TW202342064A - Circular polyribonucleotides encoding antifusogenic polypeptides - Google Patents

Abstract

The present disclosure relates, generally, to compositions and methods for producing, purifying, and using circular RNA encoding an antifusogenic polypeptide.

Description

Translated fromChinese

編碼抗融合多肽之環狀多核糖核苷酸Cyclic polyribonucleotides encoding anti-fusion polypeptides

本揭露關於環狀多核糖核苷酸，特別是編碼抗融合多肽（antifusogenic polypeptide）的環狀多核糖核苷酸。The present disclosure relates to cyclic polyribonucleotides, particularly cyclic polyribonucleotides encoding antifusogenic polypeptides.

多核苷酸和蛋白質的遞送對於廣泛的治療領域係重要的。然而，目前的遞送方式往往無效。例如，遞送短多肽（如編碼抗融合多肽的多肽）通常導致該等多肽的短半衰期和快速清除。因此，需要改進的用於遞送抗融合多肽的組成物和方法，例如，用於治療或預防病毒感染。The delivery of polynucleotides and proteins is important for a wide range of therapeutic areas. However, current delivery methods are often ineffective. For example, delivery of short polypeptides (eg, polypeptides encoding anti-fusion polypeptides) often results in a short half-life and rapid clearance of such polypeptides. Accordingly, there is a need for improved compositions and methods for delivering anti-fusion polypeptides, for example, for treating or preventing viral infections.

本揭露提供了用於產生、純化和使用編碼抗融合多肽的環狀RNA之組成物及方法。The present disclosure provides compositions and methods for generating, purifying, and using circular RNAs encoding anti-fusion polypeptides.

在一方面，本發明之特徵在於環狀多核糖核苷酸，其包括編碼抗融合多肽的多核糖核苷酸貨物（polyribonucleotide cargo）。在一些實施方式中，多核糖核苷酸貨物包括編碼抗融合多肽之表現序列。In one aspect, the invention features cyclic polyribonucleotides that include a polyribonucleotide cargo encoding an anti-fusion polypeptide. In some embodiments, the polyribonucleotide cargo includes expression sequences encoding anti-fusion polypeptides.

在一些實施方式中，多核糖核苷酸貨物包括編碼表1的多肽之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與表1的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 1-324中任一個具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the polyribonucleotide cargo includes an expressed sequence encoding a polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo includes an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of Table 1 . In some embodiments, the polyribonucleotide cargo includes a polyribonucleotide encoding a sequence that is at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) identical to any one of SEQ ID NOs: 1-324. The expression sequence of a sexual polypeptide.

在一些實施方式中，該環狀多核糖核苷酸包含連接5'外顯子片段和3'外顯子片段之剪接點。In some embodiments, the circular polyribonucleotide includes a splice junction connecting the 5' exon fragment and the 3' exon fragment.

在一些實施方式中，多核糖核苷酸貨物包括與編碼抗融合多肽的表現序列可操作地連接的IRES。該環狀多核糖核苷酸可進一步包含該IRES與該3'外顯子片段或該5'外顯子片段之間的間隔子區域。該間隔子區域的長度可以為至少5個核糖核苷酸。例如，間隔子區域的長度可以為至少10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、900、1,000個或更多個核糖核苷酸。在一些實施方式中，間隔子區域之長度為5至500個核糖核苷酸。該間隔子區域可以包括聚A、聚A-C、聚A-U或聚A-G序列。間隔子區域可為隨機序列。In some embodiments, the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding an anti-fusion polypeptide. The cyclic polyribonucleotide may further comprise a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The spacer region may be at least 5 ribonucleotides in length. For example, the length of the spacer sub-region may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more Multiple ribonucleotides. In some embodiments, the spacer region is 5 to 500 ribonucleotides in length. The spacer region may include polyA, polyA-C, polyA-U or polyA-G sequences. The spacer sub-region can be a random sequence.

在一些實施方式中，該環狀多核糖核苷酸之長度為至少500個核糖核苷酸。例如，環狀多核糖核苷酸可為至少500、600、700、800、900、1,000、2,000、3,000、4,000、5,000、6,000、7,000、8,000、9,000、10,000、15,000、20,000、25,000、30,000、35,000、40,000、45,000、50,000個或更多個核糖核苷酸。在一些實施方式中，環狀多核糖核苷酸之長度為500至20,000個核糖核苷酸。In some embodiments, the cyclic polyribonucleotide is at least 500 ribonucleotides in length. For example, the cyclic polyribonucleotide can be at least 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000 or more ribonucleotides. In some embodiments, the cyclic polyribonucleotide is 500 to 20,000 ribonucleotides in length.

在另一方面，特徵在於線性多核糖核苷酸，該線性多核糖核苷酸從5'到3'包括 (A) 3'內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 編碼該抗融合多肽的多核糖核苷酸貨物；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'內含子片段。In another aspect, characterized by a linear polyribonucleotide comprising from 5' to 3' (A) a 3' intronic segment; (B) a 3' splice site; (C) 3' exon fragment; (D) polyribonucleotide cargo encoding the anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' intron subfragment.

在一些實施方式中，多核糖核苷酸貨物包括編碼抗融合多肽之表現序列。In some embodiments, the polyribonucleotide cargo includes expression sequences encoding anti-fusion polypeptides.

在一些實施方式中，多核糖核苷酸貨物包括與編碼抗融合多肽（例如，表1的多肽）的表現序列可操作地連接的IRES。該環狀多核糖核苷酸可進一步包含該IRES與該3'外顯子片段或該5'外顯子片段之間的間隔子區域。該環狀多核糖核苷酸可進一步包括 (A)、(B)、(C)、(D)、(E)、(F) 和 (G) 中一或多個之間的間隔子區域。In some embodiments, the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding an anti-fusion polypeptide (eg, the polypeptide of Table 1). The cyclic polyribonucleotide may further comprise a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The cyclic polyribonucleotide may further include a spacer region between one or more of (A), (B), (C), (D), (E), (F) and (G).

該間隔子區域的長度可以為至少5個核糖核苷酸。例如，間隔子區域的長度可以為至少10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、900、1000個或更多個核糖核苷酸。在一些實施方式中，間隔子區域之長度為5至500個核糖核苷酸。該間隔子區域可以包括聚A、聚A-C、聚A-U或聚A-G序列。間隔子區域可為隨機序列。The spacer region may be at least 5 ribonucleotides in length. For example, the length of the spacer sub-region may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more Multiple ribonucleotides. In some embodiments, the spacer region is 5 to 500 ribonucleotides in length. The spacer region may include polyA, polyA-C, polyA-U or polyA-G sequences. The spacer sub-region can be a random sequence.

在一些實施方式中，環狀多核糖核苷酸缺少IRES。在一些實施方式中，該環狀多核糖核苷酸缺少5'帽和聚A序列中的一者或兩者。In some embodiments, the cyclic polyribonucleotide lacks an IRES. In some embodiments, the cyclic polyribonucleotide lacks one or both of the 5' cap and polyA sequence.

在一些實施方式中，環狀多核糖核苷酸包含蛋白質翻譯起始位點。在一些實施方式中，蛋白質翻譯起始位點包含科紮克（Kozak）序列。In some embodiments, the cyclic polyribonucleotide contains a protein translation initiation site. In some embodiments, the protein translation initiation site comprises a Kozak sequence.

在一些實施方式中，該線性多核糖核苷酸之長度為至少500個核糖核苷酸。例如，線性多核糖核苷酸可為至少500、600、700、800、900、1,000、2,000、3,000、4,000、5,000、6,000、7,000、8,000、9,000、10,000、15,000、20,000、25,000、30,000、35,000、40,000、45,000、50,000個或更多個核糖核苷酸。在一些實施方式中，該線性多核糖核苷酸之長度為500至20,000個核糖核苷酸。In some embodiments, the linear polyribonucleotide is at least 500 ribonucleotides in length. For example, a linear polyribonucleotide can be at least 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,00 0 , 40,000, 45,000, 50,000 or more ribonucleotides. In some embodiments, the linear polyribonucleotide is 500 to 20,000 ribonucleotides in length.

在另一方面，特徵在於編碼如本文所述之多核糖核苷酸（例如，線性或環狀多核糖核苷酸）的DNA載體。In another aspect, feature a DNA vector encoding a polyribonucleotide (eg, a linear or cyclic polyribonucleotide) as described herein.

在另一方面，特徵在於在細胞中表現抗融合多肽（例如，表1的多肽）之方法。該方法包括在適於表現抗融合多肽的條件下向細胞提供如本文所述之環狀、線性多核糖核苷酸或DNA載體。In another aspect, methods are characterized by expressing an anti-fusion polypeptide (eg, the polypeptide of Table 1) in a cell. The method involves providing a circular, linear polyribonucleotide or DNA vector as described herein to the cell under conditions suitable for expression of the anti-fusion polypeptide.

在另一方面，特徵在於由如本文所述之線性多核糖核苷酸產生環狀多核糖核苷酸之方法。該方法包括在適於該線性多核糖核苷酸自剪接以產生該環狀多核糖核苷酸的條件下提供該線性多核糖核苷酸。In another aspect, features are methods of producing cyclic polyribonucleotides from linear polyribonucleotides as described herein. The method includes providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the cyclic polyribonucleotide.

在另一方面，特徵在於一種藥物組成物，該藥物組成物包含上述實施方式中任一個的環狀多核糖核苷酸、線性多核糖核苷酸或DNA載體，以及稀釋劑、載劑或賦形劑。In another aspect, is characterized by a pharmaceutical composition comprising the cyclic polyribonucleotide, linear polyribonucleotide or DNA vector of any one of the above embodiments, and a diluent, carrier or excipient. form agent.

在另一方面，特徵在於在受試者中表現抗融合多肽（例如，表1的多肽）之方法。該方法包括以足以在受試者中產生至少500 ng/mL（例如，至少600 ng/mL、700 ng/mL、800 ng/mL、900 ng/mL、1,000 ng/mL、1,100 ng/mL、1,200 ng/mL、1,300 ng/mL、1,400 ng/mL、1,500 ng/mL、1,600 ng/mL、1,700 ng/mL、1,800 ng/mL、1,900 ng/mL、2,000 ng/mL、2,100 ng/mL、2,200 ng/mL、2,300 ng/mL、2,400 ng/mL、2,500 ng/mL、2,600 ng/mL、2,700 ng/mL、2,800 ng/mL、2,900 ng/mL、3,000 ng/mL或更多）的血清濃度的抗融合多肽（例如，表1的多肽）的量投與第一劑量的藥物組成物。In another aspect, methods are characterized by expressing an anti-fusion polypeptide (eg, a polypeptide of Table 1) in a subject. The method includes administering a drug sufficient to produce at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more) serum The concentration of the anti-fusion polypeptide (eg, the polypeptide of Table 1) is such that the first dose of the pharmaceutical composition is administered.

在一些實施方式中，該方法可進一步包括投與第二劑量的藥物組成物。該方法可進一步包括投與第三、第四、第五、第六、第七、第八、第九、第十或更多劑量的藥物組成物。In some embodiments, the method may further comprise administering a second dose of the pharmaceutical composition. The method may further comprise administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more doses of the pharmaceutical composition.

在一些實施方式中，第二劑量在藥物組成物之第一劑量後至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）投與。In some embodiments, the second dose is at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours) after the first dose of the pharmaceutical composition. hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months , eleven months, one year or more) investment.

在一些實施方式中，第二劑量在藥物組成物之第一劑量後1小時至1年（例如1小時到1天，例如一小時、兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時或一天，例如一天到一週，例如兩天、三天、四天、五天、六天或一週，例如一週到一個月，例如兩週、三週或一個月，例如一個月到一年，例如一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、九個月、十個月、十一個月或一年）投與。在一些實施方式中，第二劑量在藥物組成物之第一劑量後1天至180天（例如，1天到90天、1天到45天、1天到30天、1天到14天、1天到7天、2天到45天、2天到30天、2天到14天、2天到7天、3天到90天、3天到45天、3天到30天、3天到14天、3天到7天、4天到90天、4天到45天、4天到30天、4天到14天、4天到7天、5天到90天、5天到45天、5天到30天、5天到14天、5天到7天、6天到90天、6天到45天、6天到30天、6天到14天、6天到7天、7天到90天、7天到45天、7天到30天、7天到14天、14天到90天、14天到45天、14天到30天、21天到90天、21天到60天、21天到45天、21天到30天、30天到90天、30天到60天、30天到45天、45天到180天、45天到120天、45天到100天、45天到90天、45天到60天、60天到180天、60天到120天、60天到100天、60天到90天、90天到100天、90天到120天或90天至180天）投與。In some embodiments, the second dose is 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, Seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or a day, such as a day to a week, such as two days, three days, four days, five days, six days or a week, such as a week to a month, such as two weeks, three weeks or a month, such as a month to a year, e.g. One month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year) investment. In some embodiments, the second dose is 1 day to 180 days after the first dose of the pharmaceutical composition (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days or 90 days to 180 days) investment.

在一些實施方式中，第三劑量在藥物組成物之第二劑量後至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）投與。In some embodiments, the third dose is at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours) after the second dose of the pharmaceutical composition. hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months , eleven months, one year or more) investment.

在一些實施方式中，第三劑量在藥物組成物之第二劑量後1小時至1年（例如1小時到1天，例如一小時、兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時或一天，例如一天到一週，例如兩天、三天、四天、五天、六天或一週，例如一週到一個月，例如兩週、三週或一個月，例如一個月到一年，例如一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、九個月、十個月、十一個月或一年）投與。在一些實施方式中，第三劑量在藥物組成物之第二劑量後1天至180天（例如，1天到90天、1天到45天、1天到30天、1天到14天、1天到7天、2天到45天、2天到30天、2天到14天、2天到7天、3天到90天、3天到45天、3天到30天、3天到14天、3天到7天、4天到90天、4天到45天、4天到30天、4天到14天、4天到7天、5天到90天、5天到45天、5天到30天、5天到14天、5天到7天、6天到90天、6天到45天、6天到30天、6天到14天、6天到7天、7天到90天、7天到45天、7天到30天、7天到14天、14天到90天、14天到45天、14天到30天、21天到90天、21天到60天、21天到45天、21天到30天、30天到90天、30天到60天、30天到45天、45天到180天、45天到120天、45天到100天、45天到90天、45天到60天、60天到180天、60天到120天、60天到100天、60天到90天、90天到100天、90天到120天或90天至180天）投與。In some embodiments, the third dose is 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, Seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or a day, such as a day to a week, such as two days, three days, four days, five days, six days or a week, such as a week to a month, such as two weeks, three weeks or a month, such as a month to a year, e.g. One month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year) investment. In some embodiments, the third dose is 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days or 90 days to 180 days) investment.

在一些實施方式中，在該受試者之血清中該抗融合多肽之血清濃度小於約500 ng/mL之前投與該第二劑量。In some embodiments, the second dose is administered before the serum concentration of the anti-fusion polypeptide in the subject's serum is less than about 500 ng/mL.

在一些實施方式中，該方法在該受試者中維持至少500 ng/mL（例如，至少600 ng/mL、700 ng/mL、800 ng/mL、900 ng/mL、1,000 ng/mL、1,100 ng/mL、1,200 ng/mL、1,300 ng/mL、1,400 ng/mL、1,500 ng/mL、1,600 ng/mL、1,700 ng/mL、1,800 ng/mL、1,900 ng/mL、2,000 ng/mL、2,100 ng/mL、2,200 ng/mL、2,300 ng/mL、2,400 ng/mL、2,500 ng/mL、2,600 ng/mL、2,700 ng/mL、2,800 ng/mL、2,900 ng/mL、3,000 ng/mL或更多）的血清濃度的抗融合多肽，例如持續至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）。In some embodiments, the method maintains in the subject at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more multiple) serum concentrations of the anti-fusion polypeptide, e.g., for at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six Days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months, eleven months, a year or more).

在一些實施方式中，該方法治療或預防該受試者中的病毒感染。例如，該藥物組成物可以足以治療或預防病毒感染的量和持續時間投與給該受試者。可以將藥物組成物投與給受試者以降低病毒感染的風險。In some embodiments, the method treats or prevents viral infection in the subject. For example, the pharmaceutical composition can be administered to the subject in an amount and for a duration sufficient to treat or prevent viral infection. The pharmaceutical composition can be administered to a subject to reduce the risk of viral infection.

在一些實施方式中，該方法治療或預防人免疫缺乏病毒（HIV）感染。In some embodiments, the method treats or prevents human immunodeficiency virus (HIV) infection.

在一些實施方式中，該方法治療或預防冠狀病毒感染（例如乙型冠狀病毒（betacoronavirus）感染，例如SARS-CoV-2感染，如產生COVID-19症狀的SARS-CoV-2感染）。In some embodiments, the method treats or prevents a coronavirus infection (eg, a betacoronavirus infection, eg, a SARS-CoV-2 infection, such as a SARS-CoV-2 infection that produces symptoms of COVID-19).

在一些實施方式中，該方法治療或預防C型肝炎病毒（HCV）感染。In some embodiments, the method treats or prevents hepatitis C virus (HCV) infection.

在一些實施方式中，編碼抗融合多肽（例如，表1的多肽）的環狀多核苷酸用於減少病毒進入。In some embodiments, a circular polynucleotide encoding an anti-fusion polypeptide (eg, a polypeptide of Table 1) is used to reduce viral entry.

在另一方面，特徵在於環狀多核糖核苷酸，該環狀多核糖核苷酸包含編碼多個抗融合多肽的多核糖核苷酸貨物。該多核糖核苷酸貨物可以包含編碼抗融合多肽之表現序列。在一些實施方式中，該等抗融合多肽針對相同的病毒。可替代地，該等抗融合多肽可以針對多於一種病毒。定義In another aspect, a cyclic polyribonucleotide is characterized, the cyclic polyribonucleotide comprising a polyribonucleotide cargo encoding a plurality of anti-fusion polypeptides. The polyribonucleotide cargo may comprise an expression sequence encoding an anti-fusion polypeptide. In some embodiments, the anti-fusion polypeptides are directed against the same virus. Alternatively, the anti-fusion polypeptides may be directed against more than one virus.definition

為了促進對本揭露之理解，下面定義了多個術語。本文定義的術語具有如與本揭露相關的領域中的普通技術者通常理解的含義。術語如「一個/種（a、an）」和「該」並不旨在僅指單個實體，而係包括可以使用特定實例來說明的一般類別。術語「或」用於意指「和/或」，除非明確指出僅指替代物或者替代物相互排斥，儘管本揭露支持僅指替代物和「和/或」的定義。本文的術語用於描述特定實施方式，但它們的使用不應被視為限制，除非在申請專利範圍中列出。To facilitate understanding of the present disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by those of ordinary skill in the art relevant to this disclosure. Terms such as "a," "an" and "the" are not intended to refer only to a single entity, but rather include general categories that may be illustrated using specific examples. The term "or" is used to mean "and/or" unless it is expressly stated that only alternatives are intended or the alternatives are mutually exclusive, although this disclosure supports definitions that only alternatives and "and/or" are intended. The terms herein are used to describe particular embodiments, but their use should not be considered limiting unless set forth in the scope of the claim.

如本文所用，在值之範圍內提供的任何值都包括上限和下限、以及該上限和下限內含有的任何值。As used herein, any value provided as a range of values includes both upper and lower limits, and any values contained within such upper and lower limits.

如本文所用，術語「約」係指列舉值的 ± 10%內的值。As used herein, the term "about" means a value within ±10% of the recited value.

如本文所用，術語「載劑」係藉由對環狀多核糖核苷酸的共價修飾、經由部分或完全封裝劑或者它們的組合促進組成物（例如，環狀多核糖核苷酸）轉運或遞送到細胞中的化合物、組成物、試劑或分子。載劑之非限制性實例包括碳水化合物載劑（例如，酸酐改性的植物糖原或糖原型材料）、奈米顆粒（例如，封裝或共價連接/結合到環狀多核糖核苷酸的奈米顆粒）、脂質體、融合體、離體分化的網織紅血球、外泌體、蛋白質載劑（例如，共價連接到環狀多核糖核苷酸的蛋白質）或陽離子載劑（例如，陽離子脂質聚合物或轉染試劑）。As used herein, the term "carrier" facilitates the transport of a composition (e.g., a cyclic polyribonucleotide) by covalent modification of the cyclic polyribonucleotide, through a partial or complete encapsulant, or a combination thereof or compounds, compositions, agents or molecules delivered into cells. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified plant glycogen or glycogen-based materials), nanoparticles (e.g., encapsulated or covalently linked/conjugated to cyclic polyribonucleotides). nanoparticles), liposomes, fusions, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides) or cationic carriers (e.g., cationic lipid polymers or transfection reagents).

如本文所用，術語「環狀多核糖核苷酸」、「環狀RNA」和「circRNA」可互換使用，並且意指具有無游離端（即，無游離3'或5'端）的結構的多核糖核苷酸分子，例如通過共價或非共價鍵形成環狀或無端結構的多核糖核苷酸分子。環狀多核糖核苷酸可為例如共價閉合的多核糖核苷酸。As used herein, the terms "cyclic polyribonucleotide", "circRNA" and "circRNA" are used interchangeably and mean a structure having no free ends (i.e., no free 3' or 5' ends) Polyribonucleotide molecules, for example, polyribonucleotide molecules that form a cyclic or endless structure through covalent or non-covalent bonds. Cyclic polyribonucleotides may be, for example, covalently closed polyribonucleotides.

如本文所用，術語「環化效率」係所得環狀多核糖核苷酸相對於其非環狀起始材料的測量。As used herein, the term "cyclization efficiency" is a measurement of the resulting cyclic polyribonucleotide relative to its non-cyclic starting material.

術語「稀釋劑」意指包含非活性溶劑的媒介物，本文所述之組成物（例如，包含環狀多核糖核苷酸的組成物）可以稀釋或溶解在其中。稀釋劑可為RNA增溶劑、緩衝液、等滲劑或其混合物。稀釋劑可為液體稀釋劑或固體稀釋劑。液體稀釋劑之非限制性實例包括水或其他溶劑、增溶劑和乳化劑（諸如乙醇、異丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、二甲基甲醯胺、油類（尤其是棉籽油、花生油、玉米油、胚芽油、橄欖油、蓖麻油和芝麻油）、甘油、四氫糠醇、聚乙二醇和脫水山梨糖醇的脂肪酸酯以及1,3-丁二醇。固體稀釋劑之非限制性實例包括碳酸鈣、碳酸鈉、磷酸鈣、磷酸二鈣、硫酸鈣、磷酸氫鈣、磷酸鈉乳糖、蔗糖、纖維素、微晶纖維素、高嶺土、甘露糖醇、山梨糖醇、肌醇、氯化鈉、乾澱粉、玉米澱粉或糖粉。The term "diluent" means a vehicle containing an inactive solvent in which a composition described herein (eg, a composition comprising a cyclic polyribonucleotide) can be diluted or dissolved. The diluent can be an RNA solubilizer, a buffer, an isotonic agent, or a mixture thereof. The diluent can be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizers and emulsifiers (such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl of glycols, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan Fatty acid esters and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, Microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch or powdered sugar.

如本文所用，術語「疾病」、「病症」和「病狀」各自指亞健康狀態，例如，通常被或將會被醫療專業人員診斷或治療的狀態。As used herein, the terms "disease," "disorder," and "condition" each refer to a sub-health condition, such as a condition that is or will be typically diagnosed or treated by a medical professional.

如本文所用，術語「表現序列」係編碼產物例如肽或多肽（例如抗融合多肽）的核酸序列。編碼肽或多肽之示例性表現序列可以包括多個核苷酸三聯體，其中每一個都可以編碼胺基酸，並被稱為「密碼子」。As used herein, the term "expression sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide (eg, an anti-fusion polypeptide). Exemplary expression sequences encoding a peptide or polypeptide may include multiple nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon."

如本文所用，關於多肽或核酸序列（例如，抗融合多肽或編碼抗融合多肽的核酸序列）的術語「片段」係指多肽或核酸序列的連續的、少於全部的部分。例如，多肽或編碼多肽的核酸序列之片段，係指序列（如本文揭露的序列）之連續的、少於全部的部分（例如，整個長度的至少20%、30%、40%、50%、60%、70%、80%、90%、95%或99%）。應理解，本揭露之所有內容考慮了本文揭露的任何抗融合多肽的片段。As used herein, the term "fragment" with respect to a polypeptide or nucleic acid sequence (e.g., an anti-fusion polypeptide or a nucleic acid sequence encoding an anti-fusion polypeptide) refers to a contiguous, less than complete portion of the polypeptide or nucleic acid sequence. For example, a polypeptide or a fragment of a nucleic acid sequence encoding a polypeptide refers to a contiguous, less than the entire portion (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%). It is understood that all aspects of this disclosure contemplate fragments of any anti-fusion polypeptide disclosed herein.

如本文所用，術語「Fc結構域」係指包括至少鉸鏈結構域以及第二和第三抗體恒定結構域（C_H2和C_H3）或其功能片段（例如，能夠二聚化和結合Fc受體的片段）的多肽鏈。Fc結構域可為任何免疫球蛋白抗體同種型，包括IgG、IgE、IgM、IgA或IgD（例如IgG）。另外，Fc結構域可為IgG亞型（例如IgG1、IgG2a、IgG2b、IgG3或IgG4）（例如IgG1）。Fc結構域不包括免疫球蛋白中可作為抗原識別區的任何部分，例如可變結構域或互補決定區（CDR）。如本文所述之軛合物中的Fc結構域可以包含野生型Fc結構域序列中的改變Fc結構域和Fc受體之間相互作用的一或多個變化（例如，1-10、1-8、1-6、1-4個胺基酸取代、添加或缺失）。適合的變化的實例在本領域係已知的。除非本文另有說明，否則IgG或Fc結構域中胺基酸殘基的編號係根據抗體的EU編號系統，也稱為Kabat EU索引，如例如Kabat等人,Sequences of Proteins of Immunological Interest[具有免疫學意義的蛋白質序列], 第5版 公共衛生服務, 國家衛生研究所, 貝塞斯達, 馬里蘭州, 1991中所述As used herein, the term "Fc domain" is meant to include at least the hinge domain and the second and third antibody constant domains (_CH2 and_CH3 ) or functional fragments thereof (e.g., capable of dimerizing and binding Fc fragment of the receptor) polypeptide chain. The Fc domain can be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (eg, IgG). Additionally, the Fc domain may be of an IgG subtype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4) (eg, IgG1). The Fc domain does not include any part of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). The Fc domain in a conjugate as described herein may comprise one or more changes in the wild-type Fc domain sequence that alter the interaction between the Fc domain and the Fc receptor (e.g., 1-10, 1- 8. 1-6, 1-4 amino acid substitutions, additions or deletions). Examples of suitable variations are known in the art. Unless otherwise stated herein, the numbering of amino acid residues in an IgG or Fc domain is according to the EU numbering system for antibodies, also known as the Kabat EU index, as e.g. Kabat et al.,Sequences of Proteins of Immunological Interest [with Immunological Scientifically significant protein sequences], 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991

如本文所用，術語「GC含量」係指核酸序列中鳥嘌呤（G）和胞嘧啶（C）之百分比。用於計算GC含量的公式為 (G+C)/(A+G+C+U) × 100%（對於RNA）或 (G+C)/(A+G+C+T) × 100%（對於DNA）。同樣地，術語「尿苷含量」係指核酸序列中尿苷（U）之百分比。用於計算尿苷含量的公式為U/(A+G+C+U) × 100%。同樣地，術語「胸苷含量」係指核酸序列中胸苷（T）之百分比。用於計算胸苷含量的公式為T/(A+G+C+T) × 100%。As used herein, the term "GC content" refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula used to calculate GC content is (G+C)/(A+G+C+U) × 100% (for RNA) or (G+C)/(A+G+C+T) × 100% ( for DNA). Likewise, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula used to calculate uridine content is U/(A+G+C+U) × 100%. Likewise, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula used to calculate thymidine content is T/(A+G+C+T) × 100%.

「異源」意指發生在與天然存在的（天然的）背景不同的背景中。「異源」多核苷酸序列指示多核苷酸序列以在該序列的天然基因組中發現的方式不同的方式使用。例如，「異源啟動子」用於驅動序列的轉錄，該序列不是由該啟動子天然轉錄的序列；因此，「異源啟動子」序列通常藉由重組核酸技術包括在表現構建體中。術語「異源」也用於指被置於與另一序列的非天然存在的關係中的給定序列；例如，異源編碼或非編碼核苷酸序列通常藉由基因組轉化技術被插入基因組中，產生經基因修飾的基因組或重組基因組。"Xenologous" means occurring in a context different from that in which it occurs naturally (native). A "heterologous" polynucleotide sequence indicates that the polynucleotide sequence is used in a manner different from that found in the native genome of that sequence. For example, a "heterologous promoter" is used to drive the transcription of a sequence that is not naturally transcribed by the promoter; therefore, "heterologous promoter" sequences are often included in expression constructs by recombinant nucleic acid technology. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship to another sequence; for example, heterologous coding or non-coding nucleotide sequences are often inserted into the genome by genomic transformation techniques. , producing a genetically modified genome or a recombinant genome.

如本文所用，術語「內含子片段」係指內含子之部分，其中第一內含子片段和第二內含子片段一起形成內含子，如催化內含子。內含子片段可為內含子之5'部分（例如，催化內含子之5'部分）或內含子之3'部分（例如，催化內含子之3'部分），這樣5'內含子片段和3'內含子片段一起形成功能性內含子，如能夠催化自剪接的功能性內含子。術語內含子片段意指內含子分成兩個部分。術語內含子片段並非意在指出、暗示或表明這兩個部分或兩半長度相等。術語內含子片段與術語斷裂內含子同義使用，並且可以代替術語「半內含子」使用。As used herein, the term "intron segment" refers to the portion of an intron wherein a first intron segment and a second intron segment together form an intron, such as a catalytic intron. The intron fragment can be the 5' portion of the intron (e.g., the 5' portion of the catalytic intron) or the 3' portion of the intron (e.g., the 3' portion of the catalytic intron), such that the 5' The intron fragment and the 3' intron fragment together form a functional intron, such as a functional intron capable of catalyzing self-splicing. The term intron fragment means that the intron is divided into two parts. The term intronic segment is not intended to indicate, imply or indicate that the two parts or halves are of equal length. The term intron fragment is used synonymously with the term broken intron, and may be used instead of the term "half-intron".

如本文所用，術語「線性對應物」係具有與環狀多核糖核苷酸相同或相似的核苷酸序列（例如，100%、95%、90%、85%、80%、75%或之間的任何百分比的序列同一性）並且具有兩個游離端（即，環狀多核糖核苷酸的未環化形式（及其片段））的多核糖核苷酸分子（及其片段）。在一些實施方式中，線性對應物（例如，環化前形式）係與環狀多核糖核苷酸具有相同或相似的核苷酸序列（例如，100%、95%、90%、85%、80%、75%或其間的任何百分比序列同一性）並且具有相同或相似的核酸修飾的多核糖核苷酸分子（及其片段）並且具有兩個游離端（即，環狀多核糖核苷酸的未環化形式（及其片段））。在一些實施方式中，線性對應物係具有與環狀多核糖核苷酸相同或相似的核苷酸序列（例如，100%、95%、90%、85%、80%、75%或之間的任何百分比的序列同一性）和不同的核酸修飾或沒有核酸修飾並且具有兩個游離端（即，環狀多核糖核苷酸的未環化形式（及其片段））的多核糖核苷酸分子（及其片段）。在一些實施方式中，作為線性對應物的多核糖核苷酸分子之片段係線性對應物多核糖核苷酸分子的比該線性對應物多核糖核苷酸分子短的任何部分。在一些實施方式中，線性對應物進一步包括5'帽。在一些實施方式中，線性對應物進一步包括聚腺苷尾。在一些實施方式中，線性對應物進一步包括3' UTR。在一些實施方式中，線性對應物進一步包括5' UTR。As used herein, the term "linear counterpart" refers to a nucleotide sequence that is identical or similar to a cyclic polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or the like). A polyribonucleotide molecule (and fragments thereof) having any percent sequence identity between them) and having two free ends (i.e., an uncyclized form of a cyclic polyribonucleotide (and fragments thereof)). In some embodiments, the linear counterpart (e.g., pre-cyclized form) has the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) and have the same or similar nucleic acid modifications (and fragments thereof) and have two free ends (i.e., cyclic polyribonucleotides uncyclized form (and its fragments)). In some embodiments, the linear counterpart has the same or similar nucleotide sequence as the circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or between any percent sequence identity) and different nucleic acid modifications or polyribonucleotides without nucleic acid modifications and having two free ends (i.e., uncyclized forms of cyclic polyribonucleotides (and fragments thereof)) Molecules (and their fragments). In some embodiments, a fragment of a polyribonucleotide molecule that is a linear counterpart is any portion of a linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further includes a 5' cap. In some embodiments, the linear counterpart further includes a polyadenosine tail. In some embodiments, the linear counterpart further includes a 3' UTR. In some embodiments, the linear counterpart further includes a 5' UTR.

如本文所用，術語「線性RNA」、「線性多核糖核苷酸」和「線性多核糖核苷酸分子」可互換使用並且意指具有5'端和3'端的多核糖核苷酸分子。5'端和3'端中的一個或兩個可為游離端或者可以與另一部分連接。線性RNA包括尚未經歷環化（例如，環化前）的RNA，並且可以用作起始材料通過例如夾板連接或化學、酶促、核酶或剪接催化的環化方法進行環化。As used herein, the terms "linear RNA", "linear polyribonucleotide" and "linear polyribonucleotide molecule" are used interchangeably and mean a polyribonucleotide molecule having a 5' end and a 3' end. One or both of the 5' end and the 3' end may be free or may be connected to another moiety. Linear RNA includes RNA that has not undergone cyclization (eg, prior to cyclization) and can be used as a starting material for cyclization by, for example, splint ligation or chemical, enzymatic, ribozyme or splice-catalyzed cyclization methods.

如本文所用，術語「經修飾的核糖核苷酸」意指具有至少一個針對糖、核鹼基或核苷間鍵的修飾的核苷酸。As used herein, the term "modified ribonucleotide" means a nucleotide having at least one modification to a sugar, nucleobase, or internucleoside linkage.

如本文所用，術語「裸遞送」係用於遞送到細胞而無需載劑的幫助且無需對有助於遞送到細胞的部分的共價修飾的配製物。裸遞送配製物不含任何轉染試劑、陽離子載劑、碳水化合物載劑、奈米顆粒載劑或蛋白質載劑。例如，環狀多核糖核苷酸的裸遞送配製物係包括沒有共價修飾的環狀多核糖核苷酸並且不含載劑的配製物。As used herein, the term "naked delivery" refers to a formulation for delivery to a cell without the aid of a carrier and without covalent modification of moieties that facilitate delivery to the cell. Naked delivery formulations do not contain any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, naked delivery formulations of cyclic polyribonucleotides include formulations without covalently modified cyclic polyribonucleotides and without carriers.

如本文所用，術語「帶切口的RNA」和「帶切口的線性多核糖核苷酸」和「帶切口的線性多核糖核苷酸分子」可以互換使用並且意指由於環狀RNA切口或降解而產生的具有5'端和3'端的多核糖核苷酸分子。As used herein, the terms "nicked RNA" and "nicked linear polyribonucleotide" and "nicked linear polyribonucleotide molecule" are used interchangeably and mean that a cyclic RNA is formed due to nicking or degradation of a circular RNA. The resulting polyribonucleotide molecule has a 5' end and a 3' end.

術語「藥物組成物」旨在同樣揭露包括在藥物組成物中的環狀或線性多核糖核苷酸可用於藉由療法治療人體或動物體。因此，這意味著等同於「用於在療法中使用的多核糖核苷酸」。The term "pharmaceutical composition" is intended to also disclose that cyclic or linear polyribonucleotides included in pharmaceutical compositions can be used to treat the human or animal body by therapy. Therefore, this means the equivalent of "polyribonucleotides for use in therapy".

如本文所用，術語「多核苷酸」意指包括一或多個核酸亞基或核苷酸的分子，並且可以與「核酸」或「寡核苷酸」互換使用。多核苷酸可以包括一或多個選自腺苷（A）、胞嘧啶（C）、鳥嘌呤（G）、胸腺嘧啶（T）和尿嘧啶（U）或其變體的核苷酸。核苷酸可以包括核苷和至少1、2、3、4、5、6、7、8、9、10個或更多個磷酸（PO₃）基團。核苷酸可以包括核鹼基、五碳糖（核糖或去氧核糖）以及一或多個磷酸基團。核糖核苷酸係其中糖為核糖的核苷酸。多核糖核苷酸或核糖核酸或RNA可以指包括經由磷酸二酯鍵聚合的多個核糖核苷酸的大分子。去氧核糖核苷酸係其中糖係去氧核糖的核苷酸。如本文所用，敘述胸腺嘧啶（T）的多核糖核苷酸序列應被理解為表示尿嘧啶（U）。As used herein, the term "polynucleotide" means a molecule including one or more nucleic acid subunits or nucleotides, and may be used interchangeably with "nucleic acid" or "oligonucleotide." The polynucleotide may include one or more nucleotides selected from the group consisting of adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. Nucleotides may include nucleosides and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (_PO3 ) groups. Nucleotides may include nucleobases, a five-carbon sugar (ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotide or ribonucleic acid or RNA may refer to a macromolecule including multiple ribonucleotides polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. As used herein, a polyribonucleotide sequence reciting thymine (T) should be understood to mean uracil (U).

如本文所用，本文的術語「多核糖核苷酸貨物」包括包含至少一個多核糖核苷酸的任何序列。在實施方式中，多核糖核苷酸貨物包括一或多個表現序列，其中每個表現序列編碼多肽。在實施方式中，多核糖核苷酸貨物包括一或多個非編碼序列，如具有調控或催化功能的多核糖核苷酸。在實施方式中，多核糖核苷酸貨物包括表現序列和非編碼序列之組合。在實施方式中，多核糖核苷酸貨物包括本文所述之一或多個多核糖核苷酸序列，諸如一或多個調控元件、內部核糖體進入位點（IRES）元件或間隔子序列。As used herein, the term "polyribonucleotide cargo" herein includes any sequence comprising at least one polyribonucleotide. In embodiments, a polyribonucleotide cargo includes one or more expressed sequences, wherein each expressed sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo includes one or more non-coding sequences, such as polyribonucleotides with regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo includes a combination of expression sequences and non-coding sequences. In embodiments, the polyribonucleotide cargo includes one or more polyribonucleotide sequences described herein, such as one or more regulatory elements, internal ribosome entry site (IRES) elements, or spacer sequences.

如本文中可互換使用的，術語「聚A」和「聚A序列」係指至少5個核苷酸長並由腺苷殘基組成的核酸分子的非翻譯連續區域。在一些實施方式中，聚A序列之長度為至少10個、至少15個、至少20個、至少30個、至少40個或至少50個核苷酸。在一些實施方式中，聚A序列位於開讀框（例如編碼多肽的開讀框）的3'（例如下游），並且聚A序列位於終止元件（例如終止密碼子）的3'，使得聚A不被翻譯。在一些實施方式中，聚A序列位於終止元件的3'並且是3'非翻譯區。As used interchangeably herein, the terms "poly A" and "poly A sequence" refer to a non-translated contiguous region of a nucleic acid molecule that is at least 5 nucleotides long and consists of adenosine residues. In some embodiments, the polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, the polyA sequence is located 3' (e.g., downstream) of an open reading frame (e.g., the open reading frame encoding a polypeptide) and the polyA sequence is located 3' of a termination element (e.g., a stop codon) such that the polyA Not translated. In some embodiments, the polyA sequence is located 3' to the termination element and is the 3' untranslated region.

如本文所用，如果核酸的元件位於載體上，使得它們可以被轉錄而形成線性RNA，然後可以使用本文提供的方法將該線性RNA環化成環狀RNA，則該等元件係「可操作地相連的（operably connected）」或「可操作地連接的（operably linked）」。As used herein, nucleic acid elements are "operably linked" if they are located on a vector such that they can be transcribed to form linear RNA, which can then be circularized into circular RNA using the methods provided herein. (operably connected)" or "operably linked (operably linked)".

「多去氧核糖核苷酸」、「去氧核糖核酸」和「DNA」意指包括經由磷酸二酯鍵聚合的多個去氧核糖核苷酸的大分子。核苷酸可為一磷酸核苷或多磷酸核苷。核苷酸意指包括可檢測標籤（諸如發光標籤）或標誌物（例如，螢光團）的多磷酸去氧核糖核苷，例如三磷酸去氧核糖核苷（dNTP），其可以選自三磷酸去氧腺苷（dATP）、三磷酸去氧胞苷（dCTP）、三磷酸去氧鳥苷（dGTP）、三磷酸尿苷（dUTP）和三磷酸去氧胸苷（dTTP）dNTP。核苷酸可以包括可以摻入正在生長的核酸鏈中的任何亞基。此類亞基可為A、C、G、T或U，或對一或多個互補A、C、G、T或U有特異性或與嘌呤（即，A或G或其變體）或嘧啶（即C、T或U或其變體）互補的任何其他亞基。在一些實例中，多核苷酸係去氧核糖核酸（DNA）、核糖核酸（RNA）或其衍生物或變體。在一些情況下，多核苷酸舉幾例來說是短干擾RNA（siRNA）、微小RNA（miRNA）、質體DNA（pDNA）、短髮夾RNA（shRNA）、小核RNA（snRNA）、信使RNA（mRNA）、先質mRNA（前mRNA）、反義RNA（asRNA），並且涵蓋核苷酸序列及其任何結構實施方式，諸如單鏈、雙鏈、三鏈、螺旋、髮夾等。在一些情況下，多核苷酸分子為環狀的。多核苷酸可以具有各種長度。核酸分子可以具有至少約10個鹼基、20個鹼基、30個鹼基、40個鹼基、50個鹼基、100個鹼基、200個鹼基、300個鹼基、400個鹼基、500個鹼基、1千鹼基（kb）、2 kb、3 kb、4 kb、5 kb、10 kb、50 kb或更大的長度。可以從細胞或組織中分離多核苷酸。多核苷酸的實施方式包括分離和純化的DNA/RNA分子、合成的DNA/RNA分子以及合成的DNA/RNA類似物。"Polydeoxyribonucleotides," "deoxyribonucleic acid" and "DNA" mean macromolecules including multiple deoxyribonucleotides polymerized via phosphodiester bonds. Nucleotides may be nucleoside monophosphates or nucleoside polyphosphates. Nucleotide means a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP), including a detectable label (such as a luminescent label) or marker (eg, a fluorophore), which may be selected from three Deoxyadenosine phosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T or U, or specific for one or more complementary A, C, G, T or U or with a purine (i.e., A or G or a variant thereof) or Any other subunit complementary to a pyrimidine (i.e. C, T or U or a variant thereof). In some examples, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a derivative or variant thereof. In some cases, the polynucleotide is short interfering RNA (siRNA), microRNA (miRNA), plastid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger, to name a few RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and encompasses nucleotide sequences and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helix, hairpin, etc. In some cases, the polynucleotide molecules are circular. Polynucleotides can be of various lengths. Nucleic acid molecules can have at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases , 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb or greater in length. Polynucleotides can be isolated from cells or tissues. Embodiments of polynucleotides include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNA analogs.

多核苷酸（例如多核糖核苷酸或多去氧核糖核苷酸）的實施方式可以包括一或多種核苷酸變體，包括非標準核苷酸、非天然核苷酸、核苷酸類似物或經修飾的核苷酸。經修飾的核苷酸之實例包括但不限於二胺基嘌呤、5-氟尿嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-碘尿嘧啶、次黃嘌呤、黃嘌呤、4-乙醯胞嘧啶、5-(羧基羥基甲基)尿嘧啶、5-羧基甲基胺基甲基-2-硫代尿苷、5-羧基甲基胺基甲基尿嘧啶、二氫尿嘧啶、β-D-半乳糖基Q核苷、肌苷、N6-異戊烯基腺嘌呤、1-甲基鳥嘌呤、1-甲基肌苷、2,2-二甲基鳥嘌呤、2-甲基腺嘌呤、2-甲基鳥嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鳥嘌呤、5-甲基胺基甲基尿嘧啶、5-甲氧基胺基甲基-2-硫尿嘧啶、β-D-甘露糖基Q核苷、5'-甲氧基羧甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-D46-異戊烯基腺嘌呤、尿嘧啶-5-羥基乙酸、懷丁氧苷（wybutoxosine）、假尿嘧啶、Q核苷（queosine）、2-硫代胞嘧啶、5-甲基-2-硫尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-甲基尿嘧啶、尿嘧啶-5-羥基乙酸甲酯、尿嘧啶-5-羥基乙酸（v）、5-甲基-2-硫尿嘧啶、3-(3-胺基-3-N-2-羧基丙基)尿嘧啶、(acp3)w、2,6-二胺基嘌呤等。在一些情況下，核苷酸在其磷酸酯部分中可以包括修飾，包括對三磷酸酯部分的修飾。此類修飾之非限制性實例包括長度更長的磷酸酯鏈（例如，具有4、5、6、7、8、9、10個或更多個磷酸酯部分的磷酸酯鏈）和帶有硫醇部分（例如，α-硫代三磷酸酯和β-硫代三磷酸酯）的修飾。在實施方式中，核酸分子在鹼基部分（例如，在通常可用於與互補核苷酸形成氫鍵的一或多個原子處或者在通常不能與互補核苷酸形成氫鍵的一或多個原子處）、糖部分或磷酸酯主鏈處被修飾。在實施方式中，核酸分子含有胺修飾的基團，諸如胺基烯丙基1-dUTP（aa-dUTP）和胺基己基丙烯醯胺-dCTP（aha-dCTP），以允許共價附接胺反應性部分，諸如N-羥基琥珀醯亞胺酯（NHS）。本揭露之寡核苷酸中標準DNA鹼基對或RNA鹼基對的替代物可以提供更高的密度（以位/立方毫米計）、更高的安全性（抗天然毒素的偶然或有目的合成）、更容易辨別光程式性聚合酶（photo-programmed polymerases）或較低的二級結構。與用於從頭或擴增合成的天然和突變聚合酶相容的此類替代性鹼基對在Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. [自然化學生物學] 2012年7月;8(7):612-4中描述，其藉由援引併入本文用於所有目的。Embodiments of polynucleotides (eg, polyribonucleotides or polydeoxyribonucleotides) may include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs substances or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl Cytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β- D-galactosyl Q nucleoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenosine Purine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy Aminomethyl-2-thiouracil, β-D-mannosyl Q nucleoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46 -Isopentenyl adenine, uracil-5-glycolic acid, wybutoxosine, pseudouracil, Q nucleoside (queosine), 2-thiocytosine, 5-methyl-2-sulfide Uracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-glycolic acid methyl ester, uracil-5-glycolic acid (v), 5-methyl-2- Thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, etc. In some cases, a nucleotide may include modifications in its phosphate moiety, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and phosphate chains with sulfur Modification of the alcohol moiety (e.g., alpha-thiotriphosphate and beta-thiotriphosphate). In embodiments, the nucleic acid molecule has a base moiety (e.g., at one or more atoms that would normally be available to form hydrogen bonds with a complementary nucleotide or at one or more atoms that would normally be unable to form a hydrogen bond with a complementary nucleotide). atoms), sugar moieties, or phosphate backbones are modified. In embodiments, the nucleic acid molecules contain amine-modified groups, such as aminoallyl 1-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP), to allow for covalent attachment of amines Reactive moieties such as N-hydroxysuccinimide ester (NHS). Substitutions of standard DNA base pairs or RNA base pairs in oligonucleotides of the present disclosure can provide higher density (in bits/cubic millimeter), higher safety (against accidental or purposeful exposure to natural toxins) synthesis), easier identification of photo-programmed polymerases or lower secondary structure. Such alternative base pairs compatible with native and mutant polymerases for de novo or amplification synthesis are presented in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which is incorporated herein by reference for all purposes.

如本文所用，「多肽」意指最常藉由肽鍵連接在一起的胺基酸殘基（天然或非天然的）的聚合物。如本文所用，該術語係指任何大小、結構或功能的蛋白質、多肽和肽。多肽可以包括基因產物、天然存在的多肽、合成多肽、同系物、直系同源物、同種同源物、前述物質的片段和其他等同物、變體和類似物。多肽可為單分子或多分子複合物，如二聚體、三聚體或四聚體。它們還可以包括單鏈或多鏈多肽（如抗體或胰島素），並且可為締合的或連接的。最常見的二硫鍵存在於多鏈多肽中。術語多肽也可以適用於胺基酸聚合物，其中一或多個胺基酸殘基係對應的天然存在的胺基酸的人工化學類似物。As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) linked together most often by peptide bonds. As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologs, homologs, fragments of the foregoing and other equivalents, variants and analogs. Polypeptides can be single molecules or multi-molecular complexes, such as dimers, trimers or tetramers. They may also include single- or multi-chain polypeptides (such as antibodies or insulin), and may be associated or linked. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

如本文所用，術語「預防」意指降低發展疾病、障礙或病症（例如，病毒感染，例如HIV、SARS-CoV-2、HCV、流感或RSV）的可能性，或可替代地，降低隨後發展的疾病或障礙的症狀之嚴重程度或頻率。可以將治療劑投與於相對於一般群體成員而言發展病毒感染的風險增加的受試者，以便預防疾病或病症的發展或減輕疾病或病症的嚴重程度。可以例如在病毒感染的任何症狀或表現發展之前將治療劑作為預防劑投與。As used herein, the term "prevention" means reducing the likelihood of developing a disease, disorder or condition (e.g., a viral infection such as HIV, SARS-CoV-2, HCV, influenza, or RSV), or alternatively, reducing the subsequent development The severity or frequency of symptoms of a disease or disorder. The therapeutic agent may be administered to a subject who is at increased risk of developing a viral infection relative to members of the general population in order to prevent the development of a disease or condition or to reduce the severity of a disease or condition. The therapeutic agent may be administered as a prophylactic agent, for example, before any symptoms or manifestations of viral infection develop.

如本文所用，術語「調控元件」係修飾環狀或線性多核糖核苷酸內表現序列的表現的部分，諸如核酸序列。As used herein, the term "regulatory element" refers to a portion of a circular or linear polyribonucleotide that modifies the expression of a sequence, such as a nucleic acid sequence.

如本文所用，「間隔子」係指在兩個相鄰多核苷酸區域之間提供距離或柔性的任何連續核苷酸序列（例如，一或多個核苷酸的連續核苷酸序列）。As used herein, a "spacer" refers to any contiguous nucleotide sequence (eg, a contiguous sequence of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions.

「訊息序列」係指長度例如介於10至45個胺基酸之間的多肽序列，其存在於新生蛋白的多肽序列的N末端，將多肽序列靶向分泌途徑。"Message sequence" refers to a polypeptide sequence with a length of, for example, between 10 and 45 amino acids, which is present at the N-terminus of the polypeptide sequence of a nascent protein and targets the polypeptide sequence to the secretory pathway.

如本文所用，術語「序列同一性」係藉由使用全域或局部比對演算法對兩個肽或兩個核苷酸序列進行比對來確定的。當序列在最佳比對時（例如，當藉由程式（如GAP或BESTFIT）使用默認參數進行比對時）共用至少某個最小百分比的序列同一性時，該等序列被稱為「基本上相同的」或「基本上相似的」。GAP使用Needleman和Wunsch全域比對演算法在兩個序列的整個長度上對其進行比對，從而最大程度地增加了匹配數目並最大程度地減少了空位數目。通常，使用GAP默認參數，空位產生罰分 = 50（核苷酸）/8（蛋白質），空位延伸罰分 = 3（核苷酸）/2（蛋白質）。對於核苷酸，使用的默認得分矩陣係nwsgapdna，而對於蛋白質，默認得分矩陣係Blosum62（Henikoff和Henikoff, 1992, PNAS [美國科學院院報] 89, 915-919）。序列比對和百分比序列同一性的得分例如使用電腦程式來確定，該等電腦程式如從美國92121-3752加州聖地牙哥斯克蘭頓路9685號的阿賽樂德公司（Accelrys Inc., 9685 Scranton Road, San Diego, CA）獲得的GCG Wisconsin套裝軟體10.3版或EmbossWin 2.10.0版（使用程式「needle」）。可替代地或另外，藉由例如使用如FASTA、BLAST等的演算法對數據庫進行搜索來確定同一性百分比。序列同一性係指在序列的整個長度上的序列同一性。As used herein, the term "sequence identity" is determined by aligning two peptides or two nucleotide sequences using global or local alignment algorithms. Sequences are said to be "substantially" identical when they share at least some minimum percentage of sequence identity when optimally aligned (for example, when aligned by a program such as GAP or BESTFIT using default parameters). "Identical" or "substantially similar". GAP aligns two sequences over their entire length using the Needleman and Wunsch global alignment algorithms, thereby maximizing the number of matches and minimizing the number of gaps. Typically, using GAP default parameters, gap creation penalty = 50 (nucleotides)/8 (protein) and gap extension penalty = 3 (nucleotides)/2 (protein). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS [Proceedings of the National Academy of Sciences] 89, 915-919). Sequence alignments and percent sequence identity scores are determined, for example, using computer programs such as Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA. Road, San Diego, CA) GCG Wisconsin software suite version 10.3 or EmbossWin version 2.10.0 (using the program "needle"). Alternatively or additionally, the percent identity is determined by searching the database, for example using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

如本文所用，術語「受試者」係指生物，如動物、植物或微生物。在實施方式中，受試者係脊椎動物（例如，哺乳動物、鳥、魚、爬行動物或兩棲動物）。在實施方式中，受試者係人。在實施方式中，受試者係非人哺乳動物。在實施方式中，受試者係非人哺乳動物，諸如非人靈長類動物（例如猴、猿）、有蹄類動物（例如家牛、水牛、野牛、綿羊、山羊、豬、駱駝、美洲駝、羊駝、鹿、馬、驢）、肉食動物（例如狗、貓）、齧齒動物（例如大鼠、小鼠）或兔類動物（例如兔）。在實施方式中，受試者係鳥，諸如禽類分類群雞形目（例如雞、火雞、野雞、鵪鶉）、雁形目（例如鴨、鵝）、古顎下綱（例如鴕鳥、鴯鶓）、鴿形目（例如鴿子、野鴿）或鸚形目（例如鸚鵡）的成員。在實施方式中，該受試者係無脊椎動物，如節肢動物（例如，昆蟲、蛛形綱、甲殼動物）、線蟲、環節動物、蠕蟲、或軟體動物。在實施方式中，受試者係無脊椎動物農業有害生物或者寄生在無脊椎動物或脊椎動物宿主上的無脊椎動物。在實施方式中，受試者係植物，諸如被子植物（其可為雙子葉植物或單子葉植物）或裸子植物（例如，針葉樹、蘇鐵、買麻藤類植物、銀杏）、蕨類、馬尾植物、石鬆類或苔蘚植物。在實施方式中，受試者係真核藻類（單細胞或多細胞）。在實施方式中，受試者係具有農業或園藝重要性的植物，諸如行間作物、生產水果的植物和樹木、蔬菜、樹木以及觀賞植物（包括觀賞花、灌木、樹木、地被植物和草坪草）。As used herein, the term "subject" refers to a living organism, such as an animal, plant, or microorganism. In embodiments, the subject is a vertebrate (eg, mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, American Camels, alpacas, deer, horses, donkeys), carnivores (e.g. dogs, cats), rodents (e.g. rats, mice) or lagomorphs (e.g. rabbits). In embodiments, the subject is a bird, such as the avian taxa Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathia (e.g., ostrich, emu) , a member of the order Columbiformes (e.g. pigeons, wild pigeons) or the order Psittaciformes (e.g. parrots). In embodiments, the subject is an invertebrate, such as an arthropod (eg, insect, arachnid, crustacean), nematode, annelid, worm, or mollusk. In embodiments, the subject is an invertebrate agricultural pest or an invertebrate that is parasitic on an invertebrate or vertebrate host. In embodiments, the subject is a plant, such as an angiosperm (which may be a dicot or a monocot) or a gymnosperm (eg, conifers, cycads, cycads, ginkgo), ferns, horsetails , lycophytes or bryophytes. In embodiments, the subject is a eukaryotic algae (unicellular or multicellular). In embodiments, the subjects are plants of agricultural or horticultural importance, such as row crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, ground covers, and turfgrasses. ).

如本文所用，術語「抗融合多肽」係指抑制病毒融合相關事件（如病毒進入或病毒融合）的多肽，如10個和200個胺基酸之間的多肽。抗融合多肽包括例如表1的多肽。抗融合多肽包括多肽以及其任何生物活性片段（例如，至少10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500個胺基酸的片段）。抗融合多肽包括，例如，靶向HIV、SARS-CoV-2、HCV或RSV的多肽。在一些實施方式中，抗融合多肽包括與SEQ ID NO: 1-324中任一個具有至少70%，例如至少80%，例如至少85%（例如，至少90%、95%、97%、99%或100%）的序列同一性的多肽。抗融合多肽也指編碼抗融合多肽（例如，表1的多肽）的多核苷酸（例如多核糖核苷酸，例如環狀多核糖核苷酸）或其生物活性片段。As used herein, the term "anti-fusion polypeptide" refers to a polypeptide, such as a polypeptide between 10 and 200 amino acids, that inhibits viral fusion-related events (such as viral entry or viral fusion). Anti-fusion polypeptides include, for example, the polypeptides of Table 1. Anti-fusion polypeptides include polypeptides and any biologically active fragment thereof (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 fragments of amino acids). Anti-fusion polypeptides include, for example, polypeptides targeting HIV, SARS-CoV-2, HCV, or RSV. In some embodiments, anti-fusion polypeptides include at least 70%, e.g., at least 80%, e.g., at least 85% (e.g., at least 90%, 95%, 97%, 99%) identical to any one of SEQ ID NOs: 1-324 or 100%) sequence identity. Anti-fusion polypeptide also refers to a polynucleotide (eg, a polyribonucleotide, such as a cyclic polyribonucleotide) encoding an anti-fusion polypeptide (eg, the polypeptide of Table 1) or a biologically active fragment thereof.

如本文所用，術語「治療（「treat」和「treating」）」係指對受試者中的病毒感染（例如HIV、SARS-CoV-2、HCV、流感或RSV）進行的預防性或治療性治療。治療的效果可以包括逆轉、減輕、降低疾病或者病毒感染的一或多種症狀或表現的嚴重程度，治癒疾病或者病毒感染的一或多種症狀或表現，抑制疾病或者病毒感染的一或多種症狀或表現的進展，降低疾病或者病毒感染的一或多種症狀或表現的復發的可能性，或者與不存在治療性治療的情況下病毒感染的狀態或狀況相比，穩定（即，不惡化）病毒感染的狀態或者預防病毒感染的擴散。As used herein, the terms "treat" and "treating" refer to the prophylactic or therapeutic treatment of a viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV) in a subject treatment. The effects of treatment may include reversing, alleviating, reducing the severity of one or more symptoms or manifestations of a disease or viral infection, curing one or more symptoms or manifestations of a disease or viral infection, inhibiting one or more symptoms or manifestations of a disease or viral infection progress, reduce the likelihood of recurrence of the disease or one or more symptoms or manifestations of the viral infection, or stabilize (i.e., do not worsen) the viral infection compared to the status or condition of the viral infection in the absence of therapeutic treatment status or prevent the spread of viral infection.

如本文所用，術語「終止元件」係終止環狀或線性多核糖核苷酸中表現序列的翻譯的部分，諸如核酸序列。As used herein, the term "termination element" refers to the portion of a circular or linear polyribonucleotide that terminates translation of a sequence expressed, such as a nucleic acid sequence.

如本文所用，術語「翻譯效率」係從核糖核苷酸轉錄物產生蛋白質或肽的速率或量。在一些實施方式中，翻譯效率可以表示為給定量的編碼蛋白或肽的轉錄物產生的蛋白或肽的量，例如在給定的時間段內，例如在給定的翻譯系統（例如，無細胞的翻譯系統，像兔網織紅血球裂解物）中。As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide produced from a ribonucleotide transcript. In some embodiments, translation efficiency may be expressed as the amount of protein or peptide produced by a given amount of protein or peptide-encoding transcript, e.g., over a given period of time, e.g., in a given translation system (e.g., cell-free translation system, like rabbit reticulocyte lysate).

如本文所用，術語「翻譯起始序列」係在環狀或線性多核糖核苷酸中起始表現序列的翻譯的核酸序列。As used herein, the term "translation initiation sequence" refers to a nucleic acid sequence that initiates translation of an expression sequence in a circular or linear polyribonucleotide.

如本文所用，「載體」意指一段DNA，它係合成的（例如，使用PCR）或者取自病毒、質體或高等生物體的細胞，可以或已經將外源DNA片段插入其中以用於選殖或表現目的。在一些實施方式中，載體可以穩定地維持在生物中。可選擇標誌物載體可以包括例如複製起點、可選擇標誌物或報告基因（諸如抗生素抗性或GFP）或多選殖位點（MCS）。該術語包括線性DNA片段（例如，PCR產物、線性化質體片段）、質體載體、病毒載體、黏粒、細菌人工染色體（BAC）、酵母人工染色體（YAC）等。在一個實施方式中，本文提供的載體包括多選殖位點（MCS）。在另一實施方式中，本文提供的載體不包括MCS。As used herein, "vector" means a stretch of DNA, either synthesized (e.g., using PCR) or obtained from a virus, plasmid, or cell of a higher organism, into which a foreign DNA segment can be or has been inserted for selection. reproduction or performance purposes. In some embodiments, the vector can be stably maintained in the organism. Selectable marker vectors may include, for example, origins of replication, selectable markers or reporter genes (such as antibiotic resistance or GFP) or multiple selection sites (MCS). The term includes linear DNA fragments (e.g., PCR products, linearized plastid fragments), plastid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc. In one embodiment, vectors provided herein include a multiple selection site (MCS). In another embodiment, the vectors provided herein do not include MCS.

本發明之特徵在於含有編碼抗融合多肽的環狀多核糖核苷酸（環狀RNA）之組成物及其使用方法。本文所述之環狀多核糖核苷酸特別可用於將編碼抗融合多肽的多核苷酸貨物遞送到靶細胞。The present invention is characterized by compositions containing cyclic polyribonucleotides (circular RNA) encoding anti-fusion polypeptides and methods of using them. The cyclic polyribonucleotides described herein are particularly useful for delivering polynucleotide cargo encoding anti-fusion polypeptides to target cells.

環狀多核糖核苷酸可以包括編碼表1的多肽之多核糖核苷酸貨物。在一些實施方式中，多核糖核苷酸貨物包括編碼與表1的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 1-324中任一個具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。Cyclic polyribonucleotides may include polyribonucleotide cargos encoding the polypeptides of Table 1. In some embodiments, the polyribonucleotide cargo includes an expressed sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a polypeptide of Table 1 . In some embodiments, the polyribonucleotide cargo includes a polyribonucleotide encoding a sequence that is at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) identical to any one of SEQ ID NOs: 1-324. The expression sequence of a sexual polypeptide.

環狀多核糖核苷酸可以由先質產生，如線性去氧核糖核苷酸、環狀去氧核糖核苷酸或環狀多核糖核苷酸。Cyclic polyribonucleotides can be produced from precursors such as linear deoxyribonucleotides, cyclic deoxyribonucleotides or cyclic polyribonucleotides.

環狀多核糖核苷酸可包含，例如，連接5'外顯子片段和3'外顯子片段之剪接點。The cyclic polyribonucleotide may comprise, for example, a splice junction connecting the 5' exon fragment and the 3' exon fragment.

本文所述之線性RNA分子係編碼抗融合多肽的多核糖核苷酸。在一些實施方式中，線性RNA分子從5'到3'包括 (A) 3'催化內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 編碼抗融合多肽的多核糖核苷酸貨物（例如，編碼與編碼抗融合多肽的表現序列可操作連接的IRES的多核糖核苷酸貨物）；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'催化內含子片段。然後，催化內含子片段和剪接位點可以允許線性多核糖核苷酸自剪接，從而形成編碼抗融合多肽的環狀多核糖核苷酸。The linear RNA molecules described herein are polyribonucleotides encoding anti-fusion polypeptides. In some embodiments, the linear RNA molecule includes from 5' to 3' (A) a 3' catalytic intron segment; (B) a 3' splice site; (C) a 3' exon segment; (D) coding Polyribonucleotide cargo of an anti-fusion polypeptide (e.g., a polyribonucleotide cargo encoding an IRES operably linked to an expression sequence encoding an anti-fusion polypeptide); (E) 5' exon fragment; (F) 5 ' splice site; and (G) 5' catalytic intron fragment. The catalytic intron fragment and splice site can then allow the linear polyribonucleotide to self-splice, thereby forming a cyclic polyribonucleotide encoding an anti-fusion polypeptide.

特徵還在於使用如本文所述之環狀多核糖核苷酸之方法。例如，環狀多核糖核苷酸可以配製成用於投與給受試者（例如人受試者）的組成物（例如藥物組成物）。可以以一或多個劑量的組成物來投與藥物組成物。可將該組成物投與給受試者以治療或預防病毒感染（例如，HIV、SARS-CoV-2、HCV、流感或RSV）。多核苷酸Also characterized are methods of using cyclic polyribonucleotides as described herein. For example, the cyclic polyribonucleotide can be formulated into a composition (eg, a pharmaceutical composition) for administration to a subject (eg, a human subject). The pharmaceutical composition may be administered in one or more doses of the composition. The composition can be administered to a subject to treat or prevent viral infection (eg, HIV, SARS-CoV-2, HCV, influenza, or RSV).polynucleotide

本揭露之特徵在於編碼抗融合多肽的環狀多核糖核苷酸組成物、其用途以及製備編碼抗融合多肽的環狀多核糖核苷酸之方法。在一些實施方式中，環狀多核糖核苷酸由線性多核糖核苷酸產生（例如，藉由自剪接線性多核糖核苷酸的相容端）。在一些實施方式中，線性多核糖核苷酸從去氧核糖核苷酸模板（例如，載體、線性化載體或cDNA）轉錄。因此，本揭露之特徵在於可用於產生編碼抗融合多肽的環狀多核糖核苷酸的去氧核糖核苷酸、線性多核糖核苷酸和環狀多核糖核苷酸及其組成物。模板去氧核糖核苷酸The present disclosure features compositions of cyclic polyribonucleotides encoding anti-fusion polypeptides, uses thereof, and methods of preparing cyclic polyribonucleotides encoding anti-fusion polypeptides. In some embodiments, cyclic polyribonucleotides are generated from linear polyribonucleotides (eg, by self-splicing of compatible ends of the linear polyribonucleotides). In some embodiments, linear polyribonucleotides are transcribed from a deoxyribonucleotide template (eg, vector, linearized vector, or cDNA). Accordingly, the present disclosure features deoxyribonucleotides, linear polyribonucleotides, and cyclic polyribonucleotides and compositions thereof that can be used to generate cyclic polyribonucleotides encoding anti-fusion polypeptides.Template deoxyribonucleotide

本發明之特徵在於一種用於製備如本文所述之環狀RNA的模板去氧核糖核苷酸。在實施方式中，該去氧核糖核苷酸包含以5'至3'取向可操作地連接的以下項：(A) 3'催化內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 編碼抗融合多肽的多核糖核苷酸貨物；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'催化內含子片段。在實施方式中，該去氧核糖核苷酸包含另外的元件，例如在元件 (A)、(B)、(C)、(D)、(E)、(F) 或 (G) 中的任一個之外或之間。在實施方式中，元件 (A)、(B)、(C)、(D)、(E)、(F) 或 (G) 中的任一個被如本文所述之間隔子序列彼此隔開。The invention features a template deoxyribonucleotide for use in preparing circular RNA as described herein. In embodiments, the deoxyribonucleotide comprises the following operably linked in a 5' to 3' orientation: (A) a 3' catalytic intron fragment; (B) a 3' splice site; (C) ) 3' exon fragment; (D) polyribonucleotide cargo encoding the anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' catalytic endonuclease Intron fragment. In embodiments, the deoxyribonucleotide comprises additional elements, such as in any of elements (A), (B), (C), (D), (E), (F) or (G) outside or between one. In embodiments, any of elements (A), (B), (C), (D), (E), (F) or (G) are separated from each other by a spacer sequence as described herein.

在實施方式中，該去氧核糖核苷酸係例如環狀DNA載體、線性化DNA載體、或線性DNA（例如，cDNA，例如從DNA載體產生）。In embodiments, the deoxyribonucleotide is, for example, a circular DNA vector, a linearized DNA vector, or linear DNA (eg, cDNA, eg, generated from a DNA vector).

在一些實施方式中，該去氧核糖核苷酸進一步包括與編碼本文所述之線性RNA的序列可操作地連接的RNA聚合酶啟動子。在實施方式中，該RNA聚合酶啟動子與編碼該線性RNA的序列係異源的。在一些實施方式中，該RNA聚合酶啟動子係T7啟動子、T6啟動子、T4啟動子、T3啟動子、SP6病毒啟動子、或SP3啟動子。In some embodiments, the deoxyribonucleotide further includes an RNA polymerase promoter operably linked to a sequence encoding a linear RNA described herein. In embodiments, the RNA polymerase promoter is heterologous to the sequence encoding the linear RNA. In some embodiments, the RNA polymerase promoter is a T7 promoter, T6 promoter, T4 promoter, T3 promoter, SP6 viral promoter, or SP3 promoter.

在一些實施方式中，該去氧核糖核苷酸包含多選殖位點（MCS）。In some embodiments, the deoxyribonucleotide comprises a multiple selection site (MCS).

在一些實施方式中，使用該去氧核糖核苷酸產生大小範圍為約100至約20,000個核苷酸的環狀RNA。在一些實施方式中，該環狀RNA之大小係至少100、500、550、600、650、700、750、800、850、900、950、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、3,000、3,500、4,000、4,500、或5,000個核苷酸。在一些實施方式中，該環狀RNA之大小不多於20,000、15,000、10,000、9,000、8,000、7,000、6,000、5,000或4,000個核苷酸。線性多核糖核苷酸In some embodiments, the deoxyribonucleotides are used to generate circular RNAs ranging in size from about 100 to about 20,000 nucleotides. In some embodiments, the size of the circular RNA is at least 100, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides. In some embodiments, the circular RNA is no more than 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, or 4,000 nucleotides in size.linear polyribonucleotide

本發明之特徵還在於編碼抗融合多肽的線性多核糖核苷酸。線性多核糖核苷酸可用於產生環狀多核糖核苷酸，例如，藉由連接或剪接（例如，自剪接）線性多核糖核苷酸以產生環狀多核糖核苷酸。在實施方式中，該線性多核糖核苷酸包含以5'至3'取向可操作地連接的以下項：(A) 3'催化內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 編碼抗融合多肽的多核糖核苷酸貨物；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'催化內含子片段。在實施方式中，該線性多核糖核苷酸包含另外的元件，例如在元件 (A)、(B)、(C)、(D)、(E)、(F) 或 (G) 中的任一個之外或之間。例如，元件 (A)、(B)、(C)、(D)、(E)、(F) 或 (G) 中的任一個可被如本文所述之間隔子序列隔開。The invention also features linear polyribonucleotides encoding anti-fusion polypeptides. Linear polyribonucleotides can be used to generate cyclic polyribonucleotides, for example, by joining or splicing (eg, self-splicing) linear polyribonucleotides to generate cyclic polyribonucleotides. In embodiments, the linear polyribonucleotide comprises the following operably linked in a 5' to 3' orientation: (A) a 3' catalytic intron fragment; (B) a 3' splice site; (C ) 3' exon fragment; (D) polyribonucleotide cargo encoding the anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' catalytic endonuclease Intron fragment. In embodiments, the linear polyribonucleotide comprises additional elements, such as in any of elements (A), (B), (C), (D), (E), (F) or (G) outside or between one. For example, any of elements (A), (B), (C), (D), (E), (F) or (G) may be separated by a spacer sequence as described herein.

在某些實施方式中，本文提供了一種藉由使用本文提供的編碼抗融合多肽的去氧核糖核苷酸（例如，載體、線性化載體或cDNA）作為模板（例如，本文提供的載體、線性化載體或cDNA，它們具有位於編碼線性RNA的區域上游的RNA聚合酶啟動子）在無細胞系統中進行轉錄（例如，體外轉錄）來生成編碼抗融合多肽的線性RNA之方法。In certain embodiments, provided herein is a method by using a deoxyribonucleotide (e.g., a vector, linearized vector, or cDNA) encoding an anti-fusion polypeptide provided herein as a template (e.g., a vector, linearized vector provided herein) vector or cDNA that has an RNA polymerase promoter located upstream of the region encoding the linear RNA) is transcribed in a cell-free system (e.g., in vitro transcription) to generate a linear RNA encoding an anti-fusion polypeptide.

在實施方式中，該去氧核糖核苷酸模板被轉錄以產生含有本文所述之組分的線性RNA。在表現時，該線性多核糖核苷酸產生剪接相容的多核糖核苷酸，該剪接相容的多核糖核苷酸可自剪接以便產生環狀多核糖核苷酸。In embodiments, the deoxyribonucleotide template is transcribed to produce linear RNA containing components described herein. When expressed, the linear polyribonucleotide produces a splice-compatible polyribonucleotide that can self-splice to produce a cyclic polyribonucleotide.

