8.0 x 10 10 GC/kg, about 8.5 x 10 10 GC/kg, about 9.0 x 10 10 GC/kg, about 9.5 x 10 10 GC/kg, about 1.0 x 10 11 GC/kg, about 1.5 x 10 11 GC/kg, about 2.0 x 10 11 GC/kg, about 2.5 x 10 11 GC/kg, about 3.0 x 10 11 GC/kg, about 3.5 x 10 11 GC/kg, about 4.0 x 10 11 GC/kg kg, about 4.5 x 10 11 GC/kg, about 5.0 x 10 11 GC/kg, about 5.5 x 10 11 GC/kg, about 6.0 x 10 11 GC/kg, about 6.5 x 10 11 GC/kg, about 7.0 x 10 11 GC/kg, about 7.5 x 10 11 GC/kg, about 8.0 x 10 11 GC/kg, about 8.5 x 10 11 GC/kg, about 9.0 x 10 11 GC/kg, about 9.5 x 10 11 GC/kg , about 1.0 x 10 12 GC/kg, about 1.5 x 10 12 GC/kg, about 2.0 x 10 12 GC/kg, about 2.5 x 10 12 GC/kg, about 3.0 x 10 12 GC/kg, about 3.5 x 10 12 GC/kg, about 4.0 x 10 12 GC/kg, about 4.5 x 10 12 GC/kg, about 5.0 x 10 12 GC/kg, about 5.5 x 10 12 GC/kg, about 6.0 x 10 12 GC/kg, About 6.5 x 10 12 GC/kg, about 7.0 x 10 12 GC/kg, about 7.5 x 10 12 GC/kg, about 8.0 x 10 12 GC/kg, about 8.5 x 10 12 GC/kg, about 9.0 x 10 12 GC/kg, about 9.5 x 10 12 GC/kg, about 1.0 x 10 13 GC/kg , about 1.5 x 10 13 GC/kg, about 2.0 x 10 13 GC/kg, about 2.5 x 10 13 GC/kg, about 3.0 x 10 13 GC/kg, about 3.5 x 10 13 GC/kg, about 4.0 x 10 13 GC/kg, about 4.5 x 10 13 GC/kg, about 5.0 x 10 13 GC/kg, about 5.5 x 10 13 GC /kg, about 6.0 x 10 13 GC/kg, about 6.5 x 10 13 GC/kg, about 7.0 x 10 13 GC/kg, about 7.5 x 10 13 GC/kg, about 8.0 x 10 13 GC/kg, about 8.5 x 1013 GC/kg, about 9.0 x 1013 GC/kg, about 9.5 x 1013 GC/kg, or about 1.0 x 1014 GC/kg body weight of the subject.

在一具體實施例中，該方法進一步包含給予受試者免疫抑制協同治療(immunosuppressive co-therapy)。例如，若檢測到針對AAV衣殼的不希望的高中和抗體水準，則此種免疫抑制協同治療可在遞送所述rAAV或組成物之前開始。在某些具體實施例中，作為預防措施，亦可在遞送rAAV之前開始協同治療。在某些具體實施例中，例如，如果在治療後觀察到不希望的免疫反應，則在rAAV的遞送後開始免疫抑制協同治療。In a specific embodiment, the method further comprises administering to the subject immunosuppressive co-therapy. For example, if undesired levels of high neutralizing antibodies against the AAV capsid are detected, such immunosuppressive co-therapy can be initiated prior to delivery of the rAAV or composition. In certain embodiments, co-therapy may also be initiated prior to rAAV delivery as a precautionary measure. In certain embodiments, immunosuppressive co-therapy is initiated following delivery of rAAV, for example, if an undesired immune response is observed following treatment.

用於此類協同治療的免疫抑制劑包括但不限於醣皮質素、類固醇、抗代謝物質、T細胞抑制劑、巨環內酯類(macrolides)(例如雷帕黴素(rapamycin)或雷帕黴素類似物(rapalog))、以及細胞生長抑制劑，包括烷化劑、抗代謝物質、細胞毒性抗生素、抗體、或對免疫親和素(immunophilin)有活性的藥劑。免疫抑制劑可包括去氫皮質醇(prednisolone)、氮芥、亞硝基脲、鉑化合物、胺甲喋呤(methotrexate)、硫唑嘌呤(azathioprine)、巰基嘌呤、氟尿嘧啶、放線菌素、蒽環類(anthracycline)、絲裂黴素C、博萊黴素(bleomycin)、光輝黴素(mithramycin)、針對IL-2受體(CD25)或CD3的抗體、抗IL-2抗體、環孢菌素(ciclosporin)、他克莫司(tacrolimus)、西羅莫司(sirolimus)、IFN-β、IFN-γ、類鴉片或TNF-α(腫瘤壞死因子-α)結合劑。在某些具體實施例中，免疫抑制治療可在rAAV投予之前0、1、2、7或更多日前開始，或者在rAAV投予之後0、1、2、3、7或更多日後開始。此類治療可在同一日涉及單一藥物(例如去氫皮質醇)或兩種或多種藥物(例如去氫皮質醇、黴酚酸酯(micophenolate mofetil，MMF)及/或西羅莫司(即雷帕黴素))共同投予。此等藥物中的一種以上可於基因治療投予後以相同劑量或經調整的劑量繼續使用。視需要，此種治療可持續約1週(7日)、兩週、三週、約60日或更長時間。在某些具體實施例中，選擇無他克莫司的方案。Immunosuppressants for such co-therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T cell inhibitors, macrolides (such as rapamycin or rapamycin rapalogs), and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins. Immunosuppressants may include prednisolone, nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, actinomycin, anthracycline Anthracycline, mitomycin C, bleomycin, mithramycin, antibodies against IL-2 receptor (CD25) or CD3, anti-IL-2 antibodies, cyclosporine (ciclosporin), tacrolimus, sirolimus, IFN-beta, IFN-gamma, opioids, or TNF-alpha (tumor necrosis factor-alpha) binding agents. In certain embodiments, immunosuppressive therapy can be initiated 0, 1, 2, 7 or more days before rAAV administration, or 0, 1, 2, 3, 7 or more days after rAAV administration . Such therapy may involve a single drug (eg, prednisone) or two or more drugs (eg, prednisone, mycophenolate mofetil (MMF), and/or sirolimus (eg, Pamycin)) co-administered. One or more of these drugs may continue to be used at the same dose or at an adjusted dose after gene therapy administration. Such treatment may last for about 1 week (7 days), two weeks, three weeks, about 60 days or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

