WO2004062554A2

WO2004062554A2 - Enhancement of dopaminergic neuron generation and survital

Info

Publication number: WO2004062554A2
Application number: PCT/DK2004/000008
Authority: WO
Inventors: Alberto Martinez-Serrano; Isabel Liste; Ana Villa
Original assignee: Nsgene A/S
Priority date: 2003-01-08
Filing date: 2004-01-07
Publication date: 2004-07-29
Also published as: WO2004062554A3; WO2004062554A8

Abstract

The present invention concerns methods for enhancing the survival of neurons and especially tyrosine hydroxylase expressing neurons and dopaminergic neurons. The invention is based on the finding that Bcl-XL enhances the survival of neurons and specifically enhances the survival of TH+ and dopaminergic neurons. The present invention provides a method for producing Bcl-XL overexpressing neural cell lines expandable in vitro for cell banking. Such a cell line is able to efficiently differentiate into cells with a neuronal phenotype similar to the nigral dopaminergic neurons. Furthermore, the cells are able to survive, maintain their dopaminergic phenotype and function following transplantation and integration into the striatum.

Description

ENHANCEMENT OF NEURON GENERATION AND SURVIVAL

This application claims benefit of US 60/438,719 filed 8 January 2003 and US 60/464,546 filed 22 April 2003, which are hereby incorporated by reference in their entirety. It claims priority from Danish patent application number PA 2003 00581 filed 11 April 2003, which is incorporated by reference in its entirety. All references cited in the present application are hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention concerns methods for enhancing the survival of neurons and especially tyrosine hydroxylase expressing neurons and dopaminergic neurons. The invention furthermore concerns the cells obtained by said methods and the use of these cells especially in the treatment of neurodegenerative disorders.

BACKGROUND OF THE INVENTION

The very limited in vitro and in vivo survival of human dopaminergic (DA) cells is a major obstacle in cell biology research in Parkinson's disease (PD), and is also hampering the development of cell-based therapies for the disease (Brundin and Hagell 2001). This survival problem adds up to the well-known limitations in the supply of human dopaminergic neurons (Arenas 2002, Isacson 2002, Kim et al 2002, Dunnett and Bjόrklund 2000, Bjδrklund and Lindvall 2000, Borlongan and Sanberg 2002). The availability of a continuous source of human DA cells will not only foster the development of therapeutic strategies, but also facilitate pharmacological studies aimed at elucidating mechanisms of neurodegeneration and designing novel neuroprotective strategies (Dunnett and Bjδrklund 2000). The purpose of the present invention is to analyse the potential of human forebrain neural stem cells (hNSCs) for transgenic as well as endogenous TH expression, and to increase the efficiency of human DA neuron generation using different neuroprotective strategies aimed at enhancing cell survival.

The most common form of human DA neuron procurement is by the use of embryonic Ventral Mesencephalon (VM) tissue. However, in recent years, there has been an important effort to find alternative sources to generate DA cells in a predictable manner and in sufficient numbers (reviewed by Brundin and Hagell 2001 , Arenas 2002, Borlongan and Sanberg 2002). Rodent studies have paved the way in the discovery of procedures for generating DA neurons in vitro (Bjόrklund and Lindvall 2000). Examples of alternative tissue sources are: i) Short-term cultured VM progenitors, in which the DA phenotype was in some cases enhanced by epigenetic signals (Svendsen et al 1997, Ling et al 1998, Potter et al 1999, Rodriguez-Pallares et al 2001 , 2002, Riaz et al 2002, Studer et al 1998, 2000, Storch et al 2001). ii) Induction of the DA phenotype in cells derived from embryonic stem and neural stem cells, by either epigenetic (Matsuura et al 2001 , Daadi and Weiss 1999, Lee et al 2000, Studer et al 2000, Bjόrklund et al 2002, Kim et al 2002, Chung et al 2002), or genetic means (Wagner et al 1999, Kim et al 2002). And iii) Engineering of the DA biosynthetic pathway in neural cells (Anton et al 1994; Lundberg et al 1996; Corti et al 1996, 1999; Martinez-Serrano et al 2003 [submitted, to be published as Liste et al 2004 Human Gene Therapy]).

In the particular case of human cells, the generation of alternative tissue sources gets complicated by several factors related to species specific differences between humans and rodents: i) Availability of human DA neuron progenitor cells is much more limited (Brundin and Hagell 2001). ii) Long-term proliferation of mesencephalic neural precursors leads to a progressive reduction in the ability to generate human DA neurons (Ostenfeld et al 2002, Svendsen 2003, but see Storch et al 2001); iii) Nurr-1 over-expressing human neural stem cells, which produce a rather good percentage of DA neurons, can not be maintained in culture long-term, and their survival in vivo is compromised (Wagner et al 1999, Kele et al 2002, Arenas 2002). Nurrl over- expression has indeed been recently reported to enhance vulnerability of dopaminergic neurons against a variety of stress factors (Lee et al 2002); and iv) Engineering of the DA synthetic pathway has not been explored in detail in hNSCs (Corti et al 1996, 1999, Martinez-Serrano et al 2003 [submitted, to be published as Liste et al 2004 Human Gene Therapy], reviewed in Martinez-Serrano et al 2001).

hNSCs are promising cells for the development of neuroregenerative strategies based on cell replacement and gene therapy (Bjorklund and Lindvall 2000, Martinez-Serrano et al 2001 , Park et al 2002, Borlongan and Sanberg 2002). In this work, we have studied the capacity of different types of Nurr-1 expressing hNSCs to express transgenic TH and to differentiate along the DA phenotype pathway. To this end, we have carried out studies on established hNSC cell lines and on human neurosphere cultures derived from the human fetal CNS. Experimental evidence indicates that sudden exogenous TH over-expression is toxic in hNSCs and their derivatives. Toxicity associated to the DA phenotype is a widely accepted phenomenon, since catecholamihe metabolism results in oxidative stress for the cells (Haavik and Toska 1998, Stokes et al 1999, Olanow and Tatton 1999, Barzilai et al 2001 , Blum et al 2002). It is therefore of great interest to develop methods which not only allow the differentiation of neural progenitor cells in vitro, but do so in such a way that maximizes the survival of neuronal cells which express TH. United States Patent No. 5,851,832 (hereby incorporated by reference) describes the in vitro growth and proliferation of multipotent neural stem cells and their progeny. However, as compared with the techniques described herein, the methods described therein do not result in a population of neural cells wherein a significant percentage of the cells are TH expressing neurons. United States Patent No. 5,980,885 (hereby incorporated by reference) describes the growth factor induced proliferation of neural precursor cells in vivo. However, the methods described therein are not directed towards the in vitro proliferation of neurons and, as compared with the techniques described herein, do not result in a population of neural cells wherein a significant percentage of the cells are TH expressing neurons. United States Patent No. 5,981,165 (hereby incorporated by reference) describes the in vitro induction of dopaminergic cells. However, as compared with the techniques described herein, the methods described therein do not result in a population of neural cells wherein a significant percentage of the cells are TH expressing neurons. United States Patent No. 5,968,829 and the related United States Patent No. 6,103,530 (both hereby incorporated by reference) describe the use of Leukemia Inhibitory Factor in order to increase the rate of stem cell proliferation or neuronal differentiation. However, as compared with the techniques described herein, the methods described therein do not result in a population of neural cells wherein a significant percentage of the cells are TH expressing neurons. Similarly, United States Patent Nos. 6,040,180, 6,251 ,669, and 6,277,820 (all incorporated by reference herein) describe methods and uses, for neuronal progenitor cells or CNS stem cells. However, as compared with the techniques described herein, the methods described therein do not result in a population of neural cells wherein a significant percentage of the cells are TH expressing neurons. United States Patent No. 6,312,949 describes cells comprising an exogenous nucleic acid Nuπ that induces TH enzyme synthesis within a cell. However, the methods disclosed therein are directed to elevated TH expression within an individual cell and are distinguished from the methods described herein.

Thus, a need remains in the art for a solution to the known logistical and ethical problems of efficiently preparing sufficient numbers of well-characterized dopaminergic cells. A possible solution would be the identification of a method for producing a specific neural cell line expandable in vitro for cell banking. Such a cell line should be able to efficiently differentiate into cells with a neuronal phenotype similar to the nigral dopaminergic neurons. Furthermore, the cells should be able to survive, maintain their dopaminergic phenotype and function following transplantation and integration into the striatum.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to a method for enhancing the survival of neurons and/or of cells expressing tyrosine hydroxylase (EC 1.14.16.2), said method comprising contacting a population of cells with BCI-XL or a functional equivalent thereof wherein said population of cells is selected from the group consisting of : i. neurons or cells capable of differentiating into neurons; and ii. TH expressing cells or cells capable of differentiating into TH expressing cells.

The present inventors have studied the capacity to enhance the generation and survival of TH expressing cells and the generation and survival of neurons by neurotrophic proteins (BDNF and GDNF), an anti-oxidant protein (Cu+Zn superoxide dismutase, SODIcit) and the anti-apoptotic protein BCI-XL. Even when all of them had some interesting survival effects, BCI-XL was found to be vastly and unexpectedly superior, resulting in the generation of large numbers of neurons and in particular of TH over-expressing cells. BCI-XL over-expressing clones of hNSCs showed a marked increase in their capacity for spontaneous generation of neurons and in particular TH⁺ neurons and dopaminergic neurons. BCI-XL showed similar effects after induction of the DA phenotype in non-immortalized (growth factor expanded) human neurosphere cultures. Finally, BCI-XL showed a remarkable all-or-none effect on the survival of human TH+ neurons generated from hNSCs in vivo and showed a significant enhancement of the total number of neurons generated and surviving in vivo after grafting. It is well known that TH expression declines with the number of passages in vitro. The present inventors demonstrate that Bcl-X_L preserves the capacity for TH expression and even restores the capacity. •

By TH expression or TH⁺ is intended cells that can be identified as TH positive using immunohistochemistry with antibodies against tyrosine hydroxylase, a technique commonly used in the art for detecting TH positve cells.

By neurons is intended cells having a neuronal morphology with neurites (dendrites and/or axons). Neurons may also be defined with reference to cellular markers such as doublecortin (Dcx), Neuron Specific Enolase (NSE) and/or neurofilament. Furthermore, the results presented herein show that cells can be transformed or transduced to overexpress Bcl-X at any stage and still benefit from the survival enhancing effect of BCI-XL overexpression.

Three grounds substantiate the relevance of the results reported here: First, the magnitude of the enhancement of neuron production, and/or TH production/differentiation by BCI-XL is remarkably high, not merely a small percentage or a few-fold increase over control levels, but reached one-to-two orders of magnitude increase. This is a consistent finding in all the systems explored. Second, experiments with different designs (involving transgenic and natural TH expression, and both immortalized and non-immortalized cells) generate similar results. Thirdly, BCI-XL greatly enhanced the survival of TH⁺ neurons generated from NSCs in vivo in an all-or- none fashion. Therefore, the Bcl-X_L effects reported here are highly relevant and apply to multiple human DA neuron source cellular systems and to other cell systems involving TH expression, e.g. chromaffin cells or retinal epithelial cells such as ARPE- 19 cells. An independent enhancing effect was also seen on the number of neurons generated both in vitro and in vivo, indicating an independent survival/differentiation enhancing effect of BCI-XL on any type of neurons.

It is contemplated that the protective effect of BCI-XL can be achieved either by administering the protein as such or as a fusion protein to the cells via the growth medium or by a transgenic approach.

The methods allow the generation of large numbers of TH-expressing cells despite the documented negative effects of TH expression on survival. This is irrespective of whether the cells are obtained via spontaneous TH neuron differentiation or via transgenic TH overexpression, or whether the cells express TH even in the absence of induction (certain retinal epithelial cells, such as ARPE-19). These TH expressing cells, neurons or dopaminergic neurons may be used for therapeutic or other uses.

In a further aspect the invention relates to a composition of cells obtainable by the described method.

In a further aspect the invention relates to a composition of isolated mammalian cells overexpressing BCI-XL.

In a preferred embodiment the composition of the invention comprises neurons or cells capable of differentiating into neurons. In another preferred embodiment of the invention the composition comprises cells expressing TH or cells capable of differentiating into TH expressing cells.

Said composition of cells may in one aspect be characterised as a composition of cells overexpressing BCI-XL and of which at least 5% express tyrosine hydroxylase. Through the overexpression of BCI-XL the survival of the cells is enhanced both on a short and on a long-term basis since BCI-XL exerts it's protective effect throughout the lifecycle of the cells.

In a still further aspect the invention relates to a neural progenitor cell comprising a first heterologous expression construct comprising a first promoter capable of directing the expression of tyrosine hydroxylase or a functional equivalent thereof and a second heterologous expression construct comprising a second promoter capable of directing the expression of BCI-XL or a functional equivalent thereof. The invention also relates to compositions of neural progenitor cells comprising at least one such cell. These progenitor cells can be used for differentiation of dopaminergic neurons and will result in an enhanced survival of such neurons compared to what is obtainable from neural progenitor cells not having the transgenic BCI-XL construct.

In a further aspect the invention relates to a differentiated dopaminergic neuron comprising a first heterologous expression construct comprising a first promoter capable of directing the expression of tyrosine hydroxylase or a functional equivalent thereof and a second heterologous expression construct comprising a second promoter capable of directing the expression of BCI-XL or a functional equivalent thereof. The invention also relates to compositions of cells comprising at least one such dopaminergic neuron.

Such "double" transgenic dopaminergic neurons are less prone to oxidative stress and consequently have an enhanced survival rate compared to dopaminergic neurons, which do not overexpress BCI-XL.

In general, in the present invention, "overexpression" is intended to mean an expression level resulting in an amount of BCI-XL protein, which is at least 1.5 times higher than in non-transgenic cells. Preferably the level is at least two times higher, and it may range from 2 to 10 times higher.

In another aspect the invention relates to an implantable cell culture device, the device comprising: i) a semipermeable membrane permitting the diffusion of a biologically active agent therethrough; and ii) a composition of cells, or at least one neural progenitor cell or at least one differentiated dopaminergic neuron according to the invention.

Such an implantable cell culture device allows termination of, or adjustments to, the cell therapy protocol once the cells are implanted. This is because cells implanted into a patient's body are well isolated from the patient's own tissue. This prevents effectively migration in situ of transplanted cells and proviral integration into the host germ line cells. The cell culture device with cells can be used in replacement therapy for replacing dopaminergic neurons in patients suffering from Parkinson's disease. In another embodiment the cells in the device are genetically manipulated to overexpress and secrete therapeutically relevant amounts of a growth factor and can be used for local and sustained delivery of biologically active growth factor(s).

In another aspect the invention relates to a lentiviral vector particle, said vector particle being produced based on a lentiviral transfervector comprising a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding Bel- XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR.

The lentiviral vector particle can be used for in vivo and ex vivo transduction primarily of dopaminergic neurons and precursors to enhance the survival of these by ensuring overexpression of BCI-XL in the transduced cells.

In another aspect the invention relates to a method for enhancing the survival of TH+ cells in vivo, said method comprising administering to substantia nigra in an individual in need thereof a therapeutically effective amount of a lentiviral vector particle, said vector particle being produced based on a lentiviral transfer vector comprising a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR.

In another aspect the invention relates to a retroviral vector particle, said vector particle being produced based on a retroviral transfer vector comprising a 5' retroviral LTR, a tRNA binding, site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR. The retroviral particle in contrast to the lentiviral particle infects only dividing cells. The retrovirus can therefore be used for ex vivo gene therapy, e.g. for transducing stem cells prior to differentiation of these into dopaminergic neurons, which will have, by virtue of the overexpression of Bel- Xι_, an enhanced survival rate.

In another aspect the retrovirus can be used in a method of enhancing the survival of in vivo differentiated dopaminergic neurons. This method comprises administering to the striatum of an individual in need thereof a therapeutically effective amount of a retroviral vector particle, said vector particle being produced based on a retroviral transfer vector comprising a 5' retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR. The striatum contains cells capable of dividing and differentiating into dopaminergic neurons. By administering a retrovirus it is ensured that only de-novo differentiated dopaminergic neurons will be transduced and overexpress BCI-XL leading to enhanced survival of the said neurons.