在一些實施方式中，該線性多核糖核苷酸之長度為50至20,000個、100至20,000個、200至20,000個、300至20,000個（例如50、100、200、300、400、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、3,000、3,500、4,000、5,000、6,000、7,000、8,000、9,000、10,000、11,000、12,000、13,000、14,000、15,000、16,000、17,000、18,000、19,000或20,000個）核糖核苷酸。在實施方式中，該線性多核糖核苷酸之長度為例如至少500個、至少1,000個、至少2,000個、至少3,000個、至少4,000個或至少5,000個核糖核苷酸。環狀多核糖核苷酸In some embodiments, the length of the linear polyribonucleotide is 50 to 20,000, 100 to 20,000, 200 to 20,000, 300 to 20,000 (e.g., 50, 100, 200, 300, 400, 500, 600 , 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8, 000, 9,000, 10,000, 11,000 , 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000 or 20,000) ribonucleotides. In embodiments, the linear polyribonucleotide is, for example, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotides in length.cyclic polyribonucleotide

在一些實施方式中，本發明之特徵在於包含編碼抗融合多肽的表現序列之環狀多核糖核苷酸。在實施方式中，多核糖核苷酸包含與編碼抗融合多肽的表現序列可操作地連接的IRES。環狀多核糖核苷酸可以包括，例如，連接5'外顯子片段和3'外顯子片段之剪接點。環狀多核糖核苷酸可以包括本文所述之任一或多種元件。在一些實施方式中，環狀多核糖核苷酸包括如藉由援引以其全文併入本文的國際專利揭露號WO 2019/118919中揭露的任何特徵或特徵的任何組合。In some embodiments, the invention features a cyclic polyribonucleotide comprising an expression sequence encoding an anti-fusion polypeptide. In embodiments, the polyribonucleotide comprises an IRES operably linked to an expression sequence encoding an anti-fusion polypeptide. The cyclic polyribonucleotide may include, for example, a splice junction connecting the 5' exon fragment and the 3' exon fragment. Cyclic polyribonucleotides may include any one or more elements described herein. In some embodiments, cyclic polyribonucleotides include any feature or any combination of features as disclosed in International Patent Publication No. WO 2019/118919, which is incorporated herein by reference in its entirety.

在實施方式中，該環狀多核苷酸進一步包括多核糖核苷酸貨物。在實施方式中，多核糖核苷酸貨物包括表現（或編碼）序列、非編碼序列或表現（編碼）序列和非編碼序列的組合。在實施方式中，多核糖核苷酸貨物包括編碼多肽的表現（編碼）序列。在實施方式中，多核糖核苷酸包含與編碼多肽的表現序列可操作地連接的IRES。在一些實施方式中，IRES位於表現序列的上游。在一些實施方式中，IRES位於表現序列的下游。在一些實施方式中，環狀多核糖核苷酸進一步包括IRES與3'外顯子片段或5'外顯子片段之間的間隔子區域。間隔子區域的長度可以為例如至少5個（例如至少10個、至少15個、至少20個）核糖核苷酸。間隔子區域可以為例如5個至500個（例如，10個、20個、30個、40個、50個、60個、70個、80個、90個、100個、150個、200個、250個、300個、350個、400個、450個或500個）核糖核苷酸。在一些實施方式中，間隔子區域包括聚A序列。在一些實施方式中，間隔子區域包括聚A-C序列。在一些實施方式中，間隔子區域包括聚A-G序列。在一些實施方式中，間隔子區域包括聚A-T序列。在一些實施方式中，間隔子區域包括隨機序列。在一些實施方式中，第一退火區域和第二退火區域連接，從而形成環狀多核糖核苷酸。In embodiments, the cyclic polynucleotide further comprises a polyribonucleotide cargo. In embodiments, the polyribonucleotide cargo includes expression (or coding) sequences, non-coding sequences, or a combination of expression (coding) sequences and non-coding sequences. In embodiments, the polyribonucleotide cargo includes an expression (coding) sequence encoding a polypeptide. In embodiments, a polyribonucleotide comprises an IRES operably linked to an expression sequence encoding a polypeptide. In some embodiments, the IRES is located upstream of the expressed sequence. In some embodiments, the IRES is located downstream of the expressed sequence. In some embodiments, the cyclic polyribonucleotide further includes a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. The spacer region may be, for example, at least 5 (eg, at least 10, at least 15, at least 20) ribonucleotides in length. The spacer sub-regions may be, for example, 5 to 500 (eg, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500) ribonucleotides. In some embodiments, the spacer region includes polyA sequences. In some embodiments, the spacer region includes poly A-C sequences. In some embodiments, the spacer region includes polyA-G sequences. In some embodiments, the spacer region includes polyA-T sequences. In some embodiments, the spacer sub-region includes a random sequence. In some embodiments, the first annealed region and the second annealed region are connected to form a cyclic polyribonucleotide.

在一些實施方式中，該環狀RNA由本文所述之去氧核糖核苷酸模板或線性RNA產生。在一些實施方式中，該環狀RNA藉由本文所述方法中的任一種產生。In some embodiments, the circular RNA is generated from a deoxyribonucleotide template or linear RNA described herein. In some embodiments, the circular RNA is produced by any of the methods described herein.

在一些實施方式中，環狀多核糖核苷酸為至少約20個核苷酸、至少約30個核苷酸、至少約40個核苷酸、至少約50個核苷酸、至少約75個核苷酸、至少約100個核苷酸、至少約200個核苷酸、至少約300個核苷酸、至少約400個核苷酸、至少約500個核苷酸、至少約1,000個核苷酸、至少約2,000個核苷酸、至少約5,000個核苷酸、至少約6,000個核苷酸、至少約7,000個核苷酸、至少約8,000個核苷酸、至少約9,000個核苷酸、至少約10,000個核苷酸、至少約12,000個核苷酸、至少約14,000個核苷酸、至少約15,000個核苷酸、至少約16,000個核苷酸、至少約17,000個核苷酸、至少約18,000個核苷酸、至少約19,000個核苷酸或至少約20,000個核苷酸。In some embodiments, the cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides Nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleosides acid, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, At least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

在一些實施方式中，環狀多核糖核苷酸為500個核苷酸至20,000個核苷酸、1,000個核苷酸至20,000個核苷酸、2,000個核苷酸至20,000個核苷酸、或5,000個核苷酸至20,000個核苷酸。在一些實施方式中，環狀多核糖核苷酸為500個核苷酸至10,000個核苷酸、1,000個核苷酸至10,000個核苷酸、2,000個核苷酸至10,000個核苷酸、或5,000個核苷酸至10,000個核苷酸。In some embodiments, the cyclic polyribonucleotide is 500 to 20,000 nucleotides, 1,000 to 20,000 nucleotides, 2,000 to 20,000 nucleotides, or 5,000 nucleotides to 20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide is 500 to 10,000 nucleotides, 1,000 to 10,000 nucleotides, 2,000 to 10,000 nucleotides, or 5,000 nucleotides to 10,000 nucleotides.

作為其環化的結果，環狀多核糖核苷酸可能包含某些使其差異於線性RNA的特徵。例如，與線性RNA相比，環狀多核糖核苷酸不易被核酸外切酶降解。這樣，環狀多核糖核苷酸比線性RNA更穩定，尤其是在核酸外切酶存在下孵育時。環狀多核糖核苷酸與線性RNA相比的增加的穩定性使得環狀多核糖核苷酸作為產生多肽的細胞轉化試劑更加有用，並且與線性RNA相比，存儲更容易且時間更長。可以使用本領域標準的方法測試用核酸外切酶處理的環狀多核糖核苷酸的穩定性，該方法確定是否已經發生RNA降解（例如，藉由凝膠電泳）。此外，與線性RNA不同，當環狀多核糖核苷酸與磷酸酶（如小牛腸磷酸酶）一起孵育時，環狀多核糖核苷酸較不易去磷酸化。As a result of their cyclization, cyclic polyribonucleotides may contain certain features that distinguish them from linear RNA. For example, compared with linear RNA, cyclic polyribonucleotides are less susceptible to degradation by exonucleases. In this way, circular polyribonucleotides are more stable than linear RNA, especially when incubated in the presence of exonucleases. The increased stability of cyclic polyribonucleotides compared to linear RNA makes cyclic polyribonucleotides more useful as cell transformation reagents to produce polypeptides, and can be stored more easily and for longer periods of time than linear RNA. The stability of cyclic polyribonucleotides treated with exonucleases can be tested using methods standard in the art, which determine whether RNA degradation has occurred (eg, by gel electrophoresis). Furthermore, unlike linear RNA, cyclic polyribonucleotides are less susceptible to dephosphorylation when incubated with phosphatases such as calf intestinal phosphatase.

本文所述之環狀多核糖核苷酸及其組成物或藥物組成物可用於治療和獸醫給藥方法，以在向多個細胞提供至少兩劑環狀多核糖核苷酸後，在所述多個細胞中產生一定水平的環狀多核糖核苷酸、一定水平的與靶的結合、或一定水平的蛋白質。在一些實施方式中，環狀多核糖核苷酸在哺乳動物例如人中係非免疫性的。在一些實施方式中，環狀多核糖核苷酸能夠在來自水產養殖動物（魚、蟹、蝦、牡蠣等）的細胞、哺乳動物細胞（例如，來自寵物或動物園動物（貓、狗、蜥蜴、鳥、獅子、老虎和熊等）的細胞、來自農場或役用動物（馬、牛、豬、雞等）的細胞、人細胞）、培養的細胞、原代細胞或細胞系、幹細胞、先驅細胞、分化細胞、生殖細胞、癌細胞（例如，致瘤的、轉移的）、非致瘤細胞（正常細胞）、胎兒細胞、胚胎細胞、成年細胞、有絲分裂細胞、非有絲分裂細胞、或其任何組合中複製或者在其中複製。在一些實施方式中，本發明包括包含本文所述之環狀多核糖核苷酸的細胞，其中該細胞係來自水產養殖動物（魚、蟹、蝦、牡蠣等）的細胞、哺乳動物細胞（例如，來自寵物或動物園動物（貓、狗、蜥蜴、鳥、獅子、老虎及熊等）的細胞、來自農場或役用動物（馬、牛、豬、雞等）的細胞、人細胞、培養的細胞、原代細胞或細胞系、幹細胞、先驅細胞、分化細胞、生殖細胞、癌細胞（例如，致瘤的、轉移的）、非致瘤細胞（正常細胞）、胎兒細胞、胚胎細胞、成年細胞、有絲分裂細胞、非有絲分裂細胞、或其任何組合。在一些實施方式中，細胞被修飾以包含環狀多核糖核苷酸。The cyclic polyribonucleotides and compositions or pharmaceutical compositions thereof described herein can be used in therapeutic and veterinary administration methods to provide at least two doses of cyclic polyribonucleotides to a plurality of cells, in which A certain level of cyclic polyribonucleotide, a certain level of binding to a target, or a certain level of protein is produced in multiple cells. In some embodiments, cyclic polyribonucleotides are non-immune in mammals, such as humans. In some embodiments, the cyclic polyribonucleotide can be expressed in cells from aquaculture animals (fish, crab, shrimp, oysters, etc.), mammalian cells (e.g., from pet or zoo animals (cats, dogs, lizards, etc.) cells from birds, lions, tigers and bears, etc.), cells from farm or draft animals (horses, cows, pigs, chickens, etc.), human cells), cultured cells, primary cells or cell lines, stem cells, pioneer cells , differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof Copy or copy in it. In some embodiments, the invention includes cells comprising a cyclic polyribonucleotide described herein, wherein the cell line is derived from cells of an aquaculture animal (fish, crab, shrimp, oyster, etc.), mammalian cells (e.g., , cells from pet or zoo animals (cats, dogs, lizards, birds, lions, tigers, bears, etc.), cells from farm or draft animals (horses, cows, pigs, chickens, etc.), human cells, cultured cells , primary cells or cell lines, stem cells, pioneer cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (normal cells), fetal cells, embryonic cells, adult cells, Mitotic cells, non-mitotic cells, or any combination thereof. In some embodiments, cells are modified to contain cyclic polyribonucleotides.

在一些實施方式中，環狀多核糖核苷酸包含表現產物之序列。在一些實施方式中，環狀多核糖核苷酸包含用於與靶結合的結合位點。在一些實施方式中，經由本文所述之任何給藥方案，向多個細胞提供了環狀多核糖核苷酸。在一些實施方式中，如本文所述之環狀多核糖核苷酸在受試者中誘導響應或響應水平。在一些實施方式中，由包含在環狀多核糖核苷酸中的序列編碼的表現產物在所述多個細胞中的一或多個細胞中表現。In some embodiments, the cyclic polyribonucleotide comprises a sequence expressing a product. In some embodiments, a cyclic polyribonucleotide contains a binding site for binding to a target. In some embodiments, cyclic polyribonucleotides are provided to a plurality of cells via any of the dosing regimens described herein. In some embodiments, a cyclic polyribonucleotide as described herein induces a response or a level of response in a subject. In some embodiments, an expression product encoded by a sequence contained in a cyclic polyribonucleotide is expressed in one or more of the plurality of cells.

在一些實施方式中，環狀多核糖核苷酸具有的半衰期至少係線性對應物（例如，線性表現序列或線性多核糖核苷酸）的半衰期。在一些實施方式中，環狀多核糖核苷酸具有的半衰期相對於線性對應物的半衰期延長。在一些實施方式中，半衰期增加了約5%、10%、15%、20%、25%、30%、35%、40%、45%、50%或更多。在一些實施方式中，環狀多核糖核苷酸在細胞中的半衰期或持續性係至少約1小時，例如至少2小時、3小時、4小時、5小時、6小時、12小時、24小時、1天、2天、3天、4天、5天、6天、7天、2週、3週、4週、2個月、3個月、6個月或更長時間。在一些實施方式中，環狀多核糖核苷酸在細胞中的半衰期或持續性係約1小時至約60天，例如約1小時、2小時、6小時、12小時、18小時、24小時、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、30天、35天、40天、45天、50天、55天或60天。在一些實施方式中，環狀多核糖核苷酸在正進行細胞分裂的細胞中具有半衰期或持續性。在一些實施方式中，環狀多核糖核苷酸在分裂後的細胞中具有半衰期或持續性。在某些實施方式中，環狀多核糖核苷酸在正進行分裂的細胞中的半衰期或持久性係至少約10分鐘，例如至少約1小時，例如至少2小時、3小時、4小時、5小時6小時、12小時、24小時、1天、2天、3天、4天、5天、6天、7天、2週、3週、4週、2個月、3個月、6個月或更長時間。在某些實施方式中，環狀多核糖核苷酸在正進行分裂的細胞中的半衰期或持續性係約10分鐘至約60天，例如約1小時、2小時、3小時、4小時、5小時、6小時、7小時、8小時、9小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、24小時、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、30天或60天。In some embodiments, a cyclic polyribonucleotide has a half-life that is at least the half-life of its linear counterpart (eg, a linearly expressed sequence or a linear polyribonucleotide). In some embodiments, cyclic polyribonucleotides have a half-life that is extended relative to the half-life of their linear counterparts. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the half-life or persistence of the cyclic polyribonucleotide in the cell is at least about 1 hour, such as at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months or more. In some embodiments, the half-life or persistence of the cyclic polyribonucleotide in the cell is from about 1 hour to about 60 days, such as about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days , 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days or 60 days. In some embodiments, cyclic polyribonucleotides have a half-life or persistence in cells undergoing cell division. In some embodiments, cyclic polyribonucleotides have a half-life or persistence in post-dividing cells. In certain embodiments, the half-life or persistence of the cyclic polyribonucleotide in dividing cells is at least about 10 minutes, such as at least about 1 hour, such as at least 2 hours, 3 hours, 4 hours, 5 hours Hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months or more. In certain embodiments, the half-life or persistence of the cyclic polyribonucleotide in dividing cells is from about 10 minutes to about 60 days, such as about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days , 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 60 days.

在一些實施方式中，環狀多核糖核苷酸例如暫態地或長期地調節細胞功能。在某些實施方式中，細胞功能發生穩定改變，如持續存在以下時間的調節：至少約10分鐘，例如至少約1小時，例如至少2小時、3小時、4小時、5小時、6小時、12小時、24小時、1天、2天、3天、4天、5天、6天、7天、2週、3週、4週、2個月、3個月、6個月或更長時間。在某些實施方式中，細胞功能發生穩定改變，例如持續存在以下時間的調節：約1小時至約60天，例如約1小時至約30天，例如至少約2小時、6小時、12小時、18小時、24小時、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、30天或60天。多核苷酸的元件In some embodiments, cyclic polyribonucleotides modulate cellular function, eg, transiently or chronically. In certain embodiments, a stable change in cellular function occurs, such as a modulation that persists for at least about 10 minutes, such as at least about 1 hour, such as at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months or more . In certain embodiments, a stable change in cellular function occurs, such as a modulation that persists for a period of time from about 1 hour to about 60 days, such as from about 1 hour to about 30 days, such as at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days , 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 60 days.polynucleotide elements

本文所述之多核苷酸（例如，環狀多核糖核苷酸）可以包括本文所述之任一或多種元件和編碼抗融合多肽之表現序列。抗融合多肽Polynucleotides (eg, cyclic polyribonucleotides) described herein may include any one or more elements described herein and expression sequences encoding anti-fusion polypeptides.anti-fusion peptide

本揭露提供了編碼至少一個表現序列的環狀多核糖核苷酸，該至少一個表現序列編碼抗融合多肽。在一些實施方式中，抗融合多肽抑制病毒進入。在一些實施方式中，抗融合多肽抑制病毒融合。The present disclosure provides cyclic polyribonucleotides encoding at least one expressed sequence encoding an anti-fusion polypeptide. In some embodiments, anti-fusion polypeptides inhibit viral entry. In some embodiments, anti-fusion polypeptides inhibit viral fusion.

在一些實施方式中，抗融合多肽係包括選自表1的序列之胺基酸序列的多肽或其變體。在一些實施方式中，抗融合多肽係包括表1中序列的至少15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100個胺基酸之連續段的多肽。在一些實施方式中，抗融合多肽係包括表1中序列的至少30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%或95%胺基酸之連續段的多肽。在一些實施方式中，抗融合多肽係包括與表1的序列具有至少50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%序列同一性的序列之多肽。在一些實施方式中，抗融合多肽係表1中序列的變體，其包括不多於一個、兩個、三個、四個、五個、六個、七個、八個、九個或十個突變（例如，取代、缺失或插入）。在一些實施方式中，環狀多核糖核苷酸包含編碼多於一個抗融合多肽之表現序列。在一些實施方式中，編碼多於一個融合蛋白的環狀多核糖核苷酸減少了病毒逃逸的機會。In some embodiments, the anti-fusion polypeptide system includes a polypeptide with an amino acid sequence selected from the sequences of Table 1, or a variant thereof. In some embodiments, the anti-fusion polypeptide system includes at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of the sequences in Table 1 Or a polypeptide of a continuous stretch of 100 amino acids. In some embodiments, the anti-fusion polypeptide system includes at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, of the sequences in Table 1. A polypeptide that is a contiguous stretch of 85%, 90% or 95% amino acids. In some embodiments, anti-fusion polypeptides include sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence of Table 1 Sequence identity of a polypeptide. In some embodiments, the anti-fusion polypeptide is a variant of the sequence in Table 1, which includes no more than one, two, three, four, five, six, seven, eight, nine or ten mutations (e.g., substitutions, deletions, or insertions). In some embodiments, cyclic polyribonucleotides comprise expression sequences encoding more than one anti-fusion polypeptide. In some embodiments, cyclic polyribonucleotides encoding more than one fusion protein reduce the chance of viral escape.

在一些實施方式中，抗融合多肽靶向一或多種（例如，兩種、三種、四種或五種）病毒。抗融合多肽可能靶向的病毒包括但不限於表1中列出的所有病毒毒株。在一些實施方式中，該病毒不感染人。在一些實施方式中，該病毒感染人。 [表1].示例性抗融合多肽來源肽靶標胺基酸序列SEQ ID NO:金平菇，金頂側耳（Pleurotus citrinopileauts）抗病毒蛋白Y3煙草花葉病毒（TMV）、流感血凝素（HA）、人免疫缺乏病毒（HIV）假病毒體AACARFIDDFCDTLTPNIYRPRDNGQRCYAVNGHRCDFTVFNTNNGGNPIRASTPNCKTVLRTAANRCPTGGRGKINPNAPFLFAIDPNDGDCSTNF1達摩池蛙（Daruma pond frog），短腳赤蛙（Rana brevipoda porsa）Brevinin-1單純皰疹病毒-1（HSV-1），單純皰疹病毒-2（HSV-2）FLPVLAGIAAKVVPALFCKITKKC2歐洲普通蛙，林蛙（Rana temporaria）Temporin BCCV, FV3LLPIVGNLLKSLL3歐洲普通蛙，林蛙（Rana temporaria）Temporin G甲型流感病毒（IAV），呼吸道副流感病毒（PIV）FFPVIGRILNGIL4北美蛙，豹蛙（Rana pipiens），以及俄勒岡斑點蛙，斑點赤蛙（Rana pretiosa）Ranateurin 3PHIVGLMDTVKNVAKNLAGHMLDKLKCKITGC5人，智人（Homo sapiens）人嗜中性球肽-1（HNP-1）表現SARS-CoV-2刺突蛋白的假型病毒ACYCRIPACIAGERRYGTCIYQGRLWAFCC6人，智人HNP-2表現SARS-CoV-2刺突蛋白的假型病毒CYCRIPACIAGERRYGTCIYQGRLWAFCC7人，智人HNP-3表現SARS-CoV-2刺突蛋白的假型病毒DCYCRIPACIAGERRYGTCIYQGRLWAFCC8人，智人HNP-4表現SARS-CoV-2刺突蛋白的假型病毒VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV9人，智人人防禦肽（HD-5）BK病毒（BKV）ATCYCRTGRCATRESLSGVCEISGRLYRLCCR10人，智人HD-6呼吸道合胞病毒（RSV），人PIV（HPIV），HIV，HSV，甲型流感病毒（IAV），BKV，人腺病毒（HAdV），人乳頭瘤病毒（HPV）AFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL11兔，穴兔（Oryctolagus cuniculus）兔嗜中性球肽1（NP-1）HSV，包膜病毒VVCACRRALCLPRERRAGFCRIRGRIHPLCCRR12兔，穴兔（Oryctolagus cuniculus）兔嗜中性球防禦肽2（NP-2）RSV, HIV, HSVVVCACRRALCLPLERRAGFCRIRGRIHPLCCRR13豬嗜中性球，野豬（Sus scrofa）Protegrin 1 (PG-1)HIV，HSV-1，HSV-2，豬繁殖與呼吸綜合症病毒（PRRSV）RGGRLCYCRRRFCVCVGR14大西洋馬蹄蟹，美洲鱟（Limulus polyphemus）鱟肽（Polyphemusin）IHIVRRWCFRVCYRGFCYRKCR15大西洋馬蹄蟹，美洲鱟（Limulus polyphemus）鱟肽IIHIVRRWCFRVCYKGFCYRKCR16東南亞血細胞，中華鱟（Tachypleus tridentatus），南方鱟（Tachypleus gigas），圓尾鱟（Carcinoscorpius rotundicauda）鱟素肽（鱟素肽）I（TP1）HIV，HSV-1，HSV-2，新加坡石斑魚虹彩病毒（SGIV）KWCFRVCYRGICYRRCR17兔，穴兔（Oryctolagus cuniculus）兔嗜中性球防禦肽3a（NP-3a）RSK, HIV, HSVGICACRRRFCPNSERFSGYCRVNGARYVRCCSRR18豬嗜中性球，野豬（Sus scrofa）PG-2偽狂犬病病毒（PRV），PRRSVRGGRLCYCRRRFCICV19豬嗜中性球，野豬（Sus scrofa）PG-3PRRSVRGGGLCYCRRRFCVCVGR20豬嗜中性球，野豬（Sus scrofa）PG-4PRRSVRGGRLCYCRGWICFCVGR21大鼠，褐鼠（Rattus novegicus）大鼠NP-1，大鼠防禦肽HIV, HSVVTCYCRRTRCGFRERLSGACGYRGRIYRLCCR22大鼠，褐鼠（Rattus novegicus）大鼠NP-2HIV, HSVVTCYCRSTRCGFRERLSGACGYRGRIYRLCCR23大鼠，褐鼠（Rattus novegicus）大鼠NP-3HIV, HSVCSCRTSSCRFGERLSGACRLNGRIYRLCC24大鼠，褐鼠（Rattus novegicus）大鼠NP-4HIV, HSVACYCRIGACVSGERLTGACGLNGRIYRLCCR25澳大利亞綠樹蛙，華麗雨濱蛙（Litoria splendida），羅氏雨濱蛙（Litoria rothii）Caerin 1.1HIV，鼠白血病病毒GLLSVLGSVAKHVLPHVVPVIAEHL26澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.2HIVGLLGVLGSVAKHVLPHVVPVIAEHL27澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.3HIVGLLSVLGSVAQHVLPHVVPVIAEHL28澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.4HIVGLLSSLSSVAKHVLPHVVPVIAEHL29澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.5HIVGLLSVLGSVVKHVIPHVVPVIAEHL30澳洲橙腿蛙，黃雨濱蛙（Litoria xanthomera）Caerin 1.6HIVGLFSVLGAVAKHVLPHVVPVIAEK31澳洲橙腿蛙，黃雨濱蛙（Litoria xanthomera）Caerin 1.7HIVGLFKVLGSVAKHLLPHVAPVIAEK32澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.9HIVGLFGVLGSIAKHVLPHVVPVIAEKL-NH233澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.10HIVGLLSVLGSVAKHVLPHVVOVIAEKL-NH₂34澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.19HIVGLFKVLGSVAKHLLPHVAPIIAEKL-NH₂35澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 1.20HIVGLFGILGSVAKHVLPHVIPVVAEHL-NH236澳大利亞綠樹蛙，綠雨濱蛙（Litoria caerula）Caerin 4.1HIVGLWQKIKSAAGDLASGIVEGIKS37小鼠，小家鼠（Mus musculus）鼠β-防禦肽1（mBD-1）HIV, IAVDQYKCLQHGGFCLRSSCPSNTKLQGTCKPDKPNCCKS38小鼠，小家鼠（Mus musculus）小鼠cathelin相關抗菌肽（mCRAMP）RSVGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ39人，智人人β防禦肽3（hBD-3）HIVGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK40南方褐樹蛙，棕雨濱蛙（Litoria ewingii）Ewingiin 2.1（原名Caeridin 7.1）HIVGLLDMVTGLLGNL-NH241澳大利亞樹蛙，綠眼雨濱蛙（Litoria genimaculata）Maculatin 1.1HIV, PRVGLFGVLAKVAAHVVPAIAEHF-NH242雜種條紋鱸，條紋狼鱸（Morone saxatilis），金眼狼鱸（Morone chrysops）毒魚豆素（Piscidin）1H（Pis-1H）HIVFFHHIFRGIVHVGKTIHRLVTG43雜種條紋鱸，條紋狼鱸（Morone saxatilis），金眼狼鱸（Morone chrysops）毒魚豆素1N（Pis-1N）HIVFFHHIFRGIVHVGKTIHRLVTG44雜種條紋鱸，條紋狼鱸（Morone saxatilis），金眼狼鱸（Morone chrysops）毒魚豆素2（Pis-2）HIVFFHHIFRGIVHVGKTIHKLVTG45雜種條紋鱸，條紋狼鱸（Morone saxatilis），金眼狼鱸（Morone chrysops）毒魚豆素3（Pis-3）HIVFIHHIFRGIVHAGRSIGRFLTG46紫貽貝（Blue mussel/Mytilus edulis）Mytilin B病毒性出血性敗血症病毒（VHSV）VHSV，IPNVSCASRCKGHCRARRCGYYVSVLYRGRCYCKCLRC47地中海貽貝（Mediterranean mussel/Mytilus galloprovincialis）Mytilin CVHSV, IPNVSCASRCKSRCRARRCRYYVSVRYGWFCYCRCLRC48培菌白蟻，偽刺白蟻（Pseudocanthotermes spiniger）SpinigerinHIVHVDKKVADKVLLLKQLRIMRLLTRL49北美蛙，牛蛙（Rana catasbeiana）Ranateurin 6HIVFISAIASMLGKFL50北美蛙，牛蛙（Rana catasbeiana）Ranateurin 9HIVFLFPKITSFLSKVL51恒河猴，獼猴（Macaca mulatta）恒河猴θ防禦肽-1（RTD-1）HIVGFCRCLCRRGVCRCICTR52人，智人人β-防禦肽1（hBD-1）HIV, HSVDHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK53人，智人hBD-2HIV, HSVGIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP54鏈黴菌屬（Streptomyces）菌株AA3891Siamycin IIHIVCLGIGSCNDFAGCGYAIVCFW55北美貂蛙，貂蛙（Rana septentrionalis）Brevinin-2相關肽HIVGIWDTIKSMGKVFAGKILQNL56豚鼠（Guinea pig/Cavia porcellus）CAP11IAVGLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI57恒河猴，獼猴（Macaca mulatta）RTD-2HIVGVCRCLCRRGVCRCLCRR58恒河猴，獼猴（Macaca mulatta）RTD-3HIVGFCRCICTRGFCRCICTR59非洲爪蛙，非洲爪蟾（Xenopus laevis）蛙皮素（Magainin）1HSV-1, HSV-2GIGKFLHSAGKFGKAFVGEIMKS60非洲草藥，近緣白花蛇舌草（Oldenlandia affinis）Kalata B1 (KB1)HIVGLPVCGETCVGGTCNTPGCTCSWPVCTRN61雞，家雞（Gallus gallus domestic），鴨，綠頭鴨（Anas platyrhynchos）雞禽類β防禦肽6（AvBD-6）IAVSPIHACRYQRGVCIPGPCRWPYYRVGSCGSGLKSCCVRNRWA62雞，家雞AvBD-5IAVGLPQDCERRGGFCSHKSCPPGIGRIGLCSKEDFCCRSRWYS63中國紅腹蟾蜍，大蹼鈴蟾（Bombina maxima）Maximin H1尼帕病毒（NiV）ILGPVISTIGGVLGGLLKNL64撒哈拉蛙，撒哈拉側褶蛙（Pelophylax saharicus）Temporin-ShaHSVFLSGIVGMLGKLF65傘花堅果堇（Leonia cymosa）Cycloviolin AHIVGVIPCGESCVFIPCISAAIGCSCKNKVCYRN66傘花堅果堇（Leonia cymosa）Cycloviolin BHIVGTACGESCYVLPCFTVGCTCTSSQCFKN67傘花堅果堇（Leonia cymosa）Cycloviolin CHIVGIPCGESCVFIPCLTTVAGCSCKNKVCYRN68傘花堅果堇（Leonia cymosa）Cycloviolin DHIVGFPCGESCVFIPCISAAIGCSCKNKVCYRN69歐洲田堇菜，野生堇菜（Viola arvensis）Varv肽E（Varv E）HIVGLPICGETCVGGTCNTPGCSCSWPVCTRN70非洲草藥，近緣白花蛇舌草（Oldenlandia affinis）KB8HIVGSVLNCGETCLLGTCYTTGCTCNKYRVCTKD71Palicourea condensataPalicoureinHIVGDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN72戟葉紫羅蘭，戟葉堇菜（Viola betonicifolia）KB2HIVGLPVCGETCFGGTCNTPGCSCTWPICTRD73澳大利亞紫羅蘭，腎葉堇菜（Viola hederacea）Vhl-1HIVSISCGESCAMISFCFTEVIGCSCKNKVCYLN74熱帶樹（Tropical tree），小葉彎管花（Chassalia parvifola）環狀桿菌素CHIVGIPCGESCVFIPCITSVAGCSCKSKVCYRN75熱帶樹（Tropical tree），小葉彎管花（Chassalia parvifola）環狀桿菌素DHIVKIPCGESCVWIPCVTSIFNCKCKENKVCYHD76熱帶樹（Tropical tree），小葉彎管花（Chassalia parvifola）環狀桿菌素EHIVKIPCGESCVWIPCLTSVFNCKCENKVCYHD77熱帶樹（Tropical tree），小葉彎管花（Chassalia parvifola）環狀桿菌素FHIVKVCYRAIPCGESCVWIPCISAAIGCSCKN78羅非魚，莫三比克口孵非鯽（Oreochromis mossambicus）鐵調素1-5（TH1-5）神經壞死病毒（NNV）GIKCRFCCGCCTPGICGVCCRF79甜紫羅蘭，香堇菜（Viola odorata）環紫菌素（Cycloviolacin）O13HIVGIPCGESCVWIPCISAAIGCSCKSKVCYRN80甜紫羅蘭，香堇菜（Viola odorata）環紫菌素（Cycloviolacin）O14HIVGSIPACGESCFKGKCYTPGCSCSKYPLCAKN81甜紫羅蘭，香堇菜（Viola odorata）環紫菌素（Cycloviolacin）O24HIVGLPTCGETCFGGTCNTPGCTCDPWPVCTHN82中草藥，紫花地丁（Viola yedoensis）環紫菌素（Cycloviolacin）Y1HIVGGTIFDCGETCFLGTCYTPGCSCGNYGFCYGTN83中草藥，紫花地丁（Viola yedoensis）環紫菌素（Cycloviolacin）Y4HIVGVPCGESCVFIPCITGVIGCSCSSNVCYLN84中草藥，紫花地丁（Viola yedoensis）環紫菌素（Cycloviolacin）Y5HIVGIPCAESCVWIPCTVTALVGCSCSDKVCYN85中草藥，紫花地丁（Viola yedoensis）環紫菌素（Cycloviolacin）VY1IAVCGESCVFIPCITTVLGCSCSIKVCYKNGSIP86鏈黴菌屬菌株AA6532Siamycin IHIVCLGVGSCNDFAGCGYAVVCFW87鏈黴菌屬菌株AA6532NP-06HIVCLGVGSCNDFAGCGYAIVCFW88人，智人Retrocylin-1 (RC1)HIVGICRCICGRGICRCICGR89人，智人RC2HIVGICRCICGRRICRCICGR90人，智人RC3HIVRICRCICGRRICRCICGR91紅海龜，赤蠵龜（Caretta caretta）海龜蛋清蛋白（TEWP）棒狀病毒金迪普拉病毒QKKCPGRCTLKCGKHERPTLPYNCGKYICCVPVKVK92地中海貽貝（Mediterranean mussel/Mytilus galloprovincialis）Myticin C (Myt C)VHSVQEAQSVACTSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR93澳大利亞白唇樹蛙，白唇雨濱蛙（Litoria infrafrenata）Frenatin 2黃熱病病毒（YFV）GLLGTLGNLLNGLGL94椰子（Coconut/Cocos nucifera）椰子抗真菌肽HIVEQCREEEDDR95人，智人分泌性白血球蛋白酶抑制劑（SLPI）HIVSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPP96藍細菌，多形偽枝藻（Scytonema varium）抗病毒凝集素scytovirin（SVN）HIV-1GSGPTYCWNEANNPGGPNRCSNNKQCDGARTCSSSGFCQGTSRKPDPGPKGPTYCWDEAKNPGGPNRCSNSKQCD97藍細菌，橢孢念珠藻（Nostoc ellipsosporum）藍藻抗病毒蛋白-N（Cyanovirin-N）（CVN）HIV-1LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQPSNFIETCRNTQLAGSSELAAECKTRAQQF98藍細菌，綠色微囊藻（Microcystis viridis）綠色微囊藻凝集素（MVL）HIV-1MASYKVNIPAGPLWSNAEAQQVGPKIAAAHQGNFTGQWTTVVESAMSVVEVELQVENTGIHEFKTDVLAGPLWSND99紅藻，凋毛藻屬物種（Griffithsia sp）Griffithsin (GRFT)HIV-1SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDAIIIDGVHHGGSGGNLSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGRR100亞洲普通泥蟹，擬穴青蟹（Scylla paramamosain）擬穴青蟹抗脂多糖因子同工型1（Sp-ALF1）白斑綜合症病毒（WSSV）YETLIASVLGKLTGLWHNNSVDFMGHTCHFRRRPKVRKFKLYHEGKFWCPGWAPFEGRSRTKSRSGSSREAIKDFVRK101亞洲普通泥蟹，擬穴青蟹（Scylla paramamosain）Sp-ALF2WSSVYEALVASILGKLSGLWHSDTVDFMGHTCHIRRRPKFRKFKLYHEGKFWCPGWTHLEGNSRTKSRSGSARDAIKDFVYKA102北京鴨，綠頭鴨Apl-AvBD-16鴨肝炎病毒（DHV）FFLLFLQGAAGNSVLCRIRGGRCHVGSCHFPERHIGRCSGFQACCIRTWG103亞洲森林蠍，彼得異蠍（Heterometrus petersii）Hp1090C型肝炎病毒（HCV）IFKAIWSGIKSLF104紅沼澤螯蝦，克氏原螯蝦（Procambarus clarkii）ProcambarinWSSVHRPYCGSKGGIGGGHGGGSGGFGGGGGFGGGGLGGGKPIGIGGGGGFGGGSGFGGGVGLKPNVGGGGGFGGGGGGFGGGIGLKPNVGGGGGFGGGIGLKPNVGGGGGFGGGGGGFGGGGGGFGGGFGGGKLIGGGIGWRRWWLCRKQRLRKVNHL105蠍子，彼得異蠍Hp1036HSV-1ILGKIWEGIKSIF106蠍子，彼得異蠍Hp1239HSV-1ILSYLWNGIKSIF107小鼠，小家鼠（Mus musculus）mBD-3IAVKINNPVSCLRKGGRCWNRCIGNTRQIGSCGVPFLKCCKRK108表皮葡萄球菌（Staphylococcus epidermidis）菌株115，馬葡萄球菌（Staphylococcus equorum）菌株WS 2733微球菌素P1（MP1）HCVSCTTCVCTCSCCTT109大菱鮃（Turbot/Scophthalmus maximus）大菱鮃鐵調素-1（SmHep1P）腫大細胞病毒（MCV）QSHISLCRWCCNCCKANKGCGFCCKF110大菱鮃（Turbot/Scophthalmus maximus）SmHep2PMCVGMKCKFCCNCCNLNGCGVCCRF111奈米比亞馬杜拉放線菌（Actinomadura namibiensis）DSM 6313Labyrinthopeptin A1 (LabyA1)HIV，HSV，RSV，DENV，寨卡病毒（Zika virus，ZIKV），HCV，基孔肯雅病毒（Chikungunya virus，CHIKV），KSHV，CMVSNASVWECCSTGSWVPFTCC112奈米比亞馬杜拉放線菌LabyA2HIV, HSV, RSV, DENV, ZIKV, HCV, CHIKV, KSHV, CMVSDWSLWECCSTGSLFACC113金錢魚（Spotted scat/Scatophagus argus）SA-鐵調素2鱖魚（Siniperca chuatsi）棒狀病毒（SCRV），大口黑鱸（Micropterus salmoides）呼腸孤病毒（MsReV）NPAGCRFCCGCCPNMIGCGVCCRF114血淋巴（Hemolymph），長角血蜱（Haemaphysalis longicornis）HE防禦肽蘭加特病毒（Langat virus，LGTV）EEESEVAHLRVRRGFGCPLNQGACHRHCRSIRRRGGYCSGIIKQTCTCYRN115蠍子，馬氏中殺牛蠍（Mesobuthus martensiiKarsch）BmKDfsin4B型肝炎病毒（HBV）GFGCPFNQGQCHKHCQSIRRRGGYCDGFLKTRCVCYR116虎蝦，斑節對蝦（Penaeus monodon）環狀蝦抗脂多糖因子（cSALF）NNVECKFTVKPYLKRFQVYYKGRMWCP117人，智人肝素結合EGF樣生長因子（HB-EGF）IAVGKRKKKGKGLGKKRDPCLRKYK118嚴重急性呼吸綜合症冠狀病毒（SARS-CoV）I類融合蛋白S2SARS_WW-ISARS-CoVMWKTPTLKYFGGFNFSQIL119SARS-CoV I類融合蛋白S2SARS_WW-IISARS-CoVATAGWTFGAGAALQIPFAMQMAY120SARS-CoV I類融合蛋白S2SARS_WW-IIISARS-CoVGYHLMSFPQAAPHGVVFLHVTW121SARS-CoV I類融合蛋白S2SARS_WW-IVSARS-CoVGVFVFNGTSWFITQRNFFS122SARS-CoV I類融合蛋白S2SARS_WW-VbSARS-CoVAACEVAKNLNESLIDLQELGKYEQYIKW123CMV III類融合蛋白G_b174-200CMVWEIHHINKFAQAYSSYSRVIGGTVFA124CMV III類融合蛋白G_b233-263CMVWHSRGSTWLLYRETANLNAMLTITTARSKYPY125CMV III類融合蛋白G_b264-291CMVHFFATSTGDVVYISPFYNGTNRNASYFG126CMV III類融合蛋白G_b297-315CMVFFIFPNYTIVSDFGRPNAA127裂谷熱病毒（RFVY）I類融合蛋白G_nRVFY-6RVFYWNFFDWFSGLMWFGGPLK128RFVY I類融合蛋白G_nRVFY-6RVFYWNFFDWFSGLMSWFGGPLKTI129RFVY I類融合蛋白G_nRVFY-6RVFYSWNFFDWFSGLMSWFGGPLK130RFVY I類融合蛋白G_nRVFY-6RVFYSGSWNFFDWFSGLMSWFGG131RFVY I類融合蛋白G_nRVFY-6RVFYSGSWNFFDWFSGLMSWFGGPLKPL132IAV I類融合蛋白血凝素（HA）A/H1的殘基84-99IAVVDDGFLDIWTYNAELL133IAV I類融合蛋白HAA/H3、A/H4和A/H14的殘基84-99IAVVEDTKIDLWSYNAELL134IAV I類融合蛋白HAA/H5的殘基84-99IAVMEDGFLDVWTYNAELL135皮秦德病毒（Pichinde virus）融合蛋白複合物GP1蛋白肽4.6（GP1_143-170）皮秦德病毒（PICV）GHTLKWLLELHFNVLHVTRHIGARCKT136皮秦德病毒融合蛋白複合物GP1蛋白肽5.0（GP1_194-212）PICVHLIASLAQIIGDPKIAWVGK137皮秦德病毒融合蛋白複合物GP1蛋白肽6.0（GP1_233-251）PICVHYNFLIIQNTTWENHCTYT138皮秦德病毒融合蛋白複合物GP2蛋白肽7.0（GP2_291-307）PICVPGGYCLEQWAIIWAGIKCF139皮秦德病毒融合蛋白複合物GP2蛋白肽8.0（GP2_348-366）PICVLNLFKKTINGLISDSLVIR140HCV融合蛋白E1E1_197-214HCVVSGIYHVTNDCSNSSIVY141HCV融合蛋白E2E2_407-424HCVPSQKIQLVNTNGSWHINR142HCV融合蛋白E2E2_610-627HCVDYPYRLWHYPCTVNFTVF143HCV融合蛋白E2E2_701-718HCVYLYGIGSAVVSFAIKWEY144人，智人（成纖維細胞生長因子4訊息序列）EBHSV，IAV，痘苗病毒RRKKAAVALLPAVLLALLAP145人，智人FluPep 1 (FP1), TkipIAVWLVFFVIFYFFR146人，智人FP2IAVWLVFFVIAYFAR147人，智人FP3IAVWLVFFVIFYFFRRRKK148人，智人FP4IAVRRKKWLVFFVIFYFFR149人，智人FP7IAVRRKKIFYFFR150人，智人FP8IAVWLVFFVRRKK151人，智人FP9IAVFFVIFYRRKK152人，智人GTP酶RhoARhoARSV, HIVILMCFSIDSPDSLEN153HSV融合蛋白G_bgB64HSVTTPKFTVAWDWVPKR154HSV融合蛋白G_bgB94HSVKTTSSIEFARLQFTY155HSV融合蛋白G_bgB122HSVGHRRYFTFGGGYVYF156HSV融合蛋白G_bgB131HSVHEVVPLEVYTRHEIK157HCV非結構性蛋白NS5AC5AHCVSWLRDIWDWICEVLSDFK158人，智人密封蛋白1CL-58HCVMANAGLQLLGFILAFLGW159人，智人密封蛋白1CL-59HCVAFLGWIGAIVSTALPQWR160鼠腦cDNA噬菌體展示文庫P1西尼羅河病毒（WNV）DTRACDVIALLCHLNT161鼠腦cDNA噬菌體展示文庫P9WNVCDVIALLACHLNT162GB病毒C（GBV-C）表面糖蛋白E2P4-7HIVWDRGNVTLLCDCPNGPWVWV163GBV-C表面糖蛋白E2P6-2HIVLCDCPNGPWVWVPAFCQAVG164人，智人P5SARS-CoV-2GGGYSKAQKAQAKQAK QAQKAQKAQAKQAKQ165人，智人P5+14SARS-CoV-2GGGYSKAQKAQAKQAK QAQKAQKAQAKQAKQA QKAQKAQAKQAKQ166人，智人(P5R)_DSARS-CoV-2GGGYSRAQRAQARQAR QAQRAQRAQARQARQ167人，智人，以及家牛，普通牛（Bos taurus）乳鐵蛋白肽CMV, HIV, HSV, HPVKCFQWQRNMRKVRGPPVSCIKRDS168人，智人，以及家牛，普通牛乳鐵蛋白CMV, HIV, HSV, HPVMKLVFLVLLFLGALGLCLAGRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIRESTV169人，智人CAP37HSV-1，腺病毒GRHFCGGALIHARFVMTAASCFQ170HIV-1HIV-1 TatHIV-1GRKKRRQRRR171HIVC46HIVWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF172HIVC46mutGlycoHIVWMEWDREINNYASLIHSLIEESQNQQEKNEQELLELDKWASLWNWF173HIVC34HIVWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL174HIVC36HIVYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF175HIVT-1249HIVWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF176HIVT-649HIVWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL177HIVSC35EKHIVWEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK178HIVT-2635HIVTTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL179HIVHIV-2EHO-C46HIVWQQWERQVRFLDANITKLLEEAQIQQEKNMYELQELDKWASLWNWF180HIV衍生自分離株HIV-1 S_F2的gp41肽區域的DP178類似物HIVTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF181HIV衍生自分離株HIV-1_RF的gp41肽區域的DP178類似物HIVYTGIIYNLLEESQNQQEKNEQELLELDKW ANLWNWF182HIV衍生自分離株HIV-1_MN的gp41肽區域的DP178類似物HIVYTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF183HIV部分C34肽（最初衍生自gp41）HIVWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL184RSVDP178和/或DP107類似物RSVYTSVITIELSNIKENKCNGAKVKLIKQELDKYK185RSVDP178和/或DP107類似物RSVTSVITIELSNIKENKCNGAKVKLIKQELDKYKN186RSVDP178和/或DP107類似物RSVVITIELSNIKENKCNGAKVKLIKQELDKYKNAV187RSVDP178和/或DP107類似物RSVIALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK188HPIVDP178和/或DP107類似物HPIVVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLI189HPIVDP178和/或DP107類似物HPIVRSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV190HPIVDP178和/或DP107類似物HPIVNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKL191HPIVDP178和/或DP107類似物HPIVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI192HPIVDP178和/或DP107類似物HPIVLDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIG193HPIVDP178和/或DP107類似物HPIVDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN194HPIVDP178和/或DP107類似物HPIVPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNW195HPIVDP178和/或DP107類似物HPIVIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWH196MeVDP178和/或DP107類似物MeVHRIDLGPPISLERLDVGTNLGNAIAKLEAKELLE197MeVDP178和/或DP107類似物MeVIDLGPPISLERLDVGTNLGNAIAKLEAKELLESS198MeVDP178和/或DP107類似物MeVLGPPISLERLDVGTNLGNAIAKLEAKELLESSDQ199MeVDP178和/或DP107類似物MeVPISLERLDVGTNLGNAIAKLEAKELLESSDQILR200SIVDP178和/或DP107類似物SIVWQEWERKVDFLEENITALLEEAQIQQEKNMYELQK201SIVDP178和/或DP107類似物SIVQEWERKVDFLEENITALLEEAQIQQEKNMYELQKL202SIVDP178和/或DP107類似物SIVEWERKVDFLEENITALLEEAQIQQEKNMYELQKLN203SIVDP178和/或DP107類似物SIVWERKVDFLEENITALLEEAQIQQEKNMYELQKLNS204SIVDP178和/或DP107類似物SIVERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW205SIVDP178和/或DP107類似物SIVRKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD206SIVDP178和/或DP107類似物SIVKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV207SIVDP178和/或DP107類似物SIVVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF208SIVDP178和/或DP107類似物SIVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG209SIVDP178和/或DP107類似物SIVFLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN210HIVDP178肽（來自HIV-1_LAI分離株的gp41的AA 638-673）HIVYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF211HIVDP107肽（來自HIV-1_LAI分離株的gp41的AA 558-595）HIVNNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ212HIV衍生自HIV-1_SF2的gp41肽區域的DP178類似物HIVYTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF213HIV衍生自HIV-1_RF的gp41肽區域的DP178類似物HIVYTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF214HIV衍生自HIV-1_MN的gp41肽區域的DP178類似物HIVYTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF215RSVDP178和/或DP107類似物RSVYTSWITIELSNIKENKCNGAKWKLIKQELDKYK216RSVDP178和/或DP107類似物RSVTSWITIELSNIKENKCNGAKWKLIKQELDKYKN217RSVDP178和/或DP107類似物RSVWITIELSNIKENKCNGAKWKLIKQELDKYKNAW218RSVDP178和/或DP107類似物RSVIALLSTNKAWWSLSNGWSWLTSKWLDLKNYIDK219HPIVDP178和/或DP107類似物HPIVVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLI220HPIVDP178和/或DP107類似物HPIVRSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV221HPIVDP178和/或DP107類似物HPIVNSVALDPIDISIELNKAKSDLEESKEWIRRSNOKL222HPIVDP178和/或DP107類似物HPIVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI223HPIVDP178和/或DP107類似物HPIVLDPIDISIELNKAKSDLEESKEWIRRSNOKLDSIG224HPIVDP178和/或DP107類似物HPIVDPIDISIELNKAKSDLEESKEWIRRSNOKLDSIGN225HPIVDP178和/或DP107類似物HPIVPIDISIELNKAKSDLEESKEWIRRSNOKLDSIGNW226HPIVDP178和/或DP107類似物HPIVIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWE227MeVDP178和/或DP107類似物MeVHRIDLGPPISLERLDWGTNLGNAIAKLEAKELLE228MeVDP178和/或DP107類似物MeVIDLGPPISLERLDWGTNLGNAIAKLEAKELLESS229MeVDP178和/或DP107類似物MeVLGPPISLERLDWGTNLGNAIAKLEAKELLESSDQ230MeVDP178和/或DP107類似物MeVPISLERLDWGTNLGNAIAKLEAKELLESSDQILR231SIVDP178和/或DP107類似物SIVWQEWERKVDFLEENITALLEEAQIQQEKNMYELQK232SIVDP178和/或DP107類似物SIVQEWERKVDFLEENITALLEEAQIQQEKNMYELQKL233SIVDP178和/或DP107類似物SIVEWERKVDFLEENITALLEEAQIQQEKNMYELQKLN234SIVDP178和/或DP107類似物SIVWERKVDFLEENITALLEEAQIQQEKNMYELQKLNS235SIVDP178和/或DP107類似物SIVERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW236SIVDP178和/或DP107類似物SIVRKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD237SIVDP178和/或DP107類似物SIVKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV238SIVDP178和/或DP107類似物SIVWDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF239SIVDP178和/或DP107類似物SIVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG240SIVDP178和/或DP107類似物SIVFLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN241HIV恩夫韋地（T-1249）HIVYTSLIHSLIEESQNQQEKNEQELLELNKWA SLWNWF242HIV  HIVCLLLGTEVSEALGGAGLT243RSV  RSVDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK244亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型DITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN245RSV  RSVDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL246HIV；甲型流感  HIV；甲型流感DQYKCLQHGGFCLRSSCPSNTKLQGTCKPDKPNCCKS247RSV  RSVEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKS248RSV  RSVFDASISQVNEKINQSLAFIRKSDELLHNVNAGKST249基孔肯雅病毒；登革熱病毒  基孔肯雅病毒；登革熱病毒FLGAILKIGHALAKTVLPMVTNAFKPKQ250RSV  RSVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVN251RSV  RSVFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE252乙型流感病毒:E型肝炎病毒  乙型流感病毒；E型肝炎病毒GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDKNYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGT253登革熱病毒:寨卡病毒  登革熱病毒；寨卡病毒GFGCPLDQMQCHNHCQSVRYRGGYCTNFLKMTCKCY254HIV  HIVGIFPKIIGKGIVNGIKSLAKGVGMKVFKAGLNNIGNTGCNNRDEC255HIV  HIVHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNW256RSV  RSVIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRK257HIV  HIVIKKEIEAIKKEQEAIKKKIEAIEKEISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL258RSV  RSVINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKS259亨德拉病毒:尼帕病毒  亨德拉病毒:尼帕病毒KVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSL260HIV  HIVLGTEVSEALGGAGLTGGF261麻疹病毒:人副流感病毒3型  麻疹病毒:人副流感病毒3型LHRIDLGPPISLERLDVGTNLGNAIAKLEDAKELL262HIV  HIVLNNCLLLGTEVSEALGGA263RSV  RSVLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHN264HIV  HIVMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL265RSV  RSVNFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSD266HIV  HIVNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ267RSV  RSVPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLH268亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型PPVYTKDVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPSL269RSV  RSVPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNA270HIV  HIVRFPFHRCGAGPKLTKDLE271HIV  HIVRSQKEGLHYTCSSHFPYSQYQFWK272RSV  RSVSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAG273HIV  HIVSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL274亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型SIELNKAKSDLEESKEWIRRSNQKLDSI275HIV  HIVTTWEAWDRAIAEYAARIEALIRAAQEQQEKLEAALREL276亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VALDPIDISIELNKAKSDLEESKEWIRR277亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSD278亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI278亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VANDPIDISIELNKAKSDLEESKEWIRRSNQKLDSD280亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VANDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI281RSV  RSVVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNV282亨德拉病毒；人副流感病毒3型  亨德拉病毒；人副流感病毒3型VYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTV283HCV  HCVWVAVTPTVATRDGKLPTT284RSV  RSVYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL285HIV  HIVYTSLIHSLIEESQNQQEKNEQQLLELDKWASLWNWF286HIV-1 gp41艾博韋泰（Albuviritide）HIVAc-WEEWDREINNYT(Mpa)LIHELIEESQNQQEKNEQELL-CONH₂(Mpa=3-馬來醯亞胺丙酸)287CoV-OC43EK1CoVSLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL288CoV-OC43OC43-HR2PCoVSLDYINVTFLDLQDEMNRLQEAIKVLNQSYINLKDI289SARS-CoV-2武漢-Hu-1 HR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN290SARS-CoV-2αHR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILARLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN291SARS-CoV-2βHR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN292SARS-CoV-2γHR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN293SARS-CoV-2δHR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN294SARS-CoV-2οHR1CoVNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN295SARS-CoV-1SARS-CoV1 HR1CoVNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN296MERSMERS HR1CoVQVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQ297SARS-CoV-2武漢-Hu-1 HR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL298SARS-CoV-2αHR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL299SARS-CoV-2βHR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL300SARS-CoV-2γHR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASFVNIQKEIDRLNEVAKNLNESLIDLQEL301SARS-CoV-2δHR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL302SARS-CoV-2οHR2CoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL303SARS-CoV-1SARS-CoV1 HR2CoVVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL304MERSMERS HR2CoVVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL305SARS-CoV-2HR2ACoVDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL306SARS-CoV-2HR2BCoVVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD307SARS-CoV-2HR2CCoVFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK308SARS-CoV-2HR2全長CoVGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL309SARS-CoV-2HR2全長Fc融合CoVMGWSCIILFLVATATGVHS   GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG310SARS-CoV-2不帶有訊息肽的HR2全長Fc融合CoVGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG311HIVHIV_CHRHIVTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF312HIVT20HIVYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF313HIVT2410HIVMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL314HIVT2410_2.0HIVMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF315HIVT1144HIVTTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL316HIVT144_2.0HIVTTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASLWNWF317HIVT1249HIVWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF318HIVT1249_2.0HIVTTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF319HIVT2635HIVTTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL320HIVT2635_2.0HIVTTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF321HIVT2635_3.0HIVMTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF322HIVT290676HIVTTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL323HIVT290676_2.0HIVTTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF324In some embodiments, an anti-fusion polypeptide targets one or more (eg, two, three, four, or five) viruses. Viruses that may be targeted by anti-fusion polypeptides include, but are not limited to, all viral strains listed in Table 1. In some embodiments, the virus does not infect humans. In some embodiments, the virus infects humans. [Table1 ].Exemplary anti-fusion polypeptidesSourcepeptidetargetamino acid sequenceSEQ ID NO: Golden oyster mushroom,Pleurotus citrinopileauts antiviral protein Y3 Tobacco mosaic virus (TMV), influenza hemagglutinin (HA), human immunodeficiency virus (HIV) pseudovirions AACARFIDDFCDTLTPNIYRPRDNGQRCYAVNGHRCDFTVFNTNNGGNPIRASTPNCKTVLRTAANRCPTGGRGKINPNAPFLFAIDPNDGDCSTNF 1 Daruma pond frog,Rana brevipoda porsa Brevinin-1 Herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) FLPVLAGIAAKVVPALFCKITKKC 2 European common frog,Rana temporaria Temporin B CCV, FV3 LLPIVGNLLKSLL 3 European common frog,Rana temporaria Temporin G Influenza A virus (IAV), respiratory parainfluenza virus (PIV) FFPVIGRILNGIL 4 North American frog, leopard frog (Rana pipiens ), and Oregon spotted frog, spotted red frog (Rana pretiosa ) Ranateurin 3P HIV GLMDTVKNVAKNLAGHMLDKLKCKITGC 5Homo sapiens Human neutrophil peptide-1 (HNP-1) Pseudotyped viruses expressing SARS-CoV-2 spike protein ACYCRIPACIAGERRYGTCIYQGRLWAFCC 6 Homo sapiens HNP-2 Pseudotyped viruses expressing SARS-CoV-2 spike protein CYCRIPACIAGERRYGTCIYQGRLWAFCC 7 Homo sapiens HNP-3 Pseudotyped viruses expressing SARS-CoV-2 spike protein DCYCRIPACIAGERRYGTCIYQGRLWAFCC 8 Homo sapiens HNP-4 Pseudotyped viruses expressing SARS-CoV-2 spike protein VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV 9 Homo sapiens Human defense peptide (HD-5) BK virus (BKV) ATCYCRTGRCATRESLSGVCEISGRLYRLCCR 10 Homo sapiens HD-6 Respiratory syncytial virus (RSV), human PIV (HPIV), HIV, HSV, influenza A virus (IAV), BKV, human adenovirus (HAdV), human papillomavirus (HPV) AFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL 11 Rabbit,Oryctolagus cuniculus Rabbit neutrophil peptide 1 (NP-1) HSV, enveloped virus VVCACRRALCLPRERRAGFCRIRGRIHPLCCRR 12 Rabbit,Oryctolagus cuniculus Rabbit neutrophil defense peptide 2 (NP-2) RSV, HIV, HSV VVCACRRALCLPLERRAGFFCRIRGRIHPLCCRR 13 Pig neutrophil, wild boar (Sus scrofa ) Protegrin 1 (PG-1) HIV, HSV-1, HSV-2, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) RGGRLCYCRRRFCVCVGR 14 Atlantic horseshoe crab,Limulus polyphemus Polyphemusin I HIV RRWCFRVCYRGFCYRKCR 15 Atlantic horseshoe crab,Limulus polyphemus Limulus peptide II HIV RRWCFRVCYKGFCYRKCR 16 Southeast Asian hemocytes, Chinese horseshoe crab(Tachypleus tridentatus) , southern horseshoe crab (Tachypleus gigas ), round-tailed horseshoe crab (Carcinoscorpius rotundicauda ) Limulus peptide (Telulin peptide) I (TP1) HIV, HSV-1, HSV-2, Singapore grouper iridovirus (SGIV) KWCFRVCYRGICYRRCR 17 Rabbit,Oryctolagus cuniculus Rabbit neutrophil defense peptide 3a (NP-3a) RSK, HIV, HSV GICACRRRFCPNSERFSGYCRVNGARYVRCCSRR 18 Pig neutrophil, wild boar (Sus scrofa ) PG-2 Pseudorabies virus (PRV), PRRSV RGGRLCYCRRRFCICV 19 Pig neutrophil, wild boar (Sus scrofa ) PG-3 PRRSV RGGGLCYCRRRFCVCVGR 20 Pig neutrophil, wild boar (Sus scrofa ) PG-4 PRRSV RGGRLCYCRGWICFCVGR twenty one Rat, brown rat (Rattus novegicus ) Rat NP-1, rat defense peptide HIV, HSV VTCYCRRTRCFRERLSGACGYRGRIYRLCCR twenty two Rat, brown rat (Rattus novegicus ) Rat NP-2 HIV, HSV VTCYCRSTRCGFRERLSGACGYRGRIYRLCCR twenty three Rat, brown rat (Rattus novegicus ) Rat NP-3 HIV, HSV CSCRTSSCRFGERLSGACRLNGRIYRLCC twenty four Rat, brown rat (Rattus novegicus ) Rat NP-4 HIV, HSV ACYCRIGACVSGERLTGACGLNGRIYRLCCR 25 Australian Green Tree Frog,Litoria splendida ,Litoria rothii Caerin 1.1 HIV, murine leukemia virus GLLSVLGSVAKHVLPHVVPVIAEHL 26 Australian green tree frog,Litoria caerula Caerin 1.2 HIV GLLGVLGSVAKHVLPHVVPVIAEHL 27 Australian green tree frog,Litoria caerula Caerin 1.3 HIV GLLSVLGSVAQHVLPHVVPVIAEHL 28 Australian green tree frog,Litoria caerula Caerin 1.4 HIV GLLSSLSSVAKHVLPHVVPVIAEHL 29 Australian green tree frog,Litoria caerula Caerin 1.5 HIV GLLSVLGSVVKHVIPHVVPVIAEHL 30 Australian orange-legged frog,Litoria xanthomera Caerin 1.6 HIV GLFSVLGAVAKHVLPHVVPVIAEK 31 Australian orange-legged frog,Litoria xanthomera Caerin 1.7 HIV GLFKVLGSVAKHLLPHVAPVIAEK 32 Australian green tree frog,Litoria caerula Caerin 1.9 HIV GLFGVLGSIAKHVLPHVVPVIAEKL-NH2 33 Australian green tree frog,Litoria caerula Caerin 1.10 HIV GLLSVLGSVAKHVLPHVVOVIAEKL-NH₂ 34 Australian green tree frog,Litoria caerula Caerin 1.19 HIV GLFKVLGSVAKHLLPHVAPIIAEKL-NH₂ 35 Australian green tree frog,Litoria caerula Caerin 1.20 HIV GLFGILGSVAKHVLPHVIPVVAEHL-NH2 36 Australian green tree frog,Litoria caerula Caerin 4.1 HIV GLWQKIKSAAGDLASGIVEGIKS 37 Mouse,Mus musculus Mouse beta-defense peptide 1 (mBD-1) HIV, IAV DQYKCLQHGGFCLRSSCPSNTKLQGTCKPDKPNCCKS 38 Mouse,Mus musculus Mouse cathelin-related antimicrobial peptide (mCRAMP) RSV GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ 39 Homo sapiens Human beta defense peptide 3 (hBD-3) HIV GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK 40 Southern brown tree frog,Litoria ewingii Ewingiin 2.1 (formerly Caeridin 7.1) HIV GLLDMVTGLLGNL-NH2 41 Australian tree frog, green-eyed rain frog (Litoria genimaculata ) Maculatin 1.1 HIV, PRV GLFGVLAKVAAHVVPAIAEHF-NH2 42 Hybrid Striped Bass, Striped Bass (Morone saxatilis ), Goldeneye Bass (Morone chrysops ) Piscidin 1H (Pis-1H) HIV FFHHIFRGIVHVGKTIHRLVTG 43 Hybrid Striped Bass, Striped Bass (Morone saxatilis ), Goldeneye Bass (Morone chrysops ) Toxin 1N (Pis-1N) HIV FFHHIFRGIVHVGKTIHRLVTG 44 Hybrid Striped Bass, Striped Bass (Morone saxatilis ), Goldeneye Bass (Morone chrysops ) Pis-2 HIV FFHHIFRGIVHVGKTIHKLVTG 45 Hybrid Striped Bass, Striped Bass (Morone saxatilis ), Goldeneye Bass (Morone chrysops ) Pis-3 HIV FIHHIFRGIVHAGRSIGRFLTG 46 Blue mussel/Mytilus edulis Mytilin B Viral Hemorrhagic Septicemia Virus (VHSV) VHSV, IPNV SCASRCKGHCRARRCGYYVSVLYRGRCYCKCLRC 47 Mediterranean mussel (Mediterranean mussel/Mytilus galloprovincialis ) Mytilin C VHSV, IPNV SCASRCKSRCRARRCRYYVSVRYGWFCYCRCLRC 48Pseudocanthotermes spiniger Spinigerin HIV HVDKKVADKVLLLKQLRIMRLLTRL 49 North American frog, bullfrog (Rana catasbeiana ) Ranateurin 6 HIV FISAIASMLGKFL 50 North American frog, bullfrog (Rana catasbeiana ) Ranateurin 9 HIV FLFPKITSFLSKVL 51 Rhesus monkey, macaque (Macaca mulatta ) Rhesus theta defense peptide-1 (RTD-1) HIV GFCRCLCRRGVCRCICTR 52 Homo sapiens Human beta-defense peptide 1 (hBD-1) HIV, HSV DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK 53 Homo sapiens hBD-2 HIV, HSV GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP 54Streptomyces strain AA3891 Siamycin II HIV CLGIGGSCNDFAGCGYAIVCFW 55 North American mink frog, mink frog (Rana septentrionalis ) Brevinin-2 related peptide HIV GIWDTIKSMGKVFAGKILQNL 56 Guinea pig/Cavia porcellus CAP11 IAV GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI 57 Rhesus monkey, macaque (Macaca mulatta ) RTD-2 HIV GVCRCLCRRRGVCRCLCRR 58 Rhesus monkey, macaque (Macaca mulatta ) RTD-3 HIV GFCRCICTRGFCRCICTR 59 African clawed frog,Xenopus laevis Magainin 1 HSV-1, HSV-2 GIGKFLHSAGKFGKAFVGEIMKS 60 African herb, relatedOldenlandia affinis Kalata B1 (KB1) HIV GLPVCGETCVGGTCNTPGCTCSWPVCTRN 61 Chicken,Gallus gallus domestic , duck, mallard (Anas platyrhynchos ) Chicken Avian Beta Defense Peptide 6 (AvBD-6) IAV SPIHACRYQRGVCIPGPCRWPYYRVGSCGSGLKSCCVRNRWA 62 chicken, domestic chicken AvBD-5 IAV GLPQDCERRGGFCSHKSCPPGIGRIGLCSKEDFCCRSRWYS 63 Chinese red-bellied toad,Bombina maxima Maximin H1 Nipah virus (NiV) ILGPVISTIGGVLGGLLKNL 64 Saharan frog,Pelophylax saharicus Temporin-Sha HSV FLSGIVGMLGKLF 65Umbrella nut cymosa (Leonia cymosa) Cycloviolin A HIV GVIPCGESCVFIPCISAAIGCSCKNKVCYRN 66Umbrella nut cymosa (Leonia cymosa) Cycloviolin B HIV GTACGESCYVLPCFTVGCTCTSSQCFKN 67Umbrella nut cymosa (Leonia cymosa) Cycloviolin C HIV GIPCGESCVFIPCLTTVAGCSCKNKVCYRN 68Umbrella nut cymosa (Leonia cymosa) Cycloviolin D HIV GFPCGESCVFIPCISAAIGCSCKNKVCYRN 69 European field viola, wild viola (Viola arvensis ) Varv peptide E (Varv E) HIV GLPICGETCVGGTCNTPGCSCSWPVCTRN 70 African herb, relatedOldenlandia affinis KB8 HIV GSVLNCGETCLLGTCYTTGCTCNKYRVCTKD 71Palicourea condensata Palicourein HIV GDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN 72Viola betonicifolia KB2 HIV GLPVCGETCFGGTCNTPGCSCTWPICTRD 73 Australian violet,Viola hederacea Vhl-1 HIV SISCGESCAMISFCFTEVIGCSCKNKVCYLN 74 Tropical tree,Chassalia parvifola cyclobactin C HIV GIPCGESCVFIPCITSVAGCSCKSKVCYRN 75 Tropical tree,Chassalia parvifola cyclobactin D HIV KIPCGESCVWIPCVTSIFNCKCKENKVCYHD 76 Tropical tree,Chassalia parvifola cyclobactin E HIV KIPCGESCVWIPCLTSVNFNCKCENKVCYHD 77 Tropical tree,Chassalia parvifola cyclobactin F HIV KVCYRAIPCGESCVWIPCISAAIGCSCKN 78 Tilapia,Oreochromis mossambicus Hepcidin 1-5 (TH1-5) Neuronecrosis virus (NNV) GIKCRFCCGCCTPGICGVCCRF 79 Sweet violet,Viola odorata Cycloviolacin O13 HIV GIPCGESCVWIPCISAAIGCSCSKSKVCYRN 80 Sweet violet,Viola odorata Cycloviolacin O14 HIV GSIPACGESCFKGKCYTPGCSCSKYPLCAKN 81 Sweet violet,Viola odorata Cycloviolacin O24 HIV GLPTCGETCFGGTCNTPGCTCDPWPVCTHN 82 Chinese herbal medicine,Viola yedoensis Cycloviolacin Y1 HIV GGTIFDCGETCFLGTCYTPGCSCGNYGFCYGTN 83 Chinese herbal medicine,Viola yedoensis Cycloviolacin Y4 HIV GVPCGESCVFIPCITGVIGCSCSSNVCYLN 84 Chinese herbal medicine,Viola yedoensis Cycloviolacin Y5 HIV GIPCAESCVWIPCTVTALVGCSCSDKVCYN 85 Chinese herbal medicine,Viola yedoensis Cycloviolacin VY1 IAV CGESCVFIPCITTVLGCSCSIKVCYKNGSIP 86 Streptomyces strain AA6532 Siamycin I HIV CLGVGSCNDFAGCGYAVVCFW 87 Streptomyces strain AA6532 NP-06 HIV CLGVGSCNDFAGCGYAIVCFW 88 Homo sapiens Retrocylin-1 (RC1) HIV GICRCICGRGICRCICGR 89 Homo sapiens RC2 HIV GICRCICGRRICRCICGR 90 Homo sapiens RC3 HIV RICRCICGRRICRCICGR 91 Loggerhead sea turtle, loggerhead sea turtle (Caretta caretta ) Turtle Egg White Protein (TEWP) Rhabdovirus Kindipura virus QKKCPGRCTLKCGKHERPTLPYNCGKYICCVPVKVK 92 Mediterranean mussel (Mediterranean mussel/Mytilus galloprovincialis ) Myticin C (Myt C) VHSV QEAQSVACTSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR 93 Australian white-lipped tree frog,Litoria infrafrenata Frenatin 2 Yellow fever virus (YFV) GLLGTLGNLLNGGL 94 Coconut (Coconut/Cocos nucifera ) Coconut Antifungal Peptides HIV EQCREEEDDR 95 Homo sapiens Secretory leukocyte protease inhibitor (SLPI) HIV SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPP 96 Cyanobacteria,Scytonema varium Antiviral lectin scytovirin (SVN) HIV-1 GSGPTYCWNEANNPGGPNRCSNKQCDGARTCSSSGFCQGTSRKPDPGPKGPTYCWDEAKNPGGPNRCSNSKQCD 97 Cyanobacteria,Nostoc ellipsosporum Cyanovirin-N (CVN) HIV-1 LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQPSNFIETCRNTQLAGSSELAAECKTRAQQF 98 Cyanobacteria,Microcystis viridis Green Microcystis Lectin (MVL) HIV-1 MASYKVNIPAGPLWSNAEAQQVGPKIAAAHQGNFTGQWTTVVESAMSVVEVELQVENTGIHEFKTDVLAGPLWSND 99 Red algae,Griffithsia sp . Griffithsin (GRFT) HIV-1 SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDAIIIDGVHHGGSGGNLSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGRR 100 Asian common mud crab,Scylla paramamosain Anti-lipopolysaccharide factor isoform 1 (Sp-ALF1) of Scylla pseudocystis White spot syndrome virus (WSSV) YETLIASVLGKLTGLWHNNSVDFMGHTCHFRRRPKVRKFKLYHEGKFWCPGWAPFEGRSRTKRSGSSREAIKDFVRK 101 Asian common mud crab,Scylla paramamosain Sp-ALF2 WSSV YEALVASILGKLSGLWHSDTVDFMGHTCHIRRRPKFRKFKLYHEGKFWCPGWTHLEGNSRTKSRSGSARDAIKDFVYKA 102 Peking duck, mallard Apl-AvBD-16 Duck hepatitis virus (DHV) FFLLFLQGAAGNSVLCRIRGGRCHVGSCHFPERHIGRCSGFQACCIRTWG 103 Asian forest scorpion,Heterometrus petersii Hp1090 Hepatitis C virus (HCV) IFKAIWSGIKSLF 104 Red swamp crayfish,Procambarus clarkii Procambarin WSSV HRPYCGSKGGIGGGHGGGSGGFGGGGGFGGGGLGGGKPIGIGGGGGFGGGSGFGGGVGLKPNVGGGGGFGGGGGGFGGGIGLKPNVGGGGGFGGGIGLKPNVGGGGGFGGGGGGFGGGGGGFGGGFGGGKLIGGGIGWRRWWLCRKQRLRKVNHL 105 Scorpion, Peter the Scorpion Hp1036 HSV-1 ILGKIWEGIKSIF 106 Scorpion, Peter the Scorpion Hp1239 HSV-1 ILSYLWNGIKSIF 107 Mouse,Mus musculus mBD-3 IAV KINNPVSCLRKGGRCWNRCIGNTRQIGSCGVPFLKCCKRK 108Staphylococcus epidermidis strain 115,Staphylococcus equorum strain WS 2733 Micrococcin P1 (MP1) HCV SCTTCVCTCSCCTT 109 Turbot/Scophthalmus maximus Turbot hepcidin-1 (SmHep1P) Cytozoonvirus (MCV) QSHISLCRWCCNCCKANKGCGFCCKF 110 Turbot/Scophthalmus maximus SmHep2P MCV GMKCKFCCNCCNLNGCGVCCRF 111Actinomadura namibiensis DSM 6313 Labyrinthopeptin A1 (LabyA1) HIV, HSV, RSV, DENV, Zika virus (ZIKV), HCV, Chikungunya virus (CHIKV), KSHV, CMV SNASVWECCSTGSWVPFTCC 112Actinomyces madura in Namibia LabyA2 HIV, HSV, RSV, DENV, ZIKV, HCV, CHIKV, KSHV, CMV SDWSLWECCSTGSLFACC 113 Money fish (Spotted scat/Scatophagus argus ) SA-Hepcidin 2 Mandarin fish (Siniperca chuatsi ) rod-shaped virus (SCRV), largemouth bass (Micropterus salmoides ) reovirus (MsReV) NPAGCRFCCGCCPNMIGCGVCCRF 114 Hemolymph,Haemaphysalis longicornis HE defense peptide Langat virus (LGTV) EEESEVAHLRVRRGFGCPLNQGACHHRHCRSIRRRGGYCSGIIKQTCTCYRN 115 Scorpion,Mesobuthus martensii Karsch BmKDfsin4 Hepatitis B virus (HBV) GFGCPFNQGQCHKHCQSIRRRGGYCDGFLKTRCVCYR 116 Tiger shrimp,Penaeus monodon Ringed shrimp anti-lipopolysaccharide factor (cSALF) NNV ECKFTVKPYLKRFQVYYKGRMWCP 117 Homo sapiens Heparin-binding EGF-like growth factor (HB-EGF) IAV GKRKKKGKGLGKKRDPCLRKYK 118 Severe acute respiratory syndrome coronavirus (SARS-CoV) class I fusion protein S2 SARS_WW-I SARS-CoV MWKTPTLKYFGGFNFSQIL 119 SARS-CoV class I fusion protein S2 SARS_WW-II SARS-CoV ATAGWTFGAGAALQIPFAMQMAY 120 SARS-CoV class I fusion protein S2 SARS_WW-III SARS-CoV GYHLMSFPQAAPHGVVFLHVTW 121 SARS-CoV class I fusion protein S2 SARS_WW-IV SARS-CoV GVFVFNGTSWFITQRNFFS 122 SARS-CoV class I fusion protein S2 SARS_WW-Vb SARS-CoV AACEVAKNLNESLIDLQELGKYEQYIKW 123 CMV class III fusion protein G_b 174-200 CMV WEIHHINKFAQAYSSYSRVIGGTVFA 124 CMV class III fusion protein G_b 233-263 CMV WHSRGSTWLLYRETANLNAMLTITTARSKYPY 125 CMV class III fusion protein G_b 264-291 CMV HFFATSTGDVVYISPFYNGTNRNASYFG 126 CMV class III fusion protein G_b 297-315 CMV FFIFPNYTIVSDFGRPNAA 127 Rift Valley fever virus (RFVY) class I fusion protein G_n RVFY-6 RVFY WNFFDWFSGLMWFGGPLK 128 RFVY class I fusion protein G_n RVFY-6 RVFY WNFFDWFSGLMSWFGGPLKTI 129 RFVY class I fusion protein G_n RVFY-6 RVFY SWNFFDWFSGLMSWFGGPLK 130 RFVY class I fusion protein G_n RVFY-6 RVFY SSGSWNFFDWFSGLMSWFGG 131 RFVY class I fusion protein G_n RVFY-6 RVFY SGSWNFFDWFSGLMSWFGGPLKPL 132 IAV class I fusion protein hemagglutinin (HA) Residues 84-99 of A/H1 IAV VDDGFLDIWTYNAELL 133 IAV class I fusion protein HA Residues 84-99 of A/H3, A/H4 and A/H14 IAV VEDTKIDLWSYNAELL 134 IAV class I fusion protein HA Residues 84-99 of A/H5 IAV MEDGFLDVWTYNAELL 135 Pichinde virus fusion protein complex GP1 protein Peptide 4.6 (GP1_143-170 ) Pichinde virus (PICV) GHTLKWLLELHFNVLHVTRHIGARCKT 136 Pichinde virus fusion protein complex GP1 protein Peptide 5.0 (GP1_194-212 ) PICV HLIASLAQIIGDPKIAWVGK 137 Pichinde virus fusion protein complex GP1 protein Peptide 6.0 (GP1_233-251 ) PICV HYNFLIIQNTTWENHCTYT 138 Pichinde virus fusion protein complex GP2 protein Peptide 7.0 (GP2_291-307 ) PICV PGGYCLEQWAIIWAGIKCF 139 Pichinde virus fusion protein complex GP2 protein Peptide 8.0 (GP2_348-366 ) PICV LNLFKKTINGLISDSLVIR 140 HCV fusion protein E1 E1_197-214 HCV VSGIYHVTNDCSNSSIVY 141 HCV fusion protein E2 E2_407-424 HCV PSQKIQLVNTNGSWHINR 142 HCV fusion protein E2 E2_610-627 HCV DYPYRLWHYPCTVNFTVF 143 HCV fusion protein E2 E2_701-718 HCV YLYGIGSAVVSFAIKWEY 144 Homo sapiens (fibroblast growth factor 4 message sequence) EB HSV, IAV, vaccinia virus RRKKKAAVALLPAVLLALLAP 145 Homo sapiens FluPep 1 (FP1), Tkip IAV WLVFFVIFYFFR 146 Homo sapiens FP2 IAV WLVFFVIAYFAR 147 Homo sapiens FP3 IAV WLVFFVIFYFFRRRKK 148 Homo sapiens FP4 IAV RRKKWLVFFVIFYFFR 149 Homo sapiens FP7 IAV RRKKIFYFFR 150 Homo sapiens FP8 IAV WLVFFVRRKK 151 Homo sapiens FP9 IAV FFVIFYRRKK 152 Homo sapiens GTPase RhoA RhoA RSV, HIV ILMCFSIDSPDSLEN 153 HSV fusion protein G_b gB64 HSV TTPKFTVAWDWVPKR 154 HSV fusion protein G_b gB94 HSV KTTSSIEFARLQFTY 155 HSV fusion protein G_b gB122 HSV GHRRYFTFGGGYVYF 156 HSV fusion protein G_b gB131 HSV HEVVPLEVYTRHEIK 157 HCV nonstructural protein NS5A C5A HCV SWLRDIWDWICEVLSDFK 158 Homo sapiens sealin 1 CL-58 HCV MANAGLQLLGFILAFLGW 159 Homo sapiens sealin 1 CL-59 HCV AFLGIGAIVSTALPQWR 160 Mouse brain cDNA phage display library P1 West Nile virus (WNV) DTRACDVIALLCHLNT 161 Mouse brain cDNA phage display library P9 WNV CDVIALLACHLNT 162 GB virus C (GBV-C) surface glycoprotein E2 P4-7 HIV WDRGNVTLLCDCPNGPWVWV 163 GBV-C surface glycoprotein E2 P6-2 HIV LCDCPNGPWVWVPAFCQAVG 164 Homo sapiens P5 SARS-CoV-2 GGGYSKAQKAQAKQAK QAQKAQKAQAKQAKQ 165 Homo sapiens P5+14 SARS-CoV-2 GGGYSKAQKAQAKQAK QAQKAQKAQAKQAKQA QKAQKAQAKQAKQ 166 Homo sapiens (P5R)_D SARS-CoV-2 GGGYSRAQRAQARQAR QAQRAQRAQARQARQ 167 Man, Homo sapiens, and domestic cow,Bos taurus Lactoferrin peptide CMV, HIV, HSV, HPV KCFQWQRNMRKVRGPPVSCIKRDS 168 Humans, Homo sapiens, and domestic cattle, common cattle Lactoferrin CMV, HIV, HSV, HPV MKLVFLVLLFLGALGLCLAGRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSG AFKCLRDGAGDVAFIRESTV 169 Homo sapiens CAP37 HSV-1, adenovirus GRHFCGGALIHARFVMTAASCFQ 170 HIV-1 HIV-1 Tat HIV-1 GRKKRRQRRR 171 HIV C46 HIV WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 172 HIV C46mutGlyco HIV WMEWDREINNYASLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 173 HIV C34 HIV WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL 174 HIV C36 HIV YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 175 HIV T-1249 HIV WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF 176 HIV T-649 HIV WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL 177 HIV SC35EK HIV WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK 178 HIV T-2635 HIV TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL 179 HIV HIV-2EHO-C46 HIV WQQWERQVRFLDANITKLLEEAQIQQEKNMYELQELDKWASLWNWF 180 HIV DP178 analog derived from the gp41 peptide region of isolate HIV-1 S_F2 HIV TNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF 181 HIV DP178 analog derived from the gp41 peptide region of isolate HIV-1_RF HIV YTGIIYNLLEESQNQQEKNEQELLELDKW ANLWNWF 182 HIV DP178 analog derived from the gp41 peptide region of isolate HIV-1_MN HIV YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF 183 HIV Partial C34 peptide (originally derived from gp41) HIV WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL 184 RSV DP178 and/or DP107 analogs RSV YTSVITIELSNIKENKCNGAKVKLIKQELDKYK 185 RSV DP178 and/or DP107 analogs RSV TSVITIELSNIKENKCNGAKVKLIKQELDKYKN 186 RSV DP178 and/or DP107 analogs RSV VITIELSNIKENKCNGAKVKLIKQELDKYKNAV 187 RSV DP178 and/or DP107 analogs RSV IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK 188 HPIV DP178 and/or DP107 analogs HPIV VEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGGNLI 189 HPIV DP178 and/or DP107 analogs HPIV RSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV 190 HPIV DP178 and/or DP107 analogs HPIV NSVALDPIDISIELNKAKSDLEESKEWIRRSNQKL 191 HPIV DP178 and/or DP107 analogs HPIV ALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI 192 HPIV DP178 and/or DP107 analogs HPIV LDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIG 193 HPIV DP178 and/or DP107 analogs HPIV DPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN 194 HPIV DP178 and/or DP107 analogs HPIV PIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNW 195 HPIV DP178 and/or DP107 analogs HPIV IDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWH 196 MeV DP178 and/or DP107 analogs MeV HRIDLGPPISLERLDVGTNLGNAIAKLEAKELLE 197 MeV DP178 and/or DP107 analogs MeV IDLGPPISLERLDVGTNLGNAIAKLEAKELLESS 198 MeV DP178 and/or DP107 analogs MeV LGPPISLERLDVGTNLGNAIAKLEAKELESSDQ 199 MeV DP178 and/or DP107 analogs MeV PISLERLDVGTNLGNAIAKLEAKELESSDQILR 200 SIV DP178 and/or DP107 analogs SIV WQEWERKVDFLEENITALLEEAQIQQEKNMYELQK 201 SIV DP178 and/or DP107 analogs SIV QEWERKVDFLEENITALLEEAQIQQEKNMYELQKL 202 SIV DP178 and/or DP107 analogs SIV EWERKVDFLEENITALLEEAQIQQEKNMYELQKLN 203 SIV DP178 and/or DP107 analogs SIV WERKVDFLEENITALLEEAQIQQEKNMYELQKLNS 204 SIV DP178 and/or DP107 analogs SIV ERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW 205 SIV DP178 and/or DP107 analogs SIV RKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD 206 SIV DP178 and/or DP107 analogs SIV KVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV 207 SIV DP178 and/or DP107 analogs SIV VDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF 208 SIV DP178 and/or DP107 analogs SIV DFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG 209 SIV DP178 and/or DP107 analogs SIV FLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN 210 HIV DP178 peptide (AA 638-673 from gp41 of HIV-1_LAI isolate) HIV YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 211 HIV DP107 peptide (AA 558-595 from gp41 of HIV-1_LAI isolate) HIV NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ 212 HIV DP178 analog derived from the gp41 peptide region of HIV-1_SF2 HIV YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF 213 HIV DP178 analog derived from the gp41 peptide region of HIV-1_RF HIV YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF 214 HIV DP178 analog derived from the gp41 peptide region of HIV-1_MN HIV YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF 215 RSV DP178 and/or DP107 analogs RSV YTSWITIELSNIKENKCNGAKWKLIKQELDKYK 216 RSV DP178 and/or DP107 analogs RSV TSWITIELSNIKENKCNGAKWKLIKQELDKYKN 217 RSV DP178 and/or DP107 analogs RSV WITIELSNIKENKCNGAKWKLIKQELDKYKNAW 218 RSV DP178 and/or DP107 analogs RSV IALLSTNKAWWSLSNGWSWLTSKWLDLKNYIDK 219 HPIV DP178 and/or DP107 analogs HPIV VEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGGNLI 220 HPIV DP178 and/or DP107 analogs HPIV RSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV 221 HPIV DP178 and/or DP107 analogs HPIV NSVALDPIDISIELNKAKSDLEESKEWIRRSNOKL 222 HPIV DP178 and/or DP107 analogs HPIV ALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI 223 HPIV DP178 and/or DP107 analogs HPIV LDPIDISIELNKAKSDLEESKEWIRRSNOKLDSIG 224 HPIV DP178 and/or DP107 analogs HPIV DPIDISIELNKAKSDLEESKEWIRRSNOKLDSIGN 225 HPIV DP178 and/or DP107 analogs HPIV PIDISIELNKAKSDLEESKEWIRRSNOKLDSIGNW 226 HPIV DP178 and/or DP107 analogs HPIV IDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWE 227 MeV DP178 and/or DP107 analogs MeV HRIDLGPPISLERLDWGTNLGNAIAKLEAKELLE 228 MeV DP178 and/or DP107 analogs MeV IDLGPPISLERLDWGTNLGNAIAKLEAKELLESS 229 MeV DP178 and/or DP107 analogs MeV LGPPISLERLDWGTNLGNAIAKLEAKELESSDQ 230 MeV DP178 and/or DP107 analogs MeV PISLERLDWGTNLGNAIAKLEAKELLESSDQILR 231 SIV DP178 and/or DP107 analogs SIV WQEWERKVDFLEENITALLEEAQIQQEKNMYELQK 232 SIV DP178 and/or DP107 analogs SIV QEWERKVDFLEENITALLEEAQIQQEKNMYELQKL 233 SIV DP178 and/or DP107 analogs SIV EWERKVDFLEENITALLEEAQIQQEKNMYELQKLN 234 SIV DP178 and/or DP107 analogs SIV WERKVDFLEENITALLEEAQIQQEKNMYELQKLNS 235 SIV DP178 and/or DP107 analogs SIV ERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW 236 SIV DP178 and/or DP107 analogs SIV RKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD 237 SIV DP178 and/or DP107 analogs SIV KVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV 238 SIV DP178 and/or DP107 analogs SIV WDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF 239 SIV DP178 and/or DP107 analogs SIV DFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG 240 SIV DP178 and/or DP107 analogs SIV FLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN 241 HIV Enfuwedi (T-1249) HIV YTSLIHSLIEEESQNQQEKNEQELLELNKWA SLWNWF 242 HIV HIV CLLLGTEVSEALGGAGLT 243 RSV RSV DEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK 244 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 DITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN 245 RSV RSV DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL 246 HIV; influenza A HIV; influenza A DQYKCLQHGGFCLRSSCPSNTKLQGTCKPDKPNCCKS 247 RSV RSV EFDASISQVNEKINQSLAFIRKSDELLHNVNAGKS 248 RSV RSV FDASISQVNEKINQSLAFIRKSDELLHNVNAGKST 249 Chikungunya virus; Dengue virus Chikungunya virus; Dengue virus FLGAILKIGHALAKTVLPMVTNAFKPKQ 250 RSV RSV FPSDEFDASISQVNEKINQSLAFIRKSDELLHNVN 251 RSV RSV FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE 252 Influenza B virus: Hepatitis E virus Influenza B virus; Hepatitis E virus GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDKNYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGT 253 Dengue virus: Zika virus Dengue virus; Zika virus GFGCPLDQMQCHNHCQSVRYRGGYCTNFLKMTCKCY 254 HIV HIV GIFPKIIGKGIVNGIKSLAKGVGMKVFKAGLNNIGNTGCNNRDEC 255 HIV HIV HSLIEESQNQQEKNEQELLELDKWASLWNWFNITNW 256 RSV RSV IINFYDPLVFPSDEFDASISQVNEKINQSLAFIRK 257 HIV HIV IKKEIEAIKKEQEAIKKKIEAIEKEISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL 258 RSV RSV INFYDPLVFPSDEFDASISQVNEKINQSLAFIRKS 259 Hendra virus: Nipah virus Hendra virus: Nipah virus KVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSL 260 HIV HIV LGTEVSEALGGAGLTGGF 261 Measles virus: human parainfluenza virus type 3 Measles virus: human parainfluenza virus type 3 LHRIDLGPPISLERLDVGTNLGNAIAKLEDAKELL 262 HIV HIV LNNCLLLGTEVSEALGGA 263 RSV RSV LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHN 264 HIV HIV MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL 265 RSV RSV NFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSD 266 HIV HIV NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ 267 RSV RSV PLVFPSDEFDASISQVNEKINQSLAFIRKSDELLH 268 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 PPVYTKDVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPSL 269 RSV RSV PSDEFDASISQVNEKINQSLAFIRKSDELLHNVNA 270 HIV HIV RFPFHRCGAGPKLTKDLE 271 HIV HIV RSQKEGLHYTCSSHFPYSQYQFWK 272 RSV RSV SDEFDASISQVNEKINQSLAFIRKSDELLHNVNAG 273 HIV HIV SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL 274 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 SIELNKAKSDLEESKEWIRRSNQKLDSI 275 HIV HIV TTWEAWDRAIAEYAARIEALIRAAQEQQEKLEAALREL 276 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VALDPIDISIELNKAKSDLEESKEWIRR 277 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSD 278 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI 278 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VANDPIDISIELNKAKSDLEESKEWIRRSNQKLDSD 280 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VANDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI 281 RSV RSV VFPSDEFDASISQVNEKINQSLAFIRKSDELLHNV 282 Hendra virus; human parainfluenza virus type 3 Hendra virus; human parainfluenza virus type 3 VYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTV 283 HCV HCV WVAVTPTVATRDGKLPTT 284 RSV RSV YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL 285 HIV HIV YTSLIHSLIEEESQNQQEKNEQQLLELDKWASLWNWF 286 HIV-1 gp41 Albuviritide HIV Ac-WEEWDREINNYT(Mpa)LIHELIESQNQQEKNEQELL-CONH₂ (Mpa=3-maleimidepropionic acid) 287 CoV-OC43 EK1 CoV SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL 288 CoV-OC43 OC43-HR2P CoV SLDYINVTFLDLQDEMNRLQEAIKVLNQSYINLKDI 289 SARS-CoV-2 Wuhan-Hu-1 HR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 290 SARS-CoV-2 αHR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILARLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 291 SARS-CoV-2 βHR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 292 SARS-CoV-2 γHR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 293 SARS-CoV-2 δHR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 294 SARS-CoV-2 οHR1 CoV NVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 295 SARS-CoV-1 SARS-CoV1 HR1 CoV NVLYENQKQIANQFNKAISQIQESLTTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN 296 MERS MERS HR1 CoV QVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQ 297 SARS-CoV-2 Wuhan-Hu-1 HR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 298 SARS-CoV-2 αHR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 299 SARS-CoV-2 βHR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 300 SARS-CoV-2 γHR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASFVNIQKEIDRLNEVAKNLNESLIDLQEL 301 SARS-CoV-2 δHR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 302 SARS-CoV-2 οHR2 CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 303 SARS-CoV-1 SARS-CoV1 HR2 CoV VVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 304 MERS MERS HR2 CoV VTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL 305 SARS-CoV-2 HR2A CoV DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 306 SARS-CoV-2 HR2B CoV VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD 307 SARS-CoV-2 HR2C CoV FKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK 308 SARS-CoV-2 HR2 full length CoV GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL 309 SARS-CoV-2 HR2 full length Fc fusion CoV MGWSCIILFLVATATGVHS GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 310 SARS-CoV-2 HR2 full-length Fc fusion without message peptide CoV GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 311 HIV HIV_CHR HIV TTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 312 HIV T20 HIV YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 313 HIV T2410 HIV MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL 314 HIV T2410_2.0 HIV MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 315 HIV T1144 HIV TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL 316 HIV T144_2.0 HIV TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASLWNWF 317 HIV T1249 HIV WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF 318 HIV T1249_2.0 HIV TTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF 319 HIV T2635 HIV TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL 320 HIV T2635_2.0 HIV TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF 321 HIV T2635_3.0 HIV MTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF 322 HIV T290676 HIV TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL 323 HIV T290676_2.0 HIV TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF 324