在某些具體實施例中，協同治療可涉及組合本文提供的rAAV與配體之共同投予，該配體抑制人類新生兒Fc受體(FcRn)與免疫球蛋白G(IgG)的結合。參見例如2020年6月17日申請的US臨時專利申請號63/040,381、及2021年1月11日申請的US臨時專利申請號63/135,998、及2021年2月22日申請的US臨時專利申請號63/153,085，其每一者藉由引用併入本文。在某些具體實施例中，病毒載體被全身遞送。在某些具體實施例中，配體為肽、蛋白質、RNAi序列或小分子。在某些具體實施例中，該蛋白質為單株抗體、免疫黏附素、駱駝抗體、Fab片段、Fv片段或scFv片段。在某些具體實施例中，重組病毒載體為重組腺相關病毒、重組腺病毒、重組單純疱疹病毒、或重組慢病毒。在某些具體實施例中，該配體為單株抗體，其特異性地抑制FcRn-IgG結合而不干擾FcRn-白蛋白結合。在某些具體實施例中，該單株抗體為尼卡利單抗(Nipocalimab，M281)、洛利昔珠單抗(rozanolixizumab，UCB7665)；IMVT-1401、RVT-1401、HL161, HBM916、ARGX-113(依加替莫德(efgartigimod))、SYNT001、SYNT002、ABY-039、或DX-2507、此等的衍生物或組合。在某些具體實施例中，在病毒載體投予前一至七日遞送配體2 UPN-20-9394.P3 5 10 15 20 25 30。在某些具體實施例中，每日遞送配體。在某些具體實施例，在投予病毒載體的同一天投劑或投予配體。在某些具體實施例中，配體在載體投予後投劑1天至4週。在某些具體實施例中，配體經由與載體不同的投予途徑投劑。在某些具體實施例中，每日投劑該配體。在某些具體實施例中，該病毒載體被腹膜內、靜脈內、肌內、鼻內或鞘內投劑。在某些具體實施例中，患者被預先確定具有對載體衣殼的中和抗體力價，其在活體外測定中確定大於1:5。在某些具體實施例中，將病毒載體與抑制性配體組合遞送之前，患者之前沒有接受過基因治療，因此患者預先存在的中和抗體是野生型感染的結果。在某些具體實施例中，在病毒載體與抑制性免疫球蛋白構築體組合遞送之前，患者先前已接受過基因治療。在某些具體實施例中，方案進一步包含共同投予一種以上之：(a)類固醇或類固醇類之組合及/或(b) IgG裂解酶、(c) Fc-IgE結合的抑制劑；(d) Fc-IgM結合的抑制劑；(e) Fc-IgA結合的抑制劑；及/或(f) γ干擾素。In certain embodiments, synergistic therapy can involve co-administration of rAAV provided herein in combination with a ligand that inhibits human neonatal Fc receptor (FcRn) binding to immunoglobulin G (IgG). See, eg, US Provisional Patent Application No. 63/040,381, filed June 17, 2020, and US Provisional Patent Application No. 63/135,998, filed January 11, 2021, and US Provisional Patent Application No. 63/135,998, filed February 22, 2021 No. 63/153,085, each of which is incorporated herein by reference. In certain embodiments, viral vectors are delivered systemically. In certain embodiments, the ligand is a peptide, protein, RNAi sequence or small molecule. In certain embodiments, the protein is a monoclonal antibody, an immunoadhesin, a camelid antibody, a Fab fragment, an Fv fragment or a scFv fragment. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus, a recombinant adenovirus, a recombinant herpes simplex virus, or a recombinant lentivirus. In certain embodiments, the ligand is a monoclonal antibody that specifically inhibits FcRn-IgG binding without interfering with FcRn-albumin binding. In some specific embodiments, the monoclonal antibody is nicalimab (Nipocalimab, M281), lolixizumab (rozanolixizumab, UCB7665); IMVT-1401, RVT-1401, HL161, HBM916, ARGX- 113 (efgartigimod), SYNT001, SYNT002, ABY-039, or DX-2507, derivatives or combinations thereof. In certain embodiments, Ligand 2 UPN-20-9394.P3 5 10 15 20 25 30 is delivered one to seven days prior to viral vector administration. In certain embodiments, the ligand is delivered daily. In certain embodiments, the ligand is dosed or administered on the same day as the viral vector is administered. In certain embodiments, the ligand is administered 1 day to 4 weeks after administration of the vector. In certain embodiments, the ligand is administered via a different route of administration than the carrier. In certain embodiments, the ligand is administered daily. In certain embodiments, the viral vector is administered intraperitoneally, intravenously, intramuscularly, intranasally or intrathecally. In certain embodiments, the patient is predetermined to have a neutralizing antibody titer to the vector capsid greater than 1:5 as determined in an in vitro assay. In certain embodiments, the patient has not previously received gene therapy prior to delivery of the viral vector in combination with the inhibitory ligand, and thus the patient's pre-existing neutralizing antibodies are the result of wild-type infection. In certain embodiments, the patient has previously received gene therapy prior to delivery of the viral vector in combination with the inhibitory immunoglobulin construct. In certain embodiments, the regimen further comprises co-administering more than one of: (a) a steroid or combination of steroids and/or (b) an inhibitor of IgG lyase, (c) Fc-IgE binding; (d ) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and/or (f) gamma interferon.

