METHODS OF ATTACHING A MOLECULE-OF-INTEREST TO A MICROTUBE

A method of attaching a molecule-of-interest to a microtube, by co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and the second polymeric solution comprises the molecule-of-interest, thereby attaching the molecule-of-interest to the microtube. An electrospun microtube comprising an electrospun shell, an electrospun coat over an internal surface of the shell and a molecule-of-interest attached to the microtube. 1. A microtube comprising:an electrospun shell,an electrospun coat polymer over an internal surface of said shell and a molecule-of-interest attached to the microtube,wherein said electrospun shell is formed of a first polymeric solution comprising a first solvent and said electrospun coat is formed of a second polymeric solution comprising a second solvent,wherein said second solvent of said second polymeric solution is incapable of dissolving a polymer of said first polymeric solution,wherein said first polymeric solution solidifies faster than said second polymeric solution,wherein said second polymeric solution is capable of wetting said internal surface of said shell during or following solidification of said first polymeric solution,wherein said molecule-of-interest is selected from the group consisting of: a polypeptide, a polynucleotide, a carbohydrate, a polysaccharide, a lipid, a drug molecule, and a small molecule, andwherein said small molecule is selected from the group consisting of a nucleotide base, an amino acid, a nucleotide, an antibiotic, and a vitamin.2. The microtube of claim 1 , ...

Specific Increase of Dopamine Synthesis THrough Targeting of the Guanylate Cyclase 2C Receptor in the Treatment of Parkinson's Disease

In embodiments the invention relates to means and methods for the treatment of Parkinson's disease. In some embodiments the means and methods involve a GUCY2C agonist. The invention also relates to test systems and cells that are suited to identify new candidate compounds for the treatment of Parkinson's disease. 1. A GUCY2C agonist for use in the treatment of an individual that has Parkinson's disease.2. The GUCY2C agonist of claim 1 , further comprising administering a PDE inhibitor to the individual.3. The GUCY2C agonist of or claim 1 , wherein the individual has Parkinson's disease stage 1 claim 1 , 2 claim 1 , 3 or 4.4. The GUCY2C agonist of any one of - claim 1 , wherein the agonist is guanylin or a functional derivative thereof.5. The GUCY2C agonist of any one of - claim 1 , wherein the treatment further comprises a administering a PDE inhibitor to the individual.6. A method of increasing dopamine production by a dopaminergic cell claim 1 , the method comprising increasing signaling by Guanylate Cyclase 2C (GUCY2C) in said cell.7. A method of increasing the level of phosphorylation of Ser40 of tyrosine hydroxylase in a dopaminergic cell claim 1 , the method comprising increasing signaling by GUCY2C in said cell.8. The method of or claim 1 , wherein the signaling by GUCY2C is increased by contacting said cell with a GUCY2C agonist.9. The method of claim 8 , wherein said agonist is guanylin or a functional derivative thereof.10. The method of any one of - claim 8 , further comprising providing said cell with a PDE inhibitor.11. The method according to any one of - claim 8 , wherein the cell is a dopaminergic neuron claim 8 , preferably a midbrain dopaminergic neuron.12. An isolated or recombinant human cell comprising ectopic expression of human GUCY2C and/or ectopic expression of human tyrosine hydroxylase.13. The isolated or recombinant human cell of comprising ectopic expression of human GUCY2C and ectopic expression of human tyrosine hydroxylase.14. A ...

Benzylisoquinoline Alkaloid (BIA) Precursor Producing Microbes, and Methods of Making and Using the Same

Methods and engineered yeast cells for generating a benzylisoquinoline alkaloid product are provided herein. A method comprises providing engineered yeast cells and a feedstock to a reactor. In the reactor, the engineered yeast cells are subjected to fermentation by incubating the engineered yeast cells for a time period to produce a solution comprising the BIA product and cellular material. The solution comprises not more than one class of molecule selected from the group of protoberberine, morphinan, isopavine, aporphine, and benzylisoquinoline. Additionally, at least one separation unit is used to separate the BIA product from the cellular material to provide the product stream comprising the BIA product. 115.-. (canceled)16. An engineered non-plant cell having modifications that result in overproduction of a derivative of tyrosine , along a pathway to produce the benzylisoquinoline alkaloid product , with respect to a non-engineered non-plant cell , wherein the modifications comprise at least three modifications , wherein at least one modification of the at least three modifications is a transcriptional modulation mutation or an inactivating mutation , and wherein each modification of the at least three modifications is selected from the group consisting of: a feedback inhibition alleviating mutation , a transcriptional modulation mutation , and an inactivating mutation , wherein a first modification of the at least three modifications is associated with a first enzyme , a second modification of the at least three modifications is associated with a second enzyme , and a third modification of the at least three modifications is associated with a third enzyme , and wherein each enzyme of the first , second , and third enzymes is distinct from any other enzyme of the first , second , and third enzymes , and wherein each enzyme of the first , second , and third enzyme is selected from the group consisting of: transketolase , glucose-6-phosphate dehydrogenase , ...

PRODUCTION OF MIDBRAIN DOPAMINERGIC NEURONS AND METHODS FOR THE USE THEREOF

Methods are provided for efficient production of midbrain dopaminergic (DA) neurons. In some aspects, methods involve differentiation and selection of DA neurons for a transgenic pluripotent cell population (e.g., cells comprising a selectable marker gene). Cell populations produced by the instant methods and methods of their use are likewise provided. 144-. (canceled)45. A method for providing an enriched population of midbrain dopaminergic (DA) neurons comprising:differentiating cells of a population of human induced pluripotent cells to provide a neural lineage cell population;further differentiating cells of the neural lineage cell population to generate a cell population which includes midbrain neurons; andpurifying cells from said cell population using a transgenic screenable or selectable marker under the control of a pan-neural promoter expressed by cells of the cell population, to provide an enriched population of midbrain DA neurons;wherein the differentiation differentiation steps are carried out in a chemically defined medium that is free of feeder cells and feeder cell extracts and at least 80% of cells in the enriched population are positive for both Lmx1 and FoxA2 expression.46. The method of claim 45 , wherein the enriched population of midbrain dopaminergic (DA) neurons comprise mammalian midbrain dopaminergic (DA) neuronal cells claim 45 , at least about 85% the neuronal cells being positive for both LIM homeobox transcription factor 1 (Lmx1) and forkhead box A2 (FoxA2) expression.47. The method of claim 46 , wherein the neuronal cells comprise at least 500 claim 46 ,000 cells.48. The method of claim 47 , wherein the neuronal cells comprise at least 1 million cells.49. The method of claim 46 , wherein at least about 80% of cells in the enriched population are positive for TH expression.50. The method of claim 45 , wherein the pan-neural promoter is a TuJ-1 claim 45 , Map-2 claim 45 , Dcx claim 45 , Synapsin claim 45 , enolase 2 claim 45 , glial ...

PHARMABIOTIC TREATMENTS FOR METABOLIC DISORDERS

Herein are described pharmabiotic compositions and methods of treatment using genetically modified bacteria that include a portion or a variant of human cDNA sequence. Generally, the modified bacterium has a genetic modification that includes the introduction or inclusion of non-native DNA which contain a human cDNA sequence that can be propagated in the genetically altered bacterium. As an example, the non-native DNA can include one or more portions of human cDNA that encode the enzyme phenylalanine hydroxylase. In an embodiment, the modified bacterium can be provided to a patient as a treatment for a metabolic disorder. In a non-limiting example, a modified bacterium including human cDNA encoding the enzyme phenylalanine hydroxylase can be provided to a patient suffering from a deficiency in phenylalanine hydroxylase or suffering from a mutation to the native gene that results in an inactive form of the enzyme. As such, certain embodiments may provide methods for treating phenylketonuria using the modified bacterium. 1. A modified bacterium comprising a bacterium having a genetic modification , wherein the genetic modification encodes an enzyme that encourages conversion of phenylalanine to tyrosine.2. The modified bacterium of claim 1 , wherein the genetic modification comprises a human cDNA sequence.3. The modified bacterium of claim 1 , wherein the genetic modification encodes SEQ ID NO: 1.4. The modified bacterium of claim 1 , wherein the human cDNA sequence comprises SEQ ID NO: 2 claim 1 , SEQ ID NO: 3 claim 1 , SEQ ID NO: 4 claim 1 , SEQ ID NO: 5 claim 1 , or SEQ ID NO: 6.5Lactobacillus.. The modified bacterium of claim 1 , wherein the bacterium is of the genus6L. helveticus.. The modified bacterium of claim 5 , wherein the species is7. The modified bacterium of claim 1 , wherein the modified bacterium survives in the human gut for at least 6 hours.8. The modified bacterium of claim 1 , further comprising a second genetic modification.9. The modified ...

SYSTEMIC SYNTHESIS AND REGULATION OF L-DOPA

The present invention relates to an expression system for enzyme replacement therapy with the aim of obtaining or maintaining a steady level of L-DOPA in the blood of an individual, achieved through systemic administration of the expression system. The invention is thus useful in the treatment of catecholamine deficient disorders, such as dopamine deficient disorders including Parkinson's Disease. 1. (canceled)2. (canceled)3. (canceled)4. (canceled)5. (canceled)6. (canceled)7. (canceled)8. (canceled)9. (canceled)10. (canceled)11. (canceled)12. (canceled)13. (canceled)14. (canceled)15. (canceled)16. (canceled)17. (canceled)18. (canceled)19. (canceled)20. (canceled)21. (canceled)22. (canceled)23. (canceled)24. (canceled)25. (canceled)26. (canceled)27. (canceled)28. (canceled)29. (canceled)30. (canceled)31. (canceled)32. (canceled)33. (canceled)34. (canceled)35. (canceled)36. (canceled)37. (canceled)38. (canceled)39. (canceled)40. (canceled)41. (canceled)42. (canceled)43. (canceled)44. (canceled)45. (canceled)46. (canceled)47. (canceled)48. (canceled)49. (canceled)50. (canceled)51. (canceled)52. (canceled)53. (canceled)54. (canceled)55. (canceled)56. (canceled)57. (canceled)58. (canceled)59. (canceled)60. (canceled)61. (canceled)62. (canceled)63. (canceled)64. (canceled)65. (canceled)66. (canceled)67. (canceled)68. (canceled)69. (canceled)70. (canceled)71. (canceled)72. (canceled)73. (canceled)74. (canceled)75. (canceled)76. (canceled)77. (canceled)78. (canceled)79. (canceled)80. (canceled)81. (canceled)82. (canceled)83. (canceled)84. (canceled)85. (canceled)86. (canceled)87. (canceled)88. (canceled)89. (canceled)90. (canceled)91. (canceled)92. (canceled)93. (canceled)94. (canceled)95. (canceled)96. (canceled)97. (canceled)98. (canceled)99. (canceled)100. (canceled)101. (canceled)102. (canceled)103. (canceled)104. (canceled)105. (canceled)106. (canceled)107. (canceled)108. (canceled)109. (canceled)110. (canceled)111. (canceled)112. (canceled)113. (canceled)114. ( ...

MICROORGANISMS FOR THE PRODUCTION OF 5-HYDROXYTRYPTOPHAN

Recombinant microbial cells and methods for producing 5-hydroxytryptophan (5HTP) using such cells are described. More specifically, the recombinant microbial cell comprises an exogenous gene encoding an L-tryptophan hydroxylase, and means for providing tetrahydrobiopterin (THB). Related sequences and vectors for use in preparing such recombinant microbial cells are also described. 1. A recombinant microbial cell comprising an exogenous nucleic acid sequence encoding an L-tryptophan hydroxylase (TPH) (EC 1.14.16.4) , and exogenous nucleic acid sequences encoding enzymes of at least one pathway for producing tetrahydrobiopterin (THB).2. The recombinant microbial cell of claim 1 , comprising exogenous nucleic acid sequences encoding enzymes of a first pathway producing THB from guanosin triphosphate (GTP) claim 1 , of a second pathway regenerating THB from 4a-hydroxytetrahydrobiopterin claim 1 , or of both the first and the second pathway.3. The recombinant microbial cell of any one of the preceding claims claim 1 , comprising exogenous nucleic acid sequences encoding(a) optionally, a GTP cyclohydrolase I (EC 3.5.4.16);(b) a 6-pyruvoyl-tetrahydropterin synthase (EC 4.2.3.12); and(c) a sepiapterin reductase (EC 1.1.1.153).4. The recombinant microbial cell of any one of the preceding claims claim 1 , comprising exogenous nucleic acid sequences encoding(a) a 4a-hydroxytetrahydrobiopterin dehydratase (EC 4.2.1.96); and(b) optionally, a dihydropteridine reductase (EC 1.5.1.34).5. The recombinant microbial cell of any one of the preceding claims claim 1 , wherein each one of said exogenous nucleic acid sequences is operably linked to an inducible claim 1 , a regulated or a constitutive promoter.6. The recombinant microbial cell of any one of the preceding claims claim 1 , which comprises a mutation providing for reduced tryptophanase activity.7. The recombinant microbial cell of any one of the preceding claims claim 1 , which is derived from a microbial host cell which is a ...

