WO1997018319A1

WO1997018319A1 - Joint expression of gtp cyclohydrolase and tyrosine hydroxylase

Info

Publication number: WO1997018319A1
Application number: PCT/US1996/017696
Authority: WO
Inventors: Stuart E. Leff; Ron Mandel
Original assignee: Somatix Therapy Corporation
Priority date: 1995-11-14
Filing date: 1996-11-01
Publication date: 1997-05-22
Also published as: AU1073797A

Abstract

The invention provides novel genetically modified host cells, vectors, and methods for the treatment of Parkinson's disease and other related disease conditions in which the dopamine production is deficient. The invention relates to the surprising discovery that coexpression of tyrosine hydroxylase and GTP cyclohydrolase enables host cells to produce elevated dopamine and L-DOPA without adding tetrahydrobiopterin and that the coexpression of GTP cyclohydrolase stabilizes tyrosine hydroxylase. One embodiment of the invention is genetically modified host cells containing heterologous polynucleotide sequences encoding and capable of expressing GTP cyclohydrolase and tyrosine hydroxylase. Other embodiments of the invention include genetic construction that contain and can simultaneously express polynucleotide sequences encoding a GTP cyclohydrolase and a tyrosine hydroxylase. The invention also provides methods of producing L-DOPA or dopamine using the host cells of the invention. The invention also provides methods of treating patients of Parkinson's disease or other diseases related to a dopamine production deficiency using the vectors and methods of the invention.

Description

JOINT EXPRESSION OF GTP CYCLOHYDROLASE AND TYROSINE HYDROXYLASE

Field Of The Invention

This invention is in the field of molecular biology, more specifically, the invention is in the field of therapeutic agents for Parkinson's disease and related diseases .

Background

Parkinsonism (also known as Parkinson's Disease) is a common syndrome consisting of various combinations of symptoms such as increased muscle rigidity, resting tremor, and abnormalities of posture and gait. Without treatment, the sufferers of Parkinson's disease end up in a rigid state, incapable of taking care of themselves. The symptoms of Parkinson's disease appear to be mediated by degeneration of the pigmented dopamine-secreting neurons of the substantia nigra of the brain. Theoretically, Parkinson's disease can be treated by the addition of dopamine (3,4- dihydroxyphenethylamine) . However, dopamine is incapable of crossing the blood br <ain barrier. Therefore, instead of administering dopamine, levodopa (3-hydroxy-L- tyrosine) , which readily crosses the blood-brain barrier is used instead. Conventional treatments for Parkinson's disease with L-DOPA have proven to be inadequate for many reasons of record in the medical literature.

It is of interest to provide new methods of treating Parkinsonism and other diseases related to dopamine insufficiency by providing for a more efficient and simple methods administering dopamine or L-dopa to patients. It is also of interest to provide improved methods of producing L- dopa or dopamine through the use of genetically engineered host cells . Parkinson's disease has been suggested as a potential target for treatment by genetic therapy (using either in vivo or ex vivo genetic therapy) . However, one problem associated with the use of genetic therapy for the treatment of Parkinson' s disease and other diseases related to dopamine insufficiency is that the key enzyme in the synthesis of dopamine is tyrosine hydroxylase. Tyrosine hydroxylase requires the compound tetrahydrobiopterin the conversion of GTP to dihydroneopterin triphosphate (see Ziegler et al . , J__^ Biol. Chem. 265: 17026-17030 (1990) for an assay for GTP cyclohydrolase) . GTP cyclohydrolase catalyzes a key step in the formation of tetrahydrobiopterin (BH4, also known as (6R) -5, 6, 7, 8, -tetrahydrobiopterin) as a cofactor. The invention described herein involves the coexpression of an enzyme in the BH4 synthesis pathway and tyrosine hydroxylase. The invention relate to the unexpected discovery that the coexpression of GTP cyclohydrolase I stabilizes tyrosine hydroxylase enzyme in cells and increase dopamine and L-dopa production in the absence of added BH4.

Summary Of The Invention The invention provides novel genetically modified host cells, vectors, and methods for the treatment of Parkinson's disease and other related disease conditions in which the dopamine production is deficient. The invention relates to the surprising discovery that coexpression of tyrosine hydroxylase and GTP cyclohydrolase enables host cells to produce elevated dopamine and L-DOPA without adding tetrahydrobiopterin and that the coexpression of GTP cyclohydrolase stabilizes tyrosine hydroxylase.

One embodiment of the invention is genetically modified host cells containing heterologouε polynucleotide sequences encoding and capable of expressing GTP cyclohydrolase and tyrosine hydroxylase. The host cells may be mammalian cells or may from other animal species. In a preferred embodiment of the invention, the heterologous polynucleotide sequences encoding the GTP cyclohydrolase and tyrosine hydroxylase in the host cell are present on viral vectors.