下列實施例係為說明本發明的某些具體實施例，而非對其之限制。 實施例 實施例1：從豬組織中單離新穎的AAV衣殼及生產重組AAV載體 The following examples are intended to illustrate some specific embodiments of the present invention, but not to limit them. Example Example 1: Isolation of Novel AAV Capsids from Porcine Tissues and Production of Recombinant AAV Vectors

使用高保真度聚合酶連鎖反應(PCR)實驗規程篩選豬組織中的新穎的AAV單離株。在測試的豬組織中，小腸樣品顯示PCR陽性率(靶向一個較小且相對多樣化的區域，兩側為對於引子結合的保留區域)遠高於肝臟、心臟、肺臟及脾臟的樣品(圖3)。回收大於二十個新穎豬AAV衣殼基因(圖4)。Porcine tissues were screened for novel AAV isolates using a high-fidelity polymerase chain reaction (PCR) protocol. Of the porcine tissues tested, the small intestine samples showed a much higher rate of PCR positivity (targeting a small and relatively diverse region flanked by regions reserved for primer binding) than samples from the liver, heart, lung, and spleen (Fig. 3). More than twenty novel porcine AAV capsid genes were recovered (Figure 4).

藉由使用如先前所述的HEK293三重轉染實驗規程產生重組AAV載體，使用：一種反式質體，其編碼對於反式互補AAV2 ITR及所選擇的(豬)AAV衣殼基因之AAV2 rep基因功能；一種反式質體，其攜帶腺病毒輔助功能；以及一種順式質體，其包含AAV2 5’ ITR、CB7.CI.eGFP.WPRE.rBG表現匣、及AAV2 3’ ITR。Recombinant AAV vectors were generated by using the HEK293 triple transfection protocol as previously described using: a plastid in trans encoding the AAV2 rep gene for the complementary AAV2 ITR and selected (pig) AAV capsid genes in trans function; a plastid in trans carrying an adenovirus helper function; and a plastid in cis comprising the AAV2 5'ITR, the CB7.CI.eGFP.WPRE.rBG expression cassette, and the AAV2 3'ITR.

使用Huh7細胞在活體外測量轉導效率。AAVpoG013及AAVpoG015具有最高水準的轉導(圖5A及圖5B)並被選擇用於進一步分析。具有兩種衣殼的載體具有非常好的產率(圖6)並且在靜脈內注射後以與AAV8相當的效率轉導小鼠肝臟(圖7)。Transduction efficiency was measured in vitro using Huh7 cells. AAVpoG013 and AAVpoG015 had the highest level of transduction (Figure 5A and Figure 5B) and were selected for further analysis. The vector with both capsids had very good yields (Figure 6) and transduced mouse livers with comparable efficiency to AAV8 after intravenous injection (Figure 7).

我們亦評估AAVpoG015的血清學槪貌。初步結果顯示，即使兩個衣殼關係遠(在AAVpoG015及AAV8之間的蛋白質序列相似性僅79%)(下表)，當用來自注射AAV8載體的動物的兩個猴子血清進行測試時，AAVpoG015的中和抗體力價僅比AAV8的力價低一倍。 血清 Nab力價(Huh7) AAV8 AAVpoG015 來自注射AAV8的猴1的血清 1:2560 1:1280 來自注射AAV8的猴2的血清 1:1280 1:640 We also assessed the serological profile of AAVpoG015. Preliminary results show that even though the two capsids are distantly related (only 79% protein sequence similarity between AAVpoG015 and AAV8) (table below), when tested with two monkey sera from animals injected with the AAV8 vector, AAVpoG015 The potency of the neutralizing antibody is only twice that of AAV8. serum Nab power price (Huh7) AAV8 AAVpoG015 Sera from AAV8-injected monkey 1 1:2560 1:1280 Serum from AAV8-injected monkey 2 1:1280 1:640

我們的結果表明1)天然AAV多樣性豐富；2)豬樣品中的小腸富含天然AAV(與之前研究的人類樣品不同)；3) AAVpoG013及AAVpoG015衣殼為用於肝臟導向基因遞送的有吸引力的候選物。 實施例2：使用具有AAVpoG015衣殼的重組AAV而將轉基因遞送至非人類靈長類動物後的生物分布 Our results suggest that 1) native AAV diversity is rich; 2) the small intestine in porcine samples is enriched in native AAV (unlike previously studied human samples); 3) AAVpoG013 and AAVpoG015 capsids are attractive candidates for liver-directed gene delivery. Candidates for strength. Example 2: Biodistribution of transgenes delivered to non-human primates using recombinant AAV with AAVpoG015 capsid

