4-hydroxyphenylpyruvate dioxygenase variants

Abstract

The present invention relates to recombinant variants of the human 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme that have a decreased sensitivity to an HPD inhibitor. The invention also relates to their use in clinical applications to treat tyrosine metabolism related diseases.

Description

[0001] 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE VARIANTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to recombinant variants of the human 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme that have a decreased sensitivity to an HPD inhibitor. The invention also relates to their use in clinical applications to treat tyrosine metabolism related diseases.

[0004] BACKGROUND TO THE INVENTION

[0005] Today, most inborn errors of metabolism are still lacking proper therapy being often lifethreatening or chronically debilitating. Tyrosine is an amino acid that derives from two sources, diet and hydroxylation of phenylalanine. Its catabolism proceeds predominantly in the liver and kidneys through an enzymatic conversion into fumarate and acetoacetate. Diseases with an impaired tyrosine metabolism pathway or tyrosine-related inborn errors of metabolism are caused by a single dysfunctional enzyme of the tyrosine degradation pathway. Today, six inherited disorders of tyrosine metabolism are known, i.e. hereditary tyrosinemia (HT) type 1 , type 1 b, type 2 and type 3, hawkinsinuria, and alkaptonuria (AKU).

[0006] HT1 is the most prevalent and severe of the inherited disorders of tyrosine metabolism. HT1 is characterized by a dysfunctional fumarylacetoacetate hydrolase (FAH) enzyme, leading to a progressive loss of hepatocytes accompanied by an increased risk for hepatocellular carcinoma (HCC), renal tubular dysfunction with rickets and neurological crises. This is caused by the accumulation of the toxic upstream metabolites fumarylacetoacetate and maleylacetoacetate. These intermediates are further metabolized to the downstream toxic metabolite succinylacetone (SA) which plays a major role in the onset of neurological crises. Without proper treatment most HT1 patients die in their infancy.

[0007] AKU is a serious, autosomal recessive, multisystem disorder caused by a deficiency of the enzyme homogentisate 1 ,2-dioxygenase (HGD) which is mainly expressed in the liver and kidney cells. Loss of HGD function prevents metabolization of homogentisic acid (HGA), leading to increased levels of plasma HGA and urinary excretion. Excess HGA becomes deposited in collagenous tissues, and subsequently undergoes polymerization, principally in the cartilages of loaded joints, in a process known as ochronosis.

[0008] A number of different approaches have been used in attempts to treat patients with diseases with impaired tyrosine metabolism pathway such as HT1 and AKU. Dietary restriction of phenylalanine and tyrosine intake reduces the production and accumulation of toxic metabolites including SA and HGA, but were shown to be inadequate. Nowadays, the orphan drug NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1 ,3cyclohexanedione; nitisinone], a former herbicide and potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, is an approved treatment for HT1 and AKU. NTBC prevents early death of HT1 patients and, it is currently the only HGA lowering therapy with a potential to modify disease progression in AKU. However, although inhibition of HPD by NTBC partly corrects the biochemical deregulation by lowering SA and HGA blood and urine levels, it also leads to tyrosine accumulation in the blood, the disturbance of urinary catecholamine and serotonin metabolites and the production of alternative tyrosine-derivatives including y-glutamyltyrosine and N-acetyltyrosine. The concomitant hypertyrosinemia requires therefore lifelong dietary adjustment in order to reduce the chance to develop ocular, cutaneous and possible neurological complications. Because of these aforementioned reasons, there is currently a high need to improve current treatment approaches for diseases associated with an impaired tyrosine pathway.

[0009] SUMMARY OF THE INVENTION

[0010] As corroborated in the experimental section, the inventors of the present invention have found that one or more specific amino acid substitutions in the native human 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme result in a decreased sensitivity of the HPD enzyme against an HPD inhibitor. These recombinant human HPD enzymes are therefore interesting treatment options for the treatment of diseases associated with an impaired tyrosine metabolism pathway.

[0011] The present invention is directed in general to recombinant 4-hydroxyphenylpyruvate dioxygenase (HPD) enzymes with a decreased sensitivity to an HPD inhibitor, and the use of said enzymes for the treatment of diseases associated with an impaired tyrosine metabolism pathway.

[0012] In an aspect, the invention provides a recombinant polypeptide comprising a human HPD enzyme wherein said human HPD enzyme comprises at least one amino acid substitution as compared to the native human HPD enzyme (SEQ ID NO: 1) and wherein said at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor, for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway. More specific, the present invention provides a recombinant polypeptide comprising a human 4- hydroxyphenylpyruvate dioxygenase (HPD) enzyme for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway, wherein said HPD enzyme comprises at least one amino acid substitution as compared to the native human HPD enzyme (SEQ ID NO: 1) wherein said at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 1 17 to 177 (SEQ ID NO: 2), in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326 (SEQ ID NO: 3), or in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393 (SEQ ID NO: 4) and wherein said at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor. The human HPD enzyme of said recombinant polypeptide has thus a reduced sensitivity to an HPD inhibitor. The amino acid sequence of the native human HPD enzyme defined by amino acid positions 117 to 177 is further defined herein as SEQ ID NO: 2. The amino acid sequence of the native human HPD enzyme defined by amino acid positions 270 to 326 is further defined herein as SEQ ID NO: 3. The amino acid sequence of the native human HPD enzyme defined by amino acid positions 378 to 393 is further defined herein as SEQ ID NO: 4. In other words, the amino acid substitution in the human HPD enzyme occurs in the critical regions of the native human HPD enzyme sequence (represented by SEQ ID NO: 1) defined by amino acid positions 117 to 177 (represented by SEQ ID NO: 2), in the critical regions of the native human HPD enzyme sequence defined by amino acid positions 270 to 326 (SEQ ID NO: 3) and / or in the critical regions of the native human HPD enzyme sequence defined by amino acid positions 378 to 393 (SEQ ID NO: 4).

[0013] In some further embodiments, the amino acid substitution in the HPD enzyme occurs in the critical regions of the native human HPD enzyme sequence (represented by SEQ ID NO: 1) defined by amino acid positions 67 to 76 (represented by SEQ ID NO: 22), in the critical regions of the native human HPD enzyme sequence defined by amino acid positions 157 to 169 (SEQ ID NO: 23) and / or in the critical regions of the native human HPD enzyme sequence defined by amino acid positions 231 to 236 (represented by SEQ ID NO: 24). The amino acid sequence of the native human HPD enzyme defined by amino acid positions 67 to 76 is further defined herein as SEQ ID NO: 22. The amino acid sequence of the native human HPD enzyme defined by amino acid positions 157 to 169 is further defined herein as SEQ ID NO: 23. The amino acid sequence of the native human HPD enzyme defined by amino acid positions 231 to 236 is further defined herein as SEQ ID NO: 24.

[0014] In a further embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises a human HPD enzyme comprising at least one amino acid substitution as compared to the native human HPD enzyme (SEQ ID NO: 1) wherein said at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 261 to 326, and wherein said at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor. In some embodiments, the at least one amino acid substitution may further occur in the critical region of SEQ ID NO: 1 defined by amino acid position 67 to 76, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

[0015] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177.

[0016] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326.

[0017] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, two amino acid substitutions in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, and three amino acid substitutions in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326.

[0018] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326; and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76.

[0019] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236. In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions

[0020] 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169.

[0021] In an embodiment, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, two amino acid substitutions in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, four amino acid substitutions in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326; one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236, and one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169.

[0022] In an embodiment, the recombinant polypeptide for use as disclosed herein invention comprises a human HPD enzyme comprising amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position

[0023] 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , and (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1.

[0024] In an embodiment, the recombinant polypeptide for use as disclosed herein invention comprises a human HPD enzyme comprising amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1.

[0025] In an embodiment, the recombinant polypeptide for use as disclosed herein invention comprises a human HPD enzyme comprising amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1.

[0026] In an embodiment, the recombinant polypeptide for use as disclosed herein invention comprises a human HPD enzyme comprising amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1.

[0027] In an embodiment, the recombinant polypeptide for use as disclosed herein invention comprises a human HPD enzyme comprising amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

[0028] The recombinant polypeptide for use as disclosed herein may further comprise an amino acid substitution to a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO:1. The recombinant polypeptide for use as disclosed herein may further comprise an amino acid substitution to a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 . The recombinant polypeptide for use as disclosed herein may further comprise an amino acid substitution to a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 , or an amino acid substitution to a tyrosine at the amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1.

[0029] Another aspect of the invention provides a recombinant polypeptide comprising a human 4- hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142; at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177. Optionally, the recombinant polypeptide further comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326. Optionally, the recombinant polypeptide further comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169; and / or in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

[0030] In a further embodiment, the invention provides a recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; and (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1.

[0031] In a further embodiment, the invention provides a recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1.

[0032] In a further embodiment, the invention provides a recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1.

[0033] In a further embodiment, the invention provides a recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1.

[0034] In a further embodiment, the invention provides a recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID No: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 1 18 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1. The present invention also discloses compositions comprising a recombinant polypeptide or recombinant HPD enzyme according to any one of the described embodiments before or for use according to any one of the described embodiments before.

[0035] In another aspect of the invention, a recombinant nucleic acid molecule is disclosed, wherein the recombinant nucleic acid molecule encodes a recombinant polypeptide or recombinant HPD enzyme according to any one of the described embodiments. Also provided is a recombinant nucleic acid molecule as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway

[0036] In another aspect, a vector comprising a recombinant nucleic acid molecule according to this invention is provided. Further provided is a vector as disclosed herein for use in the treatment of a disease associated with an impaired metabolism pathway.

[0037] The present invention also discloses a genetically engineered host cell or host virus, the host cell or host virus comprising a recombinant nucleic acid molecule or a vector according to any one of the different embodiments of the invention. In some embodiments, a genetically engineered host cell comprising a recombinant nucleic acid molecule or vector as described herein is disclosed. In some embodiments, the genetically engineered host cell is a bacterial host cell, a mammalian host cell or a human host cell. In a particular embodiment, said genetically engineered host cell is a bacterial host cell. Also provided is a genetically engineered host cell or host virus as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway

[0038] The invention further also discloses a pharmaceutical composition comprising one or more genetically engineered host cells, host viruses or vectors according to any one of the different embodiments of the invention. Also provided is a pharmaceutical composition as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

[0039] In an embodiment, the recombinant polypeptide, the recombinant nucleic acid molecule, the vector or the genetically engineered host cell or host virus as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway, is in combination with an HPD inhibitor.

[0040] In another aspect, the present invention provides a method for treating diseases associated with an impaired tyrosine degradation pathway. Said method comprises administration of a pharmaceutically effective amount of a recombinant polypeptide, a recombinant nucleic acid molecule, a vector or a combination of vectors, a genetically engineered host cell or host virus, or a pharmaceutically acceptable composition according to any one of the embodiments disclosed herein, to a subject with a disease associated with an impaired tyrosine metabolism pathway.

[0041] In still another aspect of the invention, a method of producing a recombinant polypeptide comprising a human HPD enzyme with decreased sensitivity to an HPD inhibitor is disclosed. Said method comprises in vitro culturing a genetically engineered host cell according to any of the preceding embodiments under conditions in which a nucleic acid molecule encoding the recombinant polypeptide is expressed.

[0042] In another aspect, a method for identifying HPD inhibitors comprising a human HPD enzyme with decreased sensitivity to said HPD inhibitors is disclosed. Said method comprises in vitro culturing a genetically engineered host cell according to any of the preceding embodiments under conditions in which a nucleic acid molecule encoding a recombinant polypeptide of any of the preceding embodiments is expressed, exposing said recombinant polypeptide to one or more chemical compounds, and identifying one or more chemical compounds with HPD inhibition features. In a further embodiment, said method for identifying HPD inhibitors is performed in a high-throughput screening setup.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0045] Fig. 1 : Implementation of HPDAtriketoneenzymes in cell- and / or gene-based therapies restores tyrosine degradation in patients under triketone-treatment. (a) triketone inhibitors inflict a metabolic block in the tyrosine degradation pathway thereby preventing the production of toxic intermediate metabolites maleyl- and fumarylacetoacetate and the alternative toxic metabolite succinylacetone, but also causes tyrosine accumulation, (b) regular FAH-based gene therapy significantly decreases succinylacetone blood levels in FAH-deficient mice under 8 mg / L NTBC therapy and a regular diet, but does not reduce hypertyrosinemia inflicted by NTBC therapy, (c) implementation of HPDAtriketonein gene- and cell-based therapies for the treatment of tyrosine-dependent inborn errors of metabolism is expected to significantly reduce or even resolve hypertyrosinemia due to NTBC therapy, (d) FAH-based gene therapy in combination with HPDAtriketoneis expected to reduce tyrosine blood levels in FAH-deficient mice under regular diet and NTBC therapy below 200 pM (therapeutic threshold).