Inhibition of Serotonin Expression in Gut Enteroendocrine Cells Results in Conversion to Insulin-Positive Cells

Disclosed herein are methods involving the targeting of 5HT biosynthesis in gut insulin-negative cells to convert them into insulin-positive cells. Also, disclosed are methods for treating a disease or disorder in a mammal, preferably a human, associated with impaired pancreatic endocrine function, by administering a therapeutically effective amount of an enumerated active agent that reduces the expression, biosynthesis, signaling or biological activity of serotonin or increases its degradation, wherein administering comprises delivering the agent to Gut Ins− cells in the mammal. Other embodiments of the method are directed to therapy wherein an agent that significantly reduces FOXO1 expression, biosynthesis, signaling or biological activity or increases its degradation is administered in addition to the agent that reduces serotonin, or alternatively an agent that reduces FOXO1 expression is targeted to serotonin-positive gut enteroendocrine cells.

MESSENGER UNA MOLECULES AND USES THEREOF

This invention provides a range of translatable messenger UNA (mUNA) molecules. The mUNA molecules can be translated in vitro and in vivo to provide an active polypeptide or protein, or to provide an immunization agent or vaccine component. The mUNA molecules can be used as an active agent to express an active polypeptide or protein in cells or subjects. Among other things, the mUNA molecules are useful in methods for treating rare diseases. 1. A mUNA molecule , comprising one or more UNA monomers , and comprising nucleic acid monomers , wherein the mUNA molecule is translatable to express a polypeptide or protein.2. The molecule of claim 1 , wherein the molecule comprises from 200 to 12 claim 1 ,000 monomers.3. The molecule of claim 1 , wherein the molecule comprises from 200 to 4 claim 1 ,000 monomers.4. The molecule of claim 1 , wherein the molecule comprises from 1 to 8 claim 1 ,000 UNA monomers.5. The molecule of claim 1 , wherein the molecule comprises from 1 to 100 UNA monomers.6. The molecule of claim 1 , wherein the molecule comprises from 1 to 20 UNA monomers.7. The molecule of claim 1 , wherein the molecule comprises one or more modified nucleic acid nucleotides claim 1 , or one or more chemically-modified nucleic acid nucleotides.8. The molecule of claim 1 , wherein the molecule comprises a 5′ cap claim 1 , a 5′ untranslated region of monomers claim 1 , a coding region of monomers claim 1 , a 3′ untranslated region of monomers claim 1 , and a tail region of monomers.9. The molecule of claim 8 , wherein the molecule comprises a translation enhancer in a 5′ or 3′ untranslated region.10. The molecule of claim 1 , wherein the molecule is translatable in vivo.11. The molecule of claim 1 , wherein the molecule is translatable in vitro.12. The molecule of claim 1 , wherein the molecule is translatable in a mammalian cell.13. The molecule of claim 1 , wherein the molecule is translatable in a human in vivo.14. The molecule of claim 1 , wherein a translation ...

MULTIMERIC CODING NUCLEIC ACID AND USES THEREOF

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends. 1. A multimeric coding nucleic acid (MCNA) comprising two polynucleotides linked via 3′ ends via an oligonucleotide bridge comprising a stable 3-3′ inverted phosphodiester linkage such that the multimeric coding nucleic acid compound comprises two or more 5′ ends , andwherein at least one of the two polynucleotides is an encoding polynucleotide.24.-. (canceled)5. The MCNA of claim 1 , wherein the at least one encoding polynucleotide encodes a protein of interest.6. The MCNA of claim 5 , wherein the two polynucleotides are encoding polynucleotides claim 5 , and each of the two encoding polynucleotides encodes the same protein.7. The MCNA of claim 5 , wherein the two polynucleotides are encoding polynucleotides claim 5 , and each of the two encoding polynucleotides encodes a distinct protein.811.-. (canceled)12. The MCNA of claim 1 , wherein the at least one encoding polynucleotide comprises a 3′ UTR.13. (canceled)14. The MCNA of claim 12 , wherein the 3′ UTR comprises a plurality of multi-A segments with spacers in between.1526.-. (canceled)27. The MCNA of claim 1 , wherein the nucleosides comprising the oligonucleotide bridge are selected from the group consisting of 2′-OMe-A claim 1 , 2′-OMe-G claim 1 , 2′-OMe-C claim 1 , 2′-OMe-U claim 1 , 2′-F-A claim 1 , 2′-F-G claim 1 , 2′-F-C claim 1 , 2′-F-U claim 1 , LNA-A claim 1 , LNA-G claim 1 , LNA-C claim 1 , LNA-U claim 1 , N6-methyl-adenosine claim 1 , 2-thiouridine (2sU) claim 1 , 5-methyl-cytidine (5mC) claim 1 , pseudouridine (ψU) claim 1 , and 1-methyl-pseudouridine.2830.-. (canceled)31. The MCNA of claim 1 , wherein the at least one encoding polynucleotide ...

Benzylisoquinoline Alkaloid (BIA) Precursor Producing Microbes, and Methods of Making and Using the Same

Methods and engineered yeast cells for generating a benzylisoquinoline alkaloid product are provided herein. A method comprises providing engineered yeast cells and a feedstock to a reactor. In the reactor, the engineered yeast cells are subjected to fermentation by incubating the engineered yeast cells for a time period to produce a solution comprising the BIA product and cellular material. The solution comprises not more than one class of molecule selected from the group of protoberberine, morphinan, isopavine, aporphine, and benzylisoquinoline. Additionally, at least one separation unit is used to separate the BIA product from the cellular material to provide the product stream comprising the BIA product. 115-. (canceled)16. A method of producing a pharmaceutical opiate formulation , the method comprising:feeding a plurality of engineered non-plant cells in a cellular growth medium;producing a pharmaceutical opiate compound, or a precursor thereof, from the plurality of engineered non-plant cells to create a mixture comprising the plurality of engineered non-plant cells, the cellular growth medium, and the pharmaceutical opiate compound or a precursor thereof;processing the pharmaceutical opiate compound or precursor thereof; andproducing the pharmaceutical opiate formulation that comprises the pharmaceutical opiate compound.17. The method of claim 16 , wherein the pharmaceutical opiate formulation comprises an active ingredient that has 5% or greater of the pharmaceutical opiate compound.18. The method of claim 17 , wherein the active ingredient comprises the pharmaceutical opiate compound with 98% or greater purity.19. The method of claim 16 , wherein feeding comprises fermenting the plurality of engineered non-plant cells in the cellular growth medium.20. The method of claim 16 , wherein feeding occurs within a cell-culturing device that is selected from the group consisting of a batch reactor and a continuous-flow reactor.21. The method of claim 20 , wherein ...

TYROSINE PROTOTROPHY

Provided herein is a tyrosine selection marker system, and uses thereof. In some embodiments, nucleic acid constructs, vectors, host cells and related compositions and methods for generating and selecting tyrosine prototroph cells are provided. 1. A recombinant nucleic acid construct comprising a nucleotide sequence of interest , and one or both of: i) a phenylalanine hydroxylase (PAH) gene , and ii) a pterin-4-alpha-carbinolamine dehydratase (PCBD1) gene.2. The recombinant nucleic acid construct of claim 1 , wherein the nucleic acid construct further comprises a recombination target sequence.3. The recombinant nucleic acid construct of claim 2 , wherein the recombination target sequence is a FLP Recognition Target (“FRT”) claim 2 , lox claim 2 , or Bxb1 sequence.4. The recombinant nucleic acid construct of claim 1 , wherein the nucleotide sequence of interest encodes a polypeptide of interest claim 1 , encodes an RNA molecule of interest claim 1 , or contains a restriction enzyme site.5. The recombinant nucleic acid construct of claim 1 , wherein the nucleotide sequence of interest is a first nucleotide sequence of interest claim 1 , and wherein the recombinant nucleic acid construct further comprises a second nucleotide sequence of interest.6. (canceled)7. (canceled)8. The recombinant nucleic acid construct of claim 5 , wherein the first nucleotide sequence of interest encodes a first polypeptide comprising an antibody variable light (VL) region and wherein the second nucleotide sequence of interest encodes a second polypeptide comprising an antibody variable heavy (VH) region.9. A vector comprising the recombinant nucleic acid construct of .10. (canceled)11. (canceled)12. A host cell comprising the recombinant nucleic acid construct of .13. A host cell comprising the recombinant nucleic acid construct of claim 1 , wherein the recombinant nucleic acid construct is stably integrated into a chromosome of the host cell.14. The host cell of claim 12 , wherein the host ...

NOVEL VIRAL VECTOR CONSTRUCT FOR NEURON SPECIFIC OPTIMIZED CONTINUOUS DOPA SYNTHESIS IN VIVO

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. 1105-. (canceled)106. A one-vector expression system comprising:i) a first expression cassette comprising a first promoter operably linked to a nucleotide sequence encoding a GTP-cyclohydrolase 1 (GCH1) polypeptide, andii) a second expression cassette comprising a second promoter operably linked to a nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide,said one-vector expression system being capable of expressing said TH polypeptide at a higher level than said GCH1 polypeptide,wherein the vector does not contain a nucleotide sequence encoding an aromatic amino acid decarboxylase (AADC).107. The one-vector expression system of claim 106 , wherein said first promoter is a Synapsin 1 promoter.108. The one-vector expression system of claim 106 , wherein said second promoter is a constitutively active promoter selected from the group consisting of CAG claim 106 , CMV claim 106 , human UbiC claim 106 , RSV claim 106 , EF-1alpha claim 106 , SV40 claim 106 , and Mt1 promoters.109. The one-vector expression system of claim 108 , wherein said second promoter is a CMV promoter.110. The one-vector expression system of claim 106 , wherein said second expression cassette comprises an enhancer or regulator selected from the group consisting of an intron claim 106 , an insulator claim 106 , a CMV enhancer claim 106 , and a ...

ALLOSTERIC ACTIVATORS FOR TREATMENT OF PHENYLKETONURIA

A method of restoring activity in phenylalanine hydroxylase is provided. The method comprises exposing the phenylalanine hydroxylase to shikimic acid, a functionally equivalent analogue thereof, a pharmaceutically acceptable salt of shikimic acid or analogue thereof, or combinations thereof. A method of screening for allosteric activators for a target enzyme is also provided comprising the steps of: denaturing the target enzyme with a first chaotropic agent to yield denatured enzyme, incubating the denatured enzyme with a candidate compound under denaturing conditions to allow enzyme refolding, and assaying enzyme activity in the presence of enzyme substrate and a candidate compound; and if enzyme activity of the denatured enzyme was restored in step i) by at least about 10% of residual enzyme activity, denaturing the target enzyme with a second chaotropic agent to yield denatured enzyme, incubating the denatured enzyme with the candidate compound under non-denaturing conditions to allow enzyme refolding, and assaying enzyme activity in the presence of enzyme substrate and the candidate compound, wherein an increase in enzyme activity of at least about 10% of residual enzyme activity indicates that the candidate compound is an allosteric activator of the target enzyme. 1. A method of restoring phenylalanine hydroxylase activity in phenylalanine hydroxylase comprising exposing said phenylalanine hydroxylase to shikimic acid , a functionally equivalent analogue thereof , a pharmaceutically acceptable salt of shikimic acid or analogue thereof , or combinations thereof.2. The method of claim 1 , wherein the phenylalanine hydroxylase is denatured.3. The method of claim 1 , wherein the phenylalanine hydroxylase is a mutant enzyme.4. The method of claim 3 , wherein the mutant is selected from the I65T mutant and the R261Q mutant.5. The method of claim 1 , wherein the phenylalanine hydroxylase is exposed to an amount of shikimic acid or salt or analogue thereof in the range ...

STABLE EXPRESSION OF AAV VECTORS IN JUVENILE SUBJECTS

The invention relates to the use of adeno-associated virus (AAV) vectors to achieve long term expression of a transgene in the liver of a juvenile subject. The invention includes the stable long-term amelioration of disease symptoms of the subjection following a single administration of an AAV vector to a juvenile subject, wherein the AAV vector delivers the transgene to the subject's liver. 1. A method of ameliorating the symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder comprising administering to the juvenile subject a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein , wherein the expression of the therapeutic protein ameliorates the symptoms of the genetic disorder.2. A use of a therapeutic AAV virus for the preparation of a medicament for ameliorating symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder , wherein the medicament comprises a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein , wherein the expression of the therapeutic protein ameliorates the symptoms of the genetic disorder.3. A composition comprising a therapeutically effective amount of a therapeutic AAV virus encoding a therapeutic protein for use in ameliorating symptoms of a genetic disorder in a juvenile subject suffering from the genetic disorder.4. The method , use or composition of any one of - , wherein the therapeutic protein is a functional copy of a non-functional endogenous protein.5. The method , use or composition of any one of - , wherein the therapeutic protein is a modified version of the endogenous protein.6. The method , use or composition of any one of - , wherein the therapeutic protein is a heterologous protein that compensates for a non-functional endogenous protein.7. The method , use or composition of any of the preceding claims , wherein the juvenile subject is a juvenile human.8. The method claim 7 , use or ...