AAVpoG015衣殼用於包裝具有在CB7啟動子控制下的eGFP轉基因的載體基因體(AAV.CB7.CI.eGFP.WPRE.rBG)，使用如前所述的HEK293細胞的三重轉染(Lock et al. Hum. Gene Ther. 21, 1259–1271；Lock et al. Hum. Gene Ther. Methods 25, 115-125)。收穫細胞培養上清液，濃縮，並以碘克沙醇(iodixanol)梯度純化。The AAVpoG015 capsid was used to package the vector gene body (AAV.CB7.CI.eGFP.WPRE.rBG) with the eGFP transgene under the control of the CB7 promoter using triple transfection of HEK293 cells as previously described (Lock et al . Hum. Gene Ther. 21, 1259–1271; Lock et al. Hum. Gene Ther. Methods 25, 115-125). Cell culture supernatants were harvested, concentrated, and purified with an iodixanol gradient.

將純化的載體以5 x 10 13基因體拷貝(GC)/kg的劑量靜脈內遞送至恆河獼猴。十天後，犧牲動物，並藉由qPCR及RT-qPCR測定各種組織中的載體GC。以QIAamp DNA Mini Kit (QIAGEN)萃取組織基因體DNA，並使用Taqman試劑(Applied Biosystems, Life Technologies)藉由即時PCR對AAV載體基因體進行定量，其中引子/探針靶向載體的eGFP序列。在肝臟、心臟及肌肉組織中檢測到較高水準的載體GC(圖8A及圖8B)。圖9顯示投劑後的相對AST及ALT水準。與類似劑量的其它AAV血清型IV投劑三天後觀察到的明顯ALT/AST升高相反，ALT/AST水準沒有升高，表明AAVpoG015可能誘導較少的肝臟發炎。 The purified vector was delivered intravenously to rhesus monkeys at a dose of 5 x 1013 gene body copies (GC)/kg. Ten days later, animals were sacrificed, and vector GC in various tissues was determined by qPCR and RT-qPCR. Tissue genome DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN), and AAV vector genomes were quantified by real-time PCR using Taqman reagents (Applied Biosystems, Life Technologies), with primers/probes targeting the eGFP sequence of the vector. Higher levels of carrier GC were detected in liver, heart and muscle tissues (Fig. 8A and Fig. 8B). Figure 9 shows relative AST and ALT levels after dosing. In contrast to the marked ALT/AST elevation observed three days after IV administration of similar doses of other AAV serotypes, ALT/AST levels were not elevated, suggesting that AAVpoG015 may induce less liver inflammation.

3 x 10 13GC的劑量經由腦大池內(ICM)注射而投予至恆河獼猴。十五天後，犧牲動物並如上所述藉由qPCR及RT-qPCR測定載體GC水準(圖10A-圖10C)。 A dose of 3 x 1013 GC was administered to rhesus monkeys via intracisternal (ICM) injection. Fifteen days later, animals were sacrificed and vector GC levels were determined by qPCR and RT-qPCR as described above (FIGS. 10A-10C).

本說明書中列出的所有專利、專利公開案及其它出版物皆藉由引用併入本文。2021年4月27日申請的US臨時專利申請號63/180,372係藉由引用併入本文。本文引用的示於SEQ ID NO的胺基酸及核苷酸序列以及出現在標記為「21-9621PCT_ST25」的所附序列表中的胺基酸及核苷酸序列係藉由引用併入。儘管已經參考特別較佳的具體實施例描述本發明，但應當理解，可於不背離本發明的精神下進行修改。此種修改意圖落入所附申請專利範圍的範疇內。All patents, patent publications, and other publications listed in this specification are hereby incorporated by reference. US Provisional Patent Application No. 63/180,372, filed April 27, 2021, is incorporated herein by reference. The amino acid and nucleotide sequences shown in SEQ ID NO cited herein and the amino acid and nucleotide sequences appearing in the accompanying Sequence Listing labeled "21-9621PCT_ST25" are incorporated by reference. Although the invention has been described with reference to particular preferred embodiments, it should be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