在一些實施方式中，病毒係人免疫缺乏病毒（HIV）（例如，抗融合多肽抑制HIV的病毒進入）。在一些實施方式中，HIV係HIV-1。在一些實施方式中，HIV係HIV-1的毒株（例如，HIV-1_HE、HIV-1_IIIB、HIV-1_MN、HIV-1_NDK、HIV-1_NL4-3、HIV-1_RF或HIV-1_SF2）。在一些實施方式中，HIV係HIV-2。在一些實施方式中，HIV係HIV-2的毒株（例如，HIV-2_EHO或HIV-2_ROD）。在一些實施方式中，HIV係HIV假病毒體。在一些實施方式中，抗融合多肽藉由特異性結合HIV糖蛋白120（gp120）來防止HIV病毒融合。在一些實施方式中，抗融合多肽防止gp120與分化簇4（CD4）共受體的結合。在一些實施方式中，抗融合多肽降低gp120對共受體（例如，C-C趨化因子受體5型（CCR5）和C-X-C趨化因子受體4型（CXCR4））的親和力。在一些實施方式中，抗融合多肽防止gp120與共受體（例如，CCR5和CXCR4）的結合。在一些實施方式中，抗融合多肽藉由特異性結合HIV糖蛋白41（gp41）來防止HIV病毒融合。在一些實施方式中，抗融合多肽抑制HIV病毒核心進入細胞。在一些實施方式中，多核糖核苷酸貨物包括編碼SEQ ID NO: 1、5、11、13-18、22-38、40-46、49-56、58、59、61、66-78、80-85、87-91、95-100、112、113、153、163、64、168、169、171-184、211-215、242、243、247、255、256、258、261、261、265、267、271、272、274、276、286、287或312-324中任一個的多肽（例如，抑制HIV的病毒進入的多肽）之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 1、5、11、13-18、22-38、40-46、49-56、58、59、61、66-78、80-85、87-91、95-100、112、113、153、163、64、168、169、171-184、211-215、242、243、247、255、256、258、261、261、265、267、271、272、274、276、286、287或312-324中任一個的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the virus is human immunodeficiency virus (HIV) (eg, the anti-fusion polypeptide inhibits viral entry of HIV). In some embodiments, HIV is HIV-1. In some embodiments, HIV is a strain of HIV-1 (e.g., HIV-1_HE , HIV-1_IIIB , HIV-1_MN , HIV-1_NDK , HIV-1_NL4-3 , HIV-1_RF , or HIV -1_SF2 ). In some embodiments, HIV is HIV-2. In some embodiments, HIV is a strain of HIV-2 (eg, HIV-2_EHO or HIV-2_ROD ). In some embodiments, HIV is an HIV pseudovirion. In some embodiments, anti-fusion polypeptides prevent HIV viral fusion by specifically binding to HIV glycoprotein 120 (gp120). In some embodiments, the anti-fusion polypeptide prevents binding of gp120 to the cluster of differentiation 4 (CD4) co-receptor. In some embodiments, the anti-fusion polypeptide reduces the affinity of gp120 for coreceptors (eg, CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4)). In some embodiments, anti-fusion polypeptides prevent binding of gp120 to co-receptors (eg, CCR5 and CXCR4). In some embodiments, anti-fusion polypeptides prevent HIV viral fusion by specifically binding to HIV glycoprotein 41 (gp41). In some embodiments, the anti-fusion polypeptide inhibits HIV viral core entry into the cell. In some embodiments, the polyribonucleotide cargo includes encoding SEQ ID NOs: 1, 5, 11, 13-18, 22-38, 40-46, 49-56, 58, 59, 61, 66-78, 80-85, 87-91, 95-100, 112, 113, 153, 163, 64, 168, 169, 171-184, 211-215, 242, 243, 247, 255, 256, 258, 261, 261, The expression sequence of the polypeptide of any one of 265, 267, 271, 272, 274, 276, 286, 287 or 312-324 (for example, a polypeptide that inhibits viral entry of HIV). In some embodiments, the polyribonucleotide cargo includes encoding and SEQ ID NO: 1, 5, 11, 13-18, 22-38, 40-46, 49-56, 58, 59, 61, 66-78 ,80-85,87-91,95-100,112,113,153,163,64,168,169,171-184,211-215,242,243,247,255,256,258,261,261 The polypeptide of any one of , 265, 267, 271, 272, 274, 276, 286, 287 or 312-324 has at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity The expression sequence of a sexual polypeptide.

在一些實施方式中，病毒係肝炎病毒（例如，抗融合多肽抑制肝炎病毒的病毒進入）。在一些實施方式中，肝炎病毒係A型肝炎病毒（HAV）。在一些實施方式中，肝炎病毒係B型肝炎病毒（HBV）。在一些實施方式中，肝炎病毒係C型肝炎病毒（HCV）。在一些實施方式中，肝炎病毒係D型肝炎病毒（HDV）。在一些實施方式中，肝炎病毒係E型肝炎病毒（HEV）。在一些實施方式中，肝炎病毒係鴨肝炎病毒（DHV）。在一些實施方式中，多核糖核苷酸貨物包括編碼SEQ ID NO: 104、109、112、113、141-145、158-160或284中任一個的多肽（例如，抑制肝炎病毒如HCV的病毒進入的多肽）之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 104、109、112、113、141-145、158-160或284中任一個的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the virus is a hepatitis virus (eg, the anti-fusion polypeptide inhibits viral entry of a hepatitis virus). In some embodiments, the hepatitis virus is hepatitis A virus (HAV). In some embodiments, the hepatitis virus is hepatitis B virus (HBV). In some embodiments, the hepatitis virus is hepatitis C virus (HCV). In some embodiments, the hepatitis virus is hepatitis D virus (HDV). In some embodiments, the hepatitis virus is hepatitis E virus (HEV). In some embodiments, the hepatitis virus is duck hepatitis virus (DHV). In some embodiments, the polyribonucleotide cargo includes a polypeptide encoding any one of SEQ ID NO: 104, 109, 112, 113, 141-145, 158-160, or 284 (e.g., a virus that inhibits a hepatitis virus, such as HCV The expressed sequence of the incoming polypeptide). In some embodiments, the polyribonucleotide cargo includes encoding a polypeptide that is at least 85% identical (e.g., at least 90 %, 95%, 97%, 99% or 100%) sequence identity of the polypeptide.

在一些實施方式中，病毒係冠狀病毒如乙型冠狀病毒（例如，抗融合多肽抑制冠狀病毒的病毒進入）。在一些實施方式中，乙型冠狀病毒係SARS-CoV 1型（SARS-CoV-1）。在一些實施方式中，乙型冠狀病毒係SARS-CoV 2型（SARS-CoV-2）。在一些實施方式中，乙型冠狀病毒係表現SARS-CoV-2刺突蛋白的假型病毒。在一些實施方式中，多核糖核苷酸貨物包括編碼SEQ ID NO: 6-9、119-123、165-167或288-311中任一個的多肽（例如，抑制乙型冠狀病毒如SARS-CoV-1或SARS-CoV-2的病毒進入的多肽）之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 6-9、119-123、165-167或288-311中任一個的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the virus is a coronavirus, such as a betacoronavirus (eg, an anti-fusion polypeptide inhibits viral entry of a coronavirus). In some embodiments, the betacoronavirus is SARS-CoV type 1 (SARS-CoV-1). In some embodiments, the betacoronavirus is SARS-CoV type 2 (SARS-CoV-2). In some embodiments, the betacoronavirus is a pseudotyped virus expressing the SARS-CoV-2 spike protein. In some embodiments, the polyribonucleotide cargo includes a polypeptide encoding any of SEQ ID NOs: 6-9, 119-123, 165-167, or 288-311 (e.g., inhibiting a beta-coronavirus such as SARS-CoV -1 or SARS-CoV-2 viral entry polypeptide) expression sequence. In some embodiments, the polyribonucleotide cargo includes encoding a polypeptide that is at least 85% (e.g., at least 90%, The expressed sequence of a polypeptide with 95%, 97%, 99% or 100%) sequence identity.

在一些實施方式中，病毒係呼吸道合胞病毒（RSV）（例如，抗融合多肽抑制RSV的病毒進入）。在一些實施方式中，RSV係RSV A亞型（RSVA）。在一些實施方式中，RSV係RSV B亞型（RSVB）。在一些實施方式中，多核糖核苷酸貨物包括編碼SEQ ID NO: 11、13、39、112、113、153、185-188、216-219、244、246、247-249、251、252、257、259、264、266、268、270、273、282或285中任一個的多肽（例如，抑制RSV的病毒進入的多肽）之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 11、13、39、112、113、153、185-188、216-219、244、246、247-249、251、252、257、259、264、266、268、270、273、282或285中任一個的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the virus is respiratory syncytial virus (RSV) (eg, the anti-fusion polypeptide inhibits viral entry of RSV). In some embodiments, the RSV is RSV subtype A (RSVA). In some embodiments, the RSV is RSV subtype B (RSVB). In some embodiments, the polyribonucleotide cargo includes encoding SEQ ID NOs: 11, 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251, 252, The expressed sequence of the polypeptide of any one of 257, 259, 264, 266, 268, 270, 273, 282 or 285 (eg, a polypeptide that inhibits viral entry of RSV). In some embodiments, the polyribonucleotide cargo includes encoding and SEQ ID NO: 11, 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251, 252 The polypeptide of any one of , 257, 259, 264, 266, 268, 270, 273, 282 or 285 has at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity Expression sequence of polypeptide.

在一些實施方式中，病毒係流感（例如，季節性流感、大流行性流感、甲型流感、H1N1亞型流感、乙型流感、丙型流感、丁型流感）。在一些實施方式中，流感係甲型流感。在一些實施方式中，流感係乙型流感。在一些實施方式中，流感係丙型流感。在一些實施方式中，流感係丁型流感。在一些實施方式中，多核糖核苷酸貨物包括編碼SEQ ID NO: 1、4、11、245、247、253、262、269、275、277、278、279、280、281或283中任一個的多肽（例如，抑制流感的病毒進入的多肽）之表現序列。在一些實施方式中，多核糖核苷酸貨物包括編碼與SEQ ID NO: 1、4、11、245、247、253、262、269、275、277、278、279、280、281或283中任一個的多肽具有至少85%（例如，至少90%、95%、97%、99%或100%）序列同一性的多肽之表現序列。In some embodiments, the virus is influenza (eg, seasonal influenza, pandemic influenza, influenza A, H1N1 subtype, influenza B, influenza C, influenza D). In some embodiments, the influenza is influenza A. In some embodiments, the influenza is influenza B. In some embodiments, the influenza is influenza C. In some embodiments, the influenza is influenza type D. In some embodiments, the polyribonucleotide cargo includes encoding any one of SEQ ID NO: 1, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281, or 283 The expression sequence of a polypeptide (for example, a polypeptide that inhibits the entry of influenza virus). In some embodiments, the polyribonucleotide cargo includes a polyribonucleotide encoding a sequence corresponding to any of SEQ ID NO: 1, 4, 11, 245, 247, 253, 262, 269, 275, 277, 278, 279, 280, 281, or 283 The expressed sequence of a polypeptide having at least 85% (eg, at least 90%, 95%, 97%, 99%, or 100%) sequence identity.

在一些實施方式中，病毒係流感病毒（例如，抗融合多肽抑制流感的病毒進入）。在一些實施方式中，流感病毒係甲型流感病毒（IAV）。在一些實施方式中，流感病毒係乙型流感病毒（IBV）。在一些實施方式中，流感病毒係表現血凝素（HA）的流感病毒。In some embodiments, the virus is an influenza virus (eg, the anti-fusion polypeptide inhibits viral entry of influenza). In some embodiments, the influenza virus is influenza A virus (IAV). In some embodiments, the influenza virus is influenza B virus (IBV). In some embodiments, the influenza virus is a hemagglutinin (HA)-expressing influenza virus.

在一些實施方式中，病毒係單純皰疹病毒（HSV）（例如，抗融合多肽抑制HSV的病毒進入）。在一些實施方式中，HSV係HSV-1。在一些實施方式中，HSV係HSV-2。In some embodiments, the virus is herpes simplex virus (HSV) (eg, the anti-fusion polypeptide inhibits viral entry of HSV). In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2.

在一些實施方式中，病毒係人乳頭瘤病毒（HPV）（例如，抗融合多肽抑制HPV的病毒進入）。在一些實施方式中，HPV係高風險HPV毒株（例如，HPV 16、HPV 18、HPV 31、HPV 33、HPV 45、HPV 52或HPV 58）。在一些實施方式中，HPV係低風險HPV毒株（例如，HPV 6、HPV 11、HPV 42、HPV 43、HPV或44）。In some embodiments, the virus is human papillomavirus (HPV) (eg, the anti-fusion polypeptide inhibits viral entry of HPV). In some embodiments, the HPV is a high-risk HPV strain (eg, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, or HPV 58). In some embodiments, the HPV is a low-risk HPV strain (eg, HPV 6, HPV 11, HPV 42, HPV 43, HPV, or 44).

在一些實施方式中，病毒係表1中列出的任何病毒。In some embodiments, the virus is any of the viruses listed in Table 1.

在一些實施方式中，編碼抗融合多肽的核酸序列的GC含量為至少51%（例如，至少52%、53%、54%、55%、56%、57%、58%、59%或60%）。在一些實施方式中，編碼抗融合多肽的核酸序列的GC含量為至多52%、53%、54%、55%、56%、57%、58%或59%、或60%。在一些實施方式中，編碼抗融合多肽的核酸序列的GC含量為51%至60%、52%至60%、53%至60%、54%至60%、55%至60%、52%至58%、53%至58%。In some embodiments, the nucleic acid sequence encoding an anti-fusion polypeptide has a GC content of at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% ). In some embodiments, a nucleic acid sequence encoding an anti-fusion polypeptide has a GC content of at most 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%, or 60%. In some embodiments, the GC content of the nucleic acid sequence encoding the anti-fusion polypeptide is 51% to 60%, 52% to 60%, 53% to 60%, 54% to 60%, 55% to 60%, 52% to 58%, 53% to 58%.

在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量（對於RNA）或胸苷含量（對於DNA）為大於10%（例如，大於11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%或25%）。在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量（對於RNA）或胸苷含量（對於DNA）為至多30%（例如，至多29%、28%、27%、26%、25%、24%、23%、22%、21%或20%）。在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量（對於RNA）或胸苷含量（對於DNA）為20%至28%、21%至26%、10%至24%、15%至24%、20%至24%、21%至24%、22%至24%、23%至24%、10%至23%、15%至23%、20%至23%、21%至23%或22%至23%。In some embodiments, the nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content (for RNA) or a thymidine content (for DNA) of greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15 %, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%). In some embodiments, the nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content (for RNA) or a thymidine content (for DNA) of at most 30% (e.g., at most 29%, 28%, 27%, 26%, 25 %, 24%, 23%, 22%, 21% or 20%). In some embodiments, the nucleic acid sequence encoding the anti-fusion polypeptide has a uridine content (for RNA) or a thymidine content (for DNA) of 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23 % or 22% to 23%.

編碼抗融合多肽的表現序列之GC含量係指只編碼抗融合多肽的表現序列之GC含量，而沒有編碼除抗融合多肽之外的多肽之其他編碼區。同樣地，編碼抗融合多肽的表現序列之尿苷含量或胸苷係指只編碼抗融合多肽的表現序列之尿苷含量，而沒有編碼除抗融合多肽之外的多肽之其他編碼區。在一些實施方式中，編碼抗融合多肽的表現序列之GC含量或尿苷（或者胸苷）含量的計算僅考慮了從編碼抗融合多肽的開讀框起始密碼子的第一個核苷至同一開讀框終止密碼子的最後一個核苷的5'至3'方向開始的連續核酸序列。在其他實施方式中，編碼抗融合多肽的表現序列之GC含量或尿苷（或者胸苷）含量的計算僅考慮了從編碼抗融合多肽的N末端胺基酸殘基的密碼子的第一個核苷至編碼抗融合多肽的C末端胺基酸殘基的密碼子的最後一個核苷的5'至3'方向開始的連續核酸序列。The GC content of the expression sequence encoding the anti-fusion polypeptide refers to the GC content of the expression sequence encoding only the anti-fusion polypeptide without other coding regions encoding polypeptides other than the anti-fusion polypeptide. Likewise, the uridine content or thymidine content of an expression sequence encoding an anti-fusion polypeptide refers to the uridine content of an expression sequence encoding only an anti-fusion polypeptide without other coding regions encoding polypeptides other than the anti-fusion polypeptide. In some embodiments, the calculation of the GC content or uridine (or thymidine) content of the expression sequence encoding the anti-fusion polypeptide only takes into account the first nucleoside from the start codon of the open reading frame encoding the anti-fusion polypeptide to A continuous nucleic acid sequence starting from the 5' to 3' direction of the last nucleoside of the stop codon of the same open reading frame. In other embodiments, the calculation of the GC content or uridine (or thymidine) content of the expressed sequence encoding the anti-fusion polypeptide only takes into account the first codon from the N-terminal amino acid residue encoding the anti-fusion polypeptide. A contiguous nucleic acid sequence starting in the 5' to 3' direction from the last nucleoside to the codon encoding the C-terminal amino acid residue of the anti-fusion polypeptide.

在一些實施方式中，編碼抗融合多肽的核酸序列具有多於20%的尿苷含量。在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量為大於10%（例如，大於11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%或25%）。在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量為最多30%（例如，最多29%、28%、27%、26%、25%、24%、23%、22%、21%或20%）。在一些實施方式中，編碼抗融合多肽的核酸序列的尿苷含量為20%至28%、21%至26%、10%至24%、15%至24%、20%至24%、21%至24%、22%至24%、23%至24%、10%至23%、15%至23%、20%至23%、21%至23%或22%至23%。在一些實施方式中，編碼抗融合多肽的核酸序列具有20%至28%的尿苷含量。多個抗融合多肽In some embodiments, a nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content of greater than 20%. In some embodiments, the nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content of greater than 10% (e.g., greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19 %, 20%, 21%, 22%, 23%, 24% or 25%). In some embodiments, the nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content of up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 % or 20%). In some embodiments, the nucleic acid sequence encoding the anti-fusion polypeptide has a uridine content of 20% to 28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to 23%, 20% to 23%, 21% to 23% or 22% to 23%. In some embodiments, a nucleic acid sequence encoding an anti-fusion polypeptide has a uridine content of 20% to 28%.Multiple anti-fusion peptides

在一些實施方式中，環狀多核糖核苷酸編碼多個各自編碼抗融合多肽的表現序列（例如，兩個或更多個，例如2至100、2至50、2至20、2至10、5至100、5至50、5至20或5至10個表現序列）。In some embodiments, the cyclic polyribonucleotide encodes a plurality of expressed sequences (e.g., two or more, e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10) each encoding an anti-fusion polypeptide. , 5 to 100, 5 to 50, 5 to 20 or 5 to 10 performance sequences).

在一些實施方式中，環狀多核糖核苷酸編碼兩個或更多個（例如，2至100、2至50、2至20、2至10、5至100、5至50、5至20或5至10個）拷貝的相同抗融合多肽。In some embodiments, the cyclic polyribonucleotide encodes two or more (e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20 or 5 to 10) copies of the same anti-fusion polypeptide.

在一些實施方式中，環狀多核糖核苷酸編碼兩個或更多個（例如2、3、4、5、6、7、8、9或10個）不同的（例如，共用小於100%的序列同一性）抗融合多肽。在一些實施方式中，兩個或更多個不同的抗融合多肽各自選自表1的抗融合多肽。在一些實施方式中，兩個或更多個不同的抗融合多肽各自抑制不同的病毒。例如，環狀多核糖核苷酸可以編碼抑制流感的第一抗融合多肽和抑制RSV的第二抗融合多肽。環狀多核糖核苷酸可以包括抑制流感的第一抗融合多肽和抑制SARS-CoV-2的第二抗融合多肽。環狀多核糖核苷酸可以包括抑制HIV的第一抗融合多肽和抑制SARS-CoV-2的第二抗融合多肽。環狀多核糖核苷酸可以包括抑制HIV的第一抗融合多肽和抑制HCV的第二抗融合多肽。當第一或第二抗融合多肽抑制多種病毒時，第一和第二抗融合多肽可以具有不同的病毒特異性。In some embodiments, the cyclic polyribonucleotides encode two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) different (eg, share less than 100% sequence identity) anti-fusion polypeptide. In some embodiments, the two or more different anti-fusion polypeptides are each selected from the anti-fusion polypeptides of Table 1. In some embodiments, two or more different anti-fusion polypeptides each inhibit a different virus. For example, a cyclic polyribonucleotide may encode a first anti-fusion polypeptide that inhibits influenza and a second anti-fusion polypeptide that inhibits RSV. The cyclic polyribonucleotide may include a first anti-fusion polypeptide that inhibits influenza and a second anti-fusion polypeptide that inhibits SARS-CoV-2. The cyclic polyribonucleotide may include a first anti-fusion polypeptide that inhibits HIV and a second anti-fusion polypeptide that inhibits SARS-CoV-2. The cyclic polyribonucleotide may include a first anti-fusion polypeptide that inhibits HIV and a second anti-fusion polypeptide that inhibits HCV. When the first or second anti-fusion polypeptide inhibits multiple viruses, the first and second anti-fusion polypeptides can have different virus specificities.

其中環狀多核糖核苷酸編碼兩個或更多個抗融合多肽，該等抗融合多肽可以在單個開讀框或多個開讀框中編碼。The cyclic polyribonucleotide encodes two or more anti-fusion polypeptides, and the anti-fusion polypeptides can be encoded in a single open reading frame or multiple open reading frames.

在一些實施方式中，本揭露提供了包括開讀框（例如，可操作地連接到IRES的開讀框）的環狀多核糖核苷酸，其包括兩個或更多個表現序列，其中每個表現序列編碼抗融合多肽。在一些實施方式中，開讀框的翻譯產生包括兩個或更多個抗融合多肽的多肽融合物。抗融合多肽可以例如藉由本文所述之連接子（例如，由開讀框編碼的肽連接子，如下面關於肽-Fc融合所述之甘胺酸-絲胺酸連接子）連接。在一些實施方式中，抗融合多肽可以由切割結構域（例如交錯序列）隔開，例如如本文所述之。In some embodiments, the present disclosure provides circular polyribonucleotides that include an open reading frame (e.g., an open reading frame operably linked to an IRES) that includes two or more expression sequences, wherein each The expressed sequence encodes an anti-fusion polypeptide. In some embodiments, translation of the open reading frame results in a polypeptide fusion including two or more anti-fusion polypeptides. Anti-fusion polypeptides can be linked, for example, via linkers described herein (eg, a peptide linker encoded by an open reading frame, such as the glycine-serine linker described below for peptide-Fc fusions). In some embodiments, anti-fusion polypeptides may be separated by cleavage domains (eg, staggered sequences), for example, as described herein.

在一些實施方式中，本揭露提供了環狀多核糖核苷酸，其包括編碼第一抗融合多肽（例如，可操作地連接到第一IRES）的第一開讀框和編碼第二抗融合多肽（例如，可操作地連接到第二IRES）的第二開讀框。抗融合多肽-Fc融合In some embodiments, the disclosure provides a cyclic polyribonucleotide comprising a first open reading frame encoding a first anti-fusion polypeptide (e.g., operably linked to a first IRES) and a second anti-fusion polypeptide encoding The second open reading frame of the polypeptide (e.g., operably linked to a second IRES).Anti-fusion peptide-Fcfusion

在一些實施方式中，環狀多核糖核苷酸包含編碼融合蛋白（包含抗融合多肽）之表現序列。在一些實施方式中，融合蛋白包括與免疫球蛋白的Fc結構域（例如，Fc結構域的單鏈）融合的抗融合多肽。在一些實施方式中，抗融合多肽選自表1。在一些實施方式中，環狀多核糖核苷酸包含編碼多於一個融合蛋白（包含抗融合多肽）之表現序列。在一些實施方式中，環狀多核糖核苷酸包含編碼多於一個抗融合多肽之表現序列。在一些實施方式中，編碼多於一個融合蛋白的環狀多核糖核苷酸減少了病毒逃逸的機會。In some embodiments, the cyclic polyribonucleotide comprises an expressed sequence encoding a fusion protein (including an anti-fusion polypeptide). In some embodiments, the fusion protein includes an anti-fusion polypeptide fused to the Fc domain of an immunoglobulin (eg, a single chain of the Fc domain). In some embodiments, the anti-fusion polypeptide is selected from Table 1. In some embodiments, cyclic polyribonucleotides comprise expressed sequences encoding more than one fusion protein, including anti-fusion polypeptides. In some embodiments, cyclic polyribonucleotides comprise expression sequences encoding more than one anti-fusion polypeptide. In some embodiments, cyclic polyribonucleotides encoding more than one fusion protein reduce the chance of viral escape.

在一些實施方式中，Fc結構域係IgG4 Fc結構域或其片段。在一些實施方式中，Fc結構域係IgG1 Fc結構域或其片段。在一些實施方式中，Fc結構域係IgG2 Fc結構域或其片段。在一些實施方式中，Fc結構域係IgG2a Fc結構域或其片段。在一些實施方式中，Fc結構域係IgG2b Fc結構域或其片段。在一些實施方式中，Fc結構域係IgG3 Fc結構域或其片段。In some embodiments, the Fc domain is an IgG4 Fc domain or fragment thereof. In some embodiments, the Fc domain is an IgG1 Fc domain or fragment thereof. In some embodiments, the Fc domain is an IgG2 Fc domain or fragment thereof. In some embodiments, the Fc domain is an IgG2a Fc domain or fragment thereof. In some embodiments, the Fc domain is an IgG2b Fc domain or fragment thereof. In some embodiments, the Fc domain is an IgG3 Fc domain or fragment thereof.

在一些實施方式中，抗融合多肽的C-末端胺基酸殘基與Fc結構域的N-末端胺基酸殘基融合，視需要通過肽連接子。在一些實施方式中，抗融合多肽的N-末端胺基酸殘基與Fc結構域的C-末端胺基酸殘基融合，視需要通過肽連接子。在一些實施方式中，抗融合多肽和Fc結構域之間的肽連接子包括至少兩個胺基酸殘基（例如，至少3、至少4、至少5、至少6、至少7、至少8、至少9、至少10、至少15或至少20個胺基酸殘基）。在一些實施方式中，抗融合多肽和Fc結構域之間的肽連接子包括2-200個胺基酸殘基（例如，2-200、2-180、2-160、2-140、2-120、2-100、2-90、2-80、2-70、2-60、2-50、2-45、2-40、2-35、2-30、2-25、2-20、2-15、2-10、2-9、2-8、2-7、2-6、2-5、2-4、4-200、5-200、6-200、7-200、8-200、9-200、10-200、15-200、20-200、25-200、30-200、35-200、40-200、45-200、50-200、60-200、70-200、80-200、90-200、100-200、120-200、140-200、160-200或180-200個胺基酸殘基）。在一些實施方式中，肽連接子由甘胺酸（Gly）和絲胺酸（Ser）殘基組成。在一些實施方式中，肽連接子包括(GS)_x、(GGS)_x、(GGGGS)_x、(GGSG)_x或(SGGG)_x中任一個的胺基酸序列，其中x係1至50的整數（例如，1-40、1-30、1-20、1-10或1-5）。在一些實施方式中，肽連接子包括(GS)_x、(GGS)_x、(GGGGS)_x、(GGSG)_x或(SGGG)_x中任一個的胺基酸序列，其中x係1至10的整數（例如，1、2、3、4、5、6、7、8、9或10）。在一些實施方式中，肽連接子包括6至36個胺基酸。在一些實施方式中，肽連接子包括21至31個胺基酸。多核糖核苷酸貨物In some embodiments, the C-terminal amino acid residue of the anti-fusion polypeptide is fused to the N-terminal amino acid residue of the Fc domain, optionally via a peptide linker. In some embodiments, the N-terminal amino acid residue of the anti-fusion polypeptide is fused to the C-terminal amino acid residue of the Fc domain, optionally via a peptide linker. In some embodiments, the peptide linker between the anti-fusion polypeptide and the Fc domain includes at least two amino acid residues (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9. At least 10, at least 15 or at least 20 amino acid residues). In some embodiments, the peptide linker between the anti-fusion polypeptide and the Fc domain includes 2-200 amino acid residues (e.g., 2-200, 2-180, 2-160, 2-140, 2- 120, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 4-200, 5-200, 6-200, 7-200, 8- 200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200 or 180-200 amino acid residues). In some embodiments, the peptide linker consists of glycine (Gly) and serine (Ser) residues. In some embodiments, the peptide linker includes the amino acid sequence of any one of (GS)_x , (GGS)_x , (GGGGS)_x , (GGSG)_x , or (SGGG)_x , wherein x is from 1 to 50 An integer (for example, 1-40, 1-30, 1-20, 1-10, or 1-5). In some embodiments, the peptide linker includes the amino acid sequence of any one of (GS)_x , (GGS)_x , (GGGGS)_x , (GGSG)_x , or (SGGG)_x , wherein x is from 1 to 10 An integer (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker includes 6 to 36 amino acids. In some embodiments, the peptide linker includes 21 to 31 amino acids.polyribonucleotide cargo

本文所述之多核糖核苷酸貨物包括包含至少一個多核糖核苷酸的任何序列。在一些實施方式中，多核糖核苷酸貨物包括表現序列、非編碼序列、或表現序列和非編碼序列。在一些實施方式中，多核糖核苷酸貨物包括編碼抗融合多肽之表現序列。在一些實施方式中，該多核糖核苷酸貨物包括與編碼抗融合多肽的表現序列可操作地連接的IRES。在一些實施方式中，多核糖核苷酸貨物包括編碼對受試者具有生物學效應的抗融合多肽之表現序列。Polyribonucleotide cargos as described herein include any sequence comprising at least one polyribonucleotide. In some embodiments, the polyribonucleotide cargo includes expression sequences, non-coding sequences, or expression sequences and non-coding sequences. In some embodiments, the polyribonucleotide cargo includes expression sequences encoding anti-fusion polypeptides. In some embodiments, the polyribonucleotide cargo includes an IRES operably linked to an expression sequence encoding an anti-fusion polypeptide. In some embodiments, the polyribonucleotide cargo includes expression sequences encoding anti-fusion polypeptides that have a biological effect on a subject.

例如，多核糖核苷酸貨物可以包括至少約40個核苷酸、至少約50個核苷酸、至少約75個核苷酸、至少約100個核苷酸、至少約200個核苷酸、至少約300個核苷酸、至少約400個核苷酸、至少約500個核苷酸、至少約1,000個核苷酸、至少約2,000個核苷酸、至少約5,000個核苷酸、至少約6,000個核苷酸、至少約7,000個核苷酸、至少約8,000個核苷酸、至少約9,000個核苷酸、至少約10,000個核苷酸、至少約12,000個核苷酸、至少約14,000個核苷酸、至少約15,000個核苷酸、至少約16,000個核苷酸、至少約17,000個核苷酸、至少約18,000個核苷酸、至少約19,000個核苷酸、或至少約20,000個核苷酸。在一些實施方式中，多核糖核苷酸貨物包括1-20,000個核苷酸、1-10,000個核苷酸、1-5,000個核苷酸、100-20,000個核苷酸、100-10,000個核苷酸、100-5,000個核苷酸、500-20,000個核苷酸、500-10,000個核苷酸、500-5,000個核苷酸、1,000-20,000個核苷酸、1,000-10,000個核苷酸或1,000-5,000個核苷酸。For example, a polyribonucleotide cargo can include at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, At least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides Nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nuclei glycosides. In some embodiments, the polyribonucleotide cargo includes 1-20,000 nucleotides, 1-10,000 nucleotides, 1-5,000 nucleotides, 100-20,000 nucleotides, 100-10,000 nuclei nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-10,000 nucleotides, 500-5,000 nucleotides, 1,000-20,000 nucleotides, 1,000-10,000 nucleotides Or 1,000-5,000 nucleotides.

在實施方式中，多核糖核苷酸貨物包括一或多個表現（或編碼）序列，其中每個表現（或編碼）序列編碼多肽（例如抗融合多肽）。在實施方式中，多核糖核苷酸貨物包括一或多個非編碼序列。在實施方式中，多核糖核苷酸貨物完全由一或多個非編碼序列組成。在實施方式中，多核糖核苷酸貨物包括表現（或編碼）和非編碼序列的組合。In embodiments, a polyribonucleotide cargo includes one or more expression (or coding) sequences, wherein each expression (or coding) sequence encodes a polypeptide (eg, an anti-fusion polypeptide). In embodiments, the polyribonucleotide cargo includes one or more non-coding sequences. In embodiments, the polyribonucleotide cargo consists entirely of one or more non-coding sequences. In embodiments, the polyribonucleotide cargo includes a combination of expression (or coding) and non-coding sequences.

在一些實施方式中，多核糖核苷酸包含如國際專利揭露號WO 2019/118919中揭露的任何特徵或特徵的任何組合，該文獻特此藉由援引以其全文併入。多肽表現序列In some embodiments, the polyribonucleotides comprise any feature or any combination of features as disclosed in International Patent Publication No. WO 2019/118919, which document is hereby incorporated by reference in its entirety.polypeptide expression sequence

在一些實施方式中，本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸的多核糖核苷酸貨物）包括一或多個表現（或編碼）序列，其中每個表現序列編碼抗融合多肽。在一些實施方式中，環狀多核糖核苷酸包含兩個、三個、四個、五個、六個、七個、八個、九個、十個或更多個表現（或編碼）序列。In some embodiments, polyribonucleotides (e.g., polyribonucleotide cargos of cyclic polyribonucleotides) described herein include one or more expression (or coding) sequences, wherein each expression sequence Encodes anti-fusion polypeptides. In some embodiments, cyclic polyribonucleotides comprise two, three, four, five, six, seven, eight, nine, ten or more expression (or coding) sequences .

每個編碼的多肽可為直鏈或支鏈的。在各種實施方式中，多肽具有約5至約40,000個胺基酸、約15至約35,000個胺基酸、約20至約30,000個胺基酸、約25至約25,000個胺基酸、約50至約20,000個胺基酸、約100至約15,000個胺基酸、約200至約10,000個胺基酸、約500至約5,000個胺基酸、約1,000至約2,500個胺基酸或它們之間的任何範圍的長度。在一些實施方式中，長度少於約40,000個胺基酸、少於約35,000個胺基酸、少於約30,000個胺基酸、少於約25,000個胺基酸、少於約20,000個胺基酸、少於約15,000個胺基酸、少於約10,000個胺基酸、少於約9,000個胺基酸、少於約8,000個胺基酸、少於約7,000個胺基酸、少於約6,000個胺基酸、少於約5,000個胺基酸、少於約4,000個胺基酸、少於約3,000個胺基酸、少於約2,500個胺基酸、少於約2,000個胺基酸、少於約1,500個胺基酸、少於約1,000個胺基酸、少於約900個胺基酸、少於約800個胺基酸、少於約700個胺基酸、少於約600個胺基酸、少於約500個胺基酸、少於約400個胺基酸、少於約300個胺基酸或更少的多肽可能是有用的。Each encoded polypeptide may be linear or branched. In various embodiments, the polypeptide has about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any combination thereof any range of lengths in between. In some embodiments, the length is less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids. Acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids , less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 Polypeptides having less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less may be useful.

本文包括的多肽可包括天然存在的多肽或非天然存在的多肽。在一些實施方式中，該多肽係或包括參考多肽的功能性片段或變體（例如，抗融合多肽的生物活性片段或變體）。例如，該多肽可為本文所述之任一多肽的功能活性變體，例如在指定區域或整個序列上與本文所述之多肽或天然存在的多肽之序列具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或99%同一性。在一些情況下，多肽可以與目的蛋白具有至少50%（例如，至少50%、60%、70%、80%、90%、95%、97%、99%、或更大）同一性。Polypeptides included herein may include naturally occurring polypeptides or non-naturally occurring polypeptides. In some embodiments, the polypeptide is or includes a functional fragment or variant of a reference polypeptide (eg, a biologically active fragment or variant of an anti-fusion polypeptide). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein, such as at least 70%, 71%, 72% identical to the sequence of a polypeptide described herein or a naturally occurring polypeptide in a specified region or the entire sequence. %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some cases, the polypeptide may be at least 50% (eg, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater) identical to the protein of interest.

在實施方式中，多肽包括多個多肽，例如，一個多肽序列的多個拷貝、或多個不同的多肽序列。在實施方式中，多個多肽藉由連接子胺基酸或間隔胺基酸連接。In embodiments, a polypeptide includes multiple polypeptides, eg, multiple copies of one polypeptide sequence, or multiple different polypeptide sequences. In embodiments, multiple polypeptides are linked by linker amino acids or spacer amino acids.

在實施方式中，多核苷酸貨物包括編碼訊息肽的序列。已經描述了許多訊息肽序列，例如，Tat（雙精胺酸易位）訊息序列通常是含有共有SRRxFLK「雙精胺酸」模體的N末端肽序列，其用於將含有這種Tat訊息肽的折疊蛋白易位穿過脂質雙層。還參見例如，在www[dot]signalpeptide[dot]de上可公開獲得的訊息肽數據庫。訊息肽也可用於將蛋白質導向特定的細胞器；參見例如，Spdb訊息肽數據庫中揭露的實驗確定和計算預測的訊息肽，該數據庫可在proline[dot]bic[dot]nus[dot]edu[dot]sg/spdb公開獲得。In embodiments, the polynucleotide cargo includes a sequence encoding a message peptide. Many message peptide sequences have been described. For example, the Tat (twins-arginine translocation) message sequence is usually an N-terminal peptide sequence containing the consensus SRRxFLK "twins-arginine" motif, which is used to integrate this Tat message peptide. Folded proteins translocate across the lipid bilayer. See also, for example, the signal peptide database publicly available at www[dot]signalpeptide[dot]de. Message peptides can also be used to target proteins to specific organelles; see, for example, the experimentally determined and computationally predicted message peptides disclosed in the Spdb Message Peptide Database, available at proline[dot]bic[dot]nus[dot]edu[ dot]sg/spdb is publicly available.

在一些實施方式中，表現（或編碼）序列包括poly-A序列（例如，在表現序列的3'端）。在一些實施方式中，聚A序列之長度大於10個核苷酸。在一個實施方式中，聚A序列之長度大於15個核苷酸（例如，至少或大於約10、15、20、25、30、35、40、45、50、55、60、70、80、90、100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1,900、2,000、2,500、和3,000個核苷酸）。在一些實施方式中，根據國際專利公開案號WO 2019/118919 A1的[0202]-[0204]中對聚A序列的描述來設計聚A序列，將其藉由援引以其全文併入本文。在一些實施方式中，表現序列缺少聚A序列（例如，在表現序列的3'端）。In some embodiments, the presentation (or coding) sequence includes a poly-A sequence (eg, at the 3' end of the presentation sequence). In some embodiments, the polyA sequence is greater than 10 nucleotides in length. In one embodiment, the polyA sequence is greater than 15 nucleotides in length (e.g., at least or greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequences are designed according to the description of poly-A sequences in International Patent Publication No. WO 2019/118919 A1, [0202]-[0204], which is incorporated herein by reference in its entirety. In some embodiments, the presentation sequence lacks a polyA sequence (eg, at the 3' end of the presentation sequence).

在一些實施方式中，環狀多核糖核苷酸包含聚A、缺少聚A或具有經修飾的聚A以調節環狀多核糖核苷酸的一或多種特徵。在一些實施方式中，缺少聚A或具有經修飾的聚A的環狀多核糖核苷酸改善了一或多種功能特徵，例如免疫性（例如，免疫或炎性響應的一或多種標誌物的水平）、半衰期和/或表現效率。內部核糖體進入位點In some embodiments, the cyclic polyribonucleotide comprises polyA, lacks polyA, or has polyA modified to modulate one or more characteristics of the cyclic polyribonucleotide. In some embodiments, cyclic polyribonucleotides lacking polyA or having modified polyA improve one or more functional characteristics, such as immunity (e.g., one or more markers of immune or inflammatory response levels), half-life and/or performance efficiency.internal ribosome entry site

在一些實施方式中，本文所述之環狀多核糖核苷酸包含一或多個內部核糖體進入位點（IRES）元件。在一些實施方式中，IRES與一或多個表現序列可操作地連接（例如，每個IRES與一或多個表現序列可操作地連接）。在實施方式中，IRES位於異源啟動子與編碼序列的5'端之間。In some embodiments, the cyclic polyribonucleotides described herein comprise one or more internal ribosome entry site (IRES) elements. In some embodiments, an IRES is operably linked to one or more expression sequences (eg, each IRES is operably linked to one or more expression sequences). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence.

包括在多核糖核苷酸中的合適的IRES元件包括能夠接合真核核糖體的RNA序列。在一些實施方式中，IRES元件係至少約5 nt、至少約8 nt、至少約9 nt、至少約10 nt、至少約15 nt、至少約20 nt、至少約25 nt、至少約30 nt、至少約40 nt、至少約50 nt、至少約100 nt、至少約200 nt、至少約250 nt、至少約350 nt或至少約500 nt。Suitable IRES elements for inclusion in polyribonucleotides include RNA sequences capable of engaging eukaryotic ribosomes. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least About 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

在一些實施方式中，IRES元件衍生自生物體的DNA，該生物體包括但不限於病毒、哺乳動物和果蠅。這樣的病毒DNA可以衍生自但不限於小核糖核酸病毒互補DNA（cDNA）、腦心肌炎病毒（EMCV）cDNA和脊髓灰質炎病毒cDNA。在一個實施方式中，衍生IRES元件的果蠅DNA包括但不限於來自黑腹果蠅（Drosophila melanogaster）的觸角足基因。In some embodiments, IRES elements are derived from the DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA may be derived from, but is not limited to, picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

在一些實施方式中，如果存在，IRES序列係以下病毒的IRES序列：桃拉綜合症（Taura syndrome）病毒、錐獵蝽（Triatoma）病毒、泰勒氏腦脊髓炎病毒（Theiler's encephalomyelitis virus）、猿猴病毒40、紅火蟻（Solenopsis invicta）病毒1、禾穀縊管蚜（Rhopalosiphum padi）病毒、網狀內皮組織增生病毒、福爾曼脊髓灰質炎病毒（fuman poliovirus）1、普勞提婭失速腸病毒（Plautia stall intestine virus）、喀什米爾蜜蜂病毒、人鼻病毒2（HRV-2）、假桃病毒葉蟬病毒-1（Homalodisca coagulata virus-1）、人免疫缺乏病毒1型、假桃病毒葉蟬病毒-1、Himetobi P病毒、C型肝炎病毒、A型肝炎病毒、GB型肝炎病毒、口蹄疫病毒、人類腸道病毒71、馬鼻炎病毒、茶尺蠖（Ectropis obliqua）、小核糖核酸樣病毒、腦心肌炎病毒（EMCV）、果蠅C病毒、十字花科煙草病毒、蟋蟀麻痹病毒、牛病毒性腹瀉病毒1、黑皇后細胞病毒、蚜蟲致死性麻痹病毒、禽腦脊髓炎病毒（AEV）、急性蜜蜂麻痹病毒、木槿褪綠環斑病毒（Hibiscus chlorotic ringspot virus）、經典豬瘟病毒、人FGF2、人SFTPA1、人AML1/RUNX1、果蠅觸角足、人AQP4、人AT1R、人BAG-l、人BCL2、人BiP、人c-IAPl、人c-myc、人eIF4G、小鼠NDST4L、人LEF1、小鼠HIF1α、人n.myc、小鼠Gtx、人p27kipl、人PDGF2/c-sis、人p53、人Pim-l、小鼠Rbm3、果蠅reaper、犬Scamper、果蠅Ubx、人UNR、小鼠UtrA、人VEGF-A、人XIAP、薩里病毒（Salivirus）、科薩病毒（Cosavirus）、副腸孤病毒（Parechovirus）、果蠅無毛、釀酒酵母（S. cerevisiae）TFIID、釀酒酵母YAP1、人c-src、人FGF-l、猿猴小核糖核酸病毒、蕪菁皺縮病毒（Turnip crinkle virus）、愛知病毒（Aichivirus）、克羅希病毒（Crohivirus）、埃可病毒（Echovirus）11、eIF4G適配體、柯薩奇病毒（Coxsackievirus）B3（CVB3）或柯薩奇病毒A（CVB1/2）。在又另一實施方式中，IRES係柯薩奇病毒B3（CVB3）的IRES序列。在另外的實施方式中，IRES係腦心肌炎病毒的IRES序列。在另外的實施方式中，IRES係蒂勒腦脊髓炎病毒的IRES序列。In some embodiments, if present, the IRES sequence is that of the following viruses: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian virus 40. Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliosis virus, fuman poliovirus 1, Plautia stall enterovirus ( Plautia stall intestine virus), Kashmir bee virus, human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, human immunodeficiency virus type 1, pseudopeach virus leafhopper virus -1. Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot and mouth disease virus, human enterovirus 71, equine rhinitis virus, Ectropis obliqua, picornavirus, encephalomyocarditis Virus (EMCV), Drosophila C virus, cruciferous tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus (AEV), acute bee paralysis Viruses, Hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-l, human BCL2, Human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-l, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Salivirus, Cosavirus, accessory gut Parechovirus, Drosophila hairless, S. cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-l, simian picornavirus, Turnip crinkle virus, Aichivirus, Crohivirus, Echovirus 11, eIF4G aptamer, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1/2). In yet another embodiment, the IRES is the IRES sequence of coxsackievirus B3 (CVB3). In additional embodiments, the IRES is an IRES sequence of an encephalomyocarditis virus. In additional embodiments, the IRES is an IRES sequence of Tiller's encephalomyelitis virus.

與野生型IRES序列相比，IRES序列可具有經修飾的序列。在一些實施方式中，當野生型IRES的最後一個核苷酸不是胞嘧啶核酸殘基時，可以修飾野生型IRES序列的最後一個核苷酸，使得其是胞嘧啶殘基。例如，IRES序列可為CVB3 IRES序列，其中將末端腺苷殘基修飾為胞嘧啶殘基。在一些實施方式中，經修飾的CVB3 IRES可具有以下的核酸序列： TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGAT CAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTA TCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGA TGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCC TCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTG TGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAA CATTACAATTCATTGTTAAGTTGAATACAGCAAC (SEQ ID NO: 325)。The IRES sequence may have a modified sequence compared to a wild-type IRES sequence. In some embodiments, when the last nucleotide of a wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence can be modified such that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence in which the terminal adenosine residue is modified to a cytosine residue. In some embodiments, the modified CVB3 IRES can have the following nucleic acid sequence: TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGAT CAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTA TCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTTCGAAAAACCTAGTA ACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGA TGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCC TCCGGCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTG TGTCGTAACG GGCAACTCTGCAGCGGAACCGACTACTTTGGGTTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAA CATTACAATTCATTGTTAAGTTGAATACAGCAAC (SEQ ID NO: 325).

在一些實施方式中，IRES序列係腸道病毒71（EV17）IRES。在一些實施方式中，將EV17 IRES序列的末端鳥苷殘基修飾為胞嘧啶殘基。在一些實施方式中，經修飾的EV71 IRES可具有以下的核酸序列： ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTC CTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGT TCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCG GCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATA (SEQ ID NO: 326)。In some embodiments, the IRES sequence is an enterovirus 71 (EV17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES can have the following nucleic acid sequence: ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAAACCTGGCCCTGTCTTTCTTGACGAGCATTC CTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGT TCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAAACCCCCACC TGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCG GCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGGACGTGGTTT TCCTTTGAAAAACACGATGATAATA (SEQ ID NO: 326).

在一些實施方式中，多核糖核苷酸包含側接至少一個（例如2、3、4、5個或更多個）表現序列的至少一個IRES。在一些實施方式中，IRES側接至少一個（例如2、3、4、5或更多個）表現序列的兩側。在一些實施方式中，多核糖核苷酸在每個表現序列的一側或兩側包括一或多個IRES序列，導致所得的一或多種肽和或一或多種多肽的隔開。例如，本文所述之多核糖核苷酸可以包括與第一表現序列可操作地連接的第一IRES和與第二表現序列可操作地連接的第二IRES。In some embodiments, a polyribonucleotide comprises at least one IRES flanked by at least one (eg, 2, 3, 4, 5, or more) expression sequences. In some embodiments, an IRES is flanked by at least one (eg, 2, 3, 4, 5, or more) expressed sequences. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expressed sequence, resulting in spacing of the resulting peptide(s) and/or polypeptide(s). For example, a polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.

在一些實施方式中，本文所述之多核糖核苷酸包含IRES（例如，與編碼區可操作地連接的IRES）。例如，多核糖核苷酸可以包括如在以下中描述的任何IRES：Chen等人 Mol. Cell [分子細胞] 81(20):4300-18, 2021；Jopling等人 Oncogene [致癌基因] 20:2664-70, 2001；Baranick等人 PNAS [美國國家科學院院刊] 105(12):4733-38, 2008；Lang等人 Molecular Biology of the Cell [細胞分子生物學] 13(5):1792-1801, 2002；Dorokhov等人 PNAS [美國國家科學院院刊] 99(8):5301-06, 2002；Wang等人 Nucleic Acids Research [核酸研究] 33(7):2248-58, 2005；Petz等人 Nucleic Acids Research [核酸研究] 35(8):2473-82, 2007；Chen等人 Science [科學] 268:415-417, 1995；Fan等人 Nature Communication [自然通訊] 13(1):3751-3765, 2022和國際公開案號WO 2021/263124，它們各自特此藉由援引以其全文併入。訊息序列In some embodiments, the polyribonucleotides described herein comprise an IRES (eg, an IRES operably linked to a coding region). For example, the polyribonucleotide may include any IRES as described in: Chen et al. Mol. Cell 81(20):4300-18, 2021; Jopling et al. Oncogene 20:2664 -70, 2001; Baranick et al. PNAS [Proceedings of the National Academy of Sciences] 105(12):4733-38, 2008; Lang et al. Molecular Biology of the Cell [Cell Molecular Biology] 13(5):1792-1801, 2002; Dorokhov et al. PNAS [Proceedings of the National Academy of Sciences] 99(8):5301-06, 2002; Wang et al. Nucleic Acids Research [nucleic acid research] 33(7):2248-58, 2005; Petz et al. Nucleic Acids Research [Nucleic Acid Research] 35(8):2473-82, 2007; Chen et al. Science [Science] 268:415-417, 1995; Fan et al. Nature Communication [Nature Communication] 13(1):3751-3765, 2022 and International Publication No. WO 2021/263124, each of which is hereby incorporated by reference in its entirety.message sequence

在一些實施方式中，由本文揭露的環狀多核糖核苷酸表現的抗融合多肽包括分泌性蛋白，例如天然包括訊息序列的蛋白，或通常不編碼訊息序列但經修飾含有訊息序列的蛋白。在一些實施方式中，由環狀多核糖核苷酸編碼的抗融合多肽包括分泌訊息。例如，分泌訊息可為分泌性蛋白的天然編碼的分泌訊息。在另一個實例中，分泌訊息可為分泌性蛋白的經修飾的分泌訊息。在其他實施方式中，由環狀多核糖核苷酸編碼的抗融合多肽不包括分泌訊息。In some embodiments, anti-fusion polypeptides represented by cyclic polyribonucleotides disclosed herein include secreted proteins, such as proteins that naturally include a message sequence, or proteins that do not normally encode a message sequence but are modified to contain a message sequence. In some embodiments, the anti-fusion polypeptide encoded by a cyclic polyribonucleotide includes a secretion message. For example, the secretion message may be a naturally encoded secretion message for a secreted protein. In another example, the secretion message can be a modified secretion message of a secreted protein. In other embodiments, the anti-fusion polypeptide encoded by a cyclic polyribonucleotide does not include a secretion message.

在一些實施方式中，訊息序列選自SecSP38（MWWRLWWLLLLLLLLWPMVWA；SEQ ID NO: 327）；SecD4（MWWLLLL LLLLWPMVWA；SEQ ID NO: 328），gLuc（MGVKVLFALICIAVAEAK；SEQ ID NO: 329）；INHC1（MASRLTLLTLLLLLLAGDRASS；SEQ ID NO: 330）；Epo（MGVHECPAWLWLLLSLLSLPLGLPVLG；SEQ ID NO: 331）；以及IL-2（MYRMQLLSCIALSLALVTNS；SEQ ID NO: 332）。In some embodiments, the message sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 327); SecD4 (MWWLLLL LLLLWPMVWA; SEQ ID NO: 328), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO: 329); INHC1 (MASRLTLLTLLLLLLAGDRASS; SEQ ID NO: 330); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO: 331); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 332).

在一些實施方式中，環狀多核糖核苷酸編碼相同抗融合多肽的多個拷貝（例如，一、兩、三、四、五、六、七、八、九、十或更多個）。在一些實施方式中，抗融合多肽的至少一個拷貝包括訊息序列，且抗融合多肽的至少一個拷貝不包括訊息序列。在一些實施方式中，環狀多核糖核苷酸編碼多個抗融合多肽（例如，多個不同的抗融合多肽或多個具有小於100%序列同一性的抗融合多肽），其中該多個抗融合多肽中的至少一個包括訊息序列且該多個抗融合多肽中的至少一個拷貝不包括訊息序列。In some embodiments, a cyclic polyribonucleotide encodes multiple copies (eg, one, two, three, four, five, six, seven, eight, nine, ten, or more) of the same anti-fusion polypeptide. In some embodiments, at least one copy of the anti-fusion polypeptide includes a message sequence, and at least one copy of the anti-fusion polypeptide does not include a message sequence. In some embodiments, the cyclic polyribonucleotide encodes a plurality of anti-fusion polypeptides (e.g., a plurality of different anti-fusion polypeptides or a plurality of anti-fusion polypeptides with less than 100% sequence identity), wherein the plurality of anti-fusion polypeptides At least one of the fusion polypeptides includes a message sequence and at least one copy of the plurality of anti-fusion polypeptides does not include a message sequence.

在一些實施方式中，訊息序列係野生型訊息序列，其例如在內源表現時存在於對應野生型抗融合多肽的N末端。在一些實施方式中，訊息序列與抗融合多肽異源，例如當野生型抗融合多肽內源性表現時係不存在的。可以修飾編碼抗融合多肽的多核糖核苷酸序列以去除編碼野生型訊息序列的核苷酸序列和/或添加編碼異源訊息序列的序列。In some embodiments, the message sequence is a wild-type message sequence, which is present, for example, at the N-terminus of the corresponding wild-type anti-fusion polypeptide when expressed endogenously. In some embodiments, the message sequence is heterologous to the anti-fusion polypeptide, eg, is not present when the wild-type anti-fusion polypeptide is endogenously expressed. A polyribonucleotide sequence encoding an anti-fusion polypeptide can be modified to remove a nucleotide sequence encoding a wild-type message sequence and/or to add a sequence encoding a heterologous message sequence.