Other embodiments of the invention include genetic constructions that contain and can simultaneously express polynucleotide sequences encoding a GTP cyclohydrolase and a tyrosine hydroxylase . In a preferred embodiment of the invention, the genetic construction is a viral vector. Other embodiments of the invention are methods of producing L-dopa or dopamine by culturing the genetically modified host cells of the invention. These methods of production have the advantage of not requiring BH4 in order to produce substantial levels of L-dopa or dopamine.

Other embodiments of the invention include methods of treating patients of Parkinson's disease or other diseases related to a dopamine production deficiency. Embodiments of the methods of the invention include introducing into a patient a host cell that has been genetically modified to comprise heterologous polynucleotides for expression encoding GTP cyclohydrolase and tyrosine hydroxylase. Other embodiments of the invention include methods of treating a patient by administering to a patient an effective amount of a genetic construction or constructions containing polynucleotides encoding and capable of expressing GTP cyclohydrolase and tyrosine hydroxylase.

Description Of The Specific Embodiments This invention relates to the surprising discovery that by introducing genes encoding GTP cyclohydrolase and tyrosine hydroxylase into a cell so that both genes are expressed, the genetically modified cell acquires the property of producing elevated levels of dopamine and L-dopa without the addition of exogenous BH4. This discovery is particularly important for the production of L-dopa and dopamine in vivo because of the difficulties associated with introducing BH4 in vivo . The term "GTP cyclohydrolase" as used herein refers to enzymes having the biological activity of catalyzing the conversion of GTP to dihydroneopterin triphosphate (see Ziegler e t al . , J. Biol . Chem. 265: 17026-17030 (1990) for an assay for GTP cyclohydrolase) . GTP cyclohydrolase catalyzes a key step in the formation of tetrahydrobiopterin (BH4, also known as (6R) -5, 6, 7, 8, -tetrahydrobiopterin) . GTP cyclohydrolase has the enzyme convention nomenclature designation EC 3.5.4.16. Unless indicated otherwise the term "GTP cyclohydrolase" as used herein refers to any of a variety of enzymes having the desired enzymatic activity. GTP cyclohydrolases may be from a variety of organisms both eukaryotic (mammalian or otherwise) and prokaryotic. The term "polynucleotide encoding GTP cyclohydrolase", and variants of this term, refer not only to GTP cyclohydrolase genes isolated from natural sources (and cDNA derivatives thereof) , but also to various non-naturally occurring polynucleotides encoding GTP cyclohydrolase. For example, a person of ordinary skill in the art may employ the degeneracy of the genetic code in conjunction with well known DNA synthesis and DNA manipulation techniques to produce a variety of polynucleotide encoding GTP cyclohydrolase. Similarly, a person of ordinary skill in the art may readily introduce one or more mutations in GTP cyclohydrolase polynucleotide coding sequence that do not abolish the activity of the enzyme. In a preferred embodiment of the invention, the GTP cyclohydrolase encoding polynucleotide encodes a GTP cyclohydrolase that is the same as a GTP cyclohydrolase found in the species to be treated by the subject methods of treating a patient. For example, when the patient to be treated is human, a polynucleotide encoding a human GTP cyclohydrolase is used rather than a GTP cyclohydrolase from another mammal or from a non-mammalian source. GTP cyclohydrolases from a variety of sources have been purified and the genes encoding such enzymes have been isolated. For example, the DNA sequences of human GTP cyclohydrolase can be found in GenBank Database at accession numbers L27626 and L27627. A description of human GTP cyclohydrolase genes can also be found in Gutlich, et al . Biochem J. 302:215-221 (1994) . The term "tyrosine hydroxylase" as used herein refers to any of a variety of enzymes having the ability to catalyze the conversion of tyrosine to L-dopa. Tyrosine hydroxylase has the enzyme convention nomenclature designation EC 1.14.16.2. Unless indicated otherwise the term "tyrosine hydroxylase" as used herein refers to any of a variety of enzymes having the desired enzymatic activity. Tyrosine hydroxylases may be from a variety of organisms both eukaryotic (mammalian or otherwise) and prokaryotic. The term "polynucleotide encoding tyrosine hydroxylase", and variants of this term, refer not only to tyrosine hydroxylase genes isolated from natural sources (and cDNA derivatives thereof) , but also to various non-naturally occurring polynucleotides encoding tyrosine hydroxylase. For example, a person of ordinary skill in the art may employ the degeneracy of the genetic code in conjunction with well known DNA synthesis and DNA manipulation techniques to produce a variety of polynucleotide encoding tyrosine hydroxylase. Similarly, a person of ordinary skill in the art may readily introduce one or more mutations in tyrosine hydroxylase polynucleotide coding sequence that do not abolish the activity of the enzyme. In a preferred embodiment of the invention, the tyrosine hydroxylase encoding polynucleotide encodes a tyrosine hydroxylase that is the same as a tyrosine hydroxylase found in the species to be treated by the subject methods of treating a patient. For example, when the patient to be treated is human, a polynucleotide encoding a human tyrosine hydroxylase is used rather than a tyrosine hydroxylase from another mammal or from a non-mammalian source. Tyrosine hydroxylases from a variety of sources have been purified and the genes encoding such enzymes have been isolated. Tyrosine hydroxylases cloned from a number of organisms and the nucleic acid sequence coding tyrosine hydroxylases have been made publicly available. See, for example, O'Malley, et al . Biochemistry 26_ :6910-2614 (1987) .