無none

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0019

Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0020

Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0021

Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0022

Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0023

Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0024

Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0025

Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0026

Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0027

Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0028

Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0029

Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0030

Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0031

Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0032

Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0033

Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0034

Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0035

Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0036

Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0037

Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0038

Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0039

Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0040

Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0041

Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0042

Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0043

Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0044

Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0045

Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0046

Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0047

Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0048

Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0049

Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0050

Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0051

Figure 12_A0101_SEQ_0052
Figure 12_A0101_SEQ_0052

Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0053

Figure 12_A0101_SEQ_0054
Figure 12_A0101_SEQ_0054

Figure 12_A0101_SEQ_0055
Figure 12_A0101_SEQ_0055

Figure 12_A0101_SEQ_0056
Figure 12_A0101_SEQ_0056

Figure 12_A0101_SEQ_0057
Figure 12_A0101_SEQ_0057

Figure 12_A0101_SEQ_0058
Figure 12_A0101_SEQ_0058

Figure 12_A0101_SEQ_0059
Figure 12_A0101_SEQ_0059

Figure 12_A0101_SEQ_0060
Figure 12_A0101_SEQ_0060

Figure 12_A0101_SEQ_0061
Figure 12_A0101_SEQ_0061

Figure 12_A0101_SEQ_0062
Figure 12_A0101_SEQ_0062

Figure 12_A0101_SEQ_0063
Figure 12_A0101_SEQ_0063

Figure 12_A0101_SEQ_0064
Figure 12_A0101_SEQ_0064

Figure 12_A0101_SEQ_0065
Figure 12_A0101_SEQ_0065

Figure 12_A0101_SEQ_0066
Figure 12_A0101_SEQ_0066

Figure 12_A0101_SEQ_0067
Figure 12_A0101_SEQ_0067

Figure 12_A0101_SEQ_0068
Figure 12_A0101_SEQ_0068

Figure 12_A0101_SEQ_0069
Figure 12_A0101_SEQ_0069

Figure 12_A0101_SEQ_0070
Figure 12_A0101_SEQ_0070

Figure 12_A0101_SEQ_0071
Figure 12_A0101_SEQ_0071

Figure 12_A0101_SEQ_0072
Figure 12_A0101_SEQ_0072

Figure 12_A0101_SEQ_0073
Figure 12_A0101_SEQ_0073

Figure 12_A0101_SEQ_0074
Figure 12_A0101_SEQ_0074

Figure 12_A0101_SEQ_0075
Figure 12_A0101_SEQ_0075

Figure 12_A0101_SEQ_0076
Figure 12_A0101_SEQ_0076

Figure 12_A0101_SEQ_0077
Figure 12_A0101_SEQ_0077

Figure 12_A0101_SEQ_0078
Figure 12_A0101_SEQ_0078

Figure 12_A0101_SEQ_0079
Figure 12_A0101_SEQ_0079

Figure 12_A0101_SEQ_0080
Figure 12_A0101_SEQ_0080

Figure 12_A0101_SEQ_0081
Figure 12_A0101_SEQ_0081

Figure 12_A0101_SEQ_0082
Figure 12_A0101_SEQ_0082

Figure 12_A0101_SEQ_0083
Figure 12_A0101_SEQ_0083

Figure 12_A0101_SEQ_0084
Figure 12_A0101_SEQ_0084

Figure 12_A0101_SEQ_0085
Figure 12_A0101_SEQ_0085

