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Title:  Production of gabaergic cells

United States Patent:  6,602,680

Issued:  August 5, 2003

Inventors:  Rubenstein; John L. (San Francisco, CA); Mione; Marina (London, GB); Anderson; Stewart (San Francisco, CA); Stuehmer; Thorsten (San Francisco, CA); Yun; Kyuson (San Francisco, CA)

Assignee:  The Regents of the University of California (Oakland, CA)

Appl. No.:  900527

Filed:  July 5, 2001

Abstract

The invention features methods and compositions for the production of GABAergic cells, particularly GABAergic neurons. Production of GABAergic cells is accomplished by increasing activity of a Dlx gene (e.g., DLX1, DLX2, or DLX5) in an immature neuronal cell. The increase in Dlx activity causes differentiation of the immature neuronal cell into a neuronal cell exhibiting the GABAergic phenotype. The invention also encompasses use of GABAergic cells produced by the method of the invention in, for example, identification of agents that affect GABAergic cell activity and survival, and in replacement therapy.

SUMMARY OF THE INVENTION

The invention features methods and compositions for the production of GABAergic cells, particularly GABAergic neurons. Production of GABAergic cells is accomplished by increasing activity of a Dlx gene (e.g., DLX1, DLX2, or DLX5) in an immature neuronal cell. The increase in Dlx activity causes differentiation of the immature neuronal cell into a neuronal cell exhibiting the GABAergic phenotype. The invention also encompasses use of GABAergic cells produced by the method of the invention in, for example, identification of agents that affect GABAergic cell activity and survival, and in replacement therapy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated or intervening value, in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the Dlx gene" includes reference to one or more Dlx genes and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided might be different from the actual publication dates, which may need to be independently confirmed.

Definitions

By "immature cell" is meant a cell that has not yet committed to a developmental pathway, and thus manipulation of the cell to provide for an increase in DLX activity in the cell, can result in maturation of the cell toward a GABA+ phenotype. "Immature cells" thus include, but are not necessarily limited to embryonic stem (ES) cells, cells obtained from neural plate, cells obtained from early neutral tube, and the like.

By "Dlx activity" is meant activity of a Dlx polypeptide or biologically active fragment thereof, in regulating transcription of genes in the developmental pathway that leads to production of the GABA+ phenotype. Increasing Dlx activity can be accomplished by, for example, increasing expression of one or more Dlx genes (e.g., increasing expression of Dlx1, Dlx2, Dlx5, or any combination of Dlx1, Dlx2, or Dlx5) or otherwise increasing the amount of Dlx polypeptide in the cell (e.g., by introduction of a Dlx polypeptide into the cell in a manner that facilitates an increase in Dlx transcriptional activity).

By "Dlx polypeptide" is meant a polypeptide that provides for the biological activity of a Dlx family member, e.g., facilitates transcription as does a DLX family member. Of particular interest are the DLX polypeptides DLX1, DLX2, and DLX5.

By "GABA+ " is meant a neuronal cell that produces and packages gamma-aminobutyric acid (GABA) in neurotransmitter vesicles.

By "GABAergic phenotype" is meant a neuronal cell that produces and packages GABA in neurotransmitter vesicles and that may, or may not also express other features of GABA+ forebrain neurons, such as glutamic acid decarboxylase (GAD; the enzyme that synthesizes GABA), the GABA vesicular transporter, and a variety of neuropeptides (e.g., enkephalin, dynorphin, substance P, NPY, somatostatin, and nitric oxidase synthase).

By "GABAergic cell" meant a neuronal cell that is capable of producing and expressing GABA in neurotransmitter vesicles.

Overview

The invention is based on the discovery that increasing Dlx activity (e.g., by increasing activity of Dlx1, Dlx2, Dlx5, or a combination thereof, e.g., due to the induction of a transcriptional cascade) in an immature neuronal cell results in production of a GABA+ cell, e.g., a GABAergic neuron. Specifically, the inventors have shown that the DLX1, DLX2, or DLX5 proteins can induce cortical cells which ordinarily do not and would not exhibit a GABA+ phenotype to become GABA+. increasing Dlx activity in an immature neuronal cell, as well as GABAergic cells produced by the method and their use in various screening assays and in replacement therapy.

The expression of the Dlx homeobox genes is closely associated with neurons that express gamma-aminobutyric acid (GABA) in the embryonic rostral forebrain. To test whether the Dlx genes are sufficient to induce some aspects of the phenotype of GABAergic neurons, the electroporation method was used to ectopically express DLX proteins in slice cultures of the mouse embryonic cerebral cortex. This approach showed that ectopic expression of Dlx2 and Dlx5 induced the expression of glutamic acid decarboxylases (GADs), the enzymes that synthesize GABA. This method was also used to show cross-regulation between different Dlx family members. Dlx2 can induce Dlx5 expression, and Dlx1, Dlx2 and Dlx5 can induce expression from a Dlx5/6-LacZ enhancer/reporter construct.

