New enzyme composition

Abstract

The present invention provides a tyrosine hydroxylase (TH) variant lacking 60 to 120 amino acid residues at the N-terminus, and a pharmaceutical composition comprising the TH variant lacking 60 to 120 amino acid residues at the N-terminus and aromatic L-amino acid decarboxylase (AADC). The present invention also provides a nucleotide construct, a vector plasmid, a cell, or a virus comprising the TH variant or the pharmaceutical composition, and the use of the virus in the manufacture of a medicament for treating a neurodegenerative disease, such as Parkinson's disease.

Description

[Technical field]

[0001]

[0001] The present disclosure relates to a pharmaceutical composition comprising a tyrosine hydroxylase variant and an aromatic L-amino acid decarboxylase. The present disclosure further relates to a nucleotide construct comprising a polynucleotide encoding the tyrosine hydroxylase variant or a polynucleotide encoding said composition, a vector comprising said nucleotide construct, a cell prepared by transfection with said vector, and a virus comprising said nucleotide construct. The present disclosure further relates to the use of a virus in the manufacture of a medicament for treating a neurodegenerative disease (e.g., Parkinson's disease, PD), which belongs to the field of genetic engineering technology. [Background technology]

[0002]

[0002] Parkinson's disease (PD) is a severe neurodegenerative disorder characterized by cardinal symptoms including tremor, rigidity, and dyskinesia. The pathological hallmark of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) of the brain, resulting in impaired innervation of dopaminergic neurons and reduced dopamine concentrations in the striatum. Therefore, pharmacological methods that can enhance dopamine delivery to the striatum are effective therapeutic interventions for PD. Dopamine replacement therapy (i.e., oral levodopa, L-dopa) is currently the primary pharmaceutical treatment for PD. Although this dopamine replacement therapy significantly improves the quality of life of PD patients in the short term, its effectiveness decreases gradually over time. After more than 5 to 10 years, almost all PD patients eventually progress to a state that is nearly untreatable with oral L-dopa.

[0003] The aim of enzyme replacement therapy is to compensate for the reduced synthesis and secretion of dopamine caused by the degeneration of dopaminergic neurons in the SN. The mechanism underlying this therapeutic method is the delivery of genes encoding the enzymes necessary for dopamine synthesis to GABAergic neurons in the striatum, which results in the sustained de novo synthesis of dopamine in those GABAergic neurons and the release of the synthesized dopamine into the striatum. This therapy can improve dyskinesia and limit the side effects caused by the elevated levels of dopamine outside the basal ganglia. However, the elevated dopamine concentration limits the ability of tyrosine hydroxylase (TH) to synthesize ectopic dopamine by negatively regulating the activity of TH.

[0004]

[0004] Therefore, there is a substantial need to find enzyme replacement therapies with better therapeutic efficacy for the treatment of PD. Summary of the Invention

[0005]

[0005] In one aspect, the disclosure provides a tyrosine hydroxylase variant comprising the amino acid sequence set forth in SEQ ID NO:1 except for an N-terminal deletion of 60 to 120 amino acid residues, or a fragment, derivative or analog thereof having at least 80% sequence identity.

[0006]

[0006] In certain embodiments, the tyrosine hydroxylase variant comprises, except for an N-terminal deletion of 80 to 100 amino acid residues, the amino acid sequence set forth in SEQ ID NO: 1, or a fragment, derivative or analog thereof having at least 80% sequence identity.

[0007]

[0007] In certain embodiments, the tyrosine hydroxylase variant comprises, except for an N-terminal deletion of 80 to 90 amino acid residues, the amino acid sequence set forth in SEQ ID NO:1, or a fragment, derivative or analog thereof having at least 80% sequence identity.

[0008]

[0008] In certain embodiments, the tyrosine hydroxylase variant comprises the amino acid sequence set forth in SEQ ID NO:2, or a fragment, derivative or analog thereof having at least 80% sequence identity.

[0009]

[0009] In certain embodiments, the tyrosine hydroxylase variant further comprises a tag protein attached to the N-terminus or C-terminus.

[0010]

[0010] In a particular embodiment, the tag protein is HA, Myc or Flag.

[0011]

[0011] In certain embodiments, the tyrosine hydroxylase variant comprises the amino acid sequence set forth in SEQ ID NO:3.

[0012] In another aspect, the disclosure provides a composition comprising the tyrosine hydroxylase variant described above.

[0013]

[0013] In certain embodiments, the composition further comprises an aromatic L-amino acid decarboxylase.

[0014]

[0014] In a particular embodiment, the aromatic L-amino acid decarboxylase comprises an amino acid sequence set forth in any of SEQ ID NOs: 4-9, or a fragment, derivative or analog thereof having at least 80% sequence identity.

[0015]

[0015] In certain embodiments, the aromatic L-amino acid decarboxylase further comprises a tag protein attached to the N-terminus or C-terminus.

[0016]

[0016] In a specific embodiment, the tag protein is HA, Myc or Flag.

[0017]

[0017] In a particular embodiment, the aromatic L-amino acid decarboxylase has the amino acid sequence set forth in SEQ ID NO:10.

[0018]

[0018] In another aspect, the present disclosure provides a polynucleotide construct comprising a first polynucleotide encoding the tyrosine hydroxylase variant and / or a second polynucleotide encoding an aromatic L-amino acid decarboxylase as defined above.

[0019]

[0019] In certain embodiments, the first polynucleotide has a nucleotide sequence set forth in SEQ ID NO:12 or 13, or has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:12 or 13.

[0020]

[0020] In certain embodiments, the second polynucleotide has a nucleotide sequence set forth in any of SEQ ID NOs: 14-21, or has a nucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs: 14-21.

[0021]

[0021] In certain embodiments, the polynucleotide construct further comprises a promoter operably linked to the first polynucleotide and / or the second polynucleotide.

[0022]

[0022] In certain embodiments, the promoter comprises a neuron-specific promoter.

[0023]

[0023] In another aspect, the disclosure provides a vector comprising the above-described polynucleotide construct.

[0024] In certain embodiments, the first polynucleotide and the second polynucleotide are constructed in one vector or in different vectors.

[0025]

[0025] In a particular embodiment, the first polynucleotide and the second polynucleotide are constructed in a vector, and the vector further comprises a third polynucleotide inserted between the first polynucleotide and the second polynucleotide.

[0026]

[0026] In certain embodiments, the third polynucleotide encodes a self-cleavable sequence and / or an internal ribosome entry site (IRES).

[0027]

[0027] In certain embodiments, the vector is selected from the group consisting of a herpes simplex virus vector, an adenovirus vector, and an adeno-associated virus vector.

[0028]

[0028] In certain embodiments, the vector comprises a plasmid.

[0029]

[0029] In another aspect, the disclosure provides a host cell comprising or transfected with the above-described vector.

[0030]

[0030] In another aspect, the disclosure provides a virus comprising a viral genome that includes the polynucleotide construct described above or that includes a nucleic acid expressed from the polynucleotide construct described above.

[0031]

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising the virus described above and a pharma- ceutically acceptable carrier.

[0032]

[0032] In another aspect, the present disclosure provides the use of the tyrosine hydroxylase variant, the composition, the nucleotide construct, the vector, the host cell, the virus, or the pharmaceutical composition in the manufacture of a medicament for treating a neurodegenerative disease in a subject.

[0033] In certain embodiments, the neurodegenerative disease is Parkinson's disease.

[0034]

[0034] In certain embodiments, the subject is a mammal, preferably a human, rat or mouse.

[0035]

[0035] In another aspect, the present disclosure provides a method for treating a neurodegenerative disease in a subject in need of treatment, comprising the step of administering to the subject a therapeutically effective amount of the tyrosine hydroxylase variant, the composition, the nucleotide construct, the vector, the virus or the medicament.

[0036]

[0036] In certain embodiments, the neurodegenerative disease is Parkinson's disease.

[0037]

[0037] In certain embodiments, the subject is a mammal, preferably a human, rat or mouse.

[0038]

[0038] The present disclosure further provides the following embodiments.

[0039]

[0039] Embodiment 1. A tyrosine hydroxylase variant comprising a tyrosine hydroxylase having the amino acid sequence set forth in SEQ ID NO:1, but lacking 60 to 120 amino acid residues at the N-terminus.

[0040]

[0040] Embodiment 2. The tyrosine hydroxylase variant of embodiment 1, comprising a tyrosine hydroxylase having the amino acid sequence set forth in SEQ ID NO:1, but lacking 80 to 100 amino acid residues at the N-terminus.

[0041]

[0041] Embodiment 3. The tyrosine hydroxylase variant of embodiment 2, comprising a tyrosine hydroxylase having the amino acid sequence set forth in SEQ ID NO:1 but lacking 80 to 90 amino acid residues at the N-terminus.

[0042]

[0042] Embodiment 4. A tyrosine hydroxylase variant according to embodiment 2 or 3, comprising a protein having the amino acid sequence set forth in SEQ ID NO:2 or a tyrosine hydroxylase derivative having 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, preferably optionally further comprising a tag protein at the N-terminus or C-terminus, more preferably the tag protein is HA, Myc or Flag.