[0046] Fig. 2: Dose-response curve of NTBC in different human HPD variants using a in house high-throughput assay. Human HPD enzymes (wildtype or variants) are expressed in an E. coli C43 (DE3) strain through integration in a pET42b(+) plasmid. Upon induction, recombinant expressed human HPD enzymes metabolize 4-hydroxyphenylpyruvate through L-tyrosine into homogentisate that auto-oxidizes and -polymerizes into a brown ochronotic pigment quantifiable at 405 nm. Wildtype human HPD exhibits a dose-dependent decrease in metabolic activity with increasing concentrations of nitisinone as shown by the decreasing intensity of the brown pigment. Human HPD variants with decreased sensitivity towards nitisinone will also produce this brown pigment at higher nitisinone and other triketone inhibitor concentrations versus wildtype HPD.

[0047] Fig. 3. Activity and tolerance of the nine human HPD enzyme single variants in the presence of (A) 0.25 - (B) 0.5 - (C) 1 pM NTBC. Four replicates of each variant were used in the rescreening assay and challenged with the different concentrations of NTBC. Most variants still harbor 20% activity at 0.5 pM NTBC while the WT HPD is almost completely blocked. ‘Significantly higher activity than WT HPD (p<0.05); “significantly lower activity than WT HPD (p < 0.05). Abbreviations: WT: wild-type human HPD enzyme exposed to NTCB; WT-NTBC: wild-type human HPD enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0048] Fig. 4. Activity and tolerance after SSM of the nine human HPD enzyme variants in the presence of (A) 0.5 and (B) 1 pM NTBC. Four replicates of each variant were used in the rescreening assay and challenged with the different concentrations of NTBC. ‘Significantly higher activity than WT HPD (p < 0.05). Abbreviations: WT: wild type human HPD enzyme exposed to NTBC, WT-NBTC: wild-type human HPD enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0049] Fig. 5. Rescreening results after performing MSDM where all identified positions and amino acid substitutions are recombined in multiple HPD variants, i.e. M5 (M118I, T138S, N151 D, I153E, S177Q), M3 (I278V, K302R, K307N) and M8 (all positions and substitutions, also referred to as HPDAtriketone ). Four replicates of each variant were challenged at different concentrations of NTBC (A) 1 pM, (B) 2 pM and (C) 3 pM. (D) The HPD M8 variant challenged at different concentrations of NTBC while the WT version is completely blocked. * Significantly higher activity versus WT (p < 0.05). Abbreviations: WT: wild type human HPD enzyme exposed to NTBC; WT-NTBC: wild type human HPD enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0050] Fig. 6. Dose response inhibition curves of the prototypical B-triketone inhibitors (A) mesotrione (0 - 15 pM), (B) NTBC (0 - 20 pM), (C) sulcotrione (0 - 20 pM), and (D) Tembotrione (0 - 40 pM) for HPD-WT and HPD M8; (E) the calculated IC50 values for each of the HPD inhibitors.

[0051] Fig. 7. HPD WT and HPD M8 variant were challenged with different concentrations of tyrosine and with a vast concentration of NTBC at 2 pM whether to check if the new engineered variant behaves different than the WT HPD.

[0052] Fig. 8. Rescreening results of activity and tolerance of four human HPD enzyme single variants in the presence of 4 pm NTBC. Four replicates of each variant were used in the rescreening assay and challenged with 4 pM NTBC. Most variants harbor 75% activity at 4 pM NTBC while the HPD M8 is almost completely blocked (5-10 % activity). ‘Significantly higher activity than HPD M8 (p<0.05). Abbreviations: M8: human HPD M8 enzyme exposed to NTCB; M8-NTBC: human HPD M8 enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0053] Fig. 9. Rescreening results of activity and tolerance of four human HPD enzyme single variants in the presence of 5 pm NTBC. Four replicates of each variant were used in the rescreening assay and challenged with 5 pM NTBC. Most variants still harbor 25-50 % activity at 5 pM NTBC while the HPD M8 is almost completely blocked. ‘Significantly higher activity than HPD M8 (p<0.05). Abbreviations: M8: human HPD M8 enzyme exposed to NTCB; M8-NTBC: human HPD M8 enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0054] Fig. 10. Rescreening results of activity and tolerance of human HPD enzyme single variants after recombination of all positions in the presence of 6 pm NTBC. Four replicates of each variant were used in the rescreening assay and challenged with 6 pM NTBC. Most variants harbor 25-70 % activity at 6 pM NTBC while the HPD M8 is almost completely blocked. ‘Significantly higher activity than HPD M8 (p<0.05). Abbreviations: M8: human HPD M8 enzyme exposed to NTCB; M8-NTBC: human HPD M8 enzyme not exposed to NTBC, considered as 100% active; EV: empty vector control exposed to NTBC; EV-NTBC: empty vector control not exposed to NTBC.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one or ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0057] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0058] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0059] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0060] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1 % or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

[0061] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0062] Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.

[0063] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0064] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0065] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0066] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0067] Current treatment options for patients with diseases associated with an impaired tyrosine metabolism pathway, such as HT1 , AKU or HKU, are generally limited to treatment with 2-(2- nitro-4-(trifluoromethyl)benzoyl)cyclohexane-l, 3-dione, also known as nitisinone or NTBC, previously marketed as a herbicide. It is a potent inhibitor of the human HPD enzyme which is an Fe(l Independent non-heme oxygenase of critical importance in tyrosine metabolism. Triketones such as nitisinone are (ex-)herbicides and potent inhibitors of the human HPD enzyme. They are currently successfully applied in the clinic to treat HT1 patients. Furthermore, these triketones been clinically evaluated and are approved for the treatment of AKU and have been suggested to be beneficial for the treatment of HKU as well. Inhibition of the human HPD enzyme by triketones however causes an upstream metabolic block that prevents the production of the toxic metabolites and prevents severe illness and even death of the patient. However, triketone treatment does not provide a real cure because it does not correct the genetic defect or replace the defective cells and inflicts a new disease state, characterized by hypertyrosinemia with debilitating effects. The use of cell replacement or gene modification treatment is, however, hampered by its inability to correct or replace all the affected cells. Any remaining deficient cell (hepatocyte, kidney cell, ...) that is left untreated, uncorrected or not replaced, will produce toxic metabolites and puts the patient’s health at risk. This is the most pronounced in HT1 patients for which each untreated hepatocyte harbours a potential risk for progressing into a liver cancer cell due to the cell-autonomous pathophysiology of the disease. Therefore, HT1 , AKU and HKU patients need to remain under triketone treatment at all times to prevent the production of toxic metabolites by untreated defective cells (e.g. hepatocytes and / or kidney cells). This renders any therapy that aims to actually correct the genetic defect ineffective due to upstream metabolic block, resulting in a toxic accumulation of tyrosine levels in those patients. This is for example also illustrated in Fig. 1 .

[0068] With the present invention, a novel therapeutic approach for patients with diseases associated with an impaired tyrosine metabolism pathway was identified in which degradation of tyrosine can be restored. In particular, proteins or polypeptides with human HPD activity, but featuring significantly decreased sensitivity to HPD inhibitors were developed. As such, any accumulated tyrosine due to HPD inhibitor treatment is degraded by said proteins or polypeptides. As such, tyrosine metabolism pathway is completely restored.

[0069] In one aspect, the present invention provides a recombinant polypeptide comprising a human HPD enzyme, wherein said human HPD enzyme comprises at least one amino acid substitution as compared to the native human HPD enzyme, said native human HPD enzyme represented by SEQ ID NO: 1. Typical for said recombinant polypeptide is that at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor. In the context of the present invention, a decreased sensitivity to an HPD inhibitor means that at the lowest concentration of said inhibitor at which the wildtype polypeptide is almost completely blocked, the recombinant polypeptide is still active and at concentrations at which the wildtype enzyme is partially active, the recombinant polypeptide is more active. In a further particular embodiment, a decreased sensitivity to an HPD inhibitor means that at the lowest concentration of said inhibitor at which the wildtype polypeptide is completely blocked, the recombinant polypeptide at least shows 5% activity of the wildtype polypeptide. In another embodiment, decreased sensitivity to an HPD inhibitor means that at the lowest concentration of said inhibitor at which the wildtype polypeptide is completely blocked, the recombinant polypeptide at least shows 10% activity of the wildtype polypeptide.

[0070] The inventors further identified that at least one amino acid substitution preferably occurs in the critical region of the native human HPD enzyme (SEQ ID NO: 1) defined by amino acid positions 117 to 177 (SEQ ID NO: 2), in the critical region of the native human HPD enzyme defined by amino acid positions 270 to 326 (SEQ ID NO: 3), or in the critical region of the native human HPD enzyme defined by amino acid positions 378 to 393 (SEQ ID NO: 4).

[0071] In some embodiments, the at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, in the critical region of SEQ ID NO: 1 defined by amino acid positions 1 17 to 126, in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 151 , in the critical region of SEQ ID NO: 1 defined by amino acid positions 152 to 153, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236, in the critical region of SEQ ID NO: 1 defined by amino acid positions 261 to 272, in the critical region of SEQ ID NO: 1 defined by amino acid positions 274 to 283, in the critical region of SEQ ID NO: 1 defined by amino acid positions 284 to 292, in the critical region of SEQ ID NO: 1 defined by amino acid positions 293 to 303, in the critical region of SEQ ID NO: 1 defined by amino acid positions 304 to 313, in the critical region of SEQ ID NO: 1 defined by amino acid positions 323 to 326, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393 (SEQ ID NO: 4). A schematic overview of these critical regions and its sequences is provided in Table 1 .

[0072] In some embodiments, the recombinant polypeptide disclosed herein comprises at least one, preferably two or more, amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177. Optionally, the recombinant polypeptide may further comprise at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, at least one amino acid substitution in the critical region of SEQ ID NO:1 defined by amino acid positions 67 to 71 , at least one amino acid substitution in the critical region of SEQ ID NO:1 defined by amino acid positions 157 to 169, and / or at least one amino acid substitution in the critical region of SEQ ID NO:1 defined by amino acid positions 231 to 236.

[0073] In some embodiments, the recombinant polypeptides disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising at least one, preferably two or more, of the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO: 1 , (f) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (g) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 ; (h) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (i) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ;_(j) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (k) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 , (I) a tyrosine at the amino acid position corresponding to amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1 , (m) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (n) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (o) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ; or (p) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

[0074] In some embodiments, the recombinant polypeptides disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; and (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0075] In some embodiments, the recombinant polypeptides disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO: 1 , (f) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (g) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 , (h) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (i) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (j) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (k) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 , and (I) a tyrosine at the amino acid position 384 of SEQ ID NO: 1 , and wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0076] For example, the recombinant polypeptides disclosed herein comprise the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , and (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 5.

[0077] For example, the recombinant polypeptides disclosed herein comprise the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 , (g) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (h) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , and (i) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 6.

[0078] For example, the recombinant polypeptides disclosed herein comprise the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 7.

[0079] For example, the recombinant polypeptides disclosed herein comprise the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a tyrosine at the amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 8.

[0080] In some embodiments, the recombinant polypeptides disclosed herein comprise the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 1 18 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1.

[0081] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 25.

[0082] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 26.

[0083] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 27.

[0084] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 28.

[0085] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 29.

[0086] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ,. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 30.

[0087] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 31.

[0088] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 32.

[0089] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 33.

[0090] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (j) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 34.

[0091] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (k) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1. The sequence of this recombinant polypeptide is represented by SEQ ID NO: 35.

[0092] For example, the recombinant polypeptide disclosed herein comprises the following amino acid substitutions as compared to the native sequence of the human HPD enzyme (represented by SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 , (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 , (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , (d) glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 , (e) glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 , (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 . The sequence of this recombinant polypeptide is represented by SEQ ID NO: 36.

[0093] Table 1. Overview of SEQ IDs representing the human HPD enzyme, fragments of the human HPD enzyme, and mutated variants of the human HPD enzyme.

[0094] Another aspect of the invention provides a recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway, the polypeptide comprising a human HPD enzyme, said human HPD enzyme comprising at least one amino acid substitution as compared to the native human HPD enzyme (SEQ ID NO: 1) and wherein said at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor.

[0095] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, or at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393.

[0096] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393. In some embodiments, the recombinant polypeptide for use as disclosed herein further comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393.

[0097] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76.

[0098] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169.

[0099] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

[0100] In some embodiments, the recombinant polypeptide for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway as disclosed herein, comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

[0101] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 1 17 to 126; in particular an amino acid substitution to an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1.

[0102] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, in particular an amino acid substitution to a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153; in particular an amino acid substitution to an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 , and / or an amino acid substitution to an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1.