MRNA THERAPY FOR PHENYLKETONURIA

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids 1. A method of treating phenylketonuria (PKU) , comprising administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity , severity , or frequency or has delayed in onset.2. The method of claim 1 , wherein the mRNA is encapsulated within a liposome.3. The method of claim 2 , wherein the liposome comprises one or more cationic lipids claim 2 , one or more non-cationic lipids claim 2 , one or more cholesterol-based lipids and one or more PEG-modified lipids.4. The method of claim 3 , wherein the one or more cationic lipids are selected from the group consisting of C12-200 claim 3 , MC3 claim 3 , DLinDMA claim 3 , DLinkC2DMA claim 3 , cKK-E12 claim 3 , ICE (Imidazol-based) claim 3 , HGT5000 claim 3 , HGT5001 claim 3 , DODAC claim 3 , DDAB claim 3 , DMRIE claim 3 , DOSPA claim 3 , DOGS claim 3 , DODAP claim 3 , DODMA and DMDMA claim 3 , DODAC claim 3 , DLenDMA claim 3 , DMRIE claim 3 , CLinDMA claim 3 , CpLinDMA claim 3 , DMOBA claim 3 , DOcarbDAP claim 3 , DLinDAP claim 3 , DLincarbDAP claim 3 , DLinCDAP claim 3 , KLin-K-DMA claim 3 , DLin-K-XTC2-DMA claim 3 , HGT4003 claim 3 , and combinations thereof.6. The method of any one of - claim 3 , wherein the one or more non-cationic lipids are ...

Optimized microbial cells for production of melatonin and other compounds

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors. 2. The variant of claim 1 , wherein(i) the mutation in the residue corresponding to residue E147 is an amino acid substitution selected from 147K, 147R and 147H;(ii) the mutation in the residue corresponding to residue N242 is the amino acid substitution 242I; and(iii) the mutation in the residue corresponding to P244 is selected from 244C, 244D, 244L and 244Q.3. The variant of claim 2 , wherein the mutation in the residue corresponding to E147 is E147K claim 2 , the mutation in the residue corresponding to N242 is N242I claim 2 , and the mutation in the residue corresponding to P244 is P244C.4Homo sapiens. The variant of claim 3 , comprising a segment which has at least 95% sequence identity to the segment corresponding to residues E147 to T460 of TPH having the sequence of SEQ ID NO:3.5. The variant of claim 1 , which provides for a tryptophan hydroxylation activity which is at least 110% of that of the native or parent TPH.6. A nucleic acid sequence encoding the variant TPH of .7. A recombinant microbial cell comprising the nucleic acid sequence of .8. A TPH having the sequence of SEQ ID NO:13 except for E2K claim 6 , N97I and P99C mutations.9. A nucleic acid sequence encoding the TPH of .10. A recombinant microbial cell comprising the nucleic acid sequence of .11E. coli. The recombinant microbial cell of claim 10 , further comprising nucleic acid sequences encoding a pterin-4a-carbolamine dehydratase (PCD) and a GTP cyclohydrolase I (GCH1) ...

METHODS OF PRODUCING NOR-OPIOID AND NAL-OPIOID BENZYLISOQUINOLINE ALKALOIDS

A method of demethylizing an opioid to a nor-opioid is provided. The method comprises contacting an opioid with at least one enzyme. Contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid. A method of converting a nor-opioid to a nal-opioid is provided. The method comprises contacting a nor-opioid with at least one enzyme. Contacting the nor-opioid with the at least one enzyme converts the nor-opioid to a nal-opioid. 1. A method of demethylating a first opioid to a second opioid , comprising:contacting the first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to the second opioid through loss of an O-linked methyl group, andwherein the first opioid is not selected from the group consisting of codeine and thebaine.2. A method of demethylating an opioid to a nor-opioid , comprising:contacting a first opioid with at least one enzyme, wherein contacting the first opioid with the at least one enzyme converts the first opioid to a second opioid through loss of an O-linked methyl group; andcontacting the second opioid with at least one enzyme, wherein contacting the opioid with the at least one enzyme converts the second opioid to a nor-opioid through loss of an N-linked methyl group.3. A method of demethylating an opioid to a nor-opioid , comprising:contacting the opioid with at least one enzyme, wherein contacting the opioid with the at least one enzyme converts the opioid to the nor-opioid through removal of a N-linked methyl group from the opioid,wherein the opioid is not thebaine when the opioid contacts the at least one enzyme in vitro.4. (canceled)5. (canceled)6. The method of claim 1 , wherein the second opioid is produced by culturing an engineered cell comprising a coding sequence for encoding the at least one enzyme.7. The method of claim 2 , wherein the nor-opioid is produced by culturing an engineered cell comprising a coding sequence for encoding the ...

METHOD OF ATTACHING A CELL-OF-INTEREST TO A MICROTUBE

A method of attaching a cell or a membrane-coated particle-of-interest to a microtube is provided. The method comprising: co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and wherein the second polymeric solution comprises the cell or the membrane-coated particle-of-interest, thereby attaching the cell or the membrane-coated particle-of-interest to the microtube. Also provided are microtubes with attached, entrapped or encapsulated cells or membrane-coated particles and methods of using same 1. A method of attaching a cell or a membrane-coated particle-of-interest to a microtube , the method comprising:co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of said two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of said two polymeric solutions is for forming a coat over an internal surface of said shell,said first polymeric solution is selected solidifying faster than said second polymeric solution and a solvent of said second polymeric solution is incapable of dissolving said first polymeric solution and wherein said second polymeric solution comprises the cell or said membrane-coated particle-of-interest,thereby attaching the cell or said membrane-coated particle-of-interest to the microtube.2. The method of claim 1 , wherein said first solvent of said first polymeric solution evaporates faster than said second solvent of said second polymeric solution claim 1 , and wherein said second solvent of said second ...

METHODS OF ATTACHING A MOLECULE-OF-INTEREST TO A MICROTUBE

A method of attaching a molecule-of-interest to a microtube, by co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and the second polymeric solution comprises the molecule-of-interest, thereby attaching the molecule-of-interest to the microtube. An electrospun microtube comprising an electrospun shell, an electrospun coat over an internal surface of the shell and a molecule-of-interest attached to the microtube. 1. A method of attaching a molecule-of-interest to a microtube , the method comprising:co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of said two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of said two polymeric solutions is for forming a coat over an internal surface of said shell,said first polymeric solution is selected solidifying faster than said second polymeric solution and a solvent of said second polymeric solution is incapable of dissolving said first polymeric solution and wherein said second polymeric solution comprises the molecule-of-interest,thereby attaching the molecule-of-interest to the microtube.2. The method of claim 1 , wherein said first solvent of said first polymeric solution evaporates faster than said second solvent of said second polymeric solution claim 1 , and wherein said second solvent of said second polymeric solution is capable of evaporating through said internal surface of said shell.3. The method of claim 1 , wherein said polymer of said first polymeric solution ...

COMPOSITIONS AND METHODS FOR TREATING PHENYLKETONURIA

A lentiviral vector system for expressing a lentiviral particle is disclosed. The lentiviral vector system includes a therapeutic vector. The therapeutic vector comprises a phenylalanine hydroxylase (PAH) sequence for expressing at least one of PAH or a variant thereof, wherein the PAH sequence is truncated. 1. A viral vector comprising:a phenylalanine hydroxylase (PAH) sequence for expressing at least one of PAH or a variant thereof, wherein the PAH sequence is truncated.2. The viral vector of claim 1 , wherein the PAH sequence is truncated at a 3′ untranslated region (UTR) of the sequence.3. The viral vector of claim 1 , wherein the PAH sequence comprises at least one of 80% claim 1 , 85% claim 1 , 90% claim 1 , 95% claim 1 , or 100% identity with at least one of SEQ ID NO: 2 claim 1 , SEQ ID NO: 3 claim 1 , or SEQ ID NO: 4.4. The viral vector of claim 1 , further comprising:at least one small RNA sequence that is capable of binding to at least one pre-determined complementary mRNA sequence.5. The viral vector of claim 4 , wherein the at least one pre-determined complementary mRNA sequence comprises a full-length 3′ untranslated region (UTR).6. The viral vector of claim 4 , wherein the at least one pre-determined complementary mRNA sequence is a PAH mRNA sequence.7. The viral vector of claim 4 , wherein the at least one small RNA sequence comprises a shRNA.8. The viral vector of claim 4 , wherein the at least one small RNA sequence comprises a sequence having at least one of 80% claim 4 , 85% claim 4 , 90% claim 4 , 95% claim 4 , or 100% identity with at least one of SEQ ID NO: 5 or SEQ ID NO: 6.9. The viral vector of claim 4 , wherein the at least one small RNA sequence is under the control of a first promoter claim 4 , and wherein the PAH sequence is under the control of a second promoter.10. The viral vector of claim 9 , wherein the first promoter comprises a H1 promoter.11. The viral vector of claim 9 , wherein the second promoter comprises a liver-specific ...

In vitro methods for processing lignin and other aromatic compounds

Enzymes for depolymerizing lignin. The enzymes include dehydrogenases, β-etherases, and glutathione lyases. The dehydrogenases can comprise one or more or LigD, LigO, LigN, and LigL. The β-etherases can comprise one or more of LigE, LigF, LigP, and BaeA. The glutathione lyases can comprise any one or more of LigG and a number of non-stereospecific, optionally recombinant glutathione lyases derived from Sphingobium sp. SYK-6, Novosphingobium aromaticivorans, Escherichia coli, Streptococcus sanguinis, Phanerochaete chrysosporium, and other microorganisms. The enzymes can be combined in compositions and/or used in methods of processing lignin or other aromatic compounds in vitro.

GENE EXPRESSION SYSTEM AND REGULATION THEREOF

The present invention relates to a novel gene expression system comprising: a) a first nucleotide sequence encoding a fusion polypeptide of: a1) a destabilizing domain (DD) based on DHFR, and a2) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and b) a second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof. The invention also relates to use of this gene expression system together with a ligand binding to a destabilizing domain (DD) based on dihydrofolate reductase (DHFR) for treatment of diseases associated with a reduced dopamine level, such as Parkinson's disease. 1. A gene expression system comprising: a) a destabilizing domain (DD), and', 'b) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and, 'a first nucleotide sequence encoding a fusion polypeptide ofa second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof.24-. (canceled)5. A gene expression system according to claim 1 , wherein said gene expression system comprises two vectors each containing one expression cassette claim 1 , wherein:the expression cassette in the first vector comprises the first nucleotide sequence and a first promoter sequence operably linked to the first nucleotide sequence, andthe expression cassette in the second vector comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence.6. A gene expression system according to claim 1 , wherein said gene expression system comprises one vector comprising both the first nucleotide sequence and the second nucleotide sequence claim 1 , wherein the vector comprises either: ia) a promotor is operably linked to either the first or the second nucleotide sequence, and wherein the nucleotide sequence to which the promotor is linked to the other of the first ...

THERAPEUTICS FOR PHENYLKETONURIA

This invention provides a range of translatable polynucleotide and oligomer molecules for expressing a human phenylalanine hydroxylase (PAH), or a fragment thereof having PAH activity. The polynucleotide and oligomer molecules are expressible to provide the human PAH or a fragment thereof having PAH activity. The molecules can be used as active agents to express an active polypeptide or protein in cells or subjects. The agents can be used in methods for ameliorating, preventing, delaying onset, or treating a disease or condition associated with phenylketonuria, decreased metabolism of phenylalanine, or increased levels of phenylalanine in a subject. 1. A polynucleotide for expressing a human phenylalanine hydroxylase (PAH) , or a fragment thereof , wherein the polynucleotide comprises natural and chemically-modified nucleotides and is expressible to provide the human phenylalanine hydroxylase or a fragment thereof having PAH activity.2. The polynucleotide of claim 1 , wherein the polynucleotide is codon-optimized as compared to human PAH wild type mRNA.3. The polynucleotide of claim 1 , wherein the chemically-modified nucleotides are selected from5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine;5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2′-O-methyluridine, 2′-O-methyl-5-methyluridine, 2′-fluoro-2′-deoxyuridine, 2′-amino-2′-deoxyuridine, 2′-azido-2′-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine;{'sup': 1', '1', '1', '1', '1, 'pseudouridine, 2′-O-methyl-pseudouridine, N-hydroxypseudouridine, N-methylpseudouridine, 2′-O-methyl-N-methylpseudouridine, N-ethylpseudouridine, N-hydroxymethylpseudouridine, and Arauridine;'}{'sup': '6', 'N-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7- ...