In another aspect the invention relates to a packaging cell line capable of producing an infective vector particle, said vector particle comprising a retrovirally derived genome comprising a 5' retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR. This packaging cell line can be used either for in vitro production of vector particles but it may also be used for in vivo gene therapy by implanting a composition of said packaging cell line into the striatum of a subject. The presence of the packaging cell line ensures that vector particles will be provided for a longer period as compared to injection of virus.

The invention also relates to a packaging cell line capable of producing an infective vector particle, said vector particle comprising a lentivirally derived genome comprising a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR. Likewise, this packaging cell line can be used in vitro and in vivo.

In another aspect the invention relates to the use of a composition of cells according to invention for transplantation. This may be as outlined above in connection with replacement cell therapy, e.g. in the treatment of Parkinson's disease. In another aspect the invention relates to the use of a composition of cells according to the invention for drug screening and/or for gene profiling in connection with differentiation and survival of dopaminergic neurons.

In another aspect the invention relates to the use of a composition of cells according to the invention for the preparation of a medicament for the treatment of a disorder of the central nervous system.

In a further aspect the invention relates to a method of treatment of a neurological disorder comprising administering to a subject in need thereof a therapeutically effective amount of a composition of cells overexpressing BCI-XL and said cells being capable of differentiating into neurons and/or TH expressing cells. This is based on the findings of the present inventors that such cells result in a higher number of surviving neurons over non-Bcl-Xι_ expressing cells after transplantation in vivo. These BCI-XL overexpressing cells to date represent the only example of human neural stem cells capable of differentiating into a dopaminergic phenotype in vivo.

In another aspect the invention relates to a fusion protein between BCI-XL or a functional equivalent thereof and a membrane translocation signal. This fusion protein is a suitable way of administering BCI-XL to cells via an epigenetic route and thus avoid insertion of heterologous DNA into the cells. The invention also relates expression vectors coding for said fusion protein, to host cells comprising said vector as well as to methods for producing the fusion protein using the host cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Transgenic TH overexpression: Colony formation assays.

A) Experimental design: the cells are transfected and subjected to hygromycin selection for one month. Afterwards, the cultures are fixed and stained for β- galactosidase activity or immunostained for TH. Total and positive clones were subsequently quantified. B) An example of an immunostained TH⁺ clone derived from HEK293T cells. C) Quantifications of the total number of clones (open bars), and those among them positive for β-gal or TH (closed bars, percentages are given above bars), using as substrate the indicated cell lines; Hyg, empty vector (CMV-IRES-hyg, also coding for hyg^R); TH hyg, CMV-TH-IRES-hyg vector; LacZ hyg, CMV-LacZ-IRES-hyg. D) An example of negative (for TH) or positive (for β-gal) clones photographed under phase contrast (PhC) or bright-field (TH or Xgal panels). Scale bar, 100 μm Figure 2: TH over-expression induces cytotoxicity in hNS1 cells. A-D) Hoechst 33258 (blue, left panel) and TH (right panel, green) staining of transfected hNS1 cells, showing different degrees of nuclei damage and condensation/fragmentation. A is a normal nuclear ,morphology, whereas nuclear damage increases in the TH+ cells from B to D (Scale bar in A, 10 μm). Quantifications are given in the text. E) p53 upregulation in TH overexpressing hNS1 cells. Data represent the percentage of either TH+ or β-gal+ cells also expressing p53. F) BrdU incorporation assays. Data represent the percentage of either TH+ or β-gal+ cells that incorporated BrdU. G) Abnormal morphologies observed in hNS1 cells after TH overexpression. Note that the TH+ cells are single cells at this time post- transfection (8-10 days). Scale bar, 100 μm. H) Expression of GTP Cyclohydrolase I (GTP-CH I) and Aromatic Aminoacid Decarboxylase (AADC) genes in hNS1 and HEK293 cells (RT-PCR). +/- indicates presence or absence of reverse transcriptase (RT).

Figure 3: Co-expression of TH and helper genes enhances the number of TH+ hNS1 cells.

A) Histogram showing the effects of vectors coding for BCI-XL, SODIcit, GDNF, BDNF, or the empty vector, when co-transfected with the CMV- or UbiC-promoter driven, TH-coding ones (n=12), # p<0.0001 Bcl-X_Ls. all other groups, * p<0.005 different from the empty vector group (Student-t test). B) Microphotographs of the experiments shown in A, illustrating the increased number of TH+ cells after co- transfection of the indicated genes. Scale bar, 100 μm.

Figure 4: Stable overexpression of BCI-XL enhances the capacity for spontaneous TH neuron generation by hNS1 cells.

A) Western blots of naive (n) or BCI-XL (b) expressing hNS1 cells under division (Div) or differentiation (Diff) conditions. Cells were differentiated in the presence of 0.5% FBS for 12 days, at low (5%) or high (20%) O₂ tension. Blots were double-stained for BCI-XL or TH (or α-actin, as a loading control). When the same experiment was performed in the absence of serum during differentiation, no TH expression could be detected (not shown). B) Nurr-1 expression in dividing (Div) and differentiated (Diff) control hNS1 cells. Note that differentiated cells up-regulate a mature neuronal marker such as hNSE. C) Time course of appearance of TH protein in the indicated cell lines, during differentiation in low O₂ and 0,5% FBS. Blots were probed for TH and β-F1- ATP-ase as a loading control. TH expression was clearly detectable after 8 days of differentiation, and only in the BCI-XL cells. D) TH ICC in parallel cultures to those shown in A. Differentiated TH+ human neurons are illustrated only for the case of BCI- XL expressing cells, low O₂ and 0.5% FBS. Scale bar, 50 μm. E) Quantification of TH+ neuron generation rate. hNS1-hyg cells are cells stably transfected with the IRES-hyg empty vector, and hyg selected for one month (* , p<0.01 BCI-XL VS. the two other groups, Student t-test). The net increase in TH+ neuron generation rate is 15-fold.

Figure 5. Enhanced spontaneous generation of human neurons by hNS1 cells subclones expressing different levels of BCI-XL. A) BCI-XL, Nurr-1 , and β-lll-tub WB of naive hNS1 cells, or cells genetically modified for hyg^R, or BCI-XL overexpression (three representative clones: 2, 3, 5, are shown). Cells were probed under both division (Div) and differentiation (Diff) conditions (12 days in 0.5% FBS, low O₂). B) Quantification of BCI-XL overexpression levels in the different cell lines used in these assays (data from four individual WB from independent cultures were averaged, after normalizing OD values to that of the control cells. Every group was different from all the other groups, one-way ANOVA, followed by Newman-Keuls, p<0.05). C) Quantification of neuron generation rate (β-lll-tub⁺ cells) by the cell lines shown in the western blot in A. The asterisks denote a significant difference between the indicated groups (see text for details). D) Immunocytochemistry of human β-lll-tub⁺ neurons generated after differentiation of the control hNS1 cells, and BCI-XL over-expressing hNS1 subclone #5, in the same conditions as in A. Scale bar, 50 μm.

Figure 6. Enhanced spontaneous generation of TH7DA⁺ neurons by hNS1 subclones expressing different levels of BCI-XL. A) BCI-XL and TH WB of naive hNS1 cells, or cells genetically modified for hyg^R, or BCI-XL overexpression (three representative clones: #2, 3, 5 are shown). Cells were probed under both division (Div) and differentiation (Diff) conditions (12 days in 0.5% FBS, low oxygen). B) Immunocytochemistry of human TH⁺ neurons generated after differentiation of the Bcl-X hNS1 subclone #5, in the same conditions as in A. Scale bar, 50 μm. C) Double ICC for β-lll-tub and TH after differentiation of BCI-XL hNS1 subclone 5. Note co-localization of both markers, indicating the neuronal nature of TH⁺ cells. Scale bar, 20 μm. D) Dopamine ICC in differentiated cells from the BCI-XL subclone#5. (DAB-development, scale bar, 50 μm. E) TH⁺ neurons (green) produce dopamine (red), illustrating that differentiation of BCI- XL subclone#5 results in the generation of human dopaminergic neurons. Scale bar, 20 μm. F) Quantification of the rate of TH⁺ neuron generation by the different cell lines. The asterisks denote a significant difference from all the other groups (n=5, p<0.01 , Tukey test). The rate of TH⁺ neuron generation by clone #5 is 2.18% of the total number of plated cells (right axe). G) Diagram illustrating the relationship between number of TH⁺ neurons and level of BCI-XL- overexpression, supporting the notion of a threshold- or a dose response-type of effect for BCI-XL upon DA neuron generation. Figure 7: Enhancement of DA neuron generation by Bcl-X in non-immortalized neurosphere cultures of human forebrain neural precursor cells. A) Photomicrographs of TH+ human neurons after mock transfection, or after genetic modification for GFP or BCI-XL expression. Two days after transfection, the cells were differentiated for five days in the presence of a DA inductive cocktail, before fixation. B) Quantification of the rate of TH+ neuron generation by the transfected cultures Data are expressed as percentage of plated cells (100.000 cells/well, n=5, 4-6 analyzed fields per sample summing up to 800-1200 cells analyzed in total). The asterisk indicates a significant difference between the BCI-XL group and the other two groups (p<0.01 , Tukey test). C) Photomicrographs (phase contrast and fluorescence) of GFP transfected cells. Transfection efficiency, determined by GFP fluorescence after 48h following transfection, at the time when differentiation period starts, was 15%.

Figure 8: Map of the plasmid vector pCMV-BclXL-IRES-hyg used for the transfecting studies of Example 2. The polynucleotide sequence of the plasmid is set forth in SEQ ID No 3.

Figure 9: Map of representative retroviral vectors comprising an expression construct for directing the expression of BCI-XL in transduced cells. A) pFB-bclX_L-IRES-hrGFP (SEQ ID No 4); B) pFB-bcl-X_L (SEQ ID No 5); C) pLC-Bcl-X_L-Sn (SEQ ID No 6).

Figure 10. Transplantation of BCI-XL over-expressing hNSCs into the adult rat 6-OH-

DA lesioned striatum results in human TH⁺ neuron generation and survival.

A) Human-nuclei stained sections to illustrate survival of transplanted control hNS1 cells (upper row) and BCI-XL over-expressing hNS1 cells (bottom row) into the host striatum (1 month survival time). Panels are arranged from left to right to show increasing magnifications. Note the non-disruptive integration of the transplanted cells into the host parenchyma, the increased migration of BCI-XL cells, and also the preferential integration/migration through grey matter regions of the neostriatum. cc=corpus callosum, lv=lateral ventricle, fb=fiber bundles. B) Parallel sections stained for BrdU, showing confirmatory evidence to that obtained from h-nuc stained sections Scale bar, 20 μm. C) Doublecortin (Dcx) or D) hNSE stainings of parallel sections to show neurons generated from grafted control or Bcl-X_L-overexpressing hNSCs. E) A TH immunostained (DAB-development) parallel section showing a transplant of BCI-XL hNSCs. The dotted line represents the graft-host boundary. The inset shows a high magnification of a human TH⁺ neuron. Transplants of naive hNSCs cells did not contain any TH⁺ neurons (not illustrated), in spite of reasonably good survival, as shown in panels A-D. Scale bar, 100 μm. F, G) High magnification views of TH⁺ human neurons: (F) TH + h-nuclei double IF, to illustrate the human origin of TH⁺ cells (the larger photograph shows an overlay of a confocal Z-axis series, out of which a single section (one micron depth) is shown in the inset. Scale bar 10 μm; (G) TH + hNSE double IF, to illustrate the human+neuronal nature of TH⁺ cells. Scale bar, 10 μm.

Figure 11. Effects of Bcl-X and Tetrahydrobiopterin on intracellular dopamine content of hNS1 cells. Very low or not detectable DA levels were observed under normal differentiation conditions (mitogen removal and 0.5%FBS supplementation), or (mitogen removal) 0.5% FBS + [BDNF + DA + Forskolin] (called "Bradford" cocktail). Tetrahydrobiopterin (BH ) was added to the culture medium 2 h before sample collection, to activate intracellular dopamine synthesis and production.

Figure 12. Differentiated hVM cells (polyclonal cell line) display markers of mature DA neurons. Cells from the polyclonal hVM cell line at passage 6-8 were differentiated in presence of BDNF (50 ng/ml), DA (10 uM) and Forskolin (10 uM) for 7 days under low oxygen tension. A) Cultures from proliferating (Div) and differentiated cells (Diff) were lysed and assayed for the expression of different DA/neural markers by Western immunoblotting. Nuπ expression was observed both under differentiation and proliferation conditions. Expression of β-lll-tubulin, TH, AHD-2 and DAT was detected only in differentiated cells. B) Left panels: The general morphology of these cells under proliferation conditions is shown in phase contrast (Phase). Abundant immunoreactivity for β-lll-tubulin, Map-2, and GFAP can be observed in differentiated cultures. Right panels: Many TH⁺ stained neurons (accounting for around 15% of the cells in culture) can be detected by indirect immunocytochemistry of differentiated cultures. DAB ICC is shown at 40x magnification. Double immunofluorescence stainings (100x) for β-lll-tubulin/TH: note the colocalization (merge) in the same cells of β-lll-tubulin and TH (indicative of neural identity of these cells). All TH⁺ neurons in these cultures were also positive for the neuronal marker.

Figure 13. Properties of hVM cells (polyclonal cell line) change with time in culture. Photomicrographs comparing immunoreactivity for different markers at passage 8 and at passage 30. Observe the increased cell death in the later passage, and the near to complete absence of TH and β-lll-tubulin immunoreactivity, in contrast with the abundant expression detected at earlier passage.

Figure 14. hVM clone isolation/differentiation and selection. Aprox. 70 clones were isolated from the hVM polyclonal cell line (at passage 3). All these clones were differentiated at passage 4 for 7 days under the same conditions showed in Figure 12, and fixed and immunostained for TH expression. Eight of them, here represented, were selected on the basis of their TH neuron generation potential. The best clone, in terms of percentage of TH⁺ neuron generation was the clone 23 (28.7% of the total cells were TH⁺ cells).

Figure 15: BCI-XL effects on hVM polyclonal cell line (Heterogeneous Line, HL). Schematic in A: The hVM polyclonal cell line shows a diminishing potential for both neuron and TH⁺ neuron generation upon passaging (time course data not shown). The cells were infected at passage 6 with retroviral vectors coding for BCI-XL-IRES-GFP or empty-IRES-GFP, in order to test the possible effects of continued BCI-XL expression in the preservation of the potential of the cells to generate human DA neurons.

Two passages after infection the fluorescent cells were isolated by FACS, and cultured. After ten more passages, the cells were differentiated and analyzed (see part B, and text for details). B: Note (phase contrast) the amelioration of survival and decrease in cell death under division (Div) and under differentiation (Diff) in BCI-XL over-expressing cells, as compared to the control cells infected with the empty vector. See also the increased number of TH⁺ neurons in Bcl-X_L over-expressing cell cultures, in comparison with their respective controls (Quantitative data in Figure 16).

Figure 16. Maintainance of TH neuron generation potential by Bcl-X_L-overexpression in hVM cell lines.

A: Human polyclonal VM cell line (passage 6) and clone 23 (best clone selected in terms of TH generation, see Figure 13, at passage 8), were infected with retrovirus (supplied by NS-Gene) coding for: LTR-empty-IRES-rhGFP-LTR (rΦ, as a control) or LTR-Bcl-X_L-IRES-rhGFP-LTR (rBcl-X_L). The cells were allowed to proliferate for 2 more passages (P8), and trypsinised for cell sorting selection (of green flurorescent cells) by FACS. The fluorescent selected cells were proliferated for 10-12 more passages, plated and differentiated for WB (A) or ICC (B) analyses. Both cell lines are cryopreserved at different passages for further studies. The control line (rΦ), as expected shows an almost complee loss of TH and DAT expression, and also a clearly diminished β-lll-tubulin expression. In contrast, Bcl-X_L-overexpressing cells showed much higher BCI-XL, TH, β-lll-tubulin and DAT levels, similar to those observed in the heterogeneous line (HL) at passage 7 (left column). Similar results were obtained for clone C23 (represented in the right panel) also at passage 18 (#8+10) after infection. B: Examples of TH-immunoreactivity, for HL and C23, both at passage 18, after infection with the empty vector or with the BCI-XL expressing vector. Note the net increase in the number of TH⁺ neurons in both cases in the BCI-XL overexpressing lines, as compared with the corresponding empty controls. The percentage of TH⁺ neurons (expressed in relation to the total number of cells in the culture dish) is indicated in the photographs (magnification 40x in all cases).