[0103] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177; in particular an amino acid substitution to a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1.

[0104] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326; in particular an amino acid substitution to a valine at amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , and / or an amino acid substitution to a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , and / or an amino acid substitution to an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 , and / or an amino acid substitution to an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and / or an amino acid substitution to an methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 .

[0105] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393; in particular an amino acid substitution to a tyrosine at the amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1.

[0106] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76; in particular an amino acid substitution to a phenylalanine at the amino acid position 71 of SEQ ID NO: 1.

[0107] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169; in particular an amino acid substitution to a leucine at the amino acid position 164 of SEQ ID NO: 1.

[0108] In some embodiments, the recombinant polypeptide for use as disclosed herein comprises at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236; in particular an amino acid substitution to a threonine at the amino acid position 235 of SEQ ID NO: 1 .

[0109] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising at least one, preferably two or more, of the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO: 1 , (f) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (g) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 ; (h) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 , (i) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (j) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (k) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 , (I) a tyrosine at the amino acid position corresponding to amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1 , (m) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (n) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (o) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , or (p) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

[0110] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; and (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0111] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO: 1 , (f) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (g) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 , (h) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (i) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (j) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (k) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 , and (I) a tyrosine at the amino acid position 384 of SEQ ID NO: 1 , and wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0112] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0113] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0114] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0115] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; and (i) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0116] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0117] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0118] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0119] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0120] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0121] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0122] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0123] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0124] In some embodiments, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0125] For example, the recombinant polypeptides for use as disclosed herein comprise a human HPD enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): (a) an isoleucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) a glutamic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , wherein the amino acid substitutions result in decreased sensitivity of the recombinant HPD enzyme to an HPD inhibitor.

[0126] In all aspects of the invention, an HPD inhibitor is a compound that reduces the activity of the 4- hydroxyphenylpyruvate dioxygenase (HPD) enzyme. In a further embodiment, the HPD inhibitor of the invention belongs to the class of triketone. Preferably the HPD inhibitor of the invention belongs to the class of the triketone and is selected from nitisinone (also referred to as NTBC), mesotrione, sulcotrione, benzobicyclin, or tembotrione. In still another embodiment, the HPD inhibitor belongs to the class of the diketonitriles, such as isoxaflutoles and pyrazolones, such as pyrazolate, pyrazoxyfen, benzofenap, pyrasulfotole ortopramezone. In another embodiment, the HPD inhibitor of the invention is leptospermone. In yet another embodiment, the HPD inhibitor of the invention is a human HPD inhibitor, such as pyrazolone-quinazolone hybrids or pyrazole-benzimidazolone hybrids.

[0127] In specific embodiments, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , wherein at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor.

[0128] In another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , wherein at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor, and wherein said at least one amino acid substitution occurs in the critical regions of SEQ ID NO: 1 defined by amino acid positions 1 17 to 177 (SEQ ID NO: 2), in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 323 (SEQ ID NO: 3) or in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393 (SEQ ID NO: 4), in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76 (SEQ ID NO: 22), in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169 (SEQ ID NO: 23), or in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236 (SEQ ID NO: 24).

[0129] In another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , wherein at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor, and wherein the at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 1 17 to 126, in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 151 , in the critical region of SEQ ID NO: 1 defined by amino acid positions 152 to 153, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, in the critical region of SEQ ID NO: 1 defined by amino acid positions 261 to 272, in the critical region of SEQ ID NO: 1 defined by amino acid positions 274 to 283, in the critical region of SEQ ID NO: 1 defined by amino acid positions 293 to 303, in the critical region of SEQ ID NO: 1 defined by amino acid positions 304 to 313, in the critical region of SEQ ID NO: 1 defined by amino acid positions 323 to 326, in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 284 to 292.

[0130] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , wherein two or more amino acid substitutions result in decreased sensitivity to an HPD inhibitor and wherein those two or more amino acid substitutions occur in the critical region of SEQ ID NO: 1 defined by amino acid positions 1 17 to 126, in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 151 , in the critical region of SEQ ID NO: 1 defined by amino acid positions 152 to 153, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, in the critical region of SEQ ID NO: 1 defined by amino acid positions 261 to 272, in the critical region of SEQ ID NO: 1 defined by amino acid positions 274 to 283, in the critical region of SEQ ID NO: 1 defined by amino acid positions 293 to 303, in the critical region of SEQ ID NO: 1 defined by amino acid positions 304 to 313, in the critical region of SEQ ID NO: 1 defined by amino acid positions 323 to 326, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393, in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236, or in the critical region of SEQ ID NO: 1 defined by amino acid positions 284 to 292.

[0131] In yet another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , wherein one or more amino acid substitutions result in decreased sensitivity to an HPD inhibitor and wherein said one or more amino acid substitutions are selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a threonine or serine at the amino acid position corresponding to amino acid position 166 of SEQ ID NO: 1 ; (f) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (g) a valine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO: 1 ; (h) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (i) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (j) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 ; (k) a methionine at the amino acid position corresponding to amino acid position 323 of SEQ ID NO: 1 ; (I) a tyrosine at the amino acid position corresponding to amino acid position 384 of SEQ ID NO: 1 , (m) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (n) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , (o) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , or (p) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 .

[0132] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0133] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; and (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0134] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0135] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0136] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0137] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0138] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , and (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , and one or more amino acid substitutions selected from the group consisting of (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ; (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0139] In still another embodiment, the HPD enzyme of the invention has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 , said HPD enzyme comprises the following amino acid substitutions selected from the group consisting of: (a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ; (b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ; (c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ; (d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ; (e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ; (f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ; (g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 ; (h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1 , (i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1 , (j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ; (k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 , and (I) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1 , and wherein said amino acid substitutions result in decreased sensitivity to an HPD inhibitor.

[0140] For the purpose of the present invention, the HPD of the present invention may also comprise further modifications, for example, wherein some amino acids (e.g. 1 to 10 amino acids) have been replaced, added or deleted for cloning purposes, to make transit peptide fusion, and the like, which retains HPD activity, i.e. the property of catalysing the conversion of parahydroxyphenylpyruvate to homogentisate, or can be any HPD that can be further improved.

[0141] The present invention also discloses a recombinant nucleic acid molecule encoding the recombinant polypeptide or recombinant HPD enzyme according to any one of the embodiments described herein. This recombinant nucleic acid molecule is an isolated polynucleotide, or biologically active portion thereof. Also provided is the recombinant nucleic acid molecule as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

[0142] In all the aspects of the invention, the term “recombinant” encompasses polynucleotides or polypeptides that have been manipulated with respect to the native polynucleotide or polypeptide, such that the polynucleotide or polypeptide differs (e.g. in chemical composition or structure) from what is occurring in nature. In another embodiment, an “isolated” or “recombinant” polynucleotide is free of internal sequences (i.e. introns) that naturally occur in the genomic DNA of the organism from which the polynucleotide is derived. A typical example of such polynucleotide is a so-called complementary DNA (cDNA).

[0143] In some aspects, the present application is directed to a polypeptide. In some aspects, the application is directed to nucleic acid molecules. The term ‘nucleic acid’ generally encompasses DNA, RNA, and DNA / RNA hybrid molecules. The term ‘gene’ is well-known in the art and in general refers to a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and / or other functional sequence regions. Genes typically comprise a coding sequence encoding a gene product, such as an RNA molecule or a polypeptide. The term “protein” as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native proteins, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally-occurring protein parts that ensue from processing of such full-length proteins.

[0144] The term “polypeptide” as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally-occurring polypeptide parts that ensue from processing of such full-length polypeptides. The term “peptide” as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.

[0145] In another aspect, the present invention discloses a vector comprising the recombinant nucleic acid molecule as disclosed herein. Also provided is a vector as disclosed herein for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway. The vector can be selected from an adeno-associated viral vector, a lentiviral vector, an episomal vector based on e.g. the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system. Said vector can also be a non-viral vector including, but not limited to, lipid nanoparticles. Said vector can also be a donor plasmid containing homologous arms to the human HPD gene targeting human chromosome 12 (NC_000012.12) from nucleotide 121 ,834,618 until 121 ,893,520 and containing in between these homologous arms a recombinant nucleic acid sequence according to this invention. In some embodiments, said vector is an adeno-associated viral vector; even more preferred an adeno-viral pseudotype vector with liver tropism. In another embodiment, the adeno-associated viral vector is an AAV pseudotype 8 vector. Other adeno-associated viral vectors that can be used are vectors selected from AAV2, AAV5, AAV9, AAV-LK03, AAV3b, AAVrhI O, AAV-NP40 and AAV-NP59. In an even more preferred embodiment, adeno-associated viral vectors are selected from AAV-LK03, AAV3b, AAVrhI O, AAV-NP40, AAV-NP59.

[0146] A recombinant AAV vector, is defined herein as an infectious, replication-defective virus composed of an AAV protein shell, encapsidating a heterologous DNA molecule of interest which is flanked on both sides by AAV ITRs. A rAAV virion may be produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (i.e. containing a recombinant nucleic acid molecule of interest) into recombinant virion particles for subsequent gene delivery.

[0147] The term “transfection” is used herein to refer to the uptake of foreign DNA by a cell. A cell has been transfected when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. Such techniques can be used to introduce one or more exogenous DNA moieties, such as a plasmid vector and other nucleic acid molecules, into suitable host cells. The term refers to both stable and transient uptake of the genetic material.

[0148] The term “transduction” denotes the delivery of a DNA molecule to a recipient cell either in vivo of vitro, via a replication-defective viral vector, such as via a recombinant AAV virion.

[0149] In some embodiments, said vector is an adeno-associated viral vector with tissue or cell- restricted expression, for example, an adeno-associated viral vector with hepatocyte-restricted expression, such as the AAV2, AAV5, AAV8, AAV-LK02, AAV3b, AAVrhI O, AAV-NP40 or AAV- NP59 vector.

[0150] The vectors according to the different embodiments of the invention can further be used in combination with the CRISPR-Cas system for gene editing (see e.g. Yin et al., Biotechnology, 32: 551-3, 2014). For example, therapeutic CRISPR-Cas9 components can be delivered via viral vectors, such as adeno-associated viral vectors as described above, and in combination with one of the vector systems according to the present invention.

[0151] In another embodiment, the vectors according to the different embodiments of the invention can also be combined with zinc finger nuclease technology and transcription activator-like effector nuclease (TALEN) technology.

[0152] In another embodiment, the vectors of the present invention comprise the recombinant nucleic acid molecule according to the invention in combination in with any other recombinant nucleic acid molecule. An example of such a vector system could be a bicistronic IRES vector containing an internal ribosome entry site that allows the simultaneous expression of two proteins; in particular the simultaneous expression of a recombinant protein according to the present invention in combination with any other recombinant protein. Another example of such a vector system could be a polycistronic 2A vector containing one or more self-cleaving 2A linker sequences that allows the simultaneous expression of two or more proteins; in particular the simultaneous expression of a recombinant protein according to the present invention in combination with any other recombinant protein.

[0153] In a further aspect, the present invention also provides genetically engineered host cells or a host virus comprising a recombinant nucleic acid molecule or a vector according to any one of the embodiments of the invention. In some embodiments, genetically engineered host viruses comprising a recombinant nucleic acid molecule or vector as disclosed herein are provided. In some embodiments, genetically engineered host cells are provided. Preferably said genetically engineered host cell is a bacterial host cell, or a mammalian host cell, such as a human host cell. In a most preferred embodiment, said host cell is a bacterial host cell.

[0154] In a preferred embodiment, said engineered host cell is a bacterial host cell. In said aspect, the bacterial host cell of the invention can be operably linked to a promoter that is inducible by exogenous environmental conditions.

[0155] In a further embodiment, said genetically engineered host cell is a bacterial host cell wherein at least one of the nucleic acid molecules encoding a human HPD enzyme according to this invention is operably linked to a promoter that is inducible by exogenous environmental conditions. In an even further embodiment, said exogenous environmental conditions are specific conditions found in the gut of a mammal. In a further preferred embodiment, said promoter is the fumarate and nitrate reductase regulator sensor promoter (Pfnrs) is used to allow expression of the recombinant HPD gene when the bacterial cell is present in a micro-aerobic or anaerobic environment, such as for example the gut of a mammal.

[0156] In another embodiment, the genetically engineered host cell is a bacterial host cell selected from a non-pathogenic bacterium or a probiotic bacterium. In still another embodiment, the genetically engineered host cell is a bacterial host cell selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In a preferred embodiment, the bacterial host cell is E. coll Nissle. In said instance, E. coll Nissle will be modified to express the recombinant HPD enzyme in combination with HmgABC, which encodes HmgA, HmgB and HmgC enzymes of the tyrosine metabolism. In still a further embodiment, said non-pathogenic bacterium or probiotic bacterium wherein at least one of the nucleic acid molecules encoding a human HPD enzyme according to the invention is operably linked to a promoter that is inducible by environmental conditions. In still a further embodiment, said exogenous environmental conditions are specific conditions found in the gut of a mammal. In a further preferred embodiment, said promoter is the fumarate and nitrate reductase regulator sensor promoter (Pfnrs) is used to allow expression of the recombinant HPD gene when the bacterial cell is present in a micro-aerobic or anaerobic environment, such as for example the gut of a mammal.

