Note: Descriptions are shown in the official language in which they were submitted.
WO 01/19851 CA 02383076 2002-03-13 pCT/CA00/01049
DOPAMINERGIC NEURONAL SURVIVAL-PROMOTING
FACTORS AND USES THEREOF
Background of the Invention
The invention relates to compositions and methods for increasing the
survival of neurons.
The growth, survival, and differentiation of neurons in the peripheral
and central nervous systems (PNS and CNS, respectively) are dependent, in
part, on target-derived, paracrine, and autocrine neurotrophic factors.
Conversely, the lack of neurotrophic factors is thought to play a role in the
etiology of neurodegenerative diseases such as Parkinson's disease,
Alzheimer's disease, and amyotrophic lateral sclerosis (ALS or Lou Gehrig's
disease). In neuronal cultures, neurotrophic support is provided by co-
culturing
with astrocytes or by providing conditioned medium (CM) prepared from
astrocytes. Astrocytes of ventral mesencephalic origin exert much greater
efficacy in promoting the survival of ventral, mesencephalic dopaminergic
neurons, compared with astrocytes from other regions of the CNS, such as the
neostriatum and cerebral cortex. In chronic, mesencephalic cultures of 21 days
in vitro (DIV) or longer, the percentage of dopaminergic neurons increases
from 20% to 60%, coincident with proliferation of a monolayer of astrocytes.
In contrast, in conditions in which the proliferation of astrocytes was
inhibited,
dopaminergic, but not GABAergic neurons, were almost eliminated from the
cultures by 5 DIV. These results demonstrate the importance of homotypically-
derived astrocytes for the survival and development of adjacent dopaminergic
neurons, and suggest that mesencephalic astrocytes are a likely source of a
physiological, paracrine neurotrophic factor for mesencephalic dopaminergic
neurons.
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The repeated demonstration that astrocytes secrete molecules that
promote neuronal survival has made astrocytes a focus in the search for
therapeutics to treat neurodegenerative diseases. Many laboratories have
attempted to isolate astrocyte-derived neurotrophic factors, but have been
hindered by a major technical problem: serum is an essential component of the
medium for the optimal growth of primary astrocytes in culture, yet the
presence of serum interferes with the subsequent purification of factors
secreted into the conditioned medium.
Thus, there is a need to identify and purify new neurotrophic factors and
to identify new methods to produce conditioned medium that are compatible
with protein isolation techniques.
Summary of the Invention
We have isolated a spontaneously immortalized type-1 astrocyte-like
cell line, referred to as ventral mesencephalic cell line-1 (VMCL-1). This
cell
line, deposited with the American Type Culture Collection (ATCC; Manassas,
VA; ATCC Accession No: , deposit date, September 18, 2000), is derived
from the ventral mesencephalon and retains the characteristics of primary,
type-
1 astrocytes, but grows robustly in a serum-free medium. The CM prepared
from these cells contains one or more neuronal survival factors that increase
the
survival of mesencephalic dopaminergic neurons at least 3-fold, and promotes
their development as well. The potency of this neurotrophic activity and its
low degree of toxicity on dopaminergic neurons in vitro are distinguishing
features of the activity of VMCL-1 CM. Moreover, using size fractionation
techniques, we have identified activities that elute at about 14-16
kilodaltons,
18-21 kilodaltons, and 25-35 kilodaltons. The VMCL-1 immortalized cell line
does not require serum for its growth and thus allows us to identify the VMCL-
1 CM neuronal survival-promoting polypeptides.
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Using a mufti-step purification process, we identified arginine-rich
protein (having a molecular weight of approximately 20 kilodaltons) as a
protein that co-purified with the neuronal survival-promoting activity. As the
protein and the activity co-purified through five purification steps, we
conclude
that this protein is one of the factors in the VMCL-1 CM having the desired
neuronal survival-promoting activity.
Accordingly, in general, the invention features methods for increasing
the survival of neurons (e.g., dopaminergic neurons), as well as new
polypeptides exhibiting such neuronal survival-promoting activity.
In a first aspect, the invention features a pharmaceutical composition
that includes, as an active polypeptide, a substantially pure arginine-rich
protein, and a pharmaceutically acceptable carrier. In one preferred
embodiment, the arginine-rich protein is human arginine-rich protein (SEQ ID
NO: 1).
In a second aspect, the invention features a substantially pure
polypeptide having a molecular weight of about 14-16 kilodaltons that
increases the survival of dopaminergic neurons.
In a third aspect, the invention features a substantially pure polypeptide
having a molecular weight of about 18-21 kilodaltons that increases the
survival of dopaminergic neurons.
In a fourth aspect, the invention features a substantially pure polypeptide
having a molecular weight of about 25-35 kilodaltons that increases the
survival of dopaminergic neurons.
The polypeptides of the present invention can be obtained from a glial
cell line, such as VMCL-1 or another immortalized type-1 astrocyte cell line.
In preferred embodiments of the first, second, third, or fourth aspect, the
survival of dopaminergic neurons is increased at least three-fold. More
preferably, survival is increased at least four-fold, while most preferably,
survival is increased at least five-fold.
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In another aspect, the invention features a method for increasing
dopaminergic neuronal survival. The method includes contacting a
dopaminergic neuron (either in vitro or in vivo) with a polypeptide of the
first,
second, third, or fourth aspect. A preferred polypeptide is human arginine-
rich
protein. Preferably, the survival of dopaminergic neurons is increased at
least
three-fold, more preferably at least four-fold, and most preferably at least
five-
fold.
In another aspect, the invention features a method for growing
dopaminergic neurons for transplantation, including the step of culturing the
neurons, or progenitor cells thereof, with an effective amount of a
polypeptide
of the first, second, third, or fourth aspect. As above, a preferred
polypeptide is
human arginine-rich protein. In preferred embodiments, the amount is
sufficient to increase the survival of dopaminergic neurons by at least three-
fold, by at least four-fold, or even by at least five-fold.
In still another aspect, the invention features a method of treating a
patient having a disease or disorder of the nervous system, this method
includes
the step of administering to the patient a survival-promoting amount of a
substantially purified arginine-rich protein.
In yet another aspect, the invention features another method for
preventing dopaminergic neuronal cell death in a mammal. This method
includes administering to the mammal a dopaminergic neuron survival-
promoting amount of a substantially purified arginine-rich protein. A
preferred
mammal is a human.
The invention also features a method of transplanting cells into the
nervous system of a mammal, including (i) transplanting cells into the nervous
system of the mammal; and (ii) administering a dopaminergic neuronal
survival-promoting amount of arginine-rich protein (e.g., human arginine-rich
protein) to the mammal (e.g., a human) in a time window from four hours
before transplanting of the cells to four hours after transplantation of the
cells.
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In preferred embodiments, the time window is from two hours before
transplantation of the cells to two hours after transplantation of the cells.
The invention features another method of transplanting cells into the
nervous system of a mammal. In this method, the cells are contacted with
arginine-rich protein; and then transplanted into the nervous system of the
mammal. Preferably, these two steps are performed within four hours of each
other.
In yet another aspect, the invention features a method for the preparation
of a dopaminergic neuronal survival-promoting polypeptide of the present
invention, including culturing an immortalized type-1 astrocyte cell line
under
conditions permitting expression of the polypeptide.
In still another aspect, the invention features a substantially pure
composition that includes a polypeptide that increases the survival of
dopaminergic neurons, the polypeptide having a molecular weight of about 14-
16 kilodaltons, about 18-21 kilodaltons, or about 25-35 kilodaltons.
Methods for treatment of diseases and disorders using the polypeptides
or compositions of the invention are also features of the invention. For
instance, a method of treatment of a disease or disorder of the nervous system
(e.g., Parkinson's disease) can be effected with the described polypeptides.
The invention also features a method for preventing dopaminergic
neuronal cell death by administering an effective amount of a polypeptide of
the invention. Such a medicament is made by administering the polypeptide
with a pharmaceutically acceptable carrier.
The invention features the use of a polypeptide of the first, second, third,
or fourth aspect in the manufacture of a medicament.
The invention further features the use of a polypeptide as defined herein:
(1) to immunize a mammal for producing antibodies, which can optionally be
used for therapeutic or diagnostic purposes; (2) in a competitive assay to
identify or quantify molecules having receptor binding characteristics
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corresponding to those of the polypeptide; (3) for contacting a sample with a
polypeptide, as mentioned above, along with a receptor capable of binding
specifically to the polypeptide for the purpose of detecting competitive
inhibition of binding to the polypeptide; and (4) in an affinity isolation
process,
optionally affinity chromatography, for the separation of a corresponding
receptor.
