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Title:  Neural transplantation using pluripotent neuroepithelial cells
United States Patent: 
7,048,921
Issued: 
May 23, 2006

Inventors:
 Sinden; John (London, GB); Gray; Jeffrey A. (London, GB); Hodges; Helen (London, GB); Kershaw; Timothy (London, GB); Rashid-Doubell; Fiza (Oxford, GB)
Assignee:
 ReNeuron Limited (GB)
Appl. No.:
 760274
Filed:
 January 12, 2001


 

Outsourcing Guide


Abstract

The subject invention pertains to a novel method of correction of behavioral and/or psychological deficits made possible by the intracerebral transplantation of pluripotent neuroepithelial cells. Cells, cell lines, pharmaceutical preparations, medicaments, methods for the production and maintenance of the cell lines for use in the method of the invention are encompassed by the invention.

Description of the Invention

The present application relates to the correction of behavioural and/or psychological deficits by the intracerebral transplantation of neural cells, and to cells and medicaments therefor. The invention also concerns methods for the production and maintenance of the cell lines.

Behavioural and/or psychological deficits are caused by many diseases and may also be caused when the brain undergoes trauma. For example, motor dysfunction is one symptom of Parkinson's disease. As yet, in most cases, there is no satisfactory treatment available.

The present invention provides for a method of treatment of a behavioural and/or psychological deficit which comprises intracerebral transplantation of a therapeutically effective amount of pluripotent neuroepithelial cells.

The present invention is based in part on the observation that, when transplanted into a damaged or diseased brain, pluripotent neuroepithelial cells appear to respond to signals from the damaged or diseased brain by taking up a phenotype that is able to replace or compensate for functional deficits to which the damage or disease otherwise leads.

The term "pluripotent" is used herein to denote a an undifferentiated neuroepithelial cell that has the potential to differentiate into different types or different phenotypes of cell, in particular into cells having the appropriate phenotype for the intended use. The cell type or phenotype into which such a pluripotent cell finally differentiates is at least partly dependent on the conditions in which the cell exists or finds itself.

For use in the present invention the neuroepithelial cells should be capable of differentiating into cells appropriate to repair or compensate for damage or disease in the target area of the brain. It will be appreciated that cells for transplantation need not be capable of differentiation into all types or phenotypes of neural cells. The cells may, for example, be bipotent. However, a high degree of potency is generally preferred as this gives greater flexibility and potential for transplantation into different areas of the brain.

Suitable pluripotent cells include those called or known as "stem cells" and those called or known as "precursor cells".

The pluripotent neuroepithelial cells are advantageously, and will generally be, conditionally immortal.

The treatment may be carried out on any mammal but the present invention is especially concerned with the treatment of humans, especially treatment with human cells, and with human cells and cell lines.

The present invention provides a mammal which has undergone treatment according to the present invention.

The present invention provides isolated human, pluripotent neuroepithelial cells.

The present invention especially provides human, conditionally immortal pluripotent neuroepithelial cells.

The present invention further provides a conditionally immortal, pluripotent, neuroepithelial cell line, especially for therapeutic use, more especially for the treatment of a behavioural and/or psychological deficit.

The cells of the present invention are capable of correcting a behavioural or psychological deficit when implanted into a damaged part of the human brain. The term "damage" used herein includes reduction or loss of function. Damage may be caused by any of a variety of means including physical trauma, hypoxia (lack of oxygen), chemical agents, for example, damage may be caused by drug abuse, and disease. The following diseases and pathological conditions are examples of diseases or conditions which result in behavioural and/or psychological deficits which may be treated in accordance with the present invention: traumatic brain injury, stroke, perinatal ischaemia, including cerebral palsy, Alzheimer's, Pick's and related dementing neurodegenerative diseases, multi-infarct dementia, Parkinson's and Parkinson's type diseases, Huntington's disease, Korsakoff's disease and Creuzfeld-Jacob disease. Amnesia, particularly following transitory global ischaemia such as after cardiac arrest or coronary bypass surgery, may also be treated in accordance with the present invention.