DETAILED DESCRIPTION OF THE INVENTION 5

Finding an alternative source of human dopaminergic neurons other than primary fetal tissue is essential for making progress in PD research. Human dopaminergic neurons undergo continuous oxidative stress imposed by their catecholaminergic neurotransmitter phenotype (Haavik and Toska, 1998; Olanow and Tatton, 1999;

10 Stokes et al., 1999; Barzilai et al., 2001 ; Blum et al., 2001). TH activity, by itself, generates hydrogen peroxide, and the further metabolism and auto-oxidation of both L-DOPA and DA, contribute many other free radicals. Grafts of human VM tissue survive to some extent, but do so at a low rate in vivo (Lindvall et al 2003). This can be compensated by grafting cells from multiple embryos, but, in any case, its low survival

15 rate limits its application to clinical research (Arenas, 2002; Bjόrklund et al., 2003; Isacson, 2003; Lindvall et al., 2003). In alternative settings, TH⁺ neuron poor survival or teratoma formation precludes the use of NSCs or ES cells, respectively (Svendsen et al., 1997; Arenas, 2002; Bjόrklund et al., 2003; Isacson, 2003; Lindvall et al., 2003).

20 Toxicity of the DA phenotype in hNSCs

Collectively, the data reported here demonstrate the toxicity of the TH/DA phenotype in hNSCs and derivatives. These include: i) The failure to obtain stable transfectants (utilising a TH expression system that works well for HiB5 and HEK293T cells, and using both viral and cellular promoters, like CMV or hUbiC promoters). See Figure 1.

25 ii) The direct evidence of nuclear damage observed in TH transfected cells (Figure 2A- D), which is consistent with the DA effects observed by other authors in sympathetic neuron apoptosis (Zilkha-Falb et al 2000). iii) Furthermore, the increased expression of p53 in TH+ hNSCs (Figure 2E) is a direct demonstration of the toxicity associated to a sudden expression of TH and concomitant DA synthesis (reviewed by Barzilai et al

30 2002, Hahn and Weinberg 2002). In addition, iv) BrdU data (Figure 2F) expand this view on genotoxic actions of TH expression, adding an anti-proliferative effect of TH expression not previously described for hNSCs. Finally, iv) The remarkable effects of BCI-XL (compared to that of well-known neuroprotective factors, Figure 3) in dopaminergic neuron survival and differentiation shown here, also contribute to the

35 view that apoptosis-inducing stress factors compromise integrity of in vitro generated human dopaminergic neurons.

In the present work, we demonstrate that BDNF, GDNF and SODIcit, all enhance the capacity for TH over-expression of hNSCs to a moderate extent (2-fold enhancement, Figure 3). BCI-XL, in contrast, shows much more robust effects in the model system studied, both with the- CMV-vector and with a human UbiC promoter (WO 98/32869) vector for TH expression. An artifactual counteraction of unspecific apoptosis induced by lipofection does not explain BCI-XL effects on TH expression: First, LacZ transfection results in lower numbers of damaged nuclei than those generated by TH expression, and, second, BCI-XL co-transfection has no effect on the number of LacZ expressing cells, whereas it greatly enhances the generation of TH+ cells. Therefore, BCI-XL effects arise from a genuine counteraction of TH-triggered apoptosis. To obtain alternative evidence for this conclusion, we examined the effects of the pan-caspase inhibitor z-VAD-fmk, which yielded similar results to those resulting from BCI-XL overexpression.

Neuroprotective actions

Neurotrophic factors (BDNF, GDNF) and SODIcit show less efficacy than BCI-XL in transfection experiments. SODIcit acts only at a specific, discrete point in the cell death-triggering pathway, that is, dismutating the superoxide radical, and this may suffice to explain its limited efficacy. Other reactive oxygen species (hydroxyl and peroxide) may still be causing oxidative stress to the TH+ cells. The case of neurotrophic factors is more intriguing, though, since they induce expression of many survival genes, but are not as efficient as Bcl-X .

In spite of the enhancement of survival of TH transfected hNSCs, BCI-XL could not override the apparent anti-proliferative effect of continued TH expression and therefore, double transfection followed by selection yielded proliferating cultures of hygR cells that were not expressing TH, but only BCI-XL. The absence of proliferating TH+ cells in any of the experiments described here, together with the highly differentiated morphologies of the TH+ cells often seen (like those in Figures 2 and 3) indicate that TH expression is not compatible with continued proliferation of hNSCs. BrdU incorporation data (Figure 2F) provide confirmatory evidence for this. In addition, the absence of reported cell lines in the literature of hNSCs stably expressing TH comes to support these conclusions.

BCI-XL effects on transgenic TH expression were very striking. Therefore, the inventors have also studied its effects when assaying a natural or spontaneous way to obtain human TH neurons through the acquisition of a dopaminergic phenotype (in this case TH was not transfected, but only BCI-XL). It is important to remark that the cells studied here express Nun , GTP-CH-I and AADC, being negative for DBH (hNS1 cells), and also that the DA inductive protocol used on neurosphere cells results in the full expression of the DA phenotype and generation of DA neurons (Stull and lacovitti 2001). Differentiation of perpetual hNS1 cells resulted in a substantial generation of TH+ neurons only under differentiation conditions of low oxygen, presence of 0.5% FBS, and exclusively when over-expressing Bel- XL (Figures 4-6). According to the time course data in Figure 4C, BCI-XL is not protecting TH+ cells generated early during differentiation. Rather, TH+ expression is only evident later in development, ruling out a survival effect on early-generated TH+ neurons. Although these data do not exclude a slow inductive effect, the most likely explanation for the observed actions in these experiments seems to be an enhanced survival of TH+ neurons.

Data obtained from the BCI-XL over-expressing clones (Figures 5, 6) are also consistent with a survival mechanism, but add some specificity on dopaminergic neurons. BCI-XL over-expressing cells do generate more β-tub-lll+ neurons (3-4 fold increase over control cell lines, see Figure 5), although this increase does not correlate with the magnitude of BCI-XL over-expression, since all BCI-XL over- expressing clones see their neuron generation ability increased to the same extent (compare data for clones #2 and #5, for instance, Figure 6). However, TH+ neuron generation seems to depend on BCI-XL levels, suggesting a specific effect on dopaminergic neurons. Another argument for specificity is that the small net increase in total number of β-tub-lll+ neurons (4-fold) cannot explain the large increase in TH+ neurons (91 -fold).

The enhanced TH+ neuron generation rate is not merely reflecting an increase in the number of neurons generated, but BCI-XL is specifically increasing the number of TH+ neurons generated. These effects, however, do not seem to arise from an enhanced Nurrl expression, since this is very similar between control and BCI-XL over-expressing lines (Figure 5A). In a slightly different context, it is worth mentioning here that the BCI- XL overexpressing clones generate more neurons than the control cells, indicating that in v-myc immortalized hNSCs, the proposed cooperation between Bel-family members and c-Myc for cell transformation is not taking place (Cory and Adams 2002).

Experiments on non-immortalized cells (Figure 7) suggested a similar mechanism of action. In this case, epigenetically propagated human neurosphere cells of the 6wk- or 9.5wk-HFBr were transfected for BCI-XL over-expression, and differentiated under low oxygen using a TH inductive cocktail. In two independent experimental rounds, the net result was a 5.5-7 fold increase in the number of DA neurons generated, which is also consistent with increased cell survival. It should be noted that in this experiment, BCI- XL effects are under-estimated, since only 15% of the cells were transfected. Theoretically, if all cells in the culture had been transfected, the fold increase in TH/DA cells due to BCI-XL could have reached 46-fold. It is important to highlight that these latter observations, made in two strains of human neurospheres, are particularly important to rule out that the reported BCI-XL effects could be due to the presence of v-myc in the immortal cell lines. Bel proteins and c- Myc can mutually cooperate to enhance cell survival (Cory and Adams 2002), and deregulated c-Myc controls Bcl-2 and BCI-XL expression in other cell types (Eischen et al 2001). However, data from neurosphere cells obtained here clearly rule out that those events might be of any relevance for the results described in the present work.

BCI-XL effects documented here are of higher magnitude than those reported for other neuroprotective strategies utilised in rodent progenitor cells, mainly targeting oxidative stress. Thus, low oxygen (Studer et al 2000), or antioxidants like N-acetylcysteine and dipyridamole (Rodriguez-Pallares et al 2001, 2002) have been shown to exert only moderate effects on survival of rat DA neurons generated from ventral mesencephalic progenitors (2-3 fold enhancement, similar to the neurotrophic factor or SODIcit effects reported here). A link to apoptosis blockade involving BCI-XL was not established, since the underlying mechanism of action of those procedures were not investigated in detail. Low oxygen, in the present study, did not alter the outcome of TH transfection experiments (Figure 3), but was crucial for the spontaneous generation of TH neurons by BCI-XL overexpressing hNS1 cells (Figures 4-7). Interestingly, it had no effect on non BCI-XL over-expressing, control cells. The reason for this discrepancy may reside in that spontaneous differentiation should result in a more mature (and toxic) dopaminergic phenotype than TH transfection alone. It is thus conceivable to reason that it would only be under those circumstances (natural dopaminergic differentiation) when lowered oxygen tension (normoxia) becomes crucial for survival, as deduced from the work shown here.

Finally, transplants of BCI-XL overexpressing hNSCs further help to establish BCI-XL function and relevance (Fig. 10). As shown here and as reported by other workers, naive hNSCs generate very few neurons in the adult lesioned or intact striatum, and no TH-expressing neurons at all. Consistent with other studies (Fricker et al., 1999; Englund et al., 2002) neurons were only seen at or close to the implantation site. Present in vivo experiments suggest that BCI-XL over-expression helps human TH⁺ neurons to survive in a non-neurogenic site such as the adult striatum, in an all-or- none fashion. Such clear-cut data serves as a proof-of-principle evidence for BCI-XL action. No TH⁺ neurons were found in naive hNSCs transplants, as compared to hundreds of TH⁺ cells that were found in the Bcl-X ones. The number of TH⁺ neurons found was relatively small (less than 200 per animal) but this figure could be easily enhanced by transplanting more cells, possibly at multiple locations, in order to obtain a better reinnervated striatum.

Three grounds substantiate the relevance of the results reported here: First, the magnitude of the enhancement of TH production/differentiation by Bcl-X is remarkably high, not merely a small percentage or a few-fold increase over control levels, but reached one-to-two orders of magnitude increase. This is furthermore a consistent finding in all the systems explored. Second, experiments with different designs (involving transgenic and natural TH expression, and both immortalised and non- immortalised cells) generate similar results. Thirdly, Bco-X_L expression greatly enhanced the survival of human TH⁺ neurons generated from hNSCs in vivo in an all- or-none fashion. Therefore, the present inventors believe that the BCI-XL effects reported here are highly relevant and that they can be generalised to all human DA neuron source cellular systems and are also applicable in both in vivo and in vitro settings.

The results presented here on BCI-XL expand our view on how to counteract dopamine toxicity (Olanow and Tatton 1999, Blum et al 2002, Barzilai et al 2002). In one embodiment, the inventors contemplate the genetic modification of hNSCs or derivatives for the over-expression of Bcl-X as an effective means of helping the cells to cope with the toxicity of the DA phenotype. These results are of interest and of direct application for strategies aimed at the generation of human DA neurons, either for in vitro research (drug screening, gene profiling), of for intracerebral transplantation studies. Enhancing the ability of hNSCs to express TH, irrespectively of whether it is of transgenic or of endogenous origin, should help to achieve the goal of generating an unlimited supply of human TH/DA cells/neurons.

Bcl-Xι_ proteins

BCI-XL is a member of the Bcl-2 family of antiapoptotic proteins. The C terminal 21 amino acids encode a stretch of hydrophobic amino acids that are important in membrane docking: Bcl-2 resides on the cytoplasmic face of the mitochondrial outer membrane, the nuclear envelope, and the endoplasmic reticulum. Deletion of the C terminus does not abrogate Bcl-2 survival function. Most Bcl-2 homologs have this hydrophobic C terminal domain, though they are not necessarily located on membranes but are cytosolic (e.g. Bax).

The human and Rattus norvegicus Bcl-X_L genes code for a 233 amino acid protein which has a highly conserved region of high sequence similarity to Bcl-2 (amino acids 90-188 of BCI-XL). This sequence (partly) comprises the BH1 (residues 129-148), BH2 (residues 180-195) and BH3 (residues 86-100) domains. A domain known as BH4 is located in the N-terminal (amino acids 1-27, in some references only amino acids 4-

24). The C terminal comprises a transmembrane domain (amino acids 210 to 226).

The human, mouse, rat, cat, dog and pig BCI-XL proteins are highly conserved (see Table I) and it is expected that they can be used interchangeably in this group of species. Considering the small number of amino acid differences and the fact that the protein is located intracellularly, it is not expected that the BCI-XL proteins are immunogenic within this group of species.

Table I: CLUSTAL W (1.82) multiple sequence alignment of mouse, rat, pig, and human BCIXL.

Mus_musculus MSQSNRE WDFLSYKLSQKGYSWSQFSDVEENRTEAPEETEAERETPSAINGNPS HLA Rattus_norvegicus MSQSNRE WDFLSYKLSQKGYSWSQFSDVEENRTEAPEETEPERETPSAINGNPSWHLA Homo_sapiens MSQSNRE WDFLSYKLSQKGYS SQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLA Sus_scrofa MSQSNRE WDF SYK SQKGYSWSQFTDVEENRTEAPEGTESEAETPSAINGNPS H A *************************** . *********** ** * ***************

Mus_musculus DSPAV1.GATGHSSSLDAREVIPMAAVKQALREAGDEFE RYRRAFSDLTSQLHITPGTAY Rattus_norvegicus DSPAV GATGHSSSLDAREVIPMAAVKQA REAGDEFE RYRRAFSDLTSQLHITPGTAY Homo_sapiens DSPAWGATAHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAY Sus_scrofa DSPAVNGATGHSSSLDAREVIPMAAV QALREAGDEFE RYRRAFSDLTSQLHITPGTAY ********* ************************************************** Mus_musculus QSFEQVVNELFP-DGV1WGRIVAFFSFGGALCVESVDKEMQVLVSRIASWMATYL DHLEP

Rattus_norvegicus QSFEQVVNE FRDGVWWGRIVAFFSFGGALCVESVDKEMQVLVSRIASKn -ATYLNDH EP Homo_sapiens QSFEQVVNELF-^GVl^WGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEP Sus_scrofa QSFEQV NELFRDGVlWGRIVAFFSFGGALCVESVDKEMQλπ-iVSRIA WMATYLNDHLEP ****** . **************************************** . ************

Mus_musculus WIQENGG DTFVϋLYGlrø-^AAES- GQERFNRWF TGMTVAGVV GSLFSRK Rattus_norvegicus IQENGGWDTFVDLYGNN--AAESRKGQERFNR FLTGMTVAGVVLLGS FSRK Homo_sapiens WIQENGGWDTFVELYG NAAAESRKGQERFNRWF TGM VAGWLLGSLFSRK Sus_scrofa IQENGG DTFVELYGIST AAAESRKGQERF RWFLTGMTLAGVV LGS FSRK ************ . ************************** . *************

indicates positions which have a single, fully conserved residue. indicates that one of the following 'strong' groups is fully conserved:

■STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. indicates that one of the following 'weaker' groups is fully conserved:

-CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM,

HFY.