As mentioned above, the invention provides, from mammalian sources,
new dopaminergic neuronal survival factors (e.g., arginine-rich protein) that
are
distinguishable from known factors. These factors promote the survival of
dopaminergic neurons. The invention also provides processes for the
preparation of these factors, and a method for defining activity of these and
other factors. Therapeutic application of the factors is a fiuther significant
aspect of the invention.
In other aspects, the invention features a polypeptide that increases the
survival of dopaminergic neurons, the polypeptide having a molecular weight
of about 14-16 kilodaltons or 25-35 kilodaltons (relative to proteins of known
molecular weights, ranging from 15-102 kDa, run under the same conditions),
as determined using a heparin sepharose CL-6B column (Sigma Chemicals, St.
Louis, MO), and which has survival-promoting activity for dopaminergic
neurons. It will be appreciated that the molecular weight range limits quoted
are not exact, but are subject to slight variations depending upon the source
of
the particular polypeptide factor. A variation of about 10% would not, for
example, be impossible for material from another source.
In another aspect, the invention features a pharmaceutical formulation
that includes a polypeptide of the present invention formulated for
pharmaceutical use, optionally together with an acceptable diluent, Garner or
excipient and/or in unit dosage form. In using the factors of the invention,
conventional pharmaceutical practice may be employed to provide suitable
formulations or compositions. For example, it is preferred that any viral
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pathogens that may be present with the substantially pure polypeptide be
removed or inactivated, and that similar preventive measures are taken to
remove any toxic compounds that are present with the substantially pure
polypepride. In one embodiment, the pharmaceutical formulation includes cells
(e.g., dopaminergic neurons or their progenitors) for transplantation.
In another aspect, the invention features a method of transplanting cells
(e.g., dopaminergic neurons or their progenitors) into the nervous system of a
mammal. The method includes administering a polypeptide or composition of
the present invention, in a pharmaceutically acceptable carrier to the mammal
before, during, or after the cell transplantation.
It is preferred that the polypeptide or composition is administered to the
mammal in a time window from four hours before transplantation to four hours
after transplantation. More preferably, the time window is from two hours
before transplantation to two hours after transplantation. It is understood
that
the polypeptide or composition does not have to be present during the entire
time window for it to prevent or decrease cell death.
In a related aspect, the invention features another method of
transplanting cells into the nervous system of a mammal. The method includes
contacting the cells to be transplanted with a polypeptide or composition of
the
present invention, in a pharmaceutically acceptable carrier before cell
transplantation.
It is preferred that the cells to be transplanted are contacted with the
polypeptide or composition within four hours of transplantation, and, more
preferably, within two hours of transplantation. It is understood that the
polypeptide or composition does not have to be present for the entire time in
order to prevent or decrease cell death following transplantation.
Parenteral formulations may be in the form of liquid solutions or
suspensions; for oral administration, formulations may be in the form of
tablets
WO 01/19851 CA 02383076 2002-03-13 pCT/CA00/01049
or capsules; and for intranasal formulations, in the form of powders, nasal
drops, or aerosols.
Methods well known in the art for making formulations are to be found
in, for example, Remington: The Science and Practice of Pharmacy, (19th ed.)
ed. A.R. Gennaro AR., 1995, Mack Publishing Company, Easton, PA.
Formulations for parenteral administration may, for example, contain as
excipients sterile water or saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible,
biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene
copolymers may be used to control the release of the present factors. Other
potentially useful parenteral delivery systems for the factors include
ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable
infusion systems, and liposomes. Formulations for inhalation may contain as
excipients, for example, lactose, or may be aqueous solutions containing, for
example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
may be oily solutions for administration in the form of nasal drops, or as a
gel
to be applied intranasally.
The present factors can be used as the sole active agents, or can be used
in combination with other active ingredients, e.g., other growth factors which
could facilitate neuronal survival in neurological diseases, or peptidase or
protease inhibitors.
The concentration of the present factors in the formulations of the
invention will vary depending upon a number of issues, including the dosage to
be administered, and the route of administration.
In general terms, the factors of this invention may be provided in an
aqueous physiological buffer solution containing about 0.1 to 10% w/v
polypeptide for parenteral administration. General dose ranges are from about
1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from
about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage
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WO 01/19851 CA 02383076 2002-03-13 pCT/CA00/01049
to be administered is likely to depend upon the type and extent of progression
of the pathophysiological condition being addressed, the overall health of the
patient, the make up of the formulation, and the route of administration.
As indicated above, dopaminergic neurons are, in large part, prevented
from dying in the presence of the factors of the invention. Dopaminergic
neurons of the mesencephalon die in patients having Parkinson's disease. The
invention thus provides a treatment of Parkinson's disease. In addition, the
use
of the present factors in the treatment of disorders or diseases of the
nervous
system in which the loss of dopaminergic neurons is present or anticipated is
included in the invention.
The invention also features screening methods for identifying factors
that potentiate or mimic arginine-rich neuronal survival-promoting activity.
In
these screening methods for potentiators, the ability of candidate compounds
to
increase arginine-rich protein expression, stability, or biological activity
is
tested using standard techniques. A candidate compound that binds to arginine-
rich protein may act as a potentiating agent. Alternatively, a mimetic (e.g.,
a
compound that binds the arginine-rich protein receptor) is capable of acting
in
the absence of arginine-rich protein.
By "substantially pure" is meant that a polypeptide (e.g., arginine-rich
protein) has been separated from the components that naturally accompany it.
Typically, the polypeptide is substantially pure when it is at least 60%, by
weight, free from the proteins and naturally-occurring organic molecules with
which it is naturally associated. Preferably, the polypeptide is an arginine-
rich
protein that is at least 75%, more preferably at least 90%, and most
preferably
at least 99%, by weight, pure. A substantially pure arginine-rich protein may
be obtained, for example, by extraction from a natural source (e.g., a
neuronal
cell), by expression of a recombinant nucleic acid encoding an arginine-rich
protein, or by chemically synthesizing the protein. Purity can be measured by
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any appropriate method, e.g., by column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components
when it is separated from those contaminants that accompany it in its natural
state. Thus, a polypeptide which is chemically synthesized or produced in a
cellular system different from the cell from which it naturally originates
will be
substantially free from its naturally associated components. Accordingly,
substantially pure polypeptides include those which naturally occur in
eukaryotic organisms but are synthesized in E. coli or other prokaryotes.
By "polypeptide" or "protein" is meant any chain of more than two
amino acids, regardless of post-translational modification such as
glycosylation
or phosphorylation.
An arginine-rich protein that is a part of the invention includes a protein
having dopaminergic neuronal survival-promoting activity and encoded by a
nucleic acid that hybridizes at high stringency to the cDNA encoding human
arginine-rich protein. A preferred arginine-rich protein is represented by the
amino acid sequence of SEQ ID NO: 1.
Nucleic acids that are a part of the invention include those nucleic acids
encoding proteins having dopaminergic neuronal survival-promoting activity
and that hybridize at high stringency to the one of the strands of the cDNA
encoding human arginine-rich protein (SEQ ID NO: 5). A preferred nucleic
acid is represented by the nucleotide sequence of SEQ ID NO: 5.
By "substantially identical" is meant a polypeptide or nucleic acid
exhibiting at least 50%, preferably 85%, more preferably 90%, and most
preferably 95% identity to a reference amino acid or nucleic acid sequence.
For polypeptides, the length of comparison sequences will generally be at
least
16 amino acids, preferably at least 20 amino acids, more preferably at least
25 amino acids, and most preferably 35 amino acids. For nucleic acids, the
length of comparison sequences will generally be at least 50 nucleotides,
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preferably at least 60 nucleotides, more preferably at least 75 nucleotides,
and
most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis
software with the default parameters specified therein (e.g., Sequence
Analysis
Software Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This
software program matches similar sequences by assigning degrees of homology
to various substitutions, deletions, and other modifications. Conservative
substitutions typically include substitutions within the following groups:
glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
By "high stringency conditions" is meant hybridization in 2X SSC at
40°C with a DNA probe length of at least 40 nucleotides. For other
definitions
of high stringency conditions, see F. Ausubel et al., Current Protocols in
Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, NY, 1994,
hereby incorporated by reference.
By "polypeptide" or "factor" is meant a molecule having an activity that
promotes the survival (or, conversely, prevents the death) of dopaminergic
neurons in a standard cell survival assay. Compounds of the present invention
have a molecular weight of about 14-16 kilodaltons, about 18-21 kilodaltons,
or, alternatively, about 25-35 kilodaltons. Specifically excluded from the
polypeptides of the invention are glial cell-derived neurotrophic factor
(GDNF)
(Lin et al., Science 260:1130-1132, 1993), neurturin (Kotzbauer et al., Nature
384:467-470, 1996), persephin (Millbrandt et al., Neuron 20:245-253, 1998),
and artemin (Baloh et al., Neuron 21: 1291-1302, 1998).