The invention also encompasses increasing activity of transcription factors that exhibit expression patterns that are similar to the expression patterns of Dlx2 (and/or other Dlx family members), exhibit functional redundancy with Dlx1 and/or Dlx2 (e.g., are upregulated in cells that are defective for Dlx1 and/or Dlx2), and/or are expressed "upstream" of Dlx2 (and thus can induce or regulate Dlx2 expression).

Various aspects of the invention will now be described in more detail.

Source of Immature Neuronal Cells

Host cells suitable for use in the methods of the invention can be obtained from a variety of sources. These sources include, but are not necessarily limited to, naturally occurring sources, recombinantly-produced cells, cells from in vitro cultures, and the like. The host cells can be from any suitable origin, preferably mammalian, e.g., murine (mouse, rat, etc.), primate (including human), etc.

In one embodiment, the host cells are obtained from the neural plate or early neural tube of a developing embryo. To this end, two general methods can be used. The first involves using explants from the early central nervous system. Forebrain regions are preferable, but more posterior regions may also be suitable. The explants are surgically dissected from the rest of the embryo, and from other brain regions, with or without the aid of a sectioning device (e.g., vibratome). The explants are then grown in tissue culture using methods described in Anderson et al. (1997) Neuron 19:27-37; Anderson et al. (1997) Science 278:474-476; and Shimamura et al. (1997) Development 124:2709-2718.

The second method involves dissociating the explants into single cells using a titration method as used in Anderson et al. (1999) Cereb. Cortex 6:646-654. In another embodiment, the host cells are obtained from an in vitro cell culture, e.g., from a stable clone of a neural stem cell. For example, human engraftable human neural stem cells, and methods of producing such cells are described in U.S. Pat. No. 5,958,767. Mammalian neural crest stem cells and methods of obtaining the same are described in U.S. Pat. Nos. 5,589,376 and 5,824,489. Proliferated neuron progenitor cell product and methods for making same are described in U.S. Pat. No. 5,411,883.

Methods for Increasing Dlx

An increase of Dlx activity in an immature neuronal cell can be accomplished in a variety of ways. In general, increasing Dlx activity is increased by contacting an immature neuronal host cell with an agent that provides for an increase of Dlx activity in the cell (i.e., a Dlx activity-enhancing agent). Such agents include, but are not necessarily limited to, polynucleotides (e.g., DNA or RNA) encoding Dlx or a portion thereof retaining Dlx activity, Dlx polypeptides or fragments thereof retaining Dlx activity, and small molecules that cause an increase in Dlx expression or mimic Dlx activity.

It should be noted that the invention encompasses GABAergic cells produced as a direct result of contacting the immature cells with a Dlx activity-inducing agent (e.g., cells that express DLX from an introduced polynucleotide, e.g., a polynucleotide encoding DLX1, DLX2, or DLX5), as well as GABA+ cells induced by factors secreted from the cells in the culture that became GABAergic as a result of the method described herein. In addition, the invention encompasses increasing Dlx expression (e.g., expression of DLX1, DLX2, DLX5, or a combination of these genes) in addition to increasing expression of other factors that can enhance development of the GABAergic phenotype (e.g., sonic hedgehog, and the like).

Specific, non-limiting examples for increasing Dlx activity in a host cell are described in more detail below.

Dlx Polynucleotides

In one embodiment, Dlx activity in the host cell is increased by contacting the host cell with a polynucleotide (e.g., DNA or RNA) that encodes a DLX polypeptide, or a fragment thereof that retains activity as a transcriptional activator. In this context, "contacting" generally means contacting with the cell the polynucleotide so as to accomplish introduction of the polynucleotide into the host cell and expression therein to produce a polypeptide that exhibits Dlx2 activity (e.g., that provides for induction of the Dlx transcriptional cascade). The introduced polynucleotide can be maintained as an episomal element, or can be chromosomally integrated. Expression of the encoded DLX polypeptide can be either chronic or transient (e.g., short-term, not for the life of the cell).

Polynucleotides encoding Dlx genes have been described and are readily available. For example, the sequence of a polynucleotide encoding a human Dlx2 is provided at GenBank accession no. NM-- 004405 (McGuinness et al., May 7, 1999; see also, McGuinness et al. (1996) Genomics 35:473-485). The sequence of a polynucleotide encoding a mouse DLX2 is provided at GenBank accession no. NM-- 010054 (McGuinness et al. Feb. 1, 2000, see also, McGuinness et al. (1996) Genomics 35:473-485). The nucleotide (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of the human Dlx2 is provided in the Sequence Listing. The coding sequence for human Dlx2 is provided by joining 987 . . 1386, 1878 . . 2062, and 2574 . . 2975.