COMPOSITION OF ERYTHROCYTES ENCAPSULATING PHENYLALANINE HYDROXYLASE AND THERAPEUTIC USE THEREOF

The present invention relates to Enzyme Replacement Therapy (ERT) based on phenylalanine hydroxylase (PAH) and compositions intended for this use. It concerns an erythrocyte encapsulating PAH, especially in suspension in a pharmaceutically acceptable carrier or vehicle, a pharmaceutical composition comprising erythrocytes encapsulating PAH in a pharmaceutically acceptable carrier or vehicle, and such a pharmaceutical composition for use in the treatment or prevention of phenylketonuria (PKU) and/or other diseases involving a too high level of phenylalanine; the treatment or prevention may be in combination with a Phe-restricted diet. The invention particularly relates to classic PKU, variant PKU and non-PKU hyperphenylalaninemia. 119-. (canceled)20. A pharmaceutical composition comprising erythrocytes encapsulating phenylalanine hydroxylase (PAH) in a pharmaceutically acceptable carrier or vehicle.21. The composition according to claim 20 , wherein it is formulated in a dose volume comprising from about 50 to about 2000 claim 20 , preferably about 100 to about 1000 IU of PAH.22. The composition according to claim 20 , wherein it is formulated in a dose volume of about 10 to about 250 ml.23. The composition according to claim 20 , wherein the haematocrit is between about 40 and about 70%.24. The composition according to claim 20 , comprising the cofactor BH.25. An erythrocyte encapsulating phenylalanine hydroxylase (PAH).26. The erythrocyte of claim 25 , in suspension in a pharmaceutically acceptable carrier or vehicle.27. A method of treatment or prevention of phenylketonuria and/or other diseases involving a too high level of phenylalanine claim 25 , which comprises administering to a patient in need thereof of an effective amount of a pharmaceutical composition comprising erythrocytes encapsulating PAH in a pharmaceutically acceptable carrier or vehicle.28. The method of claim 27 , for the treatment or prevention of phenylketonuria (PKU) in a patient.29. The ...

Optimized Microbial Cells for Production of Melatonin and Other Compounds

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors. 1E. coliE. coli. A variant of GTP cyclohydrolase I (GCH1) having at least about 80% sequence identity to native GCH1 (SEQ ID NO:16) and comprising one or more mutations , wherein ,{'i': E. coli', 'E. coli, 'in an cell comprising a pterin-4α-carbinolamine dehydratase (PCD) and at least one of a tryptophan hydroxylase (TPH), a tyrosine hydroxylase (TH) and a phenylalanine hydroxylase (PheH), the variant provides for an increased hydroxylation activity of at least one of the TPH, TH and PheH as compared to native GCH1, and'}the mutation is not T198P.2. The variant of claim 1 , wherein at least one of the one or more mutations is in an amino acid residue in a segment selected from D97-E112 claim 1 , K121-D130 claim 1 , N170-H180 claim 1 , S193-L200 and S207-N222.3. The variant of claim 1 , wherein at least one of the one or more mutations is in an amino acid residue selected from the group consisting of D97 claim 1 , M99 claim 1 , T101 claim 1 , V102 claim 1 , A125 claim 1 , K129 claim 1 , N170 claim 1 , V179 claim 1 , T196 claim 1 , T198 claim 1 , S199 claim 1 , L200 claim 1 , S207 claim 1 , H212 claim 1 , E213 claim 1 , F214 claim 1 , L215 and H221.4. The variant of claim 1 , wherein the variant(a) comprises a mutation selected from D97V, D97L, D97A, D97T, M99C, M99T, M99V, M99L, M99I, T101I, T101V, T101L, V102M, N170K, N170D, N170L, V179A, V179M, T196I, T196V, T196L, T198I, T198V, T198S, T198L, S199Y, S199F, L200P, L200C, L200S, L200A, S207R, ...

MESSENGER UNA MOLECULES AND USES THEREOF

This invention provides a range of translatable messenger UNA (mUNA) molecules. The mUNA molecules can be translated in vitro and in vivo to provide an active polypeptide or protein, or to provide an immunization agent or vaccine component. The mUNA molecules can be used as an active agent to express an active polypeptide or protein in cells or subjects. Among other things, the mUNA molecules are useful in methods for treating rare diseases. 1. A mUNA molecule , comprising one or more UNA monomers , and comprising nucleic acid monomers , wherein the mUNA molecule is translatable to express a polypeptide or protein.2. The molecule of claim 1 , wherein the molecule comprises from 200 to 12 claim 1 ,000 monomers.3. The molecule of claim 1 , wherein the molecule comprises from 200 to 4 claim 1 ,000 monomers.4. The molecule of claim 1 , wherein the molecule comprises from 1 to 8 claim 1 ,000 UNA monomers.5. The molecule of claim 1 , wherein the molecule comprises from 1 to 100 UNA monomers.6. The molecule of claim 1 , wherein the molecule comprises from 1 to 20 UNA monomers.7. The molecule of claim 1 , wherein the molecule comprises one or more modified nucleic acid nucleotides claim 1 , or one or more chemically-modified nucleic acid nucleotides.8. The molecule of claim 1 , wherein the molecule comprises a 5′ cap claim 1 , a 5′ untranslated region of monomers claim 1 , a coding region of monomers claim 1 , a 3′ untranslated region of monomers claim 1 , and a tail region of monomers.9. The molecule of claim 8 , wherein the molecule comprises a translation enhancer in a 5′ or 3′ untranslated region.10. The molecule of claim 1 , wherein the molecule is translatable in vivo.11. The molecule of claim 1 , wherein the molecule is translatable in vitro.12. The molecule of claim 1 , wherein the molecule is translatable in a mammalian cell.13. The molecule of claim 1 , wherein the molecule is translatable in a human in vivo.14. The molecule of claim 1 , wherein a translation ...

SELECTABLE MARKERS AND RELATED METHODS

The invention relates to newly identified selectable marker systems, cells for use in a selectable marker system, and methods for using the selectable marker systems. 1. A cell , wherein said cell has been genetically engineered to disrupt expression of a protein of Table 1.2. The cell of claim 1 , wherein the protein is selected from argininosuccinate lyase claim 1 , cystathionine G-lyase claim 1 , cystathionine b-synthase claim 1 , glycine N-methyltransferase claim 1 , pyrroline-5-carboxylate synthetase claim 1 , phenylalanine 4-monooxygenase claim 1 , carbamoyl-phosphate synthase (ammonia) claim 1 , ornithine carbamoyltransferase (mitochondrial) claim 1 , ornithine transaminase (mitochondrial) claim 1 , methionine adenosyltransferase claim 1 , adenosylhomocysteinase claim 1 , propionyl-CoA carboxylase (mitochondrial) claim 1 , methylmalonyl-CoA epimerase (mitochondrial) claim 1 , methylmalonyl-CoA mutase claim 1 , myo-Inositol-1-phosphate synthase claim 1 , thymidylate synthase claim 1 , nucleoside-diphosphatase (dUDP) claim 1 , carbonate anhydrase claim 1 , adenosine kinase claim 1 , sarcosine oxidase claim 1 , alphaketobutyrate dehydrogenase (mitochondrial) claim 1 , phosphatidylserine decarboxylase claim 1 , glucose-6-phosphate isomerase claim 1 , myo-inositol 1-phosphatase claim 1 , ribonucleoside-diphosphate reductase claim 1 , and dihydropteridine reductase.3. The cell of claim 1 , wherein said cell further comprises an exogenous nucleic acid claim 1 , wherein said exogenous nucleic acid comprises an expressible nucleic acid comprising a nucleic acid encoding said protein lacking expression in said cell.4. The cell of claim 3 , wherein said exogenous nucleic acid further comprises an expressible nucleic acid encoding a product.5. A composition comprising a cell of claim 1 , wherein said cell lacks expression of a protein of Table 1 claim 1 , and an expressible nucleic acid comprising a nucleic acid encoding said protein lacking expression in said cell.67.-. ...

Process of Preparing mRNA-Loaded Lipid Nanoparticles

The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a solution of pre-formed lipid nanoparticles and mRNA. 1. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising: mixing a solution comprising pre-formed lipid nanoparticles and mRNA such that lipid nanoparticles encapsulating mRNA are formed.2. The process of claim 1 , wherein the solution comprising pre-formed lipid nanoparticles and mRNA comprises less than 10 mM citrate.3. The process of claim 1 , wherein the solution comprising pre-formed lipid nanoparticles and mRNA comprises less than 25% non-aqueous solvent.45-. (canceled)6. The process of claim 1 , comprising heating the lipid nanoparticles and mRNA to a temperature greater than ambient temperature before or after the mixing claim 1 , and wherein the temperature is or is greater than about 30° C. claim 1 , 37° C. claim 1 , 40° C. claim 1 , 45° C. claim 1 , 50° C. claim 1 , 55° C. claim 1 , 60° C. claim 1 , 65° C. claim 1 , or 70° C.78-. (canceled)9. The process of claim 1 , wherein the pre-formed lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution.10. The process of claim 1 , wherein the lipids comprise one or more cationic lipids claim 1 , one or more helper lipids claim 1 , one or more cholesterol-based lipids and PEG lipids.11. The process of claim 10 , wherein the one or more cationic lipids are selected from the group consisting of cKK-E12 claim 10 , OF-02 claim 10 , C12-200 claim 10 , MC3 claim 10 , DLinDMA claim 10 , DLinkC2DMA claim 10 , ICE (Imidazol-based) claim 10 , HGT5000 claim 10 , HGT5001 claim 10 , HGT4003 claim 10 , DODAC claim 10 , DDAB claim 10 , DMRIE claim 10 , DOSPA claim 10 , DOGS claim 10 , DODAP claim 10 , DODMA and DMDMA claim 10 , DODAC claim 10 , DLenDMA ...

BENZYLISOQUINOLINE ALKALOID (BIA) PRECURSOR PRODUCING MICROBES, AND METHODS OF MAKING AND USING THE SAME

Methods and engineered yeast cells for generating a benzylisoquinoline alkaloid product are provided herein. A method comprises providing engineered yeast cells and a feedstock to a reactor. In the reactor, the engineered yeast cells are subjected to fermentation by incubating the engineered yeast cells for a time period to produce a solution comprising the BIA product and cellular material. The solution comprises not more than one class of molecule selected from the group of protoberberine, morphinan, isopavine, aporphine, and benzylisoquinoline. Additionally, at least one separation unit is used to separate the BIA product from the cellular material to provide the product stream comprising the BIA product. 1. A method for forming a product stream having a benzylisoquinoline alkaloid product , the method comprising:(a) providing engineered non-plant cells and a feedstock including nutrients and water to a batch reactor, which engineered non-plant cells have at least one modification that results in overproduction of tyrosine with respect to a non-engineered non-plant cell, wherein the at least one modification is selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene and an inactivating mutation in an enzyme;(b) in the batch reactor, subjecting the engineered non-plant cells to fermentation by incubating the engineered non-plant cells for a time period of at least about 5 minutes to produce a solution comprising the benzylisoquinoline alkaloid product and cellular material; and(c) using at least one separation unit to separate the benzylisoquinoline alkaloid product from the cellular material to provide the product stream comprising the benzylisoquinoline alkaloid product.2. A method for forming a product stream having a benzylisoquinoline alkaloid product , the method comprising:(a) providing engineered non-plant cells and a feedstock including nutrients and water to a reactor;(b) in the reactor, subjecting ...

Genetic Construct

A genetic construct comprises a promoter operably linked to a first coding sequence, which encodes tyrosine hydroxylase (TH), and a second coding sequence, which encodes GTP cyclohydrolase 1 (GCH1), wherein the second coding sequence is 3′ to the first coding sequence, and the first and second coding sequences are part of a single operon. The genetic construct does not encode aromatic amino acid decarboxylase (AADC). 1. A genetic construct comprising a promoter operably linked to a first coding sequence , which encodes tyrosine hydroxylase (TH) , and a second coding sequence , which encodes GTP cyclohydrolase 1 (GCH1) , wherein the second coding sequence is 3′ to the first coding sequence , and the first and second coding sequences are part of a single operon , wherein the genetic construct does not encode aromatic amino acid decarboxylase (AADC).2. A genetic construct according to claim 1 , wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 1 or SEQ ID No:2 claim 1 , or a fragment or variant thereof claim 1 , and/or comprises a nucleotide sequence encoding an amino acid sequence substantially as set out in SEQ ID NO: 21 or SEQ ID No:22 claim 1 , or a fragment or variant thereof.3. A genetic construct according to claim 1 , wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO: 4 claim 1 , or a fragment or variant thereof claim 1 , and/or comprises a nucleotide sequence encoding an amino acid sequence substantially as set out in SEQ ID NO: 23 claim 1 , or a fragment or variant thereof.4. A genetic construct according to claim 1 , wherein the promoter is a constitutive promoter claim 1 , an activatable promoter claim 1 , an inducible promoter claim 1 , or a tissue-specific promoter claim 1 , optionally wherein the promoter comprises a CMV promoter or a human synapsin promoter.5. A genetic construct according to claim 1 , wherein the promoter comprises a nucleotide ...