By "composition" is meant a collection of polypeptides, including a
polypeptide of the present invention.
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By "pharmaceutically acceptable Garner" is meant a carrier that is
physiologically acceptable to the treated mammal while retaining the
therapeutic properties of the polypeptide with which it is administered. One
exemplary pharmaceutically acceptable carrier is physiological saline
solution.
Other physiologically acceptable carriers and their formulations are known to
one skilled in the art and described, for example, in Remington: The Science
and Practice of Pharmacy, (19th ed.) ed. A.R. Gennaro AR., 1995, Mack
Publishing Company, Easton, PA. It will be understood that viral pathogens
and toxic compounds that may inadvertently be included with a polypeptide or
composition if the present invention may be inactivated or removed using any
suitable method known in the art.
By a compound having "dopaminergic neuronal survival-promoting
activity" is the presence of the compound increases survival of dopaminergic
neurons by at least two-fold in a neuronal survival assay (such as the one
described herein) relative to survival of dopaminergic neurons in the absence
of
the compound. Preferably, the increase in the survival of dopaminergic
neurons is by at least three-fold, more preferably by at least four-fold, and
most
preferably by at least five-fold. The assay can be an in vitro assay or an in
vivo
assay. Preferably, the assay is an in vitro assay (see the section entitled
"cell
viability assay," infra)
The present invention provides new methods and reagents for the
prevention of neuronal cell death. The invention also provides pharmaceutical
compositions for the treatment of neurological diseases or disorders of which
aberrant neuronal cell death is one of the causes.
Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
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Detailed Description of the Invention
We have discovered that a cell line of mesencephalic origin (termed
"VMCL-1") secretes a factor that, in turn, promotes differentiation and
survival
of dopaminergic neurons. This cell line grows robustly in a serum-free .
medium. Moreover, the CM prepared from these cells contains one or more
neuronal survival factors that increase the survival of mesencephalic
dopaminergic neurons at least 3-fold, and promotes their development as well.
We purified, from the VMCL-1 cell line, a protein that we identified to
be arginine-rich protein. We purified the this protein as follows. A 3 L
volume
of VMCL-1 conditioned medium was prepared, and subjected to five sequential
steps of column chromatography. At each purification step, each column
fraction was tested for biological activity in the bioassay referred to above.
An
estimate of the effect of each fraction on neuronal survival was done at 24
hour
intervals, over a period of five days, and rated on a scale of 1-10. After the
fifth purification step, the biologically active fraction and an adjacent
inactive
fraction were analyzed by SDS-PAGE. The results of the SDS-PAGE analysis
revealed a distinctive protein band in the 20 kDa range in the lane from the
active fraction. The "active" band was excised and subjected to Cryptic
digest,
and the molecular mass and sequence of each peptide above background were
determined by mass spectrometry analysis. The following two peptide
sequences were identified: DVTFSPATIE (SEQ ID NO: 3) and QIDLSTVDL
(SEQ ID NO: 4). A search of the database identified a match for human
arginine-rich protein (SEQ ID NO: 1) and its mouse orthologue (SEQ ID NO:
2). The predicted protein encoded by the mouse EST sequence is about 95%
identical to the predicted human protein. A search of the rat EST database
revealed two sequences, one (dbEST Id: 4408547; EST name: EST348489)
having significant homology at the amino acid level to the human and mouse
proteins. The full-length rat sequence was not in the GenBank database.
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Thus, arginine-rich protein is useful as a neurotrophic factor for the
treatment of a neurodegenerative disease and for improving neuronal survival
during or following transplantation into a human. Arginine-rich protein can
also be used to improve the in vitro production of neurons for
transplantation.
In another use, arginine-rich protein allows for the identification of
compounds
that modulate or mimic its dopaminergic neuronal survival-promoting activity.
The protein can also be used to identify its cognate receptor. Each of these
uses is described in greater detail below.
Identification of molecules that modulate argLn~e-rich protein biological
activi
The effect of candidate molecules on arginine-rich protein-mediated
regulation of neuronal survival may be measured at the level of translation by
using standard protein detection techniques, such as western blotting or
immunoprecipitation with an arginine-rich protein-specific antibody.
Compounds that modulate the level of arginine-rich protein may be
purified, or substantially purified, or may be one component of a mixture of
compounds such as an extract or supernatant obtained from cells (Ausubel et
al., supra). In an assay of a mixture of compounds, arginine-rich protein
expression is measured in cells administered progressively smaller subsets of
the compound pool (e.g., produced by standard purification techniques such as
HPLC or FPLC) until a single compound or minimal number of effective
compounds is demonstrated to arginine-rich protein expression.
Compounds may also be directly screened for their ability to modulate
arginine-rich protein-mediated neuronal survival. In this approach, the amount
of neuronal survival in the presence of a candidate compound is compared to
the amount of neuronal survival in its absence, under equivalent conditions.
Again, the screen may begin with a pool of candidate compounds, from which
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one or more useful modulator compounds are isolated in a step- wise fashion.
Survival-promoting activity may be measured by any standard assay.
Another method for detecting compounds that modulate the activity of
arginine-rich protein is to screen for compounds that interact physically with
arginine-rich protein. These compounds may be detected by adapting
interaction trap expression systems known in the art. These systems detect
protein interactions using a transcriptional activation assay and are
generally
described by Gyuris et al. (Cell 75:791-803, 1993) and Field et al., (Nature
340:245-246, 1989). Alternatively, arginine-rich protein or biologically
active
fragments thereof can be labeled with l2sI Bolton-Hunter reagent (Bolton et
al.
Biochem. J. 133: 529, 1973). Candidate molecules previously arrayed in the
wells of a multi-well plate are incubated with the labeled arginine-rich
protein,
washed and any wells with labeled arginine-rich protein complex are assayed.
Data obtained using different concentrations of arginine-rich protein are used
to
calculate values for the number, affinity, and association of arginine-rich
protein with the candidate molecules.
Compounds or molecules that function as modulators of arginine-rich
protein neuronal survival-promoting activity may include peptide and non-
peptide molecules such as those present in cell extracts, mammalian serum, or
growth medium in which mammalian cells have been cultured.
A molecule that modulates arginine-rich protein expression or arginine-
rich protein-mediated biological activity such that there is an increase in
neuronal cell survival is considered useful in the invention; such a molecule
may be used, for example, as a therapeutic agent, as described below.
Therapy
The discovery of arginine-rich protein as a neurotrophic factor that
promotes the survival of dopaminergic neurons allows for its use for the
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therapeutic treatment of neurodegenerative diseases such as Parkinson's
disease.
To add arginine-rich protein to cells in order to prevent neuronal death,
it is preferable to obtain sufficient amounts of pure recombinant arginine-
rich
protein from cultured cell systems that can express the protein. Preferred
arginine-rich protein is human arginine-rich protein, but arginine-rich
protein
derived from other animals (e.8., pig, rat, mouse, dog, baboon, cow, and the
like) can also be used. Delivery of the protein to the affected tissue can
then be
accomplished using appropriate packaging or administrating systems.
Alternatively, small molecule analogs may be used and administered to act as
arginine-rich protein agonists and in this manner produce a desired
physiological effect.
Gene therapy is another potential therapeutic approach in which normal
copies of the gene encoding arginine-rich protein (or nucleic acid encoding
arginine-rich protein sense RNA) is introduced into cells to successfully
produce arginine-rich protein. The gene must be delivered to those cells in a
form in which it can be taken up and encode for sufficient protein to provide
effective neuronal survival-promoting activity.
Retroviral vectors, adenoviral vectors, adenovirus-associated viral
vectors, or other viral vectors with the appropriate tropism for neural cells
may
be used as a gene transfer delivery system for a therapeutic arginine-rich
protein gene construct. Numerous vectors useful for this purpose are generally
known (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science
244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614, 1988;
Tolstoshev and Anderson, Curr. Opin. Biotech. 1:55-61, 1990; Sharp, The
Lancet 337: 1277-1278, 1991; Cornetta et al., Nucl. Acid Res. and Mol. Biol.
36: 311-322, 1987; Anderson, Science 226: 401-409, 1984; Moen, Blood Cells
17: 407-416, 1991; Miller et al., Biotech. 7: 980-990, 1989; Le Gal La Salle
et
al., Science 259: 988-990, 1993; and Johnson, Chest 107: 77S-835, 1995).