The present invention further provides a process for the production of human, conditionally immortal, pluripotent, neuroepithelial cells which comprises the steps of:

  • (a) obtaining neuroepithelial cells from a human fetus, the cells being at a stage early enough in the developmental pathway that they have the ability to differentiate into a variety of different brain cell types,
  • (b) introducing into those cells DNA which comprises a sequence capable of causing the cells to be conditionally immortal under the control of appropriate control elements, and
  • (c) maintaining the cells in vitro under permissive conditions.

    The process may further include the step of cloning the cells to obtain one or more cell lines.

    A further aspect of the invention provides for pluripotent, neuroepithelial cells, optionally in isolated form, for therapeutic use, especially in humans. The therapeutic use may be treatment of a behavioural and/or psychological deficit.

    A further aspect of the invention provides for conditionally immortal, pluripotent, neuroepithelial cells for therapeutic use, especially in humans. The therapeutic use may be treatment of a behavioural and/or psychological deficit.

    The present invention further provides for the use of pluripotent, neuroepithelial cells, optionally in isolated form, in the manufacture of a medicament for the treatment of a behavioural and/or psychological deficit. The medicament to be administered comprises pluripotent neuroepithelial cells.

    The present invention especially provides for the use of conditionally immortal, pluripotent, neuroepithelial cells in the manufacture of a medicament for the treatment of a behavioural and/or psychological deficit. The medicament to be administered comprises conditionally immortal, pluripotent, neuroepithelial cells.

    The conditionally immortal cells according to, and used in, the present invention may be from clonal cell lines or may be of mixed population. Cells from clonal cell lines may be preferred. Cells from a single cell line may be used or a mixture of cells from two or more cell lines may be used.

    The invention further provides a pharmaceutical preparation comprising cells according to the invention and a pharmaceutically acceptable carrier.

    Transplantation of conspecific fetal neural tissue into a damaged brain has been studied in animal experiments and consequent repair has been observed at the neuroanatomical, physiological and behavioural levels (Dunnett & Bjorklund, 1994). There has been some application of this work in the treatment of the motor dysfunction of Parkinson's disease (Lindvall, 1994) but widespread use of this technique is handicapped by the need for tissue derived from conspecific fetal brain. The fetal tissue required must be specific to the type of damage one aims to repair and it must be taken at a precise, time-limited stage during brain development that differs according to both brain region and cell type. This leads to both practical and ethical problems.

    Work on fetal tissue transplant (Sinden, 1995) in certain types of damage has shown that very specific matching of cell types is required to obtain improvement in cognitive function.

    We have found that when conditionally immortal pluripotent neuroepithelial cells are implanted into a damaged brain the cells differentiate into the correct form of cell required to repair the damage and the differentiated cells are able to form the appropriate connections required to improve function. The phenotype of the differentiated cells may be the same as the phenotype of the damaged or lost cells, however, the differentiated cells may be of a different phenotype, or of a number of phenotypes. In any case, the cells take up a phenotype that is capable of functionally integrating and compensating for the damaged or lost cells. That is assisted by the propensity, that we have discovered, of the cells to migrate to, and seek out, damaged tissue.

    The use of pluripotent cells means that with one clonal cell line it is possible to repair damage in number of different areas of the brain. It also means that if more than one particular cell type is required to repair damage in a given area then a single pluripotent cell line will be capable of differentiating into the different types of cells required.

    Conditionally immortal cells are cells which are immortal under certain permissive conditions but are not immortal under nonpermissive conditions. In the present case this means that by conditionally immortalising pluripotent precursor cells extracted from fetal tissue and maintaining them under permissive conditions the development of the precursor cells may be arrested at a chosen stage and they may be propagated for long periods. Use of conditionally immortalisation allows the development of clonal lines which are readily expandable in vitro. If the conditions under which the cells are maintained are switched to nonpermissive conditions, the development of the cells is allowed to continue. If the correct conditions are provided the cells will continue to develop and will differentiate.

    Immortalised cells are usually prepared by the transduction of an oncogene into cells. There is therefore a risk of tumour formation in the long term, so such cells are not preferred for use in the present invention.