Figure 12_A0101_SEQ_0086
Figure 12_A0101_SEQ_0086

Figure 12_A0101_SEQ_0087
Figure 12_A0101_SEQ_0087

Figure 12_A0101_SEQ_0088
Figure 12_A0101_SEQ_0088

Figure 12_A0101_SEQ_0089
Figure 12_A0101_SEQ_0089

Figure 12_A0101_SEQ_0090
Figure 12_A0101_SEQ_0090

Figure 12_A0101_SEQ_0091
Figure 12_A0101_SEQ_0091

Figure 12_A0101_SEQ_0092
Figure 12_A0101_SEQ_0092

Figure 12_A0101_SEQ_0093
Figure 12_A0101_SEQ_0093

Figure 12_A0101_SEQ_0094
Figure 12_A0101_SEQ_0094

Figure 12_A0101_SEQ_0095
Figure 12_A0101_SEQ_0095

Figure 12_A0101_SEQ_0096
Figure 12_A0101_SEQ_0096

Figure 12_A0101_SEQ_0097
Figure 12_A0101_SEQ_0097

Figure 12_A0101_SEQ_0098
Figure 12_A0101_SEQ_0098

Figure 12_A0101_SEQ_0099
Figure 12_A0101_SEQ_0099

Figure 12_A0101_SEQ_0100
Figure 12_A0101_SEQ_0100

Figure 12_A0101_SEQ_0101
Figure 12_A0101_SEQ_0101

Figure 12_A0101_SEQ_0102
Figure 12_A0101_SEQ_0102

Figure 12_A0101_SEQ_0103
Figure 12_A0101_SEQ_0103

Figure 12_A0101_SEQ_0104
Figure 12_A0101_SEQ_0104

Figure 12_A0101_SEQ_0105
Figure 12_A0101_SEQ_0105

Figure 12_A0101_SEQ_0106
Figure 12_A0101_SEQ_0106

Figure 12_A0101_SEQ_0107
Figure 12_A0101_SEQ_0107

Figure 12_A0101_SEQ_0108
Figure 12_A0101_SEQ_0108

Figure 12_A0101_SEQ_0109
Figure 12_A0101_SEQ_0109

Figure 12_A0101_SEQ_0110
Figure 12_A0101_SEQ_0110

Figure 12_A0101_SEQ_0111
Figure 12_A0101_SEQ_0111

Figure 12_A0101_SEQ_0112
Figure 12_A0101_SEQ_0112

Figure 12_A0101_SEQ_0113
Figure 12_A0101_SEQ_0113

Figure 12_A0101_SEQ_0114
Figure 12_A0101_SEQ_0114

Figure 12_A0101_SEQ_0115
Figure 12_A0101_SEQ_0115

Figure 12_A0101_SEQ_0116
Figure 12_A0101_SEQ_0116

Figure 12_A0101_SEQ_0117
Figure 12_A0101_SEQ_0117

Figure 12_A0101_SEQ_0118
Figure 12_A0101_SEQ_0118

Figure 12_A0101_SEQ_0119
Figure 12_A0101_SEQ_0119

Figure 12_A0101_SEQ_0120
Figure 12_A0101_SEQ_0120

Figure 12_A0101_SEQ_0121
Figure 12_A0101_SEQ_0121

Figure 12_A0101_SEQ_0122
Figure 12_A0101_SEQ_0122

Figure 12_A0101_SEQ_0123
Figure 12_A0101_SEQ_0123

Figure 12_A0101_SEQ_0124
Figure 12_A0101_SEQ_0124

Figure 12_A0101_SEQ_0125
Figure 12_A0101_SEQ_0125

Figure 12_A0101_SEQ_0126
Figure 12_A0101_SEQ_0126

Figure 12_A0101_SEQ_0127
Figure 12_A0101_SEQ_0127

Figure 12_A0101_SEQ_0128
Figure 12_A0101_SEQ_0128

Figure 12_A0101_SEQ_0129
Figure 12_A0101_SEQ_0129

Figure 12_A0101_SEQ_0130
Figure 12_A0101_SEQ_0130

Figure 12_A0101_SEQ_0131
Figure 12_A0101_SEQ_0131

Figure 12_A0101_SEQ_0132
Figure 12_A0101_SEQ_0132

Figure 12_A0101_SEQ_0133
Figure 12_A0101_SEQ_0133

Figure 12_A0101_SEQ_0134
Figure 12_A0101_SEQ_0134

Figure 12_A0101_SEQ_0135
Figure 12_A0101_SEQ_0135

Figure 12_A0101_SEQ_0136
Figure 12_A0101_SEQ_0136

Figure 12_A0101_SEQ_0137
Figure 12_A0101_SEQ_0137

Figure 12_A0101_SEQ_0138
Figure 12_A0101_SEQ_0138

Figure 12_A0101_SEQ_0139
Figure 12_A0101_SEQ_0139

Figure 12_A0101_SEQ_0140
Figure 12_A0101_SEQ_0140

Figure 12_A0101_SEQ_0141
Figure 12_A0101_SEQ_0141

Figure 12_A0101_SEQ_0142
Figure 12_A0101_SEQ_0142

Figure 12_A0101_SEQ_0143
Figure 12_A0101_SEQ_0143

Figure 12_A0101_SEQ_0144
Figure 12_A0101_SEQ_0144

Figure 12_A0101_SEQ_0145
Figure 12_A0101_SEQ_0145

Figure 12_A0101_SEQ_0146
Figure 12_A0101_SEQ_0146

Figure 12_A0101_SEQ_0147
Figure 12_A0101_SEQ_0147

Figure 12_A0101_SEQ_0148
Figure 12_A0101_SEQ_0148

Figure 12_A0101_SEQ_0149
Figure 12_A0101_SEQ_0149

Figure 12_A0101_SEQ_0150
Figure 12_A0101_SEQ_0150

Figure 12_A0101_SEQ_0151
Figure 12_A0101_SEQ_0151

Figure 12_A0101_SEQ_0152
Figure 12_A0101_SEQ_0152

Figure 12_A0101_SEQ_0153
Figure 12_A0101_SEQ_0153

Figure 12_A0101_SEQ_0154
Figure 12_A0101_SEQ_0154

Figure 12_A0101_SEQ_0155
Figure 12_A0101_SEQ_0155

Figure 12_A0101_SEQ_0156
Figure 12_A0101_SEQ_0156

Figure 12_A0101_SEQ_0157
Figure 12_A0101_SEQ_0157

無。none.

Claims (15)