Constructs and promoters suitable for delivery of Dlx polynucleotides, and methods of constructing such, are well known in the art. Likewise, introduction of a Dlx-encoding polynucleotide into the cell can be accomplished according to methods well known in the art (e.g., through use of electroporation, microinjection, lipofection, infection with a recombinant, preferably replication-deficient, virus, and other means well known in the art). Preferably, the Dlx-encoding nucleic acid is operably linked to a promoter that facilitates a desired level of DLX polypeptide expression (e.g., a promoter derived from viruses (e.g. CMV, SV40, adenovirus), or a tissue-specific or cell type-specific (e.g., beta-actin or neuronal-specific promoter). Recombinant cells containing the Dlx-encoding nucleic acid can be selected and/or enriched via, for example, expression of a selectable marker gene present in the Dlx-encoding construct or that is present on a plasmid that is co-transfected with the Dlx-encoding construct. Typically selectable markers provide for resistance to antibiotics such as tetracycline, hygromycin, neomycin, and the like. Other markers can include thymidine kinase and the like.

In one embodiment, Dlx expression is transient, e.g., is provided for a period of time that is not permanent. In general, this can be accomplished by using a construct that remains episomal (e.g., does not become genomically integrated to any significant degree) or by providing for regulated expression that requires an exogenous regulatory factor to induce expression (e.g., detectable or significant expression occurs only the presence of the exogenous factor).

DLX Polypeptides

In another embodiment, Dlx activity is enhanced in the cell by introducing a DLX polypeptide (e.g., DLX1, DLX2, DLX5), or a biologically active fragment thereof retaining DLX activity (e.g., transcriptional activity), into the immature neuronal cell. Introduction of a DLX polypeptide can be accomplished according to methods well known in the art, e.g., microinjection, delivery using lipofection (e.g., liposomes), and the like. In one embodiment, DLX polypeptide is delivered using the Voyager system (InVitrogen).

Other Agents that Enhance Dlx Activity in a Cell

Other agents that provide for enhanced DLX activity in the cell can be readily identified and used in the methods of the invention to produce GABAergic cells. The term "DLX activity-enhancing agent" as used herein describes any molecule with the capability of enhancing or mimicking the physiological function of at least one DLX gene (e.g., DLX1, DLX2, DLX5). Such agents can include other endogenous transcription factors that induce expression of Dlx, synthetic molecules (e.g., small molecule drugs, peptides, or other synthetically produced molecules or compounds, as well as recombinantly produced gene products) as well as naturally occurring compounds (e.g., polypeptides, endogenous factors present in neuronal cells, hormones, plant extracts, and the like). For example, several secreted proteins can induce Dlx expression, including sonic hedgehog (Kohtz et al. (1998) Development 125:5079-5089); FGFs (Ferrari et al. (1999) Developmental Dynamics 216(1):10-15) and BMPs.

As is evident from the above, DLX activity enhancing agents encompass numerous chemical classes. Where the agents are synthetically produced, the agents are typically organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins or nucleic acid, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

A wide variety of in vitro assays can be used to identify DLX activity enhancing agents, including labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The screening assay can be a binding assay, wherein one or more of the molecules may be joined to a label, and the label directly or indirectly provides a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g., magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures. The purified DLX protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transcriptional regulation, etc.

A variety of other reagents may be included in the screening assays described herein. Where the assay is a binding assay, these include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. that are used to facilitate optimal protein-protein binding, protein-DNA binding, and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components is added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4oC. and 40oC. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.

Other assays of interest detect agents that mimic Dlx function. For example, candidate agents are added to a cell that lacks functional Dlx, and screened for the ability to reproduce Dlx activity in a functional assay.

Isolation and Identification of GABAergic Cells

GABAergic cells are identified and, where desirable, isolated away from GABAergic negative cells to provide for a substantially homogenous population of GABAergic cells. As used herein, the term "isolated" is meant to indicate that the GABAergic cell is in an environment different from that in which the progenitor cell or GABAergic cell naturally occurs. "Isolated" is meant to include populations that are substantially enriched for the cell of interest and/or in which the cell of interest is partially or substantially purified. As used herein, the term "substantially purified" refers to a cell or molecule (e.g., a polynucleotide or a polypeptide) that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.

The development of the GABA+ phenotype can be confirmed according to any of a variety of methods. For example, GABAergic cells produced by the method of the invention can be identified by contacting the cells with an anti-GABA antibody. Alternative methods for characterizing the properties of the GABAergic cells include assays for molecules expressed in GABAergic cells using immunohistochemistry, in situ hybridization, northern blots, or western blots. Markers for GABAergic cells include: Dlx1, Dlx5, Dlx6, Glutamic acid decarboxylase, calbinidin, neuropeptide Y, nitric oxide synthase, somatostatin, Lhx6, and NPAS1. To verify that the cells do not have properties of forebrain glutaminergic cells, markers of glutaminergic cells can be assayed using the same methods. Such markers include glutamate and Tbr1 (Bulfone et al. (1998) Neuron 21:1273-1282).