The Bcl-2 family can be defined by the presence of conserved motifs known as Bcl-2 homology domains (BH1 to BH4). Bcl-2, BCI-XL and Bcl-w contain all four BH domains, whereas the other pro-survival members contain at least BH1 and BH2.

Pro- and anti-apoptotic family members can heterodimerize: the BH1 , BH2 and BH3 domains of an anti-apoptotic member (e.g. Bcl-X_L) form a hydrophobic cleft to which a BH3 amphipathic alpha-helix can bind (Sattler et al., 1997, Science, 275: 983). This BH3 cleft coupling, reminiscent of ligand-receptor engagement, may account for all dimerization within the family.

The BCI-XL proteins used in accordance with the present invention preferably is the native protein, but it is contemplated that amino acid substitutions can be performed without substantially altering the activity of the proteins. More specifically it is contemplated that the C-terminal transmembrane domain may be removed without substantially altering the activity of the protein in the context of the present invention. It is also possible to substitute this domain with other transmembrane domains that will ensure membrane localisation of the protein.

It is also contemplated that conservative amino acid substitutions can be performed in particular in the regions located between the homology domains, BH1-BH3 and the BH4 domain. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the BCI-XL protein without altering the biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among BCI-XL proteins from different mammals are predicted to be particularly non-amenable to alteration. The same applies to conserved amino acid residues in the highly conserved BH1-BH4 domains. Amino acids for which conservative substitutions can be made are well known within the art.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the BCI-XL protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the BCI-XL coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for BCI-XL biological activity to identify mutants that retain activity. By a functional equivalent is meant a BCI-XL variant, which can compete with Bcl-X in a binding assay using an antibody against BCI-XL, preferably the antibody disclosed in example 4. Biological activity of the functional equivalent can be measured in any anti- apoptosis assay known in the art. These include, but are not limited to, the serum deprivation of the C3H 10T1/2 cell assay described in detail in PCT Publication No. WO 94/25621 , Furthermore, in vivo apoptosis inhibition may be measured by any method known in the art.

The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.

According to one embodiment of the invention, a functional variant of BCI-XL comprises all the residues identified in Table I as conserved residues, when placed in alignment with the sequences of Table I using Clustal W 1.82 with default settings. More preferably, the variant in addition to the conserved residues also comprises the strongly conserved residues in the positions marked as such in Table I, when placed in Clustal W 1.82 alignment with the other sequences of Table I. Still more preferably the variant also comprises weakly conserved residues in the positions marked as such when aligned with the sequences of Table I using Clustal W 1.82.

It is understood that the protein may also be modified by adding an affinity tag for use during preparation and purification of the protein, if it is produced recombinantly. A preferred affinity tag is a poly-his tag which does not need to be more than 6 residues long. The poly-his tag is preferably located in the N-terminal. An optional tag should not affect the biological function of the protein.

According to a preferred embodiment, the BCI-XL protein is of human origin. Other origens that can be used in the context of the present invention are generally proteins of mammalian origin, and in particular rodent (mouse or rat), simian, feline, canine, porcine, and bovine. Preferably the BCI-XL protein is from the same species as the cells, which are to be contacted with said protein. Fusion proteins

According to one embodiment of the present invention the Bcl-X_L protein is supplied to the cells via the culture medium in the same way as serum and growth factors. The advantage of this is that the cells are not transformed with a heterologous vector construct. When the treated cells are to be used for in vivo transplantation, it may be considered more safe to use this approach, since there may be unforseen disadvantages associated with constitutive expression of BCI-XL in transplanted cells.

When the BCI-XL protein is to be supplied via the growth medium, it may be advantageous to provide it with a membrane translocation signal (MTS) to ensure that the protein is taken up. One example of is the 12 amino acid MTS from Kaposi FGF-4 (SEQ ID No 2). This MTS has been used to ensure uptake of Cre recombinase in mammalian cells (Daewong et al, 2001 , Nature Biotechnology, 19:929-933). Other membrane translocation signals are known and can be readily applied by the skilled practitioner. The MTS can be linked to the N or C-terminal. Preferably the MTS is be inserted at the C-terminal end.

In addition to a MTS, any MTS-BCI-XL fusion protein may further be modified by adding an affinity tag for purification. Non-limiting examples of affinity tags include a polyhis tag and a GST tag. Preferably a short (6 residues long) polyhis tag is added. Such a tag facilitates recovery and purification of a heterlogously expressed polypeptide. It is not expected that the presence of a short polyhis tag in the N terminal will influence the function of the molecule.

Accordingly in one aspect of the invention there is provided a modified BCI-XL protein comprising a MTS and optionally an affinity tag. In a further aspect of the invention there is provided an expression vector construct coding for such a modified BCI-XL protein as well as methods for producing such a modified protein by inserting the expression vector into a host cell, culturing the host cell and recovering the protein from the culture.

As mentioned above it may also be conceivable to replace the transmembrane domain with other similar domains and obtain substantially the same effect.

Amounts of BCI-XL

When BCI-XL or the fusion protein is added to the culture medium, the amount required can be determined by a simple titration assay. Normally this will result in amount of BCI-XL being at least 0.01 ng/mL, such as at least 0.1 ng/mL, for example at least 1 ng/mL, such as at least 5 ng/mL, for example at least 10 ng/mL, such as at least 20 ng/mL, for example at least 50 ng/mL, such as at least 100 ng/mL, for example at least 500 ng/mL, such as at least 1000 ng/mL.

Nucleotide sequences

The vectors provided together with the present invention as well as those used in conjunction with the present invention comprise a sequence coding for a BCI-XL protein or one of the functional analogues described above. Nucleotide sequences coding for a specific protein can be changed substantially without changing the product encoded by the polynucleotide sequence due to the degeneracy of the genetic code.

In the context of the present invention the polynucleotide sequences coding for the BCI-XL can be changed as desired as long as the sequences encode a BCI-XL protein or one of the substituted and functionally active equivalents described above. Preferably the cDNA sequence is used. Using the cDNA sequence ensures that the pro-apoptotic splice variant Bcl-Xs is not synthesized by the cells. The cDNA sequence of Rattus norvegicus BCI-XL is set forth in SEQ ID No. 7. The cDNA sequence codes for the protein of SEQ ID No. 8.

For most purposes it is preferable to use a polynucleotide sequence from the same species as the cells into which the sequence is to be transferred.

Vectors Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a BCI-XL protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The invention in other embodiments include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses).

The advantage of using viral vectors is that these vectors result in the insertion of the genes into the genome of the host cell so that the incorporation of a selectable marker can be avoided. This makes the viral vectors especially suitable for in vivo gene therapy. Retrovirus vectors can be used for transduction of neural progenitor cells in the brain so that when these differentiate into dopaminergic neurons, the survival rate of the neurons is enhanced. Lentivirus on the other hand can be used for transducing already differentiated neurons in the brain and enhance the survival rate of these. Both types of vectors can be used with a constitutive expression of BCI-XL or a temporary expression (using an inducible promoter and/or Cre-Lox excision). Methods for preparation and in vivo administration of lentivirus to neural cells are described in US

20020037281 (Methods for transducing neural cells using lentiviral vectors).

The recombinant expression vectors of the invention comprise a nucleic acid coding for a BCI-XL protein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Promoters can be divided into constitutive and inducible/repressible promoters. A preferred group of promoters are the constitutive promoters which ensure constitutive expression of BCI-XL to have a prolonged protective effect of the protein. Examples of constitutive promoters that can be used for expression of BCI-XL include the CMV promoter, and SV40 promoter described above. Other preferred constitutive promoters are the human Ubiquitin promoter (WO 98/32869), the JeT-promoter (WO 02/12514), and the EF-1alpha promoter (Uetschi et al J Biol Chem 1989, 264:5791-5798). Various hybrid promoters comprising elements form different promoters can also be used.

In other cases it may be preferable to use an inducible promoter so that the expression of BCI-XL can be turned off at a suitable point in time. Clinical trials with in vivo gene therapy or transplantation studies may show side effects of constitutive expression of BCI-XL in neurons in the brain. Furthermore, it is contemplated that constitutive expression of BCI-XL is not necessary under all conditions. Once a suitable population of dopaminergic neurons have been generated in vivo or been transplanted into the patient, these neurons may not need overexpression of BCI-XL. For such uses an inducible promoter such as the TeT promoter or the Mx-1 promoter can be used. These promoters are activated by known drugs, the TeT promoter by Tetracyclin and the Mx-1 promoter by Interferon-alpha or Interferon-beta. Expression is simply turned off by discontinuing administration of these compounds.

One further way of ensuring temporary expression of BCI-XL is through the use of the Cre-LoxP system which results in the excision of part of the inserted DNA sequence either upon administration of Cre-recombinase to the cells (Daewoong et al, Nature Biotechnology 19:929-933) or by incorporating a gene coding for the recombinase into the virus construct (Pluck, Int J Exp Path, 77:269-278). Incorporating a gene for the recombinase in the virus construct together with the LoxP sites and a structural gene (BCI-XL in the present case) often results in expression of the structural gene for a period of approximately five days. Examples of vectors are disclosed in Figures 8 and 9. Figure 8 describes a plasmid vector used in the examples appended to the present application. It is derived from plRESIhyg (Clontech). The polynucleotide sequence of the vector is set forth in SEQ ID No 3.

Figure 9 shows three examples of retrovirus vectors. The corresponding polynucleotide sequences are set forth in SEQ ID No 4, 5, and 6.

In vivo gene therapy

Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470 and US published patent application No. 20020037281) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Nati. Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system e.g. in packaging cell line. Suitable packaging cell lines are described in e.g. US 6,218,181 (Retroviral packaging cell line), and Current Protocols in Molecular Biology laboratory handbook (Edited by: Fred M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl, John Wiley & Sons).. WO 97/44065 (Device and method for encapsulated gene therapy) describes suitable methods and capsules for gene therapy using encapsulated packaging cell lines releasing virus particles to the surrounding cells.

Cells

In the broadest aspect the invention relates to a composition of isolated animal cells overexpressing BCI-XL. Preferably, the cells are mammalian. Because of the overexpression of BCI-XL, these cells have enhanced survical and are capable of differentiating into TH expressing cells and/or neurons at higher rates.

In one embodiment, these cells once differentiated into a TH expressing phenotype are phenotypically stable. Phenotypic stability is of the utmost importance when the cells are used for transplantation, e.g. in a cell based therapy of Parkinson's Disease. In one embodiments the cells are capable of differentiating into neurons. Preferably, said neurons are TH⁺, preferably wherein said TH⁺ phenotype is stable in vitro and preferably in vivo. More preferably, said TH⁺ neurons are capable of producing dopamine in vitro, and preferably in vivo. The prior art fails to teach how to make TH positive neurons or cells capable of differentiating into TH positive neurons, which are also capable of producing dopamine in therapeutically significant amounts after transplantation.

According to a preferred embodiment the composition comprises neurons, preferably at least wherein 5% of the cells are neurons, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%.

Accrording to another preferred embodiment, the composition comprises TH expressing cells, preferably wherein at least 5% of the cells are TH⁺, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 25%.

In one embodiment, the composition comprises retinal epithelial cells, or cells derived therefrom. RPE cells express TH and produce dopamine and are therefore prone to the survival enhancing effects of BCI-XL as instantly demonstrated. A particularly preferred type of cells which is useful for encapsulated cell therapy are ARPE-19 cells or cells derived therefrom. ARPE-19 cells are a superior platform cell for encapsulated cell therapy (US 6,361 ,771).

In one embodiment, the composition comprises cells capable of producing dopamine or capable of differentiating into dopamine producing cells.

Preferably, the cells overexpressing BCI-XL contain at least two times as much Bcl-XL as corresponding cells not overexpressing BCI-XL.

The cells used in connection with the present invention include any animal cells that can differentiate into TH expressing cells, into neurons or into TH expressing, dopaminergic neurons. One preferred group of cells are embryonal stem cells that can be made to differentiate into neural progenitor cells and ultimately into neurons including dopaminergic neurons. Also included within the scope of the present invention are embryonal stem cell derived progenitors. Likewise neural stem cells can be used. Preferably these are isolated human forebrain neural stem cells (hNSCs).

Another group of cells include neural progenitor cells that can be isolated from the brain of foetuses, such as isolated embryonic ventral mesencephalon cells or ventral mesencephalon progenitor cells. Preferably, the neural progenitor cells are mammalian.

Other cells that can differentiate into neurons and dopaminergic neurons are NS4 cells (WO 01/30981) and cells originating from human neurosphere cultures.

In one embodiment of the invention the cells capable of expressing tyrosine hydroxylase are immortalised, such as being immortalised with a telomerase gene, a myc gene, a v-myc gene, a c-myc gene, a SV-40T gene.

For model studies the cells preferably are rodent (mouse, rat), porcine, canine, or non- human primate (simian, chimpanzee) although it is clear that the present invention also relates to therapeutic use in these species and in other species of animals. More preferably the cells are human.

Non-human cells can be manipulated to be less immunogenic to human beings using state of the art techniques. Such cells may be used as xenotransplants, in particular in the CNS.

TH-induction in various cell types

Methods for differentiation of TH expression in neurons are known in the art. These are described in brief in the following.

A) Expansion and differentiation of VM precursors

Attempts to increase the proportion of the expanded VM precursors that differentiate into a dopaminergic phenotype, include growing the cells in low oxygen tension (Studer et al., 2000, Storch et al., 2001) or by differentiation of cells adding 10% FCS and IL-1 (Ling et al., 1998). Further differentiation of the cells is achieved by addition of IL-11 , LIF and GDNF after culture on striatal monolayers (Storch et al., 2001). More recently, a successful method for the conversion of human mesencephalic progenitor cells into dopaminergic neuronal phenotype has been described by adding BDNF, forskolin (an activator of protein kinase-A) and dopamine to the differentiation medium (Riaz et al., 2002).

B) Differentiation of Neuronal Precursor Cells

TH expression can be induced in primary cultures from human cortex (Theophilopoulos et al., 2001), by treating the cells with dopamine, protein kinase-A activators (forskolin) and BDNF or GDNF, or in primary cultures from mouse striatum by coadministration of aFGF and either dopamine or a protein Kinase A (forskolin) or protein Kinase C (TPA) activators (Du and lacovitti, 1997, 1995).

C) Neural Stem Cells (NSCs) Midbrain dopaminergic neurons can be generated in a coordinated manner from multipotent NSCs through a process requiring both Nurrl overexpression and soluble factors derived from VM Type 1 astrocytes (Wagner et al., 1999). It has been also shown that a small proportion of NSCs from the mouse and human embryonic forebrain have the potential to differentiate into a dopaminergic phenotype, which is different from that of midbrain dopaminergic neurons (Daadi and Weiss, 1999; Storch et al., 2001).

D) Embryonic Stem Cells (ES)

It has been possible to induce differentiation of ES into dopaminergic phenotype, by stromal cell-derived inducing activity in rodent and primate cells (Kawasaki et al., 2000; 2002). Or spontaneusly, by grafting low doses of ES cells into the striatum of hemiparkinsonian rats (Bjorklund et al., 2002). In another recent work Kim et al., have developed a method based of increasing the efficiency of generation of dopamine neurons from mouse predifferentiated ES cells. These cells can functionally integrate into the host tissue, induce recovery in a rodent model of PD, and show electrophysiological properties of neurons from the midbrain (Kim et al., 2002).

One of the primary objectives of the present invention is the generation of large quantities of dopaminergic neurons, Therefore, preferably providing conditions for expression of tyrosine hydroxylase comprises induction of dopaminergic differentiation.

Induction of TH expression may be performed as in Example 2 by transducing or transfecting the population of cells with a vector comprising a heterologous expression construct comprising a promoter controlling the expression of tyrosine hydroxylase or a functional equivalent thereof.

Some cells, such as hNS1 cells spontaneously generate a small number of dopaminergic neurons under differentiation conditions.

Preferably, tyrosine hydroxylase expression is induced at low oxygen tension, such as below 10%, more preferably below 8%, more preferably below 6%, more preferably below 5%, such as below 4%, for example below 3%, such as below 2%, for example below 1 %. In the resulting population of TH expressing dopaminergic neurons the percentage of dopaminergic neurons in the resulting composition of neurons preferably is at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.

