Capsid variants and methods of using the same

A variant capsid polypeptide comprising a sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO:

• 1 and comprising a mutation selected from a mutation associated with any of VAR-1 to VAR-15, wherein the mutation is between positions 545 and 600 as compared to SEQ ID NO:
• 1, and wherein the mutation comprises:
• an insertion, e.g., an insertion of 8 or more amino acids, e.g., 8-9 amino acids, e.g., 8-11 amino acids, that correspond to an insertion between positions 584 and 587 as compared to SEQ ID NO:
• 1, and wherein the insertion comprises a polypeptide that has at least 60%, 70%, 80%, 90%, or 100% identity to SEQ ID NO:
• 42-44 or comprises a fragment of 4 or more amino acids of SEQ ID NO:
• 42-44;
• a substitution, e.g., a substitution of at least 1 or more residues, e.g., at least 1-12 residues, e.g., at least 2-12 residues, e.g., at least 3-12 residues, e.g., at least 4-12 residues, e.g., at least 8-12 residues, e.g., at least 9-12 residues, e.g., at least 10-12 residues, e.g., at least 11-12 residues, e.g., at least 12 residues that correspond to a substitution at positions between 545 and 600 as compared to SEQ ID NO:
• 1;
• or
• any combination thereof.

A variant capsid polypeptide comprising a sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO:

• 1 and comprising a mutation selected from a mutation associated with any of VAR-1 to VAR-15, wherein the mutation is between positions 548 and 554 as compared to SEQ ID NO:
• 1, and wherein the mutation comprises:
• (a) a substitution comprising the following consensus formula:
• wherein X1 is the wild type residue as set forth in SEQ ID NO:
• 1 or N and wherein X2 and X3 are, independently, selected from any amino acid;
• and
• (b) a substitution comprising
• at least 2, 3, 4, or all of the non-wild type amino acids of the consensus.

A variant capsid polypeptide comprising a sequence having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO:

• 1 and comprising a mutation selected from a mutation associated with any of VAR-1 to VAR-15, wherein the mutation is between positions 548 and 556 as compared to SEQ ID NO:
• 1, and wherein the mutation comprises:
• (a) a substitution comprising the following consensus formula:
• wherein X1 is the wild type residue as set forth in SEQ ID NO:
• 1 or N, wherein X2, X3, and X4 are, independently, selected from any amino acid, and wherein X5 is selected from H or N;
• and
• (b) a substitution comprising
• at least 2, 3, 4, or all of the non-wild type amino acids of the consensus.

The variant capsid polypeptide of any of the preceding claims, wherein the capsid polypeptide comprises:

• (a) a mutation that corresponds to a mutation at position 589, 590, 597, and 600 as compared to SEQ ID NO:
• 1;
• (b) a mutation that corresponds to a mutation at position 586 and 587 as compared to SEQ ID NO:
• 1;
• (c) a mutation that corresponds to a mutation at position 587, 591, and 595 as compared to SEQ ID NO:
• 1;
• (d) a mutation that corresponds to a mutation at position 593 and 600 as compared to SEQ ID NO:
• 1;
• (e) a mutation that corresponds to a mutation at position 547, 550, 561, 578, 581, 582, 591, 597, and 600 as compared to SEQ ID NO:
• 1;
• (f) a mutation that corresponds to a mutation at position 551, 552, 575, 578, 581, 586, 590, 593, and 598 as compared to SEQ ID NO:
• 1;
• (g) a mutation that corresponds to a mutation at position 545, 553, 554, 561, 581, 591, 597, and 600 as compared to SEQ ID NO:
• 1;
• (h) a mutation that corresponds to a mutation at position 552, 553, 554, 556, 558, 561, 575, 578, 581, 587, 591, and 597 as compared to SEQ ID NO:
• 1;
• (i) a mutation that corresponds to a mutation at position 556, 559, 561, 575, 580, 581, 586, 591, and 598 as compared to SEQ ID NO:
• 1;
• (j) a mutation that corresponds to a mutation at position 552, 559, 566, and 581 as compared to SEQ ID NO:
• 1;
• (k) a mutation that corresponds to a mutation at position 548, 549, 550, 551, 554, 556, 586, 590, 593, and 597 as compared to SEQ ID NO:
• 1;
• (l) a mutation that corresponds to a mutation at position 549, 550, 551, 554, 556, 578, 581, 589, 590, 591, 597 as compared to SEQ ID NO:
• 1;
• (m) a mutation that corresponds to a mutation at position 550, 584, 586, and 587 as compared to SEQ ID NO:
• 1;
• (n) a mutation that corresponds to a mutation at position 561, 586, 587, and 597 as compared to SEQ ID NO:
• 1;
• (o) a mutation that corresponds to a mutation at position 559, 586, and 587 as compared to SEQ ID NO:
• 1;
• or
• (p) an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1-11 amino acids, e.g., 5-11 amino acids, e.g., 8-11 amino acids, e.g., 8 amino acids, e.g., 9 amino acids, e.g., 11 amino acids that corresponds to an insertion between positions 584 and 585, or 586 and 587, as compared to SEQ ID NO:
• 1.