Another embodiment of the invention includes recombinant host cells comprising a first heterologous polynucleotide sequence encoding a GTP cyclohydrolase and a second heterologous polynucleotide sequence encoding a tyrosine hydroxylase. The two heterologous polynucleotides for expression may be on the same polynucleotide or on separate polynucleotides. Both heterologous polynucleotides are selected so as to be capable of expressing the encoded GTP cyclohydrolase and tyrosine hydroxylase genes. The term "heterologous" as used herein with respect to the polynucleotide for expression encoding GTP cyclohydrolase and tyrosine hydroxylase indicates that the entire polynucleotide for expression is not naturally found in the host cell, although significant portions of the heterologous polynucleotide may be found to occur naturally in the host cell. Thus, for example, a human GTP cyclohydrolase encoding polynucleotide encoding may be heterologous with respect to a human host cell, provided the polynucleotide sequence is non- naturally occurring or comprises additional polynucleotide sequences, e.g., a viral vector or an additional promoter sequence. Both first and second heterologous polynucleotide sequences for expression may comprise promoter sequences functional in the recombinant host cell of interest. Similarly, the heterologous polynucleotide sequences for expression may comprise additional polynucleotide sequences useful for enhancing or regulating the expression of the encoded GTP cyclohydrolase and tyrosine hydroxylase; such additional polynucleotide sequences include promoters, enhancers, regulatory protein binding sites, polyadenylation sites, and the like. These additional regulatory sequences, enhancer sequences, etc., are selected so as to be functional in the host cell of interest. Promoter sequences, enhancer sequences, regulatory sequences, and the like, as well as methods of using such sequences to modify the expression of a gene of interest are well known to the person of ordinary skill in the art and can be found in among other places, Gene Expression Technology: Methods in Enzymolocrv, Vol. 185

Goeddel, Ed. Academic Press, Inc., San Diego, CA (1991) . The heterologous polynucleotides for expression may be produced using conventional recombinant DNA techniques such as those described in Molecular Cloning: A Laboratory Manual, Sambrook, et al . , Cold Spring Harbor, Cold Spring Harbor, NY (1989) . The heterologous polynucleotide sequences in the genetically modified host cells of the invention may comprise a vector suitable or use in the selected host cell . The recombinant host cell may be prokaryotic or eukaryotic. In a preferred embodiment of the invention, the host cell used is eukaryotic, and in a more preferred embodiment of the invention, the selected host cell is a mammalian host cell. When the host cell is a mammalian host cell, the heterologous polynucleotide sequences for expression preferably comprise a viral vector. The first and second heterologous polynucleotide sequences for expression may both comprise viral vectors. Alternatively, the first heterologous polynucleotide sequence may comprise a viral vector and the second heterologous polynucleotide sequence does not comprise a viral vector. In another embodiment of the genetically modified host cells of the invention, the first heterologous polynucleotide sequence does not comprise a viral vector and the second heterologous polynucleotide sequence comprises a viral vector. In embodiments of the invention in which the genetically modified host cell is to be used to treat a patient having a dopamine production deficiency, the host cell is preferably of the same species as the patient to be treated. In such embodiments of the invention, it is more preferable to use a recombinant host cell that is derived from the actual patient to be treated so as to minimize antigenic differences, particularly MHC differences, between the genetically modified host cell and the patient to be treated.