[0157] In another aspect, the genetically engineered host cell can be a human host cell, such as for example a patient-derived stem cell. In an even more preferred embodiment, the genetically engineered host cell of the present invention is a patient-derived hepatocyte, kidney cell, induced pluripotent stem (iPS) cell or other stem cell.

[0158] In various embodiments of the invention, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may referto any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, or parenteral. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraintestinal, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophylized powders. In one embodiment, the inventive compositions are injected intravenously. In another embodiment, the inventive compositions are administered intraintestinal.

[0159] In one aspect, a pharmaceutical composition refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and / or excipient.

[0160] In another aspect, a pharmaceutical composition refers to a preparation of recombinant adenoviral vectors according to any of the embodiments, which other components such as a physiologically suitable carrier and / or excipient.

[0161] In a further aspect, the present invention provides recombinant polypeptides, recombinant HPD enzymes, recombinant nucleic acid molecules, vectors, genetically engineered host cells, genetically engineered viruses or pharmaceutically acceptable compositions as disclosed herein for use in the treatment of diseases associated with an impaired tyrosine metabolism pathway. Said inventive therapeutics may be administered by any appropriate techniques, as will be readily appreciated by those skilled in the art. For instance, any suitable gene therapeutics approach may be implemented to deliver the DNA of interest in accordance with various embodiments of the present invention. In one embodiment, an adeno-associated viral vector according to any of the embodiments is used to target to deliver the recombinant HPD enzyme to target cells (e.g. in the liver or kidney). In some embodiments, mRNA-loaded lipid nanoparticles (LNPs) are used to target the delivery of the recombinant HPD enzyme as -M- disclosed herein to target cells, such as target cells in the liver or kidney of the subject. For example, the recombinant HPD enzyme can be delivered to the liver via mRNA-loaded LNPs, and as a consequence the liver will function as a production facility that produces, uses, as well as secretes the recombinant HPD enzymes in the bloodstream of the subject.

[0162] As used herein, the terms “subject”, “individual”, or “patient” are used interchangeably throughout this specification, and typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term “non-human animals” includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chicken, amphibians, reptiles, etc. In certain embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods and uses as taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as foetuses, whether male or female, are intended to be covered.

[0163] As used throughout this specification, the terms “therapy” or “treatment” refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. The terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. The terms “therapy” or “treatment” broadly refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a disease associated with an impaired tyrosine metabolism pathway. Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and / or slow progression of the disease. The terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition. In certain embodiments, the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment. The term may encompass ex v / vo or in vivo treatments.

[0164] As already described above, the present invention provides inventive therapeutics for use in the treatment of diseases associated with an impaired tyrosine degradation pathway. Those diseases can be any diseases in which the tyrosine degradation pathway is disturbed. In a further embodiment, said diseases are selected alkaptonuria (AKU), hawkinsinuria (HKU), hereditary tyrosinemia (HT) type 1 , type 1 B, HT type 3, tyrosine hydroxylase (TH) deficiency, Parkinson’s disease, restless syndrome (RLS), tyrosinase (TYR) deficiency and / or oculocutaneous albinism (OCA). In an even more preferred embodiment, the recombinant polypeptide, the recombinant HPD enzyme, the recombinant nucleic acid molecule, the vector, the genetically engineered host cell or the pharmaceutically acceptable composition according to any one of the different embodiments of the invention is for use in the treatment of alkaptonuria (AKU), hawkinsinuria (HKU), hereditary tyrosinemia (HT) type 1 , HT type 1 b and HT type 3.

[0165] In a further aspect, the present invention provides recombinant polypeptides, recombinant HPD enzymes, recombinant nucleic acid molecules, vectors, genetically engineered host cells or pharmaceutically acceptable compositions in combination with an HPD inhibitor for use in the treatment of diseases associated with an impaired tyrosine metabolism pathway. In some of the diseases associated with an impaired tyrosine metabolism pathway, treatment with an HPD inhibitor, such as for example nitisinone, remains essential, in order to prevent accumulation of homogentisic acid (HGA) or maleyl- and fumarylacetoacetate and succinylacetone (SA) in the body. Therefore, in some aspects, the recombinant polypeptides, recombinant HPD enzymes, recombinant nucleic acid molecules, vectors, genetically engineered host cells or pharmaceutically acceptable compositions are administered in combination with an HPD inhibitor for use in the treatment of diseases associated with an impaired tyrosine metabolism pathway, such as alkaptonuria (AKU), hawkinsinuria (HKU), hereditary tyrosinemia (HT) type 1 and HT type 3.

[0166] In another aspect, a method for identifying HPD inhibitors comprising a human HPD enzyme with decreased sensitivity to said HPD inhibitors is disclosed. Said method comprises in vitro culturing a genetically engineered host cell according to any of the preceding embodiments under conditions in which a nucleic acid molecule encoding a recombinant polypeptide of any of the preceding embodiments is expressed, exposing said recombinant polypeptide to one or more chemical compound, and identifying one or more chemical compounds with HPD inhibition features. In said method, cells expressing the HPD gene will be exposed directly to the HPD inhibitor of the invention in combination with the substrate of HPD, 4-hydroxyphenylpyruvate, or indirectly via exposure to tyrosine. In a further embodiment, said method for identifying HPD inhibitors is performed in a high-throughput screening setup. EXAMPLES

[0167] Example 1 : Engineering of potent HPD inhibitor insensitive human HPD variants using directed protein evolution and evaluation of different recombinant human HPD enzymes for their sensitivity to HPD inhibitors

[0168] In this study, human HPD variants with significantly increased tolerance towards NTBC and other p-triketone inhibitors were successfully engineered

[0169] Using directed protein evolution (DPE) technology a large number of artificially engineered human HPD enzyme variants were generated and their sensitivity to an HPD inhibitor was evaluated. DPE is a technology that artificially accelerates evolution by more than six orders of magnitude through iterative rounds of mutagenesis. The design of directed evolution experiments follows the same basic evolutionary algorithm and thus relies on the simple but powerful Darwinian principles. It all begins with a parent protein, i.e. template, in the present case the parent HPD enzyme. First, the template is mutagenized to produce a library of mutant genes whereafter the proteins encoded by these mutant genes are produced and screened for their desired function. The improved proteins are subsequently selected and used for another round of DPE until the desired goal is reached and no further improvements are found or possible. The success is defined by the feasibility of the desired function and whether measurable improvements are accumulated to reach the goal.

[0170] Here, DPE is used to engineer human HPD variants with increased tolerance towards NTBC (HPDANTBC, such as the M8 variant) but without altering its catalytic function. The DPE strategy that is used herein is the KnowVolution approach, which combines random and focused mutagenesis strategies to improve enzyme properties with minimal screening efforts. The generated library is subsequently screened for tolerance to NTBC.

[0171] MATERIALS AND METHODS

[0172] Materials

[0173] All chemicals were of analytical-reagent grade or higher quality and were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), AppliChem (Darmstadt, Germany) or Carl Roth GmbH (Karlsruhe, Germany). All enzymes were purchased from New England Biolabs (Beverly, USA), Sigma Aldrich (Hamburg, Germany) or ThermoFisher Scientific (Waltham, MA, USA). NTBC was purchased from Yecuris (Tualatin, OR, USA). Oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany) or ThermoFisher Scientific (Waltham, MA, USA) in salt free form. A thermal cycler (Mastercycler gradient; Eppendorf, Hamburg, Germany) and thin-wall PCR tubes (Multi ultra-tubes; 0.2 mL; Carl Roth, Germany) were used in all PCRs. Plasmid extraction and PCR purification kits were purchased from Macherey-Nagel GmbH & Co. (Duren, Germany), Sigma Aldrich (Hamburg, Germany) and Qiagen (Hilden, Germany). DNA concentrations were quantified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA). Microtiter plates for protein expression (96-wells; V-shaped) and for absorbance measurements (96-wells, flat-bottom) (Greiner Bio-One GmbH, Frickenhausen, Germany) were incubated in a Multitron II Infers shaker (Infers AG, Bottmingen, Switzerland) or Innova™ 42R Incubator Shaker (Eppendorf, Hamburg, Germany). Fordetection of absorbance, Tecan Sunrise® orTecan Infinite® M200 pro (Tecan group Ltd, Mannedorf, Switzerland) plate readers or Victors multilabel plate reader (Perkin Elmer, Waltham, MA, USA) were used.

[0174] Bacterial strains and plasmids

[0175] E. coli DH5a (Stratagene, La Jolla, CA, USA) was used as a host for DNA maintenance and propagation, and E. coli C43 (DE3) (Lucigen Corporation, Middleton, Wl, USA) was used as a host for recombinant protein expression in all KnowVolution studies. For construction of the expression vector, the plasmid pET42b(+) (Novagen, Darmstadt, Germany) was used, containing a gene for resistance to kanamycin. A negative control, or EV, was obtained with pET42b(+) plasmid introduction in E. coli.

[0176] Gene cloning, construction of expression vectors cDNA of WT human HPD (NM_002150.2) was obtained from Geneart in a standard pMA-T- based cloning plasmid (Thermo Scientific), with an optimized codon usage for E. coli. The HPD gene was subcloned using restriction enzymes and ligase-based cloning as an Ndel-Xhol fragment in the multiple cloning site of pET42b(+), transformed into E. coli DH5a and sequenced. LB was used as the standard medium supplemented with KANA (50 pg / mL) to maintain selection pressure. DNA Sanger sequencing was conducted at GATC Biotech AG (Cologne, Germany) and sample preparation was performed according to GATC’s instructions. Sample requirement was 5 pL of purified plasmid DNA (50 - 100 ng / pL) and 5 pL of 5 pM primer of T7 FW (5’- TAA TAC GAC TCA CTA TAG GG - 3’ SEQ ID NO: 9; Tm 53.2°C, GC 40 %) or T7 RV (5’- GCT AGT TAT TGC TCA GCG GT - 3’ SEQ ID NO: 10; Tm 54.5°C, GC 47 %). BioEdit 7.2 Sequence Alignment Editor was used for all sequencing alignments.

[0177] KnowVolution of HPD

[0178] Generation of an HPD error-prone library

[0179] Primer design: PLIcing relies on the amplification of the target gene and the vector backbone by PCR using primers with complementary phosphothioester (PTO) nucleotides, containing a sulfur atom, at the 5’-end. As such, 12 PTO nucleotides (at the 5’-end of all four primers) have to be identified that will later represent the points of DNA hybridization (see Table 2). The primers were custom made and purchased from Invitrogen and the lyophilized primers are dissolved in 1x TE buffer (10 mM Tris pH 7.0, 1 mM EDTA). Table 2: Custom designed PTO primers for the HPD insert and vector backbone where nucleotide connecting phosphates contain a sulfur atom. The PTO nucleotides are located at the 5’-end of the primers.

[0180] EpPCR’. After identifying the best reaction conditions and the optimal annealing temperature for both PCR reactions, the vector fragment pET42b(+) was amplified by using template (pET42b(+)-HPD, 50 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and primers (Fw vector + Rv vector (see table 2), 0.4 pM). Following PCR conditions were applied: 98°C for 3 minutes; 98°C for 30s, 58.3°C for 30 s, 72°C for 2 minutes (25 cycles); 72°C for 5 minutes.

[0181] The Fw and Rv insert (HPD) PTO primers were used to amplify the human HPD gene and generate the epPCR libraries. The quality of the PCR products was checked by agarose gel electrophoresis.

[0182] Construction of an HPD error-prone library with PLICinq

[0183] EpPCR libraries were cloned using the PLICing method. The Dpnl digested and purified fragments were diluted or concentrated. The epPCR fragments of the insert and the vector backbone, were each digested to produce single-stranded overhangs. Subsequently, the cleaved vector backbone and cleaved insert are mixed and hybridized. The resulting epPCR HPD variant library was transformed by heat shock into chemically competent E. coli C43 (DE3) following a standard protocol and plated on LBKANA agar plates for expression, and screening and selection of the HPD variants with improved tolerance towards NTBC (vide infra). Subjected to the same procedure, a religation vector control was made by mixing the vector backbone fragment with water instead of insert, and cleavage buffer. Colonies on the EV control agar plate should not be more than 10 % of the colonies on the insert plates. Controls for Dpnl digestion were done by dilution of the respective fragment with water (no cleavage buffer added) and agar plates with Dpnl controls must be empty (< 10 colonies).

