JP-2022513750-A5 -

Dates

Publication Date

20221216

Application Date

20191209

Description

In general, the terms used in the following claims should not be interpreted as limiting the claims to specific embodiments disclosed herein and herein, but rather as encompassing all possible embodiments, along with the entire scope of equivalents for which such claims are entitled. Therefore, the claims are not limited by this disclosure. The present invention provides, for example, the following items: (Item 1) An I-OnuI homing endonuclease (HE) variant comprising one or more amino acid substitutions to a parent I-OnuI HE comprising the amino acid sequence described in Sequence ID No. 1, wherein the one or more amino acid substitutions increase the thermal stability of the I-OnuI HE variant compared to the parent I-OnuI HE variant. (Item 2) The I-OnuI HE variant according to item 1, wherein one or more of the aforementioned amino acid substitutions are located at amino acid positions selected from the group consisting of I14, A19, V116, F168, D208, N246, and L263. (Item 3) The I-OnuI HE variant according to item 1, wherein the I-OnuI HE variant includes amino acid substitutions at the amino acid positions of I14, A19, F168, D208, and N246. (Item 4) The I-OnuI HE variant according to item 1, wherein the one or more amino acid substitutions are located at amino acid positions selected from the group consisting of K108, K156, S176, E231, V261, E277, and G300. (Item 5) The I-OnuI HE variant according to item 1, wherein one or more of the aforementioned amino acid substitutions are located at amino acid positions selected from the group consisting of N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268. (Item 6) The I-OnuI HE variant according to item 1, wherein the I-OnuI HE variant comprises three or more amino acid substitutions at amino acid positions selected from the group consisting of I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300. (Item 7) The I-OnuI HE variant according to item 1, wherein the I-OnuI HE variant comprises three or more amino acid substituents at amino acid positions selected from the group consisting of I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300, and one or more amino acid substitutions are at amino acid positions selected from the group consisting of N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268. (Item 8) The I-OnuI HE variant according to item 1, wherein the I-OnuI HE variant includes five or more amino acid substitutions at amino acid positions selected from the group consisting of I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300. (Item 9) The I-OnuI HE variant described in item 1, wherein the I-OnuI HE variant comprises five or more amino acid substitutions at amino acid positions selected from the group consisting of I14, A19, K108, V116, K156, F168, S176, D208, E231, N246, V261, L263, E277, and G300, and one or more amino acid substitutions are at amino acid positions selected from the group consisting of N31, N33, K52, Y97, K124, K147, I153, K209, E264, and D268. (Item 10) The I-OnuI HE variant according to any one of items 1 to 9, wherein the I-OnuI HE variant has a TM 50 that is at least 10°C higher than the TM 50 of the parent I-OnuI HE . (Item 11) The I-OnuI HE variant according to any one of items 1 to 9, wherein the I-OnuI HE variant has a TM 50 that is at least 15°C higher than the TM 50 of the parent I-OnuI HE . (Item 12) The I-OnuI HE variant according to any one of items 1 to 9, wherein the I-OnuI HE variant has a TM 50 that is at least 20°C higher than the TM 50 of the parent I-OnuI HE . (Item 13) The I-OnuI HE variant according to any one of items 1 to 9, wherein the I-OnuI HE variant has a TM 50 that is at least 25°C higher than the TM 50 of the parent I-OnuI HE . (Item 14) The I-OnuI HE variant described in any one of items 1 to 13 targets a site in a gene selected from the group consisting of HBA, HBB, HBG1, HBG2, BCL11A, PCSK9, TCRA, TCRB, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, CIITA, AHR, PD-1, CTLA4, TIGIT, TGFBR2, LAG-3, TIM-3, BTLA, IL4R, IL6R, CXCR1, CXCR2, IL10R, IL13Rα2, TRAILR1, RCAS1R, and FAS. (Item 15) An I-OnuI homing endonuclease (HE) variant comprising one or more amino acid substitutions to the parent I-OnuI HE sequence, wherein the one or more amino acid substitutions increase the thermal stability of the I-OnuI HE variant compared to the parent I-OnuI HE variant. (Item 16) The I-OnuI HE variant described in item 15, wherein the parent I-OnuI HE amino acid sequence is described in Sequence ID No. 1. (Item 17) The I-OnuI HE variant according to item 15 or 16, wherein one or more of the amino acid substitutions are located at amino acid positions selected from the group consisting of I14, A19, V116, F168, D208, N246, and L263. (Item 18) The I-OnuI HE variant according to any one of items 15 to 17, wherein the I-OnuI HE variant includes amino acid substitutions at the amino acid positions of I14, A19, F168, D