Novel viral vector construct for neuron specific optimized continuous DOPA synthesis in vivo

Abstract

The present invention relates to a one-vector expression system comprising a sequence encoding two polypeptides, such as tyrosine hydroxylase (TH) and GTP-cyclohydrolase 1 (GCH1). The two polypeptides can be should preferentially be expressed at a ratio between 3:1 and 15:1, such as between 3:1 and 7:1. The invention is useful in the treatment of catecholamine deficient disorders, such as dopamine deficient disorders including but not limited to Parkinson's Disease. Moreover, the present invention provides a method to deliver the vector construct in order to limit the increased production of the catecholamine to the cells in need thereof.

1 - 105 . (canceled) 
     
     
         106 . A one-vector expression system comprising:
 a first and a second expression cassette,   wherein said first expression cassette comprises
 a nucleotide sequence comprising
 a first promoter sequence operably linked to 
 a first nucleotide sequence,
 said first nucleotide sequence encoding a GTP-cyclohydrolase 1 (GCH1; EC 3.5.4.16) polypeptide, and 
 
 
   wherein said second expression cassette comprises
 a nucleotide sequence comprising
 a second promoter sequence operably linked to 
 a second nucleotide sequence
 said second nucleotide sequence encoding a tyrosine hydroxylase (TH; EC 1.14.16.2) polypeptide, and 
 
 
   wherein the vector is an adeno associated vector (AAV) and   wherein said second nucleotide sequence is operably linked to a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and   wherein both said first and said second promoter are Synapsin1 promoters.   
     
     
         107 . The vector of  claim 106 , wherein the GTP-cyclohydrolase 1 (GCH1; EC 3.5.4.16) polypeptide is at least 70% identical to a polypeptide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and wherein said tyrosine hydroxylase (TH; EC 1.14.16.2) polypeptide is at least 70% identical to a polypeptide selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14. 
     
     
         108 . The vector according to  claim 106 , wherein said vector does not comprise a nucleotide sequence encoding an aromatic amino acid decarboxylase (AADC) polypeptide. 
     
     
         109 . The vector according to  claim 106 , wherein said vector has a packaging capacity selected from 1 to 40 kb, 1 to 30 kb, 1 to 20 kb, 1 to 15 kb, 1 to 10, 1 to 8 kb, 2 to 7 kb, 3 to 6 kb, or 4 to 5 kb. 
     
     
         110 . The vector according to  claim 106 , wherein said vector has a packaging capacity from 4.5 to 4.8 kb. 
     
     
         111 . The vector according to  claim 106 , wherein the adeno associated vector (AAV) is an AAV2 vector. 
     
     
         112 . The vector according to  claim 111 , wherein the capsid of the AAV2 vector is packaged in an AAV capsid other than an AAV2 capsid. 
     
     
         113 . The vector according to  claim 112 , wherein the AAV2 vector is packaged in an AAV5 capsid. 
     
     
         114 . (canceled) 
     
     
         115 . The vector according to  claim 106 , wherein said polypeptide comprises at least 50 contiguous amino acids, wherein any amino acid specified in the selected sequence is altered to a different amino acid, provided that no more than 15 of the amino acid residues in the sequence are so altered. 
     
     
         116 . The vector according to  claim 115 , wherein said polypeptide is the catalytic domain of tyrosine hydroxylase. 
     
     
         117 . The vector according to  claim 115 , wherein said polypeptide is a mutated tyrosine hydroxylase polypeptide, wherein one or more of the residues S19, S31, S40 or S404 have been altered to another amino acid residue. 
     
     
         118 . The vector according to  claim 106 , wherein said GTP-cyclohydrolase 1 (GCH1) polypeptide is at least 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. 
     
     
         119 . The vector according to  claim 106 , wherein said tyrosine hydroxylase (TH) polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 SEQ ID NO. 13 and SEQ ID NO. 14. 
     
     
         120 . The vector according to  claim 106 , wherein said first expression cassette or said second expression cassette further comprises a polyadenylation sequence. 
     
     
         121 . The vector according to  claim 106 , wherein said first expression cassette and said second expression cassette further comprises polyadenylation sequences. 
     
     
         122 . The vector according to  claim 120 , wherein said polyadenylation sequence is a SV40 polyadenylation sequence. 
     
     
         123 . The vector according to  claim 120 , wherein the 5′ of said polyadenylation sequence is operably linked to the 3′ of said first and/or said second nucleotide sequence. 
     