[0184] Generation of site saturation mutagenesis library

[0185] 22c trick primer design-. To reduce codon redundancy and subsequent screening effort, a special mixture of three primer pairs is used to obtain a balanced distribution of codon per amino acid. A degeneracy of 22 unique codons for the 20 canonical amino acids is created. This is accomplished by using two degeneracy carrying primers, one with NDT (12 unique codons - Rv: AHN) and the other with VHG (9 unique codons - Rv: CDB), and a TGG containing primer (one codon - Rv: CCA). The necessary ratio for employing the 22c-trick is 12:9:1 (NDT : VHG : TGG). By employing this technique and taking into account a 3-fold factor to achieve 95% library coverage, only 66 colonies (3 x 22) per position need to be sampled. These primers are dissolved in (DNase and RNase free) H2O according to the manufacturer’s instructions, mixed accordingly (12:9:1) and diluted until the appropriate working concentration. SSM was performed on each position individually identified after epPCR library screening, namely 118, 137, 151 , 153, 166, 177, 278, 302 and 307 of SEQ ID NO: 1 .

[0186] SSM PCR SSM on the HPD WT was employed to elucidate the role of each substitution identified in epPCR and a modified QuikChange method was used. By introducing a pre-PCR, single-primer extension stage, it overcomes the drawback of primer dimerization. The procedure consists of two stages: (I) two extension reactions are performed in separate tubes, one containing the Fw primer and the other containing the Rv primer and (II) the two reactions are mixed and the standard QuikChange method is used.

[0187] Molecular modeling

[0188] Computational analysis'. The starting structure of the human HPD was taken from the crystal structure of HPD (PDB ID: 3ISQ). HPP substrate and NTBC inhibitor were sketched and minimized in Yasara Structure Version 17.4.17. The protein residues were treated using the AMBER ff99. The ligand atoms were treated using GAFF with AM1-BCC partial charges employing particle mesh Ewald for long range electrostatic interactions and a direct force cutoff of 10.5 A. For molecular docking, crystal water molecules were deleted. The HPD structure was minimized using a water box first with steepest descent and then simulated annealing (timestep of 2 fs, atom velocities scaled down by 0.9 every 10th step) starting from 98K, 198K and 298K with a time averaged Berendsen thermostat until convergence was reached. The minimized structure of HPD was further used for molecular docking studies. A grid box of 12 A around the active site was applied by centering CO2+ion. Molecular docking calculations were performed using Autodock4.2 plug-in within Yasara with a fixed protein backbone. 100 docking runs were carried out and the docking solutions were clustered applying a RMSD cutoff of 0.5 A with the default settings provided within the YASARA dock run macro file. Conservation analysis was carried out using The Consurf Server. Graphics were prepared using Pymol, and Chimera visualization programs.

[0189] Generation of a multi-site directed mutagenesis library

[0190] After screening the SSM libraries generated for all positions, MSDM was employed to recombine all beneficial substitutions at the influential positions in order to obtain one single variant with a high tolerance towards NTBC. In this focused mutagenesis method, the modified QuikChange method with the two-step PCR was used. SDM primers with one degenerate codon were designed for each position and mixed at equal ratio’s in primers for region 1 (R1 - position 118, 138, 151 & 153, 177 of SEQ ID NO: 1 , referred to as M5) and region 2 (R2 - position 278, 302 & 307 of SEQ ID NO: 1 , referred to as M3). For the mutagenic PCR for region 1 or 2, template (pET42b(+)-HPD, 20 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and Fw R1 or R2 primer mix OR Rv R1 or R2 primer mix (0.4 pM) was used. Following PCR conditions were applied forthe first stage: 98°C for 1 minute; and 98°C for 30 s, 60°C for 30 s and 72°C for 3 minutes (3 cycles). For the second stage, after mixing the tubes containing Fw and Rv primer, following conditions were applied: 98°c for 1 minute; 98°C for 30 s, 60°C for 30 s, 72°C for 3 minutes (18 cycles); and 72°C for 8 minutes. Following the PCR, the quality of the PCR product was checked by agarose gel electrophoresis. The PCR products were digested by Dpnl (20U; NEB) at 37°C overnight to remove residual template and transformed by heat shock into E. coli C43 (DE3) for expression.

[0191] After plasmid preparation of the generated HPD M3 variant, this template was used for a second round of SDM PCR with the R1 primers. For the mutagenic PCR for region 1 , template (pET42b(+)-HPD-M3, 20 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and Fw R1 primer mix OR Rv R1 primer mix (0.4 pM) was used. Following PCR conditions were applied for the first stage: 98°C for 1 minute; and 98°C for 30 s, 60°C for 30 s and 72°C for 3 minutes (3 cycles). For the second stage: 98°c for 1 minute; 98°C for 30 s, 60°C for 30 s, 72°C for 3 minutes (18 cycles); and 72°C for 8 minutes was applied. Following the PCR, the quality of the PCR product was checked by agarose gel electrophoresis. The PCR products were digested by Dpnl (20U; NEB) at 37°C overnight to remove residual template and transformed by heat shock into E. coli C43 (DE3) for expression, and screening and selection of the ultimate HPD variant with high tolerance towards NTBC.

[0192] Focused mutagenesis on HPD lead variant

[0193] SSM based on HPD interspecies differences

[0194] 22c-trick primers were designed to employ SSM on the HPD M8 variant to elucidate the role of 15 new target positions identified after interspecies comparison. The amino acid differences between the human HPD enzyme and (I) the HPD enzymes of mice, rat, pig and cynomolgus monkey and (II) between maize were compared. When comparing human HPD with maize HPD, we selected the amino acid positions that were conserved in both human and maize HPD and were substituted by Siehl et al. in maize HPD to make the latter tolerant to triketones. Amino acid positions were also selected that are different between human and maize HPD, but after DPE of maize HPD are substituted to the amino acid present in human HPD. A modified QuikChange method was used. Forthe mutagenic PCR for each position, template (pET42b(+)- HPD M8 variant, 20 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and Fw 22c-trick primer mix OR Rv 22c-trick primer mix (0.4 pM) was used. Following PCR conditions were applied for the first stage: 98°C for 1 minute; and 98°C for 30 s, 60°C for 30 s and 72°C for 3 minutes (3 cycles). For the second stage: 98°c for 1 minute; 98°C for 30 s, 60°C for 30 s, 72°C for 3 minutes (18 cycles); and 72°C for 8 minutes. Following the PCR, the quality of the PCR products was checked by agarose gel electrophoresis. The PCR products were digested by Dpnl (20U; NEB) at 37°C overnight to remove residual template and transformed by heat shock into E. coli C43 (DE3) for expression and screening.

[0195] SDM based on HPD interspecies differences

[0196] After screening the SSM libraries generated for the aforementioned positions, SDM was employed to recombine all beneficial substitutions at the influential positions in order to obtain one single variant with a higher tolerance towards NTBC than WT HPD or HPD-M8 (also referred to as R1 / R2 or HPDAtriketone). In this focused mutagenesis method, the modified QuickChange method with the two-step PCR was used. SDM primers with one degenerate codon were designed for position 270, 323 and 384 of SEQ ID NO: 1. Each substitution was individually incorporated WT HPD and lead variant HPD. For the mutagenic PCR, template (pET42b(+)- HPD or pET42b(+)-HPD M8, 20 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and Fw primer OR Rv primer mix (0.4 pM) was used. Following PCR conditions were applied forthe first stage: 98°C for 1 minute; and 98°C for 30 s, 60°C for 30 s and 72°C for 3 minutes (3 cycles). For the second stage, after mixing the tubes containing Fw and Rv primer, following conditions were applied: 98°c for 1 minute; 98°C for 30 s, 60°C for 30 s, 72°C for 3 minutes (18 cycles); and 72°C for 8 minutes. Following the PCR, the quality of the PCR product was checked by agarose gel electrophoresis. The PCR products were digested by Dpnl (20U; NEB) at 37°C overnight to remove residual template and transformed into E. coli C43 (DE3) for expression.

[0197] High Throughput Screening (HTS) assay

[0198] For the colorimetric assay, recombinant E. coli HPD cells were induced with 1 mM IPTG, after reaching OD600 0.6-0.8 in LBKANA medium. Simultaneously, the medium was mixed with a volume of 7.5 mg / ml Tyr, and if appropriate, a triketone inhibitor. After incubation, the culture was centrifuged and OD405 of the supernatant was measured. For the HTS in 96-well MTP format, induced E. coli HPD cells, supplemented with Tyr, were added to each well of a 96-well V-bottom expression MTP. After incubation, the plate was centrifuged and supernatant was transferred into a 96-well flat bottom MTP for measurement at OD405 with an absorbance plate reader.

[0199] Cultivation and expression in 96-well plates - Screening assay

[0200] Colonies grown on LBKANA plates (after transformation of the epPCR, SSM, SDM or MSDM libraries) were transferred, by using sterile toothpicks, into a 96-well microtiter plates (conical shaped) containing 100 pL LB + 1 % glucose (non-inducing medium) supplemented with KANA (50 pg / rnL). After 16h of cultivation in a Multitron microtiter plate shaker (30°C, 900 rpm, 70% RH), 25 pL of each well was replicated into a second and thirds series of 96-well microtiter plates (conical shaped) containing 175 pL LBKANA with 1 mM IPTG for protein expression and 0.75 mg / mL Tyr in the absence or presence of NTBC. The first set of plates (referred to as the masterplate) were stored at -80°C after addition of 50 pL 50% (v / v) glycerol. The clones in the second and third expression plates were cultivated for 24h in a Multitron microtiter plate shaker at 37°C, 900 rpm and 70% RH for screening. After incubation, the culture was centrifuged at 3200 g for 10 minutes and 150 pL supernatant was transferred into a 96-well flat bottom MTP for absorbance measurement at OD405 with the Tecan® absorbance plate reader. The last column of the 96-well masterplate and expression plate is reserved for four replicates of WT human HPD (positive control) and four replicates of EV (negative control).

[0201] Rescreen inq assay

[0202] The variants of interest (defined after the screening assay) were subjected to a second round of screening by means of a rescreening assay where increasing concentrations of NTBC are used. For cell growth, the variants in the wells of the masterplate were scraped by means of a toothpick and inoculated in LBKANA + 1 % glucose (non-inducing medium) for overnight incubation at 37°C, 250 rpm and 70% RH in a shaking incubator. For expression, the overnight culture was diluted in fresh LBKANA and incubated in a flask (30°C and 250 rpm) up to an ODeoo of 0.1 using a spectrophotometer (Eppendorf Biophotometer Plus). When ODeoo reached 0.6 - 0.8, the medium was supplemented with 1 mM IPTG for protein expression and 0.75 mg / mL Tyr. 200 pL of the variant culture medium was transferred to a 96-well microtiter plate (conical shaped) and challenged by increasing concentrations of NTBC with four replicates of each variant per concentration. The WT human HPD and EV were challenged by the same increasing concentrations of NTBC. The rescreen plate was incubated at 37°C, 900 rpm and 70% RH in a Multitron II for 24h. After incubation, the culture was centrifuged at 3200 g for 10 minutes and 150 pL supernatant was transferred into a 96-well flat bottom MTP for absorbance measurement at OD405 with the Tecan® absorbance plate reader.

[0203] Dose response inhibition curves

[0204] The obtained HPD enzyme variant (M8), in conjunction with the WT HPD, was challenged by a concentration range of mesotrione (0 - 15 pM), NTBC (0 - 20 pM), sulcotrione (0 - 20 pM) and tembotrione (0 - 40 pM) with 4 replicates per concentration (n = 20). Ochronotic pigment formation and measurements were performed as previously described. The normalized OD405 (OD405-N) was calculated according to OD405-N = OD405 - OD405-EV (Eq. 1) where OD405-EV = measured OD405Of the EV. The activity percentage, Act. %, was calculated with GraphPad Prism 7 software according to the equation Act. % = (OD405-N / OD405-M) * 100 (Eq. 2), where OD405-M is the mean of OD405 measured at 0 pM (i.e. 100% activity). Whole cell dose response experiment with Tyr

[0205] The HPD M8 enzyme variant and the HPD WT enzyme were challenged with different concentrations of Tyr (0 - 0.001 - 0.01 - 0.05 - 0.1 - 0.25 - 0.5 - 1 - 2.5 - 5 - 10 mM) with 4 replicates per concentration. The same reaction was also set-up, but with the addition of 2 pM NTBC, i.e. the concentration where the WT is completely blocked and HPD M8 is 100 % active. The assay and measurements were performed as previously described.