    Conditionally immortal cells have the advantages of immortal cells in that they are "frozen" in the desired stage of development, are easily maintained and multiply well when under permissive conditions but they may be used in transplants as long as the environment into which they are transplanted has nonpermissive conditions. In the case of the cells of the present invention the gene used to confer conditional immortality should be chosen so that the conditions present in the brain will correspond to nonpermissive conditions.

    The usual way to immortalise the cells is by transduction of an oncogene. The use of conditionally immortal cells means that under nonpermissive conditions the cells do not have oncogenic properties and so this excludes any possibility of the implantation of cells leading to tumour growth.

    If non-immortal cells are used then these may be maintained in vitro in culture media with the addition of growth factors.

    The gene which is used to confer conditional immortality may be incorporated into cells after extraction from a fetal animal. Alternatively, transgenic animals, other than humans, whose neural epithelial cells comprise a gene for conferring conditional immortality may be prepared and bred. If transgenic animals are bred then the cells collected from the fetal animal tissue are already conditionally immortal and do not require further treatment.

    The cells used in the treatment of humans should preferably be derived from human cells to reduce problems with immune rejection. This requires the use of fetal tissue. The use of conditionally immortal cells means that once a population of cells has been established it is not necessary to use fetal tissue again. For example, cells are taken from a human fetus at the appropriate stage of development and the DNA necessary to cause conditional immortality in the cells is inserted. Those cells may then be propagated or they may be cloned and individual cell lines selected. Maintenance of the mixed populations and/or of selected cell lines provides a constant source of material for implantation.

    To treat a patient it is generally of assistance to know where damage has occurred in the brain. Once the existence of damage has been established, whether it be in one isolated area or in several areas, treatment by implantation of cells into the damaged area may be carried out. In many cases, however, the location and/or type of damaged tissue may be unknown or only poorly characterised. For example, neurodegenerative diseases may lead to widespread damage to different types of cells. Treatment of such damage is still possible and is assisted by the ability of pluripotent neuroepithelial cells to migrate extensively once transplanted and to seek out damaged tissue. The pluripotent cells may be transplanted at a single site, or preferably at multiple sites, and are able to migrate to the site(s) of damage and, once there, differentiate in response to the local microenvironment, into the necessary phenotype or phenotypes to improve or restore function. Post mortem analysis of the brains of rats that had received transplants of cells of a conditionally immortal pluripotent neuroepithelial cell line showed that the cells aggregated in the area of the damaged tissue, see Example 9 below, thus illustrating the propensity of the cells to establish and integrate themselves in the area of damage rather than in an area of undamaged tissue. The pluripotent nature of the cells and their propensity for damaged tissue means that treatment with cells from a single cell line of high potency is able to lead to compensation for widespread damage of a number of different cell types.

    After treatment the progress of the patient may be monitored using behavioural and/or psychological tests and/or, if desired, tests which monitor brain activity in selected areas of the brain. For example, tests for cognitive function may be performed before and after transplantation.

    Preferably, treatment will substantially correct a behavioural and/or psychological deficit. However, that may not always be possible. Treatment according to the present invention and with the cells, medicaments and pharmaceutical preparations of the invention, may lead to improvement in function without complete correction. Such improvement will be worthwhile and of value.

    The number of cells to be used will vary depending on the nature and extent of the damaged tissue. Typically, the number of cells used in transplantation will be in the range of about one hundred thousand to several million. Treatment need not be restricted to a single transplant. Additional transplants may be carried out to further improve function.

    The present invention is illustrated by work we have carried out on rats which have brain damage. In the experiments described below conditionally immortal cells used for transplantation are derived from the H-2Kb-tsA58 transgenic mouse developed by M. Noble and his associates at the Ludwig Institute for Cancer Research (Jat et al., 1991). All cells from this mouse possess a temperature-sensitive oncogene (tsA58, the temperature sensitive mutant of the SV40 large T antigen under the control of the interferon-inducible H-2Kb promotor) such that the cells divide at the permissive temperature (lower than body temperature, 33 C.) but differentiate only when restored to mouse body temperature (38 C.-39 C.). It is this feature that provides them with conditional immortality. This allowed us to clone and expand cell lines in vitro which then differentiated upon transplantation into a host brain. A number of cell lines were cloned from a population of cells taken originally from the transgenic mouse, specifically, from embryonic day 14 (E 14) hippocampus. We studied the rats, which received transplants of those cells, for at least 8 months and in no case did the cells, after transplantation, form tumours. Furthermore, in post-mortem histological preparations, the transplanted cells (marked by prior transfection with a lac-z reporter gene) have the appearance of differentiated cells appropriate to the rodent nervous system.