     
         124 . The vector according to  claim 106 , wherein said first nucleotide sequence encoding a GTP-cyclohydrolase 1 (GCH1) polypeptide comprises the sequence of SEQ ID NO. 18. 
     
     
         125 . The vector according to  claim 106 , wherein said second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide comprises the sequence of SEQ ID NO. 21. 
     
     
         126 . The vector according to  claim 106 , wherein said Woodchuck hepatitis virus post-transcriptional regulatory element comprises a sequence that has at least 85 percent, at least 90 to 95 percent, or at least 99 percent sequence identity to SEQ ID NO: 22. 
     
     
         127 . The vector according to  claim 106 , wherein said Woodchuck hepatitis virus post-transcriptional regulatory element comprises the sequence of SEQ ID NO. 22. 
     
     
         128 . The vector according to  claim 106 , wherein the expression cassettes of said vector comprises a 5′ terminal repeat and a 3′ terminal repeat. 
     
     
         129 . The vector according to  claim 128 , wherein the 5′ and 3′ terminal repeats are selected from Inverted Terminal Repeats [ITR] and Long Terminal Repeats [LTR]. 
     
     
         130 . The vector according to  claim 128 , wherein the 5′ and 3′ terminal repeats are AAV Inverted Terminal Repeats [ITR]. 
     
     
         131 . The vector according to  claim 130 , wherein said Inverted Terminal Repeats comprises the sequences of SEQ ID NO. 15 and SEQ ID NO. 16. 
     
     
         132 . The vector according to  claim 106 , wherein the TH:GCH1 ratio is selected from at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, or at least 50:1 and;
 wherein the TH:GCH1 ratio is determined using a test method selected from the group consisting of measuring enzyme activity, measuring the amount of Tetrahydrobiopterin (BH 4 ) and/or combinations thereof.   
     
     
         133 . The vector according to  claim 106 , wherein the TH:GCH1 ratio is 7:1 and;
 wherein the TH:GCH1 ratio is determined using a test method selected from the group consisting of measuring enzyme activity, measuring the amount of Tetrahydrobiopterin (BH 4 ) and/or combinations thereof.   
     
     
         134 . (canceled) 
     
     
         135 . (canceled) 
     
     
         136 . The vector according to  claim 132 , wherein the ratio is determined by the amount of mRNA transcribed. 
     
     
         137 . The vector according to  claim 132 , wherein the ratio is determined by the amount of protein expressed. 
     
     
         138 . The vector according to  claim 106 , wherein said vector comprises the nucleic acid sequence of SEQ ID NO:23. 
     
     
         139 . The vector according to  claim 106 , wherein said vector is a minimally integrating vector. 
     
     
         140 . A method of treating a disease associated with catecholamine dysfunction, in a patient in need thereof, said method comprising administering to said individual the vector according to  claim 106 . 
     
     
         141 . The method according to  claim 140 , wherein the catecholamine dysfunction is catecholamine deficiency. 
     
     
         142 . The method according to  claim 140 , wherein the catecholamine dysfunction is catecholamine excess. 
     
     
         143 . The method according to  claim 141 , wherein the catecholamine deficiency is dopamine deficiency. 
     
     
         144 . The method according to  claim 142 , wherein the catecholamine excess is dopamine excess. 
     
     
         145 . The method according to  claim 140 , wherein said disease associated with catecholamine dysfunction is a disease, disorder or damage of the central and/or peripheral nervous system. 
     
     
         146 . The method according to  claim 145 , wherein said disease, disorder or damage of the central and/or peripheral nervous system is a neurodegenerative disorder. 
     
     
         147 . The method according to  claim 140 , wherein said disease associated with catecholamine dysfunction is a disease of the basal ganglia. 
     
     
         148 . The method according to  claim 140 , wherein said disease is selected from the group consisting of Parkinson's Disease (PD), DOPA responsive dystonia, L-DOPA induced dyskinesia (LID), ADHD, schizophrenia, depression, vascular parkinsonism, essential tremor, chronic stress, genetic dopamine receptor abnormalities, chronic opoid, cocaine, alcohol or marijuana use, adrenal insufficiency, hypertension, noradrenaline deficiency, post-traumatic stress disorder, pathological gambling disorder, dementia, and Lewy body dementia. 
     
     
         149 . The method according to  claim 140 , wherein said neurodegenerative disorder is Parkinson's Disease (PD).