[0206] Purification and sequencing of HPD variants

[0207] The remaining cell suspension of the overnight culture (vide supra - rescreening assay) was further used for plasmid preparation using the Nucleospin® Plasmid DNA Purification kit from Macherey-Nagel GmbH & Co. (Duren, Germany) according to the manufacturer’s instruction. The isolated plasmid DNA was quantified at 260 nm using a Nanodrop spectrophotometer and stored at -20°c until further use. DNA Sanger sequencing was conducted at GATC Biotech AG (Cologne, Germany) with the T7 FW and RV primers and BioEdit 7.2 Sequence Alignment Editor was used for all sequencing alignments.

[0208] Statistical analysis

[0209] For blank correction, the mean OD405 of EV+NTBC is subtracted from OD405 of the selected HPD variants (epPCR, SSM, SDM, MSDM), HPD WT or HPD M8 with NTBC, HPD WT or HPD M8 without NTBC and EV with or without NTBC. OD405 values are then normalized for HPD WT or HPD M8 without NTBC. Activities of these new generated HPD variants were compared to the activity of HPD WT or HPD M8 with NTBC. A one-way ANOVA was performed followed by a post hoc test with Bonferroni correction at significance level 5 % (a = 0.05). Significant difference at p < 0.05. The data is reported as mean with 95 % confidence interval (Cl). The statistical analyses of the data were performed using GraphPad Prism 7.0.

[0210] RESULTS

[0211] KnowVolution

[0212] Screening and rescreeninq the error prone PCR mutant library of HPD

[0213] Random mutant EpPCR libraries were generated by using 0.1 mM MnCh and low fidelity DNA polymerase, with balanced deoxyribonucleotide triphosphates (dNTP). Colonies on the EV control agar plate were not more than 10 % of the colonies on the insert plates and agar plates with Dpnl controls were empty. One epPCR library was screened (~ 1700 clones) using a robust, colorimetric bacterial whole-cell HTS assay based on the expression of recombinant HPD. Briefly, when WT human HPD is expressed in bacteria they will produce a melanin-like ochronotic pigment, after addition of Tyr to the medium. This pigment is also known as pyomelanin and exhibits a characteristic brown color. The formation of pyomelanin, and as such the activity of HPD, can be spectrophotometrically determined by measuring the absorption at 405 nm. In the presence of NTBC, this ochronosis process will be reduced or even prevented when HPD activity is blocked. When an HPD variant exhibits increased tolerance towards NTBC, it will produce this brown pigment even in the presence of increasing concentrations of NTBC. Wild-type human HPD exhibits a dose-dependent decrease in metabolic activity with increasing concentrations of nitisinone as shown by the decreasing intensity of the brown pigment. Human HPD variants with decreased sensitivity towards nitisinone will also produce this brown pigment at higher nitisinone and other triketone inhibitor concentrations versus wildtype HPD (Fig. 2). The inhibitory concentration of NTBC where approximately 50 % of the WT enzyme is active, is 0.25 pM NTBC. Screening was thus performed in parallel, in the absence or presence of 0.25 pM NTBC. To obtain a higher selection pressure, screening was later performed at 0.5 pM. This lead to the selection of nine variants with potentially increased tolerance after screening 1700 mutants that harbor influential amino acid positions for NTBC tolerance.

[0214] The selected variants were subjected to a subsequent rescreening assay using a concentration range (0 - 1 pM) of NTBC to confirm previous observations of the screening assay and check for tolerance at higher concentrations. Subsequently after rescreening, these variants were sequenced to identify the beneficial amino acid substitutions. Some positions were identified multiple times, i.e. 151 and 302 (of SEQ ID NO: 1), but harbored different amino acid substitutions. The most beneficial variant, showed a 12-fold increase in tolerance to NTBC at 0.50 pM (1 pM; residual activity 4 - 10 %, see Fig. 3).

[0215] Site saturation mutagenesis

[0216] SSM studies were carried out by substitution of the nine identified positions in WT HPD with all of the other 19 canonical amino acids to identify and quantify individual contributions in order to improve NTBC resistance. The newly generated HPD variants, with new amino acids at the specified, individual positions, were again subjected to the aforementioned screening procedure (0 - 0.5 pM NTBC). To obtain 95% library coverage of the variant space, a minimum of 66 clones had to be screened per position (88 SSM clones per position were screened).

[0217] For each position individually, the best SSM variants were subjected to a rescreening assay with different concentrations of NTBC (0 - 0.5 - 1 - 2 pM). The variants giving the best improvement in tolerance were sequenced.

[0218] After epPCR the obtained positions and amino acid substitutions were M1181 / N, Q137R, N151 D, I153N, M166T, S177G, I278V, K302R, K307N (of SEQ ID NO: 1). Results showed that two positions (153 and 278) contributed more dominantly to the improved resistance towards NTBC (see fig. 4). SSM at positions 1 18, 151 , 166, 177 and 302 (of SEQ ID NO: 1) did not generate improved tolerance, while SSM at position 153 and 307 (of SEQ ID NO: 1) yielded N153E and K307P as the most beneficial. Due to a mistake with the SSM primer at position 137, an extra additional influential position and amino acid substitution was identified by coincidence, namely T138S. Finally, all beneficial amino acid substitutions that might contribute to NTBC-tolerance were recombined by multi-site directed mutagenesis. Molecular modeling

[0219] Computational modeling was carried out to understand the structural determinant for the experimentally identified positions of HPD. The human HPD is a homodimeric enzyme (PDB ID: 3ISQ) consisting of two monomeric subunits predicted by PISA server. The active center of HPD is highly conserved, and consists of CO2+ion coordinated to two histidine (H183 and H266) and one glutamic acid (E349). Molecular docking revealed that both the substrate (HPP) as well as the inhibitor (NTBC) binds close to the CO2+ion center (data not shown). We observed that the substrate HPP forms a hydrogen bond with N241 , G360, Q334 whereas NTBC forms a hydrogen bond with N241 and Q334. Other NTBC binding sites are P239, N241 , Q334, N363, F394 and L367.

[0220] Furthermore, computational analysis showed that the experimentally identified positions (M1 18, Q137, N151 , 1153, Q166, S177, I278, K302, and K307 of SEQ ID NO: 1) are scattered throughout protein surface away from the active metal center.

[0221] Two positions (Q166 and S177 of SEQ ID NO: 1) are situated at the dimeric interface while other positions are distributed over the protein surface. We found that positions M118, N151 , 1153, Q166, S177, and K307 are located in the loop region, I278 and K302 are located in the helix part, and Q137 and T138 are located in beta-sheets. Conservation analysis showed that most of the positions except K302 are non-conserved in nature and can thus be good candidates for the experiments.

[0222] After molecular modeling, SSM studies were carried out on the HPD M8 variant (vide infra) by substitution of possible influential positions in the catalytic site of HPD, i.e. positions 239, 241 , 334, 363, 364 and 367 to identify its possible individual contributions in order to improve NTBC resistance. The newly generated HPD variants, with new amino acids at specified, individual positions, were again subjected to the aforementioned screening procedure (4 - 5.5 pM NTBC). To obtain 95% library coverage of the variant space, a minimum of 66 clones had to be screened per position (88 SSM clones per position were screened). For each position individually, the best SSM variants were subjected to a rescreening assay with different concentrations of NTBC (5.5 - 7 - 8 - 9 pM). The variants giving the best improvement in tolerance were sequenced. Sequencing results showed that substitution of the WT amino acid at the computational defined positions with the other 19 non-WT amino acids through SSM resulted in non-functional enzymes (no production of pyomelanin) or functional enzymes with the original WT amino acid at the defined position.

[0223] Multi-Site-Directed mutagenesis

[0224] MSDM was performed to recombine all beneficial amino acid substitutions in one HPD enzyme variant to obtain high tolerance towards its inhibitor NTBC. The obtained positions after epPCR were divided in two regions, i.e. R1 (position 118, 138, 151 & 153, 177 of SEQ ID NO: 1 - also referred to as M5) and R2 (position 278, 302 & 307 of SEQ ID NO: 1 - also referred to as M3). PCR amplification was not influenced by the presence of multiple primers per reaction. Finally, HPD-M3 was used as a template to include all R1 positions in one single PCR reaction and eventually obtain one HPD variant containing all amino acid substitutions (M8 or R1 / R2) determined through epPCR and SSM. After transformation of the MSDM products, the resulting clones of HPD M5, M3 and M8 were subjected to a (re)screening assay at different concentrations of NTBC (2-6 pM).

[0225] As shown in figure 5 the WT HPD variant is completely blocked from 1 pM NTBC onwards. The HPD M5 variant, also referred to as the R1 variant, is 100% active at (A) 1 pM and (B) 2 pM NTBC, and (C) 30 - 40 % at 3 pM, while the M3 variant, also referred to as the R2 variant, has only (A) 75% at 1 pM and is completely blocked at (B) 2 and (C) 3 pM NTBC. (D) The final recombined variant, HPD M8, harbors still 100% metabolic activity at 4 pM NTBC, a 40-fold higher concentration NTBC at which the WT HPD is 100% blocked. M8 harbors approximately 50% activity at 5 pM and 5% at 6 pM.

[0226] The sequence of the M5 / R1 variant is represented in SEQ ID NO: 19. The sequence of the M3 / R2 variant is represented in SEQ ID NO: 20. The sequence of the recombined M8 variant, also referred to as the R1 R2 variant, is represented in SEQ ID NO: 21 (Table 3).

[0227] Table 3. Amino acid sequences of the M5 / R1 , M3 / R2 and M8 / R1 R2 variants. Dose response inhibition curves

[0228] The inhibition assay with the different triketone inhibitors was performed using IPTG-induced cultures of E. coli HPD, E. coli HPD M8 and E. coli EV mixed with Tyr and incubated for 24h at 37 °C (900 rpm, 70 % RH). Nonetheless, only NTBC is allowed to be used as a drug, and mesotrione, sulcotrione and tembotrione as herbicides due to differences in pharmacological profile in terms of magnitude, duration of activity towards HPD, and influence on plasma Tyr levels, it is still interesting to test HPD M8 tolerance towards the other triketones. In the HPD inhibitor assay wells, mesotrione, NTBC, sulcotrione and tembotrione were added in a concentration range from 0 pM - 15 pM, 0 - 20 pM, 0 - 20 pM and 0 - 40 pM respectively with 4 replicates per concentration (n = 20). Formation of the brown pyomelanin pigment was observed, and supplementing one of the inhibitors decreased pyomelanin formation with increasing concentrations. It was already visually observed that HPD M8 harbors a higher tolerance towards all triketone inhibitors than the WT version of HPD. A centrifugation step was added to avoid interference of the bacterial cells with the colorimetric quantification of the ochronotic pigment. The percentage of inhibition was calculated following Eq. 1 & Eq. 2. The calibration curves were fitted by the sigmoidal logistic four-parameter equations and graphically visualized in Fig. 6 A-D. The calculated IC50 values for each of the triketone inhibitors were 0.363 ± 0.013 pM (mesotrione), 0.195 ± 0.003 pM (NTBC), 0.164 ± 0.015 pM (sulcotrione) and 0.611 ± 0.015 pM (tembotrione) for HPD WT, and 5.103 ± 0.092 pM (mesotrione), 3.933 ± 0.499 pM (NTBC), 3.249 ± 0.397 pM (sulcotrione) and 5.504 ± 0.174 pM (tembotrione) for HPD M8 as shown in Fig. 6E and Table 4. The fold change in inhibitor tolerance was for mesotrione 14.052, NTBC 20.178, sulcotrione 19.773 and tembotrione 9.008. These IC50 values illustrate that inhibitor tolerance is not limited to NTBC only, but can be extrapolated to other triketone inhibitors as well.

[0229] Table 4. IC50 values of the engineered R1 R2 human HPDAtriketoneenzyme

[0230] Whole cell dose response experiment with tyrosine

[0231] Different concentrations of Tyr (0 - 10 mM) were tested to check if a low or high concentration of Tyr would have an effect on the production and formation of HGA and ochronotic pigment and thus indirectly on the Km value of the newly engineered variant opposed to the WT HPD. Km is defined as the affinity of an enzyme and is relative to the concentration of its substrate. It is important to check whether the rate of formation of product, HGA, will be affected by the availability of substrate and if this is altered for the HPD M8 variant. Hence, we challenged the WT HPD and HPD variant with different concentration of its indirect substrate, Tyr, by the use of our assay. We noticed that the HPD M8 variant is as responsive, with or without 2 pM NTBC, as the HPD WT enzyme regardless of variations in the concentration of its indirect substrate Tyr (see Fig. 7). If the directed evolution experiment had changed the Km value of the engineered variant, the HPD M8 enzyme reaction and ochronotic pigment production would differ from the WT, especially at low concentrations of Tyr.

