U.S. patent application number 11/918622 was filed with the patent office on 2009-12-03 for immortalization of cells including neuronal cells.
This patent application is currently assigned to The John Hopkins University. Invention is credited to Weiran Chen, Ahmet Hoke.
Application Number | 20090298095 11/918622 |
Document ID | / |
Family ID | 37115476 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090298095 |
Kind Code |
A1 |
Hoke; Ahmet ; et
al. |
December 3, 2009 |
Immortalization of cells including neuronal cells
Abstract
The instant invention provides methods for immortalizing cells.
The invention further provides immortalized cell lines, e.g.,
neuronal cell lines, and methods of using these cell lines in
screening assays.
Inventors: |
Hoke; Ahmet; (Towson,
MD) ; Chen; Weiran; (Columbia, MD) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The John Hopkins University
Baltimore
MD
|
Family ID: |
37115476 |
Appl. No.: |
11/918622 |
Filed: |
April 14, 2006 |
PCT Filed: |
April 14, 2006 |
PCT NO: |
PCT/US2006/014247 |
371 Date: |
January 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60671865 |
Apr 15, 2005 |
|
|
|
Current U.S.
Class: |
435/7.21 ;
435/325; 435/375; 435/467 |
Current CPC
Class: |
G01N 2500/10 20130101;
C12N 2799/027 20130101; G01N 33/5058 20130101; C12N 2510/04
20130101; C12N 9/1276 20130101; C12N 5/0619 20130101 |
Class at
Publication: |
435/7.21 ;
435/467; 435/375; 435/325 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12N 15/87 20060101 C12N015/87; C12N 5/00 20060101
C12N005/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The following invention was supported at least in part by
NIH Grants RO1 NS43991 and PO1 MH70056. Accordingly, the government
may have certain rights in the invention.
Claims
1. A method for generating an immortalized human cell comprising:
introducing into a cell a DNA segment encoding an oncogene;
selecting for a cell containing the DNA segment; and introducing a
hTERT into the selected cell; thereby generating an immortalized
cell.
2. The method of claim 1, wherein the DNA segment is contained in a
plasmid.
3. The method of claim 1, wherein hTERT is introduced by a
retrovirus.
4. The method of claim 1, wherein the cell is a neuronal cell.
5. The method of claim 4, wherein the neuronal cell is selected
from neuronal cells from the brain, spinal cord, and dorsal root
sensory ganglia.
6. The method of claim 1, wherein the human cell is a dorsal root
ganglia neuron.
7. The method of claim 6, wherein the dorsal root ganglion neuron
is a nociceptive dorsal root ganglion neuron.
8. The method of claim 1, wherein the cell is a glial cell.
9. The method of claim 8, wherein the glial cell is an astrocyte,
oligodendrocyte or a Schwann cell.
10. The method of claim 1, further comprises contacting the
immortalized cell with an agent that causes differentiation.
11. The method of claim 10, wherein the agent is cyclic AMP or an
analog thereof or an agent that increases intracellular cAMP
levels.
12. The method of claim 11, wherein the agent is forskolin.
13. A method of producing immortalized neuronal cells comprising:
introducing a DNA segment encoding an oncogene into a neuronal
cell; selecting for neuronal cells that contain the DNA segment;
introducing hTERT into the selected cells; and selecting for cells
that contain hTERT; thereby producing immortalized neuronal
cells.
14. The method of claim 13, wherein the DNA segment is contained in
a plasmid.
15. The method of claim 13, wherein the neuronal cells are selected
from a group consisting of neuronal cells from the brain, neuronal
cells from the spinal cord, dorsal root sensory ganglia, and
autonomic ganglia.
16. The method of claim 13, wherein the oncogene is selected from
the group consisting of Ras, Myc, Raf, and large T-Antigen.
17-28. (canceled)
29. A method of producing immortalized dorsal root ganglion
neuronal cell line comprising: introducing a plasmid comprising an
SV40 large T-antigen into dorsal root ganglion cell; selecting
dorsal root ganglion cells that contain the plasmid; introducing
hTERT into the selected cells; and selecting for cells that contain
hTERT; thereby producing immortalized dorsal root ganglion neuronal
line.