一種重組腺相關病毒(rAAV)，其具有包含衣殼蛋白的衣殼並具有包裝於該衣殼中的載體基因體，其中該衣殼蛋白具有下述序列，且該載體基因體包含非AAV核酸序列：AAVpoG015 (SEQ ID NO: 26)、AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。A recombinant adeno-associated virus (rAAV) having a capsid comprising a capsid protein and having a vector gene body packaged in the capsid, wherein the capsid protein has the following sequence, and the vector gene body comprises a non-AAV nucleic acid Sequences: AAVpoG015 (SEQ ID NO: 26), AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20) , AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42), AAVpoG024 (SEQ ID NO: 44), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 46); or a sequence that shares at least 98% or at least 99% identity with any of SEQ ID NO: 2, 4, 6, 8; Or a sequence that shares at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30; Or share at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with any of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 Sequences with % identity. 一種rAAV，其具有包含衣殼蛋白的衣殼並具有包裝於該衣殼中的載體基因體，其中該衣殼蛋白係由下述序列所編碼，且該載體基因體包含非AAV核酸序列：AAVpoG015 (SEQ ID NO: 25)、AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45共有至少70%同一性的序列。A rAAV having a capsid comprising a capsid protein and having a vector gene body packaged in the capsid, wherein the capsid protein is encoded by the following sequence, and the vector gene body comprises a non-AAV nucleic acid sequence: AAVpoG015 (SEQ ID NO: 25), AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 ( SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33 ), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO: 43), or AAVpoG025 ( The vp1, vp2, and/or vp3 sequence of SEQ ID NO: 45); or with SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27 , 29, 31, 33, 35, 37, 39, 41, 43, or 45 are sequences that share at least 70% identity. 如請求項2之rAAV，其中該序列編碼：AAVpoG015 (SEQ ID NO: 26)、AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。Such as the rAAV of claim item 2, wherein the sequence coding: AAVpoG015 (SEQ ID NO: 26), AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 ( SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18 ), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), AAVpoG023 (SEQ ID NO: 42) , AAVpoG024 (SEQ ID NO: 44), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 46); or share at least 98% with any of SEQ ID NO: 2, 4, 6, 8 % or at least 99% identical sequence; or any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 shares at least 96%, at least 97%, at least 98 % or at least 99% identical sequence; or any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 shares at least 90%, at least 95%, at least 96%, at least A sequence that is 97%, at least 98%, or at least 99% identical. 如請求項1至3中任一項之rAAV，其中該衣殼蛋白係由示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的核苷酸序列所編碼。The rAAV according to any one of claims 1 to 3, wherein the capsid protein is represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 are encoded by the nucleotide sequence. 如請求項1至4中任一項之rAAV，其中該載體基因體包含AAV反向末端重複序列及可操作地連接至調節序列的非AAV序列，該調節序列指導由非AAV核酸序列所編碼的基因產物在目標細胞中的表現。The rAAV of any one of claims 1 to 4, wherein the vector gene body comprises an AAV inverted terminal repeat sequence and a non-AAV sequence operably linked to a regulatory sequence that directs the non-AAV sequence encoded by the non-AAV nucleic acid sequence Expression of the gene product in the target cell. 如請求項1至5中任一項之rAAV，其中該載體基因體包含組織特異性啟動子，可選擇地為肝臟特異性啟動子。The rAAV according to any one of claims 1 to 5, wherein the vector gene body comprises a tissue-specific promoter, optionally a liver-specific promoter. 一種宿主細胞，其含有如請求項1至6中任一項之rAAV。A host cell containing the rAAV according to any one of claims 1 to 6. 一種醫藥組成物，其包含如請求項1至6中任一項之rAAV以及生理學上可接受的載劑、緩衝劑、佐劑、及/或稀釋劑。A pharmaceutical composition comprising the rAAV according to any one of claims 1 to 6 and a physiologically acceptable carrier, buffer, adjuvant, and/or diluent. 一種遞送轉基因至細胞之方法，該方法包含將該細胞與如請求項1至6中任一項之rAAV接觸的步驟，其中該rAAV包含該轉基因。A method of delivering a transgene to a cell, the method comprising the step of contacting the cell with the rAAV according to any one of claims 1 to 6, wherein the rAAV comprises the transgene. 一種生產包含AAV衣殼的rAAV之方法，該方法包含培養宿主細胞，該宿主細胞含有：(a)核酸，包含AAVpoG015 (SEQ ID NO: 25)、AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之AAV vp1、vp2、及/或vp3序列、或者與示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的vp1、vp2、及/或vp3核苷酸序列共有至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列；(b)功能性rep基因；(c)袖珍基因，包含AAV反向末端重複序列(ITR)及轉基因；及(d)充足的輔助功能以允許將該袖珍基因包裝至該AAV衣殼中。A method of producing rAAV comprising an AAV capsid, the method comprising culturing a host cell containing: (a) a nucleic acid comprising AAVpoG015 (SEQ ID NO: 25), AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13) , AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31), AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAV vp1, vp2, and/or vp3 sequences of AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO: 43), or AAVpoG025 (SEQ ID NO: 45), or with Shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 of the vp1, vp2, and/or vp3 nucleotide sequences share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% %, or at least 99% identical; (b) a functional rep gene; (c) a pocket gene comprising an AAV inverted terminal repeat (ITR) and a transgene; and (d) sufficient accessory functions to allow the The pocket genes are packaged into the AAV capsid. 