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Retroviral vectors are particularly well developed and have been used in
clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370, 1990; Anderson
et al., U.S. Patent No. 5,399,346). Non-viral approaches may also be employed
for the introduction of therapeutic DNA into the desired cells. For example,
arginine-rich protein may be introduced into a cell by lipofection (Felgner et
al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neurosci. Lett.
117:
259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al.,
Meth. Enzymol. 101:512, 1983), asialorosonucoid-polylysine conjugation
(Wu et al., J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem.
264:16985, 1989); or, less preferably, micro-injection under surgical
conditions
(Wolff et al., Science 247:1465, 1990).
Gene transfer could also be achieved using non-viral means requiring
infection in vitro. This would include calcium phosphate, DEAE dextran,
electroporation, and protoplast fusion. Liposomes may also be potentially
beneficial for delivery of DNA into a cell. Although these methods are
available, many of these are of lower efficiency.
Many methods for introducing vectors into cells or tissues are available
and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo
therapy,
vectors may be introduced into neural stem cells taken from the patient and
clonally propagated for autologous transplant back into that same patient.
Delivery by transfection and by liposome injections may be achieved using
methods which are well known in the art. Transplantation of normal genes into
the affected cells of a patient can also be useful therapy. In this procedure,
a
normal arginine-rich protein gene is transferred into neurons or glia, either
exogenously or endogenously to the patient. These cells are then injected into
the targeted tissue(s).
In the constructs described, arginine-rich protein cDNA expression can
be directed from any suitable promoter (e.g., the human cytomegalovirus
(CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated
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by any appropriate mammalian regulatory element. For example, if desired,
enhancers known to preferentially direct gene expression in neural cells may
be
used to direct arginine-rich protein expression. The enhancers used could
include, without limitation, those that are characterized as tissue- or cell-
s specific in their expression. Alternatively, if an arginine-rich protein
genomic
clone is used as a therapeutic construct (for example, following isolation by
hybridization with the arginine-rich protein cDNA described herein),
regulation
may be mediated by the cognate regulatory sequences or, if desired, by
regulatory sequences derived from a heterologous source, including any of the
promoters or regulatory elements described above.
RNA molecules may be modified to increase intracellular stability and
half life. Possible modifications include, but are not limited to, the
addition of
flanking sequences at the 5' andJor 3' ends of the molecule or the use of
phosphorothioate or f O-methyl rather than phosphodiesterase linkages within
the backbone of the molecule. This concept can be extended in all of these
molecules by the inclusion of nontraditional bases such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms
of adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by endogenous endonucleases.
Another therapeutic approach within the invention involves
administration of recombinant arginine-rich protein, either directly to the
site
of a potential or actual cell loss (for example, by injection) or systemically
(for
example, by any conventional recombinant protein administration technique).
An additional embodiment of the invention relates to the administration
of a pharmaceutical composition, in conjunction with a pharmaceutically
acceptable carrier, for any of the therapeutic effects discussed above. Such
pharmaceutical compositions may consist of arginine-rich protein, antibodies
to
arginine-rich protein, mimetics, or agonists of arginine-rich protein. The
compositions may be administered alone or in combination with at least one
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other agent, such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not limited to,
saline, buffered saline, dextrose, and water. The compositions may be
administered to a patient alone, or in combination with other agents, drugs or
hormones.
In one example, arginine-rich protein is administered to a subject at the
site that cells are transplanted. The administration of arginine-rich protein
can
be performed before or after the transplantation of the cells. Preferably, the
two steps are within about four hours of each other. If desirable, arginine-
rich
protein can be repeatedly administered to the subject at various intervals
before
and/or after cell transplantation. This protective administration of arginine-
rich
protein may occur months or even years after the cell transplantation.
In addition to its administration to a human or other mammal, arginine-
rich protein can also be used in culture to improve the survival of neurons
during their production any time prior to transplantation. In one example, the
cells to be transplanted are suspended in a pharmaceutical carrier that also
includes a survival-promoting amount of arginine-rich protein. Arginine-rich
protein can also be administered to the cultures earlier in the process (e.8.,
as
the neurons are first differentiating). It is understood that the neurons need
not
be primary dopaminergic neurons. Neurons (e.8., dopaminergic neurons) that
are differentiated, either in vitro or in vivo, from stem cells or any other
cell
capable of producing neurons can be cultured in the presence of arginine-rich
protein during their production and maintenance.
While human arginine-rich protein is preferred for use in the methods
described herein, arginine-rich protein has been identified in numerous
species,
including rat, mouse, and cow. One in the art will recognize that the
identification of arginine-rich protein from other animals can be readily
performed using standard methods. Any protein having dopaminergic neuronal
survival-promoting activity and encoded by a nucleic acid that hybridizes to
the
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cDNA encoding human arginine-rich protein is considered part of the
invention.
Diagnostics
Antibodies which specifically bind arginine-rich protein may be used for
the diagnosis of conditions or diseases characterized by alterations in the
levels
of arginine-rich protein, or in assays to monitor patients being treated with
arginine-rich protein. The antibodies useful for diagnostic purposes may be
prepared in the same manner as those described above for therapeutics.
Diagnostic assays for arginine-rich protein include methods which utilize the
antibody and a label to detect arginine-rich protein in human body fluids or
extracts of cells or tissues. The antibodies may be used with or without
modification, and may be labeled by joining them, either covalently or
non-covalently, with a reporter molecule. A wide variety of reporter molecules
which are known in the art may be used, several of which are described herein.
A variety of protocols including ELISA, RIA, and FACS are known in
the art for measuring arginine-rich protein art and provide a basis for
diagnosing altered or abnormal levels of arginine-rich protein expression.
Normal or standard values for arginine-rich protein expression are established
by combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to arginine-rich protein under
conditions suitable for complex formation. The amount of standard complex
formation may be quantified by various methods, but preferably by
photometric, means. Quantities of arginine-rich protein expressed in subject,
control and disease, samples from biopsied tissues are compared with the
standard values. Deviation between standard and subject values establishes the
parameters for diagnosing disease.
The nucleic acid sequences encoding arginine-rich protein may also be
used for diagnostic purposes. The nucleic acid sequences which may be used
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include antisense RNA and DNA molecules, and oligonucleotide sequences.
The nucleic acid sequences may be used to detect and quantitate gene
expression in biopsied tissues in which expression of arginine-rich protein
may
be correlated with disease. The diagnostic assay may be used to distinguish
between absence, presence, and excess expression of arginine-rich protein, and
to monitor regulation of arginine-rich protein levels during therapeutic
intervention.
Nucleic acid sequences encoding arginine-rich protein may be used for
the diagnosis of conditions or diseases which are associated with altered
expression of arginine-rich protein. The nucleic acid sequences encoding
arginine-rich protein may be used in Southern or northern analysis, dot blot,
or
other membrane-based technologies; in PCR technologies; or in dip stick, pIN,
ELISA or chip assays utilizing fluids or tissues from patient biopsies to
detect
altered arginine-rich protein expression. Such qualitative or quantitative
methods are well known in the art.
The nucleotide sequences encoding arginine-rich protein may be labeled
by standard methods, and added to a fluid or tissue sample from a patient
under
conditions suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is quantitated
and compared with a standard value. If the amount of signal in the biopsied or
extracted sample is significantly altered from that of a comparable control
sample, the nucleotide sequences have hybridized with nucleotide sequences in
the sample, and the presence of altered levels of nucleotide sequences
encoding
arginine-rich protein in the sample indicates the presence of the associated
disease. Such assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies, in clinical trials, or in
monitoring the treatment of an individual patient.
In order to provide a basis for the diagnosis of disease associated with
altered expression of arginine-rich protein, a normal or standard profile for
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expression is established. This may be accomplished by combining body fluids
or cell extracts taken from normal subjects, either animal or human, with a
sequence, or a fragment thereof, which encodes arginine-rich protein, under
conditions suitable for hybridization or amplification. Standard hybridization
may be quantified by comparing the values obtained from normal subjects with
those from an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal samples may be
compared with values obtained from samples from patients who are
symptomatic for disease. Deviation between standard and subject values is
used to establish the presence of disease.
Once disease is established and a treatment protocol is initiated,
hybridization assays may be repeated on a regular basis to evaluate whether
the
level of expression in the patient begins to approximate that which is
observed
in the normal patient. The results obtained from successive assays may be used
to show the efficacy of treatment over a period ranging from several days to
months.
The following examples are to illustrate the invention. They are not
meant to limit the invention in any way.