30. The method of claim 29, wherein hTERT is introduced by a
retrovirus.
31. An immortalized nociceptive dorsal root ganglion neuron.
32. The immortalized nociceptive dorsal root ganglion neuron of
claim 31, wherein the neuron expresses markers of nociceptive
dorsal root ganglion neurons.
33. The method of claim 24, wherein the nociceptive dorsal root
ganglion neurons express a capsaicin receptor, TRPV1,
GDNF-receptor, NGF-receptor, or a sodium channel.
34. The method of claim 24, wherein the nociceptive dorsal root
ganglion neurons respond to capsaicin by elevating intracellular
calcium flux or generate action potentials when polarized.
35. The immortalized nociceptive dorsal root ganglion neuron of
claim 33, wherein the capsaicin receptor is TRPV1.
36. The immortalized nociceptive dorsal root ganglion neuron of
claim 35, further comprising an oncogene and hTERT.
37-40. (canceled)
41. A method of identifying a modulator of pain, comprising:
contacting an immortalized dorsal root ganglion neuronal cell of
claim 29, with a candidate modulator; and determining if the
candidate modulator is binds to and/or modulates the dorsal root
ganglion neuronal cell; thereby identifying a modulator of pain.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/671,865, filed 15 Apr. 2005, the entire contents
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Neuropathic pain, also referred to as a chronic pain, is a
complex disorder resulting from injury to the nerve, spinal cord or
brain. There is evidence that nerve fibers in subjects with
neuropathic pain develop abnormal excitability, particularly
hyper-excitability, Zimmerman (2001) Eur J Pharmacol 429
(1-3):23-37. Although the American Pain Society estimates that
nearly 50 million Americans are totally or partially disabled by
pain, there are currently very few effective, well-tolerated
treatments available (Wetzel et al. (1997) Ann Pharmacother 31
(9):1082-3). Indeed, existing therapeutics cause a range of
undesirable side effects primarily due to the difficulty in
developing small-molecule drugs capable of specifically targeting
the receptor/channel of choice.
[0004] Many, relatively common clinical conditions are associated
with neuropathic pain (Berger A, et al. (2004) J Pain 5:143-149).
Traditionally, combinations of tricyclic antidepressants or
anti-epileptics along with analgesics have been used to treat
neuropathic pain (reviewed in (Mendell J et al. (2003) N Engl J Med
348:1243-1255). However, treatment of neuropathic pain is often
unsatisfactory; persistent neuropathic pain affects quality of life
and lead to significant morbidity. In recent years with the
identification of the receptor for capsaicin (Caterina M J et al.
(1997) Nature 389:816-824; Caterina, M et al. (2000) Science
288:306-313), neuropathic pain research has directed its attention
to identification of drugs that interfere with the transient
receptor potential vanilloid receptor 1 (TRPV1) physiology. Primary
effort has focused on antagonists that block nociceptive pain
sensation at the receptor level but so far, no drug has reached
clinical use (Caterina M J et al. (1997) Nature 389:816-824 and
Caterina, M J et al. (2000) Science 288:306-313)
[0005] Previous attempts at identifying TRPV1 antagonists have used
non-neuronal cell lines expressing recombinant TRPV1 and the
calcium flux induced by capsaicin as an outcome measure for high
throughput screening (HTS) (Caterina M J et al. (1997) Nature
389:816-824; Caterina, M J et al. (2000) Science 288:306-313).
Although these cells expressing recombinant TRPV1 may be useful, a
nociceptive sensory neuronal cell expressing TRPV1 might be more
relevant because the non-neuronal cell lines may lack the
appropriate intracellular signaling pathways associated with and
downstream of TRPV1 in nociceptive sensory neurons. In order to
generate tools for a more rational approach to drug screening for
neuropathic pain, it would be useful to have an immortalized DRG
sensory neuronal line with nociceptive properties. To date,
attempts to immortalize neuronal cell lines have achieved little
success.