[0043]

[0043] Embodiment 5. The tyrosine hydroxylase variant of embodiment 4, comprising a protein having the amino acid sequence set forth in SEQ ID NO:3.

[0044]

[0044] Embodiment 6. A composition comprising at least one tyrosine hydroxylase variant according to any one of embodiments 1 to 5.

[0045]

[0045] Embodiment 7. The composition of embodiment 6, further comprising an aromatic L-amino acid decarboxylase.

[0046]

[0046] Embodiment 8. The composition according to embodiment 7, wherein the aromatic L-amino acid decarboxylase is a full-length aromatic L-amino acid decarboxylase comprising a protein having an amino acid sequence set forth in any one of SEQ ID NOs: 4 to 9, or an aromatic L-amino acid decarboxylase derivative having 80% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 9, and preferably optionally further comprising a tag protein at the N-terminus or C-terminus, more preferably the tag protein is HA, Myc or Flag.

[0047]

[0047] Embodiment 9. The composition of embodiment 8, wherein the aromatic L-amino acid decarboxylase has the amino acid sequence set forth in SEQ ID NO:10.

[0048]

[0048] Embodiment 10. A nucleotide construct comprising a polynucleotide encoding a tyrosine hydroxylase variant according to any one of embodiments 1 to 5 or a polynucleotide encoding a composition according to any one of embodiments 6 to 9.

[0049]

[0049] Embodiment 11. The nucleotide construct according to embodiment 10, wherein the polynucleotide encoding the tyrosine hydroxylase variant has a nucleotide sequence as set forth in SEQ ID NO: 12 or 13 or a nucleotide sequence having more than 80% identity to SEQ ID NO: 12 or 13, and / or the polynucleotide encoding the aromatic L-amino acid decarboxylase has a nucleotide sequence as set forth in any of SEQ ID NO: 14-21 or a nucleotide sequence having more than 80% identity to any of SEQ ID NO: 14-21.

[0050]

[0050] Embodiment 12. A vector plasmid comprising the nucleotide construct of embodiment 10 or 11.

[0051]

[0051] Embodiment 13. The vector plasmid according to embodiment 12, wherein the polynucleotide encoding the tyrosine hydroxylase variant and the polynucleotide encoding the aromatic L-amino acid decarboxylase are constructed in one vector plasmid or in different vector plasmids.

[0052]

[0052] Embodiment 14. The vector plasmid of embodiment 12, which is selected from the group consisting of a herpes simplex virus vector plasmid, an adenovirus vector plasmid, and an adeno-associated virus vector plasmid.

[0053]

[0053] Embodiment 15. A cell prepared by transfection with the vector plasmid according to any one of embodiments 12 to 14.

[0054]

[0054] Embodiment 16. A virus comprising as its genome the nucleotide construct of embodiment 10 or 11.

[0055]

[0055] Embodiment 17. A pharmaceutical composition comprising the virus of embodiment 16 and a pharma- ceutically acceptable carrier.

[0056]

[0056] Embodiment 18. Use of a tyrosine hydroxylase variant according to any one of embodiments 1 to 5, a pharmaceutical composition according to any one of embodiments 6 to 9, a nucleotide construct according to embodiment 10 or 11, a vector plasmid according to any one of embodiments 12 to 14, a cell according to embodiment 15, a virus according to embodiment 16 or a pharmaceutical composition according to embodiment 17 in the manufacture of a medicament for treating a neurodegenerative disease in a subject.

[0057]

[0057] Embodiment 19. The use according to embodiment 18, wherein the neurodegenerative disease is Parkinson's disease.

[0058]

[0058] Embodiment 20. The use according to embodiment 18, wherein the subject is a mammal, preferably a human, a rat or a mouse. [Brief description of the drawings]

[0059] [Figure 1]

[0059] As described in a specific embodiment of the present disclosure, construction of a recombinant AAV vector having an expression cassette comprising a human synapsin promoter, a polynucleotide encoding an HA-tagged variant of human tyrosine hydroxylase (TH), a T2A peptide, and a Myc-tagged human aromatic L-amino acid decarboxylase (AADC), a WPRE sequence, and a human growth hormone (hGH) poly(A) signal is shown. [Diagram 2]

[0060] 1 shows a statistical quantification graph of the expression of a series of enzyme compositions including TH with N-terminal deletions and full-length AADC to promote dopamine de novo synthesis in 293 cell lines as measured by high performance liquid chromatography (HPLC). GFP indicates a negative control. WT indicates full-length or wild-type TH in a dual-enzyme composition. Isob indicates another isoform of TH. 40 indicates TH with 40 amino acids deleted at the N-terminus. 60 indicates TH with 60 amino acids deleted at the N-terminus. 80 indicates TH with 80 amino acids deleted at the N-terminus. 90 indicates TH with 90 amino acids deleted at the N-terminus. 100 indicates TH with 100 amino acids deleted at the N-terminus. 120 indicates TH with 120 amino acids deleted at the N-terminus. 150 indicates TH with 150 amino acids deleted at the N-terminus. 164 indicates TH with 164 amino acids deleted at the N-terminus. 190 indicates TH with 190 amino acids deleted at the N-terminus. Error bars represent SEM. Ns, not significant. *p<0.05 and ****p<0.0001, one-way ANOVA. [Diagram 3]

[0061] Representative immunocytochemical images of anti-TH staining in the substantia nigra / ventral tegmental area (SN / VTA) region (top) and the caudate-putamen (CP) region (bottom) of brain sections in unilateral 6-OHDA-lesioned mice, an effective model of PD symptoms. The right side was the 6-OHDA-lesioned side, and the left side was the control side. Scale bar, 1 mm. [Figure 4]

[0062] Figure 4a shows a schematic diagram of the time course of stereotaxic surgery and apomorphine rotation test. Two weeks after unilateral 6-OHDA lesion, mice were selected for significant apomorphine-induced motor asymmetry, expressed as contralateral rotations. Both groups showed statistically equivalent rotation frequencies, calculated as net rotations (contralateral to ipsilateral) per minute. Two weeks later, selected mice were intrastriatally injected with a viral vector expressing a composition containing a human TH variant with a deletion of 90 amino acids at the N-terminus and full-length human AADC (TH90del / AADC). A GFP-expressing virus was injected as a control. Four weeks after virus injection, the apomorphine-induced rotation test was performed again to evaluate the functional benefit of the treatment. Figure 4b shows significant behavioral recovery in the group with TH90del / AADC virus injection, where the number symbols indicate significant recovery from the motor asymmetry phenotype in the TH90del / AADC virus injection group compared to the control group (GFP). Asterisks indicate significant recovery from the locomotor asymmetry phenotype in the TH90del / AADC group after virus injection. Error bars represent SEM. ###p<0.001 and ****p<0.0001, Student's t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060]

[0063] The present disclosure is further described below by specific embodiments and experimental data. For purposes of clarity, specific terms are used below, but these terms are not intended to define or limit the scope of the present disclosure.

[0061]

[0064] As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or multiple cells.

[0062]

[0065] Unless otherwise specified, as used herein, the word "or" is used in its inclusive sense of "and / or" rather than in its exclusive sense of "either / or."

[0063]

[0066] Unless otherwise specified, numerical ranges set forth herein can include each and every numerical value and each subrange within the range.

[0064]

[0067] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0065]

[0068] The present disclosure provides tyrosine hydroxylase variants.

[0066]

[0069] In one aspect, the disclosure provides a tyrosine hydroxylase (TH) variant comprising the amino acid sequence set forth in SEQ ID NO: 1, except for an N-terminal deletion of 60-120 amino acid residues. In other words, the TH variants provided herein are N-terminal deletion variants of full-length TH having the amino acid sequence of SEQ ID NO: 1. The TH variant lacks 60-120 amino acid residues at the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0067]

[0070] In certain embodiments, the N-terminal deletion has a length in the range of 60-120, 70-120, 80-120, 90-120, 100-120, 60-110, 60-100, 60-90, 70-110, 80-100, or 80-90 amino acid residues. In certain embodiments, the N-terminal deletion has a length of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids. In a particular embodiment, the N-terminal deletion begins at the first amino acid residue of SEQ ID NO:1; in other words, an N-terminal deletion of 60 amino acid residues means deletion of amino acid residues 1 through 60 of SEQ ID NO:1.

[0068]

[0071] In certain embodiments, the N-terminal deletion variant of TH is the biologically active fragment of TH.The term "biologically active fragment" as used herein refers to the polypeptide fragment of a particular protein, which can maintain the complete or at least partial function of the particular protein.Generally, the biologically active fragment of TH has at least 50% biological activity of TH, preferably 60%, 70%, 80%, 90%, 95%, 99% or 100% biological activity.

[0069]

[0072] The present disclosure provides a tyrosine hydroxylase (also referred to as TH) variant, the TH variant being a human TH having the amino acid sequence set forth in SEQ ID NO: 1, except for an N-terminal deletion of 60 to 120 amino acid residues. Preferably, the TH variant is a human TH having the amino acid sequence set forth in SEQ ID NO: 1, except for an N-terminal deletion of 80 to 100 amino acid residues. More preferably, the TH variant is a human TH having the amino acid sequence set forth in SEQ ID NO: 1, except for an N-terminal deletion of 80 to 90 amino acid residues, for example, a human TH having an N-terminal deletion of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues in SEQ ID NO: 1.