Multimeric coding nucleic acid and uses thereof

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends.

Inhibition of Serotonin Expression in Gut Enteroendocrine Cells Results in Conversion to Insulin-Positive Cells

Disclosed herein are methods involving the targeting of 5HT biosynthesis in gut insulin-negative cells to convert them into insulin-positive cells. Also disclosed are methods for treating a disease or disorder in a mammal, preferably a human, associated with impaired pancreatic endocrine function, by administering a therapeutically effective amount of an enumerated active agent that reduces the expression, biosynthesis, signaling or biological activity of serotonin or increases its degradation, wherein administering comprises delivering the agent to Gut Ins− cells in the mammal. Other embodiments of the method are directed to therapy wherein an agent that significantly reduces FOXO1 expression, biosynthesis, signaling or biological activity or increases its degradation is administered in addition to the agent that reduces serotonin, or alternatively an agent that reduces FOXO1 expression is targeted to serotonin-positive gut enteroendocrine cells.

MRNA THERAPY FOR PHENYLKETONURIA

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 1. A method of treating phenylketonuria (PKU) , comprising administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity , severity , or frequency or has delayed in onset.2. The method of claim 1 , wherein the mRNA is encapsulated within aliposome.3. The method of claim 2 , wherein the liposome comprises one or more cationic lipids claim 2 , one or more non-cationic lipids claim 2 , one or more cholesterol-based lipids and one or more PEG-modified lipids.46-. (canceled)7. The method of claim 3 , wherein the one or more cholesterol-based lipids is cholesterol or PEGylated cholesterol.8. (canceled)9. The method of claim 3 , wherein the cationic lipid constitutes about 30-60% of the liposome by molar ratio.1015-. (canceled)16. The method of claim 2 , wherein the liposome has a size less than about 100 nm.17. The method of claim 1 , wherein the mRNA is administered at the effective dose ranging from about 0.1-3.0 mg/kg body weight.18. (canceled)19. The method of claim 1 , wherein the composition is administered intravenously.2023-. (canceled)24. The method of claim 1 , wherein the administering of the composition results in the expression of the PAH protein detectable in liver claim 1 , ...

VECTORS AND METHODS OF USE

This disclosure provides vectors and strategies for increasing the efficiency of gene therapy in hepatocytes. The efficiency of the delivery of a corrected gene or wild type gene is improved through the use of delivery to hepatocytes via intrahepatic (parenchyma) administration or administration via the portal vein. In addition, the corrected gene or wild type gene is delivered using an isolated exogenous nucleic acid comprising a promoter that is specifically expressed in hepatocytes. These methods and isolated exogenous nucleic acids are useful to correct gene defects in the liver such as inherited diseases of the liver. 13-. (canceled)4. A method of delivering an isolated exogenous nucleic acid coding for all or part of a polypeptide to a liver of a subject , the method comprising:administering the isolated exogenous nucleic acid to hepatocytes of the subject intrahepatically or via portal vein injection, the isolated exogenous nucleic acid comprising a nucleic acid coding for all of part of a polypeptide operably linked to a hepatocyte specific promoter and enhancer, wherein the polypeptide is a functional phenylalanine hydroxylase protein or a functional fumarylacetoacetate hydrolase protein.5. The method of claim 4 , wherein the isolated exogenous nucleic acid comprises a lentiviral vector claim 4 , an adenoviral vector claim 4 , adeno-associated viral vector or a retroviral vector.6. The method of claim 4 , wherein the isolated exogenous nucleic acid comprises a lentiviral vector.7. The method of claim 4 , wherein the hepatocyte specific promoter is an alpha1-antitrypsin (AAT) promoter.8. The method of claim 7 , wherein the polypeptide is a functional fumarylacetoacetate hydrolase protein.9. The method of claim 4 , wherein the subject is a human.10. The method of claim 9 , wherein the subject is a fetus.11. The method of claim 9 , wherein the subject is an infant or older pediatric patient.12. The method of claim 4 , wherein the isolated exogenous nucleic ...

MICROORGANISMS FOR EFFICIENT PRODUCTION OF MELATONIN AND RELATED COMPOUNDS

Recombinant microbial cells and methods for producing 5HTP, melatonin and related compounds using such cells are described. More specifically, the recombinant microbial cell may comprise exogenous genes encoding one or more of an L-tryptophan hydroxylase, a 5-hydroxy-L-tryptophan decarboxylyase, a serotonin acetyltransferase, an acetylserotonin O-methyltransferase; and means for providing tetrahydrobiopterin (THB), and can be further genetically modified to enrich one or more of tryptophan, S-adenosyl-L-methinonine and acetyl coenzyme A. Related sequences and vectors for use in preparing such recombinant microbial cells are also described. 1. A recombinant microbial cell comprising exogenous nucleic acid sequences encoding an L-tryptophan hydroxylase (TPH) (EC 1.14.16.4) , a 5-hydroxy-L-tryptophan decarboxylase (DDC) (EC 4.1.1.28) , a serotonin acetyltransferase (AANAT) (EC 2.3.1.87 or EC 2.3.1.5) , an acetylserotonin O-methyltransferase (ASMT) (EC 2.1.1.4) , and enzymes providing at least one pathway for producing tetrahydrobiopterin (THB) , wherein the recombinant microbial cell(i) further comprises a genetic modification providing for an increase in S-adenosyl-L-methinonine (SAM) production, an increase in acetyl coenzyme A (AcCoA) production, an increase in tryptophan production, or a combination of any thereof; and/or(ii) comprises an exogenous nucleic acid sequence encoding a TPH which comprises SEQ ID NO:177, SEQ ID NO:176, or functionally active variant, homolog or fragment of any thereof.2. The recombinant microbial cell of claim 1 , wherein the genetic modification in (i) comprises one or more exogenous nucleic acid sequences encoding(a) a S-adenosylmethionine synthetase (EC 2.5.1.6),(b) a ethionine resistance protein,(c) a S-adenosylhomocysteine hydrolase (EC 3.3.1.1),(d) a methionine synthase (EC 2.1.1),(e) an AcCoA synthetase (EC 6.2.1.1),(f) an acetylaldehyde dehydrogenase (EC 1.2.1.3), or(g) a combination of any two or more of (a) to (f).3. A ...

VECTORS AND METHODS OF USE

This disclosure provides vectors and strategies for increasing the efficiency of gene therapy in hepatocytes. The efficiency of the delivery of a corrected gene or wild type gene is improved through the use of delivery to hepatocytes via intrahepatic (parenchyma) administration or administration via the portal vein. In addition, the corrected gene or wild type gene is delivered using an isolated exogenous nucleic acid comprising a promoter that is specifically expressed in hepatocytes. These methods and isolated exogenous nucleic acids are useful to correct gene defects in the liver such as inherited diseases of the liver. 1. A lentiviral vector comprising an isolated exogenous nucleic acid coding for a functional phenylalanine hydroxylase protein or a functional fumarylacetoacetate hydrolase protein , the isolated exogenous nucleic acid under the control of an alpha1-antitrypsin (AAT) promoter and enhancer operably linked to the isolated exogenous nucleic acid.2. The lentiviral vector of claim 1 , further comprising a 5′ long terminal repeat (LTR) followed by a psi packaging sequence (Ψ) followed by a rev responsive element (RRE) followed by a central polypurine tract (cPPT).3. The lentiviral vector of claim 2 , further comprising a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) follow by a 3′ LTR having a U3 region that is partially or completely deleted.4. A method of delivering an isolated exogenous nucleic acid coding for all or part of a polypeptide to a liver of a subject claim 2 , the method comprising:administering the isolated exogenous nucleic acid to hepatocytes of the subject intrahepatically or via portal vein injection, the isolated exogenous nucleic acid comprising a nucleic acid coding for all of part of a polypeptide operably linked to a hepatocyte specific promoter and enhancer, wherein the polypeptide is a functional phenylalanine hydroxylase protein or a functional fumarylacetoacetate hydrolase protein.5. The method of ...

Methods of treating phenylketonuria

Provided herein are methods of treating a subject having a disease or disorder associated with a PAH gene mutation. The methods generally comprise administering to the subject a therapeutically effective does of a recombinant adeno-associated virus (rAAV) that can express a phenylalanine hydroxylase (PAH) polypeptide in a cell and thereby restore PAH gene function in the subject.

MICROBIAL APPROACH FOR THE PRODUCTION OF 5-HYDROXYTRYPTOPHAN

5-hydroxytryptophan (5-HTP), a precursor of serotonin, is produced in a microbial host cell. A modified bacterial phenylalanine 4-hydroxylase (P4H) catalyzes the tryptophan 5-hydroxylation reaction. Optionally the host cell includes a cofactor regeneration mechanism, allowing continuous production of 5-HTP without supplementation of exogenous cofactors. 1. A genetically engineered bacterial cell comprising a modified bacterial phenylalanine-4-hydroxylase (NH) having increased affinity for tryptophan compared to the corresponding wild-type P4H.2. The genetically engineered bacterial cell of which is genetically engineered to overproduce or accumulate tryptophan.3. The genetically engineered bacterial cell of further comprising a cofactor recycling system comprising at least one of a pterin-4α-carbinolamine dehydratase (PCD) and a dihydromonapterin reductase (DHMR).4E. coli. The genetically engineered bacterial cell of comprising a dihydromonapterin reductase encoded by the gene folM.5Pseudomonas, Chromobacterium, RalstoniaXanthomonas.. The genetically engineered bacterial cell of wherein the modified bacterial P4H is derived from a claim 1 , or6X. campestris. The genetically engineered bacterial cell of wherein the modified bacterial P4H comprises an amino acid mutation at any one claim 1 , any two claim 1 , or all three of amino acid positions 98 claim 1 , 179 claim 1 , and 231 of P4H claim 1 , or at any one claim 1 , any two claim 1 , or all three corresponding amino acid positions in a bacterial P4H from another species.7X. campestris. The genetically engineered bacterial cell of wherein the mutation comprises any one claim 6 , any two claim 6 , or any three mutations selected from the group consisting of L98Y claim 6 , W179F and Y231C of P4H claim 6 , or corresponding amino acid positions in a bacterial P4H enzyme from another species.8X. campestris. The genetically engineered bacterial cell of wherein the modified bacterial P4H further comprise at least one ...

Processes of preparing mrna-loaded lipid nanoparticles

The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a suspension of preformed lipid nanoparticles and mRNA.

PROCESSES FOR THE PRODUCTION OF TRYPTAMINES

Disclosed herein are prokaryotic and eukaryotic microbes, including and , genetically altered to biosynthesize tryptamine and tryptamine derivatives. The microbes of the disclosure may be engineered to contain plasmids and stable gene integrations containing sufficient genetic information for conversion of an anthranilate or an indole to a tryptamine. The fermentative production of substituted tryptamines in a whole-cell biocatalyst may be useful for cost effective production of these compounds for therapeutic use. 2. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris H.3. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris Calkyl.4. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris C.5. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris —O(P═O)(OH)and R claim 1 , R claim 1 , and Rare H.6. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris —O(P═O)(OH)and R claim 1 , R claim 1 , and Rare H.7. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris —O(P═O)(OH)and R claim 1 , R claim 1 , and Rare H.8. The compound or pharmaceutically acceptable salt of claim 1 , wherein Ris —O(P═O)(OH)and R claim 1 , R claim 1 , and Rare H.22. The pharmaceutical composition of claim 21 , wherein one of R claim 21 , R claim 21 , and Ris —O(P═O)(OH).23. The pharmaceutical composition of claim 21 , wherein Ris H or Calkyl.24. The pharmaceutical composition of claim 21 , wherein Ris Calkyl. This application is a continuation application of U.S. patent application Ser. No. 17/012,737, filed Sep. 4, 2020, which is a continuation application of International Patent Application No. PCT/US2019/021489, filed Mar. 8, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/640,443, filed Mar. 8, 2018, which applications are each incorporated herein by reference in their entirety.The instant application contains a Sequence Listing which has been ...

Production of midbrain dopaminergic neurons and methods for the use thereof

Methods are provided for efficient production of midbrain dopaminergic (DA) neurons. In some aspects, methods involve differentiation and selection of DA neurons for a transgenic pluripotent cell population (e.g., cells comprising a selectable marker gene). Cell populations produced by the instant methods and methods of their use are likewise provided.