[0006] Likewise, the ability to generate immortalized cell lines
using cells that have been historically difficult to immortalize
would be beneficial in the efforts to develop novel therapeutics
for the treatment of disease and illness.
[0007] Although neuronal cell lines have been generated in the past
these were mostly from embryonic tissues and were derived from
progenitor or stem cells (see, e.g., Bernard J (1989) Neurosci Res,
24:9-20, Evrard (1990) PNAS, 87:3062-6, Redies J (1991) Neurosci
Res 30:601-15). Also, a temperature sensitive mutant T antigen has
been used to immortalize neuronal populations, but the efficiency
of this technique has been very low (Eves (1994) Brain Res
656:396-404).
[0008] Accordingly, the need exists for effective and reliable
methods of immortalizing cells that scientists have not had success
in immortalizing with currently available methods, e.g., neuronal
cells.
SUMMARY OF THE INVENTION
[0009] The instant invention is directed to methods for making
immortalized cell lines from cells that are historically difficult
to immortalize, e.g., neuronal cells. The inventors of the instant
application have discovered a novel method for making stable
immortalized cells, e.g., neuronal cells.
[0010] Accordingly, in one aspect the instant invention provides,
methods for generating an immortalized human cell comprising
introducing into a cell a DNA segment encoding an oncogene,
selecting for a cell containing the DNA segment, and introducing
hTERT into the selected cell, thereby generating an immortalized
cell.
[0011] In one embodiment, the DNA segment is contained in a
plasmid. In another embodiment, hTERT is contained in a
plasmid.
[0012] In another embodiment, the neuronal cells are selected from
a group consisting of neuronal cells from the brain, neuronal cells
from the spinal cord, dorsal root sensory ganglia, dorsal root
ganglia neuron and autonomic ganglia. In a specific embodiment, the
neuronal cell is, for example, a nociceptive dorsal root ganglion
neuron.
[0013] In another embodiment, the cell is a glial cell, e.g., an
astrocyte, oligodendrocyte or a Schwann cell.
[0014] In another embodiment, the methods further comprises
contacting the immortalized cell with an agent that causes
differentiation and/or axon elongation. In specific embodiments,
the agent cyclic AMP or an analog thereof or an agent that
increases intracellular cAMP levels. In a specific embodiment, the
cAMP analog is forskolin.
[0015] In another aspect, the invention provides methods of
producing immortalized neuronal cells comprising introducing a DNA
segment encoding an oncogene into a neuronal cell, selecting for
neuronal cells that contain the DNA segment, introducing hTERT into
the selected cells, and selecting for cells that contain hTERT,
thereby producing immortalized neuronal cells.
[0016] In one embodiment, the DNA segment is contained in a
plasmid. In another embodiment, the hTERT is contained in a
plasmid.
[0017] In another embodiment, the neuronal cells are selected from
a group consisting of neuronal cells from the brain, neuronal cells
from the spinal cord, dorsal root sensory ganglia, dorsal root
ganglia neuron and autonomic ganglia. In a specific embodiment, the
neuronal cell is, for example, a nociceptive dorsal root ganglion
neuron.
[0018] In another embodiment, the oncogene is selected from the
group consisting of Ras, Myc, Raf, and large T-Antigen. In one
particular embodiment, the oncogene is the large T-antigen, e.g.,
the SV40 large T-antigen.
[0019] In another embodiment, the hTERT is contained in a
plasmid.
[0020] In another embodiment, the methods further comprises
contacting the immortalized cell with an agent that causes
differentiation. In specific embodiments, the agent cyclic AMP, an
analog thereof, or an agent that increases intracellular cAMP
levels. In a specific embodiment, the cAMP analog is forskolin. In
a related embodiment, the forskolin allows the immortalized neurons
to differentiate and extend axons.