[0232] Example 2: Identification and evaluation of additional beneficial mutations

[0233] Additional beneficial mutations in the HPD M8 variant were identified by performing site saturation mutagenesis on the amino acid positions 71 , 164, 235 and 286 of the HPD M8 variant. Subsequently, the variants were challenged with different concentrations of NTBC.

[0234] The amino acid sequences of the new HPD variants are disclosed in Table 5.

[0235] In a first experiment, the sensitivity of HPD M8 variants with an additional single mutation to 4 pM or 5 pM of the triketone inhibitor nitisinone (NTBC) was evaluated (Fig. 8 and 9). The following HPD enzyme variants were evaluated: M8 variant, M8 variant with L71 F substitution, M8 variant with A164L substitution, M8 variant with S235T substitution, M8 variant with L286M substitution. An empty vector was used as control, and both the M8 variant and the empty vector were also evaluated in the absence of NTBC (M8-NTBC and EV-NTBC respectively).

[0236] Figure 8 shows that most variants harbor 75% activity at 4 pM NTBC while the HPD M8 variant is almost completely blocked (only 5-10% activity) in the presence of NTBC.

[0237] Figure 9 shows that most variants still harbor 25-50% activity at 5 pM NTBC while the HPD M8 variant is almost completely blocked in the presence of NTBC.

[0238] In a next step, multiple additional mutations in the M8 variant were combined and their HPD activity was evaluated in the presence of 6 pM NTBC and compared to the HPD activity of the M8 variant.

[0239] The following HPD enzyme variants were evaluated: M8 variant, M8 variant with L71 F and S235T substitution, M8 variant with L71 F and L286M substitution, M8 variant with A164L and S235T substitution, M8 variant with A164L and L286M substitution, M8 variant with L71 F, A164L and S235T substitution, M8 variant with A164L, S235T and L286M substation, and M8 variant with L71 F, A164L, S235T and L286M substitution. An empty vector was used as control, and both the M8 variant and the empty vector were also evaluated in the absence of NTBC (M8- NTBC and EV-NTBC respectively).

[0240] Figure 10 shows that most HPD variants harbor 25-70% activity at 6 pM NTBC while the HPD M8 is almost completely blocked. Table 5. Amino acid sequences of the additional HPD variants that were created by generating additional mutations in the M8 variant. Additional mutations as compared to the M8 variant sequence are indication in bold and underlined.

[0241] Example 3: Integration of modified HPD enzymes in suitable viral vectors with liver tropism

[0242] In this example, HPDANTBCis integrated in a bicistronic AAV with liver tropism together with WT FAH and their therapeutic efficacy is evaluated in a preclinical Fah-deficient mouse model of HT1 (and / 7gc / -deficient mouse model of AKU) under NTBC treatment. At regular time points after injection, plasma SA, Tyr, Phe, and NTBC was monitored by LC-tandem-MS DBS assay. During the in vivo experiments, we noticed an influence of NTBC on the protein level of the molecular chaperone TTC36. TTC36 is a HPD-associated protein regulating its expression via PTMs as it blocks the STK33-mediated T382 phosphorylation of HPD, preventing the binding of FHA domain containing PELI1 to HPD and subsequent ubiquitylation and proteasomal degradation.

[0243] MATERIALS AND METHODS

[0244] AAV-vector construction and production

[0245] The AAV vectors were constructed with a transgene cassette encoding the gene(s) of interest, codon-optimized for Mus musculus, driven by the liver-specific thyroxine binding globulin (TBG) promotor, flanked by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and human p-globin sequences containing a polyadenylation signal (PolyA) site. The cassette is inserted between AAV2 ITRs. The bicistronic vectors harboring FAH and HPDANTBCfor multigene delivery contain a P2A linker peptide to mediate efficient, stoichiometric production of discrete protein products. Digestion ofthe transfer plasmid pAAV-TBG>Nhel / eGFP / BGIII:WPRE (Vector Builder, China), and the pMK-plasmids harboring the cDNA of the genes of interest (Geneart, Thermo Fisher Scientific), was obtained with the restriction enzymes Nhel and Bglll. Ligation products were transformed by heat shock in Stbl3 E. coli cells and LBAMPI agar plates were inoculated with the transformed bacteria for positive selection. Single colonies were picked, followed by a plasmid preparation and Sanger sequencing at Eurofins Genomics (TBG Fw primer: 5’ - AAA CTG CCA ATT CCA CTG CTG - 3’ (SEQ ID NO: 15) / WPRE Rv primer: 5’ - CAT AGC GTA AAA GGA GCA ACA - 3’ (SEQ ID NO: 16)). The packaging into capsids from AAV8, the production and purification of the various AAV2 / 8; AAV-fLuc-P2A-eGFP, AAV-FAH, pAAV-HPDwt, AAV-HPDANTBC, AAV-FAH-P2A-HPDwt and AAV-FAH-P2A-HPDANTBCvectors was outsourced to SignaGen Laboratories to obtain in vivo grade purity at > 1 E + 13 VG (see table 6) (rAAV packaging service, large scale - Catalog# SL100862). Briefly, the packaging of the ssAAV2 / 8 vectors was accomplished by co-transfection of (1) the above described transfer plasmid, (2) the chimeric packaging plasmid containing the Rep2 and Cap8 serotypes, and (3) the helper plasmid into HEK293 cells, which contain the adenovirus gene E1 +, to produce infectious AAV particles.

[0246] Table 6. Obtained viral titers of the different constructs after outsourcing it to SignaGen Laboratories.

[0247] AAV Genome copies titer

[0248] AAV8-fLUC-P2A-GFP 1 ,70E + 13 VG / mL

[0249] AAV8 - FAH 2,00E + 13 VG / mL

[0250] AAV8 - HPDwt 2,03E + 13 VG / mL

[0251] AAV8 - HPDANTBC2,00E + 13 VG / mL

[0252] AAV8 - FAH-P2A-HPDwt 1 ,71 E + 13 VG / mL

[0253] AAV8 - FAH-P2A-HPDANTBC2.36E + 13 VG / mL

[0254] AAV8 - FAH-P2A-HPDANTBC+T382A2.45E + 13 VG / mL

[0255] AAV8 - HPDANTBC+T382A1.10E + 13 VG / mL

[0256] FAH-deficient mouse model

[0257] The Fah5981SBstrain (referred to as HT1 mice), backcrossed on C57BI / 6J background and kindly provided by Markus Grompe (Oregon Health & Science University, Portland, OR, USA). The mice bear a single N-ethyl-N-nitrosourea-induced point mutation (G>A loss) leading to the splicing out of exon 7 within the Fah gene, resulting in a frameshift and subsequently the introduction of a premature stop codon at amino acid position 303. Consequently, the mice produce a truncated, unstable FAH protein that is degraded, making them a suitable model for HT1. If neonatal HT1 mice are not continuously administered NTBC, they die of acute liver failure. Therefore, all mice, except during the withdrawal experiment, received continuous NTBC treatment (8mg / L) through their drinking water. To prevent the formation and accumulation of Tyr and its toxic metabolites upon NTBC therapy discontinuation, the mice were provided ad libitum with an irradiated diet that was low in Tyr and Phe (LabDiet® 5LJ5 chow, LabDiet, St. Louis, MO, USA), which resembles the protein-restricted diet of HT1 patients.

[0258] Immune-deficient FAH-Knockout mouse model

[0259] The FRG mouse model (Yecuris), backcrossed on C57BI / 6J background, is characterized by a triple knockout of Fair1" and two immune-deficiency alleles, i.e. recombinant activation gene-2 (Rag2_ / ) and interleukin 2 receptor common gamma chain (112^'), and serves as a suitable (immunodeficient) model for HT1. As this is a knockout and not a transgenic mouse model, genotype reversion cannot occur. As in conventional Fah mice, NTBC withdrawal in FRG mice results in gradual hepatocellular injury and eventual death after 4-8 weeks. Therefore, all mice received continuous NTBC treatment (8 mg / L) through their drinking water.

[0260] Alkaptonuria mouse model

[0261] The homozygous Hgd knockout-first allele mouse (Hgd tm1 a_ / ), also backcrossed on a C57BI / 6J genetic background, is used as an animal model of human AKU (referred to as AKU mice). AKU mice are kindly provided by George Bou-Gharios (Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK). Hgd tm1 a / _mice contain an IRES:LacZ gene trap cassette and a promoter-driven neo cassette inserted into the fifth Hgd intron with the sixth exon flanked by loxP sequences. Homozygous Hgd tm1 a / _mice therefore show an AKU phenotype based on Hgd gene disruption while heterozygous Hgd tm1 a / +and homozygous Hgd tm1 a+ / +mice show a normal WT phenotype. Hgd tm1 a / _mice develop specific symptoms of AKU including blackening of urine and progressive osteoarthritis when NTBC is not administered429. Therefore, similar to the aforementioned experimental conditions of the HT1 mouse model, all mice received continuous NTBC treatment (8mg / L) through their drinking water. An irradiated diet low in Tyr and Phe (LabDiet® 5LJ5 chow, LabDiet, St. Louis, MO, USA) was provided ad libitum.

[0262] All animal procedures are carried out at the Vrije Universiteit Brussel and were approved by the Institutional Animal Ethics Committees (approved grant number 16-210-1 and 20-210-10).

[0263] Animal injections

[0264] Male and female mice of 12-weeks old are allocated randomly to two experimental groups. In the first group, mice were injected with 1012vector genomes (VG) in a volume of 100 pL diluted in PBS at room temperature. HT1 or AKU mutant control mice are injected similarly with the same volume of PBS (100 pL). After injection, all mice are maintained on NTBC throughout the experiment. Blood samples are periodically collected by venipuncture to evaluate the Tyr, Phe, SA and NTBC levels by LC-tandem MS DBS assay. Twelve weeks post injection, blood (± 800 - 1000 pL) is collected from Fair1- mice by cardiac puncture after anesthesia followed by immediate termination of the experiment by neck dislocation. Liver, kidney and brain tissue are collected. In vivo bioluminescence imaging

[0265] For the biodistribution analysis ofthe AAV-fLuc-P2A-eGFP vector containing the hepatic-specific TBG promoter, 10 months old male mice are intravenously injected into the tail vein with 100 pL of the purified AAV2 / 8-fLuc-P2A-GFP (1 x 1012VG / mouse). The mice are subjected to bioluminescence imaging (BLI) at week 2, 4 and 8 post injection using an in vivo optical imaging system (Photon I MAG ER™ Optima, Biospace, Nesles la Vallee, France). Before imaging, the mice are anesthetized with 2% isoflurane and oxygen, and are intraperitoneal injected with a D- luciferin substrate (30 mg / mL, Promega, Madison, Wl, USA) at a dose of 150 pg / g body weight whereafter images are taken during 15 minutes after injection. BLI of the individual livers is performed at week 8 post injection and in this case, all mice are euthanized by cervical dislocation and livers are dissected within 1 minute after a second D-luciferin substrate administration for ex vivo imaging of the livers. The visual output is represented as photons (ph) s-1cm-2steradian (sr)-1and displayed as a pseudo-color overlay onto a grey scale animal image using a color scale. For each time point, all images are scaled to a maximum (red) and minimum (blue).

[0266] Western blot

[0267] Frozen liver tissue samples are homogenized in a radio-immunoprecipitation assay buffer containing 1 % (w / v) protease and phosphatase inhibitor cocktail, and 1 % (w / v) EDTA 0.5 M (all from Thermo Scientific, Merelbeke, Belgium). The homogentisate is incubated on ice for 30 min and centrifuged for 15 minutes at 14,000g at 4 °C. Protein quantification is performed on the supernatant using the Pierce™bicinchoninic acid protein assay (Thermo Scientific) with BSA as reference standard.

[0268] Briefly, proteins are separated on 12% (v / v) Mini-Protean®TGX Stain-free™ gels (Bio-Rad, USA) using electrophoresis and blotted onto nitrocellulose Trans-Blot® Turbo™ Transfer Packs (BioRad, USA) using a Trans-Blot® Turbo™ transfer system (Bio-Rad, USA). Subsequently, blocking of the membranes is carried out with blocking buffer (5% (m / v) non-fatty milk in Tris-buffered saline solution (i.e. 20 mM Tris - 135 mM NaCI) containing 0.1 % (v / v) Tween-20). The membranes are incubated overnight at 4 °C with primary antibody diluted in blocking buffer followed by 1 -hour incubation at room temperature secondary antibody diluted in blocking buffer. Excessive antibody is removed by washing the membranes three times in Tris-buffered saline solution with 0.1 % (v / v) Tween-20. Detection of the stained proteins is performed by means of Pierce™ ECL Western Blotting substrate kit (Thermo Scientific, Rockford, IL, USA) and ChemiDoc MP imaging system (Bio-Rad, USA). Pixel density data was normalized by subtracting the averaged pixel density of three background areas from the sample data.

