Provided herein are biocatalysts and systems thereof for pterin-dependent enzymes and pathways and methods of making and using the same.

Provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CH1 such that they encode a fusion protein TH-CH1. Also provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding AADC is linked to the nucleotide sequence encoding TH such that they encode a fusion protein AADC-TH or TH-AADC. Further provided is a viral vector comprising such nucleotide sequences and its use in the treatment and/or prevention of Parkinson's disease.

The present invention relates to nucleic acid molecules from regions of Enterococcus spp. genomes which are associated with the production of dopamine The invention also relates to proteins encoded by such nucleic acid molecules as well as nucleic acid markers which are associated with high dopamine production. Moreover, the invention relates to uses of such molecules, including, but not limited to, transforming or transfecting cells or organisms with constructs containing the nucleic acid molecules to create cells or organisms with enhanced dopamine production. The present invention is also directed to kits for identifying bacteria which may be capable of producing dopamine based on the detection of the nucleic acid molecules.

The disclosure in some aspects, relates to nucleic acids, compositions and kits useful for gene therapy with reduced immune response to transgene products.

Provided herein are biocatalysts and systems thereof for pterin-dependent enzymes and pathways and methods of making and using the same. Provided herein in some embodiments are biocatalysts having a pterin source and a pterin-dependent enzymatic pathway biologically coupled to the pterin source. Tetrahydrobiopterin (referred to herein as BH4 or BH 4) can be the pterin source. The BH4 can be synthesized by a tetrahydrobiopterin synthesis pathway. The tetrahydrobiopterin synthesis pathway can include a GTP cyclohydrase; a pyruvoyl tetrahydropterin synthase; a sepiapterin reductase, and/or any combination thereof. The biocatalyst can further contain a pterin-dependent enzymatic pathway. The pterin-dependent enzymatic pathway can be amino acid mono-oxygenase, phenylalanine hydroxylase, tryptophan hydroxylase, tyrosine hydroxylase, nitric oxide synthase, alkylglycerol monooxygenase, and/or any combination thereof.

The present invention is directed to compositions and methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency. This invention includes a method of treating AADC deficiency in a pediatric subject, comprising the steps of: (a) providing a pharmaceutical formulation comprising an rAAV2-hAADC vector, (b) stereotactically delivering the pharmaceutical formulation to at least one target site in the brain of the subject in a dose of an amount at least about 1.8×10.sup.11 vg; wherein delivering the pharmaceutical formulation to the brain is optionally by frameless stereotaxy, and optionally wherein the dose is an amount of at least about 2.4×10.sup.11 vg and in some embodiments wherein the pharmaceutical formulation comprises a rAAV2-hAADC vector concentration of about 5.7×10.sup.11 vg/mL. This invention is also directed to methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency, wherein the method optionally further comprises the step of administering a therapeutically effective dose of dopamine-antagonist to the subject such as risperidone. This invention is also directed to methods for treating aromatic L-amino acid decarboxylase (AADC) deficiency, wherein the method optionally comprises providing a pharmaceutical formulation comprising an rAAV2-hAADC vector, and empty capsids.

The present disclosure relates to methods, formulations and devices for the delivery and therapeutic administration of polynucleotides encoding AADC. The present disclosure relates to methods, formulations and devices for the delivery and therapeutic administration of AAV vectors which include polynucleotides encoding AADC. The present disclosure relates to methods, formulations and devices for the delivery and therapeutic administration of polynucleotides encoding AADC in the treatment of neurological diseases, disorders and conditions, including Parkinson's Disease.

The disclosure relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides encoding AADC for the treatment of Parkinson's Disease.

Provided herein are engineered decarboxylase polypeptides that are useful for catalyzing the decarboxylation of amino acids such as L-tyrosine to produce tyramine or catalyzing the decarboxylation of L-DOPA to produce dopamine. Also provided are the preparation process of engineered decarboxylase polypeptides as well as reaction process under industrial-relevant conditions. The disclosure also provides polynucleotide sequences encoding engineered decarboxylase polypeptides, recombinant host cells capable of expressing engineered decarboxylase polypeptides, and methods of producing tyramine or dopamine using the engineered decarboxylase polypeptides. Compared to the wild type decarboxylase, the engineered polypeptide provided by this disclosure has better activity and/or stability. The use of the engineered polypeptides for the preparation of tyramine or dopamine reduces the production cost and has a good industrial application prospect.

The disclosure in some aspects, relates to nucleic acids, compositions and kits useful for gene therapy with reduced immune response to transgene products.