Indications to treat:

The methods, vectors and cells according to the present invention can be used in the treatment of any disorder of the CNS associated with a loss of TH-expressing neurons. Particularly preferred disorders include neurodegenrative diseases. These in turn may include a neurodegenerative disease involving lessioned and traumatic neurons, such as traumatic lessions of peripheral nerves, the medulla, the spinal chord, cerebral ischaemic neuronal damage, neuropahty, peripheral neuropathy, Alzheimer's disease, Huntingdon's disease, Parkinson's disease, amyotrophic lateral sclerosis, memory impairment connected to dementia.

According to an especially preferred embodiment, the disorder is Parkinson's disease.

According to one aspect the invention relates to a method of treatment of a neurological disorder comprising adminstering to a subject in need thereof a therapeutically effective amount of a composition of cells overexpressing BCI-XL and said cells being capable of differentiating into neurons and/or TH expressing cells.

Said neurological disorder may be a neurodegenerative disease involving lessioned and traumatic neurons, such as traumatic lessions of peripheral nerves, the medulla, the spinal chord, cerebral ischaemic neuronal damage, neuropahty, peripheral neuropathy, Alzheimer's disease, Huntingdon's disease, Parkinson's disease, amyotrophic lateral sclerosis, memory impairment connected to dementia. In a particularly preferred embodiment, the disease is Parkinson's Disease.

The transplanted cells are preferably capable of differentiating in vivo to TH⁺ neurons. More preferably said TH⁺ neurons are capable of maintaining a TH⁺ phenotype. Still more preferably, said TH⁺ neurons are capable producing dopamine in vivo. Encapsulated cells

In some embodiments of the invention, the cells or compositions of cells and in particular the packaging cell lines of the present invention are encapsulated in a microcapsule or macrocapsule prior to implantation in the brain.

One approach to encapsulating cells is called "microencapsulation", wherein tiny spheres encapsulate a microscopic droplet of a cell-containing solution (Sefton et al., Biotechnology and Bioengineering 29, pp. 1135-1143 (1987); Sugamori et al., Trans. Am. Soc. Artf. Intern. Organs 35, pp. 791-799 (1989)).

Another approach to encapsulating cells, "macroencapsulation" involves encapsulating a plurality of cells in a thermoplastic capsule. Typically this is accomplished by loading cells into a hollow fibre and then sealing the extremities. Various types of macrocapsules are known in the art. In particular, Dionne et. al. (WO 92/19195) refers to a macrocapsule having cells dispersed in a matrix and a semipermeable surface jacket, and is incorporated herein by reference. See also Aebischer, U.S. Pat. Nos. 5,158,881 , 5,283,187 and 5,284,761 which refer to a cell capsule formed by co- extruding a polymer solution and a cell suspension.

Typically, when the cells used for encapsulation and implantation are isolated directly from tissue (primary cells), they are disaggregated, washed, and then encapsulated. See, e.g., Aebischer et al., Trans. Am. Soc. Artif. Intern. Organs, 32, pp. 134-7 (1986); Altman et al., Diabetes, 35, pp. 625-33 (1986); Chang et al., U.S. Pat. No. 5,084,350); Darquay and Reach, Diabetologia, 28, pp. 776-80 (1985); Sugamori and Sefton, Trans. Am. Soc.Artif. Intern. Organs, 35, pp. 791-9 (1989).

When immortalized cells or cell lines are to be encapsulated and implanted, they are typically isolated from nutrient-rich cultures. See e.g., Aebischer et al., Biomaterials, 12, pp. 50-55 (1981); Experimental Neurology, 111 , pp. 269-75 (1981) (dopamine- secreting PC12 cells), and Ward et al, WO 93/22427 (IgG-secreting MOPC-31C cells).

Encapsulated cells are usually incubated in vitro and functionally characterized before implantation. Encapsulated cells are often cultured in a defined medium during this pre-im plantation stage. Often the medium is a balanced salt solution lacking nutrient additives (e.g. Aebisher, supra; Altman, supra; Chang et al., supra). Alternatively, encapsulated cells are incubated in a nutrient medium such as RPMI 1640, which contains various amino acids, vitamins, inorganic salts and glucose (2 g/L; 11.11 mM) (Animal Cell Culture, Eds. Pollard and Walker, Humana Press Inc., Clifton, N.J., pp. 696-700 (1990)), and is typically supplemented with 5%-15% fetal calf or horse serum.

Cells that are encapsulated and implanted in a host must undergo at least two severe changes in nutrient conditions as compared to in vitro conditions. The first occurs upon encapsulation.

Compared to in vitro conditions, cells in an encapsulated environment are nutrient depleted. This depletion is manifested in two ways. There is a nutrient gradient between the external environment and capsule interior which naturally forms across the membrane. This gradient is further accentuated because molecules do not diffuse freely . between the outside host tissue and the cells at every position within the capsule. Cells closer to the capsule surface have preferential access to nutrients diffusing across the capsule jacket. In addition, waste products of cells closer to the capsule surface are more readily eliminated.

A second severe change in the concentration of nutrients, e.g., oxygen and glucose, occurs upon implantation in a host. This is because in vitro oxygen and at least some other nutrient levels are generally much higher than occurs in vivo. Thus the driving force for diffusion of these molecules into the capsule is diminished in vivo.

In the embodiments of the present invention directed to release of viral vectors from encapsulated packaging cells in the brain, the capsule preferably is a macroporous capsule allowing the virus vectors to diffuse out of the capsule. Preferably the semipermeable membrane of the device is immunoisolatory.

In other embodiments where differentiated dopaminergic neurons are implanted, the the semipermeable membrane is microporous allowing L-DOPA and/or Dopamine to diffuse from the cells. The molecular weight cutoff of the membrane surrounding the capsule can be adapted to allow the secretion of either low molecular weight compounds (L-DOPA) or high molecular weight compounds (protein factors or virus).

In order to provide support for the cells growing in the capsule the device further comprises a matrix disposed within the semipermeable membrane. In addition the device further may comprise a tether anchor.

Methods and tools for transplantation of encapsulated cells are disclosed in US 5,487,739, US 5,676,943, US 5,908,623, US 5,653,975, US 5,643,286. References:

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EXAMPLES

The following examples are provided to illustrate specific embodiments of the present invention. The examples are included for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1, Cell cultures

Forebrain neurosphere cells derived from two human embryos of 6 and 9.5 weeks gestational age (strains 6wk-HFBr and 9.5wk-HFBr) were kindly provided by Dr. Lars Wahlberg (Karolinska Institute, Sweden), and cultured as floating aggregates in the presence of 20 ng/ml EGF, 20 ng/ml FGF-2 and 1 ng/ml LIF (Carpenter et al 1999). hNS1 (formerly called HNSC.100, a model cell line of hNSCs) is a human embryonic forebrain-derived, multipotent, clonal cell line of neural stem cells. hNS1 cells culture conditions are chemically defined HSC medium supplemented with 20 ng/ml of each EGF and FGF-2 (Villa et al. 2000). HEK293T cells (from ATCC), were used as a control non-neural cell line of human origin, and were cultured at 37°C in complete DMEM, 10% Fetal Bovine Serum (FBS, GIBCO / Life Technologies), 2mM glutamine and 100 units/ml of penicillin and 100 μg/ml streptomycin. HiB5 cells (Fredericksen et al 1988) were cultured as HEK293T cells, but at their permissive temperature of 33°C.

All cultures were maintained at 5% C0₂ and normal, atmospheric oxygen levels (resulting in 20% 0₂). When indicated, some cultures were differentiated at 5% 0₂ and 5% C0₂ in a dual control O₂/CO2 incubator (Forma).

Example 2: Expression Vectors and Transfection Expression vectors used in the present study were derived from plRESIhyg (Clontech).

p(LacZ)IREShyg: LacZ cDNA was excised as a BamHI-Notl insert from pcDNA3.1/Myc-His/lacZ (Invitrogen), and subcloned into BamHI-Notl of plRESIhyg. p(GFP)IREShyg: Renilla raniformis GFP cDNA was excised as a EcoRI-Notl insert from pFB-rhGFP (Stratagene) and subcloned into pCR2.1 (Invitrogene). From this one, a BamHI-Notl fragment was excised and subcloned into plRESIhyg. p(hTH)IRES1hyg was generated by subcloning human Tyrosine Hydroxylase I as a BamHI insert excised from pMLVTH (Lundberg et al 1996) into the BamHI site of plRESIhyg. p(BDNF)IREShyg, p(GDNF)IREShyg and p(Bcl-X_L)IREShyg have been described elsewhere (Rubio et al 1999, Villa et al 2000). p(Bcl-X_L) I REShyg contains the cDNA for Rattus norvegicus BCI-XL. p(SOD1)IREShyg was constructed after subcloning the SODIcit cDNA into the BamHI site of plRESIhyg. Human cytosolic (Cu+Zn) superoxide dismutase (SOD) 1 (GenBank X02317) was cloned by RT-PCR using human total RNA (Clontech) and primers: hSOD1c-sense GCG TGG CCT AGC GAG TTA T, hSODIc-antisense GGG CCT CAG ACT ACA TCC AA. Amplified DNA was cloned into pST1-Blue using the Perfectly Blunt Cloning Kit (Novagen, Madrid, Spain), and sequenced.

Vectors were transfected using Lipofectamine-Plus or Lipofectamine 2000 (Life Technologies), following recommendations of the supplier. When used, drug selection of stable transfectants was carried out at 50-150 mg/ml of hygromycin B (hyg, Calbiochem).

Example 3: RT-PCR

Human GTP-Cyclohydrolase I (hGTP-CH I) and human Aromatic Aminoacid Decarboxylase (hAADC) mRNAs were amplified from cellular total RNA isolated using RNAqueous (AMBION), followed by DNAse I digestion (Boehringer). Primers used were: hAADCse: 5^'-CGG CAT TGG CAG ATA CCA CT-3^', and, hAADCas: 5^'-ATT CCA CCG TGC GAG AAC AG-3^', (430 bp product); hGTP-CH I se: 5^'-ATG CAG TTC TTC ACC AAG GG-3^', and, hGTP-CH I as: 5^'-AAG GCG CTC CTG AAC TTG TA-3^' (251 bp product).

Example 4: Immunocytochemistry (ICC) and immunoblotting (western blot, WB)

At the specified time points, cultures were rinsed with PBS and fixed for 10min in freshly prepared 2% or 4% PFA in 0.1 M phosphate buffer, for b-galactosidase activity development (X-gal stain), or stored in cryoprotective solution until used for ICC. For staining, cultures were rinsed and blocked for 1 hour in 5% normal horse serum. Cultures were next incubated overnight at room temperature with monoclonal antibodies against TH (1 :1000, Sigma) or β-lll-tubulin (1 :1000, Sigma), followed by one hour incubation with biotinylated horse-anti-mouse (1 :200, BA2001 , Vector), and developed by incubation with ABC reagent (Vector) and DAB reaction. Immunofluorescent stains used FITC- (goat-anti-mouse, 1:200, Vector), Texas Red- (horse anti-mouse, 1 :200, Vector), or Cy3- (goat-anti-mouse, 1:100, Jackson ImmunoReserch) conjugated antibodies. Cell nuclei were counterstained with Hoechst 33258 at 0.2 mg/ml. In some cases, double staining for b-galactosidase or TH combined with p53 or BrdU were performed using anti-p53 monoclonal antibody from Santa Cruz (SC-98, 1:1000) or anti-BrdU monoclonal antibody (Sigma, 1 :500). For western analyses, 30 μg of protein from dividing or differentiated cultures were assayed. Samples were electrophoresed and transferred onto nitrocellulose membranes overnight. Primary antibodies were mAb anti-TH (1 :2000, Sigma), rabbit pAb anti- Bcl-X (1 :1000, Transduction Laboratories), mAb anti-Nuπ (Transduction Laboratories, 1 :1000), mAb anti-β-tubulin-lll (1:1000, Sigma), mAb anti-human Neuron Specific Enolase (hNSE, Chemicon, 1 :300), mAb anti-β-actin (1/5000, Sigma), and rabbit pAb anti- β-F1-ATPase (a gift of Prof. JM Cuezva, CBMSO, 1:10000, del Arco et al 2002). The blots were developed using horse-anti-mouse or goat-anti-rabbit antibodies conjugated to peroxidase (HAM-PO, 1:5000, Vector, GAR-PO, 1 : 10000, Nordic Immune) and developed using the ECL system (Amersham).

Example 5: Differentiation of hNSCs into TH expressing neurons hNS1 cells consistently generate a small number of TH+ neurons after differentiation under standard conditions in culture. This capacity to generate TH+ neurons has been exploited in order to study the effects of BCI-XL. Cells are differentiated on poly-L- Lysine (10 μg/ml, Sigma) coated plastic by removal of growth factors (EGF, FGF-2), and in some cases, by the addition of 0.5% heat inactivated fetal bovine serum, for 12 days or the indicated times. Human neurosphere cells were differentiated on poly-L- Lysine + laminin (2 μg/ml laminin, Sigma) coated plastic by removal of growth factors (EGF, bFGF, LIF) and in the presence of a dopaminergic-inductive cocktail (Stull and lacovitti 2001), containing 100 nM phorbol 12-myristate 13-acetate (TPA, Sigma) and 100 ng/ml acidic-FGF (Peprotech Inc.). Neurosphere cells, and some hNS1 cultures, when indicated, were differentiated under low oxygen conditions (5%) in a dual control 0₂/C0₂ incubator.

Example 6: Bcl-X over-expression in hNS1 and neurospheres hNS1 cells were transfected with p(Bcl-X_L)IREShyg and selected for one month with hygromycin (100 μg/ml), to generate a polyclonal BCI-XL over-expressing line. From this line, subclones were isolated by limiting dilution. Human neurospheres were transfected at day 0, and differentiation was started at day 2. Cells were let to differentiate for 5 days under the DA inductive conditions above.

Image and data analyses.

Analyses and photography of fluorescent or immunostained cultures was done in inverted Zeiss Axiovert 135 or Leica DM IRB (equipped with a digital camera Leica DC100) microscopes. In some experiments, digitized images were captured using Leica IM500 software, and analyses performed using NIH Image Software. Statistical tests were run using Prophet Software (NIH). Example 7A: Transgenic hTH over-expression

In a previous study to be reported elsewhere (Liste et al., 2003, to be published as Liste et al 2004 Human Gene Therapy), we have shown that when TH is over- expressed in hNSCs, it induces an upregulation of p53, a decrease in mitotic activity (BrdU incorporation assays), and a profound nuclear damage. As a result, stable and highly expressing TH⁺ hNSC lines could not be generated. These cyto- and geno-toxic effects were not present when LacZ was over-expressed in hNSCs, or when TH was expressed in other human cells (such as HEK293T) or rodent neural stem cell lines.

As an additional confirmation and extension of these data, a colony-forming assay was conducted in the present study. Cells were transfected with bi-cistronic vectors (gene of interest-IRES-hyg^R), selected for one month with 100μg/ml hygromycin, and then fixed and stained to determine the frequency of clones expressing the gene of interest (Figure 1A). As shown in Figure 1B, C, no TH⁺ clones were generated from hNS1 cells. In contrast and as expected, HiB5 and HEK293T cells yielded a high percentage of TH⁺ clones. Furthermore, hNSC cells gave a good yield of LacZ⁺ clones (Figure 1 C, D). Therefore, previous and present data point to a toxicity of TH expression in hNSCs. It is important to note at this point that hNS1 cells express both GTP- Cyclohydrolase I (GTP-CH1, responsible for BH synthesis) and Aromatic Aminoacid Decarboxylase (AADC, required for the conversion of L-DOPA to DA), thus allowing for TH being fully active, and rendering the cells with a full catecholamine synthesis pathway (Haavik and Toska, 1998; Liste et al., 2004).

In summary, the various types of experimental evidence described above are fully consistent with the view that TH expression in hNSCs triggers a cascade of oxidative stress-linked events leading to cell death and growth arrest.

