EP0445218A1

EP0445218A1 - Neural cell transplantation

Info

Publication number: EP0445218A1
Application number: EP90901107A
Authority: EP
Inventors: Dominic M. Lam
Original assignee: Baylor College of Medicine
Current assignee: Baylor College of Medicine
Priority date: 1988-11-23
Filing date: 1989-10-11
Publication date: 1991-09-11
Also published as: AU4669189A; EP0445218A4; WO1990005781A1; JPH04501960A

Abstract

Cette invention concerne des cellules et des méthodes pour leur préparation; lesdites cellules sont caractérisées par leur compatibilité avec le système nerveux central, étant capables de produire et de libérer de manière provoquée des substances neuroactives et étant arrêtées mitotiquement à une température prédéterminée. On peut présélectionner des médicaments pour leurs propriétés agonistes et antagonistes pour la production et la libération de substances neuroactives. Par ailleurs, on peut employer ces cellules en tant que source pour les substances neuroactives en des emplacements dans le système nerveux central qui ont des déficiences d'une substance neuroactive particulière et ainsi soulager partiellement ou complètement un certain trouble.This invention relates to cells and methods for their preparation; said cells are characterized by their compatibility with the central nervous system, being capable of producing and liberating in an induced manner neuroactive substances and being mitotically stopped at a predetermined temperature. Medicines can be preselected for their agonist and antagonist properties for the production and release of neuroactive substances. Furthermore, these cells can be used as a source for neuroactive substances at locations in the central nervous system which have deficiencies of a particular neuroactive substance and thus partially or completely relieve a certain disorder.

Description

NEURAL CELL TRANSPLANTATION

INTRODUCTION

Technical Field

The field of this invention relates to mutant and/or recombinant neuronal or non-neuronal cell lines for evaluating neuroactive substance synthesis and release, the effect of compounds on neuroactive substance synthesis and release, and the use of genetically engineered cells to provide neuroactive substances in a central nervous system environment.

Background Debilitating neurological disorders (such as

Parkinsonism, Huntington's chorea, Alzheimer's dementia and epilepsy) are often characterized by depletion of one or more transmitter supplies in specific neuroana- tomical loci. Such depletion is presumably due to progressive loss of neurons responsible for sythesizing the particular neuroactive substances. Therefore, alleviation of such disorders may be accomplished by replenishing transmitter-synthesizing capabilities. The epitome of this line of thinking has been the relative success in treating Parkinsonism with L-dopa

(the synthesizing precusor of dopa ine, the transmitter undergoing depletion during the course of the disease) . Unfortunately, this treatment only offers short-term recovery, since the affected individual undergoes chronic loss of dopaminergic neurons and ultimately reaches the advance stage of affliction whereby surviving dopamine-synthesizing neurons cannot adequately activate requisite dopaminergic pathways. Hence, L-dopa administration decreases in effectiveness over time.

There is, therefore, substantial interest in being able to develop model systems which could be used in the study of the synthesis of neuroactive substances, such as neurotransmitters, such as GABA (gaπuna-aminobutyric acid) dopamine, glycine, acetylcholine, norepinephrine, epinpehrine and serotonin, as well as neuroactive peptides, such as enkephalins, substance P, somatostatin, and nerve growth factor (NGF) . By providing for viable naive cells having neuroactive substance synthesis capacities, not only may the various mechanisms associated with neuroactive substance synthesis be investigated, but the cells may also find application in therapy and provide a stable supply of neuroactive substance at an ii vivo locus, where the amount of endogenously synthesized neuroactive substance is inadequate for the neurological function at the locus. Conceptually, this approach would provide for more extensive alleviation, if not elimination, of chronically debilitating movement and affective disorders.

Relevant Literature

Kuhn et al. (1983) Mol. Biol. Med. 1:335-352, describe functional expression of an MHC antigen in a host cell, while Chao et al. (1986) Science 232:418-421 describe expression of a NGF receptor on the surface of a "naive" host. The cloned rat tyrosine hydroxylase (rTH) gene has been reported by Brown et al. (1987) Biochemistry 6:5208-5212. Talave a and Basilico (1977) J. Cell Physiol. 9_2_:425-435 have described a BHK 21 Syrian hamster cell line that is blocked at 37°C, but not at 32 C, in its progression through G_j of the cell cycle. See also Greco et al. (1987) Proc. Natl. Acad. Sci. USA 8_4:1565-1569 who report the cloning of a human cDNA that complements the retention of the G- blockade in the mutant BHK21 cell line.