[0269] Isolation of RNA and Reverse Transcriptase-Polymerase Chain Reaction

[0270] All samples are collected in an RNA-protecting solution composed of RNAprotect Tissue

[0271] Reagent (Qiagen) and PBS (5:1 v / v). Total RNA is extracted from all samples using the GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich, Bornem, Belgium) according to the manufacturer’s instructions. The isolated RNA is quantified at 260 nm using a Nanodrop spectrophotometer (ThermoScientific, Wilmington, USA). Total RNA is reverse transcribed into cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad, Nazareth, Belgium) followed by cDNA purification with the GenElute PCR clean up kit (Sigma-Aldrich).

[0272] Quantitative rea-time PCR (qPCR) cDNA products are used for quantitative amplification of the target gene. All samples are done in duplicate, each run included two ‘no template controls (NTC)’ and a serial dilution of a pooled cDNA mix from all samples to estimate the qPCR efficiency. The data are only used when the calculated PCR efficiency ranged between 0.85 - 1 .15. TaqMan Fast Advanced Master Mix (see table 7, Life Technologies), 20X Assay-on-Demand Mix (Life Technologies) and cDNA are applied for RT-qPCR on a Quantstudio™ 3 system (Thermo Fisher Scientific).

[0273] For selecting reliable reference genes to normalize the qPCR data, the expression stability of five candidate genes is evaluated per experiment: glyceraldehyde-3-phosphate dehydrogenase (Gapdh), hydroxyl-methylbilane synthase (Hmbs), beta-actin (Actb), beta-2-microglobulin (B2m) and ubiquitin C (Ubc) using qbasePLUS® software (geNorm®, Biogazelle, Gent, Belgium). Results are analyzed with the 2-AACtformula and normalized against the reference gene mRNAs.

[0274] Table 7. TaqMan Gene expression assays, the accession number and amplicon length

[0275] Gene Assay on Demand ID Amplicon Length

[0276] Ttc36 Mm00507235_ml 84

[0277] Tat Mm01244282_ml 61

[0278] Gapdh Mm99999915_gl 107

[0279] Hmbs Mm01143545_ml 81

[0280] Actb Mm02619580_gl 143

[0281] Ubc Mm02525934_gl 176

[0282] B2m Mm00437762_ml 77

[0283] Site Directed Mutaqensis

[0284] For this focused mutagenesis method, the modified QuikChange method with the two-step PCR is used. An SDM primer with one degenerate codon is designed for position T382. For the mutagenic PCR template (pMK-HPDANTBCor pMK-FAH-P2A- HPDANTBC, 10 ng), dNTP mix (0.2 mM), Phusion DNA polymerase (Thermo Fisher, 2U / pL), high fidelity polymerase buffer and Fw or Rv primer (see table 8, 0.4 pM) is used. Following PCR conditions were applied for the first stage: 98°C for 1 minute; and 98°C for 30 s, 60°C for 30 s and 72°C for 3 minutes (3 cycles). For the second stage, after mixing the tubes containing Fw and Rv primer, following conditions are applied: 98°c for 1 minute; 98°C for 30 s, 60°C for 30 s, 72°C for 3 minutes (18 cycles); and

[0285] 72 C for 8 minutes. Following the PCR, the quality of the PCR product is checked by agarose gel electrophoresis. The PCR products are digested by Dpnl (20U; NEB) at 37°C for 20 minutes to remove residual template and transformed by heat shock into E. coli DH5a. After plasmid preparation, AAV vector construction and packaging is performed as previously described.

[0286] Table 8. FW and RV degenerate primer for position T382 for SDM (THR -> ALA)

[0287] RESULTS

[0288] An in vivo biodistribution study is conducted to evaluate the expression patterns of the transgene in HT1 mice with a luciferase reporter construct. The main target for gene-therapy for HT1 is the liver as HT1 has its origin there. To achieve effective liver transduction following systemic delivery the AAV serotype and a liver-specific promoter are considered. The AAV-fLUC-P2A- GFP is intravenously injected via the tail vein at a dose of 1 x 1012VG / mouse in the Fah-deficient HT1 mice under NTBC treatment. The construct is packaged using AAV serotype 8 (AAV8) to maximize liver-specific gene transfer and transduction. AAV8 harbors the ability to achieve a high level of hepatocyte gene transfer via peripheral vein administration. The liver-specific TBG promoter drives the construct and expression is further stabilized by the use of a posttranscriptional regulatory element, WPRE. It was previously demonstrated that the TBG promoter had an excellent and highly dynamic and stable activity in vivo, and has been frequently and successfully used to direct hepatic expression. BLI revealed that the AAV8-TBG- fLUC construct significantly augmented the expression of the luciferase reporter gene in the liver with no expression in other non-target tissues (data not shown). Furthermore, GFP protein expression via Western Blot on the liver lysates of HT1 mice injected with AAV8-fLUC-P2A-GFP showed that GFP was successfully expressed in the liver of all mice, except for the mice that did not receive an injection (data not shown).

[0289] In a next aspect, the therapeutic effects of AAV-FAH-P2A-HPDAntbcin a FAH-deficient HT1 mouse model under NTBC treatment will be evaluated. Fahmut / mutmice (age 8 wks) will be kept under NTBC treatment at all times and will be injected with (1) saline (sham control), (2) AAV- FAH-P2A-HPDWTand (3) AAV-FAH-P2A-HPDAntbcvectors, produced in example 1 . In addition, Fahwt / wt Httermates will be included as healthy controls. This will allow to determine the in vivo therapeutic efficiency of FAH in combination with HPDAntbcin orderto remove the metabolic block introduced by NTBC and fully restore the tyrosine catabolism pathway in transduced hepatocytes. At regular time points post-injection clinical parameters (plasma SA, tyrosine, phenylalanine and NTBC levels) will be evaluated. At 3, 6, and 12 weeks post-injection, the animals will be sacrificed and evaluation of liver injury (aspartate transaminase, AST; alanine transaminase, ALT; gamma-glutamyl transpeptidase, GGT), liver function (coagulation: partial thromboplastin time, PTT) and kidney function (serum creatinine) will performed.

[0290] Immunohistochemistry and qPCR analyses will be performed to determine the percentage of hepatocytes successfully expressing human FAH and HPDAntbc.

Claims

CLAIMS1. A recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, said human HPD enzyme comprising at least one amino acid substitution as compared to the native human HPD enzyme (SEQ ID NO: 1) and wherein said at least one amino acid substitution results in decreased sensitivity to an HPD inhibitor; for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

2. The recombinant polypeptide for use according to claim 1 , wherein at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 177 (SEQ ID NO: 2), in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326 (SEQ ID NO: 3), or in the critical region of SEQ ID NO: 1 defined by amino acid positions 378 to 393 (SEQ ID NO: 4).

3. The recombinant polypeptide for use according to claim 1 or 2, wherein at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, and at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177.

4. The recombinant polypeptide for use according to any one of claims 1 to 3, wherein at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 117 to 126, at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177, and at least one amino acid substitution occurs in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326.

5. The recombinant polypeptide for use according to any one of claims 1 to 4, wherein the humanHPD enzyme comprises the following amino acid substitutions:(a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ;(b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ;(c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ;(d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ;(e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ;(f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ;(g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ IDNO: 1 ; and(h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1.

6. The recombinant polypeptide for use according to any one of claims 1 to 5, further comprising at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, and / or at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

7. The recombinant polypeptide for use according to claim 5, wherein the human HPD enzyme further comprises:(i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1.

8. The recombinant polypeptide for use according to claim 7, wherein the human HPD enzyme further comprises:(j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ;(k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ; or(l) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

9. The recombinant polypeptide for use according to claim 7, wherein the human HPD enzyme further comprises:(j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ;(k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ; and(l) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

10. A recombinant nucleic acid molecule encoding a recombinant polypeptide as defined in any one of the preceding claims for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.11 . A vector comprising a recombinant nucleic acid molecule as defined in claim 10 for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

12. A genetically engineered host cell or host virus comprising a recombinant nucleic acid molecule as defined in claim 10 or a vector as defined in claim 11 ; preferably wherein the genetically engineered host cell is a viral host cell, a bacterial host cell, or a mammalian host cell; more preferably wherein the genetically engineered host cell is a bacterial host cell, for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

13. The recombinant polypeptide according to any one of claims 1 to 9, the recombinant nucleic acid molecule according to claim 10, the vector according to claim 11 , or the genetically engineered host cell or host virus according to claim 12, in combination with an HPD inhibitor for use in the treatment of a disease associated with an impaired tyrosine metabolism pathway.

14. The recombinant polypeptide, the recombinant nucleic acid molecule, the vector, the genetically engineered host cell or host virus, for use according to any of the preceding claims, wherein the disease associated with an impaired tyrosine metabolism pathway are selected from diseases associated with an impaired tyrosine degradation, diseases associated with impaired melanin synthesis or diseases associated with impaired dopamine synthesis; preferably selected from alkaptonuria (AKU), hawkinsinuria (HKU), hereditary tyrosinemia (HT) type 1 , HT type 1 b, HT type 3, tyrosine hydroxylase (TH) deficiency, Parkinson’s disease, restless leg syndrome (RLS), tyrosinase (TYR) deficiency and / or oculocutaneous albinism (OCA).

15. A recombinant polypeptide comprising a human 4-hydroxyphenylpyruvate (HPD) enzyme, said human HPD enzyme comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1): at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined byamino acid positions 117 to 126, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 138 to 142, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 143 to 153, and at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 173 to 177; optionally, further comprising at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 270 to 326.

16. The recombinant polypeptide according to claim 15, comprising the following amino acid substitutions as compared to the native human HPD enzyme (SEQ ID NO: 1):(a) an isoleucine or leucine at the amino acid position corresponding to amino acid position 118 of SEQ ID NO: 1 ;(b) a serine at the amino acid position corresponding to amino acid position 138 of SEQ ID NO: 1 ;(c) an aspartic acid at the amino acid position corresponding to amino acid position 151 of SEQ ID NO: 1 ;(d) an asparagine, glutamic acid or aspartic acid at the amino acid position corresponding to amino acid position 153 of SEQ ID NO: 1 ;(e) a glycine or glutamine at the amino acid position corresponding to amino acid position 177 of SEQ ID NO: 1 ;(f) a valine at the amino acid position corresponding to amino acid position 278 of SEQ ID NO: 1 ;(g) an arginine at the amino acid position corresponding to amino acid position 302 of SEQ ID NO: 1 : and(h) an asparagine or proline at the amino acid position corresponding to amino acid position 307 of SEQ ID NO: 1.

17. The recombinant polypeptide according to claim 15 or 16, further comprising at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 67 to 76, at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 157 to 169, and / or at least one amino acid substitution in the critical region of SEQ ID NO: 1 defined by amino acid positions 231 to 236.

18. The recombinant polypeptide according to claim 16, wherein the human HPD enzyme further comprises:(i) a phenylalanine at the amino acid position corresponding to amino acid position 71 of SEQ ID NO: 1.

19. The recombinant polypeptide according to claim 18, wherein the human HPD enzyme further comprises:(j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ;(k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ; or(l) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.

20. The recombinant polypeptide according to claim 18, wherein the human HPD enzyme further comprises(j) a threonine at the amino acid position corresponding to amino acid position 235 of SEQ ID NO: 1 ;(k) a leucine at the amino acid position corresponding to amino acid 164 of SEQ ID NO: 1 ; and(l) a methionine at the amino acid position corresponding to amino acid 286 of SEQ ID NO: 1.21 . A recombinant nucleic acid molecule encoding a recombinant polypeptide as defined in any one of the claims 15 to 20.

22. A vector comprising a recombinant nucleic acid molecule as defined in claim 21 .

23. A genetically engineered host cell or host virus comprising a recombinant nucleic acid molecule according to claim 21 or a vector according to claim 22; preferably wherein the genetically engineered host cell is, a bacterial host cell, a mammalian host cell or a human host cell; more preferably wherein the genetically engineered host cell is a viral host cell or a bacterial host cell.

24. A pharmaceutical composition comprising one or more genetically engineered host cells or viruses according to claim 23.

25. A method of producing a recombinant polypeptide comprising a human HPD enzyme with decreased sensitivity to an HPD inhibitor, said method comprising in vitro culturing the genetically engineered host cell of claim 12 or claim 23 under conditions in which a nucleic acid molecule encoding the recombinant polypeptide is expressed.

26. A method for identifying HPD inhibitors, said method comprising in vitro culturing the genetically engineered host cell of claim 12 or claim 23 under conditions in which a nucleic acid molecule encoding a recombinant polypeptide according to any one of claims 1 to 9 or claims 15 to 20 is expressed, exposing said recombinant polypeptide to one or more chemical compounds, and identifying one or more chemical compounds with HPD inhibition features.

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