Example l: Production and Analysis of VMCL-1 Cells
The VMCL-1 cell line was made as follows. Rat E14 mesencephalic
cells, approximately Z-3% of which are glioblasts, were incubated in medium
containing 10% (v/v) fetal bovine serum for 12 hours and subsequently
expanded in a serum-free medium, containing basic fibroblast growth factor
(bFGF) as a mitogen. After more than 15 DIV, several islets of proliferating,
glial-like cells were observed. Following isolation and passaging, the cells
(referred to herein as VMCL-1 cells) proliferated rapidly in either a serum-
free
or serum-containing growth medium. Subsequent immunocytochemical
analysis showed that they stained positive for two astrocytic markers; GFAP
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and vimentin, and negative for markers of oligodendroglial or neuronal
lineages, including A2B5, 04, GaIC and MAP2. We have deposited the
VMCL-1 cell line with the American Type Culture Collection (Manassas, VA;
ATCC Accession No: , deposit date, September 18, 2000).
Serum-free CM, prepared from the VMCL-1 cells, caused increased
survival and differentiation of E14 mesencephalic dopaminergic neurons in
culture. These actions are similar to those exerted by CM derived from
primary, mesencephalic type-1 astrocytes. The expression of mesencephalic
region-specific genes (e.g., wnt-l, en-1, en-2, pax-2, pax-5 and pax-8), was
similar between VMCL-1 cells and primary, type-1 astrocytes of E14 ventral
mesencephalic origin. In both, wnt-1 was expressed strongly, and en-1 less
strongly, supporting an expression pattern expected of their mesencephalic
origin. A chromosomal analysis showed that 70% of the cells were
heteroploid, and of these, 50% were tetraploid. No apparent decline in
proliferative capacity has been observed after more than twenty-five passages.
The properties of this cell line are consistent with those of an immortalized,
type-1 astrocyte.
The VMCL-1 cells have a distinctly non-neuronal, glial-like
morphology, but lack the large, flattened shape that is typical of type-1
astrocytes in culture. Immunocytochemical analysis demonstrated that they
stained positive for GFAP and vimentin, and negative for MAP2, A2B5 and
04. The cells were therefore not of the oligodendrocyte lineage. On the basis
of a negative reaction to A2B5 and their morphological characteristics they
were also not type-2 astrocytes. The classification that is supported by the
immunocytochemical evidence is of type-1 astrocytes, although, as noted, these
cells lack the classical morphological traits of primary type-1 astrocytes in
culture.
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Example 2: Action of VMCL-1 CM on E14 Dopamineraic Neurons in Culture
VMCL-1 CM was tested at 0, 5, 20 and 50% v/v, for its ability to
influence survival and development of E14 mesencephalic dopaminergic
neurons in culture. The cultures were primed with 10% fetal bovine serum
(FBS) for 12 hours, then grown in a serum-free growth medium thereafter, until
they were stained and analyzed after 7 DIV. There was a dose-dependent
action of the CM on the increased survival of dopaminergic neurons. The CM
increased survival by 5-fold. In contrast, there was no significant increase
in
non-dopaminergic neuronal survival. The profile of the biological action of
this putative factor is quite different from that of CM derived from the B49
glioma cell line, the source of GDNF (Lip et al., Science 260: 1130-1132).
Example 3: Gene Expression Analysis
To further investigate the similarity between the VMCL-1 cell line and
primary cultured astrocytes, we measured the expression of six marker genes
characteristic of the mesencephalic region. Analysis of wnt-1, en-1, en-2, pax-
2, pax-5, and pax-8 showed that all genes were expressed in both E13 and E14
ventral mesencephalon neural tissue, with the exception of pax-2, which was
expressed at E13 but not E14 neural tissue. Both primary astrocytes and
VMCL-1 cells expressed wnt-1 at levels comparable with those of E13 and E14
ventral mesencephalic neural tissue. The degree of expression of en-1 was
similar in primary astrocytes and VMCL-1 cells, although at a lower level
versus expressiomin E13 and E14 ventral mesencephalic tissue. In contrast,
en-2, pax-5 and pax-8 were not expressed in either primary astrocytes or
VMCL-1. Pax-2 was expressed in E13 but not E14 ventral mesencephalon,
and in primary astrocytes, but not in VMCL-1.
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Example 4: Chromosomal Ana~sis
Chromosomes were counted in 34 cells. Of these, 9 had a count of 42,
the diploid number for rat. Of the 25 cells that were heteroploid, 12/25 or
48%
were in the tetraploid range. Hyperdiploid (counts of 43-48) and hypodiploid
(counts of 39-41) cells each accounted for 20% of the population, while 12% of
the cells had structurally rearranged chromosomes.
The selective action of VMCL-1 CM in increasing the survival of
dopaminergic neurons in culture provides a potential clinical use for the
molecules) produced by this cell line. The lack of a toxic action of VMCL-1
CM at a concentration of 50% v/v indicates that the active, putative
neurotrophic factor is not toxic. The action exerted by VMCL-1 CM mirrors
almost exactly that of CM prepared from mesencephalic, primary type-1
astrocytes (Takeshima et al., J. Neurosci. 14: 4769-4779, 1994). A high degree
of specificity of the putative factor from VMCL-1 for dopaminergic neurons is
strongly indicated from the observation that general neuronal survival was not
significantly increased, while the survival of dopaminergic neurons was
increased 5-fold. We have demonstrated that primary type-1 astrocytes express
GDNF mRNA, but have not detected GDNF protein by Western blot in the
CM, at a sensitivity of 50 pg. Moreover, we have shown that under the present
experimental conditions, the increased survival of dopaminergic neurons
mediated by an optimal concentration of GDNF is never greater than 2-fold.
These observations alone indicate that the factor responsible for the
neurotrophic actions of VMCL-1 CM is not GDNF.
Examine 5: Production of Tvue-1 Astrocvte-Conditioned Medium
E16 type-1 astrocyte CM (10 L) was filtered and applied to a heparin
sepharose CL-6B column (bed volume 80 mL) which had previously been
equilibrated with 20 mM Tris-HCn (Mallinckrodt Chemical Co. Paris, K~ pH
7.6 containing 0.2 M NaCI. After washing with equilibration buffer, bound
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proteins were eluted from the column with a linear gradient of 0.2 M - 2 M
NaCI in 20 mM Tris-HCl pH 7.6 (400 mL total volume, flow rate 100 mL/hr).
Fractions were collected using a Pharmacia LKB fraction collector and
absorbance was measured at 280 nm (Sargent-Welch PU 8600 UV/VIS
Spectrophotometer). A 1 mL aliquot was taken from each fraction, pooled into
groups of four (4 mL total volume) and desalted using Centricon-10~
membrane concentrators (Millipore, Bedford, MA). Samples were diluted 1:4
in defined medium and bioassayed for dopaminergic activity. Active fractions
were pooled (80 mL total volume) and then applied to a G-75 Sephadex~
column (70 x 2.5 cm, Pharmacia Biotechnology Ltd., Cambridge, UK) which
had been pre-equilibrated with 50 mM ammonium formate pH 7.4. Proteins
were separated with the same buffer (flow rate, 75 mL/hr) and absorbance was
measured at 280 nm. A 1 mL aliquot was taken from each fraction, pooled into
groups of four (4 mL total volume), concentrated by lyopholyzation and
reconstituted in 1 mL distilled water volume. Samples were then diluted 1:4 in
defined medium for dopaminergic bioassay. Those with neurotrophic activity
were further bioassayed as individual fractions.
An important distinguishing feature of VMCL-1 CM is that it promotes
predominantly the survival of dopaminergic neurons, compared with the
survival of GABAergic, serotonergic, and other neuronal phenotypes present in
the culture. This claim of specificity is also made for GDNF. The results of
extensive testing have demonstrated, however, that the VMCL-1-derived
compound is not GDNF.
In order to express the protein, a pcDNA3-hARP expression construct,
containing the human arginine-rich protein cDNA under the control of the
CMV promoter, is transiently transfected into COS cells and the conditioned
medium tested for dopaminergic neuronal survival-promoting activity. A myc
tag can be inserted to facilitate purification and immunodetection of the
recombinant protein.
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Example 6: Isolation and Purification of a Protein having Dopaminergic
Neuronal Survival-Promoting Activity
The purification protocol was performed as follows. All salts used were
of the highest purity and obtained from Sigma Chemical Co. All buffers were
freshly prepared and filtered via 0.2 ~.M filter (GP Express vacuum-driven
system from Millipore)
Step l: Heparin-Sepharose column chromatography (4°C)
Three liters of VMCL-1 conditioned medium was diluted with an equal
volume of 20 mM sodium phosphate buffer, pH 7.2 at room temperature,
filtered, and concentrated to 550 mL volume with SK PREP/SCALE-TFF 2.5
ft2 cartridge (Millipore). The concentrated material was loaded onto a 10 mL
Heparin-Sepharose column assembled from 2 x 5 mL HiTrap Heparin columns
(Pharmacia Biotech) and pre-equilibrated with at least 100 mL of 10 mM
sodium phosphate buffer, pH 7.2 (buffer A). After the loading was complete,
the column was washed with 100 mL of buffer A. A total of 10 fractions were
eluted with buffer B (buffer A plus 1 M sodium chloride) in about 3 mL
volumes each. A 300 ~,L sample was withdrawn for analysis.