SUMMARY OF THE INVENTION

Mammalian cell lines are provided which have at least one of the following characteristics as a result of iii vitro modification: neuroactive substance synthesizing and releasing capability; compatibility with a central nervous system environment; and mitotically arrested. Available host cells are selected having one or more of the above charac¬ teristics and modified iτ\ vitro to provide the desired cell line. The cell line may then be used for evaluation of drugs affecting neuroactive substance synthesis and release, the mechanism of neuroactive substance synthesis and release, and therapeutic utilization of the cell lines as a source jLn vivo of the neuroactive substance.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, cell lines are provided which are compatible with the central nervous system and can be used for expression of synthesis and/or release of neuroactive substances. Biologically active compounds can be studied for their effect on the metabolic pathways involved with neuroactive substance synthesis and release, or the cells may serve as a source of one or more neuroactive substances at a locus deficient for such neuroactive substances in a host.

Neuroactive substances comprise neurotransmitters and neuropeptides, compounds that affect the central nervous system. For a discussion of neuroactive substances, see the Proceedings of the Retina Research Foundation Symposium, Vol. 1, ed. Dominic M-K Lam, 1988, Portfolio Publishing Co. Houston, TX.

The subject cells are characterized by being compatible with cells of the central nervous system, synthesizing and/or releasing neuroactive substance of interest, and are mitotically arrested, while functionally viable. In addition, these cells will normally be derived from cells which can be stably cultured, but will be lacking in one or more of the above characteristics. A wide variety of cell lines which are presently available or which may be developed may be employed in the subject invention. Cell lines may be obtained from such organs as the brain, eye, central and peripheral nervous systems, e.g., nerves, glial elements, etc., where the cell lines may be at an intermediate or mature level of differentiation. Usually, the cell line employed will have from zero to one of the above characteristics, where the remaining characteristics will be as a result of genetic manipulation by the introduction of a source of DNA. Illustrative cell lines include retinoblastomas, pheochromocytomas, neuroblastomas, gliomas and hybrids thereof.

Depending upon the purpose for the cell line, it may be any mammalian line of interest, such as primate, e.g. human, rodentiae, bovine, caprine, equine, canine, feline, etc.

For the most part, the cells which are employed will be compatible with the central nervous system and require one or both of the other charac¬ teristics to be provided. To that extent, the cell lines employed will be mitotically active and functionally viable and may or may not have neuroactive substance synthesis/release capability. To achieve mitotic arrest, various techniques may be employed. One technique is to use classical mutagensis. The mutagensis may be as a result of treatment with one or more chemical compounds or physical treatment. Chemical mutagents include di-(2- chloroethyl)sulfide, di-(2-chloroethyl)amine, N-methyl, N-nitroso N'-nitroguanidine, ethyl methanesulfonate, 5- bromodeoxyuridine, etc. Physical treatment may include electromagnetic irradiation, such as UV-light or X-ray radiation. Clones may then be selected which provide for mitotic arrest at physiological temperature (37°C) while capable of mitosis at a different temperature, usually at least about 3°C different, preferably at least about 4°C different.

Mitotic arrest may also be achieved by inhibiting the expression of one or more genes associated with the mitotic cycle. One technique to achieve this result is the use of antisense sequences, which serve to inhibit expression of a gene. By providing for sequences which bind to any portion of the transcript of the gene, the expression of such gene can be substantially reduced, if not completely inhibited. For example, an antisense sequence associated with the expression of the gene tsll (Greco et al. , 1987, supra), will serve to inhibit the expression of the tsll protein associated with entering the S phase of the cell cycle and thereby offer Gj blockade and mitotic arrest.

Neurotransmitters of interest include GABA dopamine, glycine, norepinephrine, epinephrine, acetylcholine, serotonin, etc. Neuropeptides include enkephalins, substanceP, somatostatin, nerve growth factor, etc. By selecting for expression of one or more genes in the metabolic path for the production of a particular neuroactive substance, particularly a rate-limiting enzyme, one can provide for expression of the neuroactive substance or enhanced expression of the neuroactive substance. In situations where the cell line has a defective metabolic pathway, one can correct the defect by introduction of the appropriate gene with the appropriate regulatory sequences under conditions for expression, whereby the neurotransmitter will then be produced.

For example, tyrosine hydroxylase is a rate limiting enzyme of the catecholamine-synthetic pathway involved in doparaine production. In order to provide for enhanced dopamine production, the cell line could be transformed with the gene, either a copy of an endogenous or exogenous gene from a different host source, which would allow for production of the desired neurotransmitter. For other neurotransmitters, genes of interest would include glutamate decarboxylase and tryptophan hydroxylase. Additionally, the genes for one or more neuroactive peptides as indicated above could be similarly introduced and expressed, in order to provide for enhanced production of these peptides.