[0070]

[0073] In one embodiment, the TH variant is a TH having an N-terminal deletion of 90 amino acid residues. In one embodiment, the TH variant comprises a protein having the amino acid sequence set forth in SEQ ID NO:2.

[0071]

[0074] The present disclosure further provides fragments, derivatives, or analogs of the TH variants provided herein, and such fragments, derivatives, or analogs substantially maintain the biological function or bioactivity of the TH variants. The term "fragment" with respect to a polypeptide sequence or polynucleotide sequence means a portion of that sequence. The term "derivative" or "analog" with respect to the polypeptides provided herein (e.g., the TH variants and AADCs provided herein) includes, but is not limited to, (i) counterpart polypeptides with one or more conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residue substitutions), or (ii) counterpart polypeptides with one or more amino acid residues having a substituent, or (iii) counterpart polypeptides in which the polypeptide is fused or attached to another compound (e.g., a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) counterpart polypeptides formed by fusing the polypeptide with additional amino acid sequences (e.g., leader sequences, secretion sequences, sequences used to purify the polypeptide, proteinogen sequences, or fusion proteins). The fragments, derivatives, and analogs thereof as defined herein are within the scope of those skilled in the art.

[0072]

[0075] In one embodiment, the fragment, derivative or analog of the TH variant comprises an amino acid sequence having at least 80% (e.g., at least 80%, 90%, 95% or 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO:1. "Percent sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are the same as the amino acid (or nucleic acid) residues in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) compared to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the reference sequence. Conservative substitutions of amino acid residues are not considered to be identical residues. Alignment for the purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished, for example, using commonly available tools such as BLASTN, BLASTp (available on the US National Center for Biotechnology Information (NCBI) website, see also SF Altschul et al., J. Mol. Biol., 215:403-410 (1990); F. Stephen et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website, see also DG Higgins et al., Methods in Enzymology, 266:383-402 (1996); MA Larkin et al., Bioinformatics (Oxford, UK), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as appropriate for the alignment, for example by selecting an appropriate algorithm.

[0073]

[0076] In certain embodiments, a fragment, derivative or analog of a TH variant provided herein comprises an amino acid sequence having at least 80% (e.g., at least 80%, 90%, 95% or 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0074]

[0077] In certain embodiments, fragments, derivatives or analogs of TH variants are formed by substitution, deletion or addition of one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues in the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, fragments, derivatives or analogs of TH variants function as proteins having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. TH variants and their fragments, derivatives or analogs have at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 99%) of the activity of proteins having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0075]

[0078] In certain embodiments, the TH variant may further comprise a tag protein. As used herein, the terms "tag protein" and "protein tag" are interchangeable and refer to a polypeptide or protein fused to a target protein by in vitro DNA recombinant technology to facilitate expression, detection, tracking or purification of the target protein. Protein tags include, but are not limited to, His6, Flag, GST, MBP, HA, GFP or Myc. In certain embodiments, the tag protein includes, but is not limited to, HA, Myc or Flag. In certain embodiments, HA comprises the amino acid sequence of SEQ ID NO: 22. In certain embodiments, Myc comprises the amino acid sequence of SEQ ID NO: 24. In certain embodiments, Flag comprises the amino acid sequence of SEQ ID NO: 26. The tag protein may be attached to the N-terminus or C-terminus of the TH variant, or its fragment, derivative or analogue. In certain embodiments, the TH variant provided herein comprises the amino acid sequence of SEQ ID NO: 3, or its fragment, derivative or analogue having at least 80% sequence identity to SEQ ID NO: 3.

[0076]

[0079] In another aspect, the disclosure further provides a composition comprising the above-described TH variant, or a fragment, derivative or analogue thereof.

[0077]

[0080] In one embodiment, the composition further comprises an aromatic L-amino acid decarboxylase (AADC), e.g., full-length AADC, or a fragment, derivative, or analog of the full-length AADC. In one embodiment, the full-length AADC comprises a protein having an amino acid sequence set forth in any of SEQ ID NOs: 4-9. In one embodiment, the fragment, derivative, or analog of full-length AADC has at least 80% (e.g., at least 80%, 90%, 95% or 99%) sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 4-9. In one embodiment, the fragment, derivative, or analog of full-length AADC is formed by substitution, deletion, or addition of one or a limited number (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of amino acid residues in the amino acid sequence set forth in any of SEQ ID NOs: 4-9. One of skill in the art will appreciate that the fragments, derivatives, and analogs of full-length AADC substantially maintain the biological function or biological activity of full-length AADC. In one embodiment, the fragment, derivative, or analog of full-length AADC functions as a protein having an amino acid sequence set forth in any of SEQ ID NOs: 4-9. A fragment, derivative or analog of full-length AADC has at least 50% (e.g., at least 60%, 70%, 80%, 85%, 90%, 95%, 99%) of the activity of a protein having an amino acid sequence set forth in any of SEQ ID NOs:4-9.

[0078]

[0081] In certain embodiments, AADC may further comprise a tag protein at the N-terminus or C-terminus, preferably including but not limited to HA, Myc or Flag. The tag protein may be attached to the N-terminus or C-terminus of AADC. In a particular embodiment, AADC has the amino acid sequence set forth in SEQ ID NO: 10 or has an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 10.

[0079]

[0082] In a particular embodiment, AADC can be any of the naturally occurring isoforms encoded by the DDC gene or its variants. Several alternative splicing transcript variants encoding different AADC isoforms have been identified in the art. In particular, the DDC gene produces seven different transcript variants encoding six different protein isoforms. Both variants 1 and 2 transcribed from the DDC gene encode AADC isoform 1. In a preferred embodiment, the full-length AADC is AADC isoform 1 (NCBI reference sequence: NP_000781.1) encoded by a polynucleotide that is the coding region of transcript variant 1 or 2 of the DDC gene. Those skilled in the art can naturally expect based on the prior art that all of these naturally occurring isoforms (e.g., AADC having an amino acid sequence described in any of SEQ ID NOs: 4-9) can be applied in the present disclosure and can achieve the same or similar effects.

[0080]

[0083] In certain embodiments, the compositions provided herein are pharmaceutical compositions. In certain embodiments, the compositions provided herein further comprise a pharma- ceutically acceptable carrier. In certain embodiments, the compositions provided herein are for therapeutic use.

[0081]

[0084] In certain embodiments, the composition provided herein is an enzyme composition.The term "enzyme composition" as used herein relates to a composition comprising AADC and a TH variant with N-terminal deletion of more than 60 and less than 120 amino acid residues (for example, 80, 90 or 100 amino acid residues).In one embodiment, the amino acid sequence of the TH variant with N-terminal deletion of 90 amino acid residues is described in SEQ ID NO:2.AADC can be the full-length AADC with its amino acid sequence described in SEQ ID NO:4.In consideration of the teachings of the present disclosure and the prior art, those skilled in the art will further understand that the TH variant with N-terminal deletion of 90 amino acid residues or full-length AADC used in the present disclosure also includes their modified forms, and such modified forms have the same or similar functions as the TH with N-terminal deletion of 90 amino acid residues or full-length AADC, despite having some differences in amino acid sequence. These modified forms include, but are not limited to, deletion, insertion and / or substitution of one or more (e.g., 1 to 5) amino acid residues, and addition of one or more (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acid residues at the C-terminus and / or N-terminus. It is well known to those skilled in the art that substitution with amino acid residues having similar or close properties, for example, substitution between isoleucine and leucine, does not change the function of the resulting protein. As another example, addition of a tag at the C-terminus and / or N-terminus that contains one or more amino acids and is convenient for purification or detection, usually does not affect the function of the resulting protein. In a specific embodiment, the "enzyme composition" used in the present disclosure may include a full-length AADC with a TH lacking 90 amino acids at the N-terminus, an HA tag at the N-terminus, and a Myc tag at the C-terminus.

[0082]

[0085] Polynucleotide Constructs

[0086] In another aspect, the present disclosure further provides a polynucleotide construct comprising a polynucleotide encoding a TH variant or a fragment, derivative or analog thereof.In certain embodiments, the polynucleotide construct further comprises a polynucleotide encoding AADC or a derivative thereof.In certain embodiments, the present disclosure provides a polynucleotide construct encoding the pharmaceutical composition or enzyme composition described above.

[0083]

[0087] The term "polynucleotide" as used herein refers to a DNA molecule or an RNA molecule. The DNA molecule includes cDNA, genomic DNA or synthetic DNA. The DNA molecule may be single-stranded or double-stranded. The sequence that codes for a mature polypeptide may be identical to the coding sequence of a particular protein or a degenerate variant thereof. A degenerate variant refers to a polynucleotide sequence that codes for a protein but differs from the coding sequence of the protein due to the degeneracy of the genetic code.