MULTIMERIC CODING NUCLEIC ACID AND USES THEREOF

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends. 142-. (canceled)43. A method of delivering a multimeric coding nucleic acid (MCNA) for in vivo protein production , comprising administering to a subject in need of delivery a MCNA , wherein the MCNA comprises two messenger RNAs (mRNAs) linked at 3′ ends , via stable linkage , such that the multimeric coding nucleic acid has two 5′ ends , and wherein the stable linkage is an oligonucleotide bridge comprising an internal 3′-to-3′ inverted phosphodiester linkage.4447-. (canceled)48. The method of claim 43 , wherein each of the mRNAs encodes a protein of interest.49. The method of claim 48 , wherein each of the mRNAs encodes a same protein.50. The method of claim 48 , wherein each of the mRNAs encodes a distinct protein.51. The method of claim 43 , wherein the mRNAs comprise a 3′ UTR.52. The method of claim 51 , wherein the 3′ UTR comprises a plurality of multi-A segments with spacers in between.53. The method of claim 43 , wherein the oligonucleotide bridge comprises nucleosides selected from the group consisting of 2′-OMe-A claim 43 , 2′-OMe-G claim 43 , 2′-OMe-C claim 43 , 2′-OMe-U claim 43 , 2′-F-A claim 43 , 2′-F-G claim 43 , 2′-F-C claim 43 , 2′-F-U claim 43 , LNA-A claim 43 , LNA-G claim 43 , LNA-C claim 43 , LNA-U claim 43 , N6-methyl-adenosine claim 43 , 2-thiouridine (2sU) claim 43 , 5-methyl-cytidine (5mC) claim 43 , pseudouridine (ψU) claim 43 , and 1-methyl-pseudouridine.54. The method of claim 43 , wherein the mRNAs comprise one or more modified nucleosides.55. The method of claim 54 , wherein the modified nucleosides are selected from the group consisting of 2′-OMe-A claim 54 , 2′-OMe-G claim 54 , 2′- ...

GENERATION OF IMPROVED HUMAN PAH FOR TREATMENT OF SEVERE PKU BY LIVER-DIRECTED GENE REPLACEMENT THERAPY

Provided herein are variant phenylalanine hydroxylase (PAH) polypeptides which are more stable and have greater activity than wild-type human PAH. Also provided are methods to treat phenylketonuria (PKU) and/or to reduce levels of phenylalanine in an individual in need thereof. Further provided herein are expression cassettes, vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions and kits for expressing the variant PAH polypeptide in an individual in need thereof. 1. A variant phenylalanine hydroxylase (PAH) polypeptide comprising two amino acid substitutions , wherein the amino acid substitutions are at sites selected from M180 , K199 , S250 , and G256 of a wild-type human PAH polypeptide.2. A variant phenylalanine hydroxylase (PAH) polypeptide comprising three amino acid substitutions , wherein the amino acid substitutions are at sites selected from M180 , K199 , S250 , and G256 of a wild-type human PAH polypeptide.3. A variant phenylalanine hydroxylase (PAH) polypeptide comprising four amino acid substitutions at sites M180 , K199 , S250 , and G256 of a wild-type human PAH polypeptide.4. The variant PAH polypeptide of any one of - , wherein the amino acid substitution comprises one of more of M180T , K199P , S250P , and G256A.5. The variant PAH polypeptide of any one of - , wherein the amino acid substitution comprises K199P , S250P , and G256A; M180T , S250P , and G256A; M180T , K199P , and G256A; or M180T , K199P , and S250P.6. The variant PAH polypeptide of any one of - , wherein the amino acid substitution comprises M180T , K199P , S250P , and G256A.7. The variant PAH polypeptide of any one of - , wherein the variant PAH polypeptide further comprises H264P , G272A , G272P , P275L , P279Q , G272P and P275L , or T323R and F327T amino acid substitutions.8. The variant PAH polypeptide of any one of - , wherein the wild-type human PAH polypeptide comprises the amino acid sequence of SEQ ID NO:1.9. The variant PAH polypeptide of any one of - , ...

MICROBIAL APPROACH FOR THE PRODUCTION OF 5-HYDROXYTRYPTOPHAN

5-hydroxytryptophan (5-HTP), a precursor of serotonin, is produced in a microbial host cell. A modified bacterial phenylalanine 4-hydroxylase (P4H) catalyzes the tryptophan 5-hydroxylation reaction. Optionally the host cell includes a cofactor regeneration mechanism, allowing continuous production of 5-HTP without supplementation of exogenous cofactors. 1. A genetically engineered bacterial cell comprising a modified bacterial phenylalanine-4-hydroxylase (P4H) wherein the modified P4H catalyzes the 5-hydroxylation of tryptophan in the presence of a tetrahydromonapterin (MH4) cofactor , and in the absence of a tetrahydrobiopterin (BH4) cofactor or 6 ,7-dimethyl-5 ,6 ,7 ,8-tetrahydropterine hydrochloride.2. The genetically engineered bacterial cell of which is genetically engineered to overproduce or accumulate tryptophan.3. The genetically engineered bacterial cell of further comprising a cofactor recycling system comprising at least one of a pterin-4α-carbinolamine dehydratase (PCD) and a dihydromonapterin reductase (DHMR).4E. coli. The genetically engineered bacterial cell of wherein the dihydromonapterin reductase is encoded by the gene folM.5Pseudomonas, RalstoniaXanthomonas.. The genetically engineered bacterial cell of wherein the modified bacterial P4H is derived from a claim 1 , or6X. campestris. The genetically engineered bacterial cell of wherein the modified bacterial P4H comprises an amino acid mutation at any one claim 1 , any two claim 1 , or all three of amino acid positions 98 claim 1 , 179 claim 1 , and 231 of P4H claim 1 , or at any one claim 1 , any two claim 1 , or all three corresponding amino acid positions in a bacterial P4H enzyme from another species.7X. campestris. The genetically engineered bacterial cell of wherein the mutation comprises any one claim 6 , any two claim 6 , or any three mutations selected from the group consisting of L98Y claim 6 , W179F and Y231C of P4H claim 6 , or corresponding amino acid positions in a bacterial P4H ...

ADENO-ASSOCIATED VIRUS COMPOSITIONS FOR RESTORING PAH GENE FUNCTION AND METHODS OF USE THEREOF

Provided herein are recombinant adeno-associated virus (rAAV) compositions that can restore phenylalanine hydroxylase (PAH) gene function in cells, and methods for using the same to treat diseases associated with reduction of PAH gene function (e.g., PKU). Also provided are nucleic acids, vectors, packaging systems, and methods for making the adeno-associated virus compositions. 1. A recombinant adeno-associated virus (rAAV) comprising:(a) an AAV capsid comprising an AAV capsid protein; and(b) an rAAV genome comprising: (i) an editing element for editing a target locus in a PAH gene, comprising at least a portion of a PAH coding sequence operably linked to a transcriptional regulatory element; (ii) a 5′ homology arm nucleotide sequence position 5′ of the editing element, having homology to a first genomic region 5′ to the target locus; and (iii) a 3′ homology arm nucleotide sequence positioned 3′ of the editing element, having homology to a second genomic region 3′ to the target locus.2. The rAAV of claim 1 , wherein:the editing element comprises a PAH coding sequence, optionally wherein:the PAH coding sequence encodes an amino acid sequence set forth in SEQ ID NO: 33;the PAH coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 53; and/orthe PAH coding sequence is silently altered, optionally wherein: the PAH coding sequence comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 28, the PAH coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 28, and/or the PAH coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 83.38-. (canceled)9. The rAAV of a claim 1 , wherein the transcriptional regulatory element is capable of mediating transcription in a hepatocyte claim 1 , a renal cell claim 1 , or a cell in the brain claim 1 , pituitary gland claim 1 , adrenal gland claim 1 , ...

TYROSINE HYDROXYLASE VARIANTS AND METHODS OF USE THEREOF

The present disclosure provides a variant tyrosine hydroxylase that provides for increased production of L-DOPA in a host cell that expresses the tyrosine hydroxylase. The present disclosure provides nucleic acids encoding the variant tyrosine hydroxylase, and host cells genetically modified with the nucleic acids. The present disclosure provides methods of making L-DOPA in a host cell. The present disclosure provides methods of making a benzylisoquinoline alkaloid (BIA), or a BIA precursor. The present disclosure provides methods of detecting L-DOPA level in a cell. The present disclosure provides methods of identifying tyrosine hydroxylase variants that provide for increased L-DOPA production; and methods of identifying gene products that provide for increased tyrosine production. 1. A variant tyrosine hydroxylase comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1 , and comprising:a) an amino acid substitution for the tryptophan at amino acid 13 of SEQ ID NO:1, or a corresponding amino acid in another tyrosine hydroxylase; and/orb) an amino acid substitution for the phenylalanine at amino acid 309 of SEQ ID NO:1, or a corresponding amino acid in another tyrosine hydroxylase.2. The variant tyrosine hydroxylase of claim 1 , wherein the variant tyrosine hydroxylase exhibits enzymatic activity that is at least 25% higher than the enzymatic activity of the tyrosine hydroxylase of SEQ ID NO:8.3. The variant tyrosine hydroxylase of claim 1 , wherein the variant tyrosine hydroxylase exhibits enzymatic activity that is at least 50% higher than the enzymatic activity of the tyrosine hydroxylase of SEQ ID NO:8.4. The variant tyrosine hydroxylase of claim 1 , wherein the variant tyrosine hydroxylase exhibits enzymatic activity that is at least 10-fold higher than the enzymatic activity of the tyrosine hydroxylase of SEQ ID NO:8.5. The variant tyrosine hydroxylase of claim 1 , wherein the ...

MULTIMERIC CODING NUCLEIC ACID AND USES THEREOF

The present invention provides, among other things, multimeric coding nucleic acids that exhibit superior stability for in vivo and in vitro use. In some embodiments, a multimeric coding nucleic acid (MCNA) comprises two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends. 1. A multimeric coding nucleic acid (MCNA) comprising two or more encoding polynucleotides linked via 3′ ends such that the multimeric coding nucleic acid compound comprises two or more 5′ ends.23-. (canceled)4. The MCNA of claim 1 , wherein each of the two or more encoding polynucleotides is a synthetic polydeoxyribonucleotide or a polyribonucleotide.5. The MCNA of claim 1 , wherein each of the two or more encoding polynucleotides encodes a protein of interest.6. The MCNA of claim 5 , wherein each of the two or more encoding polynucleotides encodes a same protein.7. The MCNA of claim 5 , wherein each of the two or more encoding polynucleotides encodes a distinct protein.8. The MCNA of claim 1 , wherein the compound comprises three or more encoding polynucleotides.911-. (canceled)1211. The MCNA of claim claim 1 , wherein the one or more of the encoding polynucleotides comprise a 3′ UTR.13. (canceled)14. The MCNA of claim 12 , wherein the 3′ UTR comprises a plurality of multi-A segments with spacers in between.1525-. (canceled)26. The MCNA of claim 1 , wherein the 3′ ends of the two or more encoding polynucleotides are linked via an oligonucleotide bridge comprising a 3′-3′ inverted phosphodiester linkage.27. The MCNA of claim 26 , wherein the nucleotides comprising the oligonucleotide bridge are selected from the group consisting of 2′-OMe-A claim 26 , 2′-OMe-G claim 26 , 2′-OMe-C claim 26 , 2′-OMe-U claim 26 , 2′-F-A claim 26 , 2′-F-G claim 26 , 2′-F-C claim 26 , 2′-F-U claim 26 , LNA-A claim 26 , LNA-G claim 26 , LNA-C claim 26 , LNA-U claim 26 , N6-methyl-adenosine claim 26 , 2-thiouridine (2sU) claim 26 , 5- ...

VECTORS WITH PROMOTER AND ENHANCER COMBINATIONS FOR TREATING PHENYLKETONURIA

A lentiviral vector system for expressing a lentiviral particle is disclosed. The lentiviral vector system includes a therapeutic vector. The lentiviral vector system produces a lentiviral particle for upregulating PAH expression in the cells of a subject afflicted with phenylketonuria (PKU). 1. A viral vector comprising a therapeutic cargo portion , wherein the therapeutic cargo portion comprises:a PAH sequence or a variant thereof,a promoter; anda liver-specific enhancer,wherein the PAH sequence or the variant thereof is operatively controlled by both the promoter and the liver-specific enhancer.2. The viral vector of claim 1 , wherein the liver-specific enhancer comprises a prothrombin enhancer.3. The viral vector of claim 2 , wherein the promoter comprises a liver-specific promoter.4. The viral vector of claim 3 , wherein the liver-specific promoter comprises a hAAT promoter.5. The viral vector of claim 1 , wherein the PAH sequence or the variant thereof is truncated.6. The viral vector of claim 5 , wherein the PAH sequence or the variant thereof is truncated at the 3′ untranslated region (UTR) of the PAH sequence or the variant thereof.7. The viral vector of claim 1 , wherein the therapeutic cargo portion further comprises a beta globin intron.8. The viral vector of claim 2 , wherein the therapeutic cargo portion further comprises at least one hepatocyte nuclear factor binding site.9. The viral vector of claim 8 , wherein the at least one hepatocyte nuclear factor binding site is disposed upstream of the prothrombin enhancer or downstream of the prothrombin enhancer.10. (canceled)11. The viral vector of claim 1 , wherein the PAH sequence or the variant thereof comprises a sequence having at least 80% identity with SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4.12. (canceled)13. The viral vector of claim 2 , wherein the prothrombin enhancer comprises a sequence having at least 80% identity with SEQ ID NO: 5.14. (canceled)15. The viral vector of claim ...