Example 7B: Transgenic hTH over-expression

In a previous study to be reported elsewhere (Martinez-Serrano et al 2003 [submitted], to be published as Liste et al 2004 Human Gene Therapy), and using the same vector for TH expression as here (p(hTH)IREShyg), we have shown that hNS1 cells can not be stably modified for the purpose of generating a DOPA/DA producing cell line. When TH expression levels are high, the modified hNSCs cannot divide in culture. In contrast, human HEK293T and rodent neural progenitors (HiB5 cells, Fredericksen et al 1988), could easily express high levels of TH, using the same vector. In addition, hNS1 cells can stably express a marker gene like LacZ (or other genes like BCI-XL, see below), using the same type of vector and promoter. Those results are suggestive of TH-induced cyto- and geno-toxicity in the hNS1 cell line (Liste et al 2004). To get further insights into the mechanistic basis for this phenomenon, three different experimental approaches were designed (Figure 2), which in turn provided consistent results with the observations and conclusions just described: A) Nuclear integrity in hNS1 cells transfected with TH or LacZ coding constructs (Figure 2A-D). We analyzed this parameter at the time point following transfection when TH+ cell loss is maximal (3 days, as determined in time course experiments, not shown). The percentage of TH+ cells showing a condensed or fragmented nuclei was 26.6±1.8% whereas β-gal+ cells showed nuclear damage in only 6±2.3% of the β-gal+ cells (pθ.001 , one-tail Student t-test). β-gal+ cells were thus significantly healthier than the TH+ ones in this respect. B) p53 upregulation after TH expression in hNS1 cells: Among other functions, p53 causes cell cycle arrest in response to DNA damage, and the data above clearly indicated that TH expression is genotoxic. The percentage of β-gal+ or TH+ cells up- regulating p53 was determined after transient transfection (48 hrs.) of p(LacZ)IREShyg or p(hTH)IREShyg constructs. The cells were stained with X-gal and immunostained for p53, or double immunostained for TH and p53. Fully consistent with nuclear integrity data above, and as shown in figure 2 E, 32.92 ± 7.99% of the TH+ cells were also p53+, in contrast to only 4.22 ± 2.72% of the β-gal+ ones (n=8, p<0.05, two-tailed Student t-test). C) Interference of TH expression with cell division (BrdU incorporation assays): In a similar experiment (transient transfection of LacZ or TH vectors), the cells were assayed for their ability to incorporate BrdU as an index of mitotic activity. In this case, the cells were pulsed with 1 μM BrdU for 8 hours one day after transfection, and the cultures were analyzed one day later. After double staining for BrdU and X-gal or TH, the percentage of mitotic cells was determined and found to be markedly reduced in the TH expressing cells (Figure 2 F, n=4, p<0.05, two-tailed Student t-test).

Several reasons may account for TH-induced toxicity and interference with cell cycle progression (reviewed by Olanow and Tatton 1999, Stokes et al 1999, Barzilai et al 2002, Blum et al 2002), including: i) The cyclo-oxygenase activity of TH (which inherently results in hydrogen peroxide production, Haavik and Toska 1998). ii) The well known oxidative stress caused by DOPA or DA, which may result in oxidative damage of lipids, proteins and DNA. iii) As demonstrated here, TH expression has cyto- and geno-toxic effects, resulting in a decreased mitotic activity or growth arrest. In fact, at ten days post-transfection, TH+ cells were single cells which morphology was either aberrant or looked highly differentiated (Figure 2G). Interestingly, all TH+ cells were always single cells, and did not generate colonies of positive cells. Taking in consideration that the cell cycle length of hNS1 cell is around 40 hrs (Villa et al 2000), the absence of colonies constitutes complementary evidence to the BrdU incorporation assays just described, and reinforce the view that TH expression interferes with cell cycle progression. For TH to be active, though, GTP-CH I (responsible for the synthesis of the cofactor tetrahydrobiopterin, BH4), is needed. AADC is also required for the conversion of L- DOPA to DA. We therefore determined whether hNS1 cells were in fact expressing these partner enzymes. As shown in Figure 2H, hNS1 cells express both human GTP- CH I and AADC, while HEK293T only express GTP-CH I. Interestingly, hNS1 and HEK293T cells yield negative and positive TH sublines, respectively, when transfected with the p(hTH)IREShyg vector and hygromycin selected (Liste et al 2004). The natural expression of these two enzymes in hNSCs completes the dopamine anabolic route, so that TH, once expressed in the cells, may be fully active, allowing for DOPA generation and fueling into the metabolic route for DA biosynthesis

Example 8: Helper genes enhance TH expression ability of established hNSCs

To identify factors that could be neuroprotective in this context, we performed a round of experiments where TH was co-expressed with neuroprotective factors such as BDNF, GDNF, SODIcit or Bcl-X_L (or with an empty vector) (Olanow and Tatton 1999, Blum et al 2002). Cultures were fixed and immunostained ten days following co- transfection. BDNF, GDNF and SODIcit expression resulted in a net increase in the number of TH+ cells (approximately two-fold, Figure 3). These results further confirm TH toxicity, since proteins with well-established neuroprotective actions could enhance TH expression. Overall, the amelioration obtained was of interesting magnitude (a net 100% or 2-fold increase).

BCI-XL, remarkably, induced a much more dramatic increase in the number of TH+ cells. Co-expression of this protein resulted in a net increase of 4-fold (or 400%) in the total number of cells obtained in this experiment (n=12). In additional control experiments, co-transfection of LacZ and BCI-XL constructs resulted in no increase of the number of β-gal+ cells (actually a reduction to 90%) as compared to co- transfection of LacZ and the empty vector (n=12). In this same experimental round, BCI-XL enhanced TH expression by 47-fold (that is, one-two orders of magnitude increase). These results, therefore, rule out unspecific Bcl-X_L effects related to the transfection procedure itself.

Using the human poly-Ubiquitin promoter for TH expression, we have reproduced the BCI-XL -mediated enhancement of TH expression. Overall, BDNF, GDNF and SOD1 co-expression also resulted in doubling of the number of TH+ cells, but BCI-XL co- transfection yielded a net 7-fold increase (Figure 3) compared to empty vector transfected controls. Since the enhancing effect of BCI-XL on TH expression was so striking, we were interested to determine if a pharmacological antiapoptotic block could also have a similar effect. To this end, we carried out parallel experiments using the broad-range, pan-caspase inhibitor z-VAD-fmk (50 μM, or DMSO as a control). Z-VAD-fmk treatment resulted in 24.04±6.55 fold increase in the number of TH+ cells, in comparison to the DMSO control (n=12), consistent with the presence of a caspase- mediated cell death process operating in the present experimental setting [Note that in this experiment there is likely a DMSO-induced toxicity associated to the treatment, which precludes conducting this assay for longer periods of time, and which probably results in an overestimation of the fold-increase in TH⁺ cell survival, in comparison to previously described experiments].

Finally, we were interested in examining the interaction between TH and BCI-XL expression, and high (20%) or low (5%) oxygen tension. The results of this experiment (n=6) indicated that low oxygen per se does not increase the rate of TH+ cells obtained by transfection (96 ± 4.2% of those at high O₂). In addition, BCI-XL effects did not depend on the oxygen tension (fold-enhancement of TH+ cells: 2.63 ± 0.47 and 3.01±0.53, at high and low oxygen, respectively, n=6).

All these results just described consistently show that a large part of cell death caused upon TH over-expression in hNS1 cells courses with an apoptotic mechanism, and more interestingly, that BCI-XL can efficiently counteract this high level of cell death. Oxygen tension had no effect in the above-described experiments, performed using proliferating cells.

Given the notorious effects of BCI-XL upon TH expression observed in co-transfection experiments, the rest of the present study focused on this particular anti-apoptotic protein. We were next interested in generating stable cell lines co-expressing both TH and BCI-XL. In three independent experimental rounds (each one performed in quadriplicate cultures), vigorously growing hygR cell lines were isolated. These cell lines expressed large amounts of BCI-XL, but none of them expressed TH, neither by ICC nor by western analysis (results not shown). These negative data are consistent with the notion that TH, in addition of being toxic to the cells, precludes cell division or induces differentiation, in a manner not overcome by BCI-XL co-expression. As mentioned above, in the transient (10 days) transfection experiments described under the previous subheading, couples or colonies of TH positive cells were never observed, neither after co-transfection of BCI-XL nor under low oxygen conditions. Also, these results fit well with the BrdU incorporation data above, leading to conclude that TH expression is interfering with cell cycle progression, or inducing differentiation. Considering that the cell cycle of hNS1 cells is of about 40 hours (Villa et al., 2000), finding small clones of positive cells was expected for a non-toxic transfected protein.

Example 9: Bcl-X_L action on spontaneous TH+ neuron generation by hNSC lines

5 The results presented above demonstrate that Bcl-X_L can counteract cell death caused by transgenic TH expression. We were therefore interested in determining if a similar neuroprotective action could also operate during the spontaneous differentiation of hNS1 cells. The capacity for TH⁺ neuron generation by hNS1 cells, although of low magnitude (less than 0.1% of total cells, see control cells data in Fig. 4

10 and 6), is nevertheless of high interest, since it indicates that the cells have the potential to differentiate along a dopaminergic pathway. In practical terms, this potential provides a baseline over which to test the putative enhancing effects of BCI- XL on dopaminergic differentiation. It is worth mentioning at this point that both proliferating and differentiated hNS1 cells do naturally express the transcription factor

15 Nurrl (Figs. 4, 5), indicating that the cells should be able to progress correctly down a dopaminergic lineage differentiation pathway. Furthermore, hNS1 cells express GTP- CH1 and AADC (Liste et al., 2004) and are DBH negative (not shown). To summarize, the model hNS1 cell line used here represents a very good experimental model system to test the questions put forward in the present study.

20

Thus, we generated stable BCI-XL over-expressing hNS1 cells to study BCI-XL enhancement of naturally generated TH⁺ neurons (under conditions of high or low oxygen, and presence or absence of FBS). As shown in the western blots of Fig. 4A (upper blot), BCI-XL over-expressing cells (labeled as "b", from BCI-XL) displayed a

25 larger amount of BCI-XL protein when compared to naive cells (labeled as "n", from naive), both under proliferation and after differentiation (verified by hNSE expression, Figure 4B). When TH expression was analyzed, a clear-cut TH signal was only detected in the case of BCI-XL over-expressing cells differentiated in low oxygen and in the presence of 0.5% FBS (Fig. 4A, C), bottom blot). Single cell level analysis by TH

30 ICC of parallel cultures revealed the presence of numerous TH⁺ neurons (which were only found under the low oxygen + 0.5% FBS conditions just mentioned). Quantitative analysis of ICC revealed over one order of magnitude increase in the number of TH⁺ neurons generated (15-fold, Figure 4D, E). To rule out that the observed effects could be due to the imposed resistance to hygromycin or to drug selection, we generated

35_. hyg^R-hNS1 cells (using the empty vector and hyg selection for one month). There were no differences between naive and hyg^R hNS1 cells in terms of TH⁺ neuron generation. Last, in the absence of. FBS, there was no detectable TH expression under any of the tested conditions, neither by WB nor by ICC. In summary, the best conditions for the generation of TH⁺ human neurons from hNS1 cells involve i) a background of increased BCI-XL, ii) differentiation in the presence of serum and iii) low oxygen tension.

To get further insights into BCI-XL effects on TH⁺ neuron generation, the time course of TH expression was studied in cultures differentiating in low oxygen + 0.5% FBS. As shown in Fig. 4C, control cells (both naive and hyg^R) did not express TH at any time point, whereas BCI-XL over-expressing cultures did, starting at day.8. These results do not support the view that BCI-XL effects could be due to enhanced survival of TH⁺ cells generated in the first few days of differentiation, which would have subsequently died in its absence. Rather, present data are suggestive of either a slow inductive effect, or the provision of a permissive cellular environment for naturally occurring TH⁺ neurons (generated over a delayed temporal window) to cope with their phenotype and survive.

The experiments reported above using the BCI-XL over-expressing polyclonal cell line let us discover means to enhance the yield of TH⁺ neurons from hNSCs. To further confirm and investigate the nature of this phenomenon on a more powerful experimental substrate, we isolated subclones from the BCI-XL over-expressing polyclonal cell line, showing various Bcl-X levels (illustrated in Figs. 5A, B, and 5A). Importantly, BCI-XL overexpression persists after differentiation. When the expression of different markers was analyzed in these subclones, it was found that β-lll-tubulin expression paralleled that of BCI-XL (Fig. 5). This was obvious from both WB inspection (Fig. 5A) and from ICC determinations (Figs. 5C and 4D). β-lll-tubulin ICC quantification after differentiation revealed a net 3 to 4-fold increase in the number of neurons generated by the BCI-XL clones when compared to the naive and hyg^R cell lines (p<0.01, Tukey test, all three BCI-XL clones vs. both control cell lines; Figs. 5C and D). The total number of neurons generated in the best case (clone #5) represented 20.2% of the total number of cells plated. The increase in total neuron production was very similar in all three clones studied, regardless of Bcl-X_L expression levels (Fig. 5C).

Working with these clones, we also found that TH expression levels and TH⁺ neuron generation after differentiation correlated with BCI-XL expression levels, in a dose response manner (Fig. 6). [Note that Nurrl expression levels were unaffected by Bcl- X overexpression or differentiation (Fig. 5A)]. Comparison of BCI-XL and TH expression levels showed a BCI-XL expression threshold, over which TH neuron generation occurred at high rates (data in Figs. 5B and 6A, F, G). TH expression by differentiated neurons, is illustrated in Fig. 6. TH expressing cells also express neuronal markers (β-lll-tub, Fig. 6C), and stain positive for the neurotransmitter of interest, dopamine (Fig. 6D, E), as expected from their gene expression profile (Nurrl , GTP-CH1 , AADC positive and DBH negative). TH⁺ neuron generation rate (Fig. 6F) by these clones accounted for up to 2.18% of the total cells plated (BCI-XL clone#5), in fact greatly exceeding the rate of TH⁺ neuron generation by serially passaged human VM cultures (approx. 0.3%, Storch et al., 2001). The net increase in TH⁺ neuron generation reached almost two orders of magnitude (91-fold, control hNS1 vs. BCI-XL clone#5). These TH⁺ cells (which were also β-lll-tub⁺ and DA+ (Figs. 6C, D)), stained negative for p53 (not shown), suggesting the absence of neuronal damage.

Example 10: BCI-XL effects on TH+ neuron generation by epigenetically expanded human neurospheres

To further extend and generalize our observations on BCI-XL effects, we were interested in determining if the same neuroprotective/inductive effects could also enhance the dopaminergic differentiation capacity of non-established, serially passaged, human neurosphere cell strains. To this end, the cells (6wkHFBr pass#6 and 9.5wkHFBr pass#18) were attached to a p-lys coated surface, and transfected with GFP or BCI-XL vectors 48h later (or mock transfected). Following transfection, we differentiated the cells in low oxygen tension for 5 days under the influence of a minimal dopaminergic inductive cocktail (TPA and aFGF). Efficiency of transfection was 12-15% (Figure 7), as determined by GFP fluorescence at 48hrs following transfection (when differentiation started). These neurosphere strains, similarly to the hNS1 cell lines, do also express the transcription factor Nurr-1 (detectable by WB, not shown). As shown in Figure 7A-B, BCI-XL greatly enhanced the number of TH+ differentiated neurons (5.5-7-fold, 9.5wk- or 6wk-HFBr cells, respectively). TH+ neuron generation increased from a baseline (mock- or GFP-transfected cells) of 1-1.5% up to 7-8.2% of the total number of cells. The statistical significance of the results, after comparing the BCI-XL transfected cells to the other two groups was equally significant for both neurosphere cell strains (p<0.01 , Tukey test, n=5). As highlighted under previous subheadings, it is also noteworthy that a TH+ neuron generation rate of 7-8% of the total number of cells in a given culture greatly exceeds the capacity of human ventral mesencephalic cultures for generation of dopaminergic neurons (0.3%, Storch et al 2001).