[0084]

[0088] In one embodiment, the polynucleotide encoding the TH variant has a nucleotide sequence set forth in SEQ ID NO: 12 or 13 or a nucleotide sequence having at least 80%, preferably at least 80%, 90%, 95%, 99% sequence identity to SEQ ID NO: 12 or 13, and / or the polynucleotide encoding AADC has a nucleotide sequence set forth in any of SEQ ID NO: 14-21 or a nucleotide sequence having at least 80%, preferably 80%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NO: 14-21. In one embodiment, the polynucleotide is a degenerate variant of SEQ ID NO: 12 or 13 and encodes the same TH variant. In one embodiment, the polynucleotide is a degenerate variant of one of SEQ ID NO: 14-21 and encodes the same AADC.

[0085]

[0089] In certain embodiments, a polynucleotide encoding a fragment, derivative or analog of a TH variant has a nucleotide sequence having at least 80%, preferably at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 12 or 13. In certain embodiments, a polynucleotide encoding a fragment, derivative or analog of AADC has a nucleotide sequence having at least 80%, preferably at least 80%, 90%, 95%, 99% identity to any of SEQ ID NOs: 14-21.

[0086]

[0090] In a particular embodiment, the polynucleotide of TH is a variant formed by substitution, deletion or addition of one or a limited number (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) of nucleotide residues or codons in the nucleotide sequence described in SEQ ID NO: 12 or 13, and functions as the polynucleotide described in SEQ ID NO: 12 or 13. The variant has at least 90% (e.g., at least 95%, 99%) sequence identity to the polynucleotide described in SEQ ID NO: 12 or 13, or at least 90% (e.g., at least 95%, 99%) biological activity of the polynucleotide described in SEQ ID NO: 12 or 13.

[0087]

[0091] In a specific embodiment, the polynucleotide encoding AADC is a variant formed by substitution, deletion or addition of one or a limited number (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) of nucleotide residues or codons in the nucleotide sequence set forth in any of SEQ ID NOs: 14 to 21, and functions as a polynucleotide set forth in any of SEQ ID NOs: 14 to 21. The variant has at least 90% (e.g., at least 95%, 99%) sequence identity to any of SEQ ID NOs: 14 to 21, or at least 90% (e.g., at least 95%, 99%) biological activity of any of SEQ ID NOs: 14 to 21.

[0088]

[0092] In a particular embodiment, the polynucleotide construct further comprises a promoter. Those skilled in the art will recognize that expression of foreign genes requires a suitable promoter, including but not limited to species-specific, inducible, tissue-specific, or cell cycle-specific promoters. Fine regulation of gene expression usually relies on a promoter that induces the initiation of RNA transcription. The promoter can be either constitutive or inducible. The promoter may be expressed in all cell types (e.g., CMV) or in a specific cell type. With respect to the central nervous system (CNS), neuron-specific promoters include but are not limited to neurofilament, synapsin, or serotonin receptor, and glia-specific promoters include but are not limited to glial fibrillary acidic protein (GFAP), S100, or glutamine synthetase. In a particular embodiment, the human synapsin promoter is used to transcribe the polynucleotide in the vector plasmid of the present disclosure, and the protein encoded by said polynucleotide is specifically expressed in neurons. One skilled in the art can of course expect that other neuron-specific promoters have corresponding functions.

[0089]

[0093] vector

[0094] In another aspect, the disclosure provides a vector comprising the above-described polynucleotide construct.

[0090]

[0095] In one embodiment, the TH variant is a human TH variant and the AADC is human AADC.

[0091]

[0096] In one embodiment, the polynucleotide encoding the TH variant (or derivative thereof) and the polynucleotide encoding AADC (or derivative thereof) of the composition may be assembled in one vector plasmid or in different vector plasmids.

[0092]

[0097] In one particular embodiment, the vector comprises three portions (from 5' to 3'):

[0098] 1) a polynucleotide encoding a TH variant, or a derivative thereof, provided herein;

[0099] 2) a T2A sequence encoding a self-cleavable peptide, and

[0100] 3) A polynucleotide encoding an AADC variant provided herein, or a derivative thereof.

[0093]

[0101] In one particular embodiment, the vector comprises three portions (from 5' to 3'):

[0102] 1) a polynucleotide encoding a TH having an N-terminal deletion of 90 amino acid residues;

[0103] 2) a T2A sequence encoding a self-cleavable peptide, and

[0104] 3) A polynucleotide encoding the full-length AADC.

[0094]

[0105] In certain embodiments, the T2A sequence comprises the nucleotide sequence of SEQ ID NO:28.

[0095]

[0106] In one specific embodiment, when a polynucleotide encoding a TH having an N-terminal deletion of 90 amino acid residues and a polynucleotide encoding a full-length AADC are present in the same vector plasmid, a T2A sequence encoding a self-cleavable peptide is added between the two polynucleotides to construct a monocistronic that synchronously expresses the two proteins.

[0096]

[0107] In another specific embodiment, an internal ribosome entry site (IRES) is added between the polynucleotide encoding TH with the 90 amino acid residue N-terminal deletion and the polynucleotide encoding full-length AADC. If the IRES nucleotide sequence is present downstream of the stop codon of the mRNA, ribosome re-entry can occur to initiate translation of the second open reading frame (ORF).

[0097]

[0108] In a particular embodiment, a polynucleotide encoding a TH having an N-terminal deletion of 90 amino acid residues and a polynucleotide encoding an AADC (e.g., full-length, or a fragment, derivative or analog thereof) can each be constructed in a different vector.

[0098]

[0109] The term "vector" as used herein relates to a molecular tool for transporting and transducing an exogenous target gene (e.g., a polynucleotide described in this disclosure) into a target cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can be a DNA vector, an RNA vector, a viral vector, a non-viral vector, a recombinant vector, or an expression vector. The term "expression vector" as used herein relates to a vector that allows the expression of an exogenous target gene after being transported or transduced into a target cell. An expression vector may provide a suitable nucleotide sequence (i.e., a promoter) that allows the initiation of transcription in the target cell. The term "viral vector" as used herein relates to an expression vector that has viral sequences, e.g., viral terminal repeat sequences. Those skilled in the art will understand that in the field of gene therapy, viral vectors are the preferred method for transducing an exogenous target gene into a target cell and expressing it in the target cell.

[0099]

[0110] In one embodiment, the vectors provided herein comprise plasmid vectors.

[0100]

[0111] In one embodiment, the vector is a viral vector.In one embodiment, the vector is selected from the group consisting of herpes simplex virus (HSV) vector, adenovirus (Ad) vector and adeno-associated virus (AAV) vector.In one embodiment, the vector can be expressed in the central nervous system.Effective expression vectors in the central nervous system (CNS) include, but are not limited to, HSV, Ad or AAV, and preferably include AAV.

[0101]

[0112] In certain embodiments, the vector comprises or is an AAV vector. AAV is a single-stranded human DNA parvovirus whose genome has a size of about 4.7 kilobases (kb). The AAV genome contains two major genes, the rep gene encoding the rep proteins (Rep76, Rep68, Rep52 and Rep40) flanked by a 5' inverted repeat (ITR) and a 3' ITR, and the cap gene encoding the AAV structural proteins (VP-1, VP-2 and VP-3). The term "AAV vector" as used herein includes any vector (e.g., a plasmid) that contains one or more heterologous sequences flanked by at least one or two AAV inverted repeats. The term "AAV ITR", which is well understood in the art, is a sequence of about 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITRs can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several short self-complementary regions, allowing intrastrand base pairing within that portion of the ITRs.

[0102]

[0113] AAV ITRs can be derived from any AAV, including but not limited to AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered.For details, see, for example, NF BERNARD et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers); Gao et al. (2004), J. Virol. 78:6381-6388.The nucleotide sequences of AAV ITR regions are known. See, e.g., RM Kotin (1994), Human Gene Therapy 5:793-801; KI Berns, "Parvoviridae and their Replication," in Fundamental Virology, 2nd Edition, edited by BN Fields and DM Knipe. An early description of the AAV1, AAV2 and AAV3 terminal repeat sequences is provided by X. Xiao (1996), "Characterization of adeno-associated virus (AAV) DNA replication and integration" (Doctoral dissertation), University of Pittsburgh, Pittsburgh, Pa., which is incorporated herein in its entirety.

[0103]

[0114] The AAV ITRs may be the native AAV ITRs, or alternatively, may be altered from the native AAV ITRs, e.g., by mutation, deletion, or insertion, so long as the altered ITRs are still capable of mediating the desired biological function, e.g., replication, viral packaging, integration, etc. The 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV vector do not necessarily need to be identical or from the same AAV serotype, so long as they function as intended, e.g., to allow excision and rescue of a sequence of interest from, and integration into, a recipient cell genome.

