TIM3 HOMING ENDONUCLEASE VARIANTS, COMPOSITIONS, AND METHODS OF USE

1 . A polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human T cell immunoglobulin mucin 3 (TIM3) gene, wherein the polypeptide binds and cleaves the polynucleotide sequence set forth in SEQ ID NO: 21.

2 .- 15 . (canceled)

16 . The polypeptide of claim 1 , wherein the HE variant comprises at least 40 or more of the following amino acid substitutions: S24C, S24A, L26M, L26F, L26H, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, Q46K, T48G, T48S, V68K, A70T, A70R, A70N, S72M, S72Q, S72A, N75D, A76Y, S78A, S78T, K80R, K80M, Q80S, I90V, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, T203V, K207R, Y223R, K225V, K227N, K229R, D236E, V238I, and L263R of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

17 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: L26M, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, T48G, V68K, A70T, S72M, N75D, A76Y, S78A, K80R, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, K229R, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

18 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: L26M, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, T48G, V68K, A70T, S72M, N75D, A76Y, S78A, K80R, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, K229R, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

19 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: L26M, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, T48G, V68K, A70T, S72M, N75D, A76Y, S78A, K80R, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, K229R, D236E, V238I, and L263R of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

20 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: L26M, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, T48G, V68K, A70T, S72M, N75D, A76Y, S78A, K80R, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203V, K207R, Y223R, K225V, K227N, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

21 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: S24C, L26F, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46K, T48S, V68K, A70R, S72Q, N75D, A76Y, K80M, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, K229R, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

22 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: S24A, L26H, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, V68K, A70N, S72A, N75D, A76Y, S78T, K80S, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203S, K207R, Y223R, K225V, K227N, K229R, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

23 . The polypeptide of claim 1 , wherein the HE variant comprises the following amino acid substitutions: L26H, N32R, K34G, S35P, S36T, V37R, G38R, S40A, E42S, G44Q, Q46S, V68K, A70N, S72A, N75D, A76Y, S78T, K80S, I90V, V116L, L138M, T143N, S159P, F 168 L, E178D, C180S, F 182 M, N184E, I186M, K189N, S190V, K191N, L192A, G193R, Q195R, Q197R, V199R, T203V, K207R, Y223R, K225V, K227N, D236E, and V238I of any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.

24 . The polypeptide of claim 1 , wherein the HE variant comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-12, or a biologically active fragment thereof.

25 . The polypeptide of claim 1 , wherein the HE variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 6-12, or a biologically active fragment thereof.

26 .- 31 . (canceled)

32 . The polypeptide of claim 1 , wherein the HE variant does not comprise S24N, L26H, Q46F, T48G, A70N, S72Q, S78E, K80R, V116L, and L263R.

33 . The polypeptide of claim 1 , wherein the HE variant does not comprise S24C, L26W, Q46G, T48Q, A70M, S72Q, S78Q, K80R, V116L, and L263R.

34 . (canceled)

35 . The polypeptide of claim 1 , further comprising a TALE DNA binding domain.

36 .- 38 . (canceled)

39 . The polypeptide of claim 35 , wherein the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 22.

40 . (canceled)

41 . The polypeptide of claim 1 , further comprising a peptide linker and Trex2 or biologically active fragment thereof.

42 .- 44 . (canceled)

45 . The polypeptide of claim 35 , wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-20, or a biologically active fragment thereof.

46 .- 54 . (canceled)

55 . A polynucleotide encoding the polypeptide of claim 1 or claim 35 .

56 . The polynucleotide of claim 55 , wherein the polynucleotide is an mRNA.

57 . The polynucleotide of claim 55 , wherein the polynucleotide is a cDNA.

58 . A vector comprising the polynucleotide of claim 55 .

59 .- 60 . (canceled)

61 . A human cell comprising the vector of claim 58 .

62 .- 138 . (canceled)