Both the first and second heterologous polynucleotide sequence for expression of the GTP cyclohydrolase and tyrosine hydroxylase, respectively, may be introduced into the host cell by a variety of methods. Such methods include calcium phosphate precipitation, protoplast fusion, electroporation, lipofection (such as the use of cationic lipids, e.g., DOTMA) BRL (Bethesda, Maryland) , transfection, packaged viral vectors, and the like. A wide variety of viral vectors may be used to introduce the heterologous polynucleotide sequences encoding GTP cyclohydrolase and tyrosine hydroxylase into the host cells of the invention or directly into patients of m vivo genetic therapy for the treatment of Parkinson's disease and related conditions attributable to dopamine production insufficiency The heterologous genes for expression may be present on the same vector or present on different viral vectors When the heterologous polynucleotide sequences for expression are present on different vectors, each vector may be of the same vector type or may be of different vector types Plasmid and phage-derived vectors may be used for the genetic modification of prokaryotic and lower eukaryotic host cells. However, when mammalian cells are used as host cells, the preferred vectors are viral vectors. Suitable viral vectors for use in modifying mammalian cells include herpes simplex virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, pseudorabies virus, alpha-herpes virus vectors, and the like. A thorough review of viral vectors, particularly viral vectors suitable for modifying neural cells, and how to use such vectors m conjunction with the expression of polynucleotides of interest can be found in the book Viral Vectors- Gene Therapy and Neuroscience

Applications Ed. Caplitt and Loewy, Academic Press, San Diego (1995) . Particularly preferred viral vectors for use in the embodiments of the invention where the host cells are mammalian cells, are retroviral vectors, particularly the retroviral vector MFG, the retroviral vector MFG-s is particularly preferred. A description of the retroviral vector MFG-s, can be found, among other places, in co-pending U.S. Patent Application No. 08/252,710, filed June 2, 1994, which is incorporated by reference to the extent that it does not directly contradict definitions of the subject invention provided herewith. Additional information on these retroviral vectors can be found, among other places m PCT Publication WO 92/07573. In a preferred embodiment of the invention, viral vectors are designed so as to co-express both GTP cyclohydrolase and tyrosine hydroxylase. Vectors with the co-expression of two genes may be obtained by inserting an IRES sequence in an intercistronic between the polynucleotide sequence encoding GTP cyclohydrolase and the polynucleotide sequence encoding tyrosine hydroxylase. IRES stand for Internal Riboso e Entry Site. The discovery and use of IRES sequences for dicistronic expression in mammalian host cells is described in, among other places, Ran et al . , Proc. Natl. Ada. Sci . USA 9 :3176-3180 (1994) .

Other embodiments of the invention include methods of producing L-dopa or dopamine by culturing the genetically modified host cells of the invention, as previously described, i.e, host cells comprising heterologous polynucleotide encoding capable of expression of GTP cyclohydrolase and a heterologous polynucleotide encoding capable of expression tyrosine hydroxylase. The subject methods of producing dopamine or L-dopa are advantageous for, among other reasons, the lack of a need to add BH4 to the cell cultures in order to obtain substantial production of L- dopa or dopamine. Furthermore, when BH4 is added to cultures of the genetically modified host cells of the invention, surprisingly high levels of L-dopa and dopamine are produced. The subject methods of dopamine and L-dopa production comprise the step of culturing the genetically modified host cell comprising a heterologous polynucleotide encoding and capable of expressing GTP cyclohydrolase and a second heterologous polynucleotide encoding and capable of expressing tyrosine hydroxylase. The host cells are cultured in a cultured medium adapted ^'for the growth of the particular host cell. After growth for a sufficient period of time, L- dopa and dopamine may be extracted from the culture medium using conventional purification techniques. The chemistry of L-dopa and dopamine are well understood, and the purification of such compounds may easily be accomplished by a person of ordinary skill in the art . Such techniques include both filtration, differential solubility, and dialysis.

Other embodiments of the invention include methods of treating a patient having a disease characterized by dopamine production deficiency. The most common disease characterized by a dopamine production deficiency is Parkinsonism; however, the subject invention may be readily adapted for the treatment of other diseases characterized by insufficiency of dopamine production. The terms "treatment" or "treating" as used herein with reference to a dopamine production deficiency disease refer to prophylaxis and to the amelioration of symptoms already present in an individual. It will be appreciated by the person of ordinary skill in the art that a treatment need not be completely effective from preventing the onset of a disease or inducing the symptoms associated with the disease, nor does a treatment need to cure a disease in order to be effective. Any reduction in the severity of the symptoms, delay an onset of symptoms, or delay in the rate of progression of severity of symptoms is desirable to a patient. The persons at risk of developing a dopamine production deficiency disease, such as Parkinson's disease may be treated prophylactically based on any variety of factors suggesting the possible onset of the disease, e.g. family history, environmental exposure to toxins, genetic markers, early symptoms, and the like.