[0104]

[0115] In certain embodiments, the AAV vector provided herein comprises an expression cassette with a suitable size for packaging into AAV viral particles.For example, the size of the expression cassette in the AAV vector can be up to the size limit of the genome size of the AAV used, for example, up to 5.2 kb.In certain embodiments, the expression cassette in the AAV vector has a size of 5.2 kb or less, about 5 kb or less, about 4.5 kb or less, about 4 kb or less, about 3.5 kb or less, about 3 kb or less, about 2.5 kb or less, for example, see JY Dong et al. (November 10, 1996).In certain embodiments, the AAV vector plasmid provided herein comprises a transgene expression cassette that is less than 5000 bp (e.g., about 4500 bp) and includes an ITR, a promoter, a WPRE and poly(A).The transgene includes the nucleotide construct provided herein.In some embodiments, the AAV vector can be recombinant. Recombinant AAV (rAAV) vectors can contain one or more heterologous sequences that are not of the same viral origin (e.g., from a non-AAV virus, or from a different serotype of AAV, or from a partially or completely synthetic sequence). In certain embodiments, the nucleotide constructs provided herein are flanked by at least one AAV ITR.

[0105]

[0116] AAV vectors can be constructed using methods known in the art. The general principles of rAAV vector construction are known in the art. For example, see Carter, 1992, Current Opinion in Biotechnology, 3:533-539; and Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:97-129. For example, heterologous sequences can be directly inserted between the ITRs of the AAV genome that has deleted the Rep gene and / or the Cap gene. It is also possible to delete other parts of the AAV genome, as long as sufficient parts of the ITRs remain to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. For example, US Pat. No. 5,173,414 and US Pat. No. 5,139,941; WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al. (1988), Molec. Cell. Biol. 8:3988~3996; Vincent et al. (1990), Vaccines 90 (Cold Spring Harbor Laboratory Press); BJ Carter (1992), Current Opinion in Biotechnology 3:533~539; N. Muzyczka (1992), Current Topics in Microbiol. and Immunol. 158:97~129; RM Kotin (1994), Human Gene Therapy 5:793-801; Schelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0106]

[0117] Alternatively, the AAV ITRs can be excised from the viral genome or from an AAV vector containing the AAV ITRs and fused 5' and 3' to the heterologous sequence using standard ligation techniques, e.g., as described in Sambrook et al., supra. AAV vectors containing AAV ITRs are commercially available and are described, e.g., in U.S. Patent No. 5,139,941.

[0107]

[0118] In a specific embodiment, the ectopic synthesis of dopamine and the expression of enzyme composition are carried out by the AAV vector in the present disclosure.However, in consideration of the present disclosure and the disclosure of the prior art, those skilled in the art will further understand that the AAV vector used in the present disclosure can also include its modified form, which includes but is not limited to the DNA sequence variant that does not affect the basic function of AAV vector, or the change of AAV serotype.

[0108]

[0119] The term "ectopic synthesis" or "de novo synthesis" as used herein refers to the initiation of production of a particular compound in cells, tissues or organs that do not naturally synthesize that compound using several techniques. In a particular embodiment, the enzyme composition used in the present disclosure may function in medium spiny neurons (MSNs) in the striatum that do not naturally synthesize dopamine, and may promote the synthesis and secretion of dopamine in that brain region, which may play an important role in alleviating PD-related phenotypes.

[0109]

[0120] Virus particles

[0121] In another aspect, the disclosure provides a cell prepared by transfection with the above-described vector (eg, a plasmid or viral vector).

[0110]

[0122] The present disclosure provides a viral particle comprising the above-described nucleotide construct as its genome.

[0111]

[0123] AAV viral particles can be produced from the AAV vector. AAV particles can be produced by introducing the AAV vector provided herein into a suitable host cell using known techniques, for example by transfection, together with other necessary mechanisms, for example, a plasmid encoding the AAV cap / rep genes, and helper genes provided by either adenovirus or herpesvirus (see, for example, MF Naso et al., BioDrugs, 31(4):317-334 (2017), which is incorporated herein in its entirety). The AAV vector can be expressed in the host cell and packaged into a viral particle.

[0112]

[0124] The AAV viral particles provided herein have a capsid protein encoded by the cap gene. In some embodiments, the capsid protein may be natural or recombinant. In some embodiments, the capsid protein may be modified, chimeric or synthetic. Modified capsids may include modifications such as insertions, additions, deletions or mutations. For example, modified capsids may incorporate detection or purification tags. Chimeric capsids include portions of two or more capsid sequences. Synthetic capsids include synthetic or artificially designed sequences. The capsid structure of AAV is also known in the art and is described in more detail in NF Bernard et al., supra.

[0113]

[0125] In some embodiments, the cap gene or capsid protein is derived from more than one AAV serotype. As used herein, the term "serotype" in reference to AAV refers to capsid protein reactivity with defined antisera. It is known in the art that various AAV serotypes are functionally and structurally related, even at the genetic level (see, for example, Blacklow, "Parvoviruses and Human Disease," edited by JR Pattison (1988), pp. 165-174; and Rose, Comprehensive Virology 3:1, 1974). However, AAV viral particles of different serotypes may have different tissue tropisms (see, for details, M Nonnenmacher et al., Gene Ther., June 2012, 19(6):649-658) and may be selected as suitable for gene therapy directed to target tissues. In some embodiments, the cap gene or capsid protein may have a specific tropism profile. The term "tropism profile" refers to the pattern of transduction of one or more target cells, tissues and / or organs. For example, a capsid protein may have a tropism profile specific to the brain, liver (e.g., hepatocytes), eye, muscle, lung, kidney, intestine, pancreas, salivary gland or synovium, or any other suitable cell, tissue or organ.

[0114]

[0126] In some embodiments, the cap gene or capsid protein is derived from any suitable AAV capsid gene or protein, for example, and without limitation, the AAV capsid gene or protein can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrhlO, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVhu44, AAVhu46, AAVhu19, AAVhu20, AAVhu23, AAVhu22, AAVhu24, AAVhu21, AAVhu27, AAVhu28, AAV hu29, AAVhu63, AAVhu64, AAVhu13, AAVhu56, AAVhu57, AAVhu49, AAVhu58, AAVhu34, AAVhu45, AAVhu47, AAVhu51, AAVhu52, AAVhu T41, AAVhu S17, AAVhu T88, AAVhu T71, AAVhu T70, AAVhu T40, AAVhu T32, AAVhu T17, AAVhu LG15, AAVhu9, AAVhu10, AAVhu11, AAVhu53, AAVhu55, AAVhu54, AAVhu7, AAVhu18, AAVhu15, AAVhu16, AAVhu25, AAVhu60, AAVch5, AAVhu3, AAVhu 1, AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAV rh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh5 7, AAVrh50, AAVrh49, AAVhu39, AAVrh58, AAVrh61, AAVrh52, AAVrh53, AAVrh51, AAVhu14, AAVhu31, AAVhu32, AAVrh34, AAVrh33, AAVrh32, Avian AAV ATCCThe capsid of AAV843 is derived from VR-865, avian AAV strain DA-1 or bovine AAV. The capsid of AAV843 is identical to the synthetic capsid AAVXL32 disclosed in WO 2019 / 241324 (incorporated herein in its entirety), and AAV843 is also disclosed, for example, in J. Xu et al., Int J Clin Exp Med, 2019, 12(8):10253-10261.

[0115]

[0127] Further examples of AAV capsid gene and protein sequences can be found in the GenBank database, GenBank Accession Nos: AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY631966; AF063497, AF085716, AF513852, AY530579, AAS99264.1, AY243022, AY243015, AY530560, AY530600, AY530611, AY530628, AY530553, AY530606, AY530583, AY530555, AY530607, AY530580, AY530569, NC 006263, NC 005889, NC 001862, AY530609, AY530581, AY530563, AY530591, AY530562, AY530584, AY530622, AY530601, AY530586, AY243021, AY530570, AY530589, AY5305 95, AY530572, AY530588, AY530575, AY530565, AY530590, AY530602, AY530566, AY530587, AY530585, AY530564, AY530592, AY530623, AY530574, AY 530593, AY530560, AY530594, AY530573, AF513852, AY530624, AY530561, AY242997, AY530625, AY530567, AY530556, AY530578, AY530568, AY5306 18, AY243020, AY530579, AY530619, AY530596, AY530612, AY243000, AY530597, AY530620, AY242998, AY530598, AY242999, AY530599, AY243016, NC 001729, NC 001401, AY243018, NC 001863, AY530608, AY243019, NC001829, AY530610, AY243017, AY243001, AY530613, AY243013, AY243002, AY5306 14, AY243003, AY695378, AY530558, AY530626, AY695376, AY695375, AY530605, AY 695374, AY530603, AY530627, AY695373, AY695372, AY530604, AY695371, AY5306 00, AY695370, AY530559, AY695377, AY243007, AY243023, AY186198, AY629583, NC See 004828, AY530629, AY530576, AY243015, AY388617, AY530577, AY530582, AY530615, AY530621, AY530617, AY530557, AY530616 or AY530554.

[0116]

[0128] In certain embodiments, the AAV viral particles contain capsid proteins from AAV9 and therefore have the serotype of AAV9. The capsid gene sequence of AAV9 is known in the art, for example, from the GenBank database, see GenBank Accession No. AY530579.