Step 2: Superose 12 column chromatography (4°C)
All of the fractions from step 1 were pooled, then concentrated to 4.5
~ mL using Centricon Plus-20 concentrator (5,000 MWCO, Millipore), loaded
onto 16 x 600 mm gel-filtration column packed with Superose 12 media (Prep
Grade, Sigma Chemical Co.) and pre-equilibrated with at least 300 mL of 20
mM sodium phosphate buffer, pH 7.2 containing 0.6 M sodium chloride (GF
buffer). The protein elution was conducted in GF buffer. Two milliliter
fractions were collected and analyzed for activity. The active protein was
eluted in a 15 mL volume after 84 m1 of GF buffer was passed through the
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column and corresponded to an approximately 20-30 kDa elution region based
on the column calibration data obtained with protein standards (Bio-Rad).
Step 3: Ceramic Hydroxyapatite column chromatography (room temperature;
FPLC system)
The active fractions from step 2 that corresponded to the 20-30 kDa
elution region were pooled and concentrated to 7.5 mL, using a Centricon
Plus-20 concentrator (5,000 MWCO), dialyzed overnight at 4°C against
2 L of
mM sodium phosphate buffer, pH 7.2 (buffer A) and loaded (via Superloop)
onto a 1 mL pre-packed ceramic hydroxyapatite (Type I, Bio-Rad) column
10 equilibrated with buffer A. After the excess of unbound protein (flow
through)
was washed off the column with buffer A, the linear gradient of buffer A
containing 1.0 M NaCI was applied from 0 to 100%. One milliliter fractions
were collected and analyzed for activity. The active protein was eluted as a
broad peak within the region of gradient corresponding to 0.4-0.8 M NaCI
concentration.
Step 4: Anion-exchange column chromatography (room temperature; FPLC
system)
The fractions corresponding to the broad peak were pooled (total volume
= 15 mL) and concentrated to 6 mL using Centricon Plus-20 (5,000 MWCO),
dialyzed overnight at 4°C against 2 L of 20 mM Tris HCl buffer, pH 7.5
(buffer
A), loaded (via Superloop) onto a 1 mL anion-exchange FPLC column (Uno,
Bio-Rad), and equilibrated with buffer A. After the excess of unbound protein
was washed off the column with buffer A, a linear gradient of 0-100% 1 M
NaCI (in buffer A) was applied. One milliliter fractions were collected and
analyzed for activity. The active protein was found in the flow-through (i.e.,
in
the unbound protein fraction).
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Step 5: BioSil 125 column chromatography (room temperature; HPLC system)
The active protein fraction from Step 4 (7 mL of total volume) was
concentrated down to nearly zero volume (about 1 ~,L) using Centricon Plus-20
concentrator (5,000 MWCO) and reconstituted in 0.6 mL of 10 mM sodium
phosphate buffer, pH 7.2. The reconstituted material (70 ~,L, analytical run)
was loaded onto BioSil 125 HPLC gel-filtration column (Bio-Rad) equilibrated
with 20 mM sodium phosphate buffer, pH 7.2 (GF buffer). The
chromatography was conducted using HP 1100 Series HPLC system
(Hewlett-Packard). The eluate was collected in 120 ~.L fractions and analyzed
for activity and protein content (SDS-PAGE). The activity was found in
fractions associated with the main 280-nm absorbance peak eluted from the
column, which was represented by a 45-kDa protein according to SDS-PAGE
analysis. Nevertheless, no activity was found in the side fractions of the
45-kDa protein peak, indicating that activity might be due to the presence of
another protein that was co-eluted with 45 kDa protein, but at much lower
concentration that could not be detected on the 12% SDS-PAGE silver-stained
gel. Therefore, the remaining concentrated material from step 5 was further
concentrated down to 80 ~,L volume using a Centricon-3 concentrator
(Millipore), and 60 ~,L was loaded and separated on the column at the same
conditions as for the above-described analytical run. Aliquots of 8 ~L were
taken from each 120 ~L fraction of the eluate and analyzed by SDS-PAGE
(12% gel) combined with silver staining. This analysis indicated that another
two additional proteins (having molecular weights of about 18 and 20 kDa)
were associated with the active fractions and co-eluted with the major 45-kDa
protein. The active fractions were dialyzed against 1 L of ammonium acetate
buffer, pH 8.0 (4°C) and combined to create two active pools, P-l and P-
2,
such that P-1 contained the 20 kDa protein and the 45 kDa protein, and P-2
contained the 18 kDa protein and the 45 kDa protein. Each pool was dried
down on SpeedVac vacuum concentrator (Savant) and separately reconstituted
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WO 01/19851 PCT/CA00/01049
in 15 ~,L 0.1 M ammonium acetate buffer, pH 6.9. Aliquots were withdrawn
from each sample and assayed for activity. Additionally, 1 ~,L aliquots were
subjected to 12% SDS-PAGE analysis followed by silver staining.
The results of the foregoing analysis clearly indicated that P-l, but not
P-2, contained the desired survival-promoting activity. In the next step, both
P-1 and P-2 were dried on SpeedVac, reconstituted (each) in 10 ~L of freshly
prepared SDS-PAGE reducing sample buffer (Bio-Rad), incubated for one
minute in a boiling water bath and loaded onto a 12% SDS-PAGE gel. After
electrophoresis was complete, the gel was fixed in methanol/acetic acid/water
solution (50:10:40) for 40 minutes at room temperature, washed three times
with nanopure water, and stained overnight with GelCode Blue Stain Reagent
(Pierce) at room temperature. After staining was completed, and the GelCode
solution was washed off the gel with nanopure water, the visible protein bands
corresponding to the 45 kDa protein (both P-1 and P-2) and the 20 kDa protein
(P-1 only) were excised from the gel with a razor blade. Each gel slice
containing a corresponding band was placed in a 1.5 mL microcentrifuge tube
until the time of in-gel digestion.
Example 7: Ana~sis of In-gel Digested Fragments by nESI-MS/MS
The protein gel bands were incubated with 100 mM ammonium
bicarbonate in 30% acetonitrile (aq.) at room temperature for 1 hour in order
to
remove the colloidal Coomassie blue stain. The destaining solution was
replaced a number of times until the dye was completely removed. The gel
pieces were then covered with deionized water (~ 200 ~L) and shaken for 10
minutes. The gel pieces were dehydrated in acetonitrile and, after removing
the
excess liquid, were dried completely on a centrifugal evaporator. The gel
bands were rehydrated with 20 ~,L of 50 mM ammonium bicarbonate, pH 8.3,
containing 200 ng of modified trypsin (Promega, Madison, WI). The gel
pieces were covered with 50 mM ammonium bicarbonate, pH 8.3
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(approximately 50 ~,L), and were incubated overnight at 37°C. The
digest
solutions were then transferred to clean eppendorf tubes and the gel pieces
were sonicated for 30 minutes in 50-100 ~,L of 5% acetic acid (aq). The
extract
solutions were combined with the digest solutions and evaporated to dryness on
a centrifugal evaporator.
The in-gel digest extracts were first analyzed by matrix-assisted laser
desorption ionization - time of flight mass spectrometry (MALDI-TOFMS)
using a Voyager Elite STR MALDI-TOFMS instrument (Applied Biosystems
Inc., Framingham, MA). The extracts were dissolved in 5 ~,L of 50%
acetonitrile, 1% acetic acid. Dihydroxybenzoic acid was used as the matrix
and spectra were acquired in positive ion, reflectron mode. Approximately one
fifth of each sample was used for this analysis. These spectra provided the
masses of the peptides in the digest extracts which were then used to search
an
in-house, non-redundant protein sequence database, a process called peptide
mass fingerprinting. The remainder of the samples were used for peptide
sequencing analysis by nanoelectrospray ionization - tandem mass
spectrometry (nESI-MS/MS). The extracts were first desalted using C 18
ZipTips (Millipore) and redissolved in 75% methanol (aq.), 0.1% acetic acid (5
~.L). Approximately one half of the samples were loaded into nanoelectrospray
glass capillaries (Micromass). nESI-MS/MS analyses were carried out using a
Q-Star quadrupole time-of flight hybrid mass spectrometer (PE SCIEX,
Concord, ON). All MS/MS analyses were carried out in positive ion mode.