One embodiment of the invention is a method of treating a patient having a disease characterized by dopamine production deficiency by introducing a genetically modified host cell into a patient, wherein the genetically modified host cell are as previously described, i.e., genetically modified host cells that comprise a heterologous polynucleotide sequence for expression encoding a GTP cyclohydrolase and a second heterologous polynucleotide sequence for expression encoding a tyrosine hydroxylase. The host cells may be of a variety of species. The host cells are preferably of the same species as the patient being treated. More preferably, the host cells are derived directly from the patient to be treated so as to minimize immune system rejection problems. The cells may be encapsulated by a polymer so as to minimize unwanted response from the patient's immune system. The host cells may be of a variety of tissue and cell types, e.g. fibroblasts, hepatocytes, keratinocytes, endothelial cells and the like. Of particular interest, is the use of host cells that are neural cells. The term "neural cell" as used herein, refers not only to neurons, but includes all cells of the mammalian central nervous system including astrocytes, microglial cells, etc. The subject method of treating the patient comprises the step of introducing the genetically modified host cells at a patient (either human or animal) . The cells may be introduced into a variety of locations within the patient. For example, the cells may be introduced intramuscularly, intraperitoneally, or directly into the central nervous system. The cells may be introduced by a variety of methods known to physicians including injections, surgical implantation, insertion through a canula, and the like. Preferably, the genetically modified host cells are introduced into a site in a patient's body that naturally contains such host cells. For example, genetically engineered neurons are preferably administered to the central nervous system as opposed to intermuscularly. However, given the fact that L-dopa is capable of crossing the blood-brain barrier, the genetically modified host cells of the invention may be introduced into parts of the body other than the central nervous system so as to achieve the desired therapeutic or prophylactic effect. The amount of genetically modified host cells introduced into the patient is an amount sufficient to treat the patient, i.e., a therapeutically effective amount. The exact amount of cells administered to a patient will vary in accordance with a number of factors, such factors include, the species of the host cell, the tissue from which the host cell is derived, the specific regulatory sequences in the genetic constructions used, body site of host cell implantation, the age and condition of the patient, the stage of the disease, other medications being taken by the patient, and the like. The pharmacology and pharmacokinetics of dopamine and L-dopa are well known. This pharmacological information can be used in conjunction with the measured dopamine and L-dopa production levels of cultures of the subject genetically modified host cells in order to optimize the amount of cells administered.

Other embodiments of the invention are in vivo genetic engineering methods for treating a dopamine production deficiency disease such as Parkinson's disease. The subject methods of in vi vo treatment comprise the step of administering an effective amount of a viral vector or vectors comprising polynucleotide sequences encoding and capable of expressing a GTP cyclohydrolase and a tyrosine hydroxylase. Such vectors are as previously described for use in preparing the genetically modified host cells of the invention. The viral vectors comprise both a GTP cyclohydrolase gene for expression and a tyrosine hydroxylase gene for expression. In an alternative embodiment of the invention two viral vectors are administered, wherein one of the viral vectors is designed for the expression of a GTP cyclohydrolase gene encoding a polynucleotide sequence and a second viral vector designed for the expression of a polynucleotide sequence encoding a tyrosine hydroxylase.

When two or more viral vectors are used, i . e . when the viral vectors used do not encode both a tyrosine hydroxylase and a GTP cyclohydrolase, the viral vectors may be the same or different from one another. For example, the GTP cyclohydrolase encoding construction may be contained within a retroviral vector and the tyrosine hydroxylase coding nucleic acid sequence may be encoded within a adenovirus vector. The choice of viral vector will depend on the particular cell type to be genetically modified. For example, retroviral vectors do not insert their polynucleotide sequence into non-replicating cells and accordingly would not be used by a person of ordinary skill in the art to genetically modify non-replicating cells. The viral vectors may be introduced into a variety of sites in the body. However, it is preferable to introduce the viral vectors at sites as close as possible to the target cells for genetic modification. The amount of viral vector or vectors administered to a patient is an amount sufficient to treat the patient, i.e., a therapeutically effective amount. The precise amount of viral vector or vectors administered to a patient will vary in accordance with a number of factors dependent upon the specific embodiment selected, such factors include, the specific virus from which the vector is derived, the specific promoter sequences used to drive gene expression, the specific patient cells to be infected, the age and condition of the patient, other medications being taken by the patient.

The invention having been described above may be better understood by reference to the following examples. The following examples are offered solely to illustrate the claimed invention and should not be interpreted as limiting the invention.