[0117]

[0129] In certain embodiments, the AAV viral particles comprise capsid proteins from one AAV serotype and AAV ITRs from a second serotype. In certain embodiments, the AAV viral particles comprise pseudotyped AAV. "Pseudotyped" AAV refers to AAV that contains a viral genome that comprises capsid proteins from one serotype and 5'-3' ITRs from a second serotype. Pseudotyped AAV is expected to have cell surface binding properties of the serotype from which the capsid proteins are derived and genetic properties that match the serotype from which the ITRs are derived.

[0118]

[0130] The rep and cap genes of AAV as well as the genome sequence of AAV are known in the art and can be found in the literature and public databases, such as the GenBank database.The following Table 1 shows some exemplary sequences of AAV genome or AAV capsid sequences, and further examples are summarized in NF Bernard et al., VIROLOGY, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers); Gao et al., (2004) J. Virol. 78:6381-6388; MF Naso et al., BioDrugs., 2017, 31(4):317-334.

[0119]

[0131] [Table 1]

[0120]

[0132] Pharmaceutical Compositions

[0133] In another aspect, the disclosure provides a pharmaceutical composition comprising the viral particle described above and a pharma- ceutically acceptable carrier.

[0121]

[0134] The term "pharmaceutical acceptable carrier" as used herein refers to any pharmaceutical carrier that can facilitate the storage and administration of the viral particles of the present disclosure to a subject, such as solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, etc. The pharmaceutical acceptable carrier may contain any suitable component, such as, without limitation, saline. Examples of saline include, without limitation, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, salt solution, and polysorbate solution.

[0122]

[0135] In certain embodiments, the pharmaceutical composition may further include additives, such as, without limitation, stabilizers, preservatives, and transfection-facilitating agents that aid in cellular uptake of the drug. Suitable stabilizers may include, without limitation, monosodium glutamate, glycine, EDTA, and albumin (e.g., human serum albumin). Suitable preservatives may include, without limitation, 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenol, thimerosal, and antibiotics. Suitable transfection-facilitating agents may include, without limitation, calcium ions.

[0123]

[0136] The pharmaceutical compositions may be suitable for administration via any appropriate route known in the art, including, without limitation, parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, intrapulmonary, intracardiac, subcutaneous, intraparenchymal, intraventricular, or intraspinal routes of administration.

[0124]

[0137] The pharmaceutical composition can be administered to a subject in the form of a formulation or preparation suitable for each administration route. Formulations suitable for administering the pharmaceutical composition can include, without limitation, solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome-based preparations, patches, implants, and suppositories.

[0125]

[0138] The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Methods of preparing these formulations or compositions include providing the exogenous nucleic acid of the present disclosure in one or more pharma- ceutically acceptable carriers and, optionally, one or more adjuvants. Methods for preparing such formulations may be found, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A. R. Gennaro (ed.), Mack Publishing Co., NJ, 1995; R. Stribling et al., Proc. Natl. Acad. Sci. USA, 89:11277-11281 (1992); T. W. Kim et al., The Journal of Gene Medicine, 7(6):749-758 (2005); S. F. Jia et al., Clinical Cancer Research, 9:3462 (2003); A. Shahiwala et al., Recent patents on drug delivery and formulation, 1:1-9 (2007); A. Barnes et al., Current Opinion in Molecular Therapeutics 2000 2:87-93 (2000), which are incorporated herein by reference in their entireties.

[0126]

[0139] Treatment methods

[0140] In another aspect, the disclosure provides a method of treating a neurodegenerative disease in a subject using said TH variant, pharmaceutical composition, nucleotide construct, vector (e.g., a plasmid or viral vector), cell, viral particle or composition (e.g., by administration of a therapeutically effective amount).

[0127]

[0141] In certain embodiments, the present disclosure provides a method for treating neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of viral particles provided herein.As used herein, the term "therapeutically effective amount" with respect to viral particles means that the amount of viral particles delivered to the subject is sufficient to produce therapeutic benefit in the subject, for example, to provide some relief, sedation or alleviation in at least one clinical symptom in the subject.For example, a therapeutically effective amount of exogenous nucleic acid can allow the delivery and expression of TH variant (or its derivative) and AADC (or its derivative) to a sufficient number of cells in the subject to produce therapeutic benefit.The therapeutic benefit can include, for example, the recovery of motor symptoms in a subject with Parkinson's disease.

[0128]

[0142] In certain embodiments, the therapeutic benefit of the viral particle, vector or composition provided herein may be tested in a PD animal model. The term "PD animal model" as used herein refers to an animal model that can simulate the critical phenotypes consistent with PD pathology (e.g., neurodegeneration of dopaminergic neurons in the substantia nigra region of the brain). In one particular embodiment, the PD animal model used in the present disclosure is a mouse strain called C57BL / 6, in which dopaminergic neurons in the unilateral substantia nigra / ventral tegmental area (SN / VTA) region are specifically killed by a toxic agent (e.g., 6-hydroxydopamine, 6-OHDA). However, those skilled in the art will further understand that the establishment of a PD animal model provides guidance and methods for the treatment of human PD. Thus, a PD model of non-human primates, which is evolutionarily closer to humans in genetic relationship, may theoretically help achieve the goal of clinical transformation. The mouse model used in the particular embodiment is only intended to illustrate that the enzyme composition of the present disclosure can improve PD dyskinesia, and does not mean that it is effective only in mice. One of skill in the art, based on their understanding of the prior art, can reasonably expect that the enzyme compositions of the present disclosure can ameliorate PD dyskinesias in other species (eg, humans).

[0129]

[0143] In certain embodiments, the viral particles provided herein are administered to the striatum of the subject's brain.

[0130]

[0144] The term "subject" as used herein refers to any human or non-human animal. The term "non-human animal" refers to all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably.

[0131]

[0145] As used herein, the terms "treatment" or "method of treating" refer to both therapeutic and prophylactic methods. Those in need of treatment may include individuals already suffering from a particular disease or individuals who may suffer from such a disease in due course.

[0132]

[0146] In one embodiment, viral particles containing a nucleotide construct comprising a polynucleotide encoding a TH variant and AADC are administered to the striatum of the subject's brain to express the TH variant and AADC nucleotides, which then cause ectopic synthesis of dopamine in the striatum, effectively reversing motor symptoms in a subject with Parkinson's disease in time.

[0133]

[0147] In certain embodiments, the striatum is the caudate-putamen (CP) region.

[0134]

[0148] In one embodiment, the present disclosure discloses the use of said TH variant, pharmaceutical composition, nucleotide construct, vector plasmid, cell, virus or composition in the manufacture of a medicament for treating a neurodegenerative disease in a subject.

[0135]

[0149] In certain embodiments, the neurodegenerative disease is Parkinson's disease.

[0136]

[0150] In certain embodiments, the subject is a mammal, preferably a human, rat or mouse.

[0137]

[0151] Advantages of the Invention

[0152] The advantage of the present disclosure is that, by utilizing the therapeutic method provided by the present disclosure, the enzyme composition for ectopic synthesis of dopamine can significantly increase the concentration of dopamine released by cells, and the concentration is significantly higher than that of other enzyme compositions.In addition, the use of AAV vector to deliver the above-mentioned foreign gene can effectively express the nucleotide construct encoding the target enzyme composition in the striatum of the brain, thereby significantly improving the disease phenotype of PD.This suggests the great value of the enzyme composition comprising AAV as the expression vector of the present disclosure for the application in gene therapy.

[0138]

[0153] Experimental methods in the following examples are conventional unless otherwise specified.

[0139]

[0154] Working Example

[0155] Methods and Materials

[0156] 1. Construction of AAV vectors expressing enzyme compositions

[0157] Polynucleotides expressing the enzyme compositions of the present disclosure and AAV vectors (Addgene:26972) were digested with endonucleases BamHI and EcoRI at 37° C. for 1 hour to obtain corresponding cohesive ends. Target fragments purified by gel recovery were ligated with T4 DNA ligase overnight at 16° C. After transformation, single bacterial colonies were picked for culture, and vector plasmids were extracted and subjected to Sanger sequencing for sequence verification.

[0140]

[0158] 2. In vitro culture and transfection of 293 cell lines

[0159] 293 cell lines were cultured in DMEM supplemented with GlutaMAX and dual antibiotics (penicillin and streptomycin) at 37°C, 5% CO2. When the density of 293 cells reached approximately 80% of the area of ​​the 6-well plate, liposomal transfection (Lipofectamine 3000 reagent) was performed. 293 cells in each well were transfected with 3μg of the corresponding plasmid and continuously cultured for 48 hours for the following experiments.

[0141]

[0160] 3. High Performance Liquid Chromatography (HPLC)

[0161] After aspirating the medium of 293 cells, the cells were washed once with warm PBS and then incubated in PBS (1 mL) for 1 h. The lysate was harvested and centrifuged at 3,000 rpm and 4°C for 10 min. The supernatant was mixed 1:1 with HClO4 (0.6 M) to a final concentration of HClO4 of 0.3 M. The thoroughly mixed sample was centrifuged at 20,000 rpm and 4°C for 15 min, and the supernatant was applied to an HPLC system equipped with an ESA Coulochem III electrochemical detector (ESA Analytical). Catecholamines were separated using an Eclipse Plus C18 reversed-phase column (3.5 μm, 2.1 × 150 mm) equilibrated in flow phase at a rate of 0.2 mL / min, followed by electrochemical detection, and dopamine concentration was calculated by integration of the specific peak.