Example 11: In vivo experiments Adult female (250-300 g) Wistar rats, housed and treated according to the guidelines of the European Community (86/609/EEC), were anaesthetized with a mix of Ketamine and Dontor, and 6-OHDA lesioned in the right median forebrain bundle (MFB) at the following coordinates (in mm): AP= -3.7; ML= - 1.6; DV (skull)= -8.8; with TB set at - 3.3. Fourteen days later the rats were grafted into the denervated striatum with cellular suspensions of naive hNS1 cells (as a control, n=6) or Bcl-X - clone 5 (n=6), at coordinates: AP = +1 ; L= -3; DV (from dura) = -4.5, with the incisor bar set at -2.3. The cells were BrdU labeled in vitro, prior to grafting (1 μM for 3 days), and a total of 400.000 cells were implanted as a single deposit (cell density of 150-200.000 cells/μl). The animals were immunosuppresed with cyclosporine A (Neoral; Novartis, 100μg/ml in drinking water, starting 48 h before grafting). Four weeks after grafting, rats were intracardially perfused with freshly-prepared, buffered 4% paraformaldehyde. Brains were postfixed for 12 h, dehydrated in 30% sucrose, and sectioned (30 μm, freezing cryotome) for free floating immunohistochemistry analyses. Serial sections were processed for TH using mouse monoclonal (Sigma, clone TH-2, 1:2000) or rabbit polyclonal anti-TH antibodies (Chemicon, AB152, 1 :1000). Monoclonal anti-human nuclei (Chemicon, MAB 1281 , 1 :500) or anti-BrdU (Sigma, clone BU33, 1 :1000) were used to detect all grafted and surviving human cells. Migrating neuroblasts were detected using anti-doublecortin antibodies (Dcx, C-18, Santa Cruz Biotechnology, Inc., 1:1000), and human neurons were specifically stained using an anti-human Neuron Specific Enolase antibody (Chemicon, MAB324, 1 :2000). For the unambiguous detection of human TH expressing neurons, double immunohistochemistry was performed combining TH antibodies with those for h-nuclei or hNSE. Secondary antibodies were biotinylated horse anti mouse (1 :200, BA2001 , Vector) (followed by ABC (Vector) and Ni-DAB reaction). For immunofluorescence (IF), secondary antibodies were Texas Red-( horse anti mouse, 1 :100, Vector) and Alexa 488- (goat anti rabbit, 1 :400; Molecular Probes Inc.).

Histological analyses Immunostained sections were viewed and analyzed on a Leica DM IRB inverted fluorescence microscope equipped with an X-Y-Z motorized stage operated by Olympus CAST-GRID software for stereological, unbiased morphometric analyses. Graft survival (on the basis of detection of h-nuclei stained cells) was analyzed by two methods: i) antero-posterior extent of grafted cells presence in coronal sections, and ii) actual graft volume measurement using stereological methods (Cavalieri estimator).

TH⁺ neurons were all counted in every graft-containing section in every animal, and total number of TH⁺ neuron was estimated on the basis of sectioning protocol. In animals grafted with naive hNS1 cells, no TH⁺ neurons were detected. For double IF analyses, sections were analyzed on a Microradiance Confocal microscope (Bio-Rad, Hercules, CA) in the Z-axis for unambiguous assignment of stained human nuclei to a TH⁺ cytoplasm. Overlapped and single Z-sections are shown in Figure 10F. In other cases, co-localization of cytoplasmic stains was performed on one-micrometer thick confocal sections (Figure 10G). Results: Bcl-X over-expressing hNSCs generate TH⁺ neurons after grafting to the adult striatum

One month following cell implantation into complete 6-OH-DA lesioned animals (mfb lesion), 3 out of 5 rats receiving control hNS1 cells, or 4 out of 6 animals receiving Bcl- XL hNS1 cells (clone #5) had surviving grafts of human cells, as determined by the presence of h-nuclei stained cells (Fig. 10A, see also other markers below). BrdU stainings of the same grafted animals provided the same information on cell survival and integration as h-nuclei data (Fig. 10B). No signs of tumor formation were observed in any transplanted animal, according to previous studies (Rubio et al., 2000; Villa et al., 2002).

hNSCs integration and survival were assessed on h-nuclei stained sections (Fig. 10A). Cell migration from the transplant core into the host parenchyma, as detected by h- nuclei immunoreactivity, reached 86±4 or 283±24 μm (control or BCI-XL cells, respectively), surrounding the implantation site (medio-lateral extent), being thus more notorious for BCI-XL cells than for control cells (Fig. 10A). Grafted cells were found to preferentially migrate through striatal gray matter, rather than invading white matter tracts (Fig. 10A, high magnification panels, and Fig. 10B).

Overall, graft survival was clearly enhanced in the case of BCI-XL cells. Antero- posterior (AP) extension of the grafts was largely enhanced for transplants of BCI-XL cells, which spanned 1.12 ±0.043 mm rostrocaudally, as compared to control cells (0.5±0.1 mm)(p<0.05, Mann-Whitney test). Total graft volume, determined following stereological procedures, was also increased from 0.02±0.01 to 0.52+0.04 mm³ (for control or BCI-XL cell implants, respectively; p<0.01, T-test). Total counts of h-nuclei⁺ cells could not be reliably determined due to the heavy, dense cellular packing at the core of the transplants (Fig. 10A).

hNSCs generate very few neurons in vivo when implanted at non-neurogenic regions, such as the striatum, as expected from a neural stem cell (Rubio et al., 2000; Martinez-Serrano et al., 2001.; Villa et al., 2002). In agreement with this background, grafted control hNS1 generated few Dcx⁺ or hNSE⁺ neurons in the present experiment. In contrast, Bcl-XL cell implants seem to be richer in Dcx⁺ and hNSE⁺ cells (Figs. 10C, D). These findings are consistent with the in vitro differentiated cells data previously described here (Fig. 5).

In vivo TH⁺ neuron generation capacity was quantified in all stained sections for every surviving graft. Whereas no TH⁺ neurons were found in any section from animals grafted with naive cells, an average of 170±16 cells/graft were quantified in BCI-XL- cells transplanted animals (Fig. 10E). The magnitude of this difference (which essentially represents an all-or-none type of situation) resembles the previously described data obtained in the in vitro experiments (Figs. 4, 6). In addition, these data further help to establish proof-of-principle of the prominent role of BCI-XL in human TH⁺ neuron generation from hNSCs, in the present case in vivo. TH⁺ neuron morphology is shown at high magnification in Figs. 10 F, G. TH⁺ neurons have been unambiguously identified as of human origin after examination of h-nuclei + TH double IF sections (illustrated in Fig. 10F). Furthermore, the human + neuronal nature of the TH⁺ cells has been established on the basis of TH/hNSE double stains (Fig. 10G).

Example 12. Human neural stem cells from forebrain (hNS1 cells) Measurements of intracellular DA content in differentiated naϊve and Bcl-X_L-over- expressing hNS1 cells.

DA/DOPAC determinations (Figure 11).

Sample preparation was done according to methods provided by Pia Wiekop (NeuroSearch A/S). Differentiation of the cells was carried out either by the removal of mitogens, or removal of mitogens and incubation with a minimal DA-inductive cocktail (Bradford). Sample analyses were performed according to her in-house methods.

Naϊve hNS1 cells or Bcl-XL-over-expressing hNS1 (clone 5) cells were differentiated for 12 days at low oxygen tension, by two different protocols. The first consisted in mitogen removal and a 12-day incubation in 0.5% fetal bovine serum (FBS) supplemented medium (standard conditions). In the second one, the cells were pre- differentiated for 5 days with 0.5% FBS medium. After that, this medium was replaced by another one containing BDNF, 50 ng/ml; DA, 10 μM; and Forskolin, 10μM (Riaz et al., 2002), for the rest of the differentiation time (7-days).

By day 12, some cells were incubated for 2 hours with BH₄ (1 mM tetrahydrobiopterin). BH₄ is an essential cofactor for aromatic amino acid hydroxylases, such as tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. BH₄ is mostly generated by GTP-cyclohydrolase I (the rate-limiting enzyme) (Zuddas et al., 2002). The samples were then lysed in 200 μl of 0.1 N percloric acid containing 0.8 mg/ml reduced glutahtione. The supernatants were collected, filtered (for analyses of intracellular DA levels) and frozen at -80°C until analysed by HPLC coupled to electrochemical detection (Lotharius et al., 2002) (kindly analyzed by Pia Wiekop at NeuroSearch A/S). Figure 11 shows the intracellular DA levels obtained by HPLC, after differentiation of control or BCI-XL overexpressing hNS1 cells (don 5). Non detectable DA levels (ND, under technical detection limit) were obseved in standard differentiation conditions. (0.5% FBS, or 0.5% FBS + BDNF, DA and Forskolin= Bradford cocktail), neither for Controls or BCI-XL overexpressing cells. However, after addition of BH₄, the DA content remarkably increased. This increase was clearly higher in the Bcl-X_L-over- expressing cells than in controls (aprox. 500 fold, over the basal level observed without BH₄).

Our results suggest that hNS1 cells (which express GTP-cyclohydrolase I) have the potential to produce dopamine, but for that, they need the exogenous addition of BH₄ as a cofactor. Most likely, endogenous GTP-cyclohydrolase I expression levels are too low as to produce enough co-factor to play its physiological role. Most importantly, the potential for DA synthesis is higher in the BCI-XL overexpressing cells after BH4 addition than in control cells, a finding consistent with the higher TH levels previously observed in these cells after differentiation.

Example 13: Human ventral mesencephalic cell lines (hVM)

METHODS hVM cell line generation/expansion and differentiation.

Human mesencephalic cells (hVM) were obtained from 10-week-old human embryonic ventral mesencephalic tissue (Lund University). Initially, different culture and subculture media (epigenetic conditions) were tested (regular neurosphere medium, HSC and ES cells media), in parallel to different immortalising retroviral vectors (coding for c-myc, ER/c-myc and v-myc). Infection efficiency was determined using retroviral vectors encoding GFP, and resulted to be in the range of 10%.

As a result of these trials, a single cell line was obtained, showing properties of truly being a proliferating cell line. These cells were established using both epigenetic mitogens in the form of EGF and bFGF, and immortalized using a retroviral vector coding for v-myc (TD1-2 virus: LTR-vmyc-SV40 promoter-neo-LTR).

These cells were propagated in HSC medium containing 20 ng/ml of EGF/bFGF, under low oxygen tension (5%) and 5% CO₂. For differentiation experiments, the cells were plated, and next day, proliferation medium was replaced with the same medium containing: brain derived neurotrophic factor (BDNF) 50 ng/ml, dopamine (DA) 10 μM and forskolin 10 μM, (Riaz et al., 2002), heretofore called differentiation medium. The cells were differentiated for 7 days, and 2/3rds of the medium were replaced every second day.

Retroviral infection and FACS selection Cells from the human polyclonal VM cell line were infected with retroviral vectors (supplied by NS-Gene) coding for: LTR-empty-IRES-rhGFP-LTR (as a control), or LTR-Bcl-XL-IRES-rhGFP-LTR. For infections, retroviral particles were added (after removal of regular proliferation medium) in DMEM/F-12 + 5% fetal bovine serum, for 4 hours. After that, the infection medium was removed and fresh HSC medium was replaced. The cells were allowed to proliferate for two more passages, and trypsinised for cell sorting selection (of fluorescent cells) by FACS. The fluorescent selected cells were plated and proliferated for under standard conditions.

Immunocytochemistry (ICC) and Immunoblotting (WB) At the specified time points, cultures were rinsed with PBS and fixed for 10 min in freshly prepared 4% PFA in 0.1 M phosphate buffer. For staining, cultures were rinsed and blocked for one hour in 10% normal horse serum. Cultures were next incubated overnight at room temperature with monoclonal antibodies against TH (1 :2000, Sigma, clone TH-2), β-lll-tubulin (1 :5000, Sigma), GFAP (1 :2000, Sigma) or MAP-2 (1 :5000, Chemicon), followed by one hour incubation with biotinylated horse anti-mouse (1 :200, BA2001 , Vector), and developed by incubation with ABC reagent (Vector) and DAB reaction. Double immunofluorescence stainings for β-lll-tubulin and TH were performed using anti-TH rabbit pAb from Chemicon (1 :1000), and β-lll-tubulin as indicated above. Secondary antibodies were Alexa 488 (goat anti rabbit, 1:400; Molecular Probes), or Texas Red- (horse anti mouse, 1 :200, Vector).

For western analyses, 30 μg of protein obtained from dividing or differentiated cultures were assayed. Samples were electrophoresed and transferred onto nitrocellulose membranes overnight. Primary antibodies were mAb anti-TH (1 :2000, Sigma), rabbit pAb anti Bcl-X_L (1 :1000, Transduction Laboratories), mAb anti Nurrl (1 :1000, Transduction Laboratories), mAb anti β-lll-tubulin (1 :1000, Sigma), mAb anti β-actin (1 :5000, Sigma), pAb anti AHD-2 (1 :1000, generous gift from Prof. J. Lipsky at Mayo Institute for Medical Education and Research, Rochester, MN), rat mAb anti DAT (1 :1000, Chemicon). The blots were developed using horse-anti-mouse or goat-anti- rabbit antibodies conjugated to peroxidase (HAM-PO, 1:5000, Vector, GAR-PO, 1 :10000, Nordic Immune) and developed using the ECL system (Amersham). Image analyses

Analyses and photography of fluorescent or immunostained cultures was done in inverted Zeiss Axiovert 135 or Leica DM IRB (equipped with a digital camera Leica DC100) microscopes. In some experiments, digitized images were captured using Leica IM500 software, and analyses performed using NIH Image software. Statistical tests were run using Prophet Software (NIH).

RESULTS

A single, polyclonal cell line was obtained from human ventral mesencephalic tissue, showing properties of truly being a proliferating cell line. These cells were established using both epigenetic mitogens in the form of EGF and bFGF, and immortalized using a retroviral vector coding for v-myc.

Initially, (at passage 6-8), its capacity for TH neuron generation after inductive differentiation was acceptable (in the range of 15% of total cells in the culture, Fig. 12). This cell line also showed expression of neuronal and true VM DA neuron markers like: β-lll-tubulin, TH, Nurrl , DAT and aldehyde dehydrogenase 2 (AHD-2), as detected by Western blotting (Figure 12A).

Neurogenic and TH⁺-neuron generation capacity was further explored by immunocytochemistry. These assays showed large numbers of β-lll-tubulin⁺, and TH⁺ neurons following differentiation. All TH⁺ cells were also β-lll-tubulin⁺, confirming their neuronal nature. Abundant cells immunoreactive for Map-2 and GFAP were also detected at these earlier passages (Figure 12B).

However, the properties of these cells gradually changed with time in culture, and the capacity for human TH neuron generation declined to less than 1 % by passage 30. Also, the proliferation capacity of the cell line declined with the time, in the end yielding cultures showing a high degree of on-going cell death (Figure 13).

In the light of these evolving properties, and since the initial cell line was polyclonal, we chose to get back to the first banked passages (number 3) to isolate clones from the heterogeneous population. We isolated around 70 clones, which were all analysed for their TH neurogenic potential. Eight of these clones were selected at passage 4 on the basis of their TH neuron generation capacity (ranging from 0 to close to 30%, Figure 14), and also considering their growth properties and the morphology of the obtained neurons (Figure 14). Unexpectedly, their capacity for TH⁺ neuron generation also declined with time (passage number) in a way similar to the polyclonal cell line, indicating that this decline was not due to the presence of clones unable to make TH⁺ neurons taking over the cultures. Rather, it seems that this decline is occurring on a cell intrinsic basis, or alternatively, that it is the result of inappropriate, suboptimal, culture conditions. It is worth to comment at this point that all these studies were performed culturing the cells in a low oxygen incubator. In terms of proliferation capacity, all clones showed a progressively impaired survival rate upon passaging, until reaching the point when, by passage 30, their continued proliferation has become rather difficult.