由多核糖核苷酸編碼的多肽（例如，抗融合多肽）可以包括將抗融合多肽引導至分泌途徑的訊息序列。在一些實施方式中，訊息序列可以引導抗融合多肽駐留在某些細胞器（例如，內質網、高爾基體或內體）中。在一些實施方式中，訊息序列引導抗融合多肽從細胞中分泌。對於分泌性蛋白，訊息序列可以在分泌後切割，從而產生成熟蛋白。在其他實施方式中，訊息序列可以嵌入細胞膜或某些細胞器中，從而產生跨膜區段，該跨膜區段將蛋白錨定至細胞膜、內質網或高爾基體。在某些實施方式中，跨膜蛋白的訊息序列係在多肽N末端的短序列。在其他實施方式中，第一跨膜結構域充當第一訊息序列，將蛋白質靶向膜。A polypeptide encoded by a polyribonucleotide (eg, an anti-fusion polypeptide) may include a message sequence that directs the anti-fusion polypeptide to the secretory pathway. In some embodiments, the message sequence can direct the anti-fusion polypeptide to reside in certain organelles (eg, endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the message sequence directs secretion of the anti-fusion polypeptide from the cell. For secreted proteins, the message sequence can be cleaved after secretion to produce the mature protein. In other embodiments, the message sequence can be embedded in the cell membrane or certain organelles, creating a transmembrane segment that anchors the protein to the cell membrane, endoplasmic reticulum, or Golgi apparatus. In certain embodiments, the message sequence of the transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain acts as a first message sequence to target the protein to the membrane.

在一些實施方式中，分泌訊息係人白血球介素-2（IL-2）分泌訊息。在一些實施方式中，IL-2分泌訊息具有與MYRMQLLSCIALSLALVTNS（SEQ ID NO: 332）具有至少90%序列同一性的胺基酸序列。在一些實施方式中，IL-2分泌訊息具有與SEQ ID NO: 332具有至少95%序列同一性的胺基酸序列。在一些實施方式中，IL-2分泌訊息具有與SEQ ID NO: 332具有至少99%序列同一性的胺基酸序列。在一些實施方式中，IL-2分泌訊息具有與SEQ ID NO: 332具有100%序列同一性的胺基酸序列。In some embodiments, the secretion message is a human interleukin-2 (IL-2) secretion message. In some embodiments, the IL-2 secretion message has an amino acid sequence that is at least 90% sequence identical to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 332). In some embodiments, the IL-2 secretion message has an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 332. In some embodiments, the IL-2 secretion message has an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 332. In some embodiments, the IL-2 secretion message has an amino acid sequence that has 100% sequence identity to SEQ ID NO: 332.

在一些實施方式中，分泌訊息係高斯螢光素酶分泌訊息。在一些實施方式中，高斯螢光素酶分泌訊息具有與MGVKVLFALICIAVAEAK（SEQ ID NO: 329）具有至少90%序列同一性的胺基酸序列。在一些實施方式中，高斯螢光素酶分泌訊息具有與SEQ ID NO: 329具有至少95%序列同一性的胺基酸序列。在一些實施方式中，高斯螢光素酶分泌訊息具有與SEQ ID NO: 329具有至少99%序列同一性的胺基酸序列。在一些實施方式中，高斯螢光素酶分泌訊息具有與SEQ ID NO: 329具有100%序列同一性的胺基酸序列。In some embodiments, the secretion message is a Gaussian luciferase secretion message. In some embodiments, the Gaussian luciferase secretion message has an amino acid sequence that is at least 90% sequence identical to MGVKVLFALICIAVAEAK (SEQ ID NO: 329). In some embodiments, the Gaussian luciferase secretion message has an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 329. In some embodiments, the Gaussian luciferase secretion message has an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 329. In some embodiments, the Gaussian luciferase secretion message has an amino acid sequence with 100% sequence identity to SEQ ID NO: 329.

在一些實施方式中，分泌訊息係EPO（例如，人EPO）分泌訊息。在一些實施方式中，EPO分泌訊息具有與MGVHECPAWLWLLLSLLSL PLGLPVLGA（SEQ ID NO: 333）具有至少90%序列同一性的胺基酸序列。在一些實施方式中，EPO分泌訊息具有與SEQ ID NO: 333具有至少95%序列同一性的胺基酸序列。在一些實施方式中，EPO分泌訊息具有與SEQ ID NO: 333具有至少99%序列同一性的胺基酸序列。在一些實施方式中，EPO分泌訊息具有與SEQ ID NO: 333具有100%序列同一性的胺基酸序列。In some embodiments, the secretion message is an EPO (eg, human EPO) secretion message. In some embodiments, the EPO secretion message has an amino acid sequence that is at least 90% sequence identical to MGVHECPAWLWLLLSLLSL PLGLPVLGA (SEQ ID NO: 333). In some embodiments, the EPO secretion message has an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 333. In some embodiments, the EPO secretion message has an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 333. In some embodiments, the EPO secretion message has an amino acid sequence that has 100% sequence identity to SEQ ID NO: 333.

在一些實施方式中，分泌訊息係野生型SARS-CoV-2分泌訊息。在一些實施方式中，野生型SARS-CoV-2分泌訊息具有與MFVFLVLLPLVSS（SEQ ID NO: 334）具有至少90%序列同一性的胺基酸序列。在一些實施方式中，野生型SARS-CoV-2分泌訊息具有與SEQ ID NO: 334具有至少95%序列同一性的胺基酸序列。在一些實施方式中，野生型SARS-CoV-2分泌訊息具有與SEQ ID NO: 334具有至少99%序列同一性的胺基酸序列。在一些實施方式中，野生型SARS-CoV-2分泌訊息具有與SEQ ID NO: 334具有100%序列同一性的胺基酸序列。In some embodiments, the secretion message is a wild-type SARS-CoV-2 secretion message. In some embodiments, the wild-type SARS-CoV-2 secretion message has an amino acid sequence with at least 90% sequence identity to MFVFLVLLPLVSS (SEQ ID NO: 334). In some embodiments, the wild-type SARS-CoV-2 secretion message has an amino acid sequence that is at least 95% sequence identical to SEQ ID NO: 334. In some embodiments, the wild-type SARS-CoV-2 secretion message has an amino acid sequence that is at least 99% sequence identical to SEQ ID NO: 334. In some embodiments, the wild-type SARS-CoV-2 secretion message has an amino acid sequence with 100% sequence identity to SEQ ID NO: 334.

在一些實施方式中，由多核糖核苷酸編碼的抗融合多肽包括分泌訊息序列、跨膜插入訊息序列或不包括訊息序列。調控元件In some embodiments, the anti-fusion polypeptide encoded by a polyribonucleotide includes a secretory message sequence, a transmembrane inserted message sequence, or does not include a message sequence.regulatory elements

在一些實施方式中，本文所述之多核糖核苷酸（例如，多核糖核苷酸的多核糖核苷酸貨物）包括一或多個調控元件。在一些實施方式中，多核糖核苷酸包括調控元件，例如修飾多核糖核苷酸內表現序列的表現的序列。In some embodiments, a polyribonucleotide (eg, a polyribonucleotide cargo) described herein includes one or more regulatory elements. In some embodiments, the polyribonucleotide includes regulatory elements, eg, sequences that modify the expression of the expressed sequence within the polyribonucleotide.

調控元件可以包括位置與編碼表現產物的表現序列相鄰的序列。調控元件可與相鄰序列可操作地連接。如與不存在調控元件時表現的產物的量相比，調控元件可以增加表現的產物的量。另外，一個調控元件可以增加串聯連接的多個表現序列表現的產物的量。因此，一種調控元件可以增強一或多個表現序列的表現。多個調控元件係熟悉該項技術者熟知的。Regulatory elements may include sequences located adjacent to the expression sequence encoding the expression product. Regulatory elements can be operably linked to adjacent sequences. The regulatory element can increase the amount of product expressed compared to the amount of product expressed in the absence of the regulatory element. Additionally, one regulatory element can increase the amount of product expressed by multiple expression sequences linked in series. Thus, a regulatory element can enhance the expression of one or more expressed sequences. A number of regulatory elements are well known to those skilled in the art.

在一些實施方式中，調控元件係翻譯調節子。翻譯調節子可以調節多核糖核苷酸中表現序列的翻譯。翻譯調節子可為翻譯強化子或翻譯抑制子。在一些實施方式中，多核糖核苷酸包含與至少一個表現序列相鄰的至少一個翻譯調節子。在一些實施方式中，多核糖核苷酸包含與每個表現序列相鄰的翻譯調節子。在一些實施方式中，翻譯調節子存在於每個表現序列的一側或兩側，導致例如一或多種肽和/或一或多種多肽的表現產物的隔開。In some embodiments, the regulatory element is a translation regulator. Translation regulators regulate the translation of sequences expressed in polyribonucleotides. A translation regulator can be a translation enhancer or a translation repressor. In some embodiments, a polyribonucleotide comprises at least one translation regulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide contains a translation regulator adjacent to each expressed sequence. In some embodiments, translational regulators are present on one or both sides of each expressed sequence, resulting in, for example, spacing of the expressed product of one or more peptides and/or one or more polypeptides.

在一些實施方式中，調控元件係微小RNA（miRNA）或miRNA結合位點。In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

調控元件的其他實例在例如國際專利公開案號WO 2019/118919的第[0154]-[0161]段中描述，該文獻特此藉由援引以其全文併入。切割結構域Other examples of regulatory elements are described, for example, in paragraphs [0154]-[0161] of International Patent Publication No. WO 2019/118919, which document is hereby incorporated by reference in its entirety.cleavage domain

本揭露之環狀多核糖核苷酸可以包括切割結構域（例如，交錯元件或切割序列）。The cyclic polyribonucleotides of the present disclosure may include cleavage domains (eg, staggered elements or cleavage sequences).

術語「交錯元件」係指在翻譯期間誘導核糖體暫停的部分，諸如核苷酸序列。在一些實施方式中，交錯元件係具有強α-螺旋傾向的胺基酸的非保守序列，接著是一致序列-D(V/I)ExNPGP，其中x = 任何胺基酸（SEQ ID NO: 335）。在一些實施方式中，交錯元件可以包括化學部分，諸如丙三醇、非核酸連接部分、化學修飾、經修飾的核酸或其任何組合。The term "staggering element" refers to a portion, such as a nucleotide sequence, that induces ribosome pausing during translation. In some embodiments, the staggered element is a non-conserved sequence of amino acids with strong alpha-helical propensity, followed by the consensus sequence - D(V/I)ExNPGP, where x = any amino acid (SEQ ID NO: 335 ). In some embodiments, interleaving elements can include chemical moieties such as glycerol, non-nucleic acid linking moieties, chemical modifications, modified nucleic acids, or any combination thereof.

在一些實施方式中，環狀多核糖核苷酸包含與表現序列相鄰的至少一個交錯元件。在一些實施方式中，環狀多核糖核苷酸包含與每個表現序列相鄰的交錯元件。在一些實施方式中，交錯元件存在於每個表現序列的一側或兩側，導致表現產物例如一或多種肽和/或一或多種多肽的隔開。在一些實施方式中，交錯元件係一或多個表現序列的一部分。在一些實施方式中，環狀多核糖核苷酸包含一或多個表現序列，並且該一或多個表現序列中的每一個藉由環狀多核糖核苷酸上的交錯元件與後繼的表現序列隔開。在一些實施方式中，交錯元件阻止 (a) 單個表現序列的兩輪翻譯或 (b) 兩個或更多個表現序列的一輪或多輪翻譯生成單個多肽。在一些實施方式中，交錯元件係與該一或多個表現序列隔開的序列。在一些實施方式中，交錯元件包括該一或多個表現序列的表現序列之一部分。In some embodiments, the cyclic polyribonucleotide contains at least one staggered element adjacent to the expressed sequence. In some embodiments, the cyclic polyribonucleotide contains staggered elements adjacent to each expressed sequence. In some embodiments, staggering elements are present on one or both sides of each expressed sequence, resulting in separation of the expressed products, such as one or more peptides and/or one or more polypeptides. In some embodiments, an interleaved element is part of one or more representation sequences. In some embodiments, the cyclic polyribonucleotide includes one or more expression sequences, and each of the one or more expression sequences is expressed by interleaving elements on the cyclic polyribonucleotide and subsequent expression. sequence separated. In some embodiments, the staggered elements prevent (a) two rounds of translation of a single expressed sequence or (b) one or more rounds of translation of two or more expressed sequences to generate a single polypeptide. In some embodiments, interleaving elements are sequences that are separated from the one or more expressed sequences. In some embodiments, an interleaved element includes a portion of a presentation sequence of the one or more presentation sequences.

在一些實施方式中，環狀多核糖核苷酸包含交錯元件。為了避免在保持滾環翻譯的同時產生連續表現產物，例如肽或多肽，可以包括交錯元件以在翻譯期間誘導核糖體暫停。在一些實施方式中，交錯元件在一或多個表現序列中的至少一個的3'端。交錯元件可以被配置為在環狀多核糖核苷酸的滾環翻譯期間使核糖體停滯。交錯元件可包括但不限於2A樣或CHYSEL（SEQ ID NO: 336）（順式作用水解酶元件）序列。在一些實施方式中，交錯元件編碼具有C-末端一致序列的序列，該一致序列係X₁X₂X₃EX₅NPGP（SEQ ID NO: 337），其中X₁不存在或者係G或H，X₂不存在或者係D或G，X₃係D或V或I或S或M，並且X₅係任何胺基酸。在一些實施方式中，該序列包含具有強α-螺旋傾向的胺基酸的非保守序列，接著是一致序列-D(V/I)EXNPGP（SEQ ID NO: 338），其中x = 任何胺基酸。交錯元件的一些非限制性實例包括GDVESNPGP（SEQ ID NO: 339）、GDIEENPGP（SEQ ID NO: 340）、VEPNPGP（SEQ ID NO: 341）、IETNPGP（SEQ ID NO: 342）、GDIESNPGP（SEQ ID NO: 343）、GDVELNPGP（SEQ ID NO: 344）、GDIETNPGP（SEQ ID NO: 345）、GDVENPGP（SEQ ID NO: 346）、GDVEENPGP（SEQ ID NO: 347）、GDVEQNPGP（SEQ ID NO: 348）、IESNPGP（SEQ ID NO: 349）、GDIELNPGP（SEQ ID NO: 350）、HDIETNPGP（SEQ ID NO: 351）、HDVETNPGP（SEQ ID NO: 352）、HDVEMNPGP（SEQ ID NO: 353）、GDMESNPGP（SEQ ID NO: 354）、GDVETNPGP（SEQ ID NO: 355）、GDIEQNPGP（SEQ ID NO: 356）和DSEFNPGP（SEQ ID NO: 357）。In some embodiments, cyclic polyribonucleotides comprise staggered elements. To avoid the generation of continuously expressed products, such as peptides or polypeptides, while maintaining rolling circle translation, staggering elements can be included to induce ribosome pausing during translation. In some embodiments, the staggered element is 3' to at least one of the one or more expression sequences. The staggered elements can be configured to stall ribosomes during rolling circle translation of circular polyribonucleotides. Interleaving elements may include, but are not limited to, 2A-like or CHYSEL (SEQ ID NO: 336) (cis-acting hydrolase element) sequences. In some embodiments, the staggered element encodes a sequence having a C-terminal consensus sequence X₁ X₂ X₃ EX₅ NPGP (SEQ ID NO: 337), wherein X₁ is absent or is G or H,_X2 is absent or is D or G,_X3 is D or V or I or S or M, and_X5 is any amino acid. In some embodiments, the sequence comprises a non-conserved sequence of amino acids with strong alpha-helical propensity, followed by the consensus sequence - D(V/I)EXNPGP (SEQ ID NO: 338), where x = any amine group acid. Some non-limiting examples of interleaved elements include GDVESNPGP (SEQ ID NO: 339), GDIEENPGP (SEQ ID NO: 340), VEPNPGP (SEQ ID NO: 341), IETNPGP (SEQ ID NO: 342), GDIESNPGP (SEQ ID NO: 342) : 343), GDVELNPGP (SEQ ID NO: 344), GDIETNPGP (SEQ ID NO: 345), GDVENPGP (SEQ ID NO: 346), GDVEENPGP (SEQ ID NO: 347), GDVEQNPGP (SEQ ID NO: 348), IESNPGP (SEQ ID NO: 349), GDIELNPGP (SEQ ID NO: 350), HDIETNPGP (SEQ ID NO: 351), HDVETNPGP (SEQ ID NO: 352), HDVEMNPGP (SEQ ID NO: 353), GDMESNPGP (SEQ ID NO: 354), GDVETNPGP (SEQ ID NO: 355), GDIEQNPGP (SEQ ID NO: 356) and DSEFNPGP (SEQ ID NO: 357).

在一些實施方式中，本文所述之交錯元件切割表現產物，諸如在本文所述之一致序列的G與P之間。作為一個非限制性實例，環狀多核糖核苷酸包含至少一個交錯元件以切割表現產物。在一些實施方式中，環狀多核糖核苷酸包含與至少一個表現序列相鄰的交錯元件。在一些實施方式中，環狀多核糖核苷酸包含在每個表現序列後的交錯元件。在一些實施方式中，環狀多核糖核苷酸包含存在於每個表現序列的一側或兩側的交錯元件，導致由每個表現序列翻譯一或多種單獨肽和/或多肽。In some embodiments, the staggered element cleavage expression products described herein, such as between G and P of the consensus sequences described herein. As a non-limiting example, cyclic polyribonucleotides contain at least one staggered element to cleave the expression product. In some embodiments, a cyclic polyribonucleotide contains staggered elements adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotide contains staggered elements following each expressed sequence. In some embodiments, cyclic polyribonucleotides comprise staggered elements present on one or both sides of each expressed sequence, resulting in the translation of one or more individual peptides and/or polypeptides from each expressed sequence.

在一些實施方式中，交錯元件包括一或多種在翻譯過程中誘導核糖體暫停的經修飾的核苷酸或非天然核苷酸。非天然核苷酸可以包括肽核酸（PNA）、𠰌啉代和鎖核酸（LNA）、以及乙二醇核酸（GNA）和蘇糖核酸（TNA）。諸如此類的實例藉由改變分子主鏈而不同於天然存在的DNA或RNA。示例性修飾可以包括對糖、核鹼基、核苷間鍵（例如，對連接磷酸酯/磷酸二酯鍵/磷酸二酯主鏈）的修飾以及可以在翻譯期間誘導核糖體暫停的它們的任何組合。本文提供的一些示例性修飾在本文其他處描述。In some embodiments, the staggered element includes one or more modified or non-natural nucleotides that induce ribosome pausing during translation. Non-natural nucleotides may include peptide nucleic acids (PNA), linoleno and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Examples such as these differ from naturally occurring DNA or RNA by altering the backbone of the molecule. Exemplary modifications may include modifications to sugars, nucleobases, internucleoside linkages (e.g., to connecting phosphate/phosphodiester bonds/phosphodiester backbones), and any of these that may induce ribosome pausing during translation. combination. Some of the exemplary modifications provided herein are described elsewhere herein.

在一些實施方式中，交錯元件以其他形式存在於環狀多核糖核苷酸中。例如，在一些示例性環狀多核糖核苷酸中，交錯元件包含環狀多核糖核苷酸中的第一表現序列的終止元件，和將終止元件與第一表現序列後繼表現的第一翻譯起始序列隔開的核苷酸間隔子序列。在一些實例中，第一表現序列的第一交錯元件在環狀多核糖核苷酸中的第一表現序列後繼表現的第一翻譯起始序列的上游（5'）。在一些情況下，第一表現序列和第一表現序列後繼表現序列係環狀多核糖核苷酸中的兩個隔開之表現序列。第一交錯元件和第一翻譯起始序列之間的距離可以使得第一表現序列及其後繼表現序列能夠連續翻譯。In some embodiments, staggered elements are present in cyclic polyribonucleotides in other forms. For example, in some exemplary cyclic polyribonucleotides, the staggering element includes a terminating element of a first expressed sequence in the cyclic polyribonucleotide, and a first translation of the terminating element subsequent to the first expressed sequence. A nucleotide spacer sequence separated by the starting sequence. In some examples, the first staggered element of the first expressed sequence is upstream (5') of the first translation initiation sequence subsequently expressed in the first expressed sequence in the circular polyribonucleotide. In some cases, the first expressed sequence and the expressed sequence subsequent to the first expressed sequence are two separate expressed sequences in a circular polyribonucleotide. The distance between the first interleaving element and the first translation initiation sequence may enable consecutive translation of the first expression sequence and its subsequent expression sequence.

在一些實施方式中，第一交錯元件包括終止元件，並將第一表現序列的表現產物與其後繼表現序列的表現產物隔開，從而產生離散的表現產物。在一些情況下，在環狀多核糖核苷酸中後繼序列的第一翻譯起始序列上游包含第一交錯元件的環狀多核糖核苷酸被連續翻譯，而在第二表現序列後繼表現序列的第二翻譯起始序列的上游包含第二表現序列的交錯元件的對應環狀多核糖核苷酸不被連續翻譯。在一些情況下，環狀多核糖核苷酸中僅存在一個表現序列，並且第一表現序列及其後繼表現序列係相同之表現序列。在一些示例性環狀多核糖核苷酸中，交錯元件包含環狀多核糖核苷酸中第一表現序列的第一終止元件，和將終止元件與下游翻譯起始序列隔開的核苷酸間隔子序列。在一些此類實例中，環狀多核糖核苷酸中第一交錯元件在第一表現序列的第一翻譯起始序列的上游（5'）。在一些情況下，第一交錯元件和第一翻譯起始序列之間的距離使得能夠連續翻譯第一表現序列和任何後繼表現序列。In some embodiments, the first interleaving element includes a termination element and separates the expression product of a first expression sequence from the expression product of a subsequent expression sequence, thereby producing discrete expression products. In some cases, a cyclic polyribonucleotide containing a first staggered element upstream of a first translation initiation sequence of a successor sequence in the cyclic polyribonucleotide is continuously translated, while a second expression sequence succeeds the expression sequence Corresponding cyclic polyribonucleotides containing staggered elements of the second expression sequence upstream of the second translation initiation sequence are not continuously translated. In some cases, only one expressed sequence is present in the cyclic polyribonucleotide, and the first expressed sequence and its subsequent expressed sequence are the same expressed sequence. In some exemplary cyclic polyribonucleotides, the staggering element includes a first termination element of the first expressed sequence in the cyclic polyribonucleotide, and a nucleotide that separates the termination element from the downstream translation initiation sequence. spacer sequence. In some such examples, the first staggered element in the cyclic polyribonucleotide is upstream (5') of the first translation initiation sequence of the first expressed sequence. In some cases, the distance between the first interleaving element and the first translation initiation sequence enables consecutive translation of the first presentation sequence and any subsequent presentation sequence.

在一些實施方式中，第一交錯元件將第一表現序列的一輪表現產物與第一表現序列的下一輪表現產物隔開，從而產生離散的表現產物。在一些情況下，在環狀多核糖核苷酸中的第一序列的第一翻譯起始序列上游包含第一交錯元件的環狀多核糖核苷酸被連續翻譯，而在對應環狀多核糖核苷酸中的第二表現序列的第二翻譯起始序列上游包含交錯元件的對應環狀多核糖核苷酸不被連續翻譯。在一些情況下，對應環狀多核糖核苷酸中第二交錯元件與第二翻譯起始序列之間的距離係環狀多聚核苷酸中第一交錯元件與第一翻譯起始序列之間的距離的至少2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍大。在一些情況下，第一交錯元件與第一翻譯起始之間的距離為至少2 nt、3 nt、4 nt、5 nt、6 nt、7 nt、8 nt、9 nt、10 nt、11 nt、12 nt、13 nt、14 nt、15 nt、16 nt、17 nt、18 nt、19 nt、20 nt、25 nt、30 nt、35 nt、40 nt、45 nt、50 nt、55 nt、60 nt、65 nt、70 nt、75 nt或更大。在一些實施方式中，第二交錯元件與第二翻譯起始之間的距離為至少2 nt、3 nt、4 nt、5 nt、6 nt、7 nt、8 nt、9 nt、10 nt、11 nt、12 nt、13 nt、14 nt、15 nt、16 nt、17 nt、18 nt、19 nt、20 nt、25 nt、30 nt、35 nt、40 nt、45 nt、50 nt、55 nt、60 nt、65 nt、70 nt、75 nt或大於第一交錯元件和第一翻譯起始之間的距離。在一些實施方式中，環狀多核糖核苷酸包含多於一個表現序列。In some embodiments, a first interleaving element separates one round of performance products of a first performance sequence from a next round of performance products of the first performance sequence, thereby producing discrete performance products. In some cases, the cyclic polyribonucleotide comprising the first staggered element upstream of the first translation initiation sequence of the first sequence in the cyclic polyribonucleotide is continuously translated while the corresponding cyclic polyribonucleotide is Corresponding cyclic polyribonucleotides containing staggered elements upstream of the second translation initiation sequence of the second expression sequence in the nucleotide are not continuously translated. In some cases, the distance between the second staggered element in the corresponding cyclic polyribonucleotide and the second translation initiation sequence is the distance between the first staggered element in the cyclic polynucleotide and the first translation initiation sequence. At least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times greater than the distance between them. In some cases, the distance between the first staggered element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt , 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or larger. In some embodiments, the distance between the second staggered element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt or greater than the distance between the first staggered element and the first translation initiation. In some embodiments, a cyclic polyribonucleotide contains more than one expressed sequence.

在國際專利公開案號WO 2019/118919的段落[0172]-[0175]中描述了交錯元件的實例，將其特此藉由援引以其全文併入。Examples of interleaved elements are described in International Patent Publication No. WO 2019/118919, paragraphs [0172]-[0175], which is hereby incorporated by reference in its entirety.

在一些實施方式中，由環狀核糖核苷酸編碼的多個抗融合多肽可以被每個抗融合多肽之間的IRES隔開（例如，每個抗融合多肽與單獨的IRES可操作地連接）。例如，環狀多核糖核苷酸可以包括與第一表現序列可操作地連接的第一IRES和與第二表現序列可操作地連接的第二IRES。所有抗融合多肽之間的IRES可為相同的IRES。不同抗融合多肽之間的IRES可以不同。In some embodiments, multiple anti-fusion polypeptides encoded by cyclic ribonucleotides can be separated by an IRES between each anti-fusion polypeptide (e.g., each anti-fusion polypeptide is operably linked to a separate IRES) . For example, a cyclic polyribonucleotide can include a first IRES operably linked to a first expressed sequence and a second IRES operably linked to a second expressed sequence. The IRES between all anti-fusion polypeptides can be the same IRES. The IRES can differ between different anti-fusion polypeptides.

在一些實施方式中，多個抗融合多肽可以被2A自切割肽隔開。例如，環狀多核糖核苷酸可編碼與編碼第一抗融合多肽、2A和第二抗融合多肽的開讀框可操作地連接的IRES。In some embodiments, multiple anti-fusion polypeptides can be separated by a 2A self-cleaving peptide. For example, the cyclic polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first anti-fusion polypeptide, 2A, and a second anti-fusion polypeptide.

在一些實施方式中，多個抗融合多肽可以被蛋白酶切割位點（例如，弗林蛋白酶切割位點）隔開。例如，環狀多核糖核苷酸可編碼與編碼第一抗融合多肽、蛋白酶切割位點（例如，弗林蛋白酶切割位點）和第二抗融合多肽的開讀框可操作地連接的IRES。In some embodiments, multiple anti-fusion polypeptides can be separated by a protease cleavage site (eg, a furin cleavage site). For example, the cyclic polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first anti-fusion polypeptide, a protease cleavage site (eg, a furin cleavage site), and a second anti-fusion polypeptide.

在一些實施方式中，多個抗融合多肽可以被2A自切割肽和蛋白酶切割位點（例如，弗林蛋白酶切割位點）隔開。例如，環狀多核糖核苷酸可編碼與編碼第一抗融合多肽、2A、蛋白酶切割位點（例如，弗林蛋白酶切割位點）和第二抗融合多肽的開讀框可操作地連接的IRES。環狀多核糖核苷酸還可以編碼與編碼第一抗融合多肽、蛋白酶切割位點（例如，弗林蛋白酶切割位點）、2A和第二抗融合多肽的開讀框可操作地連接的IRES。串聯的2A和弗林蛋白酶切割位點可稱為弗林蛋白酶-2A（其包括以任一取向排列的弗林蛋白酶-2A或2A-弗林蛋白酶）。In some embodiments, multiple anti-fusion polypeptides can be separated by a 2A self-cleaving peptide and a protease cleavage site (eg, a furin cleavage site). For example, a cyclic polyribonucleotide may encode a cyclic polyribonucleotide operably linked to an open reading frame encoding a first anti-fusion polypeptide, 2A, a protease cleavage site (e.g., a furin cleavage site), and a second anti-fusion polypeptide. IRES. The cyclic polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first anti-fusion polypeptide, a protease cleavage site (e.g., a furin cleavage site), 2A, and a second anti-fusion polypeptide. . The tandem 2A and furin cleavage sites may be referred to as furin-2A (which includes furin-2A or 2A-furin arranged in either orientation).

此外，由環狀核糖核苷酸編碼的多個抗融合多肽可以被IRES和2A序列兩者隔開。例如，IRES可以在一個抗融合多肽與第二抗融合多肽之間，而2A肽可以在第二抗融合多肽與第三抗融合多肽之間。特定IRES或2A自切割肽的選擇可用於在IRES或2A序列的控制下控制抗融合多肽的表現水平。例如，根據所選擇的IRES和/或2A肽，多肽上的表現可以更高或更低。Furthermore, multiple anti-fusion polypeptides encoded by cyclic ribonucleotides can be separated by both IRES and 2A sequences. For example, the IRES can be between one anti-fusion polypeptide and a second anti-fusion polypeptide, and the 2A peptide can be between the second anti-fusion polypeptide and a third anti-fusion polypeptide. The selection of specific IRES or 2A self-cleaving peptides can be used to control the expression level of the anti-fusion polypeptide under the control of the IRES or 2A sequence. For example, performance on the polypeptide may be higher or lower depending on the IRES and/or 2A peptide selected.

在一些實施方式中，環狀多核糖核苷酸包含至少一個切割序列。在一些實施方式中，切割序列與表現序列相鄰。在一些實施方式中，切割序列在兩個表現序列之間。在一些實施方式中，切割序列包括在表現序列中。在一些實施方式中，環狀多核糖核苷酸包含2至10個切割序列。在一些實施方式中，環狀多核糖核苷酸包含2至5個切割序列。在一些實施方式中，多個切割序列在多個表現序列之間；例如，環狀多核糖核苷酸可以包括三個表現序列和兩個切割序列，使得在每個表現序列之間存在一個切割序列。在一些實施方式中，環狀多核糖核苷酸包含切割序列，例如在犧牲型circRNA或可切割的circRNA或自切割的circRNA中。在一些實施方式中，環狀多核糖核苷酸包含兩個或更多個切割序列，導致將環狀多核糖核苷酸隔開成多個產物，例如miRNA、線性RNA、較小的環狀多核糖核苷酸等。In some embodiments, the cyclic polyribonucleotide contains at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to the expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, the cleavage sequence is included in the expression sequence. In some embodiments, the circular polyribonucleotide contains 2 to 10 cleavage sequences. In some embodiments, the circular polyribonucleotide contains 2 to 5 cleavage sequences. In some embodiments, multiple cleavage sequences are between multiple expressed sequences; for example, a circular polyribonucleotide can include three expressed sequences and two cleavage sequences such that there is one cleavage between each expressed sequence. sequence. In some embodiments, the cyclic polyribonucleotide contains a cleavage sequence, such as in a sacrificial circRNA or a cleavable circRNA or a self-cleaving circRNA. In some embodiments, the cyclic polyribonucleotide contains two or more cleavage sequences, resulting in the separation of the cyclic polyribonucleotide into multiple products, e.g., miRNA, linear RNA, smaller circular Polyribonucleotides, etc.

在一些實施方式中，切割序列包括核酶RNA序列。核酶（來自核糖核酸酶，也稱為RNA酶或催化性RNA）係催化化學反應的RNA分子。許多天然核酶催化其自身的磷酸二酯鍵之一的水解，或催化其他RNA中的鍵的水解，但也發現天然核酶催化核糖體的胺基轉移酶活性。催化性RNA可以藉由體外方法「進化」。類似於上文討論的核糖開關活性，核酶及其反應產物可以調控基因表現。在一些實施方式中，將催化性RNA或核酶置於較大的非編碼RNA中，這使得核酶以許多拷貝存在於細胞內，用於大體積分子的化學轉化之目的。在一些實施方式中，適配體和核酶都可以在相同的非編碼RNA中編碼。In some embodiments, the cleavage sequence includes a ribozyme RNA sequence. Ribozymes (from ribonucleases, also called RNases or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze the hydrolysis of one of their own phosphodiester bonds or of bonds in other RNAs, but natural ribozymes are also found to catalyze the aminotransferase activity of ribosomes. Catalytic RNA can be "evolved" through in vitro methods. Similar to the riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In some embodiments, the catalytic RNA or ribozyme is placed within a larger non-coding RNA, which allows the ribozyme to be present in many copies within the cell for the purpose of chemical transformation of large molecules. In some embodiments, both the aptamer and the ribozyme can be encoded in the same non-coding RNA.

在一些實施方式中，切割序列編碼可切割的多肽連接子。例如，多核糖核苷酸可以編碼兩個或更多個抗融合多肽，例如，其中該兩個或更多個抗融合多肽由單個開讀框（ORF）編碼。例如，兩個或更多個抗融合多肽可以由單個開讀框編碼，該開讀框的表現受IRES控制。在一些實施方式中，ORF進一步編碼多肽連接子，例如使得ORF的表現產物編碼兩個或更多個抗融合多肽，每個抗融合多肽被編碼多肽連接子（例如，5-200、5至100、5至50、5至20、50至100或50至200個胺基酸的連接子）的序列隔開。多肽連接子可以包括切割位點，例如被蛋白酶（例如，在向受試者投與多核糖核苷酸後受試者中的內源蛋白酶）識別並切割的切割位點。在這樣的實施方式中，包括兩個或更多個抗融合多肽的胺基酸序列的單一表現產物在表現時切割，從而使得該兩個或更多個抗融合多肽在表現後隔開。示例性的蛋白酶切割位點係熟悉該項技術者已知的，例如，充當被金屬蛋白酶（例如，基質金屬蛋白酶（MMP），諸如MMP 1-28中的任一或多種）、解聚素（disintegrin）和金屬蛋白酶（ADAM，諸如ADAM 2、7-12、15、17-23、28-30和33中的任一或多種）、絲胺酸蛋白酶（弗林蛋白酶）、尿激酶型纖溶酶原啟動因子、蛋白裂解酶（matriptase）、半胱胺酸蛋白酶、天冬胺酸蛋白酶或組織蛋白酶識別的蛋白酶切割位點的胺基酸序列。在一些實施方式中，蛋白酶係MMP9和/或MMP2。在一些實施方式中，蛋白酶係蛋白裂解酶。In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more anti-fusion polypeptides, e.g., wherein the two or more anti-fusion polypeptides are encoded by a single open reading frame (ORF). For example, two or more anti-fusion polypeptides can be encoded by a single open reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, e.g., such that the expression product of the ORF encodes two or more anti-fusion polypeptides, each anti-fusion polypeptide is encoded by a polypeptide linker (e.g., 5-200, 5-100 , 5 to 50, 5 to 20, 50 to 100 or 50 to 200 amino acids) sequences separated by linkers. The polypeptide linker may include a cleavage site, such as a cleavage site that is recognized and cleaved by a protease (eg, an endogenous protease in the subject following administration of the polyribonucleotide to the subject). In such embodiments, a single expression product comprising the amino acid sequences of two or more anti-fusion polypeptides is cleaved upon expression, such that the two or more anti-fusion polypeptides are separated after expression. Exemplary protease cleavage sites are known to those skilled in the art and, for example, serve as sites for cleavage by metalloproteases (e.g., matrix metalloproteinases (MMPs), such as any one or more of MMPs 1-28), disintegrins ( disintegrin) and metalloproteinases (ADAMs, such as any one or more of ADAM 2, 7-12, 15, 17-23, 28-30, and 33), serine proteases (furin), urokinase-type fibrinolysis The amino acid sequence of the protease cleavage site recognized by zymogen initiation factor, protein lytic enzyme (matriptase), cysteine protease, aspartic protease or cathepsin. In some embodiments, the protease is MMP9 and/or MMP2. In some embodiments, the protease is a protein lytic enzyme.

在一些實施方式中，本文所述之環狀多核糖核苷酸係犧牲型環狀多核糖核苷酸、可切割的環狀多核糖核苷酸或自切割的環狀多核糖核苷酸。環狀多核糖核苷酸可以遞送細胞組分，包括，例如，RNA、lncRNA、lincRNA、miRNA、tRNA、rRNA、snoRNA、ncRNA、siRNA或shRNA。在一些實施方式中，環狀多核糖核苷酸包含被以下隔開的miRNA：(i) 可自切割的元件；(ii) 切割募集位點；(iii) 可降解連接子；(iv) 化學連接子；和/或 (v) 間隔子序列。在一些實施方式中，circRNA包括被以下隔開的siRNA：(i) 可自切割的元件；(ii) 切割募集位點（例如ADAR）；(iii) 可降解連接子（例如丙三醇）；(iv) 化學連接子；和/或 (v) 間隔子序列。可自切割的元件之非限制性實例包括鎚頭結構、剪接元件、髮夾、D型肝炎病毒（HDV）、Varkud衛星（VS）和glmS核酶。翻譯起始序列In some embodiments, the cyclic polyribonucleotides described herein are sacrificial cyclic polyribonucleotides, cleavable cyclic polyribonucleotides, or self-cleaving cyclic polyribonucleotides. Cyclic polyribonucleotides can deliver cellular components including, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, cyclic polyribonucleotides comprise miRNA separated by: (i) self-cleavable elements; (ii) cleavage recruitment sites; (iii) degradable linkers; (iv) chemical linker; and/or (v) spacer sequence. In some embodiments, circRNA includes siRNA separated by: (i) a self-cleavable element; (ii) a cleavage recruitment site (e.g., ADAR); (iii) a degradable linker (e.g., glycerol); (iv) chemical linkers; and/or (v) spacer sequences. Non-limiting examples of self-cleavable elements include hammerheads, splicing elements, hairpins, hepatitis D virus (HDV), Varkud satellite (VS), andglmS ribozyme.translation initiation sequence

在一些實施方式中，本文所述之多核糖核苷酸（例如，多核糖核苷酸的多核糖核苷酸貨物）包括至少一個翻譯起始序列。在一些實施方式中，多核糖核苷酸包含與表現序列可操作地連接的翻譯起始序列。In some embodiments, a polyribonucleotide (eg, a polyribonucleotide cargo) described herein includes at least one translation initiation sequence. In some embodiments, the polyribonucleotide comprises a translation initiation sequence operably linked to the expression sequence.

在一些實施方式中，多核糖核苷酸編碼多肽並且可包括翻譯起始序列，例如起始密碼子。在一些實施方式中，翻譯起始序列包括科紮克或夏因-達爾加諾（Shine-Dalgarno）序列。在一些實施方式中，多核糖核苷酸包含與表現序列相鄰的翻譯起始序列，例如科紮克序列。在一些實施方式中，翻譯起始序列係非編碼起始密碼子。在一些實施方式中，翻譯起始序列（例如科紮克序列）存在於每個表現序列的一側或兩側，導致表現產物的隔開。在一些實施方式中，多核糖核苷酸包含與表現序列相鄰的至少一個翻譯起始序列。在一些實施方式中，翻譯起始序列為多核糖核苷酸提供構象柔性。在一些實施方式中，翻譯起始序列在多核糖核苷酸的基本上單鏈的區域內。在國際專利公開案號WO 2019/118919的段落[0163]-[0165]中描述了翻譯起始序列的其他實例，將其特此藉由援引以其全文併入。In some embodiments, a polyribonucleotide encodes a polypeptide and may include a translation initiation sequence, such as an initiation codon. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide contains a translation initiation sequence adjacent to the expression sequence, such as a Kozak sequence. In some embodiments, the translation initiation sequence is a non-coding initiation codon. In some embodiments, translation initiation sequences (eg, Kozak sequences) are present on one or both sides of each expression sequence, resulting in spacing of expression products. In some embodiments, the polyribonucleotide comprises at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the polyribonucleotide. Other examples of translation initiation sequences are described in International Patent Publication No. WO 2019/118919, paragraphs [0163]-[0165], which is hereby incorporated by reference in its entirety.

多核糖核苷酸可包括多於1個起始密碼子，諸如但不限於至少2個、至少3個、至少4個、至少5個、至少6個、至少7個、至少8個、至少9個、至少10個、至少11個、至少12個、至少13個、至少14個、至少15個、至少16個、至少17個、至少18個、至少19個、至少20個、至少25個、至少30個、至少35個、至少40個、至少50個、至少60個或多於60個起始密碼子。翻譯可以在第一個起始密碼子上起始或可以在第一個起始密碼子的下游起始。The polyribonucleotide may include more than 1 start codon, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, At least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 initiation codons. Translation can initiate on the first initiation codon or can initiate downstream of the first initiation codon.

在一些實施方式中，多核糖核苷酸可在不是第一個起始密碼子的密碼子（例如AUG）處起始。多核糖核苷酸的翻譯可以起始於替代的翻譯起始序列，如但不限於ACG、AGG、AAG、CTG/CUG、GTG/GUG、ATA/AUA、ATT/AUU、TTG/UUG。在一些實施方式中，翻譯在選擇性條件（例如，應激誘導條件）下在替代性翻譯起始序列處開始。作為非限制性實例，多核糖核苷酸的翻譯可在替代性翻譯起始序列（諸如ACG）處開始。作為另一非限制性實例，多核糖核苷酸翻譯可以在替代的翻譯起始序列CTG/CUG處開始。作為另一非限制性實例，多核糖核苷酸翻譯可以在替代的翻譯起始序列GTG/GUG處開始。作為另一非限制性實例，多核糖核苷酸可以在重複相關的非AUG（RAN）序列，如包括短段的重複RNA（例如CGG、GGGGCC、CAG、CTG）的替代的翻譯起始序列處開始翻譯。終止元件In some embodiments, the polyribonucleotide can initiate at a codon other than the first initiation codon (eg, AUG). Translation of the polyribonucleotide can be initiated from alternative translation initiation sequences such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG. In some embodiments, translation is initiated at an alternative translation initiation sequence under selective conditions (eg, stress-inducing conditions). As a non-limiting example, translation of a polyribonucleotide can initiate at an alternative translation initiation sequence, such as ACG. As another non-limiting example, polyribonucleotide translation can begin at an alternative translation initiation sequence CTG/CUG. As another non-limiting example, polyribonucleotide translation can begin at an alternative translation initiation sequence GTG/GUG. As another non-limiting example, the polyribonucleotide can be at a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA (e.g., CGG, GGGGCC, CAG, CTG) Start translating.terminating element

在一些實施方式中，本文所述之多核糖核苷酸（例如，多核糖核苷酸的多核糖核苷酸貨物）包括至少一個終止元件。在一些實施方式中，多核糖核苷酸包含與表現序列可操作地連接的終止元件。在一些實施方式中，多核苷酸缺少終止元件。In some embodiments, a polyribonucleotide (eg, a polyribonucleotide cargo) described herein includes at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to the expression sequence. In some embodiments, the polynucleotide lacks a termination element.

在一些實施方式中，多核糖核苷酸包含一或多個表現序列，並且每個表現序列可具有或可不具有終止元件。在一些實施方式中，多核糖核苷酸包含一或多個表現序列，並且表現序列缺少終止元件，使得多核糖核苷酸被連續翻譯。終止元件的排除可導致表現產物的滾環翻譯或連續表現。In some embodiments, a polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a terminating element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, and the expression sequence lacks a termination element such that the polyribonucleotide is continuously translated. Exclusion of the terminating element can result in rolling circle translation or continuous expression of the expressed product.

在一些實施方式中，環狀多核糖核苷酸包含一或多個表現序列，並且每個表現序列可具有或可不具有終止元件。在一些實施方式中，環狀多核糖核苷酸包含一或多個表現序列，並且表現序列缺少終止元件，使得環狀多核糖核苷酸被連續翻譯。由於缺少核糖體停滯或脫落，終止元件的排除可能導致表現產物例如肽或多肽的滾環翻譯或連續表現。在這樣的實施方式中，滾環翻譯通過每個表現序列來表現連續表現產物。在一些其他實施方式中，表現序列的終止元件可為交錯元件的一部分。在一些實施方式中，環狀多核糖核苷酸中的一或多個表現序列包括終止元件。然而，在環狀多核糖核苷酸中進行滾環翻譯或後繼（例如，第二、第三、第四、第五等）表現序列的表現。在此類情況下，當核糖體遇到終止元件（例如，終止密碼子）並終止翻譯時，表現產物可以從核糖體上脫落。在一些實施方式中，在核糖體例如核糖體的至少一個亞基與環狀多核糖核苷酸保持接觸時翻譯終止。In some embodiments, a cyclic polyribonucleotide includes one or more expression sequences, and each expression sequence may or may not have a terminating element. In some embodiments, the cyclic polyribonucleotides comprise one or more expression sequences, and the expression sequences lack termination elements such that the cyclic polyribonucleotides are continuously translated. Exclusion of the termination element may result in rolling circle translation or continuous expression of expressed products such as peptides or polypeptides due to lack of ribosome stalling or shedding. In such embodiments, rolling circle translation represents a continuous expression product through each expression sequence. In some other embodiments, the terminating element of the representation sequence may be part of an interleaving element. In some embodiments, one or more of the expressed sequences in the cyclic polyribonucleotide includes a termination element. However, rolling circle translation or subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences are present in circular polyribonucleotides. In such cases, the expression product can be shed from the ribosome when it encounters a termination element (e.g., a stop codon) and terminates translation. In some embodiments, translation is terminated when a ribosome, such as at least one subunit of a ribosome, remains in contact with a cyclic polyribonucleotide.

在一些實施方式中，環狀多核糖核苷酸在一或多個表現序列的端部包括終止元件。在一些實施方式中，一或多個表現序列包括兩個或更多個連續的終止元件。在這樣的實施方式中，翻譯終止並且滾環翻譯終止。在一些實施方式中，核糖體與環狀多核糖核苷酸完全脫離。在一些此類實施方式中，在環狀多核糖核苷酸中後繼（例如，第二、第三、第四、第五等）表現序列的產生可能需要核糖體在起始翻譯之前與環狀多核糖核苷酸重新接合。通常，終止元件包括發出翻譯終止信號的框內核苷酸三聯體，例如UAA、UGA、UAG。在一些實施方式中，環狀多核糖核苷酸中的一或多個終止元件係閱讀框移位的終止元件，例如但不限於，可終止翻譯的脫框（off-frame）或-1及+ 1移位的閱讀框（例如，隱藏的終止）。框移位的終止元件包括出現在表現序列的第二閱讀框和第三閱讀框中的核苷酸三聯體，TAA、TAG和TGA。框移位的終止元件可能對防止通常對細胞有害的mRNA誤讀很重要。在一些實施方式中，終止元件係終止密碼子。In some embodiments, a cyclic polyribonucleotide includes a termination element at the end of one or more expressed sequences. In some embodiments, one or more expression sequences include two or more consecutive terminating elements. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosomes are completely detached from the cyclic polyribonucleotide. In some such embodiments, generation of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in a circular polyribonucleotide may require ribosomes to interact with the circular polyribonucleotide before initiating translation. Polyribonucleotide rejoining. Typically, termination elements include in-frame nucleotide triplets that signal translation termination, such as UAA, UGA, UAG. In some embodiments, one or more termination elements in the cyclic polyribonucleotide are reading frame shifted termination elements, such as, but not limited to, off-frame or -1 and -1 that can terminate translation. +1 shifted reading frame (e.g., hidden termination). Frame-shifted termination elements include the nucleotide triplets, TAA, TAG, and TGA, occurring in the second and third reading frames of the expressed sequence. Frame-shifted termination elements may be important to prevent misreading of mRNAs that are often harmful to cells. In some embodiments, the termination element is a termination codon.

國際專利公開案號WO 2019/118919的段落[0169]-[0170]中描述了終止元件的其他實例，將其特此藉由援引以其全文併入。非翻譯區Other examples of termination elements are described in International Patent Publication No. WO 2019/118919, paragraphs [0169]-[0170], which is hereby incorporated by reference in its entirety.untranslated area

在一些實施方式中，環狀多核糖核苷酸包含非翻譯區（UTR）。包括基因的基因組區域的UTR可以轉錄但不翻譯。在一些實施方式中，UTR可以被包括在本文所述之表現序列的翻譯起始序列的上游。在一些實施方式中，UTR可以被包括在本文所述之表現序列的下游。在一些情況下，第一表現序列的一個UTR與第二表現序列的另一個UTR相同或連續或重疊。在一些實施方式中，內含子係人內含子。在一些實施方式中，內含子係全長人內含子，例如ZKSCAN1。In some embodiments, the cyclic polyribonucleotide contains an untranslated region (UTR). UTRs of genomic regions that include genes can be transcribed but not translated. In some embodiments, a UTR may be included upstream of the translation initiation sequence of the expression sequence described herein. In some embodiments, a UTR may be included downstream of an expression sequence described herein. In some cases, one UTR of the first presentation sequence is the same or continuous or overlaps with another UTR of the second presentation sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, such as ZKSCAN1.

在國際專利公開案號WO 2019/118919的段落[0197]-[201]中描述了示例性非翻譯區，將其特此藉由援引以其全文併入。Exemplary untranslated regions are described in International Patent Publication No. WO 2019/118919, paragraphs [0197]-[201], which is hereby incorporated by reference in its entirety.

在一些實施方式中，環狀多核糖核苷酸包含聚A序列。在國際專利公開案號WO 2019/118919的段落[0202]-[0205]中描述了示例性聚A序列，將其特此藉由援引以其全文併入。在一些實施方式中，環狀多核糖核苷酸缺少聚A序列。In some embodiments, the cyclic polyribonucleotide comprises polyA sequences. Exemplary polyA sequences are described in International Patent Publication No. WO 2019/118919, paragraphs [0202]-[0205], which is hereby incorporated by reference in its entirety. In some embodiments, the cyclic polyribonucleotide lacks polyA sequences.

在一些實施方式中，環狀多核糖核苷酸包含內嵌有一段或多段的腺苷和尿苷的UTR。該等AU富集簽名可能會增加表現產物的轉化率。In some embodiments, the cyclic polyribonucleotide comprises a UTR embedded with one or more segments of adenosine and uridine. These AU-enriched signatures may increase the conversion rate of expressed products.

富含UTR AU的元件（ARE）的引入、去除或修飾可用於調節環狀多核糖核苷酸的穩定性或免疫性（例如，免疫或炎性反應的一或多種標誌物的水平）。工程化特定的環狀多核糖核苷酸時，可以將ARE的一或多個拷貝引入環狀多核糖核苷酸中，並且ARE的該等拷貝可以調節表現產物的翻譯和/或產生。同樣，可以對ARE進行鑒別和去除或工程化至環狀多核糖核苷酸以調節細胞內穩定性，從而影響所得蛋白質的翻譯和產生。The introduction, removal, or modification of UTR AU-rich elements (AREs) can be used to modulate the stability or immunity of the cyclic polyribonucleotide (e.g., the levels of one or more markers of immune or inflammatory responses). When engineering a specific cyclic polyribonucleotide, one or more copies of the ARE can be introduced into the cyclic polyribonucleotide, and these copies of the ARE can modulate the translation and/or production of the expressed product. Likewise, AREs can be identified and removed or engineered into cyclic polyribonucleotides to modulate intracellular stability, thereby affecting translation and production of the resulting protein.

應當理解，可以將來自任何基因的任何UTR摻入環狀多核糖核苷酸的相應側翼區中。It is understood that any UTR from any gene can be incorporated into the corresponding flanking region of the circular polyribonucleotide.

在一些實施方式中，環狀多核糖核苷酸缺少5'-UTR，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少3'-UTR，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少聚A序列，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少終止元件，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少內部核糖體進入位點，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少帽，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸缺少5'-UTR、3'-UTR和IRES，並且能夠從其一或多個表現序列表現蛋白。在一些實施方式中，環狀多核糖核苷酸進一步包括以下序列中的一或多個：編碼一或多個miRNA的序列、編碼一或多個複製蛋白的序列、編碼外源基因的序列、編碼治療劑的序列、調控元件（例如翻譯調節子，例如翻譯強化子或抑制子）、翻譯起始序列、靶向內源基因（例如，siRNA、lncRNA、shRNA）的一或多種調控核酸和編碼治療性mRNA或蛋白質的序列。In some embodiments, a cyclic polyribonucleotide lacks a 5'-UTR and is capable of expressing protein from one or more of its expressed sequences. In some embodiments, a cyclic polyribonucleotide lacks a 3'-UTR and is capable of expressing protein from one or more of its expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence and is capable of expressing protein from one or more of its expressed sequences. In some embodiments, a cyclic polyribonucleotide lacks a termination element and is capable of expressing a protein from one or more of its expressed sequences. In some embodiments, a cyclic polyribonucleotide lacks an internal ribosome entry site and is capable of expressing protein from one or more of its expressed sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap and is capable of expressing protein from one or more of its expressed sequences. In some embodiments, a cyclic polyribonucleotide lacks a 5'-UTR, a 3'-UTR, and an IRES and is capable of expressing a protein from one or more of its expressed sequences. In some embodiments, the cyclic polyribonucleotide further includes one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding a foreign gene, Sequences encoding therapeutic agents, regulatory elements (e.g., translation regulators, such as translation enhancers or repressors), translation initiation sequences, one or more regulatory nucleic acids targeting endogenous genes (e.g., siRNA, lncRNA, shRNA) and coding Sequence of therapeutic mRNA or protein.

在一些實施方式中，環狀多核糖核苷酸缺少5'-UTR。在一些實施方式中，環狀多核糖核苷酸缺少3'-UTR。在一些實施方式中，環狀多核糖核苷酸缺少聚A序列。在一些實施方式中，環狀多核糖核苷酸缺少終止元件。在一些實施方式中，環狀多核糖核苷酸缺少內部核糖體進入位點。在一些實施方式中，環狀多核糖核苷酸缺少核酸外切酶的降解易感性。在一些實施方式中，環狀多核糖核苷酸缺少降解易感性的事實可能意味著環狀多核糖核苷酸不被核酸外切酶降解，或在僅存在核酸外切酶時被降解的有限程度例如與不存在核酸外切酶時相當或相似。在一些實施方式中，環狀多核糖核苷酸不被核酸外切酶降解。在一些實施方式中，當暴露於核酸外切酶時，環狀多核糖核苷酸降解減少。在一些實施方式中，環狀多核糖核苷酸缺少與帽結合蛋白的結合。在一些實施方式中，環狀多核糖核苷酸缺少5'帽。蛋白結合序列In some embodiments, the cyclic polyribonucleotide lacks a 5'-UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'-UTR. In some embodiments, the cyclic polyribonucleotide lacks polyA sequences. In some embodiments, the cyclic polyribonucleotide lacks a termination element. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, cyclic polyribonucleotides lack susceptibility to exonuclease degradation. In some embodiments, the fact that cyclic polyribonucleotides lack susceptibility to degradation may mean that cyclic polyribonucleotides are not degraded by exonucleases, or are degraded to a limited extent in the presence of only exonucleases. For example, the extent is comparable or similar to that in the absence of exonuclease. In some embodiments, cyclic polyribonucleotides are not degraded by exonucleases. In some embodiments, cyclic polyribonucleotide degradation is reduced when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.protein binding sequence

在一些實施方式中，環狀多核糖核苷酸包含一或多個蛋白質結合位點，使得蛋白質例如核糖體能夠結合至RNA序列中的內部位點。藉由將蛋白質結合位點（例如核糖體結合位點）工程化至環狀多核糖核苷酸中，環狀多核糖核苷酸可以逃避或更少地被宿主的免疫系統檢測到，藉由掩蔽宿主免疫系統成分中的環狀多核糖核苷酸來調節降解或調節翻譯。In some embodiments, a cyclic polyribonucleotide contains one or more protein binding sites, enabling proteins such as ribosomes to bind to internal sites in the RNA sequence. By engineering protein binding sites (e.g., ribosome binding sites) into cyclic polyribonucleotides, cyclic polyribonucleotides can escape or be less detectable by the host's immune system, by Mask cyclic polyribonucleotides in components of the host immune system to modulate degradation or modulate translation.

在一些實施方式中，環狀多核糖核苷酸包含至少一個免疫蛋白結合位點，例如用於逃避免疫反應，例如CTL（細胞毒性T淋巴細胞）反應。在一些實施方式中，免疫蛋白結合位點係結合至免疫蛋白並有助於將環狀多核糖核苷酸掩蔽為外源的核苷酸序列。在一些實施方式中，免疫蛋白結合位點係結合至免疫蛋白並有助於將環狀多核糖核苷酸隱藏為外源或外來的核苷酸序列。In some embodiments, the cyclic polyribonucleotide contains at least one immune protein binding site, eg, for evading an immune response, such as a CTL (cytotoxic T lymphocyte) response. In some embodiments, the immune protein binding site binds to the immune protein and helps mask the cyclic polyribonucleotide from foreign nucleotide sequences. In some embodiments, the immune protein binding site binds to the immune protein and helps conceal the cyclic polyribonucleotide as a foreign or foreign nucleotide sequence.

核糖體與線性RNA接合的傳統機制包括核糖體與RNA的加帽5'端的結合。從5'端，核糖體遷移到起始密碼子，於是形成第一肽鍵。根據本揭露，環狀多核糖核苷酸的翻譯的內部起始（即，不依賴帽）不需要游離端或加帽端。準確的說，核糖體結合至未加帽的內部位點，由此核糖體在起始密碼子處開始多肽延長。在一些實施方式中，環狀多核糖核苷酸包含一或多個RNA序列，該RNA序列包含核糖體結合位點，例如起始密碼子。The traditional mechanism of ribosome engagement with linear RNA involves binding of ribosomes to the capped 5' end of the RNA. From the 5' end, the ribosome migrates to the start codon and the first peptide bond is formed. According to the present disclosure, internal initiation (i.e., cap-independent) of translation of cyclic polyribonucleotides does not require free or capped ends. Specifically, the ribosome binds to an uncapped internal site, whereby the ribosome initiates polypeptide elongation at the initiation codon. In some embodiments, cyclic polyribonucleotides comprise one or more RNA sequences that comprise a ribosome binding site, such as an initiation codon.

天然5'UTR具有在翻譯起始中起作用的特徵。它們帶有類似科紮克序列的簽名，該等序列眾所周知參與核糖體起始多種基因的翻譯的過程。科紮克序列具有共有CCR(A/G)CCAUGG（SEQ ID NO: 358），其中R係起始密碼子（AUG）的三個鹼基上游的嘌呤（腺嘌呤或鳥嘌呤），後接另一個「G」。還已知5'UTR形成參與延長因子結合的二級結構。The native 5'UTR has characteristics that play a role in translation initiation. They bear signatures similar to Kozak sequences, which are known to be involved in ribosome-initiated translation of a variety of genes. The Kozak sequence has the consensus CCR(A/G)CCAUGG (SEQ ID NO: 358), in which R is a purine (adenine or guanine) upstream of the three bases of the start codon (AUG), followed by another A "G". The 5'UTR is also known to form secondary structures involved in elongation factor binding.

在一些實施方式中，環狀多核糖核苷酸編碼與蛋白質結合的蛋白質結合序列。在一些實施方式中，蛋白質結合序列靶向環狀多核糖核苷酸或將其定位至特定靶標。在一些實施方式中，蛋白結合序列特異性結合蛋白的精胺酸富集區。In some embodiments, the cyclic polyribonucleotide encodes a protein-binding sequence that binds a protein. In some embodiments, protein binding sequences target or localize cyclic polyribonucleotides to a specific target. In some embodiments, the protein binds sequence-specifically binds an arginine-rich region of the protein.

在一些實施方式中，蛋白質結合位點包括但不限於與蛋白質的結合位點，如ACIN1、AGO、APOBEC3F、APOBEC3G、ATXN2、AUH、BCCIP、CAPRIN1、CELF2、CPSF1、CPSF2、CPSF6、CPSF7、CSTF2、CSTF2T、CTCF、DDX21、DDX3、DDX3X、DDX42、DGCR8、EIF3A、EIF4A3、EIF4G2、ELAVL1、ELAVL3、FAM120A、FBL、FIP1L1、FKBP4、FMR1、FUS、FXR1、FXR2、GNL3、GTF2F1、HNRNPA1、HNRNPA2B1、HNRNPC、HNRNPK、HNRNPL、HNRNPM、HNRNPU、HNRNPUL1、IGF2BP1、IGF2BP2、IGF2BP3、ILF3、KHDRBS1、LARP7、LIN28A、LIN28B、m6A、MBNL2、METTL3、MOV10、MSI1、MSI2、NONO、NONO-、NOP58、NPM1、NUDT21、PCBP2、POLR2A、PRPF8、PTBP1、RBFOX2、RBM10、RBM22、RBM27、RBM47、RNPS1、SAFB2、SBDS、SF3A3、SF3B4、SIRT7、SLBP、SLTM、SMNDC1、SND1、SRRM4、SRSF1、SRSF3、SRSF7、SRSF9、TAF15、TARDBP、TIA1、TNRC6A、TOP3B、TRA2A、TRA2B、U2AF1、U2AF2、UNK、UPF1、WDR33、XRN2、YBX1、YTHDC1、YTHDF1、YTHDF2、YWHAG、ZC3H7B、PDK1、AKT1和任何其他結合RNA的蛋白質。間隔子序列In some embodiments, protein binding sites include, but are not limited to, binding sites with proteins such as ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2 , POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP , TIA1, TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1 and any other RNA-binding protein.spacer sequence

在一些實施方式中，本文所述之多核糖核苷酸包含一或多個間隔子序列。間隔子係指在兩個相鄰多核苷酸區域之間提供距離或柔性的任何連續核苷酸序列（例如，一或多個核苷酸的連續核苷酸序列）。間隔子可以存在於本文所述之任一核酸元件之間。間隔子還可以存在於本文所述核酸元件內。In some embodiments, the polyribonucleotides described herein comprise one or more spacer sequences. A spacer refers to any contiguous sequence of nucleotides (eg, a contiguous sequence of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacers can be present between any of the nucleic acid elements described herein. Spacers may also be present within the nucleic acid elements described herein.

例如，其中核酸包括以下元件中的任兩個或更多個：(A) 3'催化內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 多核糖核苷酸貨物；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'催化內含子片段；間隔子可存在於該等元件中的任一或多個之間。元件 (A)、(B)、(C)、(D)、(E)、(F) 或 (G) 中的任一個可被如本文所述之間隔子序列隔開。例如，在 (A) 與 (B) 之間、在 (B) 與 (C) 之間、在 (C) 與 (D) 之間、在 (D) 與 (E) 之間、在 (E) 與 (F) 之間或在 (F) 與 (G) 之間可存在間隔子。For example, wherein the nucleic acid includes any two or more of the following elements: (A) 3' catalytic intron fragment; (B) 3' splice site; (C) 3' exon fragment; (D) Polyribonucleotide cargo; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' catalytic intron fragment; spacers may be present in any of these elements or between more than one. Any of elements (A), (B), (C), (D), (E), (F) or (G) may be separated by a spacer sequence as described herein. For example, between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) There may be a spacer between (F) or between (F) and (G).

在一些實施方式中，多核糖核苷酸進一步包括 (C) 的5'外顯子片段與 (D) 的多核糖核苷酸貨物之間的第一間隔子區域。間隔子的長度可以為例如至少5個（例如，至少10個、至少15個、至少20個）核糖核苷酸。在一些實施方式中，多核糖核苷酸進一步包括 (D) 的多核糖核苷酸貨物與 (E) 的5'外顯子片段之間的第二間隔子區域。In some embodiments, the polyribonucleotide further includes a first spacer region between the 5' exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may be, for example, at least 5 (eg, at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, the polyribonucleotide further includes a second spacer region between the polyribonucleotide cargo of (D) and the 5' exon fragment of (E).

間隔子序列可用於將IRES與相鄰結構元件隔開，以保證IRES或相鄰元件的結構和功能。可以根據IRES將間隔子特異性工程化。在一些實施方式中，可以利用RNA折疊電腦軟體（如RNAFold）指導載體的各種元件（包括間隔子）的設計。Spacer sequences can be used to separate an IRES from adjacent structural elements to ensure the structure and function of the IRES or adjacent elements. Spacers can be specifically engineered according to IRES. In some embodiments, RNA folding computer software (such as RNAFold) can be used to guide the design of various elements of the vector (including spacers).

間隔子的長度可以為例如至少5個（例如，至少10個、至少15個、至少20個）核糖核苷酸。在一些實施方式中，每個間隔子區域之長度為至少5個（例如，至少10個、至少15個、至少20個）核糖核苷酸。每個間隔子區域的長度可以為例如5至500個（例如10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500個）核糖核苷酸。第一間隔子區、第二間隔子區、或第一間隔子區和第二間隔子區可包括聚A序列。第一間隔子區、第二間隔子區、或第一間隔子區和第二間隔子區可包括聚A-C序列。在一些實施方式中，第一間隔子區、第二間隔子區、或第一間隔子區和第二間隔子區包括聚A-G序列。在一些實施方式中，第一間隔子區、第二間隔子區、或第一間隔子區和第二間隔子區包括聚A-T序列。在一些實施方式中，第一間隔子區、第二間隔子區、或第一間隔子區和第二間隔子區包括隨機序列。The spacer may be, for example, at least 5 (eg, at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer region is at least 5 (eg, at least 10, at least 15, at least 20) ribonucleotides in length. The length of each spacer sub-region may be, for example, 5 to 500 (eg, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides. The first spacer region, the second spacer region, or both the first spacer region and the second spacer region may include a polyA sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region may include poly A-C sequences. In some embodiments, the first spacer region, the second spacer region, or both the first spacer region and the second spacer region include poly A-G sequences. In some embodiments, the first spacer region, the second spacer region, or both the first spacer region and the second spacer region include polyA-T sequences. In some embodiments, the first spacer region, the second spacer region, or the first and second spacer regions include a random sequence.

間隔子也可存在於本文所述之核酸區域內。例如，多核苷酸貨物區可包括一或多個間隔子。間隔子可以隔開多核苷酸貨物內的區域。Spacers may also be present within the nucleic acid regions described herein. For example, a polynucleotide cargo region may include one or more spacers. Spacers can separate regions within a polynucleotide cargo.

在一些實施方式中，間隔子序列之長度可以為，例如，至少10個核苷酸、至少15個核苷酸、或至少30個核苷酸。在一些實施方式中，間隔子序列之長度為至少7、8、9、10、11、12、13、14、15、16、17、18、19、20、25或30個核苷酸。在一些實施方式中，間隔子序列之長度為不多於100、90、80、70、60、50、45、40、35或30個核苷酸。在一些實施方式中，間隔子序列之長度為20至50個核苷酸。在某些實施方式中，間隔子序列之長度為10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個核苷酸。In some embodiments, the spacer sequence can be, for example, at least 10 nucleotides, at least 15 nucleotides, or at least 30 nucleotides in length. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the spacer sequence is 20 to 50 nucleotides in length. In certain embodiments, the length of the spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.

間隔子序列可為聚A序列、聚A-C序列、聚C序列、或聚U序列。The spacer sequence may be a polyA sequence, a polyA-C sequence, a polyC sequence, or a polyU sequence.

在一些實施方式中，間隔子序列可為聚A-T、聚A-C、聚A-G或隨機序列。In some embodiments, the spacer sequence may be polyA-T, polyA-C, polyA-G, or a random sequence.

在國際專利公開案號WO 2019/118919的段落[0293]-[0302]中描述了示例性間隔子序列，將其特此藉由援引以其全文併入。Exemplary spacer sequences are described in International Patent Publication No. WO 2019/118919, paragraphs [0293]-[0302], which is hereby incorporated by reference in its entirety.

在一些實施方式中，多核糖核苷酸包含5'間隔子序列（例如，在5'退火區域與多核糖核苷酸貨物之間）。在一些實施方式中，5'間隔子序列之長度為至少10個核苷酸。在另一實施方式中，5'間隔子序列之長度為至少15個核苷酸。在另外的實施方式中，5'間隔子序列之長度為至少30個核苷酸。在一些實施方式中，5'間隔子序列之長度為至少7、8、9、10、11、12、13、14、15、16、17、18、19、20、25或30個核苷酸。在一些實施方式中，5'間隔子序列之長度不多於100、90、80、70、60、50、45、40、35或30個核苷酸。在一些實施方式中，5'間隔子序列之長度為在20與50個核苷酸之間。在某些實施方式中，5'間隔子序列之長度為10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個核苷酸。在一個實施方式中，5'間隔子序列係聚A序列。在另一實施方式中，5'間隔子序列係聚A-C序列。在一些實施方式中，5'間隔子序列包括聚A-G序列。在一些實施方式中，5'間隔子序列包括聚A-T序列。在一些實施方式中，5'間隔子序列包括隨機序列。In some embodiments, the polyribonucleotide includes a 5' spacer sequence (eg, between the 5' annealing region and the polyribonucleotide cargo). In some embodiments, the 5' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5' spacer sequence is at least 15 nucleotides in length. In additional embodiments, the 5' spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length . In some embodiments, the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the length of the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides . In one embodiment, the 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is an A-C sequence. In some embodiments, the 5' spacer sequence includes a poly A-G sequence. In some embodiments, the 5' spacer sequence includes a polyA-T sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

在一些實施方式中，多核糖核苷酸包含3'間隔子序列（例如，在3'退火區域與多核糖核苷酸貨物之間）。在一些實施方式中，3'間隔子序列之長度為至少10個核苷酸。在另一個實施方式中，3'間隔子序列之長度為至少15個核苷酸。在另一個實施方式中，3'間隔子序列之長度為至少30個核苷酸。在一些實施方式中，3'間隔子序列之長度為至少7、8、9、10、11、12、13、14、15、16、17、18、19、20、25或30個核苷酸。在一些實施方式中，3'間隔子序列之長度不多於100、90、80、70、60、50、45、40、35或30個核苷酸。在一些實施方式中，3'間隔子序列之長度為20至50個核苷酸。在某些實施方式中，3'間隔子序列之長度為10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個核苷酸。在一個實施方式中，3'間隔子序列係聚A序列。在另一實施方式中，5'間隔子序列係聚A-C序列。在一些實施方式中，5'間隔子序列包括聚A-G序列。在一些實施方式中，5'間隔子序列包括聚A-T序列。在一些實施方式中，5'間隔子序列包括隨機序列。In some embodiments, the polyribonucleotide includes a 3' spacer sequence (eg, between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the 3' spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3' spacer sequence is at least 15 nucleotides in length. In another embodiment, the 3' spacer sequence is at least 30 nucleotides in length. In some embodiments, the 3' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length . In some embodiments, the 3' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 3' spacer sequence is 20 to 50 nucleotides in length. In certain embodiments, the length of the 3' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides . In one embodiment, the 3' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is an A-C sequence. In some embodiments, the 5' spacer sequence includes a poly A-G sequence. In some embodiments, the 5' spacer sequence includes a polyA-T sequence. In some embodiments, the 5' spacer sequence includes a random sequence.

在一個實施方式中，多核糖核苷酸包含5'間隔子序列，但不包含3'間隔子序列。在另一實施方式中，多核糖核苷酸包含3'間隔子序列，但不包含5'間隔子序列。在另一實施方式中，多核糖核苷酸既不包括5'間隔子序列，也不包括3'間隔子序列。在另一實施方式中，多核糖核苷酸不包括IRES序列。在另外的實施方式中，多核糖核苷酸不包括IRES序列、5'間隔子序列或3'間隔子序列。In one embodiment, the polyribonucleotide contains a 5' spacer sequence but not a 3' spacer sequence. In another embodiment, the polyribonucleotide contains a 3' spacer sequence but not a 5' spacer sequence. In another embodiment, the polyribonucleotide includes neither a 5' spacer sequence nor a 3' spacer sequence. In another embodiment, the polyribonucleotide does not include an IRES sequence. In additional embodiments, the polyribonucleotide does not include an IRES sequence, a 5' spacer sequence, or a 3' spacer sequence.

在一些實施方式中，間隔子序列包括至少3個核糖核苷酸、至少4個核糖核苷酸、至少5個核糖核苷酸、至少約8個核糖核苷酸、至少約10個核糖核苷酸、至少約12個核糖核苷酸、至少約15個核糖核苷酸、至少約20個核糖核苷酸、至少約25個核糖核苷酸、至少約30個核糖核苷酸、至少約40個核糖核苷酸、至少約50個核糖核苷酸、至少約60個核糖核苷酸、至少約70個核糖核苷酸、至少約80個核糖核苷酸、至少約90個核糖核苷酸、至少約100個核糖核苷酸、至少約120個核糖核苷酸、至少約150個核糖核苷酸、至少約200個核糖核苷酸、至少約250個核糖核苷酸、至少約300個核糖核苷酸、至少約400個核糖核苷酸、至少約500個核糖核苷酸、至少約600個核糖核苷酸、至少約700個核糖核苷酸、至少約800個核糖核苷酸、至少約900個核糖核苷酸或至少約100個核糖核苷酸。修飾In some embodiments, the spacer sequence includes at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides acid, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides , at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, At least about 900 ribonucleotides or at least about 100 ribonucleotides.Modify

相對於參考序列（尤其是親本多核糖核苷酸），如本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸）可以包括本揭露範圍內包括的一或多個取代、插入和/或添加、缺失和共價修飾。Relative to a reference sequence (especially a parent polyribonucleotide), a polyribonucleotide (e.g., a cyclic polyribonucleotide) as described herein may include one or more substitutions included within the scope of the present disclosure. , insertions and/or additions, deletions and covalent modifications.