[0142]

[0162] 4. Stereotactic injection

[0163] A PD mouse model was established by injecting 6-OHDA into the unilateral substantia nigra / ventral tegmental area (SN / VTA) of C57BL / 6 mice genetic background. Stereotactic injection was performed to inject 500 nL of 6-OHDA (8 mg / mL) in the unilateral SN / VTA area. As a toxic agent, 6-OHDA specifically kills dopaminergic neurons. 6-OHDA was slowly infused at a rate of 50 nL / min and delivered at AP-3.6, ML-0.5 and DV-4.3.

[0143]

[0164] In an experiment to verify the role of the enzyme composition of the present disclosure in rescuing motor asymmetry in PD model mice, the present inventors transfected AAV serotype 9 viral particles (titer: 1.95 × 10) containing a vector plasmid expressing the targeted bienzyme composition. 13 The GFP-expressing viral vector (titer: 7.78 × 10 vg / mL) was injected into the caudate putamen (CP) of the striatum. 12 vg / mL) was used as a control. Based on the mouse standard brain atlas, three suitable injection sites were selected: (1) AP0.5, ML-2.0 and DV-3.6; (2) AP0.5, ML-2.0 and DV-3.6; (3) AP-0.6, ML-2.7 and DV-3.3. The injection volume at each site was 500 nL, and the injection rate was 50 nL / min using an infusion pump.

[0144]

[0165] 5. Apomorphine Rotation Test

[0166] Apomorphine was administered subcutaneously in the neck of PD mouse models at a weight-scaled dose (10 mg / kg) before testing. Mice were placed in a 10 cm diameter cylinder for habituation and then subjected to the rotation test. Results were expressed as net rotations per minute of apomorphine-induced rotations contralateral to the 6-OHDA lesion and calculated as the difference between contralateral and ipsilateral rotations divided by the 60 min recording time.

[0145]

[0167] 6. Immune cell staining

[0168] Mice were perfused transcardially with 4% PFA in PBS. Isolated brains were fixed in 4% PFA for about 1 week and then dehydrated in 15% and 30% sucrose solutions. Cryostat sectioning was used to obtain 40 μm thick brain sections containing the brain regions of interest (SN / VTA and CP). After washing in PBS, brain sections were incubated in blocking buffer (5% BSA, 0.3% TritonX-100 in PBS) for 2 hours at room temperature. Sections were then incubated with a primary antibody (anti-TH) overnight at 4° C., followed by incubation with a secondary antibody that corresponds to the origin of the primary antibody and has a chromophore (absorption wavelength 488 nm) for 2 hours at room temperature. All images were acquired using an Olympus VS120 high-throughput fluorescent imaging system.

[0146]

[0169] 7. Sequence information involved in the experiment [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0147]

[0170] Example 1. Construction of AAV vectors expressing bienzyme compositions

[0171] As shown in Figure 1, a recombinant AAV vector was constructed to express a bienzyme composition comprising a TH variant with a 90 amino acid deletion at the N-terminus and full-length AADC. The expression of downstream genes was regulated by the synapsin promoter. The polynucleotide expressing the bienzyme composition comprises three parts (5' to 3') as shown below.

[0172] 1) a polynucleotide encoding TH with an N-terminal HA tag and an N-terminal 90 amino acid deletion as set forth in SEQ ID NO: 13;

[0173] 2) the T2A nucleotide sequence set forth in SEQ ID NO:28, which encodes a self-cleaving peptide;

[0174] 3) A polynucleotide encoding a full-length AADC tagged with a Myc tag at the C-terminus, as set forth in SEQ ID NO:21.

[0148]

[0175] The polynucleotide expressing the enzyme composition of the present disclosure was digested with endonucleases BamHI and EcoRI and subcloned into an AAV vector (Addgene:26972).

[0149]

[0176] As a control, the present inventors simultaneously constructed an AAV vector carrying the synapsin promoter to drive GFP expression.

[0150]

[0177] In order to better express the target sequence in the 293 cell line, so that the de novo dopamine synthesis ability can be conveniently compared among a series of compositions each containing TH with a specific number of amino acids deleted at the N-terminus and full-length AADC in the 293 cell line, the present inventors simultaneously constructed a group of vector plasmids having ubiquitin as a promoter, each of which is a composition containing full-length TH and full-length AADC, a composition containing another isomer of TH and full-length AADC, a composition containing TH with 40 amino acids deleted at the N-terminus (i.e., amino acid residues 41 to 528 of SEQ ID NO: 1) and full-length AADC (SEQ ID NO: 4), a composition containing TH with 60 amino acids deleted at the N-terminus (i.e., amino acid residues 61 to 528 of SEQ ID NO: 1) and full-length AADC, a composition containing TH with 80 amino acids deleted at the N-terminus (i.e., amino acid residues 81 to 528 of SEQ ID NO: 1) and full-length AADC, a composition containing TH with 90 amino acids deleted at the N-terminus (i.e., amino acid residues 91 to 928 of SEQ ID NO: 1), and full-length AADC. A composition comprising TH with 100 amino acids deleted at the N-terminus (i.e., amino acid residues 101 to 528 of SEQ ID NO: 1) and full-length AADC, a composition comprising TH with 120 amino acids deleted at the N-terminus (i.e., amino acid residues 121 to 528 of SEQ ID NO: 1) and full-length AADC, a composition comprising TH with 150 amino acids deleted at the N-terminus (i.e., amino acid residues 151 to 528 of SEQ ID NO: 1) and full-length AADC, a composition comprising TH with 164 amino acids deleted at the N-terminus (i.e., amino acid residues 165 to 528 of SEQ ID NO: 1) and full-length AADC, or a composition comprising TH with 190 amino acids deleted at the N-terminus (i.e., amino acid residues 191 to 528 of SEQ ID NO: 1) and full-length AADC. All of the series of THs with N-terminal amino acid deletions are attached to an HA tag at the N-terminus, and the C-terminus of full-length AADC is attached to a Myc tag. A GFP-expressing viral vector with ubiquitin as the promoter was constructed as a control.

[0151]

[0178] Example 2. Functional verification of enzyme compositions in in vitro cultured cell lines

[0179] In order to find the most efficient two-enzyme composition for dopamine de novo synthesis, the inventors transfected a series of two-enzyme compositions, including TH with amino acid deletion at the N-terminus and full-length AADC, into 293 cell line using liposome (Lipofectamine 3000 reagent). As a negative control, GFP expression vector was also transfected into 293 cell line. After incubation of the cultured cells at 37°C and 5% CO2 for 48 hours, the cell culture medium was replaced with PBS. After incubation in PBS for 1 hour, the supernatant PBS and cell samples were harvested, respectively.

[0152]

[0180] To detect the dopamine concentration in the PBS samples, i.e., the dopamine concentration secreted by 293 cells, high performance liquid chromatography (HPLC) was performed. The results showed that dopamine was detected in all samples harvested from 293 cells expressing a series of bienzyme compositions containing TH with amino acid deletions at the N-terminus and full-length AADC, but not in GFP-expressing samples (Figure 2). This suggests that the 293 cell line itself cannot synthesize and secrete dopamine, but when functional TH and AADC are introduced simultaneously, the cells start to synthesize and secrete dopamine, proving that the various bienzyme compositions designed by the present inventors can function normally, i.e., catalyze the de novo synthesis of dopamine.

[0153]

[0181] The results further showed that dopamine concentrations in samples from 293 cells expressing a composition comprising TH with a 90 amino acid deletion at the N-terminus and full-length AADC (90) were significantly higher than those in a composition comprising full-length TH and full-length AADC (WT), another isomer of TH and full-length AADC (Isob), a composition comprising TH with a 40 amino acid deletion at the N-terminus and full-length AADC (40), a composition comprising TH with a 60 amino acid deletion at the N-terminus and full-length AADC (60), and a composition comprising TH with a 100 amino acid deletion at the N-terminus and full-length AADC (70). and full-length AADC (100), composition containing TH with 120 amino acids deleted at the N-terminus and full-length AADC (120), composition containing TH with 150 amino acids deleted at the N-terminus and full-length AADC (150), composition containing TH with 164 amino acids deleted at the N-terminus and full-length AADC (164), and composition containing TH with 190 amino acids deleted at the N-terminus and full-length AADC (190). However, the difference was not significant when compared to the dopamine concentration in the 293 cell sample expressing TH with 80 amino acids deleted at the N-terminus and full-length AADC (80) (see FIG. 2). This data confirmed that the increase in dopamine concentration limits the ectopic dopamine synthesis ability of TH by negatively regulating its activity, and this dilemma can be solved by using a constitutively active version of TH with specific amino acid residues deleted at the N-terminus.More importantly, the inventors have found the best constitutively active type of TH with specific amino acid deletion at the N-terminus, that is, TH with 80 or 90 amino acids deleted at the N-terminus, by comparison.The two-enzyme composition provided by the present disclosure is a composition that includes TH with 80 or 90 amino acids deleted at the N-terminus and full-length AADC.This result suggests that this two-enzyme composition has better de novo dopamine synthesis ability than another type of composition that includes TH with specific deletion at the N-terminus and full-length AADC.In summary, the two-enzyme composition provided by the present disclosure can achieve the best de novo dopamine synthesis.Although TH with specific deletions at the N-terminus are known to be in a constitutively activated state, the present disclosure provides an optimized form of a constitutively activated TH variant.

