Philippe Horellou

In vivo release of DOPA and dopamine from genetically engineered cells grafted to the denervated rat striatum

Fibroblastic 3T3 and endocrine RIN cells were genetically modified by infection with a recombinant retrovirus encoding the form I of human tyrosine hydroxylase (TH) and selection in tyrosine-free medium. These cells were grafted to rats unilaterally lesioned with 6-hydroxy-dopamine. Both cell types survived implantation into the striatum, expressed TH immunoreactivity, and as assessed by microdialysis 8-9 days after implantation, secreted high amounts of DOPA and/or dopamine into the surrounding host striatum. The modified 3T3 cells secreted large amounts of DOPA that was efficiently decarboxylated to dopamine by the host striatal tissue; the newly synthesized dopamine was stored only to a limited extent in the denervated striatum. The modified RIN cells synthesized dopamine that was stored intracellularly and released in a regulated fashion. The grafted DOPA-secreting cells produced 4-5 times higher extracellular dopamine levels than the dopamine-secreting cells, and they were more efficient in reducing apomorphine-induced rotation. No effect was observed with either cell type on amphetamine-induced turning behavior.

Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease

DIRECT intracerebral gene transfer to neural cells has been demonstrated with recombinant adenovirus encoding β-galactosidase. To explore the potential of recombinant adenovirus for the therapy of neurological disease we constructed a recombinant adenovirus encoding tyrosine hydroxylase, and optimized intracerebral injection to express the gene in the striatum of unilaterally denervated rats. These animals have dopamine depletion in their lesioned striatum, causing a rotation asymmetry induced by apomorphine. One, and two weeks after intracerebral injection this sensorimotor asymmetry was decreased by the adenovirus encoding tyrosine hydroxylase, and not by a control adenovirus encoding β-galactosidase. Histological analysis showed that tyrosine hydroxylase was preferentially expressed in astrocytes.

Brain-Derived Neurotrophic Factor-Mediated Protection of Striatal Neurons in an Excitotoxic Rat Model of Huntington's Disease, as Demonstrated by Adenoviral Gene Transfer

Huntingtons disease (HD) is a genetic disorder leading to the degeneration of striatal GABA-ergic output neurons. No treatment is currently available for this devastating disorder, although several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), have been shown to be beneficial for striatal neuron survival. We analyzed the effect of adenovirus-mediated transfer of the BDNF gene in a model of HD. Using a stereological procedure, three groups of rats were given an intrastriatal injection of adenovirus encoding BDNF, beta-galactosidase, or sham surgery. Two weeks after treatment, the animals were lesioned with quinolinic acid (QUIN), a toxin that induces striatal neuron death by an excitotoxic process. One month after the lesion, histological study revealed that striatal neurons were protected only in rats treated with the BDNF adenovirus. Volume measurements showed that the QUIN-induced lesions were 55% smaller in the BDNF adenovirus-treated group than in the beta-galactosidase adenovirus-treated group (p < 0.05), and the sham-treated group (p < 0.05). To determine the survival of striatal GABA-ergic output neurons after the QUIN-induced lesion, we immunostained brain sections with DARPP-32, an antibody specific for striatal output neurons. Prior treatment with the BDNF adenovirus resulted in a cell survival of 64%, whereas that after beta-galactosidase treatment was 46% (p < 0.05), showing that the BDNF adenovirus protected the striatal neurons. These results indicate that transfer of the BDNF gene is of therapeutic value for Huntingtons disease.

Generation of DOPA-Producing Astrocytes by Retroviral Transduction of the Human Tyrosine Hydroxylase Gene:In VitroCharacterization andin VivoEffects in the Rat Parkinson Model

Astrocytes secreting high levels of L-3,4-dihydroxyphenylalanine (DOPA) have been generated by retrovirus-mediated transfer of the human tyrosine hydroxylase (TH) gene. Immature astrocytes obtained from prenatal rat brain were cocultured with TH virus producing psi-2 cells that had been pretreated with the mitosis inhibitor mitomycin-C. During the first week of coculture DOPA production gradually increased to reach a plateau after 7-9 days. At this time point virtually all cells were GFAP positive and over 80% of them expressed TH. DOPA production in the transduced astrocytes was largely independent of exogenous cofactor, and DOPA release into the medium was not influenced by addition of either KCl or tetrodotoxin or by removal of Ca2+ from the culture medium, indicating that the newly synthesized DOPA was constitutively released from the cells. Transplantation of the TH-transduced astrocytes to the striatum in unilaterally 6-hydroxydopamine lesioned rats reduced apomorphine-induced turning by about 50% at 2 weeks postgrafting. Microscopic analysis revealed that the transduced astrocytes survived very well after transplantation and that some of the grafted cells had migrated out, partly along blood vessels, into the surrounding striatum. TH expression was observed in cells with both the appearance of mature GFAP-positive astrocytes, as well as in more immature-looking cells. However, only a few percent of all transplanted cells maintained significant expression of the transgene, as determined by TH immuno-histochemistry. The results show that primary astrocytes may be highly useful as gene carriers for ex vivo gene therapy in the CNS. With future improvement in the gene transduction procedure for more efficient, sustained expression of the TH transgene in vivo, genetically engineered DOPA-producing astrocytes hold great promise as a tool to explore the potential of ex vivo gene therapy in Parkinsons disease.

A single adenovirus vector mediates doxycycline-controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors.