在一些實施方式中，環狀多核糖核苷酸包含一或多個轉錄後修飾（例如，加帽、切割、聚腺苷酸化、剪接、聚A序列、甲基化、醯化、磷酸化、離胺酸和精胺酸殘基的甲基化、乙醯化、以及硫醇基團和酪胺酸殘基的亞硝基化等）。該一或多個轉錄後修飾可為任何轉錄後修飾，諸如已經在RNA中鑒定出的多於一百種不同的核苷修飾中的任一種（Rozenski, J, Crain, P和McCloskey, J. (1999).The RNA Modification Database: 1999 update[RNA修飾數據庫:1999年更新].Nucl Acids Res[核酸研究] 27: 196-197）。在一些實施方式中，第一分離核酸包括信使RNA（mRNA）。在一些實施方式中，多核糖核苷酸包含至少一個選自下組的核苷：如國際專利公開案號WO 2019/118919 A1的[0311]中描述的那些，將其藉由援引以其全文併入本文。In some embodiments, cyclic polyribonucleotides comprise one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA sequences, methylation, chelation, phosphorylation, Methylation, acetylation of lysine and arginine residues, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).The RNA Modification Database: 1999 update [RNA modification database: 1999 update].Nucl Acids Res [Nucleic Acid Research] 27: 196-197). In some embodiments, the first isolated nucleic acid includes messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from the group consisting of those described in [0311] of International Patent Publication No. WO 2019/118919 A1, which is incorporated by reference in its entirety. Incorporated herein.

多核糖核苷酸可包括任何有用的修飾，例如針對糖、核鹼基或核苷間鍵（例如針對連接的磷酸酯/針對磷酸二酯鍵/針對磷酸二酯主鏈）。嘧啶核鹼基的一或多個原子可以被視需要取代的胺基、視需要取代的硫醇、視需要取代的烷基（例如甲基或乙基）或鹵代（例如氯代或氟代）替代或取代。在某些實施方式中，在每個糖和核苷間鍵中存在修飾（例如，一或多個修飾）。修飾可為對去氧核糖核酸（DNA）、蘇糖核酸（TNA）、乙二醇核酸（GNA）、肽核酸（PNA）、鎖核酸（LNA）或其雜交體的核糖核酸（RNA）修飾。本文描述了其他修飾。Polyribonucleotides may include any useful modification, for example to sugars, nucleobases or internucleoside linkages (eg to linked phosphates/to phosphodiester bonds/to phosphodiester backbones). One or more atoms of a pyrimidine nucleobase may be optionally substituted with an amine group, an optionally substituted thiol group, an optionally substituted alkyl group (e.g., methyl or ethyl), or halogenated (e.g., chloro or fluoro ) to replace or supersede. In certain embodiments, a modification (eg, one or more modifications) is present in each sugar and internucleoside linkage. The modification may be a ribonucleic acid (RNA) modification of deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. This article describes other modifications.

在一些實施方式中，多核糖核苷酸包含至少一個N(6)甲基腺苷（m6A）修飾以增加翻譯效率。在一些實施方式中，m6A修飾可降低環狀多核糖核苷酸的免疫性（例如，降低免疫或炎性響應的一或多種標誌物的水平）。In some embodiments, the polyribonucleotide contains at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, m6A modification can reduce the immunity of the cyclic polyribonucleotide (eg, reduce the level of one or more markers of immune or inflammatory response).

在一些實施方式中，修飾可以包括化學或細胞誘導的修飾。例如，細胞內RNA修飾的一些非限制性實例如Lewis和Pan, 「RNA modifications and structures cooperate to guide RNA-protein interactions [核糖核酸的修飾和結構共同引導核糖核酸和蛋白的相互作用]」, Nat Reviews Mol Cell Biol [自然評論：分子細胞生物學], 2017, 18:202-210所述。In some embodiments, modifications may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are Lewis and Pan, “RNA modifications and structures cooperate to guide RNA-protein interactions [RNA modifications and structures cooperate to guide RNA-protein interactions]”, Nat Reviews Mol Cell Biol, 2017, 18:202-210.

在一些實施方式中，對環狀多核糖核苷酸的核糖核苷酸的化學修飾可以增強免疫逃避。環狀多核糖核苷酸可以藉由本領域眾所周知的方法合成和/或修飾，如在Current protocols in nucleic acid chemistry [核酸化學現行方案], Beaucage, S.L等人（編輯）, 美國紐約州紐約約翰威利公司（John Wiley & Sons, Inc., New York, NY, USA）（藉由援引以其全文特此併入本文）中描述的那些方法。修飾包括例如端部修飾，如5'端修飾（磷酸化（單、二和三磷酸化）、軛合、反向連接等）、3'端修飾（軛合、DNA核苷酸、反向連接等）、鹼基修飾（例如，用穩定的鹼基、不穩定的鹼基或與擴展的親本庫鹼基配對的鹼基替代）、鹼基去除（脫鹼基核苷酸）或鹼基軛合。經修飾的核糖核苷酸鹼基還可以包括5-甲基胞苷和假尿苷。在一些實施方式中，舉幾個功能作用，鹼基修飾可以調節環狀多核糖核苷酸的表現、免疫反應、穩定性、亞細胞定位。在一些實施方式中，修飾包括雙正交核苷酸，例如非天然鹼基。參見例如，Kimoto等人, Chem Commun (Camb) [化學通訊 (劍橋)], 2017, 53:12309, DOI: 10.1039/c7cc06661a，將該文獻藉由援引以其全文特此併入。In some embodiments, chemical modification of the ribonucleotides of cyclic polyribonucleotides can enhance immune evasion. Cyclic polyribonucleotides can be synthesized and/or modified by methods well known in the art, such as in Current protocols in nucleic acid chemistry [Current Protocols in Nucleic Acid Chemistry], Beaucage, S.L et al. (Editors), John Wie, New York, NY, USA John Wiley & Sons, Inc., New York, NY, USA (hereby incorporated by reference in its entirety). Modifications include, for example, end modifications such as 5' end modifications (phosphorylation (mono, di, and triphosphorylation), conjugation, reverse ligation, etc.), 3' end modifications (conjugation, DNA nucleotides, reverse ligation, etc.) etc.), base modification (e.g., substitution with a stable base, an unstable base, or a base paired with an expanded parental library), base removal (abasilic nucleotides), or base yoke. Modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can modulate the performance, immune response, stability, and subcellular localization of cyclic polyribonucleotides, to name just a few functional roles. In some embodiments, modifications include diorthogonal nucleotides, such as non-natural bases. See, e.g., Kimoto et al., Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is hereby incorporated by reference in its entirety.

在一些實施方式中，環狀多核糖核苷酸的一或多個核糖核苷酸的糖修飾（例如在2'位置或4'位置）或糖替代以及主鏈修飾可以包括磷酸二酯鍵的修飾或替代。環狀多核糖核苷酸的特定實例包括但不限於包括經修飾的主鏈或非天然核苷間鍵（如核苷間修飾，包括磷酸二酯鍵的修飾或替代）的環狀多核糖核苷酸。具有經修飾的主鏈的環狀多核糖核苷酸尤其包括在主鏈中不具有磷原子的那些。出於本申請之目的，並且如本領域中有時提及的，在其核苷間主鏈中不具有磷原子的經修飾的RNA也可以被認為是寡核苷。在特定的實施方式中，環狀多核糖核苷酸將包括在其核苷間主鏈中具有磷原子的核糖核苷酸。In some embodiments, sugar modifications (eg, at the 2' position or 4' position) or sugar substitutions of one or more ribonucleotides of the cyclic polyribonucleotide as well as backbone modifications may include phosphodiester linkages. Modify or replace. Specific examples of cyclic polyribonucleotides include, but are not limited to, cyclic polyribonucleotides that include modified backbones or non-natural internucleoside linkages (such as internucleoside modifications, including modification or replacement of phosphodiester linkages) glycosides. Cyclic polyribonucleotides with modified backbones include especially those without phosphorus atoms in the backbone. For the purposes of this application, and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides. In specific embodiments, cyclic polyribonucleotides will include ribonucleotides having a phosphorus atom in their internucleoside backbone.

經修飾的多核糖核苷酸主鏈可以包括，例如，硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、胺基烷基磷酸三酯、甲基和其他烷基膦酸酯（如3'-伸烷基膦酸酯和手性膦酸酯）、亞膦酸酯、胺基磷酸酯（如3'-胺基胺基磷酸酯和胺基烷基胺基磷酸酯）、硫羰基胺基磷酸酯（thionophosphoramidate）、硫羰烷基膦酸酯、硫羰烷基磷酸三酯、和具有正常3'-5'鍵的硼烷磷酸酯，該等的2'-5'連接的類似物，以及具有相反極性的那些，其中相鄰的核苷單元對3'-5'至5'-3'或2'-5'至5'-2'連接。也包括各種鹽、混合鹽和游離酸形式。在一些實施方式中，環狀多核糖核苷酸可以帶負電荷或帶正電荷。Modified polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphates. Phosphonates (such as 3'-alkylenephosphonates and chiral phosphonates), phosphonites, aminophosphates (such as 3'-aminoaminophosphates and aminoalkylamine Phosphate ester), thionophosphoramidate, thionophosphoramidate, thiocarbonyl alkyl phosphonate, thiocarbonyl phosphate tryster, and borane phosphate ester with normal 3'-5' bond, the 2' of these -5'-linked analogs, as well as those with opposite polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms. In some embodiments, cyclic polyribonucleotides can be negatively or positively charged.

可摻入多核糖核苷酸中的經修飾的核苷酸可在核苷間鍵（例如磷酸酯主鏈）上被修飾。在此，在多核苷酸主鏈的上下文中，短語「磷酸酯」和「磷酸二酯」可互換使用。可以藉由用不同的取代基替代一或多個氧原子來修飾主鏈磷酸酯基團。此外，經修飾的核苷和核苷酸可以包括用如本文所述之另一個核苷間鍵合進行的對未修飾的磷酸酯部分的整體替代。經修飾的磷酸酯基團之實例包括但不限於硫代磷酸酯、亞磷酸硒酸酯、硼酸磷酸酯（boranophosphate或boranophosphate ester）、氫膦酸酯、胺基磷酸酯、二胺基磷酸酯、烷基或芳基膦酸酯和磷酸三酯。二硫代磷酸酯的兩個非連接氧都被硫替代。也可以藉由用氮（橋連的胺基磷酸酯）、硫（橋連的硫代磷酸酯）和碳（橋連的亞甲基膦酸酯）替代連接氧來修飾磷酸酯連接子。Modified nucleotides that can be incorporated into polyribonucleotides can be modified at the internucleoside linkage (eg, phosphate backbone). Here, the phrases "phosphate ester" and "phosphodiester" are used interchangeably in the context of a polynucleotide backbone. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Additionally, modified nucleosides and nucleotides may include an integral replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoselenate, boranophosphate or boranophosphate ester, hydrophosphonate, aminophosphate, diaminophosphate, Alkyl or aryl phosphonates and phosphate triesters. Both non-attached oxygens of phosphorodithioates are replaced by sulfur. Phosphate linkers can also be modified by replacing the connecting oxygen with nitrogen (bridged aminophosphate), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate).

提供α-硫代取代的磷酸酯部分以通過非天然硫代磷酸酯主鏈鍵賦予RNA和DNA聚合物穩定性。硫代磷酸酯DNA和RNA具有增強的核酸酶抗性，並因此在細胞環境中具有更長的半衰期。與環狀多核糖核苷酸連接的硫代磷酸酯有望通過減弱細胞先天性免疫分子的結合/啟動來降低先天性免疫反應。Alpha-thio-substituted phosphate moieties are provided to impart stability to RNA and DNA polymers through non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have enhanced nuclease resistance and thus a longer half-life in the cellular environment. Phosphorothioates linked to cyclic polyribonucleotides are expected to reduce innate immune responses by attenuating the binding/priming of cellular innate immune molecules.

在特定實施方式中，經修飾核苷包括α-硫代-核苷（例如，5'-0-(l-硫代磷酸)-腺苷、5'-0-(l-硫代磷酸)-胞苷(a-硫代胞苷)、5'-0-(l-硫代磷酸)-鳥苷、5'-0-(l-硫代磷酸)-尿苷或5'-0-(1-硫代磷酸)-假尿苷）。In specific embodiments, modified nucleosides include alpha-thio-nucleosides (e.g., 5'-0-(1-phosphorothioate)-adenosine, 5'-0-(1-phosphorothioate)- Cytidine (a-thiocytidine), 5'-0-(l-phosphorothioate)-guanosine, 5'-0-(l-phosphorothioate)-uridine or 5'-0-(1 -phosphorothioate)-pseudouridine).

本文描述了可根據本揭露使用的其他核苷間鍵，包括不包含磷原子的核苷間鍵。Other internucleoside linkages that may be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

在一些實施方式中，環狀多核糖核苷酸可以包括一或多種細胞毒性核苷。例如，細胞毒性核苷可以被摻入環狀多核糖核苷酸中，如雙功能修飾。細胞毒性核苷可以包括但不限於阿糖腺苷、5-氮雜胞苷、4'-硫代阿糖胞苷、環戊烯基胞嘧啶、克拉屈濱、氯法拉濱、阿糖胞苷、胞嘧啶阿糖胞苷、l-(2-C-氰基-2-去氧-β-D-阿拉伯-戊呋喃糖基)-胞嘧啶、地西他濱、5-氟尿嘧啶、氟達拉濱、氟尿苷、吉西他濱、替加氟和尿嘧啶的組合、替加氟（(RS)-5-氟-l-(四氫呋喃-2-基)嘧啶-2,4(lH,3H)-二酮）、曲沙他濱、替紮西他濱、2'-去氧-2'-亞甲基胞苷（DMDC）和6-巰基嘌呤。其他實例包括氟達拉濱磷酸酯、N4-山崳醯基-l-β-D-阿拉伯戊呋喃糖基胞嘧啶、N4-十八烷基-1-β-D-阿拉伯戊呋喃糖基胞嘧啶、N4-棕櫚醯基-l-(2-C-氰基-2-去氧-β-D-阿拉伯-戊呋喃糖基)胞嘧啶和P-4055（阿糖胞苷5'-花生酸酯）。In some embodiments, cyclic polyribonucleotides can include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into cyclic polyribonucleotides, such as bifunctional modifications. Cytotoxic nucleosides may include, but are not limited to, vidarabine, 5-azacytidine, 4'-thioarabinocytosine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine , Cytarabine, l-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludala acetabine, fluuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-di ketone), troxacitabine, tizacitabine, 2'-deoxy-2'-methylenecytidine (DMDC), and 6-mercaptopurine. Other examples include fludarabine phosphate, N4-behenyl-l-β-D-arabinopentofuranosylcytosine, N4-octadecyl-1-β-D-arabinopentofuranosylcytosine Pyrimidine, N4-palmitoyl-l-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine and P-4055 (cytarabine 5'-arachidic acid ester).

多核糖核苷酸可以沿著分子的整個長度被均一地修飾或可以不如此。例如，一或多種或所有類型的核苷酸（例如，天然存在的核苷酸、嘌呤或嘧啶，或A、G、U、C、I、pU中的任一或多種或全部）在環狀多核糖核苷酸中，或在其給定的預定序列區域中可以被均一地修飾或可以不如此。在一些實施方式中，環狀多核糖核苷酸包含假尿苷。在一些實施方式中，環狀多核糖核苷酸包含肌苷，相對於病毒RNA，肌苷可以説明免疫系統將環狀多核糖核苷酸表徵為內源性。肌苷的摻入還可以介導改善RNA穩定性/減少降解。參見例如Yu, Z等人, (2015) RNA editing by ADAR1 marks dsRNA as 「self」. [藉由ADAR1進行的RNA編輯將dsRNA標記為「自身」]Cell Res [細胞研究]. 25, 1283-1284，其藉由援引以其全文併入本文。The polyribonucleotide may be modified uniformly along the entire length of the molecule or may not be so. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or any or more or all of A, G, U, C, I, pU) are present in a cyclic The polyribonucleotide may or may not be uniformly modified within a given predetermined sequence region thereof. In some embodiments, the cyclic polyribonucleotide contains pseudouridine. In some embodiments, the cyclic polyribonucleotide contains inosine, which may account for the immune system's representation of the cyclic polyribonucleotide as endogenous relative to viral RNA. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See, e.g., Yu, Z et al., (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284 , which is incorporated herein by reference in its entirety.

在一些實施方式中，多核糖核苷酸（或其給定序列區域）中的所有核苷酸均被修飾。在一些實施方式中，修飾可以包括可增強表現的m6A；可減弱免疫反應的肌苷；可增加RNA穩定性或翻譯通讀（交錯元件）的假尿苷；可增加穩定性的m5C；以及有助於亞細胞易位（例如，核定位）的2,2,7-三甲基鳥苷。In some embodiments, all nucleotides in a polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications may include m6A to enhance performance; inosine to attenuate immune response; pseudouridine to increase RNA stability or translational readthrough (staggered elements); m5C to increase stability; and help 2,2,7-trimethylguanosine in subcellular translocation (e.g., nuclear localization).

在環狀多核糖核苷酸的各個位置上可以存在不同的糖修飾、核苷酸修飾和/或核苷間鍵合（例如主鏈結構）。熟悉該項技術者將理解，核苷酸類似物或其他一或多個修飾可以位於環狀多核糖核苷酸的任何一或多個位置，使得環狀多核糖核苷酸的功能基本上不降低。修飾也可為非編碼區域修飾。環狀多核糖核苷酸可以包含約1%至約100%的經修飾核苷酸（相對於總核苷酸含量，或相對於一或多種類型的核苷酸，即A、G、U或C中的任一或多種）或任何中間百分比（例如，1%至20%＞、1%至25%、1%至50%、1%至60%、1%至70%、1%至80%、1%至90%、1%至95%、10%至20%、10%至25%、10%至50%、10%至60%、10%至70%、10%到80%、10%至90%、10%至95%、10%至100%、20%至25%、20%至50%、20%至60%、20%至70%、20%至80%、20%至90%、20%至95%、20%至100%、50%至60%、50%至70%、50%至80%、50%至90%、50%至95%、50%至100%、70%至80%、70%至90%、70%至95%、70%至100%、80%至90%、80%至95%、80%至100%、90%至95%、90%至100%和95%至100%）。環化方法Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (eg, backbone structure) may be present at various positions of the cyclic polyribonucleotide. Those skilled in the art will understand that nucleotide analogs or other modification(s) may be located at any one or more positions of the cyclic polyribonucleotide such that the functionality of the cyclic polyribonucleotide is not substantially impaired. reduce. Modifications may also be modifications of non-coding regions. Cyclic polyribonucleotides may comprise from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., A, G, U, or any one or more of C) or any intermediate percentage (for example, 1% to 20%>, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80 %, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100 %, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100% and 95% to 100%).cyclization method

本揭露提供了產生編碼抗融合多肽（例如，表1的多肽）的環狀多核糖核苷酸的方法，包括例如重組技術或化學合成。例如，用於產生RNA環的DNA分子可以包括天然存在的原始核酸序列的DNA序列、其修飾形式、或編碼通常在自然界中不存在的合成多肽的DNA序列（例如，嵌合分子或融合蛋白）。DNA和RNA分子可以使用多種技術修飾，該等技術包括但不限於經典誘變技術和重組技術，如定點誘變、化學處理核酸分子以誘導突變、限制性酶切割核酸片段、連接核酸片段、聚合酶鏈鎖反應（PCR）擴增或誘變核酸序列的選定區域、合成寡核苷酸混合物以及連接混合物基團以「建造」核酸分子混合物及其組合。The present disclosure provides methods for producing cyclic polyribonucleotides encoding anti-fusion polypeptides (eg, the polypeptides of Table 1), including, for example, recombinant techniques or chemical synthesis. For example, DNA molecules used to generate RNA loops may include DNA sequences of naturally occurring original nucleic acid sequences, modified forms thereof, or DNA sequences encoding synthetic polypeptides that do not normally occur in nature (e.g., chimeric molecules or fusion proteins) . DNA and RNA molecules can be modified using a variety of techniques, including but not limited to classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerization Enzyme chain reaction (PCR) amplifies or mutagens selected regions of nucleic acid sequences, synthesizes mixtures of oligonucleotides, and joins mixture groups to "build" mixtures of nucleic acid molecules and their combinations.

在一些實施方式中，用於環化的線性多核糖核苷酸可以被環化或連環化。在一些實施方式中，用於環化的線性多核糖核苷酸可以在配製和/或遞送之前在體外環化。在一些實施方式中，環狀多核糖核苷酸可為與線性多核糖核苷酸的混合物。在一些實施方式中，線性多核糖核苷酸具有與環狀多核糖核苷酸相同的核酸序列。In some embodiments, linear polyribonucleotides used for cyclization can be cyclized or concatenated. In some embodiments, linear polyribonucleotides used for cyclization can be cyclized in vitro prior to formulation and/or delivery. In some embodiments, cyclic polyribonucleotides can be a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotide has the same nucleic acid sequence as the cyclic polyribonucleotide.

在一些實施方式中，使用化學方法環化或連環化用於環化的線性多核糖核苷酸以形成環狀多核糖核苷酸。在一些化學方法中，核酸（例如，用於環化的線性多核糖核苷酸）的5'-端和3'-端包括化學反應性基團，當基團彼此靠近時，可在分子的3'-端和5'-端之間形成新的共價鍵。5'-端可以含有NHS酯反應性基團，且3'-端可以含有3'-胺基末端的核苷酸，使得在有機溶劑中，線性RNA分子3'-端上的3'-胺基末端的核苷酸將在5'-NHS-酯部分上經歷親核攻擊，形成新的5'-/3'-醯胺鍵。In some embodiments, chemical methods are used to cyclize or concatenate linear polyribonucleotides for cyclization to form cyclic polyribonucleotides. In some chemical methods, the 5'- and 3'-ends of a nucleic acid (e.g., a linear polyribonucleotide used for cyclization) include chemically reactive groups that, when the groups are in close proximity to each other, can react on the molecule's A new covalent bond is formed between the 3'-end and the 5'-end. The 5'-end can contain an NHS ester reactive group, and the 3'-end can contain a 3'-amine-terminated nucleotide, such that in organic solvents, the 3'-amine on the 3'-end of the linear RNA molecule The base-terminal nucleotide will undergo nucleophilic attack on the 5'-NHS-ester moiety, forming a new 5'-/3'-amide bond.

在一些實施方式中，使用DNA或RNA連接酶將5'-磷酸化的核酸分子（例如，用於環化的線性多核糖核苷酸）酶促連接至核酸（例如，線性核酸）的3'-羥基，形成新的磷酸二酯鍵。在示例性反應中，根據製造商的方案，將用於環化的線性多核糖核苷酸與1-10單位的T4 RNA連接酶（新英格蘭生物學實驗室公司（New England Biolabs），麻塞諸塞州伊普斯威奇（Ipswich, MA））在37°C下孵育1小時。連接反應可以在存在線性核酸時發生，該線性核酸能夠與並列的5'-和3'-區域鹼基配對，以輔助酶促連接反應。在一些實施方式中，連接係夾板連接。例如，夾板連接酶，如SplintR®連接酶，可用於夾板連接，即RNA連接酶II、T4 RNA連接酶或T4 DNA連接酶。對於夾板連接，單鏈多核苷酸（夾板），如單鏈RNA，可以被設計成與線性多核糖核苷酸的兩個末端雜交，使得在與單鏈夾板雜交時可以將兩個末端並列。因此，夾板連接酶可以催化線性多核糖核苷酸並列的兩個末端的連接，生成環狀多核糖核苷酸。In some embodiments, a DNA or RNA ligase is used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., a linear polyribonucleotide for cyclization) to the 3' end of a nucleic acid (e.g., a linear nucleic acid) -hydroxyl group, forming a new phosphodiester bond. In an exemplary reaction, linear polyribonucleotides for cyclization were combined with 1-10 units of T4 RNA ligase (New England Biolabs, MA) according to the manufacturer's protocol. Ipswich, MA) and incubate for 1 hour at 37°C. Ligation reactions can occur in the presence of linear nucleic acids capable of base pairing with juxtaposed 5'- and 3'-regions to assist in the enzymatic ligation reaction. In some embodiments, the connection is a splint connection. For example, splint ligases such as SplintR® Ligase can be used for splint ligation, namely RNA Ligase II, T4 RNA Ligase or T4 DNA Ligase. For splint ligation, a single-stranded polynucleotide (splint), such as single-stranded RNA, can be designed to hybridize to both ends of a linear polyribonucleotide such that the two ends are juxtaposed when hybridized to the single-stranded splint. Therefore, splint ligase can catalyze the ligation of two juxtaposed ends of linear polyribonucleotides to generate cyclic polyribonucleotides.

在一些實施方式中，DNA或RNA連接酶用於環狀多核苷酸的合成。在一些實施方式中，用於環化的線性多核糖核苷酸的5'-端或3'-端可編碼連接酶核酶序列，使得在體外轉錄期間，所得用於環化的線性多核糖核苷酸包含活性核酶序列，該活性核酶序列能夠連接用於環化的線性多核糖核苷酸的5'-端至用於環化的線性多核糖核苷酸的3'-端。連接酶核酶可以衍生自第I組內含子、D型肝炎病毒、髮夾核酶，或者可以藉由SELEX（藉由指數富集進行的配體系統進化）進行選擇。核酶連接酶反應在0°C與37°C之間的溫度下可能需要1小時至24小時。In some embodiments, DNA or RNA ligase is used for the synthesis of circular polynucleotides. In some embodiments, the 5'-end or 3'-end of the linear polyribonucleotide for cyclization can encode a ligase ribozyme sequence, such that during in vitro transcription, the resulting linear polyribonucleotide for cyclization The nucleotide contains an active ribozyme sequence capable of linking the 5'-end of the linear polyribonucleotide for cyclization to the 3'-end of the linear polyribonucleotide for cyclization. Ligase ribozymes can be derived from group I introns, hepatitis D virus, hairpin ribozymes, or can be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 hour to 24 hours at temperatures between 0°C and 37°C.

在一些實施方式中，可藉由使用至少一個非核酸部分將用於環化的線性多核糖核苷酸環化或連環化。在一方面，該至少一個非核酸部分可與用於環化的線性多核糖核苷酸的5'末端附近和/或3'末端附近的區域或特徵反應，以環化或連環化用於環化的線性多核糖核苷酸。在另一方面，該至少一個非核酸部分可以位於或連接至或鄰近用於環化的線性多核糖核苷酸的5'末端和/或3'末端。設想的非核酸部分可為同源或異源的。作為一個非限制性實例，非核酸部分可為鍵，如疏水鍵、離子鍵、可生物降解的鍵和/或可切割的鍵。作為另一個非限制性實例，非核酸部分係連接部分。作為又一個非限制性實例，非核酸部分可為寡核苷酸或肽部分，諸如，如本文所述之適配體或非核酸連接子。In some embodiments, linear polyribonucleotides for cyclization can be cyclized or concatenated by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety can react with a region or feature near the 5' end and/or near the 3' end of the linear polyribonucleotide for cyclization to cyclize or concatenate for cyclization. ized linear polyribonucleotides. In another aspect, the at least one non-nucleic acid moiety may be located at or connected to or adjacent to the 5' end and/or 3' end of the linear polyribonucleotide used for circularization. Contemplated non-nucleic acid moieties may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a bond, such as a hydrophobic bond, an ionic bond, a biodegradable bond, and/or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety is a linker moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, such as an aptamer or non-nucleic acid linker as described herein.

在一些實施方式中，使用IVT和RNA聚合酶合成用於環化的線性多核糖核苷酸，其中用於IVT的核苷酸混合物可以含有相對於三磷酸鳥苷過量的一磷酸鳥苷，以優先產生具有5'一磷酸酯的RNA；純化的IVT產物可以使用夾板DNA環化。In some embodiments, linear polyribonucleotides for cyclization are synthesized using IVT and RNA polymerase, wherein the nucleotide mixture for IVT can contain an excess of guanosine monophosphate relative to guanosine triphosphate to RNA with a 5' monophosphate is preferentially produced; purified IVT products can be circularized using splint DNA.

在一些實施方式中，用於環化的線性多核糖核苷酸由於非核酸部分而被環化或連環化，造成位於、鄰近或連接至用於環化的線性多核糖核苷酸的5'端和3'端的原子、分子表面之間的吸引。作為一個非限制性實例，可藉由分子間作用力或分子內作用力將一或多個用於環化的線性多核糖核苷酸環化或連環化。分子間作用力之非限制性實例包括偶極-偶極力、偶極-誘導偶極力、誘導偶極-誘導偶極力、范德華力和倫敦分散力。分子內作用力之非限制性實例包括共價鍵、金屬鍵、離子鍵、共振鍵、抓氫鍵（agnostic bond）、偶極鍵、軛合、超軛合和反向鍵。In some embodiments, the linear polyribonucleotide used for cyclization is cyclized or concatenated due to a non-nucleic acid moiety such that it is located at, adjacent to, or connected to the 5' of the linear polyribonucleotide used for cyclization. The attraction between the atoms at the end and the 3' end, and the surface of the molecule. As a non-limiting example, one or more linear polyribonucleotides used for cyclization can be cyclized or concatenated by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipole bonds, conjugation, superconjugation, and reverse bonds.

在一些實施方式中，用於環化的線性多核糖核苷酸可在5'末端附近和3'末端附近包含核酶RNA序列。當序列暴露於核酶的其餘部分時，核酶RNA序列可以共價地連接至肽。在一方面，共價連接至5'末端和3'末端附近的核酶RNA序列的肽可以彼此締合，引起用於環化的線性多核糖核苷酸環化或連環化。在另一方面，肽共價地連接至核酶RNA序列5'末端和3'末端附近可以引起線性初級構建體或線性mRNA在使用本領域已知的方法（諸如但不限於蛋白連接）進行連接後環化或連環化。用於本發明之線性初級構建體或線性RNA中的核酶之非限制性實例，或摻入和/或共價地連接肽的方法的非窮舉列表在美國專利申請案號US 20030082768中描述，該專利內容藉由援引以其全文併入本文。In some embodiments, the linear polyribonucleotide used for cyclization can comprise a ribozyme RNA sequence near the 5' end and near the 3' end. The ribozyme RNA sequence can be covalently linked to the peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, peptides covalently linked to ribozyme RNA sequences near the 5' and 3' ends can associate with each other, causing cyclization or concatenation of linear polyribonucleotides for cyclization. In another aspect, covalent attachment of peptides to ribozyme RNA sequences near the 5' and 3' ends can result in ligation of linear primary constructs or linear mRNA using methods known in the art, such as, but not limited to, protein ligation. Postcyclization or serialization. Non-limiting examples of ribozymes for use in linear primary constructs or linear RNAs of the invention, or a non-exhaustive list of methods of incorporating and/or covalently linking peptides are described in US Patent Application No. US 20030082768 , the contents of this patent are incorporated herein by reference in their entirety.

在一些實施方式中，用於環化的線性多核糖核苷酸可以包括核酸的5'三磷酸酯，例如藉由使5'三磷酸酯與RNA 5'焦磷酸水解酶（RppH）或ATP二磷酸水解酶（脫磷酸酶）接觸使其轉化為5'單磷酸酯。在一些實施方式中，至少一部分線性多核糖核苷酸的5'端包括一磷酸酯部分。在一些實施方式中，使包括環狀和線性多核糖核苷酸在內的多核糖核苷酸群體在用5'核酸外切酶和/或3'核酸外切酶消化至少一部分線性多核糖核苷酸之前與RppH接觸。可替代地，將用於環化的線性多核糖核苷酸的5'三磷酸酯轉化為5'單磷酸酯可藉由兩步反應完成，該兩步反應包括：(a) 使用於環化的線性多核糖核苷酸的5'核苷酸與磷酸酶（例如，熱敏磷酸酶、蝦鹼性磷酸酶或小牛腸磷酸酶）接觸以除去所有三個磷酸酯；以及 (b) 在步驟 (a) 之後，使5'核苷酸與添加單個磷酸酯的激酶（例如，多核苷酸激酶）接觸。In some embodiments, the linear polyribonucleotide used for cyclization can include the 5' triphosphate of the nucleic acid, for example, by binding the 5' triphosphate to RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphate. Contact with phosphohydrolase (dephosphatase) converts it to 5' monophosphate. In some embodiments, at least a portion of the 5' end of the linear polyribonucleotide includes a monophosphate moiety. In some embodiments, a population of polyribonucleotides, including cyclic and linear polyribonucleotides, is digested with a 5' exonuclease and/or a 3' exonuclease at least a portion of the linear polyribonucleotide. The nucleotide was previously in contact with RppH. Alternatively, conversion of the 5' triphosphate to the 5' monophosphate of the linear polyribonucleotide used for cyclization can be accomplished by a two-step reaction including: (a) using contacting the 5' nucleotide of the linear polyribonucleotide with a phosphatase (e.g., thermosensitive phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) in step (a) The 5' nucleotide is then contacted with a kinase that adds a single phosphate (e.g., a polynucleotide kinase).

在一些實施方式中，本文提供的環化方法之環化效率為至少約10%、至少約15%、至少約20%、至少約25%、至少約30%、至少約35%、至少約40%、至少約45%、至少約50%、至少約60%、至少約70%、至少約80%、至少約90%、至少約95%或100%。在一些實施方式中，本文提供的環化方法之環化效率為至少約40%。在一些實施方式中，所提供的環化方法之環化效率介於約10%至約100%之間；例如，環化效率可為約15%、約20%、約25%、約30%、約35%、約40%、約45%、約50%、約55%、約60%、約65%、約70%、約75%、約80%、約85%、約90%、約95%和約99%。在一些實施方式中，環化效率介於約20%至約80%之間。在一些實施方式中，環化效率介於約30%至約60%之間。在一些實施方式中，環化效率為約40%。In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40 %, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or 100%. In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 40%. In some embodiments, the cyclization efficiency of the provided cyclization method is between about 10% and about 100%; for example, the cyclization efficiency can be about 15%, about 20%, about 25%, about 30% , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and about 99%. In some embodiments, the cyclization efficiency is between about 20% and about 80%. In some embodiments, the cyclization efficiency is between about 30% and about 60%. In some embodiments, the cyclization efficiency is about 40%.

在一些實施方式中，環狀多核糖核苷酸包含內部剪接元件，所述內部剪接元件在被複製時，剪接端部被連接在一起。一些實例包含具有剪接位點序列及短反向重複序列（30-40 nt）的微型內含子（＜ 100 nt），諸如AluSq2、AluJr及AluSz、側接內含子中的反向序列、側接內含子中的Alu元件以及在接近反向剪接事件的(suptable4富集模體)順式序列元件中發現的模體，諸如帶有側接外顯子的反向剪接位點（back splice site）之前（上游）或之後（下游）200 bp中的序列。在一些實施方式中，線性多核糖核苷酸包含至少一個本文其他處所述之重複核苷酸序列作為內部剪接元件。在此類實施方式中，重複核苷酸序列可以包括來自Alu家族內含子之重複序列。在一些實施方式中，剪接相關的核糖體結合蛋白質可調控環狀多核糖核苷酸的生物發生（例如，盲肌蛋白和震動蛋白（QKI）剪接因子）。In some embodiments, the cyclic polyribonucleotide contains internal splicing elements that, when replicated, ligate the splice ends together. Some examples include mini-introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt), such as AluSq2, AluJr and AluSz, inverted sequences flanking introns, flankingAlu elements in splice introns and motifs found in cis sequence elements close to back splicing events (suptable4 enriched motifs), such as back splice sites with flanking exons site) in the 200 bp before (upstream) or after (downstream) the sequence. In some embodiments, the linear polyribonucleotide contains at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repeating nucleotide sequence may include repeating sequences from Alu family introns. In some embodiments, splicing-related ribosome-binding proteins regulate the biogenesis of cyclic polyribonucleotides (e.g., blind muscle protein and shockin (QKI) splicing factors).

在一些實施方式中，線性多核糖核苷酸可包含側接環狀多核糖核苷酸的頭尾銜接點處的規範的剪接位點。In some embodiments, linear polyribonucleotides can comprise canonical splice sites at head-to-tail junctions flanking cyclic polyribonucleotides.

在一些實施方式中，線性多核糖核苷酸可包含隆起-螺旋-隆起模體，所述模體包含側接兩個3-核苷酸隆起的4-鹼基對莖。切割發生在隆起區域的一個位點處，生成末端為5'-羥基和2',3'-環狀磷酸酯的特徵片段。藉由將5'-OH基團親核攻擊到形成3',5'-磷酸二酯橋的同一分子的2',3'-環狀磷酸酯上來進行環化。In some embodiments, a linear polyribonucleotide can comprise a hump-helix-hump motif that includes a 4-base pair stem flanked by two 3-nucleotide humps. Cleavage occurs at a site in the bulge region, resulting in a characteristic fragment ending in a 5'-hydroxyl group and a 2',3'-cyclic phosphate. Cyclization occurs by nucleophilic attack of the 5'-OH group onto the 2',3'-cyclic phosphate of the same molecule forming the 3',5'-phosphodiester bridge.

在一些實施方式中，線性多核糖核苷酸可包含具有HPR元件的多聚重複RNA序列。HPR包含2',3'-環狀磷酸酯和5'-OH末端。HPR元件自處理線性線性多核糖核苷酸的5'端和3'端，由此將端連接在一起。In some embodiments, linear polyribonucleotides can comprise polyrepetitive RNA sequences with HPR elements. HPR contains 2',3'-cyclic phosphate and 5'-OH terminus. The HPR element self-processes the 5' and 3' ends of the linear polyribonucleotide, thereby joining the ends together.

在一些實施方式中，線性多核糖核苷酸可包含介導自連接的序列。在一個實施方式中，線性多核糖核苷酸可以包括HDV序列，例如，HDV複製結構域保守序列，GGCUCAUCUCGACAAGAGGCGGCAGUCCUCA GUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC（Beeharry等人2004）（SEQ ID NO: 359）或GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCGAGCC（SEQ ID NO: 360），以進行自連接。在一個實施方式中，線性多核糖核苷酸可以包括環E序列（例如在PSTVd中）以進行自連接。在另一個實施方式中，線性多核糖核苷酸可以包括自環化內含子，例如5'和3'剪接點，或自環化催化內含子，如I類、II類或III類內含子。I型內含子自剪接序列之非限制性實例可包含衍生自T4噬菌體基因td的自剪接置換內含子-外顯子序列以及眼原蟲之插入序列（IVS）rRNA。In some embodiments, linear polyribonucleotides can comprise sequences that mediate self-ligation. In one embodiment, the linear polyribonucleotide may comprise an HDV sequence, for example, the HDV replication domain conserved sequence, GGCUCAUCUCGACAAGAGGCGGCAGUCCUCA GUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al. 2004) (SEQ ID NO: 359) or GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGG AGACUGCUGGACUCGCCGCCCGAGCC (SEQ ID NO: 360) , for self-connection. In one embodiment, linear polyribonucleotides may include loop E sequences (eg, in PSTVd) for self-ligation. In another embodiment, the linear polyribonucleotide may include an autocyclizing intron, such as a 5' and 3' splice junction, or an autocyclizing catalytic intron, such as a class I, class II or class III intron. Hanzi. Non-limiting examples of type I intron self-splicing sequences may include self-splicing replacement intron-exon sequences derived from the T4 phage gene td and the insertion sequence (IVS) rRNA of Ophthalmia.

在一些實施方式中，用於環化的線性多核糖核苷酸可以包括互補序列，包括個體內含子內或側接內含子內的重複或非重複核酸序列。重複核酸序列係在線性多核糖核苷酸的區段內出現的序列。在一些實施方式中，線性多核糖核苷酸包含重複核酸序列。在一些實施方式中，重複核苷酸序列包括聚CA序列或聚UG序列。在一些實施方式中，線性多核糖核苷酸包含與線性多核糖核苷酸的另一區段中的互補重複核酸序列雜交的至少一個重複核酸序列，其雜交的區段形成內部雙鏈。在一些實施方式中，線性多核糖核苷酸包含與線性多核糖核苷酸的另一區段中的互補重複核酸序列雜交的1至10個（例如，2、3、4、5、6、7、8、9和10個）重複核酸序列，其雜交的區段形成內部雙鏈。在一些實施方式中，線性多核糖核苷酸包含與線性多核糖核苷酸的另一區段中的互補重複核酸序列雜交的2個重複核酸序列，其雜交的區段形成內部雙鏈。在一些實施方式中，兩個單獨的線性多核糖核苷酸的重複核酸序列和互補重複核酸序列雜交以生成單個環化多核糖核苷酸，雜交的區段形成內部雙鏈。在一些實施方式中，互補序列存在於用於環化的線性多核糖核苷酸的5'端和3'端。在一些實施方式中，互補序列包括約3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或更多個配對的核苷酸。In some embodiments, linear polyribonucleotides used for circularization may include complementary sequences, including repetitive or non-repetitive nucleic acid sequences within individual introns or within flanking introns. Repetitive nucleic acid sequences are sequences that occur within segments of linear polyribonucleotides. In some embodiments, linear polyribonucleotides comprise repeating nucleic acid sequences. In some embodiments, the repeating nucleotide sequences include polyCA sequences or polyUG sequences. In some embodiments, the linear polyribonucleotide comprises at least one repeating nucleic acid sequence that hybridizes to a complementary repeating nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, the linear polyribonucleotide comprises 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and 10) repeating nucleic acid sequences whose hybridized segments form internal double strands. In some embodiments, a linear polyribonucleotide comprises 2 repeating nucleic acid sequences that hybridize to a complementary repeating nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, two separate linear polyribonucleotide repeating nucleic acid sequences and a complementary repeating nucleic acid sequence hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming an internal double strand. In some embodiments, complementary sequences are present at the 5' and 3' ends of the linear polyribonucleotide used for circularization. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired cores glycosides.

在一些實施方式中，環化化學方法可用於生成環狀多核糖核苷酸。此類方法可以包括但不限於點擊化學（例如，基於炔烴和疊氮化物之方法，或可點擊的鹼基）、烯烴複分解、胺基磷酸酯連接、半胺縮醛-亞胺交聯、鹼基修飾、及其任何組合。In some embodiments, cyclization chemistries can be used to generate cyclic polyribonucleotides. Such methods may include, but are not limited to, click chemistry (e.g., alkyne- and azide-based methods, or clickable bases), olefin metathesis, aminophosphate ligation, hemiamine acetal-imine cross-linking, Base modification, and any combination thereof.

在一些實施方式中，環化酶促方法可用於生成環狀多核糖核苷酸。在一些實施方式中，連接酶，例如DNA或RNA連接酶，可用於生成環狀多核糖核苷酸或互補序列的模板、環狀多核糖核苷酸的互補鏈、或環狀多核糖核苷酸。In some embodiments, cyclization enzymatic methods can be used to generate cyclic polyribonucleotides. In some embodiments, ligases, such as DNA or RNA ligases, can be used to generate templates for cyclic polyribonucleotides or complementary sequences, complementary strands of cyclic polyribonucleotides, or cyclic polyribonucleotides. acid.

線性多核糖核苷酸的環化可以藉由本領域已知的方法完成，例如，Petkovic和Muller, 「RNA circularization strategies in vivo and in vitro [體內外RNA環化策略]」Nucleic Acids Res [核酸研究], 2015, 43(4): 2454-2465，以及Muller和Appel,「In vitro circularization of RNA [RNA的體外環化]」RNA Biol [RNA生物學], 2017, 14(8):1018-1027中描述的那些方法。Circularization of linear polyribonucleotides can be accomplished by methods known in the art, for example, Petkovic and Muller, "RNA circularization strategies in vivo and in vitro [RNA circularization strategies in vivo and in vitro]" Nucleic Acids Res [Nucleic Acid Research] , 2015, 43(4): 2454-2465, and Muller and Appel, "In vitro circularization of RNA [RNA in vitro circularization]" RNA Biol [RNA biology], 2017, 14(8):1018-1027 those methods described.

環狀多核糖核苷酸可編碼可用於複製的序列和/或模體。在國際專利公開案號WO 2019/118919的段落[0280]-[0286]中描述了示例性複製元件，該專利公開特此藉由援引以其全文併入。Cyclic polyribonucleotides may encode sequences and/or motifs that may be used for replication. Exemplary replication elements are described in International Patent Publication No. WO 2019/118919, paragraphs [0280]-[0286], which patent disclosure is hereby incorporated by reference in its entirety.

在一些實施方式中，線性多核糖核苷酸可以包括互補序列，包括個體內含子內或側接內含子內的重複或非重複核酸序列。重複核酸序列係在環狀多核糖核苷酸的區段內出現的序列。在一些實施方式中，線性多核糖核苷酸包含重複核酸序列。在一些實施方式中，重複核苷酸序列包括聚CA序列或聚UG序列。在一些實施方式中，線性多核糖核苷酸包含與線性多核糖核苷酸的另一區段中的互補重複核酸序列雜交的至少一個重複核酸序列，其雜交的區段形成內部雙鏈。在一些實施方式中，兩個單獨的線性多核糖核苷酸的重複核酸序列和互補重複核酸序列雜交以生成單個環化多核糖核苷酸，雜交的區段形成內部雙鏈。在一些實施方式中，互補序列存在於線性多核糖核苷酸的5'端和3'端。在一些實施方式中，互補序列包括約3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或更多個配對的核苷酸。In some embodiments, linear polyribonucleotides may include complementary sequences, including repeating or non-repeating nucleic acid sequences within individual introns or within flanking introns. Repetitive nucleic acid sequences are sequences that occur within segments of circular polyribonucleotides. In some embodiments, linear polyribonucleotides comprise repeating nucleic acid sequences. In some embodiments, the repeating nucleotide sequences include polyCA sequences or polyUG sequences. In some embodiments, the linear polyribonucleotide comprises at least one repeating nucleic acid sequence that hybridizes to a complementary repeating nucleic acid sequence in another segment of the linear polyribonucleotide, with the hybridized segment forming an internal double strand. In some embodiments, two separate linear polyribonucleotide repeating nucleic acid sequences and a complementary repeating nucleic acid sequence hybridize to generate a single circularized polyribonucleotide, with the hybridized segments forming an internal double strand. In some embodiments, complementary sequences are present at the 5' and 3' ends of the linear polyribonucleotide. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more paired cores glycosides.

在一些實施方式中，環化化學方法可用於生成環狀多核糖核苷酸。此類方法可以包括但不限於點擊化學（例如，基於炔烴和疊氮化物之方法，或可點擊的鹼基）、烯烴複分解、胺基磷酸酯連接、半胺縮醛-亞胺交聯、鹼基修飾、及其任何組合。In some embodiments, cyclization chemistries can be used to generate cyclic polyribonucleotides. Such methods may include, but are not limited to, click chemistry (e.g., alkyne- and azide-based methods, or clickable bases), olefin metathesis, aminophosphate ligation, hemiamine acetal-imine cross-linking, Base modification, and any combination thereof.

製備本文所述之環狀多核糖核苷酸的方法描述於以下中，例如，Khudyakov & Fields, Artificial DNA: Methods and Applications [人工DNA：方法與應用], CRC Press [CRC出版社] (2002)；Zhao, Synthetic Biology: Tools and Applications [合成生物學：工具與應用] (第一版), Academic Press [學術出版社] (2013)；Muller和Appel, RNA Biol [RNA生物學], 2017, 14(8):1018-1027；以及Egli和Herdewijn, Chemistry and Biology of Artificial Nucleic Acids [人工核酸的化學與生物學], (第一版), Wiley-VCH [威利-VCH出版社] (2012)。例如，在國際公開案號WO 2022/247943、美國專利案號US 11000547、國際公開案號2018/191722、國際公開案號WO 2019/236673、國際公開案號WO 2020/023595、國際公開案號WO 2022/204460、國際公開案號WO 2022/204464和國際公開案號WO 2022/204466中描述了製備環狀多核糖核苷酸的其他方法。Methods for preparing cyclic polyribonucleotides described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002) ; Zhao, Synthetic Biology: Tools and Applications [Synthetic Biology: Tools and Applications] (first edition), Academic Press [Academic Press] (2013); Muller and Appel, RNA Biol [RNA Biology], 2017, 14 (8):1018-1027; and Egli and Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (1st ed.), Wiley-VCH (2012) . For example, in International Publication No. WO 2022/247943, United States Patent No. US 11000547, International Publication No. 2018/191722, International Publication No. WO 2019/236673, International Publication No. WO 2020/023595, International Publication No. WO Other methods of preparing cyclic polyribonucleotides are described in International Publication No. WO 2022/204460, International Publication No. WO 2022/204464, and International Publication No. WO 2022/204466.

其他地方也描述了合成環狀多核糖核苷酸的各種方法（參見，例如，美國專利案號US 6210931、美國專利案號US 5773244、美國專利案號US 5766903、美國專利案號US 5712128、美國專利案號US 5426180、美國公開案號US 20100137407、國際公開案號WO 1992001813和國際公開案號WO 2010084371，以及Petkovic 等人, Nucleic Acids Res [核酸研究]. 43:2454-65 (2015)；將其各自內容藉由援引以其全文併入本文）。Various methods of synthesizing cyclic polyribonucleotides are also described elsewhere (see, e.g., U.S. Patent No. 6210931, U.S. Patent No. 5773244, U.S. Patent No. 5766903, U.S. Patent No. 5712128, U.S. Patent No. US 5426180, US Publication No. US 20100137407, International Publication No. WO 1992001813 and International Publication No. WO 2010084371, and Petkovic et al., Nucleic Acids Res [Nucleic Acids Res]. 43:2454-65 (2015); Their respective contents are incorporated herein by reference in their entirety).

在一些實施方式中，環狀多核糖核苷酸係純化的，例如，去除了游離核糖核酸、線性或帶切口的RNA、DNA、蛋白質等。在一些實施方式中，可以藉由本領域通常使用的任何已知方法純化環狀多核糖核苷酸。純化方法之非限制性實例包括柱層析法、凝膠切除、粒徑排阻等。產生方法無細胞系統中的產生方法In some embodiments, the cyclic polyribonucleotide is purified, for example, to remove free ribonucleic acid, linear or nicked RNA, DNA, proteins, etc. In some embodiments, cyclic polyribonucleotides can be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel excision, particle size exclusion, and the like.Production MethodsProduction Methods in Cell-Free Systems

本揭露還提供了產生環狀RNA之方法。例如，可在無細胞系統中轉錄（例如，藉由體外轉錄）去氧核糖核苷酸模板以產生線性RNA。線性多核糖核苷酸產生剪接相容的多核糖核苷酸，該多核糖核苷酸可以自剪接以產生環狀多核糖核苷酸。The present disclosure also provides methods of producing circular RNA. For example, a deoxyribonucleotide template can be transcribed (eg, by in vitro transcription) in a cell-free system to produce linear RNA. Linear polyribonucleotides produce splice-compatible polyribonucleotides that can self-splice to produce cyclic polyribonucleotides.

在一些實施方式中，本揭露提供了藉由以下來產生環狀多核糖核苷酸的方法（例如，在無細胞系統中）：提供線性多核糖核苷酸；以及在適用於剪接線性多核糖核苷酸的3'和5'剪接位點的條件下自剪接線性多核糖核苷酸；從而產生環狀多核糖核苷酸。In some embodiments, the present disclosure provides methods of generating cyclic polyribonucleotides (e.g., in a cell-free system) by: providing linear polyribonucleotides; and in a process suitable for splicing linear polyribonucleotides. Linear polyribonucleotides self-splice under the conditions of the 3' and 5' splice sites of the nucleotides; thereby producing cyclic polyribonucleotides.

在一些實施方式中，本揭露提供了藉由以下來產生環狀多核糖核苷酸的方法：提供編碼線性多核糖核苷酸的去氧核糖核苷酸；在無細胞系統中轉錄去氧核糖核苷酸以產生線性多核糖核苷酸；視需要純化剪接相容的線性多核糖核苷酸；以及在適用於剪接線性多核糖核苷酸的3'和5'剪接位點的條件下自剪接線性多核糖核苷酸，從而產生環狀多核糖核苷酸。In some embodiments, the present disclosure provides methods of generating cyclic polyribonucleotides by: providing deoxyribonucleotides encoding linear polyribonucleotides; transcribing deoxyribonucleotides in a cell-free system nucleotides to produce linear polyribonucleotides; optionally purify splice-compatible linear polyribonucleotides; and autogenize the 3' and 5' splice sites of linear polyribonucleotides under conditions suitable for splicing. Linear polyribonucleotides are spliced to produce cyclic polyribonucleotides.

在一些實施方式中，本揭露提供了藉由以下來產生環狀多核糖核苷酸的方法：提供編碼線性多核糖核苷酸的去氧核糖核苷酸；在無細胞系統中轉錄去氧核糖核苷酸以產生線性多核糖核苷酸（其中該轉錄在適用於剪接線性多核糖核苷酸的3'和5'剪接位點的條件下在溶液中發生），從而產生環狀多核糖核苷酸。在一些實施方式中，線性多核糖核苷酸包含5'斷裂內含子和3’斷裂內含子（例如，用於產生環狀多核糖核苷酸的自剪接構建體）。在一些實施方式中，線性多核糖核苷酸包含5'退火區和3'退火區。In some embodiments, the present disclosure provides methods of generating cyclic polyribonucleotides by: providing deoxyribonucleotides encoding linear polyribonucleotides; transcribing deoxyribonucleotides in a cell-free system nucleotides to produce linear polyribonucleotides (wherein this transcription occurs in solution under conditions suitable for splicing the 3' and 5' splice sites of linear polyribonucleotides), thereby producing circular polyribonucleotides glycosides. In some embodiments, the linear polyribonucleotide includes a 5' break intron and a 3' break intron (eg, a self-splicing construct used to generate cyclic polyribonucleotides). In some embodiments, the linear polyribonucleotide includes a 5' annealing region and a 3' annealing region.

用於體外轉錄和/或自剪接的合適條件可包括在一或多個方面模擬生理條件的任何條件（例如，溶液或緩衝液，如水性緩衝液或溶液）。在一些實施方式中，合適的條件包括在0.1-100 mM之間的Mg2+離子或其鹽（例如，1-100 mM、1-50 mM、1-20 mM、5-50 mM、5-20 mM或5-15 mM）。在一些實施方式中，合適的條件包括在1-1000 mM之間的K⁺離子或其鹽，如KCl（例如，1-1000 mM、1-500 mM、1-200 mM、50-500 mM、100-500 mM或100-300 mM）。在一些實施方式中，合適的條件包括在1-1000 mM之間的Cl-離子或其鹽，如KCl（例如，1-1000 mM、1-500 mM、1-200 mM、50-500 mM、100-500 mM或100-300 mM）。在一些實施方式中，合適的條件包括在0.1-100 mM之間的Mn2+離子或其鹽，如MnCl2（例如，0.1-100 mM、0.1-50 mM、0.1-20 mM、0.1-10 mM、0.1-5 mM、0.1-2 mM、0.5- 50 mM、0.5-20 mM、0.5-15 mM、0.5-5 mM、0.5-2 mM或0.1-10 mM）。在一些實施方式中，合適的條件包括二硫蘇糖醇（DTT）（例如，1-1000 μM、1-500 μM、1-200 μM、50-500 μM、100-500 μM、100-300 μM、0.1-100 mM、0.1-50 mM、0.1-20 mM、0.1-10 mM、0.1-5 mM、0.1-2 mM、0.5-50 mM、0.5-20 mM、0.5-15 mM、0.5-5 mM、0.5-2 mM或0.1-10 mM）。在一些實施方式中，合適的條件包括在0.1 mM與100 mM之間的核糖核苷三磷酸（NTP）（例如，0.1-100 mM、0.1-50 mM、0.1-10 mM、1-100 mM、1-50 mM或1-10 mM）。在一些實施方式中，合適的條件包括4至10的pH（例如，5至9的pH、6至9的pH或6.5至8.5的pH）。在一些實施方式中，合適的條件包括4°C至50°C的溫度（例如，10°C至40°C、15°C至40°C、20°C至40°C、或30°C至40°C）。Suitable conditions for in vitro transcription and/or auto-splicing may include any conditions (eg, solutions or buffers, such as aqueous buffers or solutions) that mimic physiological conditions in one or more aspects. In some embodiments, suitable conditions include Mg2+ ions or salts thereof between 0.1-100 mM (e.g., 1-100 mM, 1-50 mM, 1-20 mM, 5-50 mM, 5-20 mM or 5-15 mM). In some embodiments, suitable conditions include K⁺ ions or salts thereof, such as KCl, between 1-1000 mM (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM or 100-300 mM). In some embodiments, suitable conditions include Cl- ions or salts thereof, such as KCl, between 1-1000 mM (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM or 100-300 mM). In some embodiments, suitable conditions include Mn2+ ions or salts thereof, such as MnCl2, between 0.1-100 mM (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10 mM, 0.1 -5mM, 0.1-2mM, 0.5-50mM, 0.5-20mM, 0.5-15mM, 0.5-5mM, 0.5-2mM or 0.1-10mM). In some embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1-1000 μM, 1-500 μM, 1-200 μM, 50-500 μM, 100-500 μM, 100-300 μM ,0.1-100mM,0.1-50mM,0.1-20mM,0.1-10mM,0.1-5mM,0.1-2mM,0.5-50mM,0.5-20mM,0.5-15mM,0.5-5mM ,0.5-2 mM or 0.1-10 mM). In some embodiments, suitable conditions include ribonucleoside triphosphate (NTP) between 0.1 mM and 100 mM (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 1-100 mM, 1-50 mM or 1-10 mM). In some embodiments, suitable conditions include a pH of 4 to 10 (eg, a pH of 5 to 9, a pH of 6 to 9, or a pH of 6.5 to 8.5). In some embodiments, suitable conditions include a temperature of 4°C to 50°C (e.g., 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, or 30°C to 40°C).

在一些實施方式中，線性多核糖核苷酸從去氧核糖核酸（例如，本文所述之去氧核糖核酸，如DNA載體、線性化DNA載體、或cDNA）產生。在一些實施方式中，線性多核糖核苷酸藉由在無細胞系統中轉錄（例如，體外轉錄）從去氧核糖核酸進行轉錄。細胞中的產生方法In some embodiments, linear polyribonucleotides are produced from DNA (eg, DNA described herein, such as a DNA vector, a linearized DNA vector, or cDNA). In some embodiments, linear polyribonucleotides are transcribed from DNA by transcription in a cell-free system (eg, in vitro transcription).production methods in cells

本揭露還提供了在細胞（例如，原核細胞或真核細胞）中產生環狀RNA之方法。在一些實施方式中，向細胞提供外源多核糖核苷酸（例如，本文所述之線性多核糖核苷酸或編碼此處所述之線性多核糖核苷酸的轉錄的DNA分子）。線性多核糖核苷酸可以在細胞中從向細胞提供的外源DNA分子轉錄。線性多核糖核苷酸可以在細胞中從向該細胞暫態提供的外源重組DNA分子轉錄。在一些實施方式中，外源DNA分子不整合到細胞的基因組中。在一些實施方式中，線性多核糖核苷酸在細胞中從整合到細胞基因組中的重組DNA分子轉錄。The present disclosure also provides methods of producing circular RNA in cells (eg, prokaryotic or eukaryotic cells). In some embodiments, an exogenous polyribonucleotide (eg, a linear polyribonucleotide described herein or a transcribed DNA molecule encoding a linear polyribonucleotide described herein) is provided to the cell. Linear polyribonucleotides can be transcribed in cells from exogenous DNA molecules provided to the cell. Linear polyribonucleotides can be transcribed in a cell from exogenous recombinant DNA molecules transiently provided to the cell. In some embodiments, the exogenous DNA molecule is not integrated into the genome of the cell. In some embodiments, linear polyribonucleotides are transcribed in a cell from recombinant DNA molecules that are integrated into the genome of the cell.

在一些實施方式中，細胞係原核細胞。在一些實施方式中，包括本文所述之多核糖核苷酸的原核細胞可為細菌細胞或古菌細胞。例如，包括本文所述之多核糖核苷酸的原核細胞可為大腸桿菌（E coli）、嗜鹽古菌（例如，沃氏鹽富饒菌（Haloferax volcaniii））、鞘胺醇單胞菌（Sphingomonas）、藍菌（例如，細長聚球藻（Synechococcus elongatus）、螺旋藻（Spirulina）（節旋藻（Arthrospira））屬和集胞藻屬（Synechocystis spp.））、鏈黴菌（Streptomyces）、放線菌（actinomycetes）（例如，野村菌屬（Nonomuraea）、北裡孢菌屬（Kitasatospora）或溫雙岐菌屬（Thermobifida））、芽孢桿菌屬（Bacillus spp.）（例如，枯草芽孢桿菌（Bacillus subtilis）、炭疽芽孢桿菌（Bacillus anthracis）、蠟樣芽孢桿菌（Bacillus cereus））、β變形菌（betaproteobacteria）（例如，伯克氏菌（Burkholderia））、α變形菌（alphaproteobacterial）（例如，土壤桿菌（Agrobacterium））、假單胞菌（Pseudomonas）（例如，惡臭假單胞菌（Pseudomonas putida））和腸桿菌（enterobacteria）。原核細胞可以在培養基中生長。原核細胞可以包含在生物反應器中。In some embodiments, the cell line is a prokaryotic cell. In some embodiments, a prokaryotic cell including a polyribonucleotide described herein can be a bacterial cell or an archaeal cell. For example, a prokaryotic cell including a polyribonucleotide described herein can be E coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas ), cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) and Synechocystis spp.), Streptomyces, Actinomycetes (actinomycetes) (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis, anthrax Bacillus anthracis, Bacillus cereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacterial (e.g., Agrobacterium) , Pseudomonas (e.g., Pseudomonas putida), and enterobacteria. Prokaryotic cells can be grown in culture media. Prokaryotic cells can be contained in the bioreactor.

在一些實施方式中，細胞係真核細胞。在一些實施方式中，包括本文所述之多核糖核苷酸的真核細胞係單細胞真核細胞。在一些實施方式中，單細胞真核細胞係單細胞真菌細胞，諸如酵母細胞（例如，釀酒酵母（Saccharomyces cerevisiae）和其他酵母屬（Saccharomyces spp.）、酒香酵母屬（Brettanomyces spp.）、裂殖酵母屬（Schizosaccharomyces spp.）、有孢圓酵母屬（Torulaspora spp.）和畢赤酵母屬（Pichia spp.））。在一些實施方式中，單細胞真核細胞係單細胞動物細胞。單細胞動物細胞可為從多細胞動物分離並在培養中生長的細胞，或其子細胞。在一些實施方式中，單細胞動物細胞可以去分化。在一些實施方式中，單細胞真核細胞係單細胞植物細胞。單細胞植物細胞可為從多細胞植物中分離並在培養中生長的細胞，或其子細胞。在一些實施方式中，單細胞植物細胞可以去分化。在一些實施方式中，單細胞植物細胞來自植物愈傷組織。在實施方式中，單細胞細胞係植物細胞原生質體。在一些實施方式中，單細胞真核細胞係單細胞真核藻類細胞，如單細胞綠藻、矽藻、眼蟲、或甲藻。感興趣的單細胞真核藻類之非限制性實例包括鹽生杜氏藻（Dunaliella salina）、普通小球藻（Chlorella vulgaris）、以若夫小球藻（Chlorella zofingiensis）、雨生紅球藻（Haematococcus pluvialis）、富油新綠藻（Neochloris oleoabundans）和其他新綠藻屬（Neochloris spp.）、葡萄原管藻（Protosiphon botryoides）、布朗葡萄藻（Botryococcus braunii）、隱球菌屬（Cryptococcus spp.）、萊茵衣藻（Chlamydomonas reinhardtii）和其他衣藻屬（Chlamydomonas spp.）。在一些實施方式中，單細胞真核細胞係原生生物細胞。在一些實施方式中，單細胞真核細胞係原生動物細胞。In some embodiments, the cell line is a eukaryotic cell. In some embodiments, a eukaryotic cell line unicellular eukaryotic cell includes a polyribonucleotide described herein. In some embodiments, the unicellular eukaryotic cell line is a unicellular fungal cell, such as a yeast cell (eg, Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces cerevisiae) Schizosaccharomyces spp., Torulaspora spp., and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. Unicellular animal cells may be cells isolated from multicellular animals and grown in culture, or daughter cells thereof. In some embodiments, single-cell animal cells can be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. Unicellular plant cells may be cells isolated from multicellular plants and grown in culture, or daughter cells thereof. In some embodiments, unicellular plant cells can be dedifferentiated. In some embodiments, the single-cell plant cells are derived from plant callus. In embodiments, the single cell line is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cells are unicellular eukaryotic algal cells, such as unicellular green algae, diatoms, Euglena, or dinoflagellates. Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis ), Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii) and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell line is a protozoan cell.

在一些實施方式中，真核細胞係多細胞真核生物的細胞。例如，多細胞真核生物可選自由以下組成之群組：脊椎動物、無脊椎動物、多細胞真菌、多細胞藻類和多細胞植物。在一些實施方式中，真核生物體係人。在一些實施方式中，真核生物體係非人脊椎動物。在一些實施方式中，真核生物體係無脊椎動物。在一些實施方式中，真核生物體係多細胞真菌。在一些實施方式中，真核生物體係多細胞植物。在一些實施方式中，真核細胞係人的細胞或非人類哺乳動物的細胞，如非人類靈長類動物（例如，猴子、猿）、有蹄類動物（例如，牛科動物，包括牛、水牛、野牛、綿羊、山羊和麝牛；豬；駱駝科動物，包括駱駝、美洲駝、和羊駝；鹿，羚羊；以及馬科動物，包括馬和驢）、食肉動物（例如，狗、貓）、齧齒動物（例如，大鼠、小鼠、豚鼠、倉鼠、松鼠）或兔類動物（例如，兔、野兔）。在一些實施方式中，真核細胞係鳥的細胞，諸如禽類分類群雞形目（例如，雞、火雞、雉雞、鵪鶉）、雁形目（例如，鴨、鵝）、古顎總目（例如，鴕鳥、鴯鶓）、鴿形目（例如，鴿子、野鴿）或鸚形目（例如，鸚鵡）的成員。在一些實施方式中，真核細胞係節肢動物（例如，昆蟲、蛛形綱、甲殼動物）、線蟲、環節動物、蠕蟲或軟體動物的細胞。在實施方式中，真核細胞係多細胞植物的細胞，如被子植物（其可為雙子葉植物或單子葉植物）或裸子植物（例如，針葉樹、蘇鐵、買麻藤類植物、銀杏）、蕨類、馬尾植物、石鬆類、或苔蘚植物。在實施方式中，真核細胞係真核多細胞藻類的細胞。In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryote. For example, multicellular eukaryotes may be selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular algae, and multicellular plants. In some embodiments, the eukaryotic system is human. In some embodiments, the eukaryotic system is non-human vertebrate. In some embodiments, the eukaryotic system is an invertebrate. In some embodiments, the eukaryotic system is a multicellular fungus. In some embodiments, eukaryotic systems are multicellular plants. In some embodiments, the eukaryotic cell line is a human cell or a non-human mammal cell, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovine species, including cattle, Buffalo, bison, sheep, goats, and muskoxen; pigs; camelids, including camels, llamas, and alpacas; deer, antelope; and equids, including horses and donkeys), carnivores (e.g., dogs, cats ), rodents (e.g., rats, mice, guinea pigs, hamsters, squirrels), or lagomorphs (e.g., rabbits, hares). In some embodiments, the eukaryotic cell line is a cell of an avian species, such as the avian taxa Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Palaeognathoidea (e.g., ostrich, emu), member of the order Columbiformes (e.g., pigeons, wild pigeons), or Psittaciformes (e.g., parrots). In some embodiments, the eukaryotic cell is an arthropod (eg, insect, arachnid, crustacean), nematode, annelid, helminth, or mollusk cell. In embodiments, the eukaryotic cell line is a cell of a multicellular plant, such as an angiosperm (which may be a dicot or a monocot) or a gymnosperm (e.g., conifers, cycads, cycads, ginkgo), ferns species, horsetail plants, lycophytes, or bryophytes. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular algae.

該等真核細胞可以在培養基中生長。該等真核細胞可以包含在生物反應器中。純化的方法The eukaryotic cells can be grown in culture medium. The eukaryotic cells can be contained in a bioreactor.Purification method

本文所述之方法中可包括一或多個純化步驟。例如，在一些實施方式中，在自剪接線性多核糖核苷酸之前，線性多核糖核苷酸實質上是富集的或純的（例如，純化的）。在其他實施方式中，在自剪接線性多核糖核苷酸之前不純化線性多核糖核苷酸。在一些實施方式中，純化所得的環狀RNA。One or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotide is substantially enriched or pure (eg, purified) prior to self-splicing. In other embodiments, the linear polyribonucleotide is not purified prior to self-splicing. In some embodiments, the resulting circular RNA is purified.

純化可包括從一或多種不期望的組分（諸如任何未反應的起始材料、副產物、酶或其他反應組分）中分離或富集期望的反應產物。例如，在無細胞系統中轉錄（例如體外轉錄）後純化線性多核糖核苷酸可包括在自剪接線性多核糖核苷酸之前從DNA模板中分離或富集。剪接後環狀RNA產物的純化可用於從其對應的線性RNA中分離或富集環狀RNA。RNA的純化方法係熟悉該項技術者已知的，並且包括酶純化或藉由層析法。Purification may include isolating or enriching the desired reaction product from one or more undesirable components, such as any unreacted starting materials, by-products, enzymes, or other reaction components. For example, purifying linear polyribonucleotides after transcription in a cell-free system (eg, in vitro transcription) may include isolating or enriching the linear polyribonucleotides from the DNA template prior to self-splicing. Purification of post-splicing circRNA products can be used to isolate or enrich circRNAs from their linear RNA counterparts. Methods for purifying RNA are known to those skilled in the art and include enzymatic purification or by chromatography.

在一些實施方式中，純化方法產生具有少於50%（例如少於40%、30%、20%、10%、5%、4%、3%、2%或1%）線性多核糖核苷酸的環狀多核糖核苷酸。生物反應器In some embodiments, the purification method produces linear polyribonucleosides with less than 50% (eg, less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) Acidic cyclic polyribonucleotide.bioreactor

在一些實施方式中，產生本文所述之環狀多核糖核苷酸的任何方法都可在生物反應器中進行。生物反應器係指在其中進行化學或生物學過程的任何容器，該過程涉及生物體或衍生自此類生物體的生物化學活性物質。生物反應器可與本文所述之用於產生環狀RNA的無細胞方法相容。用於生物反應器的容器可包括培養瓶、培養皿或培養袋，它們可為一次性使用的（一次性的）、可高壓滅菌的或可滅菌的。生物反應器可以由玻璃製成，或者其也可為基於聚合物的，或者其也可以由其他材料製成。In some embodiments, any method of producing cyclic polyribonucleotides described herein can be performed in a bioreactor. Bioreactor means any vessel in which a chemical or biological process involving living organisms or biochemically active substances derived from such organisms is carried out. The bioreactor is compatible with the cell-free methods for producing circular RNA described herein. Containers for bioreactors may include culture bottles, Petri dishes, or culture bags, which may be single-use (disposable), autoclavable, or sterilizable. The bioreactor can be made of glass, or it can also be polymer-based, or it can also be made of other materials.

生物反應器之實例包括但不限於攪拌罐（例如，充分混合的）生物反應器和管式（例如，活塞流）生物反應器、氣升式生物反應器、膜攪拌罐、旋轉過濾攪拌罐、振動混合器、流體床反應器、和膜生物反應器。操作生物反應器的模式可為間歇的或連續的過程。當試劑和產物流連續地進出系統時，生物反應器係連續的。間歇式生物反應器可以具有連續的再循環流，但沒有連續的試劑進料或產物收穫。Examples of bioreactors include, but are not limited to, stirred tank (e.g., well-mixed) and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, rotary filter stirred tanks, Vibrating mixers, fluid bed reactors, and membrane bioreactors. The mode of operation of the bioreactor can be a batch or continuous process. A bioreactor is continuous when reagent and product streams flow continuously into and out of the system. Batch bioreactors can have continuous recirculation flow but no continuous reagent feed or product harvest.

本揭露之一些方法涉及大規模生產環狀多核糖核苷酸。對於大規模生產方法，該方法可以在1升（L）至50 L或更多（例如，5 L、10 L、15 L、20 L、25 L、30 L、35 L、40 L、45 L、50 L或更多）的體積中進行。在一些實施方式中，該方法可在5 L至10 L、5 L至15 L、5 L至20 L、5 L至25 L、5 L至30 L、5 L至35 L、5 L至40 L、5 L至45 L、10 L至15 L、10 L至20 L、10 L至25 L、20 L至30 L、10 L至35 L、10 L至40 L、10 L至45 L、10 L至50 L、15 L至20 L、15 L至25 L、15 L至30 L、15 L至35 L、15 L至40 L、15 L至45 L或15至50 L的體積中進行。Some methods of the present disclosure involve large-scale production of cyclic polyribonucleotides. For large-scale production methods, the method can range from 1 liter (L) to 50 L or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L , 50 L or more) in a volume. In some embodiments, the method can be performed at 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, Performed in volumes of 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L .

在一些實施方式中，生物反應器可產生至少1 g的環狀RNA。在一些實施方式中，生物反應器可以產生1-200 g環狀RNA（例如，1-10 g、1-20g、1-50 g、10-50 g、10-100 g、50-100 g、50-200 g環狀RNA）。在一些實施方式中，產生的量係測量值/升（例如，1-200 g/升）、/批或反應（例如，1-200 g/批或反應）、或/單位時間（例如，1-200 g/小時或/天）。In some embodiments, the bioreactor can produce at least 1 g of circular RNA. In some embodiments, the bioreactor can produce 1-200 g of circRNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, 50-200 g circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200 g/liter), per batch or reaction (e.g., 1-200 g/batch or reaction), or per unit time (e.g., 1 -200 g/hour or/day).

在一些實施方式中，可以串聯使用多於一個生物反應器以增加產生能力（例如，可以串聯使用一個、兩個、三個、四個、五個、六個、七個、八個、或九個生物反應器）。使用方法In some embodiments, more than one bioreactor can be used in series to increase production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine can be used in series). bioreactor).Instructions

在一些實施方式中，編碼抗融合多肽（例如，表1的多肽）的環狀多核糖核苷酸用於治療或預防病毒感染（例如，HIV、SARS-CoV-2、HCV、流感或RSV）。In some embodiments, cyclic polyribonucleotides encoding anti-fusion polypeptides (e.g., the polypeptides of Table 1) are used to treat or prevent viral infections (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV) .