The variant capsid polypeptide of any of the preceding claims, wherein the capsid polypeptide comprises:

• (a) a mutation that corresponds to a Q589G, A590P, T597H, and V600A mutation as compared to SEQ ID NO:
• 1;
• (b) a mutation that corresponds to a G586T and N587G mutation as compared to SEQ ID NO:
• 1;
• (c) a mutation that corresponds to a N587T, A591T, and V595L mutation as compared to SEQ ID NO:
• 1;
• (d) a mutation that corresponds to a A593E and V600I mutation as compared to SEQ ID NO:
• 1;
• (e) a mutation that corresponds to a S547T, T550D, D561S, S578D, T581D, N582T, A591T, T597H, and V600A mutation as compared to SEQ ID NO:
• 1;
• (f) a mutation that corresponds to a N551D, V552A, Q575P, S578T, T581D, G586S, A590P, A593E, and Q598L mutation as compared to SEQ ID NO:
• 1;
• (g) a mutation that corresponds to a Q545I, D553V, I554L, D561S, T581D, A591T, T597H, and V600A mutation as compared to SEQ ID NO:
• 1;
• (h) a mutation that corresponds to a V552T, D553I, I554L, K556N, M558L, D561S, Q575E, S578V, T581D, N587G, A591T, and T597N mutation as compared to SEQ ID NO:
• 1;
• (i) a mutation that corresponds to a K556N, I559L, D561S, Q575A, S580A, T581D, G586Q, A591T, and Q598L mutation as compared to SEQ ID NO:
• 1;
• (j) a mutation that corresponds to a V552A, I559L, R556K, and T581D mutation as compared to SEQ ID NO:
• 1;
• (k) a mutation that corresponds to a E548N, K549A, T550N, N551D, I554L, K556H, G586S, A590P, A593E, and T597H mutation as compared to SEQ ID NO:
• 1;
• (l) a mutation that corresponds to a K549A, T550N, N551D, I554L, K556N, S578T, T581N, Q589G, A590P, A591T, and T597H mutation as compared to SEQ ID NO:
• 1;
• (m) a mutation of T550N, G586P, and N587A, and an insertion between residues 584 and 585 as compared to SEQ ID NO:
• 1, wherein the insertion comprises a polypeptide of RARLDETA (SEQ ID NO:
• 42), or a fragment of at least 4, at least 5, at least 6, or at least 7 amino acids thereof;
• (n) a mutation of D561C, N587A, and T597N, and an insertion between residues 586 and 587 as compared to SEQ ID NO:
• 1, wherein the insertion comprises a polypeptide of GLRAEQTRP (SEQ ID NO:
• 43), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof;
• or
• (o) a mutation of I559L and N587A, and an insertion between residues 586 and 587 as compared to SEQ ID NO:
• 1, wherein the insertion comprises a polypeptide of TNLARGETARP (SEQ ID NO:
• 44), or a fragment of at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

A variant capsid polypeptide, comprising (a) a polypeptide of any one of SEQ ID NO:

• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 14, SEQ ID NO:
• 15, SEQ ID NO:
• 16, SEQ ID NO:
• 17, SEQ ID NO:
• 18, SEQ ID NO:
• 19, SEQ ID NO:
• 20, SEQ ID NO:
• 21, SEQ ID NO:
• 22, SEQ ID NO:
• 23, SEQ ID NO:
• 24, SEQ ID NO:
• 25, or SEQ ID NO:
• 26, (b) the VP2 or VP3 sequence of any one of SEQ ID NO:
• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 14, SEQ ID NO:
• 15, SEQ ID NO:
• 16, SEQ ID NO:
• 17, SEQ ID NO:
• 18, SEQ ID NO:
• 19, SEQ ID NO:
• 20, SEQ ID NO:
• 21, SEQ ID NO:
• 22, SEQ ID NO:
• 23, SEQ ID NO:
• 24, SEQ ID NO:
• 25, or SEQ ID NO:
• 26, (c) a polypeptide comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, wherein said sequence comprises at least one (e.g., one, two, three or more, e.g., all) of the mutation differences associated with any of SEQ ID NO:
• 12 through SEQ ID NO:
• 26, relative to SEQ ID NO:
• 1;
• or (d) a polypeptide having at least 1, but no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 3, or no more than 2 amino acid mutations relative to the polypeptide of (a) or (b), wherein said polypeptide comprises at least one (e.g., one, two, three or more, e.g., all) of the mutation differences associated with any of SEQ ID NO:
• 12 through SEQ ID NO:
• 26, relative to SEQ ID NO:
• 1.

A variant capsid polypeptide comprising:

• an amino acid sequence that has 95% or more amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs:
• 12-26 and;
• has at least 80% of the mutations in said amino acid sequence of one of SEQ ID NO:
• 12-26 as compared to SEQ ID NO:
• 1.

A variant capsid polypeptide comprising:

• an amino acid sequence that has less than 95% amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs:
• 12-26 and;
• has at least 80% of the mutations in said amino acid sequence of one of SEQ ID NO:
• 12-26 as compared to SEQ ID NO:
• 1.

A variant capsid polypeptide comprising:

• an amino acid sequence that has 95% or more amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs:
• 12-26 and;
• has less than 80% of the mutations in said amino acid sequence of one of SEQ ID NO:
• 12-26 as compared to SEQ ID NO:
• 1.

A variant capsid polypeptide having:

• (a) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 597, wherein the mutation is a T597N mutation as compared to SEQ ID NO:
• 1;
• (b) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 550, wherein the mutation is a T550N mutation as compared to SEQ ID NO:
• 1;
• (c) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1 and comprising a mutation at three consecutive positions between 549 and 551 as compared to SEQ ID NO:
• 1, wherein the mutation is an alanine (A), an asparagine (N), and an aspartic acid (D) mutation, respectively;
• (d) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1 and comprising a mutation at four consecutive positions between 548 and 551 as compared to SEQ ID NO:
• 1, wherein the mutation is an asparagine (N), an alanine (A), an asparagine (N), and an aspartic acid (D) mutation, respectively;
• (e) comprising at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1 and comprising a mutation between positions 548 and 554 as compared to SEQ ID NO:
• 1, wherein the mutation is a substitution comprising the consensus formula X1-A-N-D-X2-X3-L, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2 and X3 are each, independently, selected from any amino acid;
• and
• optionally wherein X2 and X3 are the wild type residues as set forth in SEQ ID NO:
• 1;
• or
• (e) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1 and comprising a mutation between positions 548 and 55 as compared to SEQ ID NO:
• 1, wherein the mutation is a substitution comprising the consensus formula X1-A-N-D-X2-X3-L-X4-X5, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2, X3, and X4 are each, independently, selected from any amino acid;
• optionally wherein X2, X3, and X4 are the wild type residues as set forth in SEQ ID NO:
• 1;
• and
• wherein X5 is selected from H or N.