[0021] In another embodiment, the immortalized nociceptive dorsal
root ganglion neurons maintain the biochemical and
electrophysiological properties of primary neurons. In a related
embodiment, the nociceptive dorsal root ganglion neurons express a
capsaicin receptor, TRPV1, GDNF-receptor, NGF-receptor, or a sodium
channel. In a specific embodiment, the nociceptive dorsal root
ganglion neurons express capsaicin receptor TRPV1. In another
related embodiment, the nociceptive dorsal root ganglion neurons
respond to capsaicin by elevating intracellular calcium flux or
generate action potentials when polarized.
[0022] In another embodiment, the immortalized nociceptive dorsal
root ganglion neurons express one or more axonal markers, e.g.,
neurofilament or .beta.III tubulin.
[0023] In another aspect, the invention provides methods of
producing immortalized dorsal root ganglion neuronal cell line
comprising, introducing a plasmid comprising an SV40 large
T-antigen into dorsal root ganglion cell, selecting dorsal root
ganglion cells that contain the plasmid, introducing hTERT into the
selected cells, and selecting for cells that contain hTERT, thereby
producing immortalized dorsal root ganglion neuronal line.
[0024] In a related embodiment, hTERT is contained in a
plasmid.
[0025] In another aspect, the invention provides immortalized
nociceptive dorsal root ganglion neurons.
[0026] In another embodiment, the immortalized nociceptive dorsal
root ganglion neurons maintain the biochemical and
electrophysiological properties of primary neurons. In a related
embodiment, the nociceptive dorsal root ganglion neurons express a
capsaicin receptor, TRPV1, GDNF-receptor, NGF-receptor, or a sodium
channel. In a specific embodiment, the nociceptive dorsal root
ganglion neurons express capsaicin receptor TRPV1. In another
related embodiment, the nociceptive dorsal root ganglion neurons
respond to capsaicin by elevating intracellular calcium flux or
generate action potentials when polarized.
[0027] In a specific embodiment, the invention provides
immortalized nociceptive dorsal root ganglion neurons comprising an
oncogene and hTERT. In exemplary embodiments, the oncogene is
selected from the group consisting of Ras, Myc, Raf, and large
T-Antigen. In one particular embodiment, the oncogene is the large
T-antigen, e.g., the SV40 large T-antigen.
[0028] The invention further provides methods for screening for
modulators of neuronal cells. These modulators are useful in, for
example, the treatment and prevention of pain. In one embodiment,
the invention provides high throughput methods of screening using
the immortalized cells produced by the methods of the
invention.
DESCRIPTION OF THE DRAWINGS
[0029] FIGS. 1A-EC depict immortalized DRG neuronal cells extend
neurites, express neuronal markers and generate action potentials
after differentiation. Phase contrast images of undifferentiated
(A) and differentiated (B) 50B11 cells show extension of axons 4
hours after differentiation with forskolin. After differentiation,
50B11 cells stain with anti-neurofilament (C) and
anti-.beta.III-tubulin antibodies. DAPI counterstain shows nuclei.
Scale bar=20 .mu.m. A representative action potential is seen in a
differentiated 50B11 cell after application of a depolarizing
current (E).
[0030] FIGS. 2A-C depict 50B11 neuronal line expresses markers of
small diameter sensory neurons. Immunofluorescence images of 50B11
cells stained with fluorescently tagged IB4 (A) and anti-CGRP (B).
Nuclei are counterstained with DAPI. Scale bar=20 .mu.m. Changes in
expression of mRNA for p75, Trk-A, c-ret and GFRa-1 in the presence
of forskolin (FSK), NGF and GDNF compared to baseline levels of
undifferentiated 50B11 cells (C). (n=6-8/group; error bars denote
standard error of mean; *=p<0.05 compared to baseline;
**=p<0.05 FSK+GDNF versus FSK+NGF)
[0031] FIGS. 3A-C depict 50B11 neuronal line expresses nociceptive
markers and respond to capsaicin. Immunofluorescence image of 50B11
cells stained with anti-TRPV-1 antibody (A). Nuclei are
counterstained with DAPI. Scale bar=25 .mu.m. Fold change in TRPV-1
mRNA in response to differentiation and neurotrophic factor
treatment is seen (13). (n=6-8/group; error bars denote standard
error of mean; *=p<0.05 compared to baseline; **=p<0.05
PSK+GDNF versus FSK+NGF) Measurements of intracellular calcium
levels of undifferentiated 50B11 with vehicle control treatment
(red line), after differentiation with forskolin with (green) and
without (blue) capsazepin pretreatment (C). Single arrow indicate
the time at which capsazepin was added and double arrows indicate
the time at which capsaicin was added.