EXjkMP ES

Example 1

A full-length cDNA encoding an isoform of human GTP cyclohydrolase I was isolated. GTP cyclohydrolase is not expressed in fibroblasts and is expressed at very low levels in non-monoamine synthesizing neurons. The cDNA was isolated using PCR amplification from a "Quick clone" liver cDNA library. The primers used for cloning were based on published sequence information, and they included sequences encoding restriction sites Ncol at the 5' end and Bell and Bgll at the 3' end as determined from published sequence information. 50 bp of 3' non-coding sequence is included in the clone. The cDNA obtained from the library was 810bp in length. The isolated clone sequenced in its entirety in both strands after cloning into the "TA" : vector pCRII. An Ncol- Bglll fragment of this cDNA was cloned into the retrovirus vector MFG-S at the Ncol and BamHI sites. The resultant plasmid was referred to as MFG-s-hGTPCH I. 10μg of a plasmid MFG-s-hGTPCH I was co-transfected with lμg pSV2neo into psi- CRIP3.1. Transformants were selected from resistance on G418. A population of G418 resistant CRIP cells were generated and screened as candidates for the high level production of MFG-s-hGTPCH I. In addition a portion of the transfected cells were plated at a 20 fold lower density as selection was initiated. These plates were allowed to grow in G418 selection until numerous colonies were observed on 150 mm plates. 72 colonies were picked and replated in 24 well plates. These replated colonies were expanded so as to enable 52 "clones" of producers to be isolated. More than 30 of these clones were screened as described in Example 2.

Example 2

The MFG-s-hGTPCH I retroviral particles from the producer of populations described in Example 1 used to transduce monkey dermal fibroblasts (MDFs) that were previously transduced with the vector MFG-s-hTH2 (MFG-s-hTH2 is a retroviral genetic construct comprising retroviral vector MFG-s and the human tyrosine hydroxylase 2 gene inserted for expression) . The ability of these doubly transduced MDFs to secrete L-DOPA in the media in different concentrations of added BH4 was examined. MDF-TH + lacZ transduced cells secreted measurable L-DOPA only when 30- lOOμM BH4 was added; amounts of L-DOPA produced were 46 (for 30 μM BH4) and 178 ng/ml (for 100 μM BH4) . MDF cells transduced with both MFG-s-hGTPCH I and MFG-s-hTH2 produced 120 ng/ml L-DOPA in the absence of added BH4 ; these values rose to 290 and 524 ng/ml in 30 and 100 μM BH4 respectively. These studies showed that coexpression of hTH and GTPCHI in a portion of the MDF cells gave rise to cultures in which L- DOPA secretion in the absence of added BH4 could be observed.

Example 3

Clones of MFG-s-hGTPCH I producers were screened to identify those that had the highest transducing titres. BH4- independent L-DOPA secretion assay in hTH expressing target fibroblasts was used for the screening. Four clones were re- screened after showing initial high L-DOPA stimulating values in a quick screen using MDF-TH cells. These four viral supernatants were compared to supernatants taken from a producer population. Results were consistent between the two target cells. Minimal differences in the four clones were observed. All L-DOPA values that were only slightly (much less than two-fold) higher than the L-DOPA observed with cells transduced with the producer pops. The addition of lOOμM BH4 elevated levels further; therefore, the TH enzyme is not being saturated with BH4. L-DOPA values in the cell media of FDF (feline dermal fibroblass) cells that coexpress recombinant tyrosine hydroxylase and GTP cyclohydrolase grown in the absence of added BH4 was comparable to levels observed in FDF-TH cells cultured in the presence of lOOμM BH4. In comparison, MFG-s-hGTPCH I transduced MDF-TH cells yield L- DOPA at levels 20-30% of that we observe in normal MDF-TH cells assayed in the presence of 100 micromolar BH4. Thus, although no direct measurements of intracellular BH4 were taken in the GTPCH-I expressing cells, it is possible to calculate that the MDF's are producing 15-50 micromolar and that the FDFs are synthesizing 100 micromolar BH4.

Example 4

Experiments were performed to determine if cells that have been co-transduced to express TH and GTPCH I enzymes can produce measurable levels of L-DOPA and/or dopamine (PA) after intracerebral grafting. Experiments were conducted in rats. Fibroblasts that were transduced with either MFG-S-TH2 alone, or MFG-S-TH2 + MFG-s-GTPCH, were grafted in the striatum and animals were allowed to survive for one or two weeks. A portion of the animals were perfused with fixative and brain sections were processed for anti-tyrosine hydroxylase immunohistochemistry. At both survival times, only grafts that contained fibroblasts that were co- transduced with MFG-s-TH² and MFG-s-GTPCH I showed visible anti-TH immunoreactivity. Fibroblasts that are transduced with MFG-S-TH2 alone did not stain. These data suggest that the co-expression of GTPCH leads to elevated BH4 levels and that this BH4 helps stabilize and activate the intracellular. L-DOPA content and in vivo microdialysis studies will be conducted to more directly address this question.