The collision gas was nitrogen and the collision energy was 40-60 eV. MS/MS
spectra were typically acquired every second over a period of two minutes.
The MS/MS spectra were used to search an in-house non-redundant protein
sequence database using partial sequence tags (i.e., only the peptide mass and
a
few fragment ions are used to search the database). If the protein was not
identified by this procedure then the amino acid sequences of two or more
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peptides were determined as fully as possible from the MS/MS spectra. These
sequences were used to carry out BLAST searches on NCBI's protein,
nucleotide and EST sequence databases.
The results of the analysis identified the 45 kDa protein in both P-1
(active) and P-2 (inactive) as glia-derived nexin, and the 20 kDa protein in P-
1
as arginine-rich protein. Therefore, arginine-rich protein is likely to be a
main
protein that is responsible for activity observed in P-1, while the necessity
of
the presence of nexin for activity cannot be excluded.
Example 8: Generation of Immortalized Cell Line from Human Mesencephalic
Tissue
Using the methods described herein, one may produce a type-1
astrocyte-derived cell line, having the same or similar neuronal survival-
promoting activity, from aborted human tissue. In humans, the corresponding
gestational age of E14 is approximately 9-10 weeks, although other ages are
also likely to be successful. The human compound is identified using standard
protein purification techniques, as described herein.
To induce a spontaneous immortalization of human fetal astrocytes,
ventral mesencephalic tissue is dissected from human fetal brain. The
dissection is preferably performed under sterile conditions in salt solution
(e.g.,
Hank's balanced salt solution (HBSS)), at pH 7.4. The ventral mesencephalon
(VM) with the floor plate intact, is localized, micro-dissected in a culture
dish
in fresh, salt solution, thoroughly cleared of non-neural tissue, and stored
in salt
solution. After tissue collection, the salt solution is removed, and the
tissue
rinsed with two changes of growth medium (e.g., N2), then dispersed in 2.0 mL
of growth medium, which is used in all subsequent procedures. The tissue is
then triturated. About 10-15 strokes are needed to disperse the cells
completely. The cells are centrifuged (1,000 rpm, 2 minutes), the medium
aspirated, and the pellet dispersed in growth medium. The cells are counted
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WO 01/19851 PCT/CA00/01049
using a hemocytometer, and the density adjusted approximately 2.5 x 105
cells/mL. The cells are then dispersed in cultures dishes previously coated
polyornithine ( 15 mg/mL) and fibronectin ( 1.0 mg/mL), at a density of 5 .0 x
104 cells/cm2. The dishes are transferred to the incubator (37°C, 5%
C02,
100% humidity). bFGF ( 10 ng/mL) is added daily, and the medium changed
every second day. At 8-12 DIV, when the cultures are approximately 50%
confluence, the cells are disturbed for 5 days. These conditions have been
shown previously to cause a small percentage of the expanding astrocytes to
become spontaneously immortalized.
Alternatively, a human mesencephalic type-1 astrocyte cell line may be
established from primary cultures by transforming the cells with a DNA
construct containing the oncogenic early region of SV40, under the
transcriptional control of a human GFAP promoter, and a selectable marker
(e.g., pPGK-neo, which contains the marine phosphoglycerate kinase gene
promoter). The transformants are selected with 6418 and cloned. It has been
previously demonstrated that other transformed astrocytes retained
characteristics consistent with the phenotype of type 1 astrocytes, including
GFAP immunoreactivity and a high affinity uptake mechanism for GABA that
is inhibitable by beta alanine (Radany et al. Proc. Natl. Acad. Sci. U S A
89:6467-6471, 1992).
The foregoing results were obtained with the following methods.
Mesencephalic Cultures
Primary mesencephalic cell culture was prepared from timed-pregnant
Sprague-Dawley rats (Taconic Farms; Germantown, NY). as described
previously (Shimoda et al., Brain Res. 586:319-323, 1992 ; Takeshima et al.,
J.
Neurosci. 14:4769-4779, 1994; Takeshima et al., Neuroscience. 60:809-823,
1994; Takeshima et al., J. Neurosci. Meth. 67:27-41, 1996). The dissected
tissue was collected and pooled in oxygenated, cold (4°C), HBSS or
medium
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containing 10% fetal bovine serum (Biofluids Laboratories, Rockville, MD),
depending on the purpose of the experiment. Pregnant rats were killed by
exposure to COZ on the fourteenth gestational day (i.e., E14), the abdominal
region was cleaned with 70% EtOH, a laparotomy was performed, and the
fetuses collected and pooled in cold Dulbecco's phosphate-buffered saline
(DPBS), pH 7.4, without Ca2+ or Mg2+. The intact brain was then removed, a
cut was made between the diencephalon and mesencephalon, and the tectum
slit medially and spread out laterally. The ventral, medial 1.0 mm3 block of
tissue comprising the mesencephalic dopaminergic region was isolated.
Dissected tissue blocks were pooled in cold (4°C), oxygenated
medium. The
tissue was triturated without prior digestion. Alternatively, the cells were
incubated in L-15 growth medium containing papain (Sigma Chemical Co.), 10
U/mL, at 37°C, for 15 minutes, washed (3 x 2 mL) with medium, and
triturated
using only the needle and syringe. The dispersed cells were transferred to 1.5
mL Eppendorf tubes (1.0 mL / tube), and centrifuged at 600 g for 2 minutes.
The use of higher centrifugation speeds for longer periods results in
contamination of the cultures with debris and, as a result, suboptimal growth
of
the cells. The medium was carefully aspirated, and the cells resuspended in
fresh medium and counted using a hemocytometer. All procedures, from
laparotomy to plating were completed within 2 hours. In a typical experiment,
one litter of 10-15 fetuses yielded 1.0 x 105 cells/fetus, or 1.0 x 106 - 1.5
x 106
cells/litter.
Mesencephalic Microisland Cultut~es
To make mesencephalic microisland cultures, cells were prepared as
described above, and resuspended at a final density of 5.0 x 105/mL. A 25 ~.L
droplet of the suspension (1.25 x 104 cells) was plated using a 100~,L pipette
onto 8-well chamber slides coated with poly-D-lysine (50 wg/mL). The area of
the droplet was ~ 12.5 mm2, for a final mean cell density of 1.0 x 105/cm2.
The
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droplet was dispensed uniformly, and the pipette tip withdrawn vertically, to
avoid smearing. The area occupied by the microisland culture remained
uniform for the duration of the culture. The cultures were incubated for 30
minutes at 37°C, in 5% COZ at 100% humidity, to allow the cells to
attach, and
375 ~L of growth medium was then added to each well. The medium was
changed after the first 12 hours, and approximately half of the medium was
changed every second day thereafter.
Cell Viability Assay
A two-color fluorescence cell viability assay kit (Live/Dead
Viability/Cytotoxicity Assay Kits, #L-3224, Molecular Probes, Inc., Eugene,
OR) was used to identify live and dead cells prior to plating and in cultures.
Live and dead cells fluoresced green and red, respectively, giving two
positive
indicators of viability. Ethidium homodimer and calcein-AM were diluted with
DPBS to give final concentrations of 3.8 ~,M and 2.0 ~,M, respectively.
Evaluation of cell viability was done before plating. A cell suspension was
incubated for 15 minutes with an equal volume of dye (typically 20 ~,L) on
glass slides at room temperature in a dark, humid chamber, coverslipped, and
then examined with a fluorescent microscope using FITC optics. Cell viability
just before plating was about 95%.
Culture Medium
The serum-free medium used consisted of equal volumes of Dulbecco's
modified Eagle medium (DMEM) and Ham's F-12 (Gibco, Grand Island, N.Y.;
320-1320AJ), 1.0 mg/mL bovine albumin fraction V (Sigma Chemical Co.; A-
4161), 0.1 ~,g /mL apo-transferrin (Sigma; T-7786), S ~,g/mL insulin (Sigma; I-
1882), 30 nM L-thyroxine (Sigma; T-0397) , 20 nM progesterone (Sigma; P-
6149), 30 nM sodium selenite (Sigma; S-5261), 4.5 mM glutamine (Gibco,
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320-5039AF), 100 U/mL penicillin, and 100 ~g/mL streptomycin (Gibco; P-
100-1-91).
Preparation of Conditioned Medium from hMCL-1 Cell Line
In preparing conditioned medium from the VMCL-1 cell line, Z.0 x 106
cells were plated in a 15 cm uncoated culture dish, in 20 mL of growth medium
containing 1.0% of FBS. At 80% confluence, the medium was aspirated and
the cells washed once with serum-free medium. 20 mL of serum-free N2
medium without albumin was added, and conditioning allowed to continue for
48 hours. During this time, the cells usually expanded to 100% confluence.