However, recently, we have discovered that the genetic modification of human neural stem cells (hNSCs) for BCI-XL expression changes this scenario. BCI-XL has the capacity to maintain (along the time in culture) the dopaminergic potential of hVM polyclonal and clonal cultures. We have found that Bcl-X_L has prominent effects enhancing the capacity of human VM cell lines for healthy proliferation in culture (results not shown, but see morphology of cultures in Figure 15). Possibly, as a consequence of this (but it could also be due to other operating mechanisms), BCI-XL expression has a major impact on their capacity for differentiation into dopaminergic human ventral mesencephalic neurons.

Our initial results indicate that early passage polyclonal or clonal human VM cell lines (v-myc immortalized), once infected with retroviral vectors coding for BCI-XL+GFP or only for GFP (see experimental design in Figure 15A), showed that the capacity for differentiation towards generation of human DA neurons is preserved in both polyclonal lines and clones (Figure 16). Our results show that these cells recover their capacity for TH, DAT and β-lll-tubulin expression after differentiation, as opposite to control, GFP-transfected cells (observed by ICC and western blot analyses of differentiated cells, Figure 16).

Interestingly, a similar activity has been detected in cells at high passage number, but in a "recover" mode. That is, cells that had already lost their capacity for DA human neuron generation, can, in two passages, recover their capacity upon expression of BCI-XL.

At present, we are analyzing at molecular and cellular levels, which are the key BCI-XL effects controlling proliferation, cell death and differentiation in these modified cells. Also, transplantation experiments of human VM cells (polyclonal and clonal), modified for the expression of BCI-XL are currently underway.

From these evidence, obtained using multiple and independent model systems, both in vitro and in vivo, we conclude that enhancing BCI-XL expression is important for the generation of a continuous source of human TH/DA neurons from hNSCs (either of forebrain or midbrain origin). These findings may facilitate the development of drug screening and cell replacement activities, in order to discover new therapeutics strategies for Parkinson^'s Disease.

Claims

1. A method for enhancing the survival of neurons and/or of cells expressing tyrosine hydroxylase (EC 1.14.16.2) (TH⁺), said method comprising contacting a population of cells with BCI-XL or a functional equivalent thereof wherein said population of cells is selected from the group consisting of: i. neurons or cells capable of differentiating into neurons; and ii. TH expressing cells or cells capable of differentiating into TH expressing cells.

2. The method of claim 1 , comprising i. providing a population of cells capable of expressing tyrosine hydroxylase; ii. contacting said population of cells with BCI-XL or a functional equivalent thereof; and iii. providing conditions for expression of tyrosine hydroxylase.

3. The method of claim 1 , wherein the BCI-XL is provided to the population of cells via a growth medium.

4. The method of claim 3, wherein the amount of BCI-XL is at least 0.01 ng/mL, such as at least 0.1 ng/mL, for example at least 1 ng/mL, such as at least 5 ng/mL, for example at least 10 ng/mL, such as at least 20 ng/mL, for example at least 50 ng/mL, such as at least 100 ng/mL, for example at least 500 ng/mL, such as at least 1000 ng/mL.

5. The method of claim 3, wherein Bcl-X_L is fused to a membrane translocation signal sequence capable of directing the said protein to the nucleus of the cells.

6. The method of claim 5, wherein the fusion protein further comprises a nuclear localisation signal and optionally an affinity tag, such as a polyHis tag.

7. The method according to claim 1, wherein the BCI-XL is provided by transfecting/transducing the cells with a vector capable of directing the expression of BCI-XL in the cells.

8. The method of claim 7, wherein the heterologous expression of BCI-XL is at least two times the background expression.

9. The method of claim 7, wherein the expression is controlled by an inducible promoter, such as the TET promoter, the Mx1 promoter, Ecdisone responsive promoter, promoters sensing oxygen level related signals, the EF-1aplha promoter.

10. The method of claim 7, wherein the expression of BCI-XL is controlled by a constitutive promoter, such as the CMV promoter, the human UbiC promoter, the JeT promoter, the RSV promoter, the promoter for Brain Creatine Kinase, the promoter for RNA polymerase2 subunit A.

11. The method of claim 7, wherein the vector is a viral vector.

12. The method of claim 7, wherein the vector is an adenovirus.

13. The method of claim 7, wherein the vector is stably integrated into the genome of the host cell.

14. The method of claim 13, wherein the vector is a retrovirus, a lentivirus, or an adeno-associated virus.

15. The method of claim 14, wherein the lentivirus comprises 5' and/or 3' LTRs from a virus selected from the group consisting of HIV, HIV-1 , HIV-2, FIV, and SIV.

16. The method of claim 7, wherein the vector comprises sequences allowing selective excision of at least part of the vector, such as Cre-loxP excision.

17. The method of claim 7, wherein the vector is a plasmid.

18. The method according to any of the preceding claims 7 to 17, wherein the vector carries at least one selectable marker.

19. The method according to any of the preceding claims, wherein the Bcl-X is human BCI-XL (SEQ ID No. 1) or a functional equivalent therof.

20. The method according to any of the preceding claims, wherein the BCI-XL is Rattus norvegicus BCI-XL (SEQ ID No. 8) or a functional equivalent thereof.

21. The method according to any of the preceding claims, wherein the Bcl-X is Mus musculus BCI-XL (SEQ ID No. 9) or a functional equivalent thereof.

22. The method according to any of the preceding claims, wherein the BCI-XL is Sus scrofa BCI-XL (SEQ ID No. 10) or a functional equivalent thereof.

23. The method according to any of the preceding claims, wherein a functional equivalent of BCI-XL comprises all the conserved residues (*) of Table I, preferably wherein said functional equivalent at the positions identified as strongly conserved (:) in Table I also comprises residues identified in Table I as strongly conserved, preferably wherein said functional equivalent at the positions identified as weakly conserved (.) in Table I also comprises residues identified in Table I as weakly conserved.

24. The method according to any of the preceding claims, wherein the BCI-XL is essentially non-immunogenic in the species from which the cells originate.

25. The method according to any of the preceding claims, wherein the cells expressing or being capable of expressing tyrosine hydroxylase originate from isolated embryonic stem cells.

26. The method according to any of the preceding claims, wherein the cells expressing or being capable of expressing tyrosine hydroxylase are immortalised, such as being immortalised with a telomerase gene, a myc gene, a v-myc gene, a c-myc gene, a SV-40T gene.

27. The method according to any of the preceding claims, wherein the cells are neural stem cells (hNSC lines).

28. The method of claim 27, wherein the cells are isolated human forebrain neural stem cells.

29. The method of claim 27, wherein the cells are isolated embryonic ventral mesencephalon cells or ventral mesencephalon progenitor cells.

30. The method of claim 27, wherein the cells originate from growth factor expanded cultures, such as human neurosphere cultures.

31. The method according to any of the preceding claims, wherein the cells are neural progenitor ceils.

32. The method according to claim 1 , wherein the cells expressing TH are chromaffin cells.

33. The method according to claim 1 , wherein the TH expressing cells are retinal pigment epithelial cells.

34. The method of claim 1, wherein the cells are rodent, porcine, canine, or simian.

35. The method of claim 1 , wherein the cells are human.

36. The method according to any of the preceding claims, further comprising providing conditions for expression of tyrosine hydroxylase.

37. The method of claim 36, wherein providing conditions for expression of tyrosine hydroxylase comprises induction of dopaminergic differentiation.

38. The method according to any of the preceding claims, wherein the cells comprise a heterologous expression construct comprising a promoter controlling the expression of tyrosine hydroxylase or a functional equivalent thereof.

39. The method according to any of the preceding claims, wherein tyrosine hydroxylase expression is induced by treating cells with one or more of FCS, lnterleukin-1 , LIF, GDNF, BDNF, forskolin, dopamine, protein Kinase A or protein Kinase C aktivators, and aFGF.

40. The method according to any of the preceding claims, wherein tyrosine hydroxylase expression is induced by at low oxygen tension, such as below atmospheric oxygen level.

41. The method according to claim 40, wherein low oxygen tension is below 10%, more preferably below 8%, more preferably below 6%, more preferably below

5%, such as below 4%, for example below 3%, such as below 2%, for example below 1 %.

42. The method, according to any of the preceding claims, wherein dopaminergic differentiation is induced in the presence of serum, by using conditioned medium, by modulating the cellular levels of c-AMP and/or by modulating cellular levels of activated PKC levels.

43. The method according to any of the preceding claims 36 to 42, wherein the percentage of dopaminergic neurons in the resulting composition of neurons is at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.

44. A composition of cells obtainable by the method of any of the preceding claims.

45. A composition of isolated mammalian cells overexpressing BCI-XL.

46. The composition of claim 45, comprising cells capable of differentiating into neurons.

47. The composition of claim 46, wherein the neurons are TH⁺, preferably wherein said TH⁺ phenotype is stable in vitro and preferably in vivo.

48. The composition of claim 47, wherein said TH⁺ neurons are capable of producing dopamine in vitro, and preferably in vivo.

49. The composition of claim 45, comprising neural progenitor cells.

50. The composition of claim 45, comprising neural stem cells.

51. The composition of claim 50, wherein the cells are immortalised, such as being immortalised with a telomerase gene, a myc gene, a v-myc gene, a c-myc gene, or with SV-40T.

52. The composition of claim 45, comprising cells originating from growth factor expanded cultures, such as neurospheres.

53. The composition of claim 45, comprising neurons, preferably wherein at least 5% of the cells are neurons, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%.

54. The composition of claim 45, comprising TH expressing cells, preferably wherein at least 5% of the cells are TH+, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 25%.

55. The composition of claim 45, comprising forebrain derived progenitor cells.

56. The composition of claim 45, comprising mesencephalic derived progenitor cells.

57. The composition of claim 45, comprising embryonal stem cells and/or embryonal stem cells derived progenitor cells.

58. The composition of claim 45, comprising retinal epithelial cells, or cells derived therefrom.

59. The composition of claim 58, comprising ARPE-19 cells or cells derived therefrom.

60. The composition of claim 45, comprising cells capable of producing dopamine or capable of differentiating into dopamine producing cells.

61. The composition of any of the preceding claims 45 to 60, comprising non- human primate cells.

62. The composition of any of the preceding claims 45 to 60, comprising rodent cells.

63. The composition of any of the preceding claims 45 to 60, comprising human cells.

64. The composition of any of the preceding claims 45 to 60, comprising pig cells.

65. The composition of claim 64, wherein the cells have been manipulated to be less immunogenic to humans.

66. The composition of claim 45, wherein the cells overexpressing BCI-XL contain at least two times as much Bcl-X_L as corresponding cells not overexpressing BCI- XL-

67. A neural progenitor cell comprising a first heterologous expression construct comprising a first promoter capable of directing the expression of tyrosine hydroxylase or a functional equivalent thereof and a second heterologous expression construct comprising a second promoter capable of directing the expression of BCI-XL or a functional equivalent thereof.

68. A differentiated dopaminergic neuron comprising a first heterologous expression construct comprising a first promoter capable of directing the expression of tyrosine hydroxylase or a functional equivalent thereof and a second heterologous expression construct comprising a second promoter capable of directing the expression of BCI-XL or a functional equivalent thereof.

69. An implantable cell culture device, the device comprising: i) a semipermeable membrane permitting the diffusion of a biologically active protein therethrough; and ii) a composition of cells according to any of the preceding claims 44 to

66 or a composition comprising at least one cell according to any of the preceding claims 67 or 68.

70. The device of claim 69, wherein the semipermeable membrane is immunoisolatory.

71. The device of claim 69, wherein the semipermeable membrane is microporous.

72. The device of claim 69, wherein the biologically acitve protein is a growth factor.

73. The device of claim 69, wherein the device further comprises a matrix disposed within the semipermeable membrane.

74. The device of claim 69, wherein the device comprises ARPE-19 cells.

75. The device of claim 69, wherein the device further comprises a tether anchor.

76. A lentiviral vector particle, said vector particle being produced based on a lentiviral transfer vector comprising a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR.

77. A method enhancing the survival of TH+ cells in vivo, said method comprising administering to substantia nigra in an individual in need thereof a therapeutically effective amount of a lentiviral vector particle, said vector particle being produced based on a lentiviral transfer vector comprising a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR.

78. A retroviral vector particle, said vector particle being produced based on a retroviral transfer vector comprising a 5' retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR.

79. A method of enhancing the survival of in vivo differentiated dopaminergic neurons, said method comprising administering to the striatum of an individual in need thereof a therapeutically effective amount of a retroviral vector particle, said vector particle being produced based on a retroviral transfer vector comprising a 5' retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR.

80. The method of claim 79, wherein said vector particle is administered by administering to said individual a composition of packaging cells capable of producing said vector particle.

81. The method of claim 80, wherein said composition of packaging cells are administered in an implantable cell culture device, the device comprising a semipermeable membrane permitting the diffusion of a growth factor therethrough and said composition of packaging cells.

82. A packaging cell line capable of producing an infective vector particle, said vector particle comprising a retrovirally derived genome comprising a 5' retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' retroviral LTR.

83. A packaging cell line capable of producing an infective vector particle, said vector particle comprising a lentivirally derived genome comprising a 5' lentiviral

LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding BCI-XL or a functional equivalent thereof, an origin of second strand DNA synthesis and a 3' lentiviral LTR.

84. Use of the cells according to any of the claims 44 to 68 for transplantation.

85. Use of the cells according to any of claims 44 to 68 for drug screening.

86. Use of the cells according to any of claims 44 to 68 for gene profiling.

87. Use of the cells according to any of the claims 44 to 68 for the preparation of a medicament for the treatment of a CNS disorder.

88. The use of claim 83, wherein the CNS disorder is a neurodegenerative disease.

89. The use of claim 88, wherein the neurodegenerative disease is a neurodegenerative disease involving lessioned and traumatic neurons, such as traumatic lessions of peripheral nerves, the medulla, the spinal chord, cerebral ischaemic neuronal damage, neuropahty, peripheral neuropathy, Alzheimer's disease, Huntingdon's disease, Parkinson's disease, amyotrophic lateral sclerosis, memory impairment connected to dementia.

90. The use of claim 88, wherein the neurodegenerative disease is Parkinson's Disease.

91. A method of treatment of a neurological disorder comprising adminstering to a subject in need thereof a therapeutically effective amount of a composition of cells overexpressing BCI-XL said cells further being capable of differentiating into neurons and/or TH expressing cells.

92. The method of claim 91 , wherein the neurological disorder is a neurodegenerative disease involving lessioned and traumatic neurons, such as traumatic lessions of peripheral nerves, the medulla, the spinal chord, cerebral ischaemic neuronal damage, neuropahty, peripheral neuropathy, Alzheimer's disease, Huntingdon's disease, Parkinson's disease, amyotrophic lateral sclerosis, memory impairment connected to dementia.

93. The method of claim 91 , wherein the composition of cells are capable of 5 differentiating in vivo to TH⁺ neurons.

94. The method of claim 93, wherein said TH⁺ neurons are capable of maintaining a TH⁺ phenotype.

10 95. The method of claim 93, wherein said TH⁺ neurons are capable producing dopamine.

96. The method of claim 91 , wherein the neudegenerative disorder is Parkinson's Disease.

15.

97. The method of claim 91 , wherein the cells overexpressing BCI-XL comprise cells according to any of claims 44 to 68

98. A fusion protein comprising the amino acid sequence of BCI-XL (SEQ ID No 1) 20 or a functional equivalent thereof and a membrane translocation signal.

99. The protein of claim 98, wherein the membrane translocation signal is the MTS from Kaposi FGF-4 as set forth in SEQ ID No 2.

25 100. The protein of claim 98, further comprising a polyhis tag, preferably wherein the polyhistag is from 6 to 9 residues, more preferably wherein the polyhistag is 6 residues long.

101. The protein of claim 98, wherein the MTS is located in the C-terminal of the 30 BCL-XL protein.

102. An expression vector comprising a polynucleotide sequence coding for the fusion protein of any of the preceding claims 98-101 as well as a promoter sequence capable of directing the expression of said fusion protein in a host

35 cell.

103. A host cell comprising the expression vector of claim 102.

104. The host cell of claim 103, selected from the group comprising E. coli, yeast, CHO cells, BHK cells.

105. A method of producing a fusion protein of any of the preceding claims 98-101 comprising culturing the cell of claim 103, and recovering the protein from the culture.