Methods and compositions for modulating serotonin levels

The present application in one aspect provides methods and compositions that are fine-tuned to modulate brain and peripheral serotonin levels, which in turn would lead to prevention and treatment of various diseases associated with a reduced level of brain serotonin. In another aspect, there are provided methods of assessing the peripheral or placenta level of serotonin and associated risks based on the peripheral placental level of TPH1.

GENE THERAPY FOR TREATING PHENYLKETONURIA

Compositions and regimens useful in treating phenylketonuria are provided. The compositions include recombinant adeno-associated virus (rAAV) with a transthyretin enhancer and promoter driving expression of a human phenylalanine hydroxylase. 1. A recombinant adeno-associated virus (rAAV) useful as a liver-directed therapeutic for phenylketonuria (PKU) , said rAAV comprising an AAV capsid , and a vector genome packaged therein , said vector genome comprising:(a) an AAV 5′ inverted terminal repeat (ITR) sequence;(b) a promoter;(c) a codon optimized sequence encoding a human phenylalanine hydroxylase (PAH);(d) an AAV 3′ ITR.2. The rAAV according to claim 1 , wherein the coding sequence of (c) is SEQ ID NO: 1.3. The rAAV according to claim 1 , wherein the rAAV capsid is an AAV8 capsid.4. The rAAV according to claim 1 , wherein the promoter is TBG promoter or TBG-S 1 promoter.5. The rAAV according to claim 1 , wherein the promoter is A1AT promoter.6. The rAAV according to claim 1 , wherein the promoter is LSP promoter.7. The rAAV according to claim 1 , wherein the promoter is TTR promoter.8. The rAAV according to claim 1 , wherein the AAV 5′ ITR and/or AAV3′ ITR is from AAV2.9. The rAAV according to claim 1 , wherein the vector genome further comprises a polyA.10. The rAAV according to claim 1 , wherein the polyA is from bGH.11. The rAAV according to claim 1 , further comprising a WPRE.12. The rAAV according to claim 1 , further comprising an intron.13. The rAAV according to claim 12 , wherein the intron is from human beta globin IVS2 or SV40.14. The rAAV according to claim 1 , further comprising an enhancer.15. The rAAV according to claim 14 , wherein the enhancer is an APB enhancer claim 14 , ABPS enhancer claim 14 , an alpha mic/bik enhancer claim 14 , TTR enhancer claim 14 , en34 claim 14 , or ApoE enhancer.16. The rAAV according to claim 1 , wherein the vector genome is about 3 kilobases to about 5.5 kilobases in size.17. An aqueous suspension suitable for ...

GENE SEQUENCE CONSTRUCT USED FOR TREATMENT OF CENTRAL NERVOUS SYSTEM DISEASES

A gene sequence construct used for the treatment of central nervous system diseases: by means of the construction of an auto-processing expression vector, tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on may be simultaneously expressed; proteins are connected by means of an auto-processing unit (APU); the use of a viral vector to introduce the construct into a target cell may ultimately result in the high-efficiency expression of tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on having independent functions, being used in the prevention or treatment of Parkinson's disease, Alzheimer's disease and other neurodegenerative diseases. 1. A gene sequence construct , characterized in that the construct comprises two or more nucleotide sequences that are related to the treatment of a central nervous system disease , and wherein the two or more nucleotide sequences are linked by an auto-processing unit (APU).2. The gene sequence construct of claim 1 , characterized in that said auto-processing unit (APU) comprises an N-terminal auto-processing domain and/or a C-terminal auto-processing domain.3. The gene sequence construct of claim 2 , characterized in that said N-terminal auto-processing domain comprises Intein claim 2 , B-type bacterial intein-like domain (BIL) claim 2 , Furin sequence claim 2 , or a derivative thereof.4. (canceled)5. The gene sequence construct of claim 1 , characterized in that said two or more nucleotide sequences related to the central nervous system disease comprise two or more of the nucleotide sequences of tyrosine hydroxylase (TH) claim 1 , GTP-cyclohydrolase I (GCH1) claim 1 , aromatic amino acid dopa decarboxylase (AADC) claim 1 , or a nervous system growth factor;wherein the two or more nucleotide sequences are linked by an auto-processing unit (APU).6. The gene sequence construct of claim 5 , characterized in that the ...

Method of grafting genetically modified cells to treat defects, disease or damage or the central nervous system

Methods of genetically modifying donor cells by gene transfer for grafting into the central nervous system to treat diseased or damaged cells are disclosed. The modified donor cells produce a molecule capable of affecting the recovery of cells in the CNS. Methods and vectors for carrying out gene transfer and grafting are described.

Compositions, methods, and devices for dialysis

Compositions of peritoneal dialysis solutions and metabolizing enzymes, and their uses to treat disorders associated with elevated levels of metabolites are disclosed. Animal models of hyperoxalemia are also disclosed.

Tyrosine prototrophy

Provided herein is a tyrosine selection marker system, and uses thereof. In some embodiments, nucleic acid constructs, vectors, host cells and related compositions and methods for generating and selecting tyrosine prototroph cells are provided.

Inhibition of serotonin expression in gut enteroendocrine cells results in conversion to insulin-positive cells

AAV-mediated delivery of DNA to cells of the nervous system

Methods and compositions for treating phenylketonuria

FIELD: medicine, pharmaceutics. SUBSTANCE: declared invention refers to medicine and aims at treating phenylketonuria. What is declared is a dosage form containing a) phenylalanine-4-hydroxylase and/or phenylalanine ammonia-lyase and b) at least one peptidase, wherein a), b) or both a) and b) have an enteric coating. EFFECT: declared invention is effective in treating phenylketonuria. 13 cl, 4 tbl, 4 ex РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) 2 496 489 (13) C2 (51) МПК A61K 31/195 (2006.01) A61P 43/00 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (21)(22) Заявка: 2009148036/15, 23.05.2008 (24) Дата начала отсчета срока действия патента: 23.05.2008 (73) Патентообладатель(и): ДЗЕ БОРД ОФ РИДЖЕНТС ОФ ДЗЕ ЮНИВЕРСИТИ ОФ ТЕХАС СИСТЕМ (US) R U Приоритет(ы): (30) Конвенционный приоритет: 24.05.2007 US 60/931,645 (72) Автор(ы): МАТАЛОН Рубен (US) (43) Дата публикации заявки: 27.06.2011 Бюл. № 18 C 2 2 4 9 6 4 8 9 (85) Дата начала рассмотрения заявки PCT на национальной фазе: 24.12.2009 (86) Заявка PCT: US 2008/006655 (23.05.2008) R U C 2 (56) Список документов, цитированных в отчете о поиске: KIM W. Trends in ehzyme therapy for phenylkettonuria. Mol. Ther. 2004 Aug; 10(2):220224. WO 03093436 A2, 13.11.2003. НИКИТИН И.Г. и др. Пегилированные лекарственные препараты: современное состояние проблемы и перспективы // Вирусные гепатиты, информационный бюллетень. 2001, №3 (13). RU 2279883 C2, 20.07.2006. (54) СПОСОБЫ И КОМПОЗИЦИИ ДЛЯ ЛЕЧЕНИЯ ФЕНИЛКЕТОНУРИИ (87) Публикация заявки РСТ: WO 2008/153771 (18.12.2008) Адрес для переписки: 129090, Москва, ул. Б. Спасская, 25, стр.3, ООО "Юридическая фирма Городисский и Партнеры" (57) Реферат: Заявленное изобретение относится к области медицины и предназначено для лечения фенилкетонурии. Заявлена пероральная дозированная форма, содержащая: a) фенилаланин-4-гидроксилазу и/или фенилаланинаммиак-лиазу; и b) по меньшей мере одну пептидазу, где a), b) или оба a) и b) имеют растворяющееся в кишечнике ...

MRNA therapy for phenylketonuria

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

Tyrosine prototrophy

Provided herein is a tyrosine selection marker system, and uses thereof. In some embodiments, nucleic acid constructs, vectors, host cells and related compositions and methods for generating and selecting tyrosine prototroph cells are provided.

mRNA therapy for phenylketonuria

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

MRNA therapy for phenylketonuria

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

ADENOVIRUS VECTORS FOR THE TRANSFER OF STRANGE GENES IN CELLS OF THE CENTRAL NERVOUS SYSTEM, PARTICULARLY IN THE BRAIN.

EL INVENTO SE REFIERE A UN VECTOR DNA RECOMBINANTE CARACTERIZADO EN QUE ES CAPAZ DE DIRIGIR LA PRESENTACION Y/O TRANSCRIPCION DE UNA SECUENCIA NUCLEOTIDA SELECCIONADA EN LAS CELULAS DEL SISTEMA NERVIOSO CENTRAL Y EN QUE COMPRENDE (I) AL MENOS PARTE DE UN GENOMA DE UN ADENOVIRUS, INCLUYENDO LAS REGIONES REQUERIDAS PARA QUE EL ADENOVIRUS PENETRE EN LAS CELULAS NORMALMENTE INFECTABLES POR ESE ADENOVIRUS Y (II) INJERTARLO DENTRO DE DICHA PARTE DE GENOMA DE UN ADENOVIRUS BAJO EL CONTROL DE UN PROMOTOR, BIEN PRESENTE O TAMBIEN INSERTADO DENTRO DE DICHA PARTE DE GENOMA Y OPERATIVO EN DICHAS CELULAS. ESTE VECTOR RECOMBINANTE ENCUENTRA EMPLEO PARTICULAR EN EL TRATAMIENTO DE ENFERMEDADES DEL SISTEMA NERVIOSO CENTRAL, TAMBIEN EN TERAPIA GENICA.

In vitro induction of dopaminergic cells

A culture method for inducing the expression of tyrosine hydroxylase in neural cells is provided. Mammalian CNS neural cells are cultured in the presence of a fibroblast growth factor and at least one selected from a member of the transforming growth factor beta family, a feeder layer bed of cells, and cell conditioned medium. Cells cultured as provided above may be transplanted to provide dopaminergic cells to a patient. The cells may also be used in methods for drug screening.

REV-ERB Use of REV-ERB for treating affective and addictive disorders

본 발명은 일주기적 조절 기작에 관여하는 생체시계 유전자와 중뇌 도파민 합성 시스템과의 관련성에 관한 것이다.　본 발명에 따르면, 생체시계 단백질인　 REV-ERBα 단백질 및 이를 코딩하는 유전자를 이용하여 도파민 시스템 조절이상에서 기인하는 뇌질환을 예방 및/또는 치료하기 위한 신규한 약물후보물질을 스크리닝할 수 있다.　또한, 본 발명은 핵수용체인 REV-ERBα 단백질 및 이를 코딩하는 유전자를 억제하여, 도파민 시스템 조절이상에서 기인하는 뇌질환의 예방 및/또는 치료에 효과가 있는 약학조성물을 제공할 수 있다. The present invention relates to a relationship between a biological clock gene involved in a periodic regulation mechanism and a midbrain dopamine synthesis system. According to the present invention, it is possible to screen a novel drug candidate for preventing and / or treating a brain disease caused by dopamine system control abnormality using a REV-ERBα protein, which is a biological clock protein, and a gene encoding the same. Further, the present invention can provide a pharmaceutical composition which is effective for prevention and / or treatment of brain diseases caused by dopamine system control abnormality by suppressing REV-ERB alpha protein and a gene encoding the same.

Mrna therapy for phenylketonuria.

La presente invención proporciona, entre otras cosas, métodos para tratar la fenilcetonuria (PKU) que incluyen administrar a un sujeto que necesita un tratamiento, una composición que comprende un ARNm que codifica la fenilalanina hidroxilasa (PAH) en una dosis eficaz y un intervalo de administración de modo que al menos un síntoma o característica de la PKU se reduzca en intensidad, gravedad o frecuencia, o tenga aparición retrasada; en algunas modalidades, el ARNm se encapsula en un liposoma que comprende uno o más lípidos catiónicos, uno o más lípidos no catiónicos, uno o más lípidos a base de colesterol y uno o más lípidos modificados con PEG.