在一些實施方式中，編碼抗融合多肽（例如，表1的多肽）的環狀多核苷酸用於減少病毒進入。In some embodiments, a circular polynucleotide encoding an anti-fusion polypeptide (eg, a polypeptide of Table 1) is used to reduce viral entry.

在一些實施方式中，編碼抗融合多肽（例如，表1的多肽）的環狀多核苷酸可投與給受試者以降低病毒感染（例如，HIV、SARS-CoV-2、HCV、流感或RSV）的風險。In some embodiments, a cyclic polynucleotide encoding an anti-fusion polypeptide (e.g., a polypeptide of Table 1) can be administered to a subject to reduce viral infection (e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV) risk.

例如，可向受試者投與如本文所述之環狀多核糖核苷酸（例如，在藥物組成物中）。在一些實施方式中，受試者係脊椎動物（例如哺乳動物、鳥、魚、爬行動物或兩棲動物）。在一些實施方式中，受試者係人。在一些實施方式中，受試者係非人哺乳動物。在實施方式中，受試者係非人哺乳動物，諸如非人靈長類動物（例如猴、猿）、有蹄類動物（例如家牛、水牛、綿羊、山羊、豬、駱駝、美洲駝、羊駝、鹿、馬、驢）、肉食動物（例如狗、貓）、齧齒動物（例如大鼠、小鼠）或兔類動物（例如兔）。在實施方式中，受試者係鳥，諸如禽類分類群雞形目（例如雞、火雞、野雞、鵪鶉）、雁形目（例如鴨、鵝）、古顎下綱（例如鴕鳥、鴯鶓）、鴿形目（例如鴿子、野鴿）或鸚形目（例如鸚鵡）的成員。在實施方式中，受試者係無脊椎動物，諸如節肢動物（例如昆蟲、蜘蛛、甲殼類動物）、線蟲、環節動物、蠕蟲或軟體動物。For example, a cyclic polyribonucleotide as described herein can be administered to a subject (eg, in a pharmaceutical composition). In some embodiments, the subject is a vertebrate (eg, mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is human. In some embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpacas, deer, horses, donkeys), carnivores (e.g. dogs, cats), rodents (e.g. rats, mice) or lagomorphs (e.g. rabbits). In embodiments, the subject is a bird, such as the avian taxa Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathia (e.g., ostrich, emu) , a member of the order Columbiformes (e.g. pigeons, wild pigeons) or the order Psittaciformes (e.g. parrots). In embodiments, the subject is an invertebrate, such as an arthropod (eg, insect, spider, crustacean), nematode, annelid, worm, or mollusk.

在一些實施方式中，本揭露提供了一種藉由向受試者提供本文所述之組成物或配製物來改變受試者之方法。在一些實施方式中，組成物或配製物係或包括核酸分子（例如，本文所述之DNA分子或RNA分子），並且向真核受試者提供多核苷酸。在一些實施方式中，組成物或配製物係或包括包含本文所述之核酸的真核或原核細胞。In some embodiments, the present disclosure provides a method of altering a subject by providing the subject with a composition or formulation described herein. In some embodiments, a composition or formulation is or includes a nucleic acid molecule (eg, a DNA molecule or an RNA molecule as described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, a composition or formulation is or includes a eukaryotic or prokaryotic cell comprising a nucleic acid described herein.

在一些實施方式中，本揭露提供了一種藉由向有需要的受試者提供本文所述之組成物或配製物來治療受試者的病毒感染之方法。在一些實施方式中，組成物或配製物係或包括核酸分子（例如，本文所述之DNA分子或RNA分子），並且向真核受試者提供多核苷酸。在一些實施方式中，組成物或配製物係或包括包含本文所述之核酸的真核或原核細胞。在一些實施方式中，多核糖核苷酸的提供量和持續時間足以治療例如需要治療的受試者中的病毒感染。In some embodiments, the present disclosure provides a method of treating a viral infection in a subject by providing a composition or formulation described herein to the subject in need thereof. In some embodiments, a composition or formulation is or includes a nucleic acid molecule (eg, a DNA molecule or an RNA molecule as described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, a composition or formulation is or includes a eukaryotic or prokaryotic cell comprising a nucleic acid described herein. In some embodiments, the polyribonucleotide is provided in an amount and for a duration sufficient to treat, for example, a viral infection in a subject in need of treatment.

在一些實施方式中，該方法可用於治療或預防HIV。例如，在一些實施方式中，環狀多核糖核苷酸編碼靶向HIV的抗融合多肽，並且該組成物可用於治療或預防HIV。In some embodiments, the method can be used to treat or prevent HIV. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting HIV, and the composition can be used to treat or prevent HIV.

在一些實施方式中，該方法可用於治療或預防SARS-CoV-2。例如，在一些實施方式中，環狀多核糖核苷酸編碼靶向SARS-CoV-2的抗融合多肽，並且該組成物可用於治療或預防SARS-CoV-2。In some embodiments, the method can be used to treat or prevent SARS-CoV-2. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting SARS-CoV-2, and the composition can be used to treat or prevent SARS-CoV-2.

在一些實施方式中，該方法可用於治療或預防HCV。例如，在一些實施方式中，環狀多核糖核苷酸編碼靶向HCV的抗融合多肽，並且該組成物可用於治療或預防HCV。In some embodiments, the method can be used to treat or prevent HCV. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting HCV, and the composition can be used to treat or prevent HCV.

在一些實施方式中，該方法可用於治療或預防RSV。例如，在一些實施方式中，環狀多核糖核苷酸編碼靶向RSV的抗融合多肽，並且該組成物可用於治療或預防RSV。給藥方法In some embodiments, the method can be used to treat or prevent RSV. For example, in some embodiments, the cyclic polyribonucleotide encodes an anti-fusion polypeptide targeting RSV, and the composition can be used to treat or prevent RSV.Dosing method

本文揭露了一種在向細胞提供環狀多核糖核苷酸的至少兩個劑量或組成物後，在細胞中產生一定水平的編碼抗融合多肽（例如，表1的多肽）的環狀多核糖核苷酸或表現一定水平的抗融合多肽（例如，表1的多肽）的給藥方法。本文揭露了一種在向受試者提供（例如，投與）環狀多核糖核苷酸的至少兩個劑量或組成物後，在受試者（例如，哺乳動物，例如，人）中產生一定水平的環狀多核糖核苷酸或表現一定水平的抗融合多肽（例如，表1的多肽，例如表2-4中任一個的多肽）的給藥方法。該組成物包含編碼如本文所述之抗融合多肽的環狀多核糖核苷酸。給藥方法可以包括投與兩個或更多個劑量的環狀多核糖核苷酸組成物，例如在短時間段內或在延長時間段內。在一些實施方式中，含有環狀多核糖核苷酸的組成物進一步包含藥學上可接受的載劑或賦形劑。環狀多核糖核苷酸編碼抗融合多肽，其可以在細胞中表現，例如在投與後。Disclosed herein is a method that produces a certain level of a cyclic polyribonucleotide encoding an anti-fusion polypeptide (e.g., the polypeptide of Table 1) in a cell after providing at least two doses or compositions of the cyclic polyribonucleotide to the cell. Methods of administering polypeptides or anti-fusion polypeptides (e.g., the polypeptides of Table 1) that exhibit certain levels. Disclosed herein is a method for producing a certain amount in a subject (e.g., a mammal, e.g., a human) after providing (e.g., administering) at least two doses or compositions of a cyclic polyribonucleotide to the subject. A method of administering a cyclic polyribonucleotide at a certain level or expressing a certain level of an anti-fusion polypeptide (e.g., a polypeptide of Table 1, e.g., a polypeptide of any one of Tables 2-4). The composition includes a cyclic polyribonucleotide encoding an anti-fusion polypeptide as described herein. Methods of administration may include administration of two or more doses of the cyclic polyribonucleotide composition, for example, over a short period of time or over an extended period of time. In some embodiments, the cyclic polyribonucleotide-containing composition further includes a pharmaceutically acceptable carrier or excipient. The cyclic polyribonucleotide encodes an anti-fusion polypeptide, which can be expressed in the cell, for example, upon administration.

本文所述之方法可包括以足以在受試者中產生至少500 ng/mL（例如，至少600 ng/mL、700 ng/mL、800 ng/mL、900 ng/mL、1,000 ng/mL、1,100 ng/mL、1,200 ng/mL、1,300 ng/mL、1,400 ng/mL、1,500 ng/mL、1,600 ng/mL、1,700 ng/mL、1,800 ng/mL、1,900 ng/mL、2,000 ng/mL、2,100 ng/mL、2,200 ng/mL、2,300 ng/mL、2,400 ng/mL、2,500 ng/mL、2,600 ng/mL、2,700 ng/mL、2,800 ng/mL、2,900 ng/mL、3,000 ng/mL或更多）的血清濃度的抗融合多肽的量投與第一劑量的該藥物組成物。The methods described herein may include administering a drug at a concentration sufficient to produce in a subject at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more The first dose of the pharmaceutical composition is administered in an amount greater than) the serum concentration of the anti-fusion polypeptide.

在一些實施方式中，該方法可進一步包括投與第二劑量的藥物組成物。該方法可進一步包括投與第三、第四、第五、第六、第七、第八、第九、第十或更多劑量的藥物組成物。在一些實施方式中，後續劑量有助於在受試者中維持至少500 ng/mL（例如，至少600 ng/mL、700 ng/mL、800 ng/mL、900 ng/mL、1,000 ng/mL、1,100 ng/mL、1,200 ng/mL、1,300 ng/mL、1,400 ng/mL、1,500 ng/mL、1,600 ng/mL、1,700 ng/mL、1,800 ng/mL、1,900 ng/mL、2,000 ng/mL、2,100 ng/mL、2,200 ng/mL、2,300 ng/mL、2,400 ng/mL、2,500 ng/mL、2,600 ng/mL、2,700 ng/mL、2,800 ng/mL、2,900 ng/mL、3,000 ng/mL或更多）的血清濃度的抗融合多肽。在一些實施方式中，在受試者中的抗融合多肽之血清濃度降至500 ng/mL以下之前投與後續劑量。In some embodiments, the method may further comprise administering a second dose of the pharmaceutical composition. The method may further comprise administering a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more doses of the pharmaceutical composition. In some embodiments, subsequent doses help maintain at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL) in the subject , 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL , 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more) of the serum concentration of the anti-fusion peptide. In some embodiments, subsequent doses are administered before the serum concentration of the anti-fusion polypeptide in the subject falls below 500 ng/mL.

在一些實施方式中，提供多個劑量以在細胞、組織或受試者中產生一定水平的該組成物或表現一定水平的該抗融合多肽。在一些實施方式中，提供多個劑量以在一段時間內在細胞、組織或受試者中產生或維持一定水平的該組成物或者產生或維持一定水平的該抗融合多肽，例如至少20、30、40、50、60、70、80、90、100、120、150天，或至少1、2、3、4、5、6、7、8、9、10、12、15、18、21或24個月，或至少1、2、3、4或5年。In some embodiments, multiple doses are provided to produce a certain level of the composition or to express a certain level of the anti-fusion polypeptide in a cell, tissue or subject. In some embodiments, multiple doses are provided to produce or maintain a certain level of the composition or to produce or maintain a certain level of the anti-fusion polypeptide in a cell, tissue or subject over a period of time, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21 or 24 months, or at least 1, 2, 3, 4 or 5 years.

在一些實施方式中，第二劑量在藥物組成物之第一劑量後至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）投與。In some embodiments, the second dose is at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours) after the first dose of the pharmaceutical composition. hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months , eleven months, one year or more) investment.

在一些實施方式中，第二劑量在藥物組成物之第一劑量後1小時至1年（例如1小時到1天，例如一小時、兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時或一天，例如一天到一週，例如兩天、三天、四天、五天、六天或一週，例如一週到一個月，例如兩週、三週或一個月，例如一個月到一年，例如一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、九個月、十個月、十一個月或一年）投與。在一些實施方式中，第二劑量在藥物組成物之第一劑量後1天至180天（例如，1天到90天、1天到45天、1天到30天、1天到14天、1天到7天、2天到45天、2天到30天、2天到14天、2天到7天、3天到90天、3天到45天、3天到30天、3天到14天、3天到7天、4天到90天、4天到45天、4天到30天、4天到14天、4天到7天、5天到90天、5天到45天、5天到30天、5天到14天、5天到7天、6天到90天、6天到45天、6天到30天、6天到14天、6天到7天、7天到90天、7天到45天、7天到30天、7天到14天、14天到90天、14天到45天、14天到30天、21天到90天、21天到60天、21天到45天、21天到30天、30天到90天、30天到60天、30天到45天、45天到180天、45天到120天、45天到100天、45天到90天、45天到60天、60天到180天、60天到120天、60天到100天、60天到90天、90天到100天、90天到120天或90天至180天）投與。In some embodiments, the second dose is 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, Seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or a day, such as a day to a week, such as two days, three days, four days, five days, six days or a week, such as a week to a month, such as two weeks, three weeks or a month, such as a month to a year, e.g. One month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year) investment. In some embodiments, the second dose is 1 day to 180 days after the first dose of the pharmaceutical composition (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days or 90 days to 180 days) investment.

在一些實施方式中，第三劑量在藥物組成物之第二劑量後至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）投與。In some embodiments, the third dose is at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours) after the second dose of the pharmaceutical composition. hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months , eleven months, one year or more) investment.

在一些實施方式中，第三劑量在藥物組成物之第二劑量後1小時至1年（例如1小時到1天，例如一小時、兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時或一天，例如一天到一週，例如兩天、三天、四天、五天、六天或一週，例如一週到一個月，例如兩週、三週或一個月，例如一個月到一年，例如一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、九個月、十個月、十一個月或一年）投與。在一些實施方式中，第三劑量在藥物組成物之第二劑量後1天至180天（例如，1天到90天、1天到45天、1天到30天、1天到14天、1天到7天、2天到45天、2天到30天、2天到14天、2天到7天、3天到90天、3天到45天、3天到30天、3天到14天、3天到7天、4天到90天、4天到45天、4天到30天、4天到14天、4天到7天、5天到90天、5天到45天、5天到30天、5天到14天、5天到7天、6天到90天、6天到45天、6天到30天、6天到14天、6天到7天、7天到90天、7天到45天、7天到30天、7天到14天、14天到90天、14天到45天、14天到30天、21天到90天、21天到60天、21天到45天、21天到30天、30天到90天、30天到60天、30天到45天、45天到180天、45天到120天、45天到100天、45天到90天、45天到60天、60天到180天、60天到120天、60天到100天、60天到90天、90天到100天、90天到120天或90天至180天）投與。In some embodiments, the third dose is 1 hour to 1 year (e.g., 1 hour to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six hours, Seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or a day, such as a day to a week, such as two days, three days, four days, five days, six days or a week, such as a week to a month, such as two weeks, three weeks or a month, such as a month to a year, e.g. One month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year) investment. In some embodiments, the third dose is 1 day to 180 days (e.g., 1 day to 90 days, 1 day to 45 days, 1 day to 30 days, 1 day to 14 days, 1 day to 7 days, 2 days to 45 days, 2 days to 30 days, 2 days to 14 days, 2 days to 7 days, 3 days to 90 days, 3 days to 45 days, 3 days to 30 days, 3 days to 14 days, 3 days to 7 days, 4 days to 90 days, 4 days to 45 days, 4 days to 30 days, 4 days to 14 days, 4 days to 7 days, 5 days to 90 days, 5 days to 45 days, 5 days to 30 days, 5 days to 14 days, 5 days to 7 days, 6 days to 90 days, 6 days to 45 days, 6 days to 30 days, 6 days to 14 days, 6 days to 7 days, 7 days to 90 days, 7 days to 45 days, 7 days to 30 days, 7 days to 14 days, 14 days to 90 days, 14 days to 45 days, 14 days to 30 days, 21 days to 90 days, 21 days to 60 days, 21 days to 45 days, 21 days to 30 days, 30 days to 90 days, 30 days to 60 days, 30 days to 45 days, 45 days to 180 days, 45 days to 120 days, 45 days to 100 days, 45 days to 90 days, 45 days to 60 days, 60 days to 180 days, 60 days to 120 days, 60 days to 100 days, 60 days to 90 days, 90 days to 100 days, 90 days to 120 days or 90 days to 180 days) investment.

在一些實施方式中，在該受試者之血清中抗融合多肽之血清濃度小於約500 ng/mL之前投與該第二劑量。In some embodiments, the second dose is administered before the serum concentration of the anti-fusion polypeptide in the subject's serum is less than about 500 ng/mL.

在一些實施方式中，該方法在該受試者中維持至少500 ng/mL（例如，至少600 ng/mL、700 ng/mL、800 ng/mL、900 ng/mL、1,000 ng/mL、1,100 ng/mL、1,200 ng/mL、1,300 ng/mL、1,400 ng/mL、1,500 ng/mL、1,600 ng/mL、1,700 ng/mL、1,800 ng/mL、1,900 ng/mL、2,000 ng/mL、2,100 ng/mL、2,200 ng/mL、2,300 ng/mL、2,400 ng/mL、2,500 ng/mL、2,600 ng/mL、2,700 ng/mL、2,800 ng/mL、2,900 ng/mL、3,000 ng/mL或更多）的血清濃度的抗融合多肽，例如持續至少一小時（例如，至少兩小時、三小時、四小時、五小時、六小時、七小時、八小時、九小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、一天、兩天、三天、四天、五天、六天、一週、兩週、三週、一個月、兩個月、三個月、四個月、五個月、六個月、七個月、八個月、十個月、十一個月、一年或更長時間）。In some embodiments, the method maintains in the subject at least 500 ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL or more multiple) serum concentrations of the anti-fusion polypeptide, e.g., for at least one hour (e.g., at least two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days, three days, four days, five days, six Days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, ten months, eleven months, a year or more).

一種投與多個劑量的本文所述核酸分子（例如，環狀多核糖核苷酸）的組成物之方法包括在一段時間內向細胞、組織或受試者（例如哺乳動物）提供兩種或更多種組成物。根據某些實施方式，多個劑量的本文所述核酸分子的組成物可以以確定的時間過程投與給受試者。根據本發明這一方面的方法包括順序地向受試者投與多個劑量的本文所述核酸分子（例如，環狀多核糖核苷酸、線性多核糖核苷酸、環狀多去氧核糖核苷酸、線性多去氧核糖核苷酸）的組成物（例如，在藥物或獸醫組成物中）。如本文所用，「順序投與」意指在由預定間隔（例如，按小時、天、周或月）隔開的不同時間點（例如，在不同的一天），將本文所述核酸分子的組成物的每一劑量投與給受試者。在一些實施方式中，本發明提供的方法包括順序地向受試者投與單個初始劑量的本文所述核酸分子的組成物，接著是一或多個第二劑量的該組成物，視需要接著是一或多個第三劑量的該組成物。A method of administering multiple doses of a composition of a nucleic acid molecule (e.g., a cyclic polyribonucleotide) described herein includes providing two or more doses to a cell, tissue, or subject (e.g., a mammal) over a period of time. Various compositions. According to certain embodiments, multiple doses of a composition of nucleic acid molecules described herein can be administered to a subject over a defined time course. Methods according to this aspect of the invention include sequentially administering to a subject multiple doses of a nucleic acid molecule described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides, cyclic polydeoxyribose Nucleotides, linear polydeoxyribonucleotides) (e.g., in pharmaceutical or veterinary compositions). As used herein, "sequential administration" means that compositions of the nucleic acid molecules described herein are administered at different points in time (e.g., on different days) separated by predetermined intervals (e.g., by hours, days, weeks, or months). Each dose of the substance is administered to the subject. In some embodiments, methods provided herein include sequentially administering to a subject a single initial dose of a composition of a nucleic acid molecule described herein, followed by one or more second doses of the composition, followed as appropriate. is one or more third doses of the composition.

術語「初始劑量」、「第二劑量」和「第三劑量」係指本文所述核酸分子的組成物投與的時間次序。因此，「初始劑量」係在治療方案開始時投與的劑量；「第二劑量」係在初始劑量後投與的劑量；「第三劑量」係在第二劑量之後投與的劑量。初始劑量、第二劑量和第三劑量都可以包含相同量的本文所述核酸分子的組成物，並且在某些實施方式中，在投與頻率方面可以彼此不同。在某些實施方式中，在治療過程中，初始、第二和/或第三劑量中包含的本文所述核酸分子的組成物的量彼此不同（例如，適當地上調或下調）。在某些實施方式中，在治療方案開始時作為「負荷劑量」投與一或多個（例如，2個、3個、4個或5個）劑量，接著是以較小的頻率投與的後續劑量（例如，「維持劑量」）。The terms "initial dose," "second dose," and "third dose" refer to the temporal sequence of administration of the compositions of nucleic acid molecules described herein. Thus, an "initial dose" is the dose administered at the beginning of the treatment regimen; a "second dose" is the dose administered after the initial dose; and a "third dose" is the dose administered after the second dose. The initial dose, the second dose, and the third dose may each contain the same amount of a composition of the nucleic acid molecules described herein, and, in certain embodiments, may differ from each other in frequency of administration. In certain embodiments, the amounts of compositions of nucleic acid molecules described herein included in the initial, second, and/or third doses differ from one another (eg, are appropriately up- or down-regulated) during treatment. In certain embodiments, one or more (eg, 2, 3, 4, or 5) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by less frequent doses. Subsequent doses (e.g., "maintenance doses").

在某些實施方式中，每個第二和/或第三劑量在緊臨的前一劑量之後投與。如本文所用，短語「緊臨的前一劑量」意指在多次投與的次序中，該劑量的本文所述核酸分子的組成物係在投與次序中緊接著的劑量之前投與給受試者，沒有中間劑量。在某些實施方式中，在緊臨的前一劑量後每天、每2天、3天、4天、5天、6天或7天投與每個第二和/或第三劑量。在某些實施方式中，在緊臨的前一劑量後每0.5週、1週、2週、3週或4週投與每個第二和/或第三劑量。In certain embodiments, each second and/or third dose is administered immediately after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means that, in a sequence of multiple administrations, that dose of a composition of a nucleic acid molecule described herein is administered immediately before the dose that immediately precedes the sequence of administrations. subjects, there were no intermediate doses. In certain embodiments, each second and/or third dose is administered every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediately preceding dose. In certain embodiments, each second and/or third dose is administered every 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose.

根據本發明這一方面的方法可包括向受試者投與任意數目的第二和/或第三劑量的本文所述核酸分子的組成物。例如，在某些實施方式中，僅向受試者投與單個第二劑量。在其他實施方式中，向受試者投與兩個或更多個（例如，2、3、4、5、6、7、8個或更多個）第二劑量。同樣，在某些實施方式中，僅向受試者投與單個第三劑量。在其他實施方式中，向受試者投與兩個或更多個（例如，2、3、4、5、6、7、8個或更多個）第三劑量。Methods according to this aspect of the invention may comprise administering to the subject any number of second and/or third doses of a composition of nucleic acid molecules described herein. For example, in certain embodiments, only a single second dose is administered to the subject. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the subject. Likewise, in certain embodiments, only a single third dose is administered to the subject. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the subject.

在某些實施方式中，向受試者投與第二和/或第三劑量的頻率可以在治療方案的過程中變化。投與頻率也可以在治療過程中調整。In certain embodiments, the frequency with which the second and/or third doses are administered to the subject can vary over the course of the treatment regimen. Frequency of administration can also be adjusted during treatment.

在一些實施方式中，該方法包括向細胞、組織或受試者（例如，哺乳動物，例如，人）提供（例如，投與）至少第一組成物和第二組成物。在一些實施方式中，該方法進一步包括提供（例如，投與）第三組成物、第四組成物、第五組成物、第六組成物、第七組成物、第八組成物、第九組成物、第十組成物或更多組成物。在一些實施方式中，在細胞壽命的持續時間內提供額外的組成物。在一些實施方式中，當細胞、組織或受試者從組成物中獲益時，提供（例如投與）額外的組成物。In some embodiments, the method includes providing (eg, administering) at least a first composition and a second composition to a cell, tissue, or subject (eg, a mammal, eg, a human). In some embodiments, the method further includes providing (e.g., administering) a third component, a fourth component, a fifth component, a sixth component, a seventh component, an eighth component, a ninth component object, the tenth component or more components. In some embodiments, additional compositions are provided for the duration of the cell's lifetime. In some embodiments, additional compositions are provided (eg, administered) when the cell, tissue, or subject benefits from the composition.

在一些實施方式中，多次給藥方案中的第一組成物包括第一量的本文揭露的核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，多次給藥方案中的第二組成物包括第二量的本文揭露的核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，多次給藥方案中的第三組成物、第四組成物、第五組成物、第六組成物、第七組成物、第八組成物、第九組成物、第十組成物或更多組成物包含第三、第四、第五、第六、第七、第八、第九、第十量或更多量的本文所揭露核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第二量與核酸分子（例如，環狀多核糖核苷酸）的第一量相同。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第三量與核酸分子（例如，環狀多核糖核苷酸）的第一量相同。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第四、第五、第六、第七、第八、第九、第十量或更多量與核酸分子（例如，環狀多核糖核苷酸）的第一量相同。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第二量小於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第三量小於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第四、第五、第六、第七、第八、第九、第十量或更多量小於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第二量大於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第三量大於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，核酸分子（例如，環狀多核糖核苷酸）的第四、第五、第六、第七、第八、第九、第十量或更多量大於核酸分子（例如，環狀多核糖核苷酸）的第一量。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量變化不多於第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量的1%、5%、10%、15%、20%或25%。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量少不多於1%、5%、10%、15%、20%或25%。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量高0.1倍至1000倍。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量高0.1倍、1倍、5倍、10倍、100倍或1000倍。在一些實施方式中，後續組成物（例如，在第一組成物後投與的組成物）的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量高0.1倍、1倍、5倍、10倍、100倍或1000倍。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量低0.1倍至1000倍。在一些實施方式中，第二組成物的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量低0.1倍、1倍、5倍、10倍、100倍或1000倍。在一些實施方式中，後續組成物（例如，在第一組成物後投與的組成物）的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量低0.1倍、1倍、5倍、10倍、100倍或1000倍。在一些實施方式中，後續組成物（例如，在一定量的核酸分子（例如，環狀多核糖核苷酸）的第一組成物之後）的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量高或低0.1倍至1000倍。在一些實施方式中，後續組成物（例如，在一定量的核酸分子（例如，環狀多核糖核苷酸）的第一組成物之後）的核酸分子（例如，環狀多核糖核苷酸）的量比第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量高或低0.1倍、1倍、5倍、10倍、100倍或1000倍。例如，第一組成物包含1倍的核酸分子（例如，環狀多核糖核苷酸），第二組成物與第一組成物相比包含5倍的核酸分子（例如，環狀多核糖核苷酸），並且第三組成物與第一組成物相比包含0.2倍的核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，與第一組成物的核酸分子（例如，環狀多核糖核苷酸）的量相比，第二組成物包含至少5倍的核酸分子（例如，環狀多核糖核苷酸）。In some embodiments, the first composition of the multiple dosing regimen includes a first amount of a nucleic acid molecule disclosed herein (eg, a cyclic polyribonucleotide). In some embodiments, the second composition in the multiple dosing regimen includes a second amount of a nucleic acid molecule disclosed herein (eg, a cyclic polyribonucleotide). In some embodiments, the third component, the fourth component, the fifth component, the sixth component, the seventh component, the eighth component, the ninth component, the tenth component in the multiple dosing regimen The composition or compositions comprise a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of a nucleic acid molecule disclosed herein (e.g., a cyclic polyribonucleic acid glycosides). In some embodiments, the second amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is the same as the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is the same as the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amounts of the nucleic acid molecule (e.g., cyclic polyribonucleotide) are identical to the nucleic acid molecule (e.g., , the first amount of cyclic polyribonucleotide) is the same. In some embodiments, the second amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is less than the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is less than the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amounts of the nucleic acid molecule (e.g., cyclic polyribonucleotide) are less than the nucleic acid molecule (e.g., , the first amount of cyclic polyribonucleotides). In some embodiments, the second amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is greater than the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the third amount of nucleic acid molecules (eg, cyclic polyribonucleotides) is greater than the first amount of nucleic acid molecules (eg, cyclic polyribonucleotides). In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amounts of the nucleic acid molecule (e.g., cyclic polyribonucleotide) are greater than the nucleic acid molecule (e.g., , the first amount of cyclic polyribonucleotides). In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the second composition does not vary more than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition 1%, 5%, 10%, 15%, 20% or 25%. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the second composition is approximately less than the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition. at 1%, 5%, 10%, 15%, 20% or 25%. In some embodiments, the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the second composition is 0.1 times greater than the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the first composition to 1000 times. In some embodiments, the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the second composition is 0.1 times greater than the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the first composition , 1x, 5x, 10x, 100x or 1000x. In some embodiments, a subsequent composition (e.g., a composition administered after a first composition) has a greater amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the first composition (e.g., , cyclic polyribonucleotide), the amount is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times higher. In some embodiments, the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the second composition is 0.1 times lower than the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the first composition to 1000 times. In some embodiments, the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the second composition is 0.1 times lower than the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the first composition , 1x, 5x, 10x, 100x or 1000x. In some embodiments, a subsequent composition (e.g., a composition administered after a first composition) has a greater amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) than the first composition (e.g., , cyclic polyribonucleotide), the amount is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times lower. In some embodiments, subsequent compositions (e.g., following a first composition of an amount of nucleic acid molecules (e.g., cyclic polyribonucleotides)) of nucleic acid molecules (e.g., cyclic polyribonucleotides) The amount is 0.1 to 1000 times higher or lower than the amount of nucleic acid molecules (for example, cyclic polyribonucleotides) of the first composition. In some embodiments, subsequent compositions (e.g., following a first composition of an amount of nucleic acid molecules (e.g., cyclic polyribonucleotides)) of nucleic acid molecules (e.g., cyclic polyribonucleotides) The amount is 0.1 times, 1 times, 5 times, 10 times, 100 times or 1000 times higher or lower than the amount of nucleic acid molecules (eg, cyclic polyribonucleotides) of the first composition. For example, the first composition contains 1 times as many nucleic acid molecules (e.g., cyclic polyribonucleotides), and the second composition contains 5 times as many nucleic acid molecules (e.g., cyclic polyribonucleotides) as the first composition. acid), and the third composition contains 0.2 times more nucleic acid molecules (eg, cyclic polyribonucleotides) than the first composition. In some embodiments, the second composition includes at least 5 times the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) compared to the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides) of the first composition. acid).

在一些實施方式中，第一組成物比第二組成物包含更高量的核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，第一組成物比第三、第四、第五、第六、第七、第八、第九或第十組成物包含更高量的核酸分子（例如，環狀多核糖核苷酸）。In some embodiments, the first composition contains a higher amount of nucleic acid molecules (eg, cyclic polyribonucleotides) than the second composition. In some embodiments, the first composition includes a higher amount of nucleic acid molecules (e.g., cyclic polyribose) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition. nucleotides).

在一些實施方式中，以如本文所述之多次給藥方案投與的編碼抗融合多肽的核酸分子（例如，環狀多核糖核苷酸）的多種（例如，兩種或更多種）組成物係相同的組成物。在一些實施方式中，以如本文所述之多次給藥方案投與的編碼抗融合多肽的核酸分子（例如，環狀多核糖核苷酸）的多種（例如，兩種或更多種）組成物係不同的組成物。在一些實施方式中，相同的組成物包含編碼相同的抗融合多肽的核酸分子（例如，環狀多核糖核苷酸）。在一些實施方式中，不同的組成物包含編碼不同的抗融合多肽的核酸分子（例如，環狀多核糖核苷酸），或其組合。In some embodiments, multiple (e.g., two or more) nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding anti-fusion polypeptides are administered in a multiple dosing regimen as described herein The composition is the same composition. In some embodiments, multiple (e.g., two or more) nucleic acid molecules (e.g., cyclic polyribonucleotides) encoding anti-fusion polypeptides are administered in a multiple dosing regimen as described herein Compositions are different compositions. In some embodiments, the same composition includes nucleic acid molecules (eg, cyclic polyribonucleotides) encoding the same anti-fusion polypeptide. In some embodiments, different compositions comprise nucleic acid molecules (eg, cyclic polyribonucleotides) encoding different anti-fusion polypeptides, or combinations thereof.

在一些實施方式中，在多次給藥方案中，投與本文提供的核酸分子（例如，環狀多核糖核苷酸）的方法包括向有需要的受試者多次（多個劑量）投與核酸分子，例如至少3、4、5、6、7、8、9、10、12、15、20、30、40、50、60、100、150、200或500次投與，間隔1天至56天（如約49天、42天、35天、28天、21天、14天或7天）。在一些實施方式中，在多次給藥方案中，本文提供的方法包括向有需要的受試者至少3次投與核酸分子，間隔約7天。在一些實施方式中，在接受投與多個劑量的本文提供的核酸分子（例如，至少3、4、5、6、7、8或9個劑量）的受試者中，在最後一個劑量後超過1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、18或20週的時間段內，抗融合多肽（例如，血漿抗融合多肽）的水平保持在小於50%、40%、30%、20%或10%變化的水平。在一些實施方式中，在接受投與多個劑量的本文提供的核酸分子（例如，至少3、4、5、6、7、8或9個劑量）的受試者中，在第二、第三、第四、第五、第六、第七、第八或最後一個劑量後超過1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、18、19或20週的時間段內，抗融合多肽的水平（例如，血漿抗融合多肽水平）維持在第一水平，其中第一水平高於在第一個劑量後不久測得的抗融合多肽的水平（例如，在第一個劑量後約12、24、36或48小時測量的水平）。在一些實施方式中，在接受投與多個劑量的本文提供的核酸分子（例如，至少3個劑量，間隔約7天）的受試者中，在第二、第三、第四、第五、第六、第七、第八或最後一個劑量後超過8、9、10、11、12、13、14、15、16、17、18、19或20週的時間段內，抗融合多肽的水平（例如，血漿抗融合多肽水平）維持在第一水平，其中第一水平高於在第一個劑量後不久測得的抗融合多肽的水平（例如，在第一個劑量後約12、24、36或48小時測量的水平）。遞送方法In some embodiments, methods of administering a nucleic acid molecule (e.g., a cyclic polyribonucleotide) provided herein comprise administering multiple times (multiple doses) to a subject in need thereof, in a multiple dosing regimen. with nucleic acid molecules, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 administrations separated by 1 day to 56 days (such as approximately 49 days, 42 days, 35 days, 28 days, 21 days, 14 days or 7 days). In some embodiments, methods provided herein include administering a nucleic acid molecule to a subject in need thereof at least 3 times, spaced about 7 days apart, in a multiple dosing regimen. In some embodiments, in a subject who receives administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), after the last dose Over a period of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 weeks, fusion polypeptide) remains at a level that changes by less than 50%, 40%, 30%, 20% or 10%. In some embodiments, in a subject who receives administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), in the second, third 3. More than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, after the fourth, fifth, sixth, seventh, eighth or last dose. The level of the anti-fusion polypeptide (e.g., plasma anti-fusion polypeptide level) is maintained at a first level for a period of 15, 16, 18, 19, or 20 weeks, wherein the first level is greater than when measured shortly after the first dose The level of the anti-fusion polypeptide (e.g., levels measured approximately 12, 24, 36, or 48 hours after the first dose). In some embodiments, in a subject who receives administration of multiple doses of a nucleic acid molecule provided herein (e.g., at least 3 doses spaced about 7 days apart), the second, third, fourth, fifth , the sixth, seventh, eighth or last dose for a period of more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks, the anti-fusion polypeptide The level (e.g., plasma anti-fusion polypeptide level) is maintained at a first level, wherein the first level is greater than the level of the anti-fusion polypeptide measured shortly after the first dose (e.g., approximately 12, 24 , 36 or 48 hour measured levels).Delivery method

編碼本文所述抗融合多肽（例如，表1的多肽）的環狀多核糖核苷酸可以包含在具有或不具有載劑的藥物組成物中。Cyclic polyribonucleotides encoding anti-fusion polypeptides described herein (eg, the polypeptides of Table 1) may be included in pharmaceutical compositions with or without a carrier.

本文所述之藥物組成物可為配製的，例如包含載劑（諸如藥物載劑和/或聚合物載劑，例如脂質體），並藉由已知方法遞送至有需要的受試者（例如人或非人農業動物或家畜，例如牛、狗、貓、馬、家禽）。此類方法包括但不限於轉染（例如，脂質介導的陽離子聚合物、磷酸鈣、樹狀聚合物）；電穿孔或其他破壞膜的方法（例如，核轉染）、病毒遞送（例如，慢病毒、反轉錄病毒、腺病毒、AAV）、顯微注射、微粒轟擊（「基因槍」）、fugene、直接聲波載入、細胞擠壓、光轉染、原生質體融合、刺穿感染、磁轉染、外泌體介導的轉移、脂質奈米顆粒介導的轉移、及其任何組合。遞送方法也描述於例如Gori等人, Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy [人類基因治療用CRISPR/Cas9基因組編輯技術的傳遞和特異性].Human Gene Therapy [人類基因治療].2015年7月, 26(7): 443-451. doi:10.1089/hum.2015.074；和Zuris等人, Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editingin vitroandin vivo[陽離子脂質介導的蛋白質遞送能夠在體外和體內進行有效的基於蛋白質的基因組編輯].Nat Biotechnol [自然生物技術]. 2014年10月30日;33(1):73-80。Pharmaceutical compositions described herein may be formulated, for example, containing a carrier (such as a pharmaceutical carrier and/or a polymeric carrier, such as liposomes), and delivered to a subject in need thereof by known methods (e.g., human or non-human agricultural animals or livestock, such as cattle, dogs, cats, horses, poultry). Such methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., Lentivirus, retrovirus, adenovirus, AAV), microinjection, particle bombardment ("gene gun"), fugene, direct sonic loading, cell extrusion, phototransfection, protoplast fusion, puncture infection, magnetic Transfection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Delivery methods are also described, for example, in Gori et al., Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy . July 2015, 26(7): 443-451. doi:10.1089/hum.2015.074; and Zuris et al., Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editingin vitro andin vivo [cationic lipids Mediated protein delivery enables efficient protein-based genome editing in vitro and in vivo]. Nat Biotechnol. 2014Oct 30;33(1):73-80.

在一些實施方式中，環狀多核糖核苷酸可以在「裸」遞送配製物中遞送。裸遞送配製物在不借助載劑並且不對環狀多核糖核苷酸進行共價修飾或者不部分或完全封裝環狀多核糖核苷酸的情況下將環狀多核糖核苷酸遞送至細胞。In some embodiments, cyclic polyribonucleotides can be delivered in a "naked" delivery formulation. Naked delivery formulations deliver the cyclic polyribonucleotide to cells without the aid of a carrier and without covalently modifying the cyclic polyribonucleotide or partially or completely encapsulating the cyclic polyribonucleotide.

裸遞送配製物係不含載劑的配製物，並且其中環狀多核糖核苷酸沒有結合有助於遞送至細胞的部分的共價修飾並且環狀多核糖核苷酸未被部分或完全封裝。在一些實施方式中，沒有與有助於遞送至細胞的部分結合的共價修飾的環狀多核糖核苷酸可為未與有助於遞送至細胞的部分（諸如蛋白質、小分子、顆粒、聚合物、或生物聚合物）共價結合的多核糖核苷酸。在一些實施方式中，可以將環狀多核糖核苷酸與魚精蛋白或魚精蛋白鹽（例如硫酸魚精蛋白）一起在遞送配製物中遞送。Naked delivery formulations are formulations that do not contain a carrier and in which the cyclic polyribonucleotide has no covalent modifications that incorporate moieties that facilitate delivery to cells and the cyclic polyribonucleotide is not partially or completely encapsulated . In some embodiments, the covalently modified cyclic polyribonucleotide that is not associated with a moiety that facilitates delivery to the cell can be a covalently modified cyclic polyribonucleotide that is not associated with a moiety that facilitates delivery to the cell (such as a protein, small molecule, particle, Polymer, or biopolymer) covalently bound polyribonucleotides. In some embodiments, cyclic polyribonucleotides can be delivered in a delivery formulation with protamine or a protamine salt (eg, protamine sulfate).

沒有與有助於遞送至細胞的部分結合的共價修飾的多核糖核苷酸可以不含經修飾的磷酸基團。例如，沒有與有助於遞送至細胞的部分結合的共價修飾的多核糖核苷酸可以不含硫代磷酸酯、硒代磷酸酯、硼代磷酸鹽、硼代磷酸酯、氫磷酸酯、胺基磷酸酯、二胺基磷酸酯、烷基或芳基膦酸酯或磷酸三酯。Polyribonucleotides that are not covalently modified to be associated with a moiety that facilitates delivery to cells may not contain modified phosphate groups. For example, a polyribonucleotide that is not covalently modified with a moiety that facilitates delivery to a cell may be free of phosphorothioates, selenophosphates, borophosphates, borophosphates, hydrogen phosphates, Amino phosphates, diamino phosphates, alkyl or aryl phosphonates or phosphate triesters.

在一些實施方式中，裸遞送配製物可以不含以下任何或全部物質：轉染試劑、陽離子載劑、碳水化合物載劑、奈米顆粒載劑或蛋白質載劑。例如，裸遞送配製物可以不含植物糖原辛烯基琥珀酸酯、植物糖原β-糊精、酸酐改性的植物糖原β-糊精、脂轉染胺（lipofectamine）、聚乙烯亞胺、聚(三甲烯亞胺)、聚(四甲烯亞胺)、聚丙烯亞胺、胺基糖苷-聚胺、雙去氧-二胺基-b-環糊精、精胺、亞精胺、聚甲基丙烯酸(2-二甲基胺基)乙酯、聚(離胺酸)、聚(組胺酸)、聚(精胺酸)、陽離子化明膠、樹狀聚合物、殼聚糖、l,2-二油醯基-3-三甲基銨-丙烷（DOTAP）、N-[1-(2,3-二油醯基氧基)丙基]-N,N,N-三甲基氯化銨（DOTMA）、l-[2-(油醯基氧基)乙基]-2-油烯基-3-(2-羥乙基)咪唑啉鎓氯化物（DOTIM）、2,3-二油醯基氧基-N-[2(精胺甲醯胺基)乙基]-N,N-二甲基-l-三氟乙酸丙銨（DOSPA）、3B-[N-(N\N'-二甲基胺基乙烷)-胺基甲醯基]膽固醇鹽酸鹽（DC-膽固醇HCl）、雙十七烷基醯胺基甘胺醯亞精胺（DOGS）、N,N-二硬脂基-N,N-二甲基溴化銨（DDAB）、N-(l,2-二肉豆蔻基氧基丙-3-基)-N,N-二甲基-N-羥乙基溴化銨（DMRIE）、N,N-二油烯基-N,N-二甲基氯化銨（DODAC）、人血清白蛋白（HSA）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）、或球蛋白。In some embodiments, naked delivery formulations may be free of any or all of the following: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation may be free of phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylene subsides Amine, poly(trimethylenimine), poly(tetramethylenimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine Amine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine acid), poly(histidine acid), poly(arginine acid), cationized gelatin, dendrimers, chitopolymer Sugar, l,2-dioleyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N- Trimethylammonium chloride (DOTMA), l-[2-(Oleyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminemethyloxy)ethyl]-N,N-dimethyl-l-propylammonium trifluoroacetate (DOSPA), 3B-[N -(N\N'-dimethylaminoethane)-aminoformyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylglycerol-spermidine (DOGS) , N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxypropan-3-yl)-N,N-dimethyl -N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein ( LDL), high-density lipoprotein (HDL), or globulin.

裸遞送配製物可以包含非載劑賦形劑。在一些實施方式中，非載劑賦形劑可以包括不表現出活性細胞穿透作用的非活性成分。在一些實施方式中，非載劑賦形劑可以包括緩衝劑，例如PBS。在一些實施方式中，非載劑賦形劑可為溶劑、非水性溶劑、稀釋劑、懸浮助劑、界面活性劑、等滲劑、增稠劑、乳化劑、防腐劑、聚合物、肽、蛋白質、細胞、透明質酸酶、分散劑、制粒劑、崩解劑、黏合劑、緩衝劑、潤滑劑、或油。Naked delivery formulations may contain non-carrier excipients. In some embodiments, non-carrier excipients may include inactive ingredients that do not exhibit active cell penetration. In some embodiments, non-carrier excipients may include buffers such as PBS. In some embodiments, non-carrier excipients can be solvents, non-aqueous solvents, diluents, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, Proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers, lubricants, or oils.

在一些實施方式中，裸遞送配製物可以包含稀釋劑，諸如腸胃外可接受的稀釋劑。稀釋劑（例如，腸胃外可接受的稀釋劑）可為液體稀釋劑或固體稀釋劑。在一些實施方式中，稀釋劑（例如，腸胃外可接受的稀釋劑）可為RNA增溶劑、緩衝劑或等滲劑。RNA增溶劑之實例包括水、乙醇、甲醇、丙酮、甲醯胺和2-丙醇。緩衝液之實例包括2-(N-𠰌啉代)乙磺酸（MES）、Bis-Tris、2-[(2-胺基-2-側氧基乙基)-(羧甲基)胺基]乙酸（ADA）、N-(2-乙醯胺基)-2-胺基乙烷磺酸（ACES）、哌𠯤-N,N′-雙(2-乙磺酸)（PIPES）、2-[[1,3-二羥基-2-(羥甲基)丙-2-基]胺基]乙磺酸（TES）、3-(N-𠰌啉代)丙烷磺酸（MOPS）、4-(2-羥乙基)-1-哌𠯤乙磺酸（HEPES）、Tris、Tricine、Gly-Gly、Bicine或磷酸鹽。等滲劑之實例包括甘油、甘露醇、聚乙二醇、丙二醇、海藻糖或蔗糖。In some embodiments, naked delivery formulations may include a diluent, such as a parenterally acceptable diluent. The diluent (eg, a parenterally acceptable diluent) may be a liquid diluent or a solid diluent. In some embodiments, the diluent (eg, a parenterally acceptable diluent) can be an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers include 2-(N-𠰌lino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-side oxyethyl)-(carboxymethyl)amino ] Acetic acid (ADA), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2 -[[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-𠰌lino)propanesulfonic acid (MOPS), 4 -(2-Hydroxyethyl)-1-piperidineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine or phosphate. Examples of isotonic agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose or sucrose.

在一些實施方式中，配製物包括細胞穿透劑。在一些實施方式中，配製物係局部配製物，並且包括細胞穿透劑。細胞穿透劑可以包括有機化合物，諸如具有一或多個羥基官能基的醇。在一些情況下，細胞穿透劑包括醇，諸如但不限於一元醇、多元醇、不飽和脂族醇和脂環醇。細胞穿透劑可以包括以下的一或多種：甲醇、乙醇、異丙醇、苯氧乙醇、三乙醇胺、苯乙醇、丁醇、戊醇、鯨蠟醇、乙二醇、丙二醇、變性醇、苯甲醇（特別是變性醇）、二醇、硬脂醇、鯨蠟硬脂醇、薄荷醇、聚乙二醇（PEG）-400、乙氧基化脂肪酸或羥乙基纖維素。在某些實施方式中，細胞穿透劑包括乙醇。細胞穿透劑可以包括如WO 2020/180751或WO 2020/180752中所述之任何量或任何配製物中的任何細胞滲透劑，將其特此藉由援引以其全文併入。In some embodiments, the formulation includes a cell-penetrating agent. In some embodiments, the formulation is topical and includes a cell penetrating agent. Cell penetrating agents may include organic compounds such as alcohols having one or more hydroxyl functional groups. In some cases, cell-penetrating agents include alcohols such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols. Cell-penetrating agents may include one or more of the following: methanol, ethanol, isopropyl alcohol, phenoxyethanol, triethanolamine, phenylethanol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzene Methanol (especially denatured alcohol), glycols, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycol (PEG)-400, ethoxylated fatty acids or hydroxyethyl cellulose. In certain embodiments, the cell-penetrating agent includes ethanol. The cell-penetrating agent may include any cell-penetrating agent in any amount or in any formulation as described in WO 2020/180751 or WO 2020/180752, which are hereby incorporated by reference in their entirety.

在一些實施方式中，如本文揭露的藥物製劑、如本文揭露的藥物組成物、如本文揭露的藥物原料藥或如本文揭露的藥物成品藥處於腸胃外核酸遞送系統中。腸胃外核酸遞送系統可以包括如本文揭露的藥物製劑、如本文揭露的藥物組成物、所揭露的藥物原料藥或如本文揭露的藥物成品藥和腸胃外可接受的稀釋劑。在一些實施方式中，腸胃外核酸遞送系統中的如本文揭露的藥物製劑、如本文揭露的藥物組成物、如本文揭露的藥物原料藥或如本文揭露的藥物成品藥不含任何載劑。In some embodiments, a pharmaceutical formulation as disclosed herein, a pharmaceutical composition as disclosed herein, a pharmaceutical drug substance as disclosed herein, or a finished pharmaceutical product as disclosed herein is in a parenteral nucleic acid delivery system. The parenteral nucleic acid delivery system may include a pharmaceutical formulation as disclosed herein, a pharmaceutical composition as disclosed herein, a pharmaceutical bulk drug as disclosed, or a finished pharmaceutical product as disclosed herein, and a parenterally acceptable diluent. In some embodiments, a pharmaceutical formulation as disclosed herein, a pharmaceutical composition as disclosed herein, a pharmaceutical bulk drug as disclosed herein, or a finished pharmaceutical product as disclosed herein in a parenteral nucleic acid delivery system does not contain any carrier.

本揭露進一步關於包含本文所述之環狀多核糖核苷酸的宿主或宿主細胞。在一些實施方式中，宿主或宿主細胞係脊椎動物、哺乳動物（例如人）或其他生物體或細胞。The present disclosure further relates to a host or host cell comprising a cyclic polyribonucleotide described herein. In some embodiments, the host or host cell is a vertebrate, mammalian (eg, human), or other organism or cell.

在一些實施方式中，與由參考化合物（例如，對應於所述環狀多核糖核苷酸的線性多核苷酸）引發的響應相比，環狀多核糖核苷酸具有降低的宿主免疫系統的不期望響應或不能產生該不期望響應。在實施方式中，環狀多核糖核苷酸在宿主中係非免疫性的。一些免疫反應包括但不限於體液免疫反應（例如，免疫原特異性抗體的產生）和細胞介導的免疫反應（例如，淋巴細胞增殖）。In some embodiments, the cyclic polyribonucleotide has a reduced response of the host immune system compared to a response elicited by a reference compound (e.g., a linear polynucleotide corresponding to the cyclic polyribonucleotide). The response is not expected or cannot be produced. In embodiments, the cyclic polyribonucleotide is non-immune in the host. Some immune responses include, but are not limited to, humoral immune responses (e.g., production of immunogen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation).

在一些實施方式中，使宿主或宿主細胞接觸（例如，遞送至或投與至）環狀多核糖核苷酸。在一些實施方式中，宿主係哺乳動物，如人類。可以在投與後的任何時間測量宿主中環狀多核糖核苷酸或線性多核糖核苷酸、表現產物或兩者的量。在某些實施方式中，測定培養物中宿主生長的時程。如果在環狀多核糖核苷酸或線性多核糖核苷酸的存在下生長增加或減少，則環狀多核糖核苷酸或表現產物或兩者都被認為在增加或減少宿主的生長方面係有效的。In some embodiments, a host or host cell is contacted with (eg, delivered to or administered to) a cyclic polyribonucleotide. In some embodiments, the host is a mammal, such as a human. The amount of cyclic or linear polyribonucleotides, expressed product, or both, in the host can be measured at any time after administration. In certain embodiments, the time course of host growth in culture is determined. If growth is increased or decreased in the presence of cyclic polyribonucleotides or linear polyribonucleotides, then either the cyclic polyribonucleotide or the expression product, or both, is considered to increase or decrease the growth of the host. Effective.

將如本文所述之環狀多核糖核苷酸分子遞送至細胞、組織或受試者的方法包括向該細胞、組織或受試者投與如本文所述之藥物組成物、藥物原料藥或藥物成品藥。A method of delivering a cyclic polyribonucleotide molecule as described herein to a cell, tissue or subject includes administering to the cell, tissue or subject a pharmaceutical composition, pharmaceutical drug substance or drug as described herein. Pharmaceutical products.

在一些實施方式中，細胞係真核細胞。在一些實施方式中，細胞係哺乳動物細胞。在一些實施方式中，細胞係有蹄類動物細胞。在一些實施方式中，細胞係動物細胞。在一些實施方式中，細胞係免疫細胞。在一些實施方式中，組織係結締組織、肌肉組織、神經組織或上皮組織。在一些實施方式中，組織係器官（例如，肝、肺、脾、腎等）。In some embodiments, the cell line is a eukaryotic cell. In some embodiments, the cell line is a mammalian cell. In some embodiments, the cell line is an ungulate cell. In some embodiments, the cells are animal cells. In some embodiments, the cell line is an immune cell. In some embodiments, the tissue is connective tissue, muscle tissue, neural tissue, or epithelial tissue. In some embodiments, the tissue is an organ (eg, liver, lung, spleen, kidney, etc.).

在一些實施方式中，遞送方法係體內方法。例如，如本文所述之環狀多核糖核苷酸的遞送方法包括向有需要的受試者腸胃外投與，將如本文所述之藥物組成物、藥物原料藥或藥物成品藥經腸胃外投與給有需要的受試者。再如，將環狀多核糖核苷酸遞送至受試者的細胞或組織的方法包括將如本文所述之藥物組成物、藥物原料藥或藥物成品藥腸胃外投與至該細胞或組織。在一些實施方式中，環狀多核糖核苷酸的量在受試者中有效地引發生物反應。在一些實施方式中，環狀多核糖核苷酸的量有效地對受試者的細胞或組織具有生物學作用。在一些實施方式中，如本文所述之藥物組成物、藥物原料藥或藥物成品藥包含載劑。在一些實施方式中，如本文所述之藥物組成物、藥物原料藥或藥物成品藥包含稀釋劑而不含任何載劑。In some embodiments, the delivery method is in vivo. For example, the delivery method of the cyclic polyribonucleotide as described herein includes parenteral administration to a subject in need thereof, parenterally administering a pharmaceutical composition, pharmaceutical raw material or finished pharmaceutical product as described herein. Give to subjects in need. As another example, a method of delivering a cyclic polyribonucleotide to a cell or tissue of a subject includes parenterally administering a pharmaceutical composition, drug substance, or finished drug as described herein to the cell or tissue. In some embodiments, the amount of cyclic polyribonucleotide is effective to elicit a biological response in a subject. In some embodiments, the amount of cyclic polyribonucleotide is effective to have a biological effect on the cells or tissues of the subject. In some embodiments, a pharmaceutical composition, pharmaceutical substance, or finished pharmaceutical product as described herein includes a carrier. In some embodiments, a pharmaceutical composition, drug substance, or finished pharmaceutical product as described herein contains a diluent without any carrier.

在一些實施方式中，腸胃外投與藥物組成物、藥物原料藥或藥物成品藥。在一些實施方式中，藥物組成物、藥物原料藥或藥物成品藥通過靜脈內、動脈內、腹膜內、皮內、顱內、鞘內、淋巴內、皮下或肌內投與。在一些實施方式中，腸胃外投與係靜脈內、肌內、眼、皮下、皮內或局部投與。In some embodiments, a pharmaceutical composition, drug substance, or finished drug is administered parenterally. In some embodiments, the pharmaceutical composition, drug substance, or finished drug is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphally, subcutaneously, or intramuscularly. In some embodiments, parenteral administration is intravenous, intramuscular, ocular, subcutaneous, intradermal, or topical.

在一些實施方式中，如本文所述之藥物組成物、藥物原料藥或藥物成品藥經肌內投與。在一些實施方式中，如本文所述之藥物組成物、藥物原料藥或藥物成品藥經皮下投與。在一些實施方式中，如本文所述之藥物組成物、藥物原料藥或藥物成品藥局部投與。在一些實施方式中，該藥物組成物、藥物原料藥或藥物成品藥經氣管內投與。In some embodiments, a pharmaceutical composition, pharmaceutical substance, or finished pharmaceutical product as described herein is administered intramuscularly. In some embodiments, a pharmaceutical composition, pharmaceutical substance, or finished pharmaceutical product as described herein is administered subcutaneously. In some embodiments, a pharmaceutical composition, pharmaceutical substance, or finished pharmaceutical product as described herein is administered topically. In some embodiments, the pharmaceutical composition, pharmaceutical raw material or finished pharmaceutical product is administered intratracheally.

在一些實施方式中，該藥物組成物、藥物原料藥或藥物成品藥藉由注射投與。投與可為全身投與或局部投與。在一些實施方式中，如本文所述之任何遞送方法均用載劑進行。在一些實施方式中，無需借助於載劑或細胞穿透劑即可進行如本文所述之任何遞送方法。In some embodiments, the pharmaceutical composition, pharmaceutical raw material or finished pharmaceutical product is administered by injection. Administration can be systemic or local. In some embodiments, any delivery method as described herein is performed with a carrier. In some embodiments, any of the delivery methods described herein can be performed without the aid of carriers or cell-penetrating agents.

在一些實施方式中，在投與步驟後至少1天、至少2天、至少3天、至少4天或至少5天在細胞、組織或受試者中檢測到環狀多核糖核苷酸或從該環狀多核糖核苷酸翻譯的產物。在一些實施方式中，在投與步驟之前評價細胞、組織或受試者中環狀多核糖核苷酸或從該環狀多核糖核苷酸翻譯的產物的存在。在一些實施方式中，在投與步驟之後評價細胞、組織或受試者中環狀多核糖核苷酸或從該環狀多核糖核苷酸翻譯的產物的存在。配製物In some embodiments, the cyclic polyribonucleotide is detected in the cell, tissue, or subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days after the administering step or from The product of translation of this cyclic polyribonucleotide. In some embodiments, the cell, tissue, or subject is evaluated for the presence of a cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide prior to the step of administering. In some embodiments, the cell, tissue, or subject is assessed for the presence of a cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide following the step of administering.formulation

在本揭露之一些實施方式中，本文所述之環狀多核糖核苷酸可被配製在組成物中，例如用於遞送到細胞、植物、無脊椎動物、非人脊椎動物或人受試者的組成物，例如農業、獸醫或藥物組成物。在一些實施方式中，環狀多核糖核苷酸被配製在藥物組成物中。在一些實施方式中，組成物包含環狀多核糖核苷酸和稀釋劑、載劑、佐劑或它們的組合。在特定實施方式中，組成物包含本文所述之環狀多核糖核苷酸和載劑或不含任何載劑的稀釋劑。在一些實施方式中，包括環狀多核糖核苷酸和不含任何載劑的稀釋劑的組成物用於將環狀多核糖核苷酸裸遞送給受試者。In some embodiments of the present disclosure, the cyclic polyribonucleotides described herein can be formulated in compositions, for example, for delivery to cells, plants, invertebrates, non-human vertebrates, or human subjects compositions, such as agricultural, veterinary or pharmaceutical compositions. In some embodiments, cyclic polyribonucleotides are formulated in pharmaceutical compositions. In some embodiments, a composition includes a cyclic polyribonucleotide and a diluent, carrier, adjuvant, or combinations thereof. In certain embodiments, a composition includes a cyclic polyribonucleotide described herein and a carrier or diluent without any carrier. In some embodiments, a composition comprising a cyclic polyribonucleotide and a diluent without any carrier is used to deliver the cyclic polyribonucleotide naked to a subject.

藥物組成物可視需要包含一或多種額外活性物質，例如治療和/或預防活性物質。藥物組成物可以視需要包含充當本文所述之組成物（例如，包含環狀多核糖核苷酸的組成物）的媒介物或介質的非活性物質，諸如由美國食品和藥品管理局（United States Food and Drug Administration）（FDA）批准並且在非活性成分數據中列出的任一種非活性成分。本發明之藥物組成物可為無菌的和/或無熱原的。可在以下文獻中找到藥劑的配製和/或生產中的總體考慮：例如，Remington: The Science and Practice of Pharmacy [雷明頓：藥物科學與實踐] 第21版, Lippincott Williams & Wilkins [利平科特•威廉斯和威爾金斯出版公司], 2005 （藉由援引併入本文）。非活性物質之非限制性實例包括溶劑、水性溶劑、非水性溶劑、分散介質、稀釋劑、分散體、懸浮助劑、界面活性劑、等滲劑、增稠劑、乳化劑、防腐劑、聚合物、肽、蛋白質、細胞、透明質酸酶、分散劑、制粒劑、崩解劑、黏合劑、緩衝劑（例如，磷酸鹽緩衝鹽水（PBS））、潤滑劑、油、及其混合物。The pharmaceutical compositions may optionally contain one or more additional active substances, for example therapeutic and/or prophylactic active substances. Pharmaceutical compositions may optionally contain inactive substances that serve as vehicles or mediators for the compositions described herein (e.g., compositions containing cyclic polyribonucleotides), such as those approved by the United States Food and Drug Administration. Any inactive ingredient approved by the Food and Drug Administration (FDA) and listed in the Inactive Ingredients Data. The pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical products may be found in, for example, Remington: The Science and Practice of Pharmacy 21st Edition, Lippincott Williams & Wilkins Williams & Wilkins Publishers], 2005 (incorporated herein by reference). Non-limiting examples of inactive substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers substances, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrating agents, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

儘管本文提供的藥物組成物之描述主要針對適合於投與至人的藥物組成物，但是熟悉該項技術者應理解，此類組成物通常適合於投與至任何其他動物，例如非人動物，例如非人哺乳動物。為了使組成物適合於投與於各種動物而對適合於投與於人的藥物組成物的修飾係熟知的，並且普通獸醫藥理師可僅通過普通實驗（如果有的話）來設計和/或進行此種修飾。我們設想向其投與藥物組成物的受試者包括但不限於人類和/或其他靈長類動物；哺乳動物，包括商業相關的哺乳動物，如牛、豬、馬、綿羊、貓、狗、小鼠和/或大鼠；和/或鳥類，包括商業相關的鳥類，如家禽、雞、鴨、鵝和/或火雞。Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, For example, non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals are well known and can be devised and/or devised by the ordinary veterinary pharmacist by mere ordinary experimentation, if any. Make this modification. Subjects to which we contemplate administering pharmaceutical compositions include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, Mice and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese and/or turkeys.

本文所述之藥物組成物的配製物可藉由藥理學領域中已知的或以後開發的任何方法來製備。通常，這樣的製備方法包括以下步驟：使活性成分與賦形劑和/或一或多種其他輔助成分結合，並且然後，如果必要和/或期望的話，將產品分開、成形和/或包裝。Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the field of pharmacology. Typically, such preparation methods include the steps of combining the active ingredient with the excipient and/or one or more other accessory ingredients and then, if necessary and/or desired, dividing, shaping and/or packaging the product.

在一些實施方式中，關於製劑中存在的環狀多核糖核苷酸分子的量之參考標準係占藥物製劑中的總核糖核苷酸分子至少30%（w/w）、40%（w/w）、50%（w/w）、60%（w/w）、70%（w/w）、80%（w/w）、85%（w/w）、90%（w/w）、91%（w/w）、92%（w/w）、93%（w/w）、94%（w/w）、95%（w/w）、96%（w/w）、97%（w/w）、98%（w/w）、99%（w/w）、99.1%（w/w）、99.2%（w/w）、99.3%（w/w）、99.4%（w/w）、99.5%（w/w）、99.6%（w/w）、99.7%（w/w）、99.8%（w/w）、99.9%（w/w）、或100%（w/w）的分子。In some embodiments, the reference standard for the amount of cyclic polyribonucleotide molecules present in the formulation is at least 30% (w/w), 40% (w/ w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w) , 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97 % (w/w), 98% (w/w), 99% (w/w), 99.1% (w/w), 99.2% (w/w), 99.3% (w/w), 99.4% ( w/w), 99.5% (w/w), 99.6% (w/w), 99.7% (w/w), 99.8% (w/w), 99.9% (w/w), or 100% (w /w) numerator.

在一些實施方式中，製劑中存在的線性多核糖核苷酸分子的量之參考標準係存在不多於1 ng/ml、5 ng/ml、10 ng/ml、15 ng/ml、20 ng/ml、25 ng/ml、30 ng/ml、35 ng/ml、40 ng/ml、50 ng/ml、60 ng/ml、70 ng/ml、80 ng/ml、90 ng/ml、100 ng/ml、200 ng/ml、300 ng/ml、400 ng/ml、500 ng/ml、600 ng/ml、1 µg/ ml、10 µg/ml、50 µg/ml、100 µg/ml、200 g/ml、300 µg/ml、400 µg/ml、500 µg/ml、600 µg/ml、700 µg/ml、800 µg/ml、900 µg/ml、1 mg/ml、1.5 mg/ml或2 mg/ml的線性多核糖核苷酸分子。In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the formulation is present at no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml. ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 1 µg/ ml, 10 µg/ml, 50 µg/ml, 100 µg/ml, 200 g/ ml, 300 µg/ml, 400 µg/ml, 500 µg/ml, 600 µg/ml, 700 µg/ml, 800 µg/ml, 900 µg/ml, 1 mg/ml, 1.5 mg/ml or 2 mg/ ml of linear polyribonucleotide molecules.

在一些實施方式中，關於製劑中存在的線性多核糖核苷酸分子的量之參考標準係占藥物製劑中的總核糖核苷酸分子不多於0.5%（w/w）、1%（w/w）、2%（w/w）、5%（w/w）、10%（w/w）、15%（w/w）、20%（w/w）、25%（w/w）、30%（w/w）、40%（w/w）、50%（w/w）的線性多核糖核苷酸分子。In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the formulation is no more than 0.5% (w/w), 1% (w) of the total ribonucleotide molecules in the pharmaceutical formulation. /w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w ), 30% (w/w), 40% (w/w), 50% (w/w) linear polyribonucleotide molecules.

在一些實施方式中，關於製劑中存在的帶切口多核糖核苷酸分子的量之參考標準係占藥物製劑中的總核糖核苷酸分子不多於0.5%（w/w）、1%（w/w）、2%（w/w）、5%（w/w）、10%（w/w）、或15%（w/w）的帶切口多核糖核苷酸分子。In some embodiments, the reference standard for the amount of nicked polyribonucleotide molecules present in the formulation is no more than 0.5% (w/w), 1% ( w/w), 2% (w/w), 5% (w/w), 10% (w/w), or 15% (w/w) of nicked polyribonucleotide molecules.

在一些實施方式中，關於製劑中存在的組合的帶切口和線性多核糖核苷酸分子的量之參考標準係占藥物製劑中的總核糖核苷酸分子不多於0.5%（w/w）、1%（w/w）、2%（w/w）、5%（w/w）、10%（w/w）、15%（w/w）、20%（w/w）、25%（w/w）、30%（w/w）、40%（w/w）、50%（w/w）的組合的帶切口和線性多核糖核苷酸分子。在一些實施方式中，藥物製劑係最終環狀多核糖核苷酸成品藥的中間體藥物製劑。在一些實施方式中，藥物製劑係原料藥或活性藥物成分（API）。在一些實施方式中，藥物製劑係用於投與於受試者的成品藥。In some embodiments, the reference standard for the amount of combined nicked and linear polyribonucleotide molecules present in the formulation is no more than 0.5% (w/w) of the total ribonucleotide molecules in the pharmaceutical formulation. , 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25 % (w/w), 30% (w/w), 40% (w/w), 50% (w/w) combinations of nicked and linear polyribonucleotide molecules. In some embodiments, the pharmaceutical formulation is an intermediate pharmaceutical formulation to the final cyclic polyribonucleotide finished drug product. In some embodiments, the pharmaceutical formulation is a drug substance or active pharmaceutical ingredient (API). In some embodiments, the pharmaceutical formulation is a finished drug for administration to a subject.

在一些實施方式中，對環狀多核糖核苷酸的製劑（在減少線性RNA之前、期間或之後）進一步處理以基本上去除DNA、蛋白質污染物（例如，細胞蛋白質（諸如宿主細胞蛋白質）或蛋白質工藝雜質）、內毒素、單核苷酸分子、和/或工藝相關雜質。In some embodiments, the preparation of cyclic polyribonucleotides (before, during or after linear RNA reduction) is further processed to substantially remove DNA, protein contaminants (e.g., cellular proteins (such as host cell proteins) or protein process impurities), endotoxins, single nucleotide molecules, and/or process-related impurities.

在一些實施方式中，本文揭露的藥物配製物可以包含：(i) 本文揭露的化合物（例如，環狀多核糖核苷酸）；(ii) 緩衝液；(iii) 非離子洗滌劑；(iv) 張度劑；和/或 (v) 穩定劑。在一些實施方式中，本文揭露的藥物配製物係穩定的液體藥物配製物。在一些實施方式中，本文揭露的藥物配製物包含魚精蛋白或魚精蛋白鹽（例如，硫酸魚精蛋白）。防腐劑In some embodiments, pharmaceutical formulations disclosed herein can include: (i) a compound disclosed herein (e.g., cyclic polyribonucleotides); (ii) a buffer; (iii) a non-ionic detergent; (iv) ) tonicity agent; and/or (v) stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations. In some embodiments, pharmaceutical formulations disclosed herein comprise protamine or a protamine salt (eg, protamine sulfate).Preservatives

本文提供的組成物或藥物組成物可以包含用於單次投與的材料，或者可以包含用於多次投與的材料（例如，「多劑量」套組（kit））。多核糖核苷酸可以線性或環狀形式存在。該組成物或藥物組成物可以包含一或多種防腐劑，諸如硫柳汞或2-苯氧乙醇。防腐劑可用於防止使用過程中的微生物污染。合適的防腐劑包括：氯化苄烷銨、硫柳汞、氯丁醇、對羥基苯甲酸甲酯、對羥基苯甲酸丙酯、苯乙醇、依地酸二鈉、山梨酸、Onamer M或熟悉該項技術者已知的其他試劑。在眼科產品中，例如，此類防腐劑可以以0.004%至0.02%的含量採用。在本文所述之組成物中，防腐劑（例如，氯化苄烷銨）可以按重量計以0.001%至小於0.01%，例如0.001%至0.008%，較佳的是約0.005%的含量採用。The compositions or pharmaceutical compositions provided herein may contain materials for a single administration, or may contain materials for multiple administrations (eg, a "multi-dose" kit). Polyribonucleotides can exist in linear or cyclic form. The composition or pharmaceutical composition may contain one or more preservatives, such as thimerosal or 2-phenoxyethanol. Preservatives can be used to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenethyl alcohol, disodium edetate, sorbic acid, Onamer M or those familiar with this Other reagents known to the skilled person. In ophthalmic products, for example, such preservatives may be used at levels of 0.004% to 0.02%. In the compositions described herein, the preservative (eg, benzalkonium chloride) may be employed at a level by weight of 0.001% to less than 0.01%, such as 0.001% to 0.008%, preferably about 0.005%.

多核糖核苷酸可易受周圍環境中可能豐富的RNA酶的影響。本文提供的組成物可以包含抑制RNA酶活性的試劑，從而防止多核糖核苷酸降解。在一些情況下，該組成物或藥物組成物包含熟悉該項技術者已知的任何RNA酶抑制劑。可替代地或另外，可以在無RNA酶的環境中製備本文提供的組成物中的多核糖核苷酸和細胞穿透劑和/或藥學上可接受的稀釋劑或載劑、媒介物、賦形劑或其他試劑。該組成物可以在無RNA酶的環境中配製。Polyribonucleotides can be susceptible to RNases that may be abundant in the surrounding environment. The compositions provided herein may include agents that inhibit RNase activity, thereby preventing polyribonucleotide degradation. In some cases, the composition or pharmaceutical composition includes any RNase inhibitor known to those skilled in the art. Alternatively or additionally, the polyribonucleotides and cell-penetrating agents and/or pharmaceutically acceptable diluents or carriers, vehicles, excipients and/or pharmaceutically acceptable diluents or carriers in the compositions provided herein may be prepared in an RNase-free environment. excipients or other reagents. The composition can be formulated in an RNase-free environment.

在一些情況下，本文提供的組成物可為無菌的。可以在本領域已知的合適的媒介物中將組成物配製成無菌溶液或懸浮液。可以藉由常規的已知滅菌技術對組成物進行滅菌，例如，可以對組成物進行無菌過濾。鹽In some cases, the compositions provided herein can be sterile. The compositions may be formulated as sterile solutions or suspensions in suitable vehicles known in the art. The composition can be sterilized by conventional known sterilization techniques, for example, the composition can be sterile filtered.salt

在一些情況下，本文提供的組成物或藥物組成物包含一或多種鹽。為了控制張度，本文提供的組成物可以包含生理鹽諸如鈉鹽。其他鹽可以包括氯化鉀、磷酸二氫鉀、磷酸氫二鈉和/或氯化鎂等。在一些情況下，該組成物與一或多種藥學上可接受的鹽一起配製。該一或多種藥學上可接受的鹽可以包括無機離子，諸如鈉、鉀、鈣、鎂離子等的那些鹽。此類鹽可以包括與無機酸或有機酸的鹽，該無機酸或有機酸諸如鹽酸、氫溴酸、磷酸、硝酸、硫酸、甲磺酸、對甲苯磺酸、乙酸、富馬酸、琥珀酸、乳酸、苦杏仁酸、蘋果酸、檸檬酸、酒石酸或馬來酸。多核糖核苷酸可以線性或環狀形式存在。緩衝劑/pHIn some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. To control tonicity, the compositions provided herein may contain physiological salts such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and/or magnesium chloride, among others. In some cases, the compositions are formulated with one or more pharmaceutically acceptable salts. The one or more pharmaceutically acceptable salts may include inorganic ions, such as those salts of sodium, potassium, calcium, magnesium ions, and the like. Such salts may include salts with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid , lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid. Polyribonucleotides can exist in linear or cyclic form.Buffer/pH

本文提供的組成物或藥物組成物可以包含一或多種緩衝劑，諸如Tris緩衝劑；硼酸鹽緩衝劑；琥珀酸鹽緩衝劑；組胺酸緩衝劑（例如，含氫氧化鋁佐劑）；或檸檬酸鹽緩衝劑。在一些情況下，包含5-20 mM範圍內的緩衝劑。The compositions or pharmaceutical compositions provided herein may include one or more buffers, such as Tris buffer; borate buffer; succinate buffer; histidine buffer (e.g., aluminum hydroxide-containing adjuvant); or Citrate buffer. In some cases, buffers in the range of 5-20 mM are included.