    The cloned cell lines show the potential, in vitro, to differentiate into more than one phenotype, e.g., into both astroglial and neuronal phenotypes, see Example 4 below.

    The lesion-and-behaviour model in which we have demonstrated that cloned cell lines are able to restore function in the damaged brain is one that we have previously studied intensively using fetal conspecific transplants (see Sinden et al., 1995). It utilises rats in which the technique for four-vessel occlusion (4 VO), simulating human heart attack, causes relatively circumscribed and specific damage to the CA 1 pyramidal cells of the dorsal hippocampus, along with a cognitive deficit manifest as difficulty in locating a submerged and invisible platform in a swimming pool. This lesion and behaviour model provides a model of cognitive dysfunction occurring as a consequence of a common form of brain damage, i.e., transient loss of blood supply to the brain, for example, as may occur during cardiac arrest.

    We have previously demonstrated that, for fetal cell-suspension transplants to restore performance in this task, they must be highly specific to the damage caused by 4VO: transplants containing CA 1 pyramidal cells are effective; transplants containing cholinergic cells from the basal forebrain, granule cells from the dentate gyrus, or even a different class of pyramidal cells (CA3) from the hippocampus are ineffective. Examples 5 to 8 below described experiments in which both the clonal cell lines and a mixed population of E 14 hippocampal neuroepithelial cells taken from the H-2Kb-tsA58 transgenic mouse provide effective transplants for restoration of cognitive function in this model.

    We have found that two of the three clonal cell lines tested, the MHP36 cell line (previously known as the C36 cell line) and the MHP3 cell line are as effective at restoring cognitive function as fetal rat transplants containing CA 1 pyramidal cells. The third cell line tested, the MHP15 cell line (previously known as the C15 cell line) leads to an improvement of function but does not show as great an improvement as MHP36 and MHP3. The chances that we happened to pick upon cell lines that would differentiate into CA 1 pyramidal cells, irrespective of the nature of the host brain environment, are small. Thus it appears that the cell lines are capable of responding to damage-associated signals so as to differentiate into cells, of one or more types, that are able to re-establish the necessary connections and restore the function(s) discharged by the damaged tissue. It is this capacity that provides both a strategy and a material basis for transplant therapies with which to target a wide range of behavioural and psychological deficits consequent upon an equally wide range of forms of damage to the human brain, while circumventing the ethical and practical problems associated with the use of human fetal tissues.

    The two cell lines which have shown the greatest ability, so far, to restore function are both FGF2-responsive, i.e., they substantially increase their proliferation in both permissive and non-permissive culture in the presence of that growth factor, whereas the third cell line is only slightly responsive. Cells and cell lines which show significantly increased proliferation in response to the addition of a growth factor to their culture environment are therefore generally preferred. The cells may be tested under permissive conditions and/or nonpermissive conditions. Cells showing the greatest increase in proliferation are generally most preferred. The growth factor used to test the cells should preferably be appropriate to the area of the brain in which the cells are intended for use, i.e., a growth factor secreted in that area. For example, cells intended for the repair of tissue in the hippocampus may be tested with FGF2 (also known as bFGF). Cells responsive to FGF2 are generally preferred. Other mitogenic growth factors may be used in testing, including EGF and NGF.

    The invention therefore provides a method of testing comprising maintaining a population of cells of a conditionally immortal pluripotent neuroepithelial cell line in vitro and culturing portions of the cells under permissive conditions, in the presence and absence of a growth factor, for example, FGF2, and determining the proliferation of the cells. Preferably the cells are also tested under nonpermissive conditions. Those cells which are responsive, i.e., show significantly increased proliferation in the presence of the growth factor under both permissive conditions, and preferably also under nonpermissive conditions, appear to be cells which are especially suitable for use in the treatment of the invention. The growth factor may, for example, be used at a concentration of 10 ng/ml.