如請求項10之方法，其中該vp1、vp2、及/或vp3序列編碼：AAVpoG015 (SEQ ID NO: 26)、AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。The method as claimed in item 10, wherein the vp1, vp2, and/or vp3 sequence coding: AAVpoG015 (SEQ ID NO: 26), AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4), AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16) , AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO: 40), The sequence of AAVpoG023 (SEQ ID NO: 42), AAVpoG024 (SEQ ID NO: 44), or AAVpoG025 (SEQ ID NO: 46); or share at least 98% with any of SEQ ID NO: 2, 4, 6, 8 % or at least 99% identical sequence; or any one of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 shares at least 96%, at least 97%, at least 98 % or at least 99% identical sequence; or any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, or 46 shares at least 90%, at least 95%, at least 96%, at least A sequence that is 97%, at least 98%, or at least 99% identical. 一種組成物，其包含如請求項10或11之方法所生產的rAAV的儲料(stock)。A composition comprising a stock of rAAV produced by the method of claim 10 or 11. 一種質體，其包含：AAVpoG015 (SEQ ID NO: 25)、AAVpoG001 (SEQ ID NO: 1)、AAVpoG002 (SEQ ID NO: 3)、AAVpoG003 (SEQ ID NO: 5)、AAVpoG004 (SEQ ID NO: 7)、AAVpoG005 (SEQ ID NO: 9)、AAVpoG006 (SEQ ID NO: 11)、AAVpoG007 (SEQ ID NO: 13)、AAVpoG008 (SEQ ID NO: 15)、AAVpoG009 (SEQ ID NO: 17)、AAVpoG012 (SEQ ID NO: 19)、AAVpoG013 (SEQ ID NO: 21)、AAVpoG014 (SEQ ID NO: 23)、AAVpoG016 (SEQ ID NO: 27)、AAVpoG017 (SEQ ID NO: 29)、AAVpoG018 (SEQ ID NO: 31)、AAVpoG019 (SEQ ID NO: 33)、AAVpoG020 (SEQ ID NO: 35)、AAVpoG021 (SEQ ID NO: 37)、AAVpoG022 (SEQ ID NO: 39)、AAVpoG023 (SEQ ID NO: 41)、AAVpoG024 (SEQ ID NO: 43)、或AAVpoG025 (SEQ ID NO: 45)之vp1、vp2、及/或vp3核苷酸序列；或與示於SEQ ID NO: 1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、或45的vp1、vp2、及/或vp3核苷酸序列共有至少70%同一性的序列。A plastid comprising: AAVpoG015 (SEQ ID NO: 25), AAVpoG001 (SEQ ID NO: 1), AAVpoG002 (SEQ ID NO: 3), AAVpoG003 (SEQ ID NO: 5), AAVpoG004 (SEQ ID NO: 7 ), AAVpoG005 (SEQ ID NO: 9), AAVpoG006 (SEQ ID NO: 11), AAVpoG007 (SEQ ID NO: 13), AAVpoG008 (SEQ ID NO: 15), AAVpoG009 (SEQ ID NO: 17), AAVpoG012 (SEQ ID NO: 19), AAVpoG013 (SEQ ID NO: 21), AAVpoG014 (SEQ ID NO: 23), AAVpoG016 (SEQ ID NO: 27), AAVpoG017 (SEQ ID NO: 29), AAVpoG018 (SEQ ID NO: 31) , AAVpoG019 (SEQ ID NO: 33), AAVpoG020 (SEQ ID NO: 35), AAVpoG021 (SEQ ID NO: 37), AAVpoG022 (SEQ ID NO: 39), AAVpoG023 (SEQ ID NO: 41), AAVpoG024 (SEQ ID NO: 43), or the vp1, vp2, and/or vp3 nucleotide sequence of AAVpoG025 (SEQ ID NO: 45); or shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, The vp1, vp2, and/or vp3 nucleotide sequences of 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45 share at least 70% identity sexual sequence. 如請求項12之質體，其中該vp1、vp2、及/或vp3核苷酸序列編碼：AAVpoG015 (SEQ ID NO: 26)、AAVpoG001 (SEQ ID NO: 2)、AAVpoG002 (SEQ ID NO: 4)、AAVpoG003 (SEQ ID NO: 6)、AAVpoG004 (SEQ ID NO: 8)、AAVpoG005 (SEQ ID NO: 10)、AAVpoG006 (SEQ ID NO: 12)、AAVpoG007 (SEQ ID NO: 14)、AAVpoG008 (SEQ ID NO: 16)、AAVpoG009 (SEQ ID NO: 18)、AAVpoG012 (SEQ ID NO: 20)、AAVpoG013 (SEQ ID NO: 22)、AAVpoG014 (SEQ ID NO: 24)、AAVpoG016 (SEQ ID NO: 28)、AAVpoG017 (SEQ ID NO: 30)、AAVpoG018 (SEQ ID NO: 32)、AAVpoG019 (SEQ ID NO: 34)、AAVpoG020 (SEQ ID NO: 36)、AAVpoG021 (SEQ ID NO: 38)、AAVpoG022 (SEQ ID NO: 40)、AAVpoG023 (SEQ ID NO: 42)、AAVpoG024 (SEQ ID NO: 44)、或AAVpoG025 (SEQ ID NO: 46)之vp1、vp2、及/或vp3序列；或與SEQ ID NO: 2、4、6、8之任一者共有至少98%或至少99%同一性的序列；或與SEQ ID NO: 12、14、16、18、20、22、24、26、28、30之任一者共有至少96%、至少97%、至少98%或至少99%同一性的序列；或與SEQ ID NO: 32、34、36、38、40、42、44、或46之任一者共有至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性的序列。The plastid of claim 12, wherein the vp1, vp2, and/or vp3 nucleotide sequence codes: AAVpoG015 (SEQ ID NO: 26), AAVpoG001 (SEQ ID NO: 2), AAVpoG002 (SEQ ID NO: 4) , AAVpoG003 (SEQ ID NO: 6), AAVpoG004 (SEQ ID NO: 8), AAVpoG005 (SEQ ID NO: 10), AAVpoG006 (SEQ ID NO: 12), AAVpoG007 (SEQ ID NO: 14), AAVpoG008 (SEQ ID NO: 16), AAVpoG009 (SEQ ID NO: 18), AAVpoG012 (SEQ ID NO: 20), AAVpoG013 (SEQ ID NO: 22), AAVpoG014 (SEQ ID NO: 24), AAVpoG016 (SEQ ID NO: 28), AAVpoG017 (SEQ ID NO: 30), AAVpoG018 (SEQ ID NO: 32), AAVpoG019 (SEQ ID NO: 34), AAVpoG020 (SEQ ID NO: 36), AAVpoG021 (SEQ ID NO: 38), AAVpoG022 (SEQ ID NO : 40), AAVpoG023 (SEQ ID NO: 42), AAVpoG024 (SEQ ID NO: 44), or the vp1, vp2, and/or vp3 sequence of AAVpoG025 (SEQ ID NO: 46); or with SEQ ID NO: 2, Any of 4, 6, 8 shares at least 98% or at least 99% identical sequence; or any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or share at least 96%, at least 97%, at least 98%, or at least 99% identity; or share at least A sequence that is 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. 一種培養的宿主細胞，其含有如請求項13或14之質體。A cultured host cell containing the plastid according to claim 13 or 14.
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