本文提供的組成物或藥物組成物可以具有約5.0至約8.5、約6.0至約8.0、約6.5至約7.5、或約7.0至約7.8的pH。該組成物或藥物組成物可以具有約7的pH。多核糖核苷酸可以線性或環狀形式存在。洗滌劑/界面活性劑The compositions or pharmaceutical compositions provided herein can have a pH of about 5.0 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.8. The composition or pharmaceutical composition may have a pH of about 7. Polyribonucleotides can exist in linear or cyclic form.Detergent/Surfactant

根據預期的投與途徑，本文提供的組成物或藥物組成物可以包含一或多種洗滌劑和/或界面活性劑，例如聚氧乙烯脫水山梨糖醇酯界面活性劑（通常稱為「妥文（Tween）」），例如聚山梨醇酯20和聚山梨醇酯80；以DOWFAX™商標出售的環氧乙烷（EO）、環氧丙烷（PO）和/或環氧丁烷（BO）的共聚物，諸如線性EO/PO嵌段共聚物；重複的乙氧基(氧基-l,2-乙二基)基團的數目可以變化的辛基酚聚醚，例如辛基酚聚醚-9（Triton X-100或叔辛基苯氧基聚乙氧基乙醇）；(辛基苯氧基)聚乙氧基乙醇（IGEPAL CA-630/NP-40）；磷脂，諸如磷脂醯膽鹼（卵磷脂）；壬基酚聚氧乙烯醚，諸如Tergitol™ NP系列；衍生自月桂醇、鯨蠟醇、硬脂醇和油醇的聚氧乙烯脂肪醚（稱為Brij界面活性劑），諸如三乙二醇單月桂基醚（Brij 30）；和脫水山梨糖醇酯（通常稱為「SPAN」），諸如脫水山梨糖醇三油酸酯（Span 85）和脫水山梨糖醇單月桂酸酯、辛基酚聚醚（諸如辛基酚聚醚9（Triton X-100）或叔辛基苯氧基聚乙氧基乙醇）、十六烷基三甲基溴化銨（「CTAB」）或去氧膽酸鈉。該一或多種洗滌劑和/或界面活性劑可僅以痕量存在。在一些情況下，該組成物可以包含小於各1 mg/ml的辛基酚聚醚-10和聚山梨醇酯80。在本文中可使用非離子界面活性劑。界面活性劑可以按其「HLB」（親水/親脂平衡值）進行分類。在一些情況下，界面活性劑的HLB為至少10、至少15和/或至少16。多核糖核苷酸可以線性或環狀形式存在。稀釋劑Depending on the intended route of administration, the compositions or pharmaceutical compositions provided herein may include one or more detergents and/or surfactants, such as polyoxyethylene sorbitan ester surfactant (commonly known as "Tourvin"). Tween), such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO) sold under the trademark DOWFAX™ octylphenol polyethers, such as linear EO/PO block copolymers; octylphenol polyethers with varying numbers of repeating ethoxy (oxy-1,2-ethylenediyl) groups, such as octylphenol polyether-9 (Triton lecithin); nonylphenol polyoxyethylene ethers, such as the Tergitol™ NP series; polyoxyethylene fatty ethers (called Brij surfactants) derived from lauryl, cetyl, stearyl and oleyl alcohol, such as triethyl alcohol Diol monolauryl ether (Brij 30); and sorbitan esters (commonly known as "SPAN"), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, octyl phenol polyethers (such as octylphenoxypolyether 9 (Triton Sodium cholate. The detergent and/or surfactant(s) may be present in only trace amounts. In some cases, the composition may contain less than 1 mg/ml each of Octylphenol-10 and Polysorbate 80. Nonionic surfactants may be used herein. Surfactants can be classified according to their "HLB" (hydrophile/lipophile balance value). In some cases, the surfactant has an HLB of at least 10, at least 15, and/or at least 16. Polyribonucleotides can exist in linear or cyclic form.Thinner

在一些實施方式中，本揭露之組成物包含環狀多核糖核苷酸和稀釋劑。在一些實施方式中，本揭露之組成物包含線性多核糖核苷酸和稀釋劑。In some embodiments, compositions of the present disclosure include cyclic polyribonucleotides and a diluent. In some embodiments, compositions of the present disclosure comprise linear polyribonucleotides and a diluent.

稀釋劑可為非載劑賦形劑。非載劑賦形劑用作組成物（諸如，如本文所述之環狀多核糖核苷酸）的媒介物或介質。非載劑賦形劑用作組成物（諸如，如本文所述之線性多核糖核苷酸）的媒介物或介質。非載劑賦形劑之非限制性實例包括溶劑、水性溶劑、非水溶劑、分散介質、稀釋劑、分散劑、助懸劑、界面活性劑、等滲劑、增稠劑、乳化劑、防腐劑、聚合物、肽、蛋白質、細胞、透明質酸酶、分散劑、制粒劑、崩解劑、黏合劑、緩衝劑（例如，磷酸鹽緩衝鹽水（PBS））、潤滑劑、油及其混合物。非載劑賦形劑可為經美國食品和藥物管理局（FDA）批准並列在非活性成分數據庫中的不表現出細胞穿透作用的任一種非活性成分。非載劑賦形劑可為適於投與於非人動物（例如，適合獸醫用途）的任何非活性成分。為了使組成物適於向各種動物投與，對適於向人投與的組成物的修飾得到很好的理解，並且普通技術的獸醫藥理師可以僅通過普通的實驗（如果有）來設計和/或進行此類修飾。The diluent can be a non-carrier excipient. Non-carrier excipients serve as vehicles or media for compositions such as cyclic polyribonucleotides as described herein. Non-carrier excipients serve as vehicles or media for compositions such as linear polyribonucleotides as described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives agents, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and their mixture. A non-carrier excipient can be any inactive ingredient approved by the U.S. Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database that does not exhibit cell-penetrating effects. A non-carrier excipient can be any inactive ingredient suitable for administration to non-human animals (eg, suitable for veterinary use). In order to make the compositions suitable for administration to a variety of animals, modifications of the compositions suitable for administration to humans are well understood and can be devised and designed by a veterinary pharmacist of ordinary skill only by ordinary experimentation, if any. /or make such modifications.

在一些實施方式中，環狀多核糖核苷酸可以以裸遞送配製物的形式遞送，諸如包含稀釋劑。裸遞送配製物將環狀多核糖核苷酸遞送至細胞，無需借助於載劑並且無需對環狀多核糖核苷酸、加帽的多核糖核苷酸或其複合物進行修飾或部分或完全封裝。In some embodiments, cyclic polyribonucleotides can be delivered in a naked delivery formulation, such as containing a diluent. Naked delivery formulations deliver cyclic polyribonucleotides to cells without the aid of a carrier and without modification or partial or complete modification of the cyclic polyribonucleotides, capped polyribonucleotides, or complexes thereof Encapsulation.

裸遞送配製物係不含載劑的配製物並且其中環狀多核糖核苷酸沒有結合有助於遞送至細胞的部分的共價修飾，或者沒有對環狀多核糖核苷酸的部分或完全封裝。在一些實施方式中，沒有結合有助於遞送至細胞的部分的共價修飾的環狀多核糖核苷酸係未與蛋白質、小分子、顆粒、聚合物或生物聚合物共價結合的多核糖核苷酸。沒有結合有助於遞送至細胞的部分的共價修飾的環狀多核糖核苷酸不含經修飾的磷酸基團。例如，沒有結合有助於遞送至細胞的部分的共價修飾的環狀多核糖核苷酸不含硫代磷酸酯、硒代磷酸酯、硼代磷酸鹽、硼代磷酸酯、氫磷酸酯、胺基磷酸酯、二胺基磷酸酯、烷基或芳基膦酸酯或磷酸三酯。Naked delivery formulations are formulations that do not contain a carrier and in which the cyclic polyribonucleotide has no covalent modifications to incorporate moieties that facilitate delivery to cells, or no partial or complete modification of the cyclic polyribonucleotide. Encapsulation. In some embodiments, a covalently modified cyclic polyribonucleotide that is not bound to a moiety that facilitates delivery to a cell is a polyribose that is not covalently bound to a protein, small molecule, particle, polymer, or biopolymer. Nucleotides. Covalently modified cyclic polyribonucleotides that are not bound to a moiety that facilitates delivery to cells do not contain modified phosphate groups. For example, covalently modified cyclic polyribonucleotides that do not incorporate moieties that facilitate delivery to cells do not contain phosphorothioates, selenophosphates, borophosphates, borophosphates, hydrogen phosphates, Amino phosphates, diamino phosphates, alkyl or aryl phosphonates or phosphate triesters.

在一些實施方式中，裸遞送配製物不含以下任何或全部：轉染試劑、陽離子載劑、碳水化合物載劑、奈米顆粒載劑或蛋白質載劑。在一些實施方式中，裸遞送配製物不含植物糖原辛烯基琥珀酸酯、植物糖原β-糊精、酸酐改性的植物糖原β-糊精、脂轉染胺、聚乙烯亞胺、聚(三甲烯亞胺)、聚(四甲烯亞胺)、聚丙烯亞胺、胺基糖苷-聚胺、雙去氧-二胺基-b-環糊精、精胺、亞精胺、聚甲基丙烯酸(2-二甲基胺基)乙酯、聚(離胺酸)、聚(組胺酸)、聚(精胺酸)、陽離子明膠、樹狀聚合物、殼聚糖、l,2-二油醯基-3-三甲基銨-丙烷（DOTAP）、N-[1-(2,3-二油醯基氧基)丙基]-N,N,N-三甲基氯化銨（DOTMA）、l-[2-(油醯基氧基)乙基]-2-油烯基-3-(2-羥乙基)咪唑鎓氯化物（DOTIM）、2,3-二油醯基氧基-N-[2(精胺甲醯胺基)乙基]-N,N-二甲基-l-三氟乙酸丙銨（DOSPA）、3B-[N-(N\N'-二甲基胺基乙烷)-胺基甲醯基]膽固醇鹽酸鹽（DC-膽固醇HCl）、雙十七烷基醯胺基甘胺醯亞精胺（DOGS）、N,N-二硬脂基-N,N-二甲基溴化銨（DDAB）、N-(l,2-二肉豆蔻基氧基丙-3-基)-N,N-二甲基-N-羥乙基溴化銨（DMRIE）、N,N-二油烯基-N,N-二甲基氯化銨（DODAC）、人血清白蛋白（HSA）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）或球蛋白。In some embodiments, naked delivery formulations do not contain any or all of the following: transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. In some embodiments, the naked delivery formulation does not contain phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine, polyethylene sulfide Amine, poly(trimethylenimine), poly(tetramethylenimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine Amine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine acid), poly(histidine acid), poly(arginine acid), cationic gelatin, dendrimers, chitosan , l,2-dioleyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-tri Methyl ammonium chloride (DOTMA), l-[2-(oleyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolium chloride (DOTIM), 2, 3-dioleyloxy-N-[2(sperminemethyloxy)ethyl]-N,N-dimethyl-l-propylammonium trifluoroacetate (DOSPA), 3B-[N-( N\N'-dimethylaminoethane)-aminoformyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylglycerol-spermidine (DOGS), N ,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxypropan-3-yl)-N,N-dimethyl- N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density lipoprotein (LDL) , high-density lipoprotein (HDL) or globulin.

在某些實施方式中，裸遞送配製物包括非載劑賦形劑。在一些實施方式中，非載劑賦形劑包括不表現出細胞穿透作用的非活性成分。在一些實施方式中，非載劑賦形劑包括緩衝液，例如PBS。在一些實施方式中，非載劑賦形劑係溶劑、非水溶劑、稀釋劑、助懸劑、界面活性劑、等滲劑、增稠劑、乳化劑、防腐劑、聚合物、肽、蛋白質、細胞、透明質酸酶、分散劑、制粒劑、崩解劑、黏合劑、緩衝劑、潤滑劑或油。In certain embodiments, naked delivery formulations include non-carrier excipients. In some embodiments, non-carrier excipients include inactive ingredients that do not exhibit cell penetration. In some embodiments, non-carrier excipients include buffers such as PBS. In some embodiments, the non-carrier excipients are solvents, non-aqueous solvents, diluents, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, polymers, peptides, proteins , cells, hyaluronidase, dispersants, granulating agents, disintegrating agents, binders, buffers, lubricants or oils.

在一些實施方式中，裸遞送配製物包括稀釋劑。稀釋劑可為液體稀釋劑或固體稀釋劑。在一些實施方式中，稀釋劑係RNA增溶劑、緩衝液或等滲劑。RNA增溶劑之實例包括水、乙醇、甲醇、丙酮、甲醯胺和2-丙醇。緩衝液之實例包括2-(N-𠰌啉代)乙磺酸（MES）、Bis-Tris、2-[(2-胺基-2-側氧基乙基)-(羧甲基)胺基]乙酸（ADA）、N-(2-乙醯胺基)-2-胺基乙烷磺酸（ACES）、哌𠯤-N,N′-雙(2-乙磺酸)（PIPES）、2-[[1,3-二羥基-2-(羥甲基)丙-2-基]胺基]乙磺酸（TES）、3-(N-𠰌啉代)丙烷磺酸（MOPS）、4-(2-羥乙基)-1-哌𠯤乙磺酸（HEPES）、Tris、Tricine、Gly-Gly、Bicine或磷酸鹽。等滲劑之實例包括甘油、甘露醇、聚乙二醇、丙二醇、海藻糖或蔗糖。載劑In some embodiments, naked delivery formulations include diluents. The diluent can be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizer, buffer, or isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers include 2-(N-𠰌lino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-side oxyethyl)-(carboxymethyl)amino ] Acetic acid (ADA), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2 -[[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-𠰌lino)propanesulfonic acid (MOPS), 4 -(2-Hydroxyethyl)-1-piperidineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine or phosphate. Examples of isotonic agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose or sucrose.carrier

在一些實施方式中，本揭露之組成物包含環狀多核糖核苷酸和載劑。在一些實施方式中，本揭露之組成物包含線性多核糖核苷酸和載劑。In some embodiments, compositions of the present disclosure comprise cyclic polyribonucleotides and a carrier. In some embodiments, compositions of the present disclosure comprise linear polyribonucleotides and a carrier.

在某些實施方式中，組成物在囊泡或其他基於膜的載劑中包括如本文所述之環狀多核糖核苷酸。在某些實施方式中，組成物在囊泡或其他基於膜的載劑中包括如本文所述之線性多核糖核苷酸。In certain embodiments, compositions include cyclic polyribonucleotides as described herein in a vesicle or other membrane-based vehicle. In certain embodiments, compositions include linear polyribonucleotides as described herein in a vesicle or other membrane-based vehicle.

在其他實施方式中，組成物在細胞、囊泡或其他基於膜的載劑中或經由細胞、囊泡或其他基於膜的載劑包括環狀多核糖核苷酸。在其他實施方式中，組成物在細胞、囊泡或其他基於膜的載劑中或經由細胞、囊泡或其他基於膜的載劑包括線性多核糖核苷酸。在一個實施方式中，組成物在脂質體或其他類似囊泡中包括環狀多核糖核苷酸。在一個實施方式中，組成物在脂質體或其他類似囊泡中包括線性多核糖核苷酸。脂質體係球形囊泡結構，該等球形囊泡結構由圍繞內部水性隔室的單層或多層的脂質雙層和相對不可滲透的外部親脂性磷脂雙層構成。脂質體可為陰離子的、中性的或陽離子的。脂質體具有生物相容性，無毒，可以遞送親水性和親脂性藥物分子，保護其貨物免受血漿酶的降解，並將其負載運輸穿過生物膜和血腦屏障（BBB）（有關綜述，參見，例如，Spuch和Navarro, Journal of Drug Delivery [藥物遞送雜誌], 第2011卷, 文章ID 469679, 第12頁, 2011. doi:10.1155/2011/469679）。In other embodiments, the compositions include cyclic polyribonucleotides in or via cells, vesicles, or other membrane-based vehicles. In other embodiments, the compositions include linear polyribonucleotides in or via cells, vesicles, or other membrane-based vehicles. In one embodiment, the composition includes cyclic polyribonucleotides in liposomes or other similar vesicles. In one embodiment, the composition includes linear polyribonucleotides in liposomes or other similar vesicles. Lipid systems are spherical vesicle structures composed of a single or multi-layered lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, and can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their payload across biological membranes and the blood-brain barrier (BBB) (for review, See, e.g., Spuch and Navarro, Journal of Drug Delivery, Volume 2011, Article ID 469679, Page 12, 2011. doi:10.1155/2011/469679).

囊泡可以由若干種不同類型的脂質製成；然而，磷脂最常用於生成作為藥物載劑的脂質體。用於製備多層囊泡脂質的方法係本領域已知的（參見，例如美國專利案號6,693,086，其中涉及多層囊泡脂質製備的教導藉由援引併入本文）。儘管當脂質膜與水溶液混合時，囊泡形成可為自發的，但也可以藉由經由使用均質器、超音波儀或擠壓設備以振盪的形式施加力來加快囊泡形成（關於綜述，參見例如，Spuch和Navarro, Journal of Drug Delivery [藥物遞送雜誌], 第2011卷, 文章ID 469679, 第12頁, 2011. doi:10.1155/2011/469679）。可以藉由擠出藉由具有減小尺寸的過濾器來製備擠出的脂質，如Templeton等人, Nature Biotech [自然 生物技術], 15:647-652, 1997中所述，該文獻關於擠出脂質製備的傳授內容藉由援引併入本文。Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, eg, U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings regarding the preparation of multilamellar vesicle lipids). Although vesicle formation can be spontaneous when lipid membranes are mixed with aqueous solutions, vesicle formation can also be accelerated by applying force in an oscillatory manner through the use of homogenizers, sonicators, or extrusion devices (for a review, see For example, Spuch and Navarro, Journal of Drug Delivery, Volume 2011, Article ID 469679, Page 12, 2011. doi:10.1155/2011/469679). Extruded lipids can be prepared by extrusion through a filter of reduced size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, on extrusion The teachings of lipid preparation are incorporated herein by reference.

在某些實施方式中，本揭露之組成物包含環狀多核糖核苷酸和脂質奈米顆粒，例如本文所述之脂質奈米顆粒。在某些實施方式中，本揭露之組成物包含線性多核糖核苷酸和脂質奈米顆粒。脂質奈米顆粒係為如本文所述之環狀多核糖核苷酸分子提供生物相容性和可生物降解的遞送系統的載劑的另一個實例。脂質奈米顆粒係為如本文所述之線性多核糖核苷酸分子提供生物相容性和可生物降解的遞送系統的載劑的另一個實例。奈米結構化的脂質載劑（NLC）係經修飾的固體脂質奈米顆粒（SLN），該等經修飾的固體脂質奈米顆粒保留了SLN的特徵、改善了藥物穩定性和負載能力、並且防止了藥物洩漏。聚合物奈米顆粒（PNP）係藥物遞送的重要部分。該等奈米顆粒可以有效地指導藥物遞送至特定靶標並且提高藥物穩定性和藥物可控釋放。也可以採用脂質-聚合物奈米顆粒（PLN），即一種組合了脂質體和聚合物的新型載劑。該等奈米顆粒具有PNP和脂質體的互補優勢。PLN由核殼結構構成；聚合物核提供了穩定的結構，而磷脂殼提供了良好的生物相容性。因此，這兩種組分增加了藥物封裝率、促進了表面修飾、並且防止了水溶性藥物的洩漏。對於綜述，參見，例如，Li等人 2017, Nanomaterials [奈米材料] 7, 122；doi:10.3390/nano7060122。In certain embodiments, compositions of the present disclosure comprise cyclic polyribonucleotides and lipid nanoparticles, such as the lipid nanoparticles described herein. In certain embodiments, compositions of the present disclosure include linear polyribonucleotides and lipid nanoparticles. Lipid nanoparticles are another example of a vehicle that provides a biocompatible and biodegradable delivery system for cyclic polyribonucleotide molecules as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for linear polyribonucleotide molecules as described herein. Nanostructured lipid carriers (NLC) are modified solid lipid nanoparticles (SLN). These modified solid lipid nanoparticles retain the characteristics of SLN, improve drug stability and loading capacity, and Prevents drug leakage. Polymer nanoparticles (PNPs) are an important part of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLN), a new type of carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNP and liposomes. PLN is composed of a core-shell structure; the polymer core provides a stable structure, while the phospholipid shell provides good biocompatibility. Therefore, these two components increase the drug encapsulation efficiency, promote surface modification, and prevent the leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.

載劑的其他非限制性實例包括碳水化合物載劑（例如，酸酐改性的植物糖原或糖原型材料）、蛋白質載劑（例如，與環狀多核糖核苷酸共價連接的蛋白質或與線性多核糖核苷酸共價連接的蛋白質）或陽離子載劑（例如，陽離子脂聚合物或轉染試劑）。碳水化合物載劑之非限制性實例包括植物糖原辛烯基琥珀酸酯、植物糖原β-糊精和酸酐改性的植物糖原β-糊精。陽離子載劑之非限制性實例包括脂轉染胺、聚乙烯亞胺、聚(三甲烯亞胺)、聚(四甲烯亞胺)、聚丙烯亞胺、胺基糖苷-聚胺、雙去氧-二胺基-b-環糊精、精胺、亞精胺、聚甲基丙烯酸(2-二甲基胺基)乙酯、聚(離胺酸)、聚(組胺酸)、聚(精胺酸)、陽離子明膠、樹狀聚合物、殼聚糖、l,2-二油醯基-3-三甲基銨-丙烷（DOTAP）、N-[1-(2,3-二油醯基氧基)丙基]-N,N,N-三甲基氯化銨（DOTMA）、l-[2-(油醯基氧基)乙基]-2-油烯基-3-(2-羥乙基)咪唑鎓氯化物（DOTIM）、2,3-二油醯基氧基-N-[2(精胺甲醯胺基)乙基]-N,N-二甲基-l-三氟乙酸丙銨（DOSPA）、3B-[N-(N,N'-二甲基胺基乙烷)-胺基甲醯基]膽固醇鹽酸鹽（DC-膽固醇HCl）、雙十七烷基醯胺基甘胺醯亞精胺（DOGS）、N,N-二硬脂基-N,N-二甲基溴化銨（DDAB）、N-(l,2-二肉豆蔻基氧基丙-3-基)-N,N-二甲基-N-羥乙基溴化銨（DMRIE）和N,N-二油烯基-N,N-二甲基氯化銨（DODAC）。蛋白質載劑之非限制性實例包括人血清白蛋白（HSA）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）或球蛋白。Other non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified plant glycogen or glycogen), protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides or to linear polyribonucleotide covalently linked proteins) or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, and anhydride-modified phytoglycogen beta-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethylenimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, bis-ethylenimine Oxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine acid), poly(histidine acid), poly (arginine), cationic gelatin, dendrimer, chitosan, l,2-dioleyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-di Oleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleyloxy)ethyl]-2-oleyl-3- (2-Hydroxyethyl)imidazolium chloride (DOTIM), 2,3-dioleyloxy-N-[2(spermineformamide)ethyl]-N,N-dimethyl- l-propylammonium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-aminoformyl]cholesterol hydrochloride (DC-cholesterol HCl), double ten Heptadylglycinolspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyl Oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC ). Non-limiting examples of protein carriers include human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

外泌體也可用作本文所述之環狀RNA組成物或製劑的藥物遞送媒介物。外泌體可用作本文所述之線性多核糖核苷酸組成物或製劑的藥物遞送媒介物。對於綜述，參見Ha等人 2016年7月.Acta Pharmaceutica Sinica B [藥學學報英文版] 第6卷, 第4期, 第287-296頁；doi.org/10.1016/j.apsb.2016.02.001。Exosomes can also be used as drug delivery vehicles for the circRNA compositions or formulations described herein. Exosomes can be used as drug delivery vehicles for the linear polyribonucleotide compositions or formulations described herein. For a review, see Ha et al. 2016 July. Acta Pharmaceutica Sinica B Volume 6, Issue 4, pages 287-296; doi.org/10.1016/j.apsb.2016.02.001.

離體分化的紅血球也可以用作本文所述之環狀RNA組成物或製劑的載劑。離體分化的紅血球也可用作本文所述之線性多核糖核苷酸組成物或製劑的載劑。參見，例如，國際專利公開案號WO 2015/073587；WO 2017/123646；WO 2017/123644；WO 2018/102740；WO 2016/183482；WO 2015/153102；WO 2018/151829；WO 2018/009838；Shi等人 2014.Proc Natl Acad Sci USA [美國國家科學院院刊]. 111(28): 10131–10136; 美國專利9,644,180；Huang等人 2017.Nature Communications [自然-通訊] 8: 423；Shi等人 2014. Proc Natl Acad Sci USA [美國國家科學院院刊].111(28): 10131–10136。Ex vivo differentiated red blood cells may also be used as carriers for the circRNA compositions or formulations described herein. Ex vivo differentiated erythrocytes may also be used as carriers for the linear polyribonucleotide compositions or formulations described herein. See, for example, International Patent Publication Nos. WO 2015/073587; WO 2017/123646; WO 2017/123644; WO 2018/102740; WO 2016/183482; WO 2015/153102; WO 2018/151829; WO 2018/009838; Shi et al. 2014. Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences]. 111(28): 10131–10136; U.S. Patent 9,644,180; Huang et al. 2017. Nature Communications [Nature - Communications] 8: 423; Shi et al. 2014 . Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences]. 111(28): 10131–10136.

例如國際專利公開案號WO 2018/208728中所述之融合體組成物也可以用作載劑遞送本文所述之環狀多核糖核苷酸分子。例如WO 2018/208728中所述之融合體組成物也可以用作載劑遞送本文所述之線性多核糖核苷酸分子。For example, the fusion composition described in International Patent Publication No. WO 2018/208728 can also be used as a carrier to deliver the cyclic polyribonucleotide molecules described herein. Fusion compositions such as those described in WO 2018/208728 may also be used as carriers to deliver linear polyribonucleotide molecules described herein.

病毒體和病毒樣顆粒（VLP）也可用作載劑將本文所述之環狀多核糖核苷酸分子遞送至靶向的細胞。病毒體和病毒樣顆粒（VLP）也可用作載劑將本文所述之線性多核糖核苷酸分子遞送至靶向的細胞。Virions and virus-like particles (VLPs) can also be used as vehicles to deliver the cyclic polyribonucleotide molecules described herein to targeted cells. Virions and virus-like particles (VLPs) can also be used as vehicles to deliver the linear polyribonucleotide molecules described herein to targeted cells.

例如國際專利公開案號WO 2011/097480、WO 2013/070324、WO 2017/004526或WO 2020/041784中所述之植物奈米囊泡和植物信使包（PMP）也可用作載劑遞送本文所述之環狀RNA組成物或製劑。植物奈米囊泡和植物信使包（PMP）也可用作載劑遞送本文所述之線性多核糖核苷酸組成物或製劑。For example, plant nanovesicles and plant messenger packages (PMP) described in International Patent Publication Nos. WO 2011/097480, WO 2013/070324, WO 2017/004526 or WO 2020/041784 can also be used as carriers to deliver the materials disclosed herein. Said circular RNA composition or preparation. Plant nanovesicles and plant messenger packages (PMPs) can also be used as vehicles to deliver the linear polyribonucleotide compositions or formulations described herein.

微泡也可以用作載劑遞送本文所述之環狀多核糖核苷酸分子。微泡也可用作載劑遞送本文所述之線性多核糖核苷酸分子。參見，例如，US7115583；Beeri, R.等人，Circulation. [循環]2002年10月1日;106(14):1756-1759；Bez, M.等人，Nat Protoc. [自然實驗手冊] 2019年4月；14(4): 1015–1026; Hernot, S.等人，Adv Drug Deliv Rev. [高級藥物遞送綜述] 2008年6月30日; 60(10): 1153–1166; Rychak, J.J.等人，Adv Drug Deliv Rev. [高級藥物遞送綜述] 2014年6月; 72: 82-93。在一些實施方式中，微泡係白蛋白包被的全氟化碳微泡。Microvesicles can also be used as vehicles to deliver the cyclic polyribonucleotide molecules described herein. Microvesicles can also be used as vehicles to deliver the linear polyribonucleotide molecules described herein. See, e.g., US7115583; Beeri, R. et al., Circulation. [Circulation] 2002 Oct 1;106(14):1756-1759; Bez, M. et al., Nat Protoc. [Natural Experiments Handbook] 2019 Apr;14(4):1015–1026; Hernot, S. et al., Adv Drug Deliv Rev. 2008Jun 30;60(10):1153–1166; Rychak, J.J. et al., Adv Drug Deliv Rev. 2014 Jun;72: 82-93. In some embodiments, the microvesicles are albumin-coated perfluorocarbon microvesicles.

包含本文所述之環狀多核糖核苷酸的載劑可以包含多個顆粒。該等顆粒可具有30至700奈米的中值粒度（例如30至50、50至100、100至200、200至300、300至400、400至500、500至600、600至700、100至500、50至500或200至700奈米）。可以優化顆粒之大小以有利於包括環狀多核糖核苷酸在內的有效負載沈積到細胞中。環狀多核糖核苷酸沈積到某些細胞類型中可有利於不同的粒度。例如，可以優化粒度以使環狀多核糖核苷酸沈積到抗原呈現細胞中。可以優化粒度以使環狀多核糖核苷酸沈積到樹突細胞中。另外，可以優化粒度以使環狀多核糖核苷酸沈積到引流淋巴結細胞中。脂質奈米顆粒A carrier comprising a cyclic polyribonucleotide described herein may comprise a plurality of particles. The particles may have a median particle size of 30 to 700 nanometers (e.g., 30 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to 500, 50 to 500 or 200 to 700 nm). The size of the particles can be optimized to facilitate deposition of payloads, including cyclic polyribonucleotides, into cells. Deposition of cyclic polyribonucleotides into certain cell types can favor different particle sizes. For example, the particle size can be optimized to deposit cyclic polyribonucleotides into antigen-presenting cells. Particle size can be optimized to enable deposition of cyclic polyribonucleotides into dendritic cells. Additionally, the particle size can be optimized to enable deposition of cyclic polyribonucleotides into draining lymph node cells.lipid nanoparticles

本揭露提供的組成物、方法和遞送系統可以採用本文所述之任何合適的載劑或遞送形式，在某些實施方式中包括脂質奈米顆粒（LNP）。在一些實施方式中，脂質奈米顆粒包括一或多種離子脂質，如非陽離子脂質（例如，中性或陰離子或兩性離子脂質）；一或多種軛合脂質（諸如WO 2019217941的表5中描述的PEG軛合的脂質或軛合至聚合物的脂質；其藉由援引以其全文併入本文）；一或多種固醇（例如，膽固醇）。The compositions, methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery form described herein, including, in certain embodiments, lipid nanoparticles (LNPs). In some embodiments, lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (eg, neutral or anionic or zwitterionic lipids); one or more conjugated lipids (such as those described in Table 5 of WO 2019217941 PEG-conjugated lipid or lipid conjugated to a polymer; which is incorporated herein by reference in its entirety); one or more sterols (eg, cholesterol).

可用於奈米顆粒形成的脂質（例如，脂質奈米顆粒）包括例如WO 2019217941的表4中描述的那些，其藉由援引併入本文—例如，含脂質的奈米顆粒可包括WO 2019217941的表4中的一或多種脂質。脂質奈米顆粒可以包括另外的要素，諸如聚合物，諸如藉由援引併入的WO 2019217941的表5中描述的聚合物。Lipids that can be used in nanoparticle formation (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941, which is incorporated herein by reference—for example, lipid-containing nanoparticles can include those described in Table 4 of WO 2019217941 One or more lipids in 4. Lipid nanoparticles may include additional elements, such as polymers, such as those described in Table 5 of WO 2019217941, which is incorporated by reference.

在一些實施方式中，軛合脂質，當存在時，可以包括以下的一或多種：PEG-二醯基甘油（DAG）（諸如l-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯甘油（PEG-DMG））、PEG-二烷氧基丙基（DAA）、PEG-磷脂、PEG-神經醯胺（Cer）、聚乙二醇化磷脂醯乙醇胺（PEG-PE）、PEG琥珀酸二醯基甘油（PEGS-DAG）（諸如4-0-(2',3'-二(十四烷醯氧基)丙基-l-0-(w-甲氧基(聚乙氧基)乙基)丁二酸酯（PEG-S-DMG））、PEG二烷氧基丙基胺基甲酸酯、N-(羰基-甲氧基聚乙二醇2000)-1 ,2-二硬脂醯-sn-甘油-3-磷酸乙醇胺鈉鹽，以及在WO 2019051289的表2中描述的那些（藉由援引併入）和前述的組合。In some embodiments, the conjugated lipid, when present, can include one or more of the following: PEG-digylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3 -Dimyristylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), pegylated phospholipid ethanolamine (PEG-PE) , PEG diacylglycerol succinate (PEGS-DAG) (such as 4-0-(2',3'-bis(tetradecanyloxy)propyl-l-0-(w-methoxy(poly Ethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1, 2-Distearyl-sn-glycerol-3-phosphoethanolamine sodium salt, as well as those described in Table 2 of WO 2019051289 (incorporated by reference) and combinations of the foregoing.

在一些實施方式中，可摻入脂質奈米顆粒中的固醇包括膽固醇或膽固醇衍生物中的一或多種，諸如藉由援引併入的WO 2009/127060或US 2010/0130588中的那些。另外的示例性固醇包括植物固醇，包括藉由援引併入本文的Eygeris等人(2020)，dx.doi.org/10.1021/acs.nanolett.0c01386中描述的那些。In some embodiments, sterols that may be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO 2009/127060 or US 2010/0130588, incorporated by reference. Additional exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.

在一些實施方式中，脂質顆粒包括可電離脂質、非陽離子脂質、抑制顆粒聚集的軛合脂質和固醇。該等組分的量可以獨立地變化，以獲得所需特性。例如，在一些實施方式中，脂質奈米顆粒包括可電離脂質、非陽離子脂質、軛合脂質和甾醇，該可電離脂質的量為總脂質的約20 mol %至約90 mol %（在其他實施方式中，該可電離脂質可為脂質奈米顆粒中存在的總脂質的20%-70%（mol）、30%-60%（mol）或40%-50%（mol）；約50 mol%至約90 mol%）；該非陽離子脂質的量為總脂質的約5 mol %至約30 mol %；該軛合脂質的量為總脂質的約0.5 mol%至約20 mol%，該甾醇的量為總脂質的約20 mol%至約50 mol%。總脂質與核酸的比率可以根據需要變化。例如，總脂質與核酸（品質或重量）的比率可為約10 : 1至約30 : 1。In some embodiments, lipid particles include ionizable lipids, noncationic lipids, conjugated lipids that inhibit particle aggregation, and sterols. The amounts of these components can be varied independently to obtain the desired properties. For example, in some embodiments, the lipid nanoparticles include ionizable lipids, noncationic lipids, conjugated lipids, and sterols in an amount of about 20 mol % to about 90 mol % of the total lipids (in other embodiments In the method, the ionizable lipid can be 20%-70% (mol), 30%-60% (mol) or 40%-50% (mol) of the total lipids present in the lipid nanoparticles; about 50 mol% to about 90 mol%); the amount of the noncationic lipid is about 5 mol% to about 30 mol% of the total lipid; the amount of the conjugated lipid is about 0.5 mol% to about 20 mol% of the total lipid, and the amount of the sterol From about 20 mol% to about 50 mol% of the total lipids. The ratio of total lipids to nucleic acids can be varied as desired. For example, the ratio of total lipids to nucleic acids (mass or weight) can be from about 10:1 to about 30:1.

在一些實施方式中，脂質與核酸的比率（品質/品質比率；w/w比）可處於約1 : 1至約25 : 1、約10 : 1至約14 : 1、約3 : 1至約15 : 1、約4 : 1至約10 : 1、約5 : 1至約9 : 1或約6 : 1至約9 : 1之範圍內。可以調節脂質和核酸的量以提供所需的N/P比，例如3、4、5、6、7、8、9、10或更高的N/P比。通常，脂質奈米顆粒配製物的總脂質含量可在約5 mg/ml至約30 mg/mL之範圍內。In some embodiments, the ratio of lipids to nucleic acids (mass/mass ratio; w/w ratio) can range from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acids can be adjusted to provide a desired N/P ratio, such as an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10, or higher. Typically, the total lipid content of lipid nanoparticle formulations can range from about 5 mg/ml to about 30 mg/mL.

可用於（例如，與其他脂質組分組合）形成用於遞送本文所述之組成物，例如本文所述之核酸（例如，RNA（例如，環狀多核糖核苷酸、線性多核糖核苷酸））的脂質奈米顆粒的脂質化合物的一些非限制性實例包括：(i)Can be used (e.g., in combination with other lipid components) to form compositions for delivery of a nucleic acid (e.g., RNA (e.g., cyclic polyribonucleotide, linear polyribonucleotide) described herein). )) Some non-limiting examples of lipid compounds for lipid nanoparticles include: (i)

在一些實施方式中，包括式 (i) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(ii)In some embodiments, LNPs comprising Formula (i) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (ii)

在一些實施方式中，包括式 (ii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(iii)In some embodiments, LNPs comprising formula (ii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (iii)

在一些實施方式中，包括式 (iii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(iv)(v)In some embodiments, LNPs comprising formula (iii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (iv) (v)

在一些實施方式中，包括式 (v) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(vi)In some embodiments, LNPs comprising formula (v) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (vi)

在一些實施方式中，包括式 (vi) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(vii)(viii)In some embodiments, LNPs comprising formula (vi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (vii) (viii)

在一些實施方式中，包括式 (viii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(ix)In some embodiments, LNPs comprising formula (viii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (ix)

在一些實施方式中，包括式 (ix) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(x) 其中 X¹係O、NR¹或直接鍵，X²係C2-5伸烷基，X³係C(=O)或直接鍵，R¹係H或Me，R³係C1-3烷基，R²係C1-3烷基，或R²與它所附接的氮原子和X²的1-3個碳原子一起形成4-員、5-員或6-員環，或X¹係NR¹，R¹和R²與它們所附接的氮原子一起形成5-員或6-員環，或R²與R³和它們所附接的氮原子一起形成5-員、6-員或7-員環，Y¹係C2-12伸烷基，Y²選自（以任一取向），（以任一取向），（以任一取向）， n為0至3，R⁴為C1-15烷基，Z¹為C1-6伸烷基或直接鍵，（以任一取向）或不存在，條件是如果Z¹係直接鍵，則Z²不存在； R⁵係C5-9烷基或C6-10烷氧基，R⁶係C5-9烷基或C6-10烷氧基，W係亞甲基或直接鍵，並且R⁷係H或Me，或其鹽，條件是如果R³和R²係C2烷基，X¹係O，X²係直鏈C3伸烷基，X³係C(=0)，Y¹係直鏈Ce伸烷基，(Y²)n-R⁴係，R⁴係直鏈C5烷基，Z¹係C2伸烷基，Z²不存在，W係亞甲基，並且R⁷係H，則R⁵和R⁶不是Cx烷氧基。In some embodiments, LNPs comprising formula (ix) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.⁽ x⁾ Where X¹ is O, NR¹ or a direct bond,^X2 is a C2-5 alkylene group, Alkyl group, R² is a C1-3 alkyl group, or R² together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X² form a 4-membered, 5-membered or 6-membered ring, or X¹ series NR¹ , R¹ and R² together with the nitrogen atom to which they are attached form a 5-membered or 6-membered ring, or R² together with R³ and the nitrogen atom to which they are attached form a 5-membered or 6-membered ring -membered or 7-membered ring, Y¹ is C2-12 alkylene group, Y² is selected from (in any orientation), (in any orientation), (in any orientation), n is 0 to 3, R⁴ is C1-15 alkyl, Z¹ is C1-6 alkylene or direct bond, (in either orientation) or absent, provided that if Z¹ is a direct bond, Z² is absent; R⁵ is C5-9 alkyl or C6-10 alkoxy, R⁶ is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R⁷ is H or Me, or a salt thereof, provided that if R³ and R² are C2 alkyl, X¹ is O, and X² is straight Chain C3 alkylene group, X³ system is C (=0), Y¹ system is straight chain Ce alkylene group, (Y² )nR⁴ system , R⁴ is a linear C5 alkyl group, Z¹ is a C2 alkylene group, Z² does not exist, W is a methylene group, and R⁷ is H, then R⁵ and R⁶ are not Cx alkoxy groups.

在一些實施方式中，包括式 (xii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(xi)In some embodiments, LNPs comprising formula (xii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (xi)

在一些實施方式中，包括式 (xi) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。其中R=(xii)(xiii)(xiv)In some embodiments, LNPs comprising formula (xi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. where R= (xii) (xiii) (xiv)

在一些實施方式中，LNP包括具有式 (xiii) 的化合物和具有式 (xiv) 的化合物。(xv)In some embodiments, LNPs include compounds of formula (xiii) and compounds of formula (xiv). (xv)

在一些實施方式中，包括式 (xv) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(xvi)In some embodiments, LNPs comprising Formula (xv) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (xvi)

在一些實施方式中，包括具有式 (xvi) 配製物的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。(xvii)其中X=(xviii)(a)(xviii)(b)(xix)In some embodiments, LNPs comprising formulations of Formula (xvi) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. (xvii) where X= (xviii)(a) (xviii)(b) (xix)

在一些實施方式中，用於形成用於遞送本文所述之組成物，例如本文所述之核酸（例如，RNA（例如，環狀多核糖核苷酸、線性多核糖核苷酸））的脂質奈米顆粒的脂質化合物藉由以下反應之一製成：+(xx)(a)+(xx)(b)。In some embodiments, lipids are used to form lipids for delivering compositions described herein, such as nucleic acids (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) described herein. The lipid compounds of the nanoparticles are made by one of the following reactions:+ (xx)(a)+ (xx)(b).

在一些實施方式中，包括式 (xxi) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。在一些實施方式中，式 (xxi) 的LNP係WO 2021113777描述的LNP（例如，式 (1) 的脂質，諸如WO 2021113777的表1的脂質）。(xxi) 其中 每個n獨立地是2-15的整數；L₁和L₃各自獨立地是-OC(O)-*或-C(O)O-*，其中「*」表示與R₁或R₃的附接點； R₁和R₃各自獨立地是直鏈或支鏈C₉-C₂₀烷基或C₉-C₂₀烯基，其視需要被選自由以下組成之群組的一或多個取代基取代：側氧基、鹵代、羥基、氰基、烷基、烯基、醛、雜環基烷基、羥基烷基、二羥基烷基、羥基烷基胺基烷基、胺基烷基、烷基胺基烷基、二烷基胺基烷基、(雜環基)(烷基)胺基烷基、雜環基、雜芳基、烷基雜芳基、炔基、烷氧基、胺基、二烷基胺基、胺基烷基羰基胺基、胺基羰基烷基胺基、(胺基羰基烷基)(烷基)胺基、烯基羰基胺基、羥基羰基、烷氧基羰基、胺基羰基、胺基烷基胺基羰基、烷基胺基烷基胺基羰基、二烷基胺基烷基胺基羰基、雜環基烷基胺基羰基、(烷基胺基烷基)(烷基)胺基羰基、烷基胺基烷基羰基、二烷基胺基烷基羰基、雜環基羰基、烯基羰基、炔基羰基、烷基亞碸、烷基亞碸烷基、烷基磺醯基和烷基碸烷基；以及 R₂選自由以下組成之群組：In some embodiments, LNPs comprising Formula (xxi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNP of formula (xxi) is an LNP described in WO 2021113777 (eg, a lipid of formula (1), such as the lipids of Table 1 of WO 2021113777). (xxi) where each n is independently an integer from 2 to 15; L₁ and L₃ are each independently -OC(O)-* or -C(O)O-*, where "*" means the same as R₁ or the point of attachment of R₃ ; R₁ and R₃ are each independently a linear or branched C₉ -C₂₀ alkyl or a C₉ -C₂₀ alkenyl group, which is optionally selected from the group consisting of One or more substituents: pendant oxy, halogen, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl , aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkyne group, alkoxy group, amino group, dialkylamino group, aminoalkylcarbonylamino group, aminocarbonylalkylamino group, (aminocarbonylalkyl)(alkyl)amine group, alkenylcarbonylamino group , hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl , (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylene Tris, alkylprenylene, alkylsulfonyl and alkylprenyl; and R₂ is selected from the group consisting of:

在一些實施方式中，包括式 (xxii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。在一些實施方式中，式 (xxii) 的LNP係WO 2021113777描述的LNP（例如，式 (2) 的脂質，諸如WO 2021113777的表2的脂質）。(xxii) 其中 每個n獨立地是1-15的整數； R₁和R₂各自獨立地選自由以下組成之群組：R₃選自由以下組成之群組：。In some embodiments, LNPs comprising Formula (xxii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNP of formula (xxii) is an LNP described in WO 2021113777 (eg, a lipid of formula (2), such as the lipids of Table 2 of WO 2021113777). (xxii) where each n is independently an integer from 1 to 15; R₁ and R₂ are each independently selected from the group consisting of: R₃ is selected from the group consisting of: .

在一些實施方式中，包括式 (xxiii) 的LNP用於將本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）組成物遞送至細胞。在一些實施方式中，式 (xxiii) 的LNP係WO 2021113777描述的LNP（例如，式 (3) 的脂質，諸如WO 2021113777的表3的脂質）。(xxiii) 其中 X選自-O-、-S-或-OC(O)-*，其中*表示與R₁的附接點； R₁選自由以下組成之群組：且R₂選自由以下組成之群組：In some embodiments, LNPs comprising Formula (xxiii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells. In some embodiments, the LNP of formula (xxiii) is an LNP described in WO 2021113777 (eg, a lipid of formula (3), such as the lipids of Table 3 of WO 2021113777)._(xxiii ) wherein And R₂ is selected from the group consisting of:

在一些實施方式中，本文所述之組成物（例如，核酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）或蛋白質）在包括可電離脂質的LNP中提供。在一些實施方式中，可電離脂質係十七烷-9-基8-((2-羥乙基)(6-側氧基-6-(十一烷氧基)己基)胺基)辛酸酯（SM-102）；例如，如US9,867,888（藉由援引以其全文併入本文）的實例1中所述。在一些實施方式中，可電離脂質係9Z,12Z)-3-((4,4-雙(辛氧基)丁醯基)氧基)-2-((((3-(二乙基胺基)丙氧基)羰基)氧基)甲基)丙基十八碳-9,12-二烯酸酯（LP01），例如，如WO 2015/095340（藉由援引以其全文併入本文）的實例13中合成的。在一些實施方式中，可電離脂質係二((Z)-非-2-烯-1-基)9-((4-二甲胺基)丁醯基)氧基)十七烷二酸酯（L319），例如，如US 2012/0027803（藉由援引以其全文併入本文）的實例7、8和9中合成的。在一些實施方式中，可電離脂質係1,1'-((2-(4-(2-((2-(雙(2-羥基十二烷基)胺基)乙基)(2-羥基十二烷基)胺基)乙基)哌𠯤-1-基)乙基)氮烷二基)雙(十二烷-2-醇)（C12-200），例如，如WO 2010/053572（藉由援引以其全文併入本文）的實例14和實例16中合成的。在一些實施方式中，可電離脂質係咪唑膽固醇酯（ICE）脂質(3S,10R,13R,17R)-10,13-二甲基-17-((R)-6-甲基庚-2-基)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-十四氫-1H-環戊烯並[a]菲-3-基3-(1H-咪唑-4-基)丙酸酯，例如來自WO 2020/106946（藉由援引以其全文併入本文）的結構 (I)。In some embodiments, the compositions described herein (eg, nucleic acids (eg, cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs that include ionizable lipids. In some embodiments, the ionizable lipid system heptadecan-9-yl 8-((2-hydroxyethyl)(6-sideoxy-6-(undecyloxy)hexyl)amino)octanoic acid Ester (SM-102); for example, as described in Example 1 of US 9,867,888 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid system 9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-(((3-(diethylamino) Propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (LP01), for example, as exemplified in WO 2015/095340 (incorporated herein by reference in its entirety) Synthesized in 13. In some embodiments, the ionizable lipid system bis((Z)-non-2-en-1-yl)9-((4-dimethylamino)butyl)oxy)heptadecanedioate (L319 ), for example, as synthesized in Examples 7, 8 and 9 of US 2012/0027803 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid system 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl))(2-hydroxy Dodecyl)amino)ethyl)piperidin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as in WO 2010/053572 ( synthesized in Examples 14 and 16, which are incorporated herein by reference in their entirety. In some embodiments, the ionizable lipid system is imidazole cholesteryl ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2- base)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3 -(1H-imidazol-4-yl)propionate, for example structure (I) from WO 2020/106946 (incorporated herein by reference in its entirety).

在一些實施方式中，可電離脂質可為陽離子脂質、可電離陽離子脂質，例如可以根據pH以帶正電荷的形式或中性形式存在的陽離子脂質，或可以容易地質子化的含胺脂質。在一些實施方式中，陽離子脂質係例如在生理條件下能夠帶正電的脂質。示例性的陽離子脂質包括一或多個帶有正電荷的胺基。在一些實施方式中，脂質顆粒包括與中性脂質、可電離含胺脂質、可生物降解的炔脂質、類固醇、包括多不飽和脂質的磷脂、結構脂質（例如固醇）、PEG、膽固醇和聚合物軛合脂質中的一或多種配製的陽離子脂質。在一些實施方式中，陽離子脂質可為可電離的陽離子脂質。如本文揭露的示例性陽離子脂質可具有超過6.0的有效pKa。在實施方式中，脂質奈米顆粒可包括具有與第一陽離子脂質不同的有效pKa（例如，大於第一有效pKa）的第二陽離子脂質。脂質奈米顆粒可包括40莫耳%至60莫耳%的封裝在脂質奈米顆粒內或與脂質奈米顆粒締合的陽離子脂質、中性脂質、類固醇、聚合物軛合的脂質和治療劑，例如本文所述之核酸（例如，RNA（例如，環狀多核糖核苷酸、線性多核糖核苷酸））。在一些實施方式中，核酸與陽離子脂質共同配製。核酸可以吸附到LNP（例如包括陽離子脂質的LNP）的表面。在一些實施方式中，核酸可以封裝在LNP（例如包括陽離子脂質的LNP）中。在一些實施方式中，脂質奈米顆粒可包括靶向部分，例如用靶向劑包被的靶向部分。在實施方式中，LNP配製物係生物可降解的。在一些實施方式中，包括一或多種本文所述之脂質（例如式 (i)、(ii)、(ii)、(vii) 和/或 (ix)）的脂質奈米顆粒封裝至少1%、至少5%、至少10%、至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少80%、至少90%、至少92%、至少95%、至少97%、至少98%或100%的RNA分子。In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged form or a neutral form depending on the pH, or an amine-containing lipid that can be readily protonated. In some embodiments, cationic lipids are, for example, lipids capable of being positively charged under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, lipid particles include neutral lipids, ionizable amine-containing lipids, biodegradable acetylenic lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymeric One or more formulated cationic lipids among the conjugated lipids. In some embodiments, the cationic lipid can be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein may have an effective pKa in excess of 6.0. In embodiments, the lipid nanoparticle may include a second cationic lipid having a different effective pKa than the first cationic lipid (eg, greater than the first effective pKa). Lipid nanoparticles may include 40 molar % to 60 molar % of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and therapeutic agents encapsulated within or associated with the lipid nanoparticles , such as nucleic acids (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) as described herein. In some embodiments, nucleic acids are co-formulated with cationic lipids. Nucleic acids can be adsorbed to the surface of LNPs (eg, LNPs including cationic lipids). In some embodiments, nucleic acids can be encapsulated in LNPs (eg, LNPs including cationic lipids). In some embodiments, lipid nanoparticles can include a targeting moiety, such as a targeting moiety coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, lipid nanoparticles including one or more lipids described herein (e.g., formulas (i), (ii), (ii), (vii), and/or (ix)) encapsulate at least 1%, At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97 %, at least 98% or 100% RNA molecules.

可用於脂質奈米顆粒配製物中的示例性可電離脂質包括但不限於藉由援引併入本文的WO 2019051289的表1中所列的那些。另外的示例性脂質包括但不限於下式中的一或多種：US 2016/0311759的X；US 20150376115或US 2016/0376224中的I；US 20160151284的I、II或III；US 20170210967的I、IA、II或IIA；US 20150140070的I-c；US 2013/0178541的A；US 2013/0303587或US 2013/0123338的I；US 2015/0141678的I；US 2015/0239926的II、III、IV或V；US 2017/0119904的I；WO 2017/117528的I或II；US 2012/0149894的A；US 2015/0057373的A；WO 2013/116126的A；US 2013/0090372的A；US 2013/0274523的A；US 2013/0274504的A；US 2013/0053572的A；WO 2013/016058的A；WO 2012/162210的A；US 2008/042973的I；US 2012/01287670的I、II、III或IV；US 2014/0200257的I或II；US 2015/0203446的I、II或III；US 2015/0005363的I或III；US 2014/0308304的I、IA、IB、IC、ID、II、IIA、IIB、IIC、IID或III-XXIV；US 2013/0338210；WO 2009/132131的I、II、III或IV；US2012/01011478的A；US 2012/0027796的I或XXXV；US 2012/0058144的XIV或XVII；US 2013/0323269；US 2011/0117125的I；US 2011/0256175的I、II或III；US 2012/0202871的I、II、III、IV、V、VI、VII、VIII、IX、X、XI、XII；US 2011/0076335的I、II、III、IV、V、VI、VII、VIII、X、XII、XIII、XIV、XV或XVI；US 2006/008378的I或II；US 2013/0123338的I；US 2015/0064242的I或X-A-Y-Z；US 2013/0022649的XVI、XVII或XVIII；US 2013/0116307的I、II或III；US 2013/0116307的I、II或III；US 2010/0062967的I或II；US 2013/0189351的I-X；US 2014/0039032的I；US 2018/0028664的V；US 2016/0317458的I；US 2013/0195920的I；US 10,221,127的5、6或10；WO 2018/081480的III-3；WO 2020/081938的I-5或I-8；US 9,867,888的18或25；US 2019/0136231的A；WO 2020/219876的II；US 2012/0027803的1；US 2019/0240349的OF-02；US 10,086,013的23；Miao等人(2020)的cKK-E12/A6；WO 2010/053572的C12-200；Dahlman等人(2017)的7C1；Whitehead等人的304-O13或503-O13；US 9,708,628的TS-P4C2；WO 2020/106946的I；WO 2020/106946的I；和WO 2021/113777的 (1)、(2)、(3) 或 (4)。示例性脂質進一步包括WO 2021/113777的表1-16中任一個的脂質。Exemplary ionizable lipids useful in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: , II or IIA; I-c of US 20150140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV or V of US 2015/0239926; US I or II of WO 2017/117528; A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372; A of US 2013/0274523; A of US 2013/0274504; A of US 2013/0053572; A of WO 2013/016058; A of WO 2012/162210; I of US 2008/042973; I, II, III or IV of US 2012/01287670; US 2014 /0200257 of I or II; US 2015/0203446 of I, II or III; US 2015/0005363 of I or III; US 2014/0308304 of I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV; US 2013/0338210; I, II, III or IV of WO 2009/132131; A of US2012/01011478; I or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; US 2013 /0323269; I of US 2011/0117125; I, II or III of US 2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US 2011/0076335; I or II of US 2006/008378; I of US 2013/0123338; US I or X-A-Y-Z of 2015/0064242; I - -3; I-5 or I-8 of WO 2020/081938; 18 or 25 of US 9,867,888; A of US 2019/0136231; II of WO 2020/219876; 1 of US 2012/0027803; OF of US 2019/0240349 -02; 23 of US 10,086,013; cKK-E12/A6 of Miao et al. (2020); C12-200 of WO 2010/053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al. ; TS-P4C2 of US 9,708,628; I of WO 2020/106946; I of WO 2020/106946; and (1), (2), (3) or (4) of WO 2021/113777. Exemplary lipids further include the lipids of any of Tables 1-16 of WO 2021/113777.

在一些實施方式中，可電離脂質係MC3 (6Z,9Z,28Z,31Z)-三十七烷-6,9,28,31-四烯-19-基-4-(二甲基胺基)丁酸酯（DLin-MC3-DMA或MC3），例如，如WO 2019051289 A9（藉由援引以其全文併入）的實例9中所述。在一些實施方式中，可電離脂質係脂質ATX-002，例如，如WO 2019051289 A9（藉由援引以其全文併入）的實例10中所述。在一些實施方式中，可電離脂質係(13Z,16Z)-A,A-二甲基-3-壬基二十二-13,16-二烯-1-胺（化合物32），例如，如WO 2019051289 A9（藉由援引以其全文併入）的實例11中所述。在一些實施方式中，可電離脂質係化合物6或化合物22，例如，如WO 2019051289 A9（藉由援引以其全文併入）的實例12中所述。In some embodiments, the ionizable lipid system MC3 (6Z,9Z,28Z,31Z)-triacontan-6,9,28,31-tetraen-19-yl-4-(dimethylamino) Butyrate (DLin-MC3-DMA or MC3), for example, is described in Example 9 of WO 2019051289 A9 (incorporated by reference in its entirety). In some embodiments, the ionizable lipid-based lipid ATX-002 is, for example, as described in Example 10 of WO 2019051289 A9 (incorporated by reference in its entirety). In some embodiments, the ionizable lipid system (13Z,16Z)-A,A-dimethyl-3-nonyldodecano-13,16-diene-1-amine (Compound 32), for example, as As described in Example 11 of WO 2019051289 A9 (incorporated by reference in its entirety). In some embodiments, ionizable lipid-based Compound 6 or Compound 22 is, for example, as described in Example 12 of WO 2019051289 A9 (incorporated by reference in its entirety).

示例性非陽離子脂質包括但不限於二硬脂醯-sn-甘油基-磷酸乙醇胺、二硬脂醯磷脂醯膽鹼（DSPC）、二油醯磷脂醯膽鹼（DOPC）、二棕櫚醯磷脂醯膽鹼（DPPC）、二油醯磷脂醯膽鹼（DOPG）、二棕櫚醯磷脂醯甘油（DPPG）、二油醯磷脂醯乙醇胺（DOPE）、棕櫚醯油醯磷脂醯膽鹼（POPC）、棕櫚醯油醯磷脂醯乙醇胺（POPE）、二油醯-磷脂醯乙醇胺4-(N-馬來醯亞胺甲基)-環己烷-1-甲酸酯（DOPE-mal）、二棕櫚醯磷脂醯乙醇胺（DPPE）、二肉豆蔻醯磷酸乙醇胺（DMPE）、二硬脂醯-磷脂醯-乙醇胺（DSPE）、單甲基-磷脂醯乙醇胺（諸如16-O-單甲基PE）、二甲基-磷脂醯乙醇胺（諸如16-O-二甲基PE）、18-1-反式PE、1-硬脂醯-2-油醯-磷脂醯乙醇胺（SOPE）、氫化大豆磷脂醯膽鹼（HSPC）、卵磷脂醯膽鹼（EPC）、二油醯磷脂醯絲胺酸（DOPS）、神經鞘磷脂（SM）、二肉豆蔻醯磷脂醯膽鹼（DMPC）、二肉豆蔻醯磷脂醯甘油（DMPG）、二硬脂醯磷脂醯甘油（DSPG）、二芥醯基磷脂醯膽鹼（DEPC）、棕櫚醯油醯磷脂醯甘油（POPG）、二反油烯醯-磷脂醯乙醇胺（DEPE）、卵磷脂、磷脂醯乙醇胺、溶血卵磷脂、溶血磷脂醯乙醇胺、磷脂醯絲胺酸、磷脂醯肌醇、鞘磷脂、卵鞘磷脂（ESM）、腦磷脂、心磷脂、磷脂酸、腦苷脂、雙十六烷基磷酸酯、溶血磷脂醯膽鹼、二亞油醯基磷脂醯膽鹼或其混合物。應當理解，也可以使用其他二醯基磷脂醯膽鹼和二醯基磷脂醯乙醇胺磷脂。該等脂質中的醯基基團較佳的為衍生自具有C10-C24碳鏈的脂肪酸的醯基基團，例如月桂醯基、肉豆蔻醯基、棕櫚醯基、硬脂醯基或油醯基。在某些實施方式中，另外的示例性脂質包括但不限於藉由援引併入本文的Kim等人(2020) dx.doi.org/10.1021/acs.nanolett.0c01386描述的那些。在一些實施方式中，此類脂質包括發現會改善用mRNA進行肝轉染的植物脂質（例如DGTS）。Exemplary noncationic lipids include, but are not limited to, distearyl-sn-glyceryl-phosphoethanolamine, distearyl phosphatidylcholine (DSPC), dioleyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine Choline (DPPC), dioleyl phosphatidyl choline (DOPG), dipalmityl phosphatidyl glycerol (DPPG), dioleyl phosphatidyl ethanolamine (DOPE), palmit oleyl phosphatidyl choline (POPC), palm Phospholipid ethanolamine (POPE), dioleyl-phospholipid ethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phospholipid Dimethylethanolamine (DPPE), dimyristylphosphoethanolamine (DMPE), distearyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethylPE), dimethyl Phospholipidylethanolamine (such as 16-O-dimethylPE), 18-1-trans PE, 1-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine ( HSPC), lecithin choline (EPC), dioleyl phosphatidyl serine (DOPS), sphingomyelin (SM), dimyristyl phosphatidylcholine (DMPC), dimyristyl phosphatidyl glycerol (DMPG), distearyl phosphatidyl glycerol (DSPG), disteyl phosphatidyl choline (DEPC), palmityl phosphatidyl glycerol (POPG), disteyl phospholipid glycerol (DEPE) , Lecithin, Phosphatidylethanolamine, Lysolecithin, Lysophosphatidylethanolamine, Phosphatidyl serine, Phosphatidyl inositol, Sphingomyelin, Egg Sphingomyelin (ESM), Cephalin, Cardiolipin, Phosphatidic acid, Cerebroside , dishexadecyl phosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine or mixtures thereof. It should be understood that other diylphospholipids, acylcholine and diylphospholipids, ethanolamine phospholipids may also be used. The acyl group in these lipids is preferably a acyl group derived from a fatty acid having a C10-C24 carbon chain, such as lauryl, myristyl, palmityl, stearyl or oleyl. base. In certain embodiments, additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference. In some embodiments, such lipids include plant lipids (eg, DGTS) found to improve liver transfection with mRNA.

適合用於脂質奈米顆粒中的非陽離子脂質的其他實例包括但不限於非磷脂質，例如像硬脂胺、十二烷基胺、十六烷基胺、乙醯基棕櫚酸酯、蓖麻酸甘油酯、硬脂酸十六烷基酯、肉豆蔻酸異丙酯、兩性丙烯酸聚合物、三乙醇胺-月桂基硫酸鹽、烷基-芳基硫酸鹽、聚乙氧基化脂肪酸醯胺、雙十八烷基二甲基溴化銨、神經醯胺、鞘磷脂等。其他非陽離子脂質在WO 2017/099823或美國專利公開US 2018/0028664中有描述，其內容藉由援引以其全文併入本文。Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, cetylamine, acetyl palmitate, castor Glyceryl acid ester, cetyl stearate, isopropyl myristate, amphoteric acrylic polymer, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, polyethoxylated fatty acid amide, Dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017/099823 or US Patent Publication US 2018/0028664, the contents of which are incorporated herein by reference in their entirety.

在一些實施方式中，非陽離子脂質係油酸或藉由援引以其全文併入的US 2018/0028664的式I、II或IV的化合物。非陽離子脂質可以占例如脂質奈米顆粒中存在的總脂質的0%-30%（莫耳）。在一些實施方式中，非陽離子脂質含量係脂質奈米顆粒中存在的總脂質的5%-20%（莫耳）或10%-15%（莫耳）。在實施方式中，可電離脂質與中性脂質的莫耳比為約2 : 1至約8 : 1（例如，約2 : 1、3 : 1、4 : 1、5 : 1、6 : 1、7 : 1或8 : 1）。In some embodiments, the noncationic lipid is oleic acid or a compound of Formula I, II or IV of US 2018/0028664, which is incorporated by reference in its entirety. Noncationic lipids may comprise, for example, 0%-30% molar of the total lipids present in the lipid nanoparticles. In some embodiments, the noncationic lipid content is 5%-20% (mol) or 10%-15% (mol) of the total lipids present in the lipid nanoparticles. In embodiments, the molar ratio of ionizable lipid to neutral lipid is from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1).

在一些實施方式中，脂質奈米顆粒不包括任何磷脂。In some embodiments, lipid nanoparticles do not include any phospholipids.

在一些方面，脂質奈米顆粒可進一步包括如固醇的組分以提供膜完整性。可用於脂質奈米顆粒中的一種示例性固醇係膽固醇及其衍生物。膽固醇衍生物之非限制性實例包括極性類似物，諸如5a-膽甾烷醇、53-糞甾烷醇、膽甾醇基-(2^,-羥基)-乙基醚、膽甾醇基-(4'-羥基)-丁基醚和6-酮膽甾烷醇；非極性類似物，諸如5a-膽甾烷、膽甾烯酮、5a-膽甾烷酮、5p-膽甾烷酮和膽甾醇癸酸酯；及其混合物。在一些實施方式中，膽固醇衍生物係極性類似物，例如，膽甾醇基-(4'-羥基)-丁基醚。示例性的膽固醇衍生物在PCT公開WO 2009/127060和美國專利公開US 2010/0130588中有描述，其各自藉由援引以其全文併入本文。In some aspects, lipid nanoparticles can further include components such as sterols to provide membrane integrity. An exemplary sterol-based cholesterol and its derivatives useful in lipid nanoparticles. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholestyl-(^2' -hydroxy)-ethyl ether, cholestyl-(4' -Hydroxy)-butyl ether and 6-ketocholestanol; nonpolar analogs such as 5a-cholestane, cholestanone, 5a-cholestanone, 5p-cholestanone, and cholestanoldecane Acid esters; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009/127060 and United States Patent Publication US 2010/0130588, each of which is incorporated herein by reference in its entirety.

在一些實施方式中，提供膜完整性的組分，如固醇，可以包括脂質奈米顆粒中存在的總脂質的0%-50%（莫耳）（例如，0%-10%、10%-20%、20%-30%、30%-40%或40%-50%）。在一些實施方式中，此類組分係脂質奈米顆粒的總脂質含量的20%-50%（莫耳）、30%-40%（莫耳）。In some embodiments, components that provide membrane integrity, such as sterols, can include 0%-50% (mol) of the total lipids present in the lipid nanoparticles (e.g., 0%-10%, 10% -20%, 20%-30%, 30%-40% or 40%-50%). In some embodiments, such components are 20%-50% (mol), 30%-40% (mol) of the total lipid content of the lipid nanoparticles.

在一些實施方式中，脂質奈米顆粒可包括聚乙二醇（PEG）或軛合的脂質分子。通常，該等用於抑制脂質奈米顆粒的聚集和/或提供空間穩定。示例性的軛合脂質包括但不限於PEG-脂質軛合物、聚㗁唑啉（POZ）-脂質軛合物、聚醯胺-脂質軛合物（諸如ATTA-脂質軛合物）、陽離子聚合物脂質（CPL）軛合物及其混合物。在一些實施方式中，軛合脂質分子係PEG-脂質軛合物，例如(甲氧基聚乙二醇)軛合脂質。In some embodiments, lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. Typically, these serve to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyethazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymers Phytolipid (CPL) conjugates and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as (methoxypolyethylene glycol) conjugated lipid.

示例性的PEG-脂質軛合物包括但不限於PEG-二醯基甘油（DAG）（諸如l-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯甘油（PEG-DMG））、PEG-二烷氧基丙基（DAA）、PEG-磷脂、PEG-神經醯胺（Cer）、聚乙二醇化磷脂醯乙醇胺（PEG-PE）、PEG琥珀酸二醯基甘油（PEGS-DAG）（諸如4-0-(2',3'-二(十四烷醯氧基)丙基-1-0-(w-甲氧基(聚乙氧基)乙基)丁二酸酯（PEG-S-DMG））、PEG二烷氧基丙基胺基甲酸酯、N-(羰基-甲氧基聚乙二醇2000)-l,2-二硬脂醯-sn-甘油-3-磷酸乙醇胺鈉鹽或其混合物。另外的示例性PEG-脂質軛合物描述於以下中：例如，US 5,885,613、US 6,287,591、US 2003/0077829、US 2003/0077829、US 2005/0175682、US 2008/0020058、US 2011/0117125、US 2010/0130588、US 2016/0376224、US 2017/0119904、US 2018/0028664和WO 2017/099823，所有該等的內容藉由援引以其全文併入本文。在一些實施方式中，PEG-脂質係US 2018/0028664的式III、III-a-I、III-a-2、III-b-1、III-b-2或V的化合物，其內容藉由援引以其全文併入本文。在一些實施方式中，PEG-脂質具有US 20150376115或US 2016/0376224的式II，兩者的內容藉由援引以其全文併入本文。在一些實施方式中，PEG-DAA軛合物可為例如PEG-二月桂基氧基丙基、PEG-二肉豆蔻基氧基丙基、PEG-二棕櫚基氧基丙基或PEG-二硬脂基氧基丙基。PEG-脂質可為以下的一或多種：PEG-DMG、PEG-二月桂基甘油、PEG-二棕櫚醯甘油、PEG-二硬脂基甘油、PEG-二月桂基甘油脂醯胺、PEG-二肉豆蔻基甘油脂醯胺、PEG-二棕櫚醯甘油脂醯胺、PEG-二硬脂基甘油脂醯胺、PEG-膽固醇（1-[8'-(膽甾-5-烯-3[β]-氧基)甲醯胺基-3',6'-二氧雜辛基]胺基甲醯基-[ω]-甲基-聚(乙二醇)）、PEG-DMB（3,4-雙十四烷氧基苄基-[ω]-甲基-聚(乙二醇)醚）和1,2-二肉豆蔻醯基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000]。在一些實施方式中，PEG-脂質包括PEG-DMG、1,2-二肉豆蔻醯基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000]。在一些實施方式中，PEG-脂質包括選自以下的結構：Exemplary PEG-lipid conjugates include, but are not limited to, PEG-digylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG- DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), pegylated phospholipid ethanolamine (PEG-PE), PEG diacylglycerol succinate ( PEGS-DAG) (such as 4-0-(2',3'-bis(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedi acid ester (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearyl-sn- Glyceryl-3-phosphoethanolamine sodium salt or mixtures thereof. Additional exemplary PEG-lipid conjugates are described in: e.g., US 5,885,613, US 6,287,591, US 2003/0077829, US 2003/0077829, US 2005/0175682, US 2008/0020058, US 2011/0117125, US 2010/0130588, US 2016/0376224, US 2017/0119904, US 2018/0028664 and WO 2017/099823, all of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2 or V of US 2018/0028664, the contents of which are incorporated by reference. The entire contents of which are incorporated herein. In some embodiments, the PEG-lipid has Formula II of US 20150376115 or US 2016/0376224, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA The conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl or PEG-distearyloxypropyl. PEG- The lipid can be one or more of the following: PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitylglycerol, PEG-distearylglycerol, PEG-dilaurylglycerol, PEG-dimyristylglycerol Glycerolipidamide, PEG-dipalmityl glycerolamide, PEG-distearylglycerolamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]- Oxy)formamide-3',6'-dioxaoctyl]aminoformyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-bis Tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether) and 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N-[methoxy( polyethylene glycol)-2000]. In some embodiments, PEG-lipids include PEG-DMG, 1,2-dimyristyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes a structure selected from:

在一些實施方式中，與PEG以外的分子軛合的脂質也可用於代替PEG-脂質。例如，聚㗁唑啉（POZ）-脂質軛合物、聚醯胺-脂質軛合物（諸如ATTA-脂質軛合物）和陽離子聚合物脂質（GPL）軛合物可用於代替PEG-脂質或與PEG-脂質一起使用。In some embodiments, lipids conjugated to molecules other than PEG may also be used in place of PEG-lipids. For example, polyethazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates can be used in place of PEG-lipid or For use with PEG-lipids.

示例性的軛合脂質，即PEG-脂質、（POZ）-脂質軛合物、ATTA-脂質軛合物和陽離子聚合物-脂質在WO 2019051289 A9的表2中列出的PCT和LIS專利申請中有描述，其全部的內容藉由援引以其全文併入本文。Exemplary conjugated lipids, namely PEG-lipid, (POZ)-lipid conjugate, ATTA-lipid conjugate and cationic polymer-lipid are in the PCT and LIS patent applications listed in Table 2 of WO 2019051289 A9 are described, the entire contents of which are incorporated herein by reference in their entirety.