[0154]

[0182] Example 3. Construction of PD model mice

[0183] Eight-week-old C57BL / 6 mouse strain was selected to construct the PD model. Stereotactic injection of 500 nL of 6-OHDA (8 mg / mL) was performed into the unilateral SN / VTA region according to the mouse standard brain atlas. 6-OHDA is a toxic drug that specifically kills dopaminergic neurons. Two weeks later, mice were subcutaneously injected with apomorphine in the neck at a weighted dose (10 mg / kg), and then rotation tests were performed. Mice with a phenotype of apomorphine-induced motor asymmetry, which showed rotations contralateral to the 6-OHDA lesion, were selected for subsequent experiments.

[0155]

[0184] Immunocytochemical staining assays of cryostat brain sections from mice with motor asymmetry were performed, which showed that TH-positive staining signals were detected in both the SN / VTA and striatal CP regions contralateral to the 6-OHDA lesion, but not in the ipsilateral region to the lesion, as controls in PD mice (see Figure 3). This result indicated that 6-OHDA caused an effective lesion to dopaminergic neurons in the SN / VTA that project to the CP.

[0156]

[0185] In summary, we successfully established a PD mouse model for subsequent rescue experiments.

[0157]

[0186] Example 4. Phenotypic rescue of PD mouse models by bienzyme compositions

[0187] For in vivo expression in PD mice, a vector plasmid expressing a composition containing TH with a 90 amino acid deletion at the N-terminus and full-length AADC (TH90del / AADC) was transfected with AAV serotype 9 viral particles (titer: 1.95 × 10 13As a control, a GFP-expressing plasmid was packaged into AAV particles (GFP, titer: 7.78 × 10 12 The antibody was packaged in a 100 µg / mL IgG antibody.

[0158]

[0188] The PD mouse model successfully constructed in Example 3 was used to carry out phenotype rescue experiments according to the workflow shown in Figure 4a. AAV packaging of TH90del / AADC or GFP was intrastriatal injected using a stereotaxic apparatus at three appropriate assigned injection sites selected based on the mouse standard brain atlas. At each site, 500 nL of virus was injected. Four weeks after virus administration, apomorphine-induced rotation test was performed by subcutaneous injection of apomorphine in the neck, with the dose measured by body weight (10 mg / kg). Rescue efficacy was shown as the reduction in net rotations per minute of apomorphine-induced rotations on the side opposite to the 6-OHDA lesion, which was calculated by dividing the difference between contralateral and ipsilateral rotations by the 60-minute recording time. The results showed a significant decrease in net rotations per minute of contralateral rotations in PD mice injected with TH90del / AADC viral vectors compared with the net rotations before virus injection and the net rotations of the control group (GFP) in the apomorphine-induced motor asymmetry test 4 weeks after virus administration (Figure 4b). In summary, it was shown that the specific bienzyme composition of the present disclosure (i.e., TH90del / AADC) can effectively function in vivo and increase dopamine concentration in the striatal CP region innervated by dopaminergic neurons in the SN, thereby significantly improving apomorphine-induced motor asymmetry in PD mouse models. Therefore, the bienzyme composition (TH90del / AADC) provided by the present disclosure has a potential therapeutic effect on PD.

[0159]

[0189] Although the enzyme composition used in the embodiments and / or examples is of human origin, those skilled in the art would naturally expect that a human or mouse bienzyme composition would have superior therapeutic efficacy in mouse models or human clinical trials, because based on the disclosure of the present disclosure, the protein homology of TH or AADC between humans and mice is 83% or 89%, respectively.

[0160]

[0190] In summary, although the inventor has described the detailed description of the present disclosure, the scope of the present disclosure goes beyond the description. Those skilled in the art will understand that the scope of the present disclosure includes modified and altered embodiments, and those embodiments fall within the protection scope of the present disclosure.

Claims

1. A composition comprising a tyrosine hydroxylase variant comprising an amino acid sequence set forth in SEQ ID NO:1, except for an N-terminal deletion of 80 to 100 amino acid residues, or an amino acid sequence having at least 95% sequence identity thereto, and an aromatic L-amino acid decarboxylase.

2. 2. The composition of claim 1, wherein the tyrosine hydroxylase variant comprises the amino acid sequence set forth in SEQ ID NO:1, except for an N-terminal deletion of 80-90 amino acid residues, or an amino acid sequence having at least 95% sequence identity thereto.

3. 3. The composition of claim 1 or 2, wherein the tyrosine hydroxylase variant comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 95% sequence identity thereto.

4. 4. The composition of claim 3, wherein the tyrosine hydroxylase variant further comprises a tag protein attached to the N-terminus or C-terminus.

5. The composition of claim 4 , wherein the tag protein is HA, Myc or Flag.

6. The composition of any one of claims 1 to 5, wherein the tyrosine hydroxylase variant comprises the amino acid sequence set forth in SEQ ID NO:

3.

7. The composition according to any one of claims 1 to 6, wherein the aromatic L-amino acid decarboxylase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 4 to 9, or an amino acid sequence having at least 90% sequence identity thereto.

8. 8. The composition of claim 7, wherein the aromatic L-amino acid decarboxylase further comprises a tag protein attached to the N-terminus or C-terminus.

9. The composition of claim 8, wherein the tag protein is HA, Myc or Flag.

10. The composition of any one of claims 1 to 9, wherein the aromatic L-amino acid decarboxylase has the amino acid sequence set forth in SEQ ID NO:

10.

11. 11. A polynucleotide construct comprising a first polynucleotide encoding a tyrosine hydroxylase variant as defined in any one of claims 1 to 6 and a second polynucleotide encoding an aromatic L-amino acid decarboxylase as defined in any one of claims 7 to 10.

12. The polynucleotide construct of claim 11, wherein the first polynucleotide has a nucleotide sequence set forth in SEQ ID NO: 12 or 13, or has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 or 13.

13. The polynucleotide construct of claim 11 or 12, wherein the second polynucleotide has a nucleotide sequence set forth in any one of SEQ ID NOs: 14 to 21 or has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14 to 21.

14. The polynucleotide construct of any one of claims 11 to 13, further comprising a promoter operably linked to the first polynucleotide and / or the second polynucleotide.

15. The polynucleotide construct of claim 14 , wherein the promoter comprises a neuron-specific promoter.

16. A vector comprising the polynucleotide construct according to any one of claims 11 to 15.

17. 17. The vector of claim 16, wherein the first polynucleotide and the second polynucleotide are constructed in one vector or in different vectors.

18. The vector of claim 17, wherein the first polynucleotide and the second polynucleotide are assembled in a vector, and the vector further comprises a third polynucleotide inserted between the first polynucleotide and the second polynucleotide.

19. 19. The vector of claim 18, wherein the third polynucleotide encodes a self-cleavable sequence and / or an internal ribosome entry site (IRES).

20. The vector according to any one of claims 16 to 19, which is selected from the group consisting of herpes simplex virus vectors, adenovirus vectors and adeno-associated virus vectors.

21. A host cell comprising or transfected with a vector according to any one of claims 16 to 20.

22. 16. A virus comprising a viral genome comprising a polynucleotide construct according to any one of claims 11 to 15 or comprising a nucleic acid expressed from a polynucleotide construct according to any one of claims 11 to 15.

23. A tyrosine hydroxylase variant comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:

3.

24. 24. A polynucleotide construct comprising a polynucleotide encoding a tyrosine hydroxylase variant as defined in claim 23.

25. A vector comprising the polynucleotide construct of claim 24.

26. 26. A host cell comprising or transfected with the vector of claim 25.

27. 25. A virus comprising the polynucleotide construct of claim 24 or comprising a nucleic acid expressed from the polynucleotide construct of claim 24.

28. 23. A pharmaceutical composition comprising the virus of claim 22 and a pharma- ceutically acceptable carrier.

29. 27. Use of a composition according to any one of claims 1 to 10, a tyrosine hydroxylase variant according to claim 23, a polynucleotide construct according to any one of claims 11 to 15 and 24, a vector according to any one of claims 16 to 20 and 25, a host cell according to claim 21 or 26, a virus according to claim 22 or 27, or a pharmaceutical composition according to claim 28 in the manufacture of a medicament for treating a neurodegenerative disease in a subject.

30. 30. The use according to claim 29, wherein the neurodegenerative disease is Parkinson's disease.

31. 31. The use according to claim 29 or 30, wherein the subject is a mammal.

32. 32. The use according to claim 31 , wherein the mammal is a human, a rat or a mouse.

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