    The temperature-sensitive oncogene which confers conditional immortality upon cells derived from the H-2Kb-tsA58 transgenic mouse can be introduced into human cells in vitro. Well known techniques for the introduction of exogeneous DNA exists and these may be used, for example, the gene may be introduced by transfection of the cells. Normal screening techniques for checking that the gene has been incorporated may be used, for example, Southern blotting may be used to screen for DNA insertion sites. In some cases markers may be used or, if the ts SV40 large T antigen gene is used then cells may be screened at the permissive temperature for expression of SV40 as described in Example 4.

    It should be understood that although the experiments described in the Examples below have been carried out using the ts SV40 large T antigen gene to confer conditional immortality on the cells, any other gene which is capable of causing conditional immortality may be used. Such genes may be constructed from known oncogenes. For example, a conditionally immortal gene has been constructed from the c-myc oncogene and is described by Hoshimaiuaru et al, 1996.

    In the experiments on rats which are described in Examples 6, 7 and 8 below conditionally immortal pluripotent cells have been used to repair a very specific type of damage. The uses of cells according to the invention are not limited to repair of that particular type of damage. Transplantation into any area of the brain is envisaged with consequent improvement in function.

    The part of the fetal brain from which the neuroepithelial cells are taken and the precise time (stage and development) may vary. If pluripotent cells are desired then the cells must, however, be taken at a point early enough in the developmental pathway that they have the ability to differentiate into the desired variety of different types and/or phenotypes of brain cell types. For example, in the case of cells taken from the embryonic mouse hippocampus the cells may be taken on embryonic day 14 to 15. Human cells may be taken at the equivalent developmental stage. For example, cells may be taken from human fetuses at about 8 weeks.

    Cells which have been removed may be screened in vitro to ensure that they are still able to differentiate, in particular, to differentiate into the appropriate type or phenotype of cell. Different areas of the brain when damaged may produce different signals, for example, growth factors, and/or different types of damage may cause different signals. The ability to differentiate may be determined in vitro in the presence of the appropriate signal, for example, the appropriate growth factor. Example 4 below describes a procedure in which the ability of cells to differentiate into neuronal and glial phenotypes may be shown.

    Some behavioural and/or psychological deficits are caused by the absence of one or more chemicals in an otherwise healthy brain. It has previously been proposed that transplants of transgenic cells could be used to supply the missing chemicals. The present invention is not specifically concerned with such problems. Although the cells of the present invention may be genetically modified to include extra genes which express desired products, this will not usually be necessary because the cells used according to the present invention, once transplanted, differentiate and then function fully as replacements for cells which have been lost or damaged. The cells achieve functional integration and replace or compensate for the missing or damaged cells. They become a functional part of the brain rather than being merely a sophisticated method of drug administration. Genetic modification of the cells will therefore usually be restricted to the insertion of genes necessary for conditional immortalisation and cloning. Genes required for cloning may be, for example, a gene providing resistance to a selected antibiotic to enable selection. Genetic modification to enable secretion of pharmacologic agents is not preferred.

    Methods for transplantation of cells into humans and animals are known to those in the art and are described in the literature in the art. The term "transplantation" used herein includes the transplantation of cells which have been grown in vitro, and may have been genetically modified, as well as the transplantation of material extracted from another organism. Cells may be transplanted by implantation by means of microsyringe infusion of a known quantity of cells in the target area where they would normally disperse around the injection site. They may also be implanted into ventricular spaces in the brain. If implanted into the neonate then they may disperse throughout the entire brain.
     

  • Claim 1 of 17 Claims

    1. A method for treating brain damage in a mammal, said method comprising intracerebrally transplanting pluripotent, nestin-positive, neuroepithelial cells into the damaged part of the brain of said mammal, wherein said cells have been genetically modified to be conditionally immortal, wherein said cells are immortal prior to said transplanting and differentiate after said transplanting, and wherein said transplanting improves brain function of said mammal.

    ____________________________________________
    If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

     

     

         
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