ALANINE-GLYOXYLATE AMINOTRANSFERASE THERAPEUTICS
The present invention relates to an alanine glyoxylate aminotransferase (AGXT) I340M therapeutic for use as a medicament or in a method of treatment, for example in the treatment of an AGXT-responsive condition. The AGXT I340M therapeutic is an AGXT I340M protein comprising an amino acid sequence, which, when optimally aligned with SEQ ID NO: 2, comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2, a nucleic acid molecule encoding such an AGXT I340M protein, or a virion of a viral gene therapy vector comprising such a nucleic acid molecule. The AGXT I340M therapeutic has a higher specific activity as compared to other AGXT alleles and may therefore be advantageously used in the treatment of primary hyperoxaluria type I.
1 - 17 . (canceled)
18 . A method of treating an condition responsive to alanine-glyoxylate aminotransferase (AGXT), comprising administering to subject an effective amount of an AGXT I340M therapeutic.
19 . The method according to claim 18 , wherein the AGXT-responsive condition is primary hyperoxaluria type I.
20 . The method according to claim 18 , wherein the AGXT I340M therapeutic is selected from the group consisting of:
(a) an AGXT I340M protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2;
(b) a nucleic acid molecule comprising an nucleotide sequence encoding an AGXT I340M protein as defined in (a); and,
(c) a virion of a viral gene therapy vector comprising a nucleic acid molecule as defined in (b).
21 . The method according to claim 20 , wherein the AGXT I340M protein comprises an amino acid substitution selected from the group consisting of:
(i) a leucine at a position corresponding to position 11 in SEQ ID NO: 2;
(ii) an arginine at a position corresponding to position 41 in SEQ ID NO: 2;
(iii) an isoleucine at a position corresponding to position 152 in SEQ ID NO: 2;
(iv) an arginine at a position corresponding to position 170 in SEQ ID NO: 2;
(v) a threonine at a position corresponding to position 244 in SEQ ID NO: 2;
(vi) a threonine at a position corresponding to position 294 in SEQ ID NO: 2;
(vii) an isoleucine at a position corresponding to position 326 in SEQ ID NO: 2
(viii) combinations of one or more of the substitutions amino acid (ii) to (vii); and,
(ix) a combination of the substitutions (i) and (vi).
22 . The method according to claim 18 , wherein the AGXT I340M protein is selected from the group consisting of:
(a) an AGXT I340M protein comprising the amino acid sequence of SEQ ID NO: 2; and,
(b) an AGXT I340M protein comprising the amino acid sequence of SEQ ID NO: 2 and having one or more amino acid substitutions selected from the group consisting of:
(i) a leucine at a position corresponding to position 11 in SEQ ID NO: 2;
(ii) an arginine at a position corresponding to position 41 in SEQ ID NO: 2;
(iii) an isoleucine at a position corresponding to position 152 in SEQ ID NO: 2;
(iv) an arginine at a position corresponding to position 170 in SEQ ID NO: 2;
(v) a threonine at a position corresponding to position 244 in SEQ ID NO: 2;
(vi) a threonine at a position corresponding to position 294 in SEQ ID NO: 2;
(vii) an isoleucine at a position corresponding to position 326 in SEQ ID NO: 2
(viii) combinations of one or more of the substitutions amino acid (ii) to (vii); and,
(ix) a combination of the substitutions (i) and (vi).
23 . The method according to claim 18 , wherein the AGXT I340M therapeutic is a nucleic acid molecule comprising a nucleotide sequence encoding an AGXT I340M protein selected from the group consisting of:
(a) an AGXT I340M protein comprising the amino acid sequence of SEQ ID NO: 2; and,
(b) an AGXT I340M protein comprising the amino acid sequence of SEQ ID NO: 2 and having one or more amino acid substitutions selected from the group consisting of:
(i) a leucine at a position corresponding to position 11 in SEQ ID NO: 2;
(ii) an arginine at a position corresponding to position 41 in SEQ ID NO: 2;
(iii) an isoleucine at a position corresponding to position 152 in SEQ ID NO: 2;
(iv) an arginine at a position corresponding to position 170 in SEQ ID NO: 2;
(v) a threonine at a position corresponding to position 244 in SEQ ID NO: 2;
(vi) a threonine at a position corresponding to position 294 in SEQ ID NO: 2;
(vii) an isoleucine at a position corresponding to position 326 in SEQ ID NO:
(viii) combinations of one or more of the substitutions amino acid (ii) to (vii); and,
(ix) a combination of the substitutions (i) and (vi).
24 . A nucleic acid construct comprising a nucleotide sequence coding for a AGXT I340M protein comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2,
wherein the nucleotide sequence is operably linked to a promoter for expression in human cells and, optionally, wherein the promoter is not a promoter from a human AGXT gene.
25 . The nucleic acid construct according to claim 24 , wherein the promoter is a liver-specific promoter.
26 . The nucleic acid construct according to claim 25 , wherein the liver-specific promoter is selected from the group consisting of an α1-anti-trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an AAT promoter combined with the mouse albumin gene enhancer (Ealb) element and an apolipoprotein E promoter.
27 . The nucleic acid construct according to claim 24 , wherein the promoter comprises the sequence of SEQ ID NO: 3.
28 . The nucleic acid construct according to claim 24 , wherein the construct is a viral gene therapy vector.
29 . The nucleic acid construct according to claim 28 , wherein the viral gene therapy vector comprises a parvoviral vector.
30 . A parvoviral virion comprising a nucleic acid construct according to claim 24 .
31 . A pharmaceutical composition comprising:
(a) an AGXT I340M protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2;
(b) a nucleic acid molecule comprising an nucleotide sequence encoding an AGXT I340M protein of (a);
(c) a virion of a viral gene therapy vector comprising a nucleic acid molecule of (b);
(d) a nucleic acid construct comprising a nucleotide sequence coding for a AGXT I340M protein comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2, wherein the nucleotide sequence is operably linked to a promoter for expression in human cells and, optionally, wherein the promoter is not a promoter from a human AGXT gene; or
(e) parvoviral virion comprising the nucleic acid construct of (d), and
a pharmaceutically acceptable carrier.
32 . A method for treating a condition responsive to AGXT comprising administering to a subject in need thereof an effective amount of an AGXT I340M therapeutic selected from the group consisting of:
(a) an AGXT I340M protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2;
(b) a nucleic acid molecule comprising an nucleotide sequence encoding an AGXT I340M protein of (a);
(c) a virion of a viral gene therapy vector comprising a nucleic acid molecule of (b);
(d) a nucleic acid construct comprising a nucleotide sequence coding for a AGXT I340M protein comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 when optimally aligned over their entire length using the GAP program, and wherein the AGXT I340M protein comprises a methionine at a position corresponding to position 340 in SEQ ID NO: 2, wherein the nucleotide sequence is operably linked to a promoter for expression in human cells and, optionally, wherein the promoter is not a promoter from a human AGXT gene; and
(e) parvoviral virion comprising the nucleic acid construct of (d).