To introduce the DNA sequence of interest into the host cell line, various techniques may be employed. One may use bare DNA which may solely involve the DNA sequence of interest and a marker and transform the cell line by the calcium phosphate precipitation method or the like. (Graham and Van der Eb (1973) Virology 5_2:456-467) . Alternatively, one may transfect, transduce or transform the host cell with a vector capable of stable maintenance in the host cell or one that will integrate into a chromosome of the cell. Various techniques for introducing the DNA of interest into the host cell exist, such as contacting with an invasive bacterium, virus, or liposomes, electroporation, sonication/scrape loading, carrier- mediated endocytosis, etc.

The DNA constructs which are employed will have a transcription cassette and a marker for selection. The marker will provide for selection, frequently based on stress, such as resistance to antibiotics, toxins, or the like. Selection can be achieved with G418, chloramphenicol, etc. An expression cassette is provided, where the gene providing for the resistance is normally under the constitutive control of a promoter functioning in the host cell. The marker will normally be joined to a transcription cassette comprising a promoter functional in the host cell, the DNA sequence to be transcribed, and a transcription termination region.

For the transcription cassette, the choice of promoter will vary, depending upon the level of transcription desired. For example, where antisense is used as exemplified by mitotic arrest, a strong promoter will be employed, so as to provide a high level of the antisense sequence. Where neuroactive substance release is of interest, the level of production of the neuroactive substance will determine the choice of the promoter.

A large number of promoters for mammalian cells have been reported in the literature. These include the beta-actin promoter, immunoglobin promoter, globin promoter, 5S RNA promoter, long terminal repeats, or late or early promoters of such viruses as Siman virus, adenovirus, papilloma virus, and the like.

Frequently, the two cassettes will be joined together with a replication system functioning in a prokaryote, particularly E. coli for repetitive cloning. In some instances, it may be desirable to remove the prokaryotic sequences, before introducing the construct into the mammalian host cell. In addition, it may be useful to provide for a replication system which is functional in the mammalian host, so as to provide for stable maintenance. Various replication systems exist, usually viral replication systems, derived from such viruses as those indicated above for the various promoters. Other functional elements may also be present in the construct, where a particular function is desired.

Once the composite construct has been prepared, it may be introduced into the host cell. Following introduction, these cells would then be selected for resistance to one or more agents, which will indicate the presense of the construct in the host cell. These cells may then be screened for the production of the desired neuroactive substance. Assays may be performed by providing for radiolabeled metabolic intermediates or directly assaying for the neuroactive substance by providing immunoassays, or the like. In order to provide for a standard, intracellular alkaline phosphatase activity may also be measured to normalize the values of the various cells.

Clones from the primary screening will then be selected for the desired production of the neuroactive substance and may be repetitively cloned to establish the maintenance of neuroactive substance synthesis and reproducibility. In addition, the cells may be screened in response to known induction mechanisms of neuroactive substance release, such as induction by secretagogues, elevation of extracellular potassium in the presence of calcium, or elevation of cytoplasmic levels of calcium.

Following the secondary screening, each clone is expanded into single-cell or monoclonal cultures.

The resulting cell lines will now be used for studying the mechanism of synthesis and release of neuroactive substance. In addition, the cell lines may be used for the evaluation of various agonist or antagonist properties of compounds affecting the production and/or release.of the neuroactive substance. By having a temperature sensitive mutant clone, which is mitotically arrested at physiological temperature, but capable of proliferation at a different temperature, the cells may be continuously expanded, until employed in vitro and thereby under the conditions of mitotic arrest. The cells may be transplanted into a locus of the central nervous system, particularly the brain, which is deficient in one or more neurotransmitters which may be supplied by the subject cells. Surgical transplantation is well known and has been described for transplanting of syngeneic adrenal cells for production of dopamine. The particular number of cells, the manner in which the cells are introduced at a desired site, ancillary materials which may be employed, are all within the skill of the art and do not require exemplification here.

The following examples are offered by way of illustration and not by way of limitation.

Example

BHK21 Syrian hamster cell line is employed as the host employing a mutant strain which is blocked at 37°C in progression through G^_ of the cell cycle. This mutant tsll shows no metabolic deficits while blocked in G but only progresses through the cell cycle at

33°C. This cell line and the following human cell line are treated in substantially the same way to achieve production of dopamine.