Uses of GABAergic Cells Produced by the Methods of the Invention

The GABAergic neuronal cells produced by the methods of the invention can be used in a variety of applications both in vitro and in vivo. Examples of such uses are described below.

Screening Assays

In one embodiment, the GABAergic neuronal cells can be used in screening assays to identify agents that modulate an activity of such cells. In general, such screening assays involve contacting a candidate agent with a GABAergic cell produced according to the invention, and detecting an alteration in the an activity or phenotype of interest. Applications of this approach would include, but are not necessarily limited to, assays to identify: (1) agents that increase or decrease the response of GABAergic cells to neurotoxic agents; including the Huntington protein; (2) agents that alter the physiological properties of GABAergic cells (e.g., the expression of neurotransmitter/modulator receptors or pumps, ion channels, signal transduction machinery); (3) agents that alter the growth and/or branching of the axon or dendrites of GABAergic cells; and/or (4) agents that alter the interactions of GABAergic cells with other neurons (e.g., glutaminergic).

Reagents to Facilitate Research

In another embodiment, the GABAergic cells produced according to the invention are extremely useful as reagents. For example, GABAergic cells of the invention can be used at the bench to study, for example, the effect of various endogenous or exogenous factors upon activity of GABAergic cells and/or to further understand molecular pathways active in GABAergic cells. This utility is of paramount importance give the difficulty in obtaining specific types of neuronal cells for study, particularly where the experiments require a homogenous population of GABAergic cells or require large numbers of cells.

In addition, the GABAergic cells as well as the Dlx family of transcription factors, can be used to facilitate identification of other molecules required for GABAergic neuronal development, function and survival. This can be accomplished by, for example, using polynucleotide arrays and other methods (e.g., subtractive hybridization) to identify genes that are regulated by Dlx, e.g., exhibit increased expression upon contacting a GABAergic cell with one of these transcriptional factors or other activity-enhancing agent. This can be accomplished by, for example, comparing gene expression profiles in wild-type and mutant transgenic mice, and/or by comparing gene expression profiles in neuroepithelial stem cells that are express differing levels of Dlx.

Transplantation

In another embodiment, the GABAergic cells produced according to the invention can be used in ex vivo replacement or supplemental therapy in subjects having a condition or disease caused by or associated with a defect in GABAergic neurons. Exemplary conditions or diseases for which supplemental or replacement therapy can be useful include, but are not necessarily limited to, epilepsies, neuropsychiatric disorders (e.g., schizophrenia), Huntington's disease, and Alzheimer's disease.

In one embodiment, the GABAergic cell is derived from a donor of the same species as the recipient, more preferably from a donor having a compatible complement of MHC molecules. Where possible, it may be preferred to produce GABAergic cells from the individual who will receive the transplant (e.g., to provide an autologous transplant). For example, stem cells may be obtained from the individual's subependymal zone.

In general, after production according to the invention, the GABAergic cells are expanded in vitro, and are implanted into the subject by methods well known in the art. The number of cells implanted is a number of cells sufficient to provide for the adequate repair of the neuronal defect or deficiency. The number cells to be transplanted can be determined based upon such factors as severity of the defect in the subject and/or the percentage of cells that survive implantation. Preferably the cells are implanted in an area of dense vascularization, and in a manner that minimizes evidence of surgery in the subject. There is evidence that immature forebrain interneurons are capable of widespread migration within the cerebral cortex (Wichterle et al. (1999) Nat. Neurosci. 2:461-466; and Anderson et al. (1997) Science 278:474-476), indicating that transplants of such cells disperse throughout the recipient's cerebral cortex. The engraftment of the cells can be monitored using standard CNS imaging methods (e.g., MRI) and by examining the subject for classic signs of graft rejection, i.e., inflammation and/or exfoliation at the site of implantation, and fever. Migrating immature interneurons are likely postmitotic, and thus would not pose a risk if treatment is continued.

Claim 1 of 7 Claims

That which is claimed is:

1. A method of producing a GABAergic cell in vitro, the method comprising:

introducing into an immature neuronal cell a polynucleotide encoding a human DLX2 polypeptide, said introducing allowing for expression of DLX2 in the cell;

wherein expression of DLX2 in the cell results in development of a GABAergic phenotype in the cell, and wherein the GABAergic phenotype is selected from gamma amino butyric acid production, expression of GAD65 , and expression of GAD67.



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