Ex vivo gene transfer is emerging as a promising therapeutic approach to human neurodegenerative diseases. By combining efficient methodologies for cell amplification and gene delivery, large numbers of cells can be generated with the capacity to synthesize therapeutic molecules. These cells can then be transplanted into the degenerating central nervous system (CNS). Applying this approach to human diseases will require the development of suitable cellular vehicles, as well as safe gene delivery systems capable of tightly controlled transgene expression. For such brain repair technologies, human neural progenitors may be extremely valuable, because of their human CNS origin and developmental potential. We have used these cells to develop a system for the regulated expression of a gene of therapeutic potential. We report the construction of a single adenovirus encoding human tyrosine hydroxylase 1 (hTH-1) under the negative control of the tetracycline-based gene regulatory system. Human neural progenitors infected with this vector produced large amounts of hTH-1. Most importantly, doxycycline allowed a reversible switch of transgene transcription both in vitro and in vivo. This system may be applied to the development of therapies for human neurodegenerative diseases.

Behavioural Effect of Engineered Cells that Synthesize l-dopa or Dopamine after Grafting into the Rat Neostriatum.

Cell lines in which tyrosine hydroxylase was introduced either by infection or transfection were used in grafting experiments in a rat model of Parkinsons disease obtained by unilateral lesion of the substantia nigra. A neuroblastoma NS20 Y cell line which synthesizes only L‐DOPA and a neuroendocrine AtT‐20 cell line which produces dopamine were obtained. They were grafted into denervated striata and their ability to compensate for the dopaminergic deficit was studied. Both modified cell types displayed a rapid partial reversal of apomorphine‐induced turning behaviour. No effect was observed with the control unmodified cell lines. We discuss the usefulness of engineered cell lines to address the fundamental issues in grafting experiments and more particularly in the therapy of Parkinsons disease.

ADENOVIRUS IN THE BRAIN : RECENT ADVANCES OF GENE THERAPY FOR NEURODEGENERATIVE DISEASES

Adenovirus is an efficient vector for neuronal gene therapy due to its ability to infect post-mitotic cells, its high efficacy of cell transduction and its low pathogenicity. Recombinant adenoviruses encoding for therapeutical agents can be delivered in vivo after direct intracerebral injection into specific brain areas. They can be transported in a retrograde manner from the injection site to the projection cell bodies offering promising applications for the specific targeting of selected neuronal populations not easily accessible by direct injection, such as the motor neurons in the spinal cord. Adenoviral vectors are also efficient tools for the ex vivo gene therapy, that is, the genetical modification of cells prior to their transplantation into the nervous system. Recently, the efficacy of the adenovirus as a gene vector system has been demonstrated in several models of neurodegenerative diseases including Parkinsons disease (PD) and motor neuron diseases. In rat models of PD, adenoviruses encoding for either tyrosine hydroxylase, superoxide dismutase or glial-derived neurotrophic factor improved the survival and the functional efficacy of dopaminergic cells. Similarly, the intramuscular injection of an adenovirus encoding for neurotrophin-3 had substantial therapeutic effects in a mutant mouse model of motor neuron degenerative disease. However, although adenoviruses are highly attractive for neuronal gene transfer, they can trigger a strong inflammatory reaction leading in particular to the destruction of infected cells. The recent development of new generations of adenoviral vectors could shed light on the nature of the immune reaction caused by adenoviral vectors in the brain. The use of these new vectors, combined with that of neurospecific and regulatable promoters, should improve adenovirus gene transfer into the central nervous system.

Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms

A single human gene has been described to encode multiple tyrosine hydroxylase (TH) mRNAs. The study of this variation has been extended by S1 mapping experiments and by analysis of the 5’region of the TH gene. Four different mRNAs were found to originate solely from alternative splicing of two exons. Comparison of the 5’flanking regions of human and rat genes discloses several highly conserved segments, likely to play an important role in the regulation of TH gene expression.

Gene therapy for Parkinson's disease

Gene therapy is a potentially powerful approach to the treatment of neurological diseases. The discovery of neurotrophic factors inhibiting neurodegenerative processes and neurotransmitter-synthesizing enzymes provides the basis for current gene therapy strategies for Parkinsons disease. Genes can be transferred by viral or nonviral vectors. Of the various possible vectors, recombinant retroviruses are the most efficient for genetic modification of cells in vitro that can thereafter be used for transplantation (ex vivo gene therapy approach). Recently, in vivo gene transfer to the brain has been developed using adenovirus vectors. One of the advantages of recombinant adenovirus is that it can transduced both quiescent and actively dividing cells, thereby allowing both direct in vivo gene transfer and ex vivo gene transfer to neural cells. Probably because the brain is partially protected from the immune system, the expression of adenoviral vectors persists for several months with little inflammation. Novel therapeutic tools, such as vectors for gene therapy have to be evaluated in terms of efficacy and safety for future clinical trials. These vectors still need to be improved to allow long-term and possibly regulatable expression of the transgene.

Multiple Human Tyrosine Hydroxylase Enzymes, Generated Through Alternative Splicing, Have Different Specific Activities in Xenopus Oocytes

Abstract: A single human tyrosine hydroxylase (HTH) gene has been shown previously to generate four species of mRNA by alternative splicing. The four different HTH mRNAs were independently synthesized in vitro, using the SP6 transcription system. Each of these mRNA species was able to direct the synthesis of an active form of TH following injection into Xenopus oocytes. Quantitation of synthesized HTH polypeptides allowed the determination of the relative specific activity of each individual HTH form. A significant difference in specific activity was found between each form, suggesting that alternative splicing may play a role in regulating HTH activity in vivo.