The hamster cell line and a neural glial origin cell line, such as Y79 human retinoblastoma, are employed for transfection. The entire rat tyrosine hydroxylase gene is cotranfected with pSV2neo into each host under conditions described previously (Graham and Van der Eb, 1973, supra). The cells are screened on selective medium for resistance to G418. Surviving cells are then screened for hybridization with the rat tyrosine hydroxylase gene, with a probe of about lOOnt, where the rat gene is readily distinguished from the human and hamster genes. Both Southerns and Northerns are used for screening the clones and those clones having a strong hybridizing signals selected for further study. The cells are then lysed and western blots performed to determine the level of tyrosine hydroxylase product.

The cells are then screened for tyrosine hydroxylase activity.

Finally, the response to changes in extracellular and intracellular potassium and calcium is determined to evaluate the inducible release of catecholamines. The clones which fulfill the indicated characteristics are expanded and recloned to homogeneity. One clone is selected for further modification to provide for mitotic arrest.

The human tsll gene is modified, so at least a portion of the open reading frame is in the antisense direction. The tsll 5*-non-coding and 3'-non-coding regions are retained. The selected clone is transfected as described above with the antisense construct. Clones are then screened for expression of the antisense RNA transcript using Northerns and mitotic arrest evaluated by maintaining cells at 37°C. These cells are further evaluated to determine whether their metabolism is impaired and longevity evaluated for at least three months. Finally, the continued expression of tyrosine hydroxylase is evaluated by employing western blots and neurochemical assays.

The cell clone fulfilling the above characteristics is expanded and recloned to homogeneity. The cells are then transplanted to the brain region basal ganglia of a mammalian host, e.g., MPTP-treated Macaques, by surgical implantation, where the brain region has an established dopamine deficit. The effect of the presence of the cells on Parkinsonian symptoms is then evaluated using known neurological criteria.

It is evident from the above results that novel cell lines can be provided which can be used for studying various attributes of the central nervous system associated with the production and release of neuroactive substance. In addition, these cells can serve as an JLΠ vivo source of the neuroactive substance, where the neuroactive substance are in deficit. Furthermore, these cells will be regulatable in accordance with substantially normal responses to lesions, so that the presence of the subject cells at a site of the central nervous system will not provide for unregulated production of the neuroactive substance. These cells will be safe in being non-tumorous and protected from reversion to a malignant state.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary,skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A cell line characterized by being: (1) mammalian; (2) compatible with cells of the central nervous system;

(3) mitotically arrested;

(4) capable of neuroactive substance synthesis and release; wherein at least one of the above characteristics is imparted by an in vitro modification of a precursor cell.

2. A cell line according to Claim 1, wherein said precursor cell is of neuronal cell origin.

3. A cell line according to Claim 1, wherein said precursor cell is a non-neuronal cell line.

4. A cell line according to Claim 1, wherein said mitotically arrested characeristic is as a result of inhibiting the expression of a protein necessary for progression through mitosis.

5. A cell line according to Claim 1, wherein said mitotically arrested characteristic is as a result of chemical or physical mutagenesis, or viral infection.

6. A cell line according to Claim 5, wherein said mutagenesis results in a temperature sensitive mutation being silent at a temperature other than physiological temperature.

7. A cell line according to Claim 1, wherein said characteristic of being capable of neuroactive substance synthesis and release is enhanced as a result of transformation of said precursor cell line with a gene encoding an enzyme in the metabolic pathway for production of said neuroactive substance.

8. A cell line according to Claim 7, wherein said neutrotransmitter is dopamine.

9. A cell line according to Claim 7, wherein said mitotically arrested characteristic is as a result of transformation of said precursor line with an antisense sequence from the tsll gene.

10. A cell line according to Claim 7, wherein said cell line is a mouse cell line.

11. A cell line according to Claim 8, wherein said cell line is transformed with human genomic DNA and selected for said neuroactive substance synthesis and release.

12. A cell line according to Claim 11, wherein said neuroactive substance is a neurotransmitter.

13. A method for determining the effect of a compound on the neuroactive substance release or synthesis of a neuronal cell characterized by being:

(1) mammalian;

(2) compatible with cells of the central nervous system;

(3) mitotically arrested;

(4) capable of neuroactive substance synthesis and release; wherein at least one of the above characteristics is imparted by an ii vitro modification of a precursor cell, said method comprising: contacting said cell with said compound under conditions where said compound binds to said cell; and determining the effect of said compound on at least one of neuroactive substance release or synthesis.

14. A method according to Claim 13, wherein said neuronal cell is transfected with a construct to provide enhanced production of tyrosine hydroxylase.

15. A method according to Claim 13, wherein said neuroactive substance is a neurotransmitter.