Example 5

Genetic constructs for the linked co-expression of hTH and hGTPCH I was prepared. The construct was referred to as MFG-s-hTH2-ires-GTPCH I. MFG-s-hTH2-ires-GTPCH I consists of the human tyrosine hydroxylase II gene and the human GTP cyclohydrolase I gene inserted into the retroviral vector MFG-s. The 2 human genes are separated by an internal ribosome entry site (IRES) . IRES sequences and their use are described in, among, other place, Ghattas et al . , Mol . Cell . Biol . 11:5848-5959 (1991) . MFG-s-hTH2-ires-GTPCH I was co- transfected into psi-CRIP packaging cells with pSV2neo and populations. Clones were isolated after G418 selection. Viral supernatants from these cells have been screened for their ability to yield L-DOPA secreting FDF targets (in the presence or absence of added BH4) . In preliminary screening of populations and greater than 30 clones, relatively low functional titres have been observed. These titres were assayed by transducing target fibroblasts and measuring L- DOPA secretion in the presence or absence of BH4, and by staining transduced target cells for TH immunoreactivity. TABLE 1

L-DOPA IN TH and GTPCH Transduαion Samples

Sample PsxxArca L-DOPA Cocc Cell No. Fl=_d co_αc

Mo (ngΛnJ⁾ 10 c 5 (ug/lGaS/24i_r)

1 N£DF-CTPCHC2u£s)0uMBK

2 DF-GT?Ch'C21ωs) 0 uM BH4

3 SmF-CπTCHr2 i) 2 uM BH4

4 >.{DF-GTPCH(2iιiis) 30 uM B H4

5 N£DFX7TPCH(2iώ) 100 M BH4 5 MDF-TH-GTPCH Ou BK4

7 MDF-TH-C PCK OsM 3 K4

3 MDF-Tri-GTPCH 2uM 3H4

9 NiDF- H-σπ H 30 S< 3H4

10 MDF-TH -GTPCH IOOUM BH4

11 DF-TK-L15Z OuV B H4

12 SCDF-TH-Li .0aMBK4

14 .MDF-TH- a^z SOu BH-J

15 '.(UF-TΗ-Ligz 1 OOu 3 K4 lό S{ F-LO5TO- M BK

17 £DF-Lagz0-MBH4

IS MDr- g?.2u 3K4

20 ^V.CDF- z ICOOM 3K4

TABLE 2A

IiJDOPA.I :TH:^'aιtd GTECH.Thtπsdπciioπ Samples

τmρV_ No

MDF 95-3. OuM BH4 •

MDF 95-3. 100uW BH4 -

MDF215S7-TH. Ouλf BH4

MDF-216S7-TH, OuM BH4

5 MDF 21687-TH. 100uM BH4 ό MDF 16S7-TH, 100uM BH4

7 MDF-TH-GTPCH Qooc 1 , OuM BH4

S MDF-TH-GTPCH Cloae 1 . OuM BH4

9 MDF-TK-GTPCH Clone 3,0uM 3H4

10 MDF-TH-GTPCH Clσαc 3, OuM BH4

1 1 MOF-TH-C-TPCH CIODC 6.0uM BH4

L2 MDF-TH-GTPCH Ciooώ ό.OuM BH4

13 MDF-TH-GTPCH Qooc 9.0αM BH4

14 MDF-TH-GTPCH Qooc 9.0uM B K4

15 MDF-TH-GTPCH OOOC lO.OuM BH4 16 MDF-TH-GT?CK Oooc 10.0uM BH4 17 MDF-TH-GTPCH Oo * 11. OuM BH4 I S MDF-TH-GTPCH Oooc 12J0uM B H4 L9 MDF-TK-GTTCH Qooc 12,0uM BH-* 20 UDF-TH-GTPCH Oooc 13 OuM 3H4 21 MDF-TH-GTPCH Oooc 1 , OuM 3 H4 23 MDF-TH-GIPCH Oon U.OuM 3H4 24 Diaαπ 25 Dαica + scrum + cofa or

TABLE 2B

L-DOPA IN TH and GTPCH Transdπctioπ Samples

PsikArεa L-DOPΛCco

So (ng/ml)

MDF2l«7.TH-GTPCH X2 MDF I6Ϊ7-TH-GTPCH X2 MDF 216S7-TH-GTPCH X4 MDF 216S7-TH-GTPCH X

5 MDF 216S7-TH-GTPCH X5 6 MDF 21637-TH-GTPCH X5 7 MDF 1687-TK-GTPCK X7 S N DF 21687-TK-GTPCri X7 9 MDF 215S7 -TK-GTPCH X 15 10 MDF21637-TH-GTPCHX1 i 1 MDr 21637-TH-GTPCH Xiό