A variant capsid polypeptide having:

• (a) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 597, wherein the mutation is a T597N mutation as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1;
• (b) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 550, wherein the mutation is a T550N mutation as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1;
• (c) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at three consecutive positions between 549 and 551 as compared to SEQ ID NO:
• 1, wherein the three consecutive mutations are an alanine (A), an asparagine (N), and an aspartic acid (D) mutation, respectively and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1;
• (d) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at four consecutive positions between 548 and 551 as compared to SEQ ID NO:
• 1, SEQ ID NO:
• 1, wherein the three consecutive mutations are an asparagine (N), an alanine (A), an asparagine (N), and an aspartic acid (D) mutation, respectively and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1;
• (e) having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation comprising a mutation between positions 548 and 554 as compared to SEQ ID NO:
• 1, wherein the mutation is a substitution comprising the consensus formula X1-A-N-D-X2-X3-L, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2 and X3 are each, independently, selected from any amino acid;
• optionally wherein X2 and X3 are the wild type residues as set forth in SEQ ID NO:
• 1;
• and
• at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1;
• or
• (f) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation comprising a mutation between positions 548 and 556 as compared to SEQ ID NO:
• 1, wherein the mutation is a substitution comprising the consensus formula X1-A-N-D-X2-X3-L-X4-X5, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2, X3, and X4 are each, independently, selected from any amino acid;
• optionally wherein X2, X3, and X4 are the wild type residues as set forth in SEQ ID NO:
• 1;
• wherein X5 is selected from H or N;
• and
• at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1.

A variant capsid polypeptide comprising:

• (a) an asparagine (N) mutation at a position corresponding to a position 597 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus);
• (b) an asparagine (N) mutation at a position corresponding to a position 550 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus);
• (c) an alanine (A), an asparagine (N), and an aspartic acid (D) mutation at a position corresponding to three consecutive positions between positions 549 and 551 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus);
• (d) an asparagine (N), an alanine (A), an asparagine (N), and an aspartic acid (D) mutation at a position corresponding to four consecutive positions between positions 548 and 551 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus);
• (e) the consensus formula X1-A-N-D-X2-X3-L, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2 and X3 are each, independently, selected from any amino acid;
• optionally wherein X2 and X3 are the wild type residues as set forth in SEQ ID NO:
• 1;
• and
• wherein the consensus formula is at a position corresponding to a position 548 and 554 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus);
• or
• (f) the consensus formula X1-A-N-D-X2-X3-L-X4-X5, wherein
• X1 is the wild type residue as set forth in SEQ ID NO:
• 1;
• optionally wherein X1 is N;
• wherein X2, X3, and X4 are each, independently, selected from any amino acid;
• optionally wherein X2, X3, and X4 are the wild type residues as set forth in SEQ ID NO:
• 1;
• wherein X5 is selected from H or N;
• and
• wherein the consensus formula is at a position corresponding to a position 548 and 556 of SEQ ID NO:
• 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus).

A variant capsid polypeptide having:

• (a) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 591, wherein the mutation is an A591T substitution as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1, and wherein a virus comprising said capsid has increased retina transduction following intravitreal administration;
• (b) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 597, wherein the mutation is a T597N substitution as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1, and wherein a virus comprising said capsid has increased retina transduction following intravitreal administration;
• (c) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 591, wherein the mutation is an A591T substitution as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1, and wherein a virus comprising said capsid has increased anterior eye region (e.g., trabecular meshwork and/or schlemm's canal) transduction following intravitreal or intracameral administration;
• or
• (d) at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:
• 1, and comprising a mutation at position 597, wherein the mutation is a T597N substitution as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1, and wherein a virus comprising said capsid has increased anterior eye region (e.g., trabecular meshwork and/or schlemm's canal) transduction following intravitreal or intracameral administration.

A variant capsid polypeptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to VP1, VP2, or VP3 sequence of SEQ ID NO:

• 1, and comprising mutations at any two positions selected from positions:
• 556, 561, 581, and 591;
• or any three positions selected from positions:
• 556, 561, 581, and 591;
• or optionally 556, 561, 581, and 591, wherein the mutation comprises any two mutations selected from mutations:
• K566N, D561S, T581D, and A591T;
• or any three mutations selected from mutations:
• K566N, D561S, T581D, and A591T;
• or optionally K566N, D561S, T581D, and A591T mutations as compared to SEQ ID NO:
• 1, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional mutations, but fewer than 40, 39, 38, 37, 36, 35 mutations as compared to SEQ ID NO:
• 1.