The medium was aspirated, pooled in 50 mL tubes, centrifuged (15,000 rpm for
minutes) and subsequently pooled in a 1.0 L plastic bottle. Usually 5 mL of
each batch of CM was filter-sterilized using a 0.22 ~,m filter, stored at
aliquots
of 5 mL, at -70°C, and used to determine neurotrophic potency, before
being
pooled with the larger store of CM. If desired, VMCL-1 CM can be made in
15 large quantities using standard industrial cell culture techniques known to
those
in the art.
Production of Conditioned Medium for Type-1 Astrocytes
Type-1 astrocytes were prepared as follows. E16 rat fetal brain stem
was dissected in cold DPBS, and the mesencephalic region transferred to
20 astrocyte culture medium (DMEM/Ham's F-12, 1:1, 15% FBS, 4.0 mM
glutamine, 30 nM sodium selenite, penicillin, and streptomycin). Cells were
dispersed by trituration in 2 mL of fresh medium using an 18-gauge needle
fitted to a syringe. Cells were centrifuged for 5 minutes at 2,000 rpm in a
centrifuge, re-suspended in medium, and triturated again. The final cell
pellet
was dispersed and plated at a density of 1 x 106 cells / 75 cmz flask in 15 mL
of
medium. Cells were incubated at 37°C in an atmosphere of 5% carbon
dioxide
and 95% air for 24 hours, and unattached cells were removed by aspiration.
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Cells were cultured for an additional nine days, and flasks were then shaken
vigorously for 16 hours to remove any contaminating cell types. Astrocyte
monolayers were washed three times with DPBS, trypsinized and replated
(density of 1 x 106 cells/flask). At this time, a small proportion of the
cells were
plated onto eight-well chamber slides (Nunc Inc., Naperville, IL); these
sister
cultures were treated as described for the flask cultures. At confluence, the
medium was replaced with medium containing 7.5% FBS and cells were
incubated for 48 hours. At the next exchange, defined serum-free medium
(DMEM/Ham's F-12, 1:1, 4.0 mM glutamine, 30 nM sodium selenite,
penicillin 100 U/mL and streptomycin 100 mg/mL) was added and cells were
incubated for a further 48 hours. Medium was replaced and, after five days,
conditioned medium was harvested and mixed with leupeptin (10 mM:
Bachem, Torrance, CA) and 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride (1.0 mM: ICN Biochemicals, Aurora, OH) to inhibit proteolysis.
At the time of harvesting, astrocyte monolayers cultured on chamber slides
were immunostained in order to assess the culture phenotype.
Culturing of hMCL-1 Cells
1n culturing VMCL-1 and preparing VMCL-1 CM, 2.0 x 106 cells were
plated in a 15-cm uncoated culture dish, in 20 mL growth medium initially
containing 10% FBS. At 80% confluence, the medium was aspirated and the
cells washed once with serum-free medium. Usually 20 mL of serum-free
medium without albumin was added, and conditioning allowed to continue for
48 hours. N2 medium proved to be particularly suitable for use to collect
conditioned medium. During these 48 hours, the cells usually expanded to
100% confluence. The medium was aspirated, pooled in 50 mL tubes,
centrifuged (15,000 ipm, 20 min) and pooled in a 1.0 L plastic bottle.
Approximately 5 mL of each batch of CM was sterilized using a 0.22 mm filter,
stored at aliquots of 0.5 mL, at -70°C, and used to determine
neurotrophic
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potency, before being pooled with the larger store of CM. The VMCL-1 cell
line has now been passaged greater than 50 times.
Immunocytochemistry
For MAP2 and TH immunocytochemistry, the cultures were washed
(2 x 250 ~L) with cold DPBS, fixed with 4% formaldehyde in PBS for 10
minutes, permeabilized using 1% CH3COOH/95% EtOH at -20°C, for 5
minutes, and then washed (3 x 250 ~,L) with PBS. Non-specific binding was
blocked with 1% bovine serum albumin in PBS (BSA-PBS) for 15 minutes.
Anti-TH antibody (50 ~,L) (Boehringer-Mannheim, Indianapolis, IN), or anti-
MAP2 antibody (Boehringer-Mannheim) was applied to each well, and the
slides incubated in a dark humid box at room temperature for 2 hours. Control
staining was done using mouse serum at the same dilution as the anti-TH
antibody. After washing (2 x 250 ~.L) with PBS, anti-mouse IgG-FITC (50 ~,L)
was applied, and the slides incubated for an additional 1 hour. After washing
with PBS (2 x 250 ~,L), excess fluid was aspirated, the chamber walls removed,
and a single drop of VectaShield mounting medium (Vector Laboratories,
Burlingame, CA) applied, followed by a cover glass, which was sealed with
nail polish. In some experiments, TH was identified using biotinylated,
secondary antibodies, and the nickel-enhanced, diaminobenzidine (DAB)
reaction product was developed using the biotinylated peroxidase-avidin
complex (ABC kit; Vector Laboratories).
For glial fibrillary acidic protein (GFAP, Boehringer-Mannheim,
#814369), fixation and permeabilization were done in one step using 5%
CH3COOH/95% C2H50H at -20°C. The subsequent procedures were the
same
as those used to visualize TH. For A2B5 and 04, the cultures were washed
with cold DPBS (2 x 250 ~,L) and blocked with 1% BSA-PBS for 10 minutes.
The A2B5 antibody (50 wL) was applied to each well, and incubated for 1
hour. After washing with DPBS (2 x 250 ~,L), the secondary antibody, anti-
IgM-FITC, was applied for 30 minutes. The cells were then washed with
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DPBS (2 x 250~,L). To counter-stain cell nuclei, cells were incubated with 0.5
~,g/mL of nucleic acid dye H33258 (Hoechst, Kansas City, MO) in 10 mM
sodium bicarbonate for 15 minutes at room temperature, then rinsed in PBS for
2 x 10 minutes. After a final washing with cold DPBS (2 x 250 ~,L), they were
mounted as described above.
RT PCR Analysis
Total RNA was extracted from rat E 13 or E 14 ventral mesencephalic
tissue or from 1 x 109 astrocytes or from 1 x 109 VMCL-1 cells using RNA-
STAT reagent (TelTest, University of Maryland, Baltimore, MD). First strand
cDNA was generated from RNA and amplified by polymerase chain reaction
using the manufacturer's procedures.
Reaction products were resolved by 2% agarose gel electrophoresis to
determine size and relative abundance of fragments. PCR results for (3-actin
and GAPDH were included as controls to confirm equal loading of cDNA.
Chromosomal Analysis
The cells were grown in DMEMlF-12 1:1 medium supplemented with
2.5% FBS, D-glucose (2.5 g/L) and ITS supplement, diluted 1:100. Twenty-
four hours later, subcultures at metaphase stage were arrested with colchicine
(10 ~,g/mL). The cells were trypsinized and subjected to hypotonic shock (75
mM KCl). The cells were then fixed in three changes of MeOH/CH3COOH,
3:1, and air-dried. The cells were then stained using 4% Geisma, and
microscopically examined.
Deposit
Applicant has made a deposit of at least 25 vials containing cell line
VMCL-1 with the American Type Culture Collection, Manassas VA, 20110
U.S.A., ATCC Deposit No. The cells were deposited with the ATCC
on September 18, 2000. This deposit of VMCL-1 will be maintained in the
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ATCC depository, which is a public depository, for a period of 30 years, or 5
years after the most recent request, or for the effective life of the patent,
whichever is longer, and will be replaced if it becomes nonviable during that
period. Additionally, Applicant has satisfied all the requirements of 37
C.F.R.
~ ~ 1.801-1.809, including providing an indication of the viability of the
sample.
Applicant imposes no restrictions on the availability of the deposited
material
from the ATCC. Applicant has no authority, however, to waive any restrictions
imposed by law on the transfer of biological material or its transportation in
commerce. Applicant does not waive any infringement of its rights granted
under this patent.
Other Embodiments
All publications and patents mentioned in the above specification are
herein incorporated by reference. Various modifications and variations of the
described method and system of the invention will be apparent to those skilled
in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with specific
preferred embodiments, it should be understood that the invention as claimed
should not be unduly limited to
such specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention which are obvious to those skilled in
molecular biology or related fields are intended to be within the scope of the
invention.
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SEQUENCE LISTING
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CA 02383076 2002-03-13
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SUBSTITUTE SHEET (RULE 26)