12 MDF 216S7-TH -GTPCH X 16 13 MDF 216S7-TH-GTPCH X25 14 MDF 215S7.TH -GTPCH X25

15 VtDP 216S7-TH-GTPCH X26

16 MDF 21657 -TK-GTPCH X26

17 ^"MDF 216S7-TH-GTP H X27

13 MDF 216S7-TH-GTPC-Ϊ X27

19 MDF 21SS7-7H-GTPCK X2S

20 MDF 216S7-TH-GTPCH X2S

21 SCDF !5$7-TK,03K4

22 MDF 21657-TH, 03K4

23 MDF236S7-TH.1003H4

24 MDF 21 S7-TH. ICO 3H

25 MDF 21637-TH-GTPCH , PO?

26 SDFDF-TH. Cnp 10-13

27 SDFDF-TK. C-.o 10-34

N^'O-Ϊ. Tb- ludiv viGii-Li SLan r s ii-^' o^:: during iz± IΓ.JI LIC TABLE 3

L-DOPA in TH Transduction Samples

Si-riD.ε - Sarnole Dϋ πoαoπ PcaX n-z L-DOPA Co-c F'.Γ.ΞJ cone

(1:10 Dilution)

20.23 1345 93.29 92.97 2327 25.S2 1173ι

S 124.20 9 2049 10 20.59 π 101.54

12 121 13 17.53 14 1759 15 99.9! 15 l02.Sc 17 ._- IS 19 11272 20 !07';

VOTΞS s-Ti.-ir. ;or l ncv o;^' MrG-C. °CM C I? orixluccrs t'cl'.ir,'-.- i i 3; i ^ )

:est b> L-ar.du ιn.5.-DP- 1 ri uπd MDT-TH (l-dup2 in 0 oi MiiluM n:oρ;.r^τ.r. i

TABLE 4

NOTES: sataiog te clone. of-J-CFG -GTPCH CRL? producers (clunu 7^, 25.33.3S . o?)

tut by t_TLQiducing FDF-TH nd MDF-TH (l-dopa Li 0 or ICOμM iopⁱcriaϊ

TABLE 5

L-DOPA IN TH Transdαction Samples

NOTES:

2 -r-ij ll (or 24 h:s

Incorporation by Reference All patents, patents applications, and publications cited are incorporated herein by reference.

Equivalents

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the above- described makes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

Claims

What is claimed is: 1. A recombinant host cell comprising a first

heterologous polynucleotide sequence, wherein the first heterologous polynucleotide sequence is capable of expression GTP cyclohydrolase, a second heterologous polynucleotide sequence, wherein the second heterologous polynucleotide is capable of expressing tyrosine hydroxylase.

2. A recombinant host cell according to claim 1, wherein said host cell is a mammalian cell.

3. A recombinant host cell according to claim 2, wherein said host cell is a fibroblast cell.

4. A recombinant host cell according to claim 2, wherein said host cell is a neural cell.

5. A recombinant host cell according to claim 2, wherein the first and second heterologous polynucleotide sequences comprise a viral vector.

6. A recombinant host cell according to claim 5, wherein at least one of the viral vectors is a retroviral vector.

7. A recombinant host cell according to claim 6, wherein the retroviral vector is an MFG vector.

8. A recombinant host cell according to claim 5, wherein at least one of the viral vectors an adenovirus vector.

9. A recombinant host cell according to claim 2, wherein said first and second heterologous polynucleotides are on a single genetic construction.

10. A recombinant host cell according to claim 9, wherein said genetic construction comprises a viral vector.

11. A recombinant host cell according to claim 10, wherein the viral vector is a retroviral vector.

12. A genetic construction for expression of

heterologous genes comprising in functional combination,

a polynucleotide encoding a GTP cyclohydrolase, and a polynucleotide encoding a tyrosine hydroxylase.

13. A genetic construction according to claim 12, wherein the genetic construction comprises a viral vector.

14. A genetic construction according to claim 13, wherein the viral vector is a retroviral vector.

15. A method of producing L-dopa or dopamine, said method comprising the step of culturing a host cell according to claim 1 in a culture medium, and extracting L-dopa or dopamine from said culture medium.

16. A method of treating a patient having a dopamine production deficiency, said method comprising the step of introducing a host cell according to claim 1 into the body of the patient.

17. A method according to claim 16, wherein said host cells are introduced into the brain of the patient.

18. A method of treating a patient having a dopamine production deficiency, said method comprising the step of administering a genetic construction according to claim 12.