In vitro Induction of dopaminergic cells

神经细胞中酪氨酸羟化酶的体外表达可通过将神经细胞与一种培养基接触进行诱导,该培养基至少含有成纤维细胞生长因子族中一员,并配合条件培养基或转化生长因子β族的分子。本方法诱导从正常情况下非多巴胺能的神经组织,如纹状体和皮层中所得的神经细胞,使之表达酪氨酸羟化酶。这些细胞可用于治疗需要多巴胺能的细胞的患者的神经障碍。

Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof

本发明提供了一种用于产生5‑羟基色氨酸的基因工程菌及其构建方法与应用，首先对大肠杆菌中色氨酸合成途径和整个氨基酸代谢网络中与色氨酸相关的代谢流进行分析重构，加强了5‑HTP前体物色氨酸的供应。之后又引入人源2型色氨酸羟基化酶突变体和四氢蝶呤合成与再生途径，打通色氨酸胞内羟化反应，得到了一株遗传背景清晰、能通过微生物直接发酵法高效稳定生产5‑HTP的无质粒基因工程菌，具有良好的应用价值。

Method for cell selection

本文描述了生产细胞和用于基于编码缺乏功能性N末端调节域的苯丙氨酸羟化酶(PAH)的序列和编码GTP环化水解酶1(GCH1)的序列的组合鉴定、选择或培养包含酪氨酸营养缺陷型选择标记系统的生产细胞的方法。还描述了制备生产细胞和用所述生产细胞制备产物的方法。

MULTIMER CODING NUCLEIC ACID AND ITS USES

Compositions, methods and devices for dialysis

Gene therapy compositions and methods for treating parkinson's disease

A method of improving motor function and reducing dyskinesia in a subject suffering from a neurodegenerative disease or a disease where endogenous dopamine levels are reduced in the subject comprising administering an effective amount of a viral vector comprising a nucleic acid construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1), (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC), or any combination thereof to the subject.

Selectable markers and related methods

The invention relates to newly identified selectable marker systems, cells for use in a selectable marker system, and methods for using the selectable marker systems.

Microbial approach for the production of 5-hydroxytryptophan

5-hydroxytryptophan (5-HTP), a precursor of serotonin, is produced in a microbial host cell. A modified bacterial phenylalanine 4-hydroxylase (P4H) catalyzes the tryptophan 5 -hydroxylation reaction. Optionally the host cell includes a cofactor regeneration mechanism, allowing continuous production of 5-HTP without supplementation of exogenous cofactors.

Methods of producing nor-opioid and nal-opioid benzylisoquinoline alkaloids

【課題】オピオイドを脱メチル化してノル-オピオイドにする方法を提供する。【解決手段】オピオイドを脱メチル化してノル-オピオイドにする方法であって、以下の工程：第1オピオイドを少なくとも1種の酵素と接触させる工程であって、第1オピオイドを該少なくとも1種の酵素と接触させることが、O-結合型メチル基の喪失によって第1オピオイドを第2オピオイドに変換する、工程;および第2オピオイドを少なくとも1種の酵素と接触させる工程であって、該オピオイドを該少なくとも1種の酵素と接触させることが、N-結合型メチル基の喪失によって第2オピオイドをノル-オピオイドに変換する、工程を含む、前記方法である。【選択図】図２６

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PHENILKETONURIA

1. Способ лечения субъекта, страдающего фенилкетонурией, причем способ включает введение одного или нескольких из фенилаланин-4-гидроксилазы или фенилаланинаммиаклиазы субъекту в условиях, эффективных для доставки фенилаланин-4-гидроксилазы и/или фенилаланинаммиаклиазы в тонкий кишечник субъекта. ! 2. Способ по п.1, дополнительно включающий введение пептидазы субъекту в условиях, эффективных для доставки пептидазы в желудок и/или тонкий кишечник субъекта. ! 3. Способ по п.2, где пептидаза представляет собой одну или несколько дипептидаз. !4. Способ по п.2, где пептидазу вводят одновременно или последовательно с одной или обеими из фенилаланин-4-гидроксилазы или фенилаланинаммиаклиазы. ! 5. Способ по п.2, где пептидазу и одну или обе из фенилаланин-4-гидроксилазы или фенилаланинаммиаклиазы вводят в отдельных составах или в одном составе. ! 6. Способ по п.5, где составы покрыты растворяющимся в кишечнике покрытием. ! 7. Способ по п.5, где одну или обе из фенилаланин-4-гидроксилазы или фенилаланинаммиаклиазы включают в состав с растворяющимся в кишечнике покрытием в качестве одного состава или отдельных составов. ! 8. Способ по п.5, где составы представляют собой составы с замедленным высвобождением. ! 9. Способ по п.1, где фенилаланин-4-гидроксилаза представляет собой полноразмерную или укороченную рекомбинантную фенилаланин-4-гидроксилазу. ! 10. Способ по п.1, где фенилаланин-4-гидроксилаза представляет собой фенилаланин-4-гидроксилазу млекопитающего. ! 11. Способ по п.10, где млекопитающим является человек. ! 12. Способ по п.1, где фенилаланин-4-гидроксилаза является тиолированной в области, богатой серином/треонином/тирозином. ! 13. РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) 2009 148 036 (13) A (51) МПК A61K 31/195 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ, ПАТЕНТАМ И ТОВАРНЫМ ЗНАКАМ (12) ЗАЯВКА НА ИЗОБРЕТЕНИЕ (21)(22) Заявка: 2009148036/15, 23.05.2008 (71) Заявитель(и): ДЗЕ БОРД ОФ РИДЖЕНТС ОФ ДЗЕ ЮНИВЕРСИТИ ОФ ТЕХАС СИСТЕМ (US) Приоритет(ы): (30) ...

Construct

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

Compositions, methods, and devices for dialysis

Sustained release formulations

The invention relates to tyrosine hydroxylase inhibitor compositions and methods thereof. Specifically, the invention relates to a sustained release formulation of a tyrosine hydroxylase inhibitor, particularly α-methyl-para-tyrosine.

In vitro induction of dopaminergic cells

The expression of thyrosine hydroxylase in neural cells in vitro can be induced by contacting neural cells with a culture medium comprising at least one member of the fibroblast growth factor family in combination with conditioned medium or molecules of the transforming growth factor beta family. The method induces neural cells obtained from normally non-dopaminergic neural tissue such as the striatum and the cortex to express tyrosine hydroxylase. The cells can be used to treat neurological disorders in patients requiring dopaminergic cells.

Polymer code nucleic acid and application thereof

本发明尤其提供了多聚体编码核酸，其显示出用于体内和体外使用的优异稳定性。在一些实施例中，多聚体编码核酸(MCNA)包含经由3'末端连接的两种或更多种编码多核苷酸，使得多聚体编码核酸化合物包含两个或更多个5'末端。

tyrosine prototrophy

Mrna therapy for phenylketonuria

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids

Gene construct

本发明涉及基因构建体、表达盒和包含此类构建体和盒的重组载体用于基因治疗的用途，以及用于治疗神经退行性疾病如帕金森病(PD)的方法。该构建体包含与编码酪氨酸羟化酶(TH)的第一编码序列和编码GTP环化水解酶1(GCH1)的第二编码序列可操作地连接的启动子。第二编码序列位于第一编码序列的3’方向，并且第一编码序列和第二编码序列是单个操纵子的一部分，其中基因构建体不编码芳香族氨基酸脱羧酶(AADC)。将构建体递送至个体的脑脊液(CSF)。

Compositions and methods for making benzylisoquinoline alkaloids, morphinan alkaloids, thebaine, and derivatives thereof

Disclosed herein are methods that may be used for the synthesis of benzylisoquinoline alkaloids (BIAs) such as alkaloid morphinan. The methods disclosed can be used to produce thebaine, oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, and/or buprenorphine. Compositions and organisms useful for the synthesis of BIAs, including thebaine synthesis polypeptides, purine permeases, and polynucleotides encoding the same, are provided.

Therapeutics for phenylketonuria

Systemic synthesis and regulation of l-dopa

The present invention relates to an expression system for enzyme replacement therapy with the aim of obtaining or maintaining a steady level of L-DOPA in the blood of an individual, achieved through systemic administration of the expression system. The invention is thus useful in the treatment of catecholamine deficient disorders, such as dopamine deficient disorders including Parkinson's Disease.

Compositions, methods, and devices for dialysis

Method for inducing dna synthesis in neurons

A method is provided for inducing DNA synthesis in a differentiated neuron, which includes obtaining a vector comprising nucleic acid encoding an E2F regulator and/or an E1A regulator, wherein the vector can be used to express the nucleic acid in a differentiated neuron, and transfecting a differentiated neuron with the vector. According to certain embodiments of the invention, a method for integrating DNA encoding a desired protein in a differentiated neuron is provided that includes obtaining a vector comprising nucleic acid encoding an E2F regulator and/or an E1A regulator, wherein the vector can be used to express the nucleic acid in a neuron, obtaining DNA encoding a desired protein, and cotransfecting a differentiated neuron with the vector and the DNA encoding the desired protein such that the DNA encoding the desired protein is integrated in the differentiated neuron and the desired protein is produced.

Methods of treating phenylketonuria

Provided herein are methods of treating phenylketonuria by normalizing levels of amino acids, neurotransmitters, and neurotransmitter metabolites in a subject having phenylketonuria.

Adeno-associated virus packaging systems

Provided herein is a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV). The dual vector transfection system generally comprises: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising an rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene.

Generation of improved human pah for treatment of severe pku by liver-directed gene replacement therapy

本文提供了變異體苯丙胺酸羥化酶(PAH)多肽，其比野生型人類PAH更穩定並且具有更大的活性。還提供了在有需要的個體中治療苯丙酮尿症(PKU)和/或降低苯丙胺酸量的方法。本文還提供了在有需要的個體中表現所述變異體PAH多肽的表現盒、載體(例如，rAAV載體)、病毒顆粒、醫藥組成物和套組。

Closed-ended dna vectors and uses thereof for expressing phenylalanine hydroxylase (pah)

Described herein are ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette comprises a codon optimized nucleic acid sequence encoding a PAH protein, in combination with particular promoter sequences and cis-regulatory elements. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Also provided herein are methods and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).

Microorganisms for the production of melatonin

Recombinant microbial cells and methods for producing melatonin and related compounds using such cells are described. More specifically, the recombinant microbial cell may comprise exogenous genes encoding one or more of an L-tryptophan hydroxylase, a 5-hydroxy-L- tryptophan decarboxylyase, a serotonin acetyltransferase, an acetylserotonin O- methyltransferase; an L-tryptophan decarboxy-lyase, and a tryptamine-5-hydroxylase, and means for providing tetrahydrobiopterin (THB). Related sequences and vectors for use in preparing such recombinant microbial cells are also described.

Recombinant host cells with improved production of l-dopa, dopamine, (s)-norcoclaurine or derivatives thereof.

The present invention relates to a recombinant microbial host cell comprising an operative biosynthetic metabolic pathway capable of producing one or more compounds selected from the group consisting of L-dopa, dopamine, (S)-Norcoclaurine and derivatives thereof; said pathway comprising a heterologous L-tyrosine hydroxylase (TyrH) converting L-Tyrosine into L-dopa capable of increasing the cell production of the Compound compared to a reference L-tyrosine hydroxylase having the sequence set forth in SEQ ID NO: 58.

Isolated nucleic acid molecule encoding gp75, a method for preparing an amino acid sequence from the same, and a method for producing a vaccine

Human pah expression cassette for treatment of pku by liver-directed gene replacement therapy

Provided herein are expression cassettes for expressing a transgene in a liver cell, wherein the transgene encodes a PAH polypeptide. Also provided are methods to treat phenylketonuria (PKU) and/or to reduce levels of phenylalanine in an individual in need thereof. Further provided herein are vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions and kits for expressing a PAH polypeptide in an individual in need thereof.

SYSTEMIC SYNTHESIS AND REGULATION OF L-DOPA

Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain

The invention concerns a recombinant DNA vector characterized in that it is capable of directing the expression an/or transcription of a selected nucleotide sequence in the cells of the central nervous system and in that it comprises (i) at least part of the genome of an adenovirus, including the regions required for that adenovirus to penetrate into the cells normally infectable by that adenovirus and (ii) being inserted into said part of genome of an adenovirus under the control of a promoter, either present or also inserted into said genome part and operative in said cells. This recombinant vector finds particular use in the treatment of diseases of the central nervous system, also in gene therapy.

In vitro induction of dopaminergic cells

The expression of thyrosine hydroxylase in neural cells in vitro can be induced by contacting neural cells with a culture medium comprising at least one member of the fibroblast growth factor family in combination with conditioned medium or molecules of the transforming growth factor beta family. The method induces neural cells obtained from normally non-dopaminergic neural tissue such as the striatum and the cortex to express tyrosine hydroxylase. The cells can be used to treat neurological disorders in patients requiring dopaminergic cells.