在一些實施方式中，PEG或軛合脂質可包括脂質奈米顆粒中存在的總脂質的0%-20%（莫耳）。在一些實施方式中，PEG或軛合脂質的含量為脂質奈米顆粒中存在的總脂質的0.5%-10%或2%-5%（莫耳）。可電離脂質、非陽離子脂質、固醇和PEG軛合脂質的莫耳比可以根據需要變化。例如，脂質顆粒可包括按組成物的莫耳或總重量計30%-70%的可電離脂質，按組成物的莫耳或總重量計0%-60%的膽固醇，按組成物的莫耳或總重量計0%-30%的非陽離子脂質和按組成物的莫耳或總重量計1%-10%的軛合脂質。較佳的是，該組成物包括按組成物的莫耳或總重量計30%-40%的可電離脂質，按組成物的莫耳或總重量計40%-50%的膽固醇，以及按組成物的莫耳或總重量計10%-20%的非陽離子脂質。在一些其他實施方式中，組成物係按組成物的莫耳或總重量計50%-75%的可電離脂質，按組成物的莫耳或總重量計20%-40%的膽固醇，和按組成物的莫耳或總重量計5%-10%的非陽離子脂質，以及按組成物的莫耳或總重量計1%-10%的軛合脂質。該組成物可含有按組成物的莫耳或總重量計60%-70%的可電離脂質，按組成物的莫耳或總重量計25%-35%的膽固醇，以及按組成物的莫耳或總重量計5%-10%的非陽離子脂質。該組成物還可含有按組成物的莫耳或總重量計至多90%的可電離脂質和按組成物的莫耳或總重量計2%至15%的非陽離子脂質。該配製物也可為脂質奈米顆粒配製物，例如包括按組成物的莫耳或總重量計8%-30%的可電離脂質，按組成物的莫耳或總重量計5%-30%的非陽離子脂質，以及按組成物的莫耳或總重量計0%-20%的膽固醇；按組成物的莫耳或總重量計4%-25%的可電離脂質，按組成物的莫耳或總重量計4%-25%的非陽離子脂質，按組成物的莫耳或總重量計2%至25%的膽固醇，按組成物的莫耳或總重量計10%至35%的軛合脂質，以及按組成物的莫耳或總重量計5%的膽固醇；或按組成物的莫耳或總重量計2%-30%的可電離脂質，按組成物的莫耳或總重量計2%-30%的非陽離子脂質，按組成物的莫耳或總重量計1%至15%的膽固醇，按組成物的莫耳或總重量計2%至35%的軛合脂質，以及按組成物的莫耳或總重量計1%-20%的膽固醇；或按組成物的莫耳或總重量計甚至高達90%的可電離脂質和按組成物的莫耳或總重量計2%-10%的非陽離子脂質，或按組成物的莫耳或總重量計甚至100%的陽離子脂質。在一些實施方式中，脂質顆粒配製物包括莫耳比為50 : 10 : 38.5 : 1.5的可電離脂質、磷脂、膽固醇和聚乙二醇化脂質。在一些其他實施方式中，脂質顆粒配製物包括莫耳比為60 : 38.5 :  1.5的可電離脂質、膽固醇和聚乙二醇化脂質。In some embodiments, PEG or conjugated lipids may comprise 0%-20% molar of the total lipids present in the lipid nanoparticles. In some embodiments, the amount of PEG or conjugated lipid is 0.5%-10% or 2%-5% molar of the total lipids present in the lipid nanoparticles. The molar ratios of ionizable lipids, non-cationic lipids, sterols and PEG-conjugated lipids can be varied as desired. For example, the lipid particles may include 30% to 70% ionizable lipid, based on moles or total weight of the composition, 0% to 60% cholesterol, based on moles or total weight of the composition. or 0% to 30% by total weight of noncationic lipids and 1% to 10% by mole or total weight of the composition of conjugated lipids. Preferably, the composition includes 30% to 40% by mole or total weight of the composition of ionizable lipids, 40% to 50% by mole or total weight of the composition of cholesterol, and a composition of 10%-20% non-cationic lipids based on the mole or total weight of the substance. In some other embodiments, the composition is 50%-75% ionizable lipids, based on moles or total weight of the composition, 20%-40% cholesterol, based on moles or total weight of the composition, and 5% to 10% by mole or total weight of the composition of noncationic lipids, and 1% to 10% by mole or total weight of the composition of conjugated lipids. The composition may contain from 60% to 70% by mole or total weight of the composition of ionizable lipids, from 25% to 35% by mole or total weight of the composition of cholesterol, and by mole or total weight of the composition. Or 5%-10% non-cationic lipids by total weight. The composition may also contain up to 90% by mole or total weight of the composition of ionizable lipids and from 2% to 15% by mole or total weight of the composition of non-cationic lipids. The formulation may also be a lipid nanoparticle formulation, for example, including 8%-30% by mole or total weight of the composition of ionizable lipid, 5%-30% by mole or total weight of the composition. of non-cationic lipids, and 0% to 20% cholesterol, based on moles of the composition or total weight; 4% to 25% ionizable lipids, based on moles of the composition or total weight, based on the moles of the composition or 4% to 25% by total weight of non-cationic lipids, 2% to 25% by moles or total weight of the composition cholesterol, 10% to 35% conjugated by moles or total weight of the composition Lipids, and 5% cholesterol, based on the moles or total weight of the composition; or 2% to 30% ionizable lipids, based on the moles or total weight of the composition2 % to 30% noncationic lipids, 1% to 15% cholesterol by mole or total weight of the composition, 2% to 35% conjugated lipids by mole or total weight of the composition, and 1%-20% cholesterol by moles or total weight of the composition; or even up to 90% ionizable lipids and 2%-10% by moles or total weight of the composition % non-cationic lipids, or even 100% cationic lipids on a molar or total weight basis of the composition. In some embodiments, the lipid particle formulation includes ionizable lipid, phospholipid, cholesterol, and pegylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation includes ionizable lipid, cholesterol, and pegylated lipid in a molar ratio of 60:38.5:1.5.

在一些實施方式中，脂質顆粒包含可電離脂質、非陽離子脂質（例如，磷脂）、固醇（例如，膽固醇）和PEG化脂質，其中對於可電離脂質，脂質的莫耳比在20至70莫耳百分比之範圍內，目標為40-60，非陽離子脂質的莫耳百分比在0至30之範圍內，目標為0至15，固醇的莫耳百分比在20至70之範圍內，目標為30至50，並且PEG化脂質的莫耳百分比在1至6之範圍內，目標為2至5。In some embodiments, the lipid particles comprise ionizable lipids, noncationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids, wherein for the ionizable lipids, the molar ratio of the lipids is between 20 and 70 molar The molar percentages of noncationic lipids range from 0 to 30, with a target of 0 to 15, and the molar percentages of sterols range from 20 to 70, with a target of 30. to 50, and the molar percentage of PEGylated lipids ranged from 1 to 6, with a target of 2 to 5.

在一些實施方式中，脂質顆粒包括莫耳比為50 : 10 : 38.5 : 1.5的可電離脂質/非陽離子脂質/固醇/軛合脂質。In some embodiments, the lipid particles include ionizable lipid/noncationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

在一方面，本揭露提供了包括磷脂、卵磷脂、磷脂醯膽鹼和磷脂醯乙醇胺的脂質奈米顆粒配製物。In one aspect, the present disclosure provides lipid nanoparticle formulations including phospholipids, lecithin, phosphatidylcholine, and phosphatidylcholine.

在一些實施方式中，還可以包括一或多種另外的化合物。那些化合物可以單獨投與，或者另外的化合物可以包括在本發明之脂質奈米顆粒中。換言之，除核酸或至少第二核酸之外，脂質奈米顆粒可含有不同於第一核酸的其他化合物。非限制性地，其他另外的化合物可以選自由以下組成之群組：小的或大的有機分子或無機分子、單糖、二糖、三糖、寡糖、多糖、肽、蛋白質、其肽類似物和衍生物、肽模擬物、核酸、核酸類似物和衍生物、由生物材料製成的提取物，或其任何組合。In some embodiments, one or more additional compounds may also be included. Those compounds can be administered alone, or additional compounds can be included in the lipid nanoparticles of the invention. In other words, in addition to the nucleic acid or at least the second nucleic acid, the lipid nanoparticle may contain other compounds than the first nucleic acid. Without limitation, other additional compounds may be selected from the group consisting of: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs thereof and derivatives, peptide mimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

在一些實施方式中，LNP包括可生物降解的、可電離的脂質。在一些實施方式中，LNP包括(9Z,l2Z)-3-((4,4-雙(辛氧基)丁醯基)氧基)-2-((((3-(二乙基胺基)丙氧基)羰基)氧基)甲基)丙基 十八碳-9,l2-二烯酸酯，也稱為3-((4,4-雙(辛氧基)丁醯基)氧基)-2-((((3-(二乙基胺基)丙氧基)羰基)氧基)甲基)丙基 (9Z,l2Z)-十八碳-9,l2-二烯酸酯)或另一種可電離脂質。參見，例如WO 2019/067992、WO/2017/173054、WO 2015/095340、和WO 2014/136086，以及其中提供的參考文獻的脂質。在一些實施方式中，在LNP脂質的上下文中術語陽離子和可電離係可互換的，例如，其中可電離脂質根據pH係陽離子的。In some embodiments, LNPs include biodegradable, ionizable lipids. In some embodiments, the LNP includes (9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((3-(diethylamino)propanyl) Oxy)carbonyl)oxy)methyl)propyloctadeca-9,l2-dienoate, also known as 3-((4,4-bis(octyloxy)butyl)oxy)-2 -((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,l2Z)-octadeca-9,l2-dienoate) or another Ionizable lipids. See, for example, lipids in WO 2019/067992, WO/2017/173054, WO 2015/095340, and WO 2014/136086, and the references provided therein. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, for example, where the ionizable lipid is cationic according to the pH system.

在一些實施方式中，LNP配製物的平均LNP直徑可以在數十nm和數百nm之間，例如藉由動態光散射（DLS）測量的。在一些實施方式中，LNP配製物的平均LNP直徑可以為約40 nm至約150 nm，諸如約40 nm、45 nm、50 nm、55 nm、60 nm、65 nm、70 nm、75 nm、80 nm、85 nm、90 nm、95 nm、100 nm、105 nm、110 nm、115 nm、120 nm、125 nm、130 nm、135 nm、140 nm、145 nm或150 nm。在一些實施方式中，LNP配製物的平均LNP直徑可為約50 nm至約100 nm、約50 nm至約90 nm、約50 nm至約80 nm、約50 nm至約70 nm、約50 nm至約60 nm、約60 nm至約100 nm、約60 nm至約90 nm、約60 nm至約80 nm、約60 nm至約70 nm、約70 nm至約100 nm、約70 nm至約90 nm、約70 nm至約80 nm、約80 nm至約100 nm、約80 nm至約90 nm或約90 nm至約100 nm。在一些實施方式中，LNP配製物的平均LNP直徑可為約70 nm至約100 nm。在一個特定實施方式中，LNP配製物的平均LNP直徑可為約80 nm。在一些實施方式中，LNP配製物的平均LNP直徑可為約100 nm。在一些實施方式中，LNP配製物的平均LNP直徑範圍為約l mm至約500 mm、約5 mm至約200 mm、約10 mm至約100 mm、約20 mm至約80 mm、約25 mm至約60 mm、約30 mm至約55 mm、約35 mm至約50 mm，或約38 mm至約42 mm。In some embodiments, the average LNP diameter of the LNP formulation can be between tens and hundreds of nm, such as measured by dynamic light scattering (DLS). In some embodiments, the LNP formulation can have an average LNP diameter of about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the LNP formulation can have an average LNP diameter of about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the LNP formulation can have an average LNP diameter from about 70 nm to about 100 nm. In a specific embodiment, the LNP formulation may have an average LNP diameter of about 80 nm. In some embodiments, the LNP formulation can have an average LNP diameter of about 100 nm. In some embodiments, the LNP formulation has an average LNP diameter in the range of about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

在一些情況下，LNP可為相對均質的。多分散性指數可用於指示LNP的均質性，例如脂質奈米顆粒的粒度分佈。小的（例如，小於0.3）多分散性指數通常指示窄的粒度分佈。LNP的多分散指數可為約0至約0.25，諸如0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25。在一些實施方式中，LNP的多分散指數可為約0.10至約0.20。In some cases, LNPs can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of LNPs, such as the particle size distribution of lipid nanoparticles. A small (eg, less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of LNP can be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the LNP can have a polydispersity index of about 0.10 to about 0.20.

LNP的ζ電位可用於指示組成物的電動電位。在一些實施方式中，ζ電位可以描述LNP的表面電荷。具有相對低電荷（正電荷或負電荷）的脂質奈米顆粒通常是期望的，因為更高電荷的物質可能不理想地與體內的細胞、組織和其他元素相互作用。在一些實施方式中，LNP的ζ電位可為約-10 mV至約+20 mV、約-10 mV至約+15 mV、約-10 mV至約+10 mV、約-10 mV至約+5 mV、約-10 mV至約0 mV、約-10 mV至約-5 mV、約-5 mV至約+20 mV、約-5 mV至約+15 mV、約-5 mV至約+10 mV、約-5 mV至約+5 mV、約-5 mV至約0 mV、約0 mV至約+20 mV、約0 mV至約+15 mV、約0 mV至約+10 mV、約0 mV至約+5 mV、約+5 mV至約+20 mV、約+5 mV至約+15 mV或約+5 mV至約+10 mV。The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of the LNP. Lipid nanoparticles with a relatively low charge (either positive or negative) are generally desirable because higher-charged substances may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, approximately -10 mV to approximately 0 mV, approximately -10 mV to approximately -5 mV, approximately -5 mV to approximately +20 mV, approximately -5 mV to approximately +15 mV, approximately -5 mV to approximately +10 mV , about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

蛋白質和/或核酸的封裝效率描述了相對於所提供的初始量，在製備後被封裝或以其他方式與LNP締合的蛋白質和/或核酸的量。封裝效率理想的是較高（例如，接近100%）。封裝效率可以例如藉由比較在用一或多種有機溶劑或洗滌劑破碎脂質奈米顆粒之前和之後含有脂質奈米顆粒的溶液中蛋白質或核酸的量來測量。陰離子交換樹脂可用於測量溶液中游離蛋白質或核酸（例如RNA）的量。螢光可用於測量溶液中游離蛋白質和/或核酸（例如RNA）的量。對於本文所述之脂質奈米顆粒，蛋白質和/或核酸的封裝效率可為至少50%，例如50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%。在一些實施方式中，封裝效率可為至少80%。在一些實施方式中，封裝效率可為至少90%。在一些實施方式中，封裝效率可為至少95%。Encapsulation efficiency of protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. Packaging efficiency is ideally high (eg, close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disrupting the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (such as RNA) in a solution. Fluorescence can be used to measure the amount of free protein and/or nucleic acid (such as RNA) in a solution. For lipid nanoparticles described herein, the encapsulation efficiency of proteins and/or nucleic acids can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, packaging efficiency can be at least 80%. In some embodiments, packaging efficiency can be at least 90%. In some embodiments, packaging efficiency can be at least 95%.

LNP可以視需要包括一層或多層包衣。在一些實施方式中，LNP可以配製在具有包衣的膠囊、膜或片劑中。包含本文所述之組成物的膠囊、膜或片劑可具有任何可用的尺寸、拉伸強度、硬度或密度。The LNP may optionally include one or more coatings. In some embodiments, LNPs may be formulated in capsules, films, or tablets with coatings. Capsules, films or tablets containing the compositions described herein can be of any available size, tensile strength, hardness or density.

WO 2020/061457和WO 2021/113777（它們各自藉由援引以其全文併入本文）教導了LNP的另外的示例性脂質、配製物、方法和表徵。其他示例性的脂質、配製物、方法和LNP表徵由Hou等人Lipid nanoparticles for mRNA delivery [用於mRNA遞送的脂質奈米顆粒]. Nat Rev Mater [自然評論材料] (2021). doi.org/10.1038/s41578-021-00358-0傳授，其藉由援引以其全文併入本文（參見例如Hou等人的圖2的示例性脂質和脂質衍生物）。WO 2020/061457 and WO 2021/113777 (each of which is incorporated herein by reference in its entirety) teaches additional exemplary lipids, formulations, methods, and characterization of LNPs. Additional exemplary lipids, formulations, methods, and LNP characterizations are provided by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater [Nature Reviews Materials] (2021). doi.org/ 10.1038/s41578-021-00358-0, which is incorporated herein by reference in its entirety (see, eg, Hou et al., Figure 2 for exemplary lipids and lipid derivatives).

在一些實施方式中，使用Lipofectamine MessengerMax（賽默飛世爾公司（Thermo Fisher））或TransIT-mRNA轉染試劑（米盧斯生物公司（Mirus Bio））進行體外或離體細胞脂質轉染。在某些實施方式中，使用GenVoy_ILM可電離脂質混合物（精密奈米系統（Precision NanoSystems））配製LNP。在某些實施方式中，使用2,2‐二亞油烯基‐4‐二甲基胺基乙基‐[1,3]‐二氧戊環（DLin‐KC2‐DMA）或二亞油烯基甲基‐4‐二甲基胺基丁酸酯（DLin-MC3-DMA或MC3）配製LNP，其配製和體內用途在Jayaraman等人Angew Chem Int Ed Engl [德國應用化學] 51(34):8529-8533 (2012)中傳授，藉由援引以其全文併入本文。In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinolene is used The formulation and in vivo use of LNPs formulated with methyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3) is described in Jayaraman et al. Angew Chem Int Ed Engl [Applied Chemistry] 51(34): 8529-8533 (2012), which is incorporated by reference in its entirety.

優化用於遞送CRISPR-Cas系統（例如Cas9-gRNA RNP、gRNA、Cas9 mRNA）的LNP配製物在兩者均藉由援引併入的WO 2019067992和WO 2019067910中有描述，並且可用於遞送本文所述之環狀多核糖核苷酸和線性多核糖核苷酸。LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both incorporated by reference, and can be used to deliver as described herein cyclic polyribonucleotides and linear polyribonucleotides.

可用於遞送核酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）的另外的特定LNP配製物在兩者均藉由援引併入的US 8158601和US 8168775中有描述，其包括帕替西蘭（patisiran）中使用的以名稱ONPATTRO銷售的配製物。Additional specific LNP formulations useful for delivering nucleic acids (eg, cyclic polyribonucleotides, linear polyribonucleotides) are described in US 8158601 and US 8168775, both incorporated by reference, including The formulation sold under the name ONPATTRO is used in patisiran.

在實施方式中，編碼本文所述之蛋白質或多肽的至少部分（例如，抗原的部分）的多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）被配製在LNP中，其中：(a) LNP包括陽離子脂質、中性脂質、膽固醇和PEG脂質，(b) LNP的平均粒度為80 nm和160 nm之間，以及 (c) 多核糖核苷酸。在實施方式中，在LNP中配製的多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）係疫苗。In embodiments, a polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) encoding at least a portion (e.g., a portion of an antigen) of a protein or polypeptide described herein is formulated in In LNP, where: (a) LNP includes cationic lipids, neutral lipids, cholesterol and PEG lipids, (b) the average particle size of LNP is between 80 nm and 160 nm, and (c) polyribonucleotides. In embodiments, the polyribonucleotides (eg, cyclic polyribonucleotides, linear polyribonucleotides) formulated in LNP are vaccines.

多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）LNP的示例性給藥可包括約0.1、0.25、0.3、0.5、1、2、3、4、5、6、8、10或100 mg/kg（RNA）。在一些實施方式中，本文所述之多核糖核苷酸（例如，環狀多核糖核苷酸、線性多核糖核苷酸）抗原的組成物的劑量為30-200 mcg之間，例如30 mcg、50 mcg、75 mcg、100 mcg、150 mcg或200 mcg。套組Exemplary administration of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10 or 100 mg/kg (RNA). In some embodiments, the dosage of the composition of the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) antigen described herein is between 30-200 mcg, such as 30 mcg , 50 mcg, 75 mcg, 100 mcg, 150 mcg or 200 mcg.set

在一些方面，本揭露提供了套組。在一些實施方式中，套組包括 (a) 編碼抗融合多肽（例如，表1的多肽）的環狀多核糖核苷酸或本文所述之藥物組成物，以及視需要 (b) 資訊材料。資訊材料可為描述性的、指導性的、行銷性的或是與本文所述方法和/或用於本文所述方法的藥物組成物或環狀多核糖核苷酸的用途相關的其他材料。藥物組成物或環狀多核糖核苷酸可包含用於單次投與（例如，單劑量形式）的材料，或可包含用於多次投與（例如，「多劑量」套組）的材料。In some respects, this disclosure provides a package. In some embodiments, the kit includes (a) a cyclic polyribonucleotide encoding an anti-fusion polypeptide (e.g., a polypeptide of Table 1) or a pharmaceutical composition described herein, and optionally (b) informational materials. Informational material may be descriptive, instructional, marketing, or other material related to the methods described herein and/or the use of pharmaceutical compositions or cyclic polyribonucleotides for the methods described herein. A pharmaceutical composition or cyclic polyribonucleotide may contain materials for a single administration (e.g., a single dose form) or may contain materials for multiple administrations (e.g., a "multi-dose" set) .

套組之資訊材料的形式不受限制。在一個實施方式中，資訊材料可包括關於藥物組成物、藥物原料藥或藥物成品藥的生產之資訊，藥物組成物、藥物原料藥或藥物成品藥的分子量，濃度，有效期，批次或生產地點資訊等。在一個實施方式中，資訊材料涉及用於投與藥物組成物劑型之方法。在一個實施方式中，資訊材料涉及用於投與環狀多核糖核苷酸劑型之方法。There are no restrictions on the format of the information materials in the package. In one embodiment, the information material may include information about the production of the pharmaceutical composition, drug substance or finished drug, the molecular weight, concentration, expiration date, batch or production location of the pharmaceutical composition, drug substance or finished drug. Information, etc. In one embodiment, the informational material relates to methods for administering pharmaceutical composition dosage forms. In one embodiment, the informational material relates to methods for administering cyclic polyribonucleotide dosage forms.

除了本文所述之藥物組成物和環狀多核糖核苷酸的劑型之外，該套組還可包括其他成分，如溶劑或緩衝液、穩定劑、防腐劑、調味劑（例如，苦味拮抗劑或增甜劑）、香料、染料或著色劑（例如，用於將套組中的一或多種組分著色或染色）、或其他化妝品成分、和/或用於治療本文所述病症或障礙的第二藥劑。可替代地，套組中可包括其他成分，但在與本文所述藥物組成物或環狀多核糖核苷酸不同的組成物或容器中。在這樣的實施方式中，套組可包括將本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）和其他成分混合，或者將本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）與其他成分一起使用的說明書。In addition to the pharmaceutical compositions and dosage forms of the cyclic polyribonucleotides described herein, the kit may also include other ingredients such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), fragrances, dyes or colorants (e.g., used to color or color one or more components of the kit), or other cosmetic ingredients, and/or used to treat the conditions or disorders described herein Second potion. Alternatively, other ingredients may be included in the kit, but in different compositions or containers than the pharmaceutical compositions or cyclic polyribonucleotides described herein. In such embodiments, the kit may include a pharmaceutical composition or nucleic acid molecule described herein (e.g., a cyclic polyribonucleotide) mixed with other ingredients, or a pharmaceutical composition or nucleic acid molecule described herein. (e.g., cyclic polyribonucleotides) Instructions for use with other ingredients.

在一些實施方式中，將套組的組分儲存在惰性條件下（例如，在氮氣或另一種惰性氣體諸如氬氣下）。在一些實施方式中，將套組的組分儲存在無水條件下（例如，用乾燥劑）。在一些實施方式中，將組分儲存在遮光容器，如琥珀色小瓶中。In some embodiments, the components of the kit are stored under inert conditions (eg, under nitrogen or another inert gas such as argon). In some embodiments, the components of the kit are stored under anhydrous conditions (eg, with a desiccant). In some embodiments, the components are stored in light-shielded containers, such as amber vials.

本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）的劑型可以以任何形式，例如液體、乾燥或凍乾形式提供。較佳的是本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）係基本上純的和/或無菌的。當在液體溶液中提供本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）時，液體溶液較佳的是為水溶液，其中較佳的是無菌水溶液。當本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）以乾燥形式提供時，通常藉由添加合適的溶劑來重構。套組中可以視需要提供溶劑，例如無菌水或緩衝液。Dosage forms of pharmaceutical compositions or nucleic acid molecules (eg, cyclic polyribonucleotides) described herein may be provided in any form, such as liquid, dried, or lyophilized form. Preferably, the pharmaceutical compositions or nucleic acid molecules (eg, cyclic polyribonucleotides) described herein are substantially pure and/or sterile. When a pharmaceutical composition or nucleic acid molecule (eg, a cyclic polyribonucleotide) described herein is provided in a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. When the pharmaceutical compositions or nucleic acid molecules (eg, cyclic polyribonucleotides) described herein are provided in dry form, they are typically reconstituted by the addition of a suitable solvent. Optionally, solvents such as sterile water or buffers are provided in the kit.

該套組可包括用於含有本文所述劑型的組成物的一或多個容器。在一些實施方式中，套組含有用於組成物和資訊材料的單獨容器、分隔物或隔室。例如，藥物組成物或環狀多核糖核苷酸可以裝在瓶子、小瓶或注射器中，並且資訊材料可以裝在塑膠套管（plastic sleeve）或包中。在其他實施方式中，套組的單獨元件包含在單個未分開的容器中。例如，本文所述之藥物組成物或核酸分子（例如，環狀多核糖核苷酸）的劑型包含在瓶、小瓶或注射器中，其上附有標籤形式之資訊材料。在一些實施方式中，套組包括多個（例如，一包）單獨的容器，每個容器含有一或多個單位劑型的本文所述之藥物組成物或環狀多核糖核苷酸。例如，該套組包括多個注射器、安瓿、箔包或泡罩包裝，每個含有單個單位劑量的本文所述之劑型。The kit may include one or more containers for compositions containing dosage forms described herein. In some embodiments, a kit contains separate containers, dividers, or compartments for compositions and informational materials. For example, the pharmaceutical composition or cyclic polyribonucleotide may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or bag. In other embodiments, the individual elements of the kit are contained in a single, undivided container. For example, a dosage form of a pharmaceutical composition or nucleic acid molecule (eg, a cyclic polyribonucleotide) described herein is contained in a bottle, vial, or syringe with information material in the form of a label attached thereto. In some embodiments, a kit includes a plurality (eg, a pack) of individual containers, each container containing one or more unit dosage forms of a pharmaceutical composition or cyclic polyribonucleotide described herein. For example, the kit includes a plurality of syringes, ampoules, foil packs, or blister packs, each containing a single unit dose of a dosage form described herein.

套組的容器可為氣密的、防水的（例如，不受水分或蒸發變化的影響）和/或不透光的。The container of the set may be airtight, waterproof (eg, not affected by changes in moisture or evaporation), and/or light-tight.

該套組視需要包括適用於劑型的使用的裝置，例如注射器、移液管、鑷子、量勺、拭子（例如，棉簽或木簽）或任何這樣的裝置。The kit optionally includes devices suitable for use with the dosage form, such as syringes, pipettes, tweezers, measuring spoons, swabs (eg, cotton swabs or wooden swabs) or any such device.

本發明之套組可包括不同強度的劑型，以向受試者提供適用於本文所述之起始期方案、誘導期方案或維持期方案中的一或多個的劑量。可替代地，套組可包括刻痕片劑，以允許使用者根據需要投與分劑量。實例Kits of the invention may include dosage forms of varying strengths to provide a subject with a dose suitable for use in one or more of the initiation, induction or maintenance regimens described herein. Alternatively, the kit may include scored tablets to allow the user to administer divided doses as desired.Example

提出以下實例是為了向熟悉該項技術者提供可以如何使用、製備和評價本文所述之組成物和方法的描述，並且旨在純粹作為本揭露之示例，而不旨在限制諸位發明人認為是其發明的範圍。實例1：哺乳動物細胞中由RNA表現抗融合多肽The following examples are set forth to provide those skilled in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated, and are intended purely as illustrations of the present disclosure and are not intended to limit what the inventors believe to be the the scope of its invention.Example1 :Expression of anti-fusion polypeptides fromRNAin mammalian cells

該實例展示了編碼一或多個OC43-HR2P和EK1肽的一或多個開讀框（ORF）在Huh-7細胞中的表現。This example demonstrates the expression of one or more open reading frames (ORFs) encoding one or more OC43-HR2P and EK1 peptides in Huh-7 cells.

在該實例中，設計了編碼一個OC43-HR2P肽（SEQ ID NO: 289）、一個EK1肽（SEQ ID NO: 288）、多個OC43-HR2P肽、多個EK1肽以及OC43-HR2P肽、肽類似物和EK1肽的組合的環狀RNA。環狀RNA被設計成包括IRES、分泌訊息、弗林蛋白酶位點、一或多個OC43-HR2P肽、類似物和/或EK1序列和間隔子元件。根據製造商的說明，使用Lipofectamine MessengerMax（英傑公司（Invitrogen） LMRNA001）將環狀RNA轉染到Huh-7細胞中。In this example, peptides encoding an OC43-HR2P peptide (SEQ ID NO: 289), an EK1 peptide (SEQ ID NO: 288), multiple OC43-HR2P peptides, multiple EK1 peptides, and OC43-HR2P peptides were designed. Cyclic RNAs of combinations of analogs and EK1 peptides. Circular RNAs are designed to include an IRES, secretion message, furin site, one or more OC43-HR2P peptides, analogs, and/or EK1 sequence and spacer elements. Circular RNA was transfected into Huh-7 cells using Lipofectamine MessengerMax (Invitrogen LMRNA001) according to the manufacturer's instructions.

在一項研究中，在一個時間過程內體外監測肽表現。實例2：對MERS-CoV S蛋白介導的細胞間融合的抑制In one study, peptide performance was monitored in vitro over a time course.Example2:Inhibition of cell-to-cell fusion mediated byMERS-CoV S protein

為了確定293T靶細胞的情況下對MERS-CoV融合的抑制，使用了MERS-CoV S蛋白介導的細胞間融合測定法。該實例使用用編碼EGFP（293T/EGFP）的質體pAAV-IRES-EGFP或編碼MERS-CoV S蛋白和EGFP（293T/MERS/EGFP）的pAAV-IRES-MERS-EGFP轉染的293T細胞，並在含有10% FBS的DMEM中於37°C培養48小時。將根據實例1製備的表現MERS-CoV受體DPP4的Huh-7細胞（5 × 10⁴個）在96孔板中於37°C孵育5小時，接著分別加入1 × 10⁴個293T/EGFP或293T/MERS/EGFP細胞。To determine the inhibition of MERS-CoV fusion in the context of 293T target cells, a MERS-CoV S protein-mediated cell-to-cell fusion assay was used. This example uses 293T cells transfected with plasmid pAAV-IRES-EGFP encoding EGFP (293T/EGFP) or pAAV-IRES-MERS-EGFP encoding MERS-CoV S protein and EGFP (293T/MERS/EGFP), and Incubate in DMEM containing 10% FBS for 48 hours at 37°C. Huh-7 cells (5 × 10⁴ cells) expressing MERS-CoV receptor DPP4 prepared according to Example 1 were incubated in a 96-well plate at 37°C for 5 hours, and then 1 × 10⁴ cells of 293T/EGFP or 293T/EGFP were added respectively. 293T/MERS/EGFP cells.

在37°C共培養4小時後，在倒置螢光顯微鏡（Nikon Eclipse Ti-S）下計數與Huh-7細胞融合或未與其融合的293T/MERS/EGFP細胞（293T/EGFP細胞用作陰性對照）。將融合細胞視為比未融合細胞大2倍或更多的細胞，並將融合細胞中的螢光強度差異與未融合細胞中的螢光強度差異進行比較。細胞間融合的抑制百分比可以使用以下公式計算：(1−(E−N)/(P−N)) × 100。「E」代表實驗組中細胞間融合之百分比。「P」代表陽性對照組中細胞間融合的百分比，在陽性對照組中沒有添加circRNA。「N」係陰性對照組中細胞間融合的百分比，在陰性對照組中293T/MERS/EGFP細胞被293T/EGFP細胞替代。可以使用CalcuSyn軟體計算50%抑制的濃度（IC₅₀）。共培養可以在37°C下持續48小時，並且可以測量例如合胞體的形成。細胞內S蛋白ELISA可適用於在病毒激發後兩天測量抗病毒活性。實例3：對假型SARS-CoV-2和MERS-CoV感染的抑制After co-culture for 4 hours at 37°C, 293T/MERS/EGFP cells fused with or without Huh-7 cells were counted under an inverted fluorescence microscope (Nikon Eclipse Ti-S) (293T/EGFP cells were used as a negative control ). Treat fused cells as cells that are 2 times or more larger than unfused cells, and compare the difference in fluorescence intensity in fused cells to the difference in fluorescence intensity in unfused cells. The percent inhibition of cell-to-cell fusion can be calculated using the following formula: (1−(E −N )/(P −N )) × 100. "E " represents the percentage of cell-to-cell fusion in the experimental group. “P ” represents the percentage of cell-to-cell fusion in the positive control group, in which circRNA was not added. "N " is the percentage of cell-to-cell fusion in the negative control group, in which 293T/MERS/EGFP cells were replaced by 293T/EGFP cells. CalcuSyn software can be used to calculate the concentration of 50% inhibition (IC₅₀ ). Co-culture can be continued at 37°C for 48 hours, and syncytia formation can be measured, for example. Intracellular S protein ELISA can be adapted to measure antiviral activity two days after viral challenge.Example3: Inhibition of pseudotypedSARS-CoV-2andMERS-CoVinfections

藉由將293T細胞與質體pNL4-3.luc.RE（編碼Env缺陷型的表現螢光素酶的HIV-1）和pcDNA3.1-MERS-CoV-S質體共轉染，製備分別攜帶SARS-CoV-2或MERS-CoV S蛋白的SARS或MERS假病毒以及表現螢光素酶作為報告分子的缺陷型HIV-1基因組。為了檢測表現的肽對SARS或MERS假病毒感染的抑制活性，分別將已經用本發明之circRNA轉染和沒有用其轉染的經ACE2轉染的293T（293T/ACE2）細胞和Huh-7細胞（96孔板中每孔10⁴個）用SARS或MERS-CoV假病毒感染。在感染之後，將培養物在感染後12小時用新鮮培養基進行重新補料，並再孵育72小時。將細胞用PBS洗滌，並使用螢光素酶套組（普洛麥格公司（Promega））中包含的裂解試劑裂解。將細胞裂解物的等分試樣轉移到96孔Costar平底光度計板（康寧柯斯達公司（Corning Costar）），接著加入螢光素酶底物（普洛麥格公司）。在Ultra 384光度計（美國帝肯公司（Tecan US））中立即確定相對光單位。實例4：SARS-CoV-2抗融合多肽的表現By co-transfecting 293T cells with plasmids pNL4-3.luc.RE (encoding Env-deficient HIV-1 expressing luciferase) and pcDNA3.1-MERS-CoV-S plasmids, we prepared plasmids carrying respectively SARS-CoV-2 or MERS-CoV S protein SARS or MERS pseudoviruses and defective HIV-1 genomes expressing luciferase as a reporter molecule. In order to detect the inhibitory activity of the expressed peptides against SARS or MERS pseudovirus infection, ACE2-transfected 293T (293T/ACE2) cells and Huh-7 cells that have been transfected with the circRNA of the present invention and have not been transfected with it were respectively used. (10⁴ per well in a 96-well plate) were infected with SARS or MERS-CoV pseudoviruses. Following infection, cultures were re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h. Cells were washed with PBS and lysed using the lysis reagent included in the luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well Costar flat-bottom luminometer plates (Corning Costar), followed by addition of luciferase substrate (Promega). Relative light units were determined immediately in an Ultra 384 photometer (Tecan US).Example4: Performance ofSARS-CoV-2anti-fusion peptides

該實例展示了來自環狀RNA的SARS-CoV-2抗融合多肽表現。This example demonstrates the performance of SARS-CoV-2 anti-fusion peptides derived from circular RNA.

基於圖1所示的HR2區域設計了幾種SARS-CoV-2抗融合多肽（圖2和3）。將環狀RNA設計成包括IRES和編碼SARS-CoV-2抗融合多肽的核苷酸序列。在本實例中，DNA構建體被設計成包括間隔子元件和多核苷酸貨物。構建體被設計成包括IRES和ORF的組合作為多核苷酸貨物。ORF被設計成包括IL-2分泌訊息序列、編碼SARS-CoV-2抗融合多肽的核苷酸序列和編碼帶有GGGGS肽連接子的HiBiT標籤的核苷酸序列。IRES係EMCV。Several SARS-CoV-2 anti-fusion peptides were designed based on the HR2 region shown inFigure1 (Figures2 and3 ). The circular RNA is designed to include an IRES and a nucleotide sequence encoding a SARS-CoV-2 anti-fusion polypeptide. In this example, the DNA construct is designed to include spacer elements and polynucleotide cargo. Constructs are designed to include a combination of IRES and ORF as polynucleotide cargo. The ORF was designed to include an IL-2 secretion message sequence, a nucleotide sequence encoding a SARS-CoV-2 anti-fusion polypeptide, and a nucleotide sequence encoding a HiBiT tag with a GGGGS peptide linker. IRES is EMCV.

所用的所有構建體的胺基酸和核酸序列如下所示。The amino acid and nucleic acid sequences of all constructs used are shown below.

N-末端IL-2分泌訊息以大寫顯示（20 AA或60個核苷酸）粗體= 弗林蛋白酶粗體和斜體= 帶有G4S肽連接子的HiBiT標籤 HR2全長 ATGTATAGAATGCAGCTGCTGTCTTGTATTGCTCTTTCTCTGGCTCTTGTGACTAATTCTagactgaggagaggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaagaagaactggataaatattttaagaatcatacttctcctgacgttgatctgggtgatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagactgaacgaagttgctaagaatctgaacgaatctcttattgatcttcaggaacttggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc(SEQ ID NO: 378) MYRMQLLSCIALSLALVTNSRLRRGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSVSGWRLFKKIS(SEQ ID NO: 379) HR2A ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTTTCTCTTGCTCTGGTTACTAATTCTagactgaggagagatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagacttaacgaagttgctaaaaatctgaacgaatctctgattgatctgcaggaactgggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc(SEQ ID NO: 380) MYRMQLLSCIALSLALVTNSRLRRDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSVSGWRLFKKIS(SEQ ID NO: 381) HR2C ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTTGTTACTAATTCTagactgaggagatttaaaaatcatacttctcctgacgttgatctgggtgatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagactgaacgaagttgctaaaggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc(SEQ ID NO: 382) MYRMQLLSCIALSLALVTNSRLRRFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKGGGGSVSGWRLFKKIS(SEQ ID NO: 383) HR2B ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTGGTTACTAATTCTaggctgagaagagttgttattggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaggaagaactggataagtattttaaaaatcacacttctcctgatggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc(SEQ ID NO: 384) MYRMQLLSCIALSLALVTNSRLRRVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDGGGGSVSGWRLFKKIS(SEQ ID NO: 385) EK1 ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTGTCTCTGGCTCTTGTTACTAATTCTagactgaggagatctcttgatcagattaacgttacttttctggatctggaatacgaaatgaaaaagctggaagaagctattaaaaagcttgaagaatcttatattgatctgaaagaactgggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc(SEQ ID NO: 386) MYRMQLLSCIALSLALVTNSRLRRSLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKELGGGGSVSGWRLFKKIS(SEQ ID NO: 387)N-terminal IL-2 secretion message shown in upper case (20 AA or 60 nucleotides)bold = furinbold and italics = HiBiT-tagged HR2 with G4S peptide linker Full-length ATGTATAGAATGCAGCTGCTGTCTTGTATTGCTCTTTCTCTGGCTCTTGTGACTAATTCTagactgaggaga ggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaagaagaactggata aatattttaagaatcatacttctcctgacgttgatctgggtgatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagactgaacgaagttgctaagaatctgaacgaatctcttattgatcttcaggaacttggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 378) MYRMQLLSCIALSLALVTNSRLRR GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSVSGWRLFKKIS (SEQ ID NO: 379) HR2A ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTTTCTCTTGCTCTGGTTACTAATTCTagactgaggaga gatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagacttaacgaagttgctaaaaatctgaacgaatctctgattgatctgcaggaactgggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 380) MYRMQLLSCIALSLALVTNSRLRR DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGGGGSVSGWRLFKKIS (SEQ ID NO: 381) HR2C ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTTGTTACTAATTCTagactgaggaga tttaaaaatcatacttctcctgacgttgatctgggtgatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagactgaacgaagttgctaaaggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 382) MYRMQLLSCIALSLALVTNSRLRR FKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKGGGGSVSGWRLFKKIS (SEQ ID NO: 383) HR2B ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTGGTTACTAATTCTaggctgagaaga gttgttattggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaggaagaactggataagtattttaaaaatcacacttctcctgatggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 384) MYRMQLLSCIALSLALVTNSRLRR VVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDGGGGSVSGWRLFKKIS (SEQ ID NO: 385) EK1 ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTGTCTCTGGCTCTTGTTACTAATTCTagactgaggaga tctcttgatcagattaacgttacttttctggatctggaatacgaaatgaaaaagctggaagaagctattaaaaagcttgaagaatcttatattgatctgaaagaactgggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO : 386) MYRMQLLSCIALSLALVTNSRLRR SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKELGGGGSVSGWRLFKKIS (SEQ ID NO: 387)

使用本文所述之方法藉由自剪接來產生環狀RNA。在存在7.5 mM的NTP的情況下，藉由體外轉錄使用T7 RNA聚合酶從包括以上列出的模體的DNA模板合成未經修飾的線性RNA。藉由用DNase處理除去模板DNA。用RNA純化套組（新英格蘭生物學實驗室公司（New England Biolabs），T2050）純化合成的線性RNA。自剪接發生在轉錄期間。藉由尿素聚丙烯醯胺凝膠電泳（尿素-PAGE）或逆相柱層析法純化編碼抗融合肽的環狀RNA。Circular RNA is generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from DNA templates including the motifs listed above in the presence of 7.5 mM NTP. Template DNA is removed by treatment with DNase. Synthetic linear RNA was purified using an RNA purification kit (New England Biolabs, T2050). Self-splicing occurs during transcription. The circRNA encoding the anti-fusion peptide is purified by urea-polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

為了測量SARS-CoV-2抗融合多肽的表現，使用脂轉染胺將0.4 pmol環狀RNA遞送到HEK293細胞。48小時後測量表現。各種多肽在HEK293細胞中表現。圖2中顯示了總表現（ng/ml和nM）。 [表2]：HR2構建體表現  表現（ng/mL）表現（nM）HR2全長69.28.4HR2A51.3HR2C1.40.4HR2B00EK10.10實例5：對假型SARS-CoV-2感染的抑制To measure the performance of SARS-CoV-2 anti-fusion peptides, 0.4 pmol circular RNA was delivered to HEK293 cells using lipofectamine. Performance was measured after 48 hours. Various peptides are expressed in HEK293 cells. Overall performance (ng/ml and nM) is shown in Figure 2. [Table2 ]:HR2construct performancePerformance (ng/mL)Performance (nM) HR2 full length 69.2 8.4 HR2A 5 1.3 HR2C 1.4 0.4 HR2B 0 0 EK1 0.1 0Example5: Inhibition of pseudotypedSARS-CoV-2infection

該實例展示了由環狀RNA表現的抗融合多肽對假型SARS-CoV2-感染的抑制。This example demonstrates the inhibition of pseudotyped SARS-CoV2-infection by anti-fusion polypeptides expressed by circular RNAs.

將環狀RNA設計成包括內部核糖體進入位點（IRES）和編碼SARS-CoV-2的抗融合多肽的核苷酸序列。在本實例中，DNA構建體被設計成包括間隔子元件，以及EMCV IRES和ORF的組合作為多核苷酸貨物。ORF被設計成包括IL-2分泌訊息序列和編碼HR2全長抗融合多肽的核苷酸序列，以及編碼HiBiT肽標籤的核苷酸序列。ORF還被設計成包括IL-2分泌訊息序列和編碼HR2全長抗融合多肽的核苷酸序列，而沒有編碼HiBiT肽標籤的核苷酸序列。The circular RNA was designed to include an internal ribosome entry site (IRES) and a nucleotide sequence encoding an anti-fusion polypeptide of SARS-CoV-2. In this example, the DNA construct was designed to include spacer elements, as well as a combination of EMCV IRES and ORF as polynucleotide cargo. The ORF was designed to include the IL-2 secretion message sequence and the nucleotide sequence encoding the HR2 full-length anti-fusion polypeptide, as well as the nucleotide sequence encoding the HiBiT peptide tag. The ORF was also designed to include the IL-2 secretion message sequence and the nucleotide sequence encoding the HR2 full-length anti-fusion polypeptide, without the nucleotide sequence encoding the HiBiT peptide tag.

使用本文所述之方法藉由自剪接來產生環狀RNA。在存在7.5 mM的NTP的情況下，藉由體外轉錄使用T7 RNA聚合酶從包括以上列出的模體的DNA模板合成未經修飾的線性RNA。藉由用DNase處理除去模板DNA。用RNA純化套組（新英格蘭生物學實驗室公司（New England Biolabs），T2050）純化合成的線性RNA。自剪接發生在轉錄期間。藉由尿素聚丙烯醯胺凝膠電泳（尿素-PAGE）或逆相柱層析法純化編碼HR2全長抗融合多肽的環狀RNA。Circular RNA is generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from DNA templates including the motifs listed above in the presence of 7.5 mM NTP. Template DNA is removed by treatment with DNase. Synthetic linear RNA was purified using an RNA purification kit (New England Biolabs, T2050). Self-splicing occurs during transcription. The circRNA encoding HR2 full-length anti-fusion polypeptide was purified by urea-polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

為了檢測表現的多肽對SARS假病毒感染的抑制活性，分別將已經用環狀RNA轉染和沒有用其轉染的經ACE2轉染的293T（293T/ACE2）細胞（96孔板中每孔10⁴個）用SARS-CoV-2假病毒感染。單獨的轉染試劑（不含環狀RNA）用作對照（「模擬品」）。在感染之後，將培養物在感染後12小時用新鮮培養基進行重新補料，並再孵育72小時。將細胞用PBS洗滌，並使用螢光素酶套組（普洛麥格公司（Promega））中包含的裂解試劑裂解。將細胞裂解物的等分試樣轉移到96孔Costar平底光度計板（康寧柯斯達公司），接著加入螢光素酶底物（普洛麥格公司）。在Ultra 384光度計（美國帝肯公司）中立即確定相對光單位。In order to test the inhibitory activity of the expressed polypeptides against SARS pseudovirus infection, ACE2-transfected 293T (293T/ACE2) cells that had been transfected with circular RNA and those that had not been transfected with it were separately transfected (10 cells per well in a 96-well plate).⁴ ) infected with SARS-CoV-2 pseudovirus. Transfection reagent alone (without circRNA) was used as a control ("mock"). Following infection, cultures were re-fed with fresh medium 12 h post-infection and incubated for an additional 72 h. Cells were washed with PBS and lysed using the lysis reagent included in the luciferase kit (Promega). Aliquots of cell lysates were transferred to 96-well Costar flat-bottom photometer plates (Corning Costar), followed by addition of luciferase substrate (Promega). Relative light units were determined immediately in an Ultra 384 photometer (Tecan, USA).

使用編碼本文所述抗融合多肽的環狀多核糖核苷酸測定使用奧密克戎和德爾塔假病毒時的體外抑制融合功效。顯示在細胞中表現的HR2全長抗融合多肽（圖4A）以及HR2全長抗融合多肽和與HiBiT標籤軛合的HR2全長抗融合多肽抑制德爾塔和奧密克戎毒株的融合（圖4A）。細胞活力沒有變化（藉由cellTiter glo測量）（圖4B），表明螢光素酶傳訊的減少（圖4A）歸因於對病毒融合的抑制。In vitro fusion inhibition efficacy using Omicron and Delta pseudoviruses was determined using cyclic polyribonucleotides encoding anti-fusion polypeptides described herein. HR2 full-length anti-fusion polypeptide expressed in cells (Fig.4A ) and HR2 full-length anti-fusion polypeptide conjugated to a HiBiT tag were shown to inhibit fusion of Delta and Omicron strains (Fig.4A ). There was no change in cell viability (measured by cellTiter glo) (Figure4B ), indicating that the reduction in luciferase signaling (Figure4A ) was attributable to inhibition of viral fusion.

為了測量多肽在體內的表現，通過靜脈內注射將120 pmol配製成脂質奈米顆粒的環狀RNA遞送給小鼠。藉由Nano-Glo® HiBiT胞外檢測系統（#N3030，普洛麥格公司）10%血清來測量表現。如表3和圖5所示，抗融合多肽在6小時高度表現，並在24小時顯著降低。 [表3]：HR2表現  6小時（ng/mL）24小時（ng/mL）HR2全長HiBiT24.80.2無HiBiT0.020.01實例6：對假型SARS-CoV-2感染的抑制To measure the in vivo performance of the peptide, 120 pmol of circRNA formulated into lipid nanoparticles was delivered to mice via intravenous injection. Performance was measured by Nano-Glo® HiBiT Extracellular Detection System (#N3030, Promega Corporation) 10% serum. As shown in Table 3 andFigure5 , the anti-fusion polypeptide was highly expressed at 6 hours and significantly reduced at 24 hours. [Table3 ]:HR2performance6hours (ng/mL)24hours (ng/mL) HR2 full length HiBiT 24.8 0.2 No HiBiT 0.02 0.01Example6: Inhibition of pseudotypedSARS-CoV-2infection

本實例展示了使用抗融合多肽的假型SARS-CoV2-感染。This example demonstrates pseudotyped SARS-CoV2-infection using anti-fusion peptides.

SARS-CoV-2的抗融合多肽係基於SARS-CoV-2刺突多肽的HR2、HR2A、HR2B和HR2C區域以及EK1多肽建立的（圖2）。The anti-fusion peptide of SARS-CoV-2 was established based on the HR2, HR2A, HR2B and HR2C regions of the SARS-CoV-2 spike peptide and the EK1 polypeptide (Figure2 ).

進行功能測定法以測量EK-1和HR2A多肽的體外假病毒中和作用。HR2A多肽顯示出對武漢和奧密克戎毒株的功效（圖6A和6B）。Functional assays were performed to measure in vitro pseudovirus neutralization of EK-1 and HR2A polypeptides. HR2A peptides showed efficacy against Wuhan and Omicron strains (Figures6A and6B ).

使用HR2A-C和HR2全長多肽進行進一步的多肽實驗。對於陰性對照，使用IPB19，一種HIV肽；對於陽性對照，使用ACE-Fc，一種直接結合受體的抗體。使用4倍連續稀釋（8次稀釋）和HEK293 ACE2細胞製備多肽（起始稀釋10 μM）。所有多肽都顯示出抑制奧密克戎和WT毒株，表4中顯示了IC50值。 [表4]：IC50值IC50 (µM)IC50 (nM)IC50 (ng/ml)奧密克戎野生型奧密克戎野生型奧密克戎野生型HR2全長0.026250.174226.3174.2215.31428.4HR2A--37.7614.2158.12579.6EK-1--64.8329.3272.21383.1HR2B--Neg.Neg.Neg.Neg.HR2C--Neg.Neg.Neg.Neg.IPB19--1046.0Neg.4393.2Neg.ACE-Fc--4.38.4    Further peptide experiments were performed using HR2A-C and HR2 full-length peptides. For negative controls, IPB19, an HIV peptide, was used; for positive controls, ACE-Fc, an antibody that directly binds to the receptor, was used. Peptides were prepared using 4-fold serial dilutions (8 dilutions) and HEK293 ACE2 cells (starting dilution 10 μM). All peptides were shown to inhibit Omicron and WT strains, with IC50 values shown in Table 4. [Table4 ]:IC50valueIC50 (µM)IC50 (nM)IC50 (ng/ml) Omicron Wild type Omicron Wild type Omicron Wild type HR2 full length 0.02625 0.1742 26.3 174.2 215.3 1428.4 HR2A - - 37.7 614.2 158.1 2579.6 EK-1 - - 64.8 329.3 272.2 1383.1 HR2B - - Neg. Neg. Neg. Neg. HR2C - - Neg. Neg. Neg. Neg. IPB19 - - 1046.0 Neg. 4393.2 Neg. ACE-Fc - - 4.3 8.4

還測試了全長HR2 Fc融合，並且其顯示維持對奧密克戎和武漢毒株的抑制活性。Full-length HR2 Fc fusions were also tested and shown to maintain inhibitory activity against the Omicron and Wuhan strains.

HR2全長多肽（「HR2完全」）顯示成功抑制奧密克戎BA.4/BA.5、SARS CoV-1和武漢毒株的融合（圖7A、7B和8A-8D）。實例7：HIV抗融合多肽的表現The HR2 full-length peptide ("HR2 complete") was shown to successfully inhibit the fusion of Omicron BA.4/BA.5, SARS CoV-1, and the Wuhan strain (Figures7A,7B,and8A-8D ).Example7: Performance ofHIVanti-fusion polypeptides

該實例展示了來自環狀RNA的HIV抗融合多肽表現。This example demonstrates the expression of HIV anti-fusion peptides from circular RNAs.

設計了幾種HIV抗融合多肽（圖9）。將環狀RNA設計成包括IRES和編碼HIV抗融合多肽的核苷酸序列。在本實例中，DNA構建體被設計成包括間隔子元件和多核苷酸貨物（圖9）。構建體被設計成包括IRES和ORF的組合作為多核苷酸貨物。ORF被設計成包括IL-2分泌訊息序列（SEQ ID NO: 332）、編碼HIV抗融合多肽的核苷酸序列和編碼帶有GGS或GGGGS肽連接子的HiBiT標籤（具有序列VSGWRLFKKIS（SEQ ID NO: 362））的核苷酸序列。IRES係EMCV，抑或是經修飾的CVB3。Several HIV anti-fusion peptides were designed (Figure9 ). The circular RNA is designed to include an IRES and a nucleotide sequence encoding an HIV anti-fusion polypeptide. In this example, the DNA construct was designed to include spacer elements and polynucleotide cargo (Figure9 ). Constructs are designed to include a combination of IRES and ORF as polynucleotide cargo. The ORF was designed to include an IL-2 secretion message sequence (SEQ ID NO: 332), a nucleotide sequence encoding an HIV anti-fusion polypeptide and a HiBiT tag encoding a GGS or GGGGS peptide linker (having the sequence VSGWRLFKKIS (SEQ ID NO : 362)) nucleotide sequence. IRES is EMCV or modified CVB3.

使用本文所述之方法藉由自剪接來產生環狀RNA。在存在7.5 mM的NTP的情況下，藉由體外轉錄使用T7 RNA聚合酶從包括以上列出的模體的DNA模板合成未經修飾的線性RNA。藉由用DNase處理除去模板DNA。用RNA純化套組（新英格蘭生物學實驗室公司（New England Biolabs），T2050）純化合成的線性RNA。自剪接發生在轉錄期間。藉由尿素聚丙烯醯胺凝膠電泳（尿素-PAGE）或逆相柱層析法純化編碼抗融合肽的環狀RNA。Circular RNA is generated by self-splicing using the methods described herein. Unmodified linear RNA was synthesized by in vitro transcription using T7 RNA polymerase from DNA templates including the motifs listed above in the presence of 7.5 mM NTP. Template DNA is removed by treatment with DNase. Synthetic linear RNA was purified using an RNA purification kit (New England Biolabs, T2050). Self-splicing occurs during transcription. Circular RNA encoding the anti-fusion peptide is purified by urea-polyacrylamide gel electrophoresis (urea-PAGE) or reverse-phase column chromatography.

為了測量HIV抗融合多肽的表現，使用脂轉染胺將0.4 pmol環狀RNA遞送到HEK293細胞。48小時後測量表現。如圖10A和10B所示，多肽在HEK293細胞中表現。如圖11A和11B所示，在環狀RNA和DNA質體之間的表現係相當的。圖12中顯示了各種多肽的總表現（ng/mL和nM）。HIV多肽和核酸序列T20YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 313) tatacttctctgatccactctctgatcgaggaatctcagaaccagcaggagaagaacgaacaggaactgctggaactggataagtgggcttctctgtggaactggttc (with EMCV IRES) (SEQ ID NO: 363) OR tacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 364)T1249WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 318) tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgcagaagctggataagtgggcttctctgtgggagtggttc (with EMCV IRES) (SEQ ID NO: 365) OR tggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 366)T1144TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL (SEQ ID NO: 316) actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctgagagctctgcaggaacagcaggaaaagaacgaagctgctctgagagaactg (with EMCV IRES) (SEQ ID NO: 367) OR accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 368)1144-2-0TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 317) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 369)T_2410MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL (SEQ ID NO: 314) atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctg (with modified CVB3 IRES) (SEQ ID NO: 370)T_2410_2.0MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 315) atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 371)1249_2.0TTWQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 319) accacctggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 372)290676TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELREL (SEQ ID NO: 323) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaacgaggccgagctgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 373)290676-2-0TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELRELDKWASLWNWF (SEQ ID NO: 324) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagccaggagcagcaagagaagaacgaggccgagctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 374)2635TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL (SEQ ID NO: 320) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 375)2635_2.0TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 321) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 376)2635_3.0MTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 322) atgacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (SEQ ID NO: 377)其他實施方式To measure the performance of HIV anti-fusion peptides, 0.4 pmol circular RNA was delivered to HEK293 cells using lipofectamine. Performance was measured after 48 hours. As shownin Figures10A and10B , the polypeptide was expressed in HEK293 cells. As shown inFigures11A and11B , the performance between circular RNA and DNA plasmids is comparable. The overall performance (ng/mL and nM) of various peptides is shown inFigure12 .HIVpolypeptide and nucleic acid sequenceT20 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 313) tatacttctctgatccactctctgatcgaggaatctcagaaccagcaggagaagaacgaacaggaactgctggaactggataagtgggcttctgtggaactggttc (with EMCV IRES) (SEQ ID NO: 363) OR tacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 364)T1249 WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 318 ) tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgcagaagctggataagtgggcttctctgtggggagtggttc (with EMCV IRES) (SEQ ID NO: 365) OR tggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaaga acgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 366)T1144 TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL (SEQ ID NO: 316) actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctg agagctctgcaggaacagcaggaaaagaacgaagctgctctgagagaactg (with EMCV IRES) (SEQ ID NO: 367 ) OR accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcggggagctg (with modified CVB3 IRES) (SEQ ID NO: 368)1144-2-0 TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALRELDKWASL WNWF (SEQ ID NO: 317) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagcagcaagagaagaacgaggccgccctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 369)T_2410 MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL (SEQ ID NO: 314) atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctg (with modified CVB3 IRES) (SEQ ID NO: 370)T_2410_2.0 MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWF (SEQ ID NO: 315) atgacctggatggagtgggaccgggagatcaacaattacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 371)1249_2.0 TTW QEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 319) accacctggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgcagaagctggacaagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 372)290676 TTWEAWDRAIAEYAARIEALIRASQEQQEKNEAELR EL (SEQ ID NO: 323) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagcccaggagcagcaagagaagaacgaggccgagctgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 373)290676-2-0 TTWEAWDRAIAEYAARIEALIRASQEQQ EKNEAELRELDKWASLWNWF (SEQ ID NO: 324) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccagcccaggagcagcaagagaagaacgaggccgagctgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 374)2635 TTWEAWDRAIAEYAARI EALIRAAQEQQEKNEAALREL (SEQ ID NO: 320) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 375)2635_2.0 TTWEAWDRAIA EYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 321) accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 376)2635_3.0 MTWEAWDRAIAEYAARIEA LIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 322) atgacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagcagcaagagaagaacgaggccgcactgcggggagctggacaagtgggccagcctgtggaggactttc (SEQ ID NO: 377)Other embodiments

雖然已經結合本發明之特定實施方式描述了本發明，但是應當理解，能夠進行進一步的修改，並且本申請旨在涵蓋總體上遵循本發明之原理並且包括在本發明所屬領域內的已知或常規實踐內的與本發明之此類偏離的本發明之任何變化、使用或改編，並且可應用於在上文中闡述的基本特徵，並且遵循申請專利範圍的範圍。其他實施方式在申請專利範圍中。While this invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications are possible and this application is intended to cover any known or customary art that generally adheres to its principles and includes those within the art to which this invention belongs. Any variations, uses or adaptations of the invention within the scope of such departures from the present invention and applicable to the essential characteristics set forth hereinabove and within the scope of the claimed claims. Other implementations are within the scope of the patent claims.

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[圖1]係顯示了各種冠狀病毒S蛋白的蛋白質結構域或區域以及HR1和HR2區域的序列之示意圖。[Figure1 ] is a schematic diagram showing the protein domains or regions of various coronavirus S proteins and the sequences of the HR1 and HR2 regions.

[圖2]係顯示了衍生自SARS CoV-2的HR2區域的各種抗融合多肽和序列之示意圖。[Figure2 ] shows a schematic diagram of various anti-fusion peptides and sequences derived from the HR2 region of SARS CoV-2.

[圖3]係顯示了多ORF抗融合多肽構建體的示例性實施方式之示意圖。[Fig. 3 ] is a schematic diagram showing an exemplary embodiment of a multi-ORF anti-fusion polypeptide construct.

[圖4A和4B]係顯示了使用奧密克戎和德爾塔假病毒時對融合的抑制效力之圖。圖4A顯示了使用HR2全長或帶有HiBiT標籤的HR2全長對德爾塔和奧密克戎的抑制%。圖4B顯示了抗融合多肽與模擬對照相比的相對表現。[Figures4A and4B ] are graphs showing the inhibitory potency against fusion using Omicron and Delta pseudoviruses.Figure4A shows the % inhibition of Delta and Omicron using HR2 full length or HiBiT-tagged HR2 full length.Figure4B shows the relative performance of anti-fusion polypeptides compared to mock controls.

[圖5]係顯示了在6小時和24小時帶有和不帶有HiBiT標籤的HR2全長抗融合多肽的體內表現之圖。[Fig.5 ] is a graph showing the in vivo performance of HR2 full-length anti-fusion polypeptide with and without HiBiT tag at 6 hours and 24 hours.

[圖6A和6B]係顯示了使用HR2A多肽體外中和SARS CoV-2的武漢和奧密克戎毒株的假病毒之圖。圖6A顯示了使用HR2A中和SARS CoV-2的武漢毒株。圖6B顯示了使用HR2A中和SARS CoV-2的奧密克戎毒株。[Figures6Aand6B ] Figures showing the use of HR2A polypeptides to neutralize pseudoviruses of the Wuhan and Omicron strains of SARS CoV-2 in vitro.Figure6A shows the use of HR2A to neutralize the Wuhan strain of SARS CoV-2.Figure6B shows the use of HR2A to neutralize the Omicron strain of SARS CoV-2.

[圖7A和7B]係顯示了對SARS CoV-2假病毒奧密克戎BA4和BA.5（圖7A）或SARS CoV-1假病毒（圖7B）毒株的抑制率（%）之圖。[Figures7A and7B ] A graph showing the inhibition rate (%) against SARS CoV-2 pseudovirus Omicron BA4 and BA.5 (Figure7A ) or SARS CoV-1 pseudovirus (Figure7B ) strains .

[圖8A-8D]係顯示了使用全長HR2（HR2完全（HR2Complete））對SARS CoV-2假病毒的抑制率（%）之圖。圖8A顯示了對武漢毒株的抑制。圖8B顯示了對奧密克戎BA.4和BA.5毒株的抑制。圖8C顯示了對奧密克戎BA.1毒株的抑制。圖8D顯示了對SARS CoV-1假病毒的抑制。[Figures8A-8D ] are graphs showing the inhibition rate (%) of SARS CoV-2 pseudovirus using full-length HR2 (HR2Complete).Figure8A shows inhibition of the Wuhan strain.Figure8B shows inhibition of Omicron BA.4 and BA.5 strains.Figure8C shows inhibition of the Omicron BA.1 strain.Figure8D shows inhibition of SARS CoV-1 pseudovirus.

[圖9]係顯示了各種HIV抗融合多肽的構建體設計和序列之示意圖。[Fig.9 ] is a schematic diagram showing the construct design and sequences of various HIV anti-fusion polypeptides.

[圖10A和10B]係顯示了來自環狀RNA的各種HIV抗融合多肽的表現的圖（圖10A）和表格（圖10B）。[Figures10A and10B ] A graph (Figure10A ) and a table (Figure10B ) showing the performance of various HIV anti-fusion polypeptides derived from circular RNAs are shown.

[圖11A和11B]係顯示了來自環狀RNA（圖11A）或質體DNA（圖11B）的各種HIV抗融合多肽的表現之圖。[Figures11A and11B ] Figures showing the expression of various HIV anti-fusion polypeptides derived from circular RNA (Figure11A ) or plastid DNA (Figure11B ).

[圖12]係顯示了來自環狀RNA的各種HIV抗融合多肽的表現的表格。[Fig.12 ] A table showing the performance of various HIV anti-fusion polypeptides derived from circular RNA.

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TW202342064A_111149837_SEQL.xmlTW202342064A_111149837_SEQL.xml

Claims (36)

Translated fromChinese

一種環狀多核糖核苷酸，該環狀多核糖核苷酸包含編碼抗融合多肽的多核糖核苷酸貨物。A cyclic polyribonucleotide comprising a polyribonucleotide cargo encoding an anti-fusion polypeptide.如請求項1所述之環狀多核糖核苷酸，其中該多核糖核苷酸貨物包括編碼該抗融合多肽之表現序列。The cyclic polyribonucleotide of claim 1, wherein the polyribonucleotide cargo includes an expression sequence encoding the anti-fusion polypeptide.如請求項1或2所述之環狀多核糖核苷酸，其中該環狀多核糖核苷酸包含連接5'外顯子片段和3'外顯子片段之剪接點。The cyclic polyribonucleotide as described in claim 1 or 2, wherein the cyclic polyribonucleotide includes a splice junction connecting the 5' exon fragment and the 3' exon fragment.如請求項2或3所述之環狀多核糖核苷酸，其中該多核糖核苷酸貨物包括與編碼該抗融合多肽的該表現序列可操作地連接的IRES。The cyclic polyribonucleotide of claim 2 or 3, wherein the polyribonucleotide cargo includes an IRES operably linked to the expression sequence encoding the anti-fusion polypeptide.如請求項4所述之環狀多核糖核苷酸，其中該環狀多核糖核苷酸進一步包含該IRES與該3'外顯子片段或該5'外顯子片段之間的間隔子區域。The cyclic polyribonucleotide as claimed in claim 4, wherein the cyclic polyribonucleotide further comprises a spacer region between the IRES and the 3' exon fragment or the 5' exon fragment. .如請求項5所述之環狀多核糖核苷酸，其中該間隔子區域之長度為至少5個核糖核苷酸。The cyclic polyribonucleotide as claimed in claim 5, wherein the length of the spacer region is at least 5 ribonucleotides.如請求項6所述之環狀多核糖核苷酸，其中該間隔子區域之長度為5至500個核糖核苷酸。The cyclic polyribonucleotide as claimed in claim 6, wherein the spacer region has a length of 5 to 500 ribonucleotides.如請求項5-7中任一項所述之環狀多核糖核苷酸，其中該間隔子區域包含聚A、聚A-C、聚A-U或聚A-G序列。The cyclic polyribonucleotide as described in any one of claims 5-7, wherein the spacer region includes polyA, polyA-C, polyA-U or polyA-G sequences.如請求項1-8中任一項所述之環狀多核糖核苷酸，其中該環狀多核糖核苷酸之長度為至少500個核糖核苷酸。The cyclic polyribonucleotide as described in any one of claims 1-8, wherein the length of the cyclic polyribonucleotide is at least 500 ribonucleotides.如請求項9所述之環狀多核糖核苷酸，其中該環狀多核糖核苷酸之長度為500至20,000個核糖核苷酸。The cyclic polyribonucleotide as claimed in claim 9, wherein the length of the cyclic polyribonucleotide is 500 to 20,000 ribonucleotides.一種線性多核糖核苷酸，該線性多核糖核苷酸從5'到3'包括 (A) 3'內含子片段；(B) 3'剪接位點；(C) 3'外顯子片段；(D) 編碼該抗融合多肽的多核糖核苷酸貨物；(E) 5'外顯子片段；(F) 5'剪接位點；和 (G) 5'內含子片段。A linear polyribonucleotide comprising (A) a 3' intron fragment from 5' to 3'; (B) a 3' splice site; (C) a 3' exon fragment ; (D) polyribonucleotide cargo encoding the anti-fusion polypeptide; (E) 5' exon fragment; (F) 5' splice site; and (G) 5' intron fragment.如請求項11所述之線性多核糖核苷酸，其中該多核糖核苷酸貨物包括編碼該抗融合多肽之表現序列。The linear polyribonucleotide of claim 11, wherein the polyribonucleotide cargo includes an expression sequence encoding the anti-fusion polypeptide.如請求項12所述之線性多核糖核苷酸，其中該多核糖核苷酸貨物包括與編碼該抗融合多肽的該表現序列可操作地連接的IRES。The linear polyribonucleotide of claim 12, wherein the polyribonucleotide cargo includes an IRES operably linked to the expression sequence encoding the anti-fusion polypeptide.如請求項11-13中任一項所述之線性多核糖核苷酸，其中該環狀多核糖核苷酸進一步包括 (A)、(B)、(C)、(D)、(E)、(F) 和 (G) 中一或多個之間的間隔子區域。The linear polyribonucleotide as described in any one of claims 11-13, wherein the cyclic polyribonucleotide further includes (A), (B), (C), (D), (E) , a spacer subregion between one or more of (F) and (G).如請求項14所述之線性多核糖核苷酸，其中該間隔子區域之長度為至少5個核糖核苷酸。The linear polyribonucleotide of claim 14, wherein the length of the spacer region is at least 5 ribonucleotides.如請求項15所述之線性多核糖核苷酸，其中該間隔子區域之長度為5至500個核糖核苷酸。The linear polyribonucleotide as claimed in claim 15, wherein the spacer region has a length of 5 to 500 ribonucleotides.如請求項14-16中任一項所述之線性多核糖核苷酸，其中該間隔子區域包含聚A、聚A-C、聚A-U或聚A-G序列。The linear polyribonucleotide as described in any one of claims 14-16, wherein the spacer region includes polyA, polyA-C, polyA-U or polyA-G sequences.如請求項11-17中任一項所述之線性多核糖核苷酸，其中該線性多核糖核苷酸之長度為至少500個核糖核苷酸。The linear polyribonucleotide as described in any one of claims 11-17, wherein the length of the linear polyribonucleotide is at least 500 ribonucleotides.如請求項18所述之線性多核糖核苷酸，其中該線性多核糖核苷酸之長度為500至20,000個核糖核苷酸。The linear polyribonucleotide of claim 18, wherein the length of the linear polyribonucleotide is 500 to 20,000 ribonucleotides.一種DNA載體，該DNA載體編碼如請求項11-19中任一項所述之線性多核糖核苷酸。A DNA vector encoding the linear polyribonucleotide described in any one of claims 11-19.一種在細胞中表現抗融合多肽之方法，該方法包括在適於表現該抗融合多肽的條件下向該細胞提供如請求項1-10中任一項所述之環狀多核糖核苷酸、如請求項11-19中任一項所述之線性多核糖核苷酸或如請求項20所述之DNA載體。A method for expressing an anti-fusion polypeptide in a cell, the method comprising providing the cell with a cyclic polyribonucleotide as described in any one of claims 1-10, under conditions suitable for expressing the anti-fusion polypeptide, The linear polyribonucleotide as described in any one of claims 11-19 or the DNA vector as described in claim 20.一種由如請求項11-20中任一項所述之線性多核糖核苷酸產生環狀多核糖核苷酸之方法，該方法包括在適於該線性多核糖核苷酸自剪接以產生該環狀多核糖核苷酸的條件下提供該線性多核糖核苷酸。A method for producing a cyclic polyribonucleotide from a linear polyribonucleotide as described in any one of claims 11-20, the method comprising self-splicing the linear polyribonucleotide to produce the The linear polyribonucleotide is provided under the conditions of cyclic polyribonucleotide.一種藥物組成物，該藥物組成物包含如請求項1-10中任一項所述之環狀多核糖核苷酸、如請求項11-19中任一項所述之線性多核糖核苷酸或如請求項20所述之DNA載體以及稀釋劑、載劑或賦形劑。A pharmaceutical composition comprising the cyclic polyribonucleotide as described in any one of claims 1-10, and the linear polyribonucleotide as described in any one of claims 11-19 Or the DNA carrier and diluent, carrier or excipient as described in claim 20.一種在受試者中表現抗融合多肽之方法，該方法包括以足以在該受試者中產生至少500 ng/mL的血清濃度的該抗融合多肽的量投與第一劑量的如請求項23所述之藥物組成物。A method of expressing an anti-fusion polypeptide in a subject, the method comprising administering a first dose of claim 23 in an amount sufficient to produce a serum concentration of the anti-fusion polypeptide in the subject of at least 500 ng/mL The pharmaceutical composition.如請求項24所述之方法，該方法進一步包括投與第二劑量的該藥物組成物。The method of claim 24, further comprising administering a second dose of the pharmaceutical composition.如請求項25所述之方法，其中該第二劑量在該藥物組成物的該第一劑量後至少1天投與。The method of claim 25, wherein the second dose is administered at least 1 day after the first dose of the pharmaceutical composition.如請求項26所述之方法，其中該第二劑量在該藥物組成物的該第一劑量後1天至90天投與。The method of claim 26, wherein the second dose is administered from 1 day to 90 days after the first dose of the pharmaceutical composition.如請求項25-27中任一項所述之方法，其中在該受試者之血清中該抗融合多肽之血清濃度小於約500 ng/mL之前投與該第二劑量。The method of any one of claims 25-27, wherein the second dose is administered before the serum concentration of the anti-fusion polypeptide in the subject's serum is less than about 500 ng/mL.如請求項28所述之方法，其中該方法在該受試者中維持至少500 ng/mL的血清濃度的該抗融合多肽。The method of claim 28, wherein the method maintains a serum concentration of the anti-fusion polypeptide in the subject of at least 500 ng/mL.如請求項24-29中任一項所述之方法，其中該方法治療或預防該受試者中的病毒感染。The method of any one of claims 24-29, wherein the method treats or prevents viral infection in the subject.如請求項30所述之方法，其中該藥物組成物以足以治療或預防該病毒感染的量和持續時間投與給該受試者。The method of claim 30, wherein the pharmaceutical composition is administered to the subject in an amount and for a duration sufficient to treat or prevent the viral infection.如請求項24-31中任一項所述之方法，其中該方法減少病毒進入。The method of any one of claims 24-31, wherein the method reduces virus entry.一種環狀多核糖核苷酸，該環狀多核糖核苷酸包含編碼多個抗融合多肽的多核糖核苷酸貨物。A cyclic polyribonucleotide comprising a polyribonucleotide cargo encoding a plurality of anti-fusion polypeptides.如請求項33所述之環狀多核糖核苷酸，其中該多核糖核苷酸貨物包括編碼該等抗融合多肽之表現序列。The cyclic polyribonucleotide of claim 33, wherein the polyribonucleotide cargo includes expression sequences encoding the anti-fusion polypeptides.如請求項33或34所述之環狀多核糖核苷酸，其中該等抗融合多肽針對相同的病毒。The cyclic polyribonucleotide as claimed in claim 33 or 34, wherein the anti-fusion polypeptides are directed against the same virus.如請求項33或34所述之環狀多核糖核苷酸，其中該等抗融合多肽針對多於一種病毒。The cyclic polyribonucleotide as claimed in claim 33 or 34, wherein the anti-fusion polypeptides are directed against more than one virus.

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