[0032] FIGS. 4A-B depict Capsaicin induced neurotoxicity. 50B11
cells were grown in 96-well plates and treated with varying doses
of capsaicin (A) and capsaicin plus capsazepin (B) for 24 hours;
cellular ATP levels were measured and expressed as a percentage of
control cultures. (n=8/condition, error bars denote standard error
of mean, *=p<0.05 compared to controls)
[0033] FIGS. 5A-B depict ddC induced neurotoxicity and rescue by
GPI-1046. 50B11 cells were grown in 96-well plates and treated with
varying doses of ddC and ddC plus GPI-1046 (A) for 24 hours;
cellular ATP levels were measured and expressed as a percentage of
control cultures. (n=8/condition, error bars denote standard error
of mean, *=p<0.05 compared to controls). In validation
experiments, 50B11 cells were differentiated and allowed to extend
their neurites 24 hours. Then they were treated with ddC or ddC
plus GPI-1046 for another 24 hours, and total neurite lengths were
measured (B). (n=6/condition, error bars denote standard error of
mean, *=p<0.05 compared to controls).
DETAILED DESCRIPTION OF THE INVENTION
[0034] To obtain immortalized cells, e.g., dorsal root ganglion
cells, the inventors developed a method that reproducibly yields
clonal lines of cells, e.g., dorsal root ganglion cells, by
introducing an oncogene and hTERT into the cells.
[0035] The methods of the invention are particularly useful in
creating immortalized cells from cells that are known to be
difficult to immortalize. Specifically, the methods of the
invention can be used with any cell type, but are particularly
useful in cells that have been historically difficult to
immortalize, e.g., neuronal cells.
[0036] As used herein, the term "vector" refers to a polynucleotide
construct designed for transduction/transfection of one or more
cell types including for example, "expression vectors" which are
designed for expression of a nucleotide sequence in a host cell,
such as a neuronal cell.
[0037] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. These
terms include a single-, double- or triple-stranded DNA, genomic
DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and
pyrimidine bases, or other natural, chemically, biochemically
modified, non-natural or derivatized nucleotide bases. The backbone
of the polynucleotide can comprise sugars and phosphate groups (as
may typically be found in RNA or DNA), or modified or substituted
sugar or phosphate groups. Alternatively, the backbone of the
polynucleotide can comprise a polymer of synthetic subunits such as
phosphoramidates and thus can be a oligodeoxynucleoside
phosphoramidate (P--NH2) or a mixed phosphoramidate-phosphodiester
oligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;
Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et
al. (1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate
linkage can be used in place of a phosphodiester linkage. Braun et
al. (1988) J. Immunol. 141: 2084-9; Latimer et al. (1995) Molec.
Immunol. 32:1057-1064. In addition, a double-stranded
polynucleotide can be obtained from the single stranded
polynucleotide product of chemical synthesis either by synthesizing
the complementary strand and annealing the strands under
appropriate conditions, or by synthesizing the complementary strand
de novo using a DNA polymerase with an appropriate primer.
[0038] The term "oncogene" as used herein is intended to mean a
gene whose action promotes cell proliferation. Oncogenes are
altered forms of proto-oncogenes and are often expressed in
cancerous cells. Exemplary oncogenes include large T antigen, myc,
abl, ras, and raf.
[0039] "Schwann cell" is a cell of neural crest origin that forms a
continuous envelope around each peripheral nerves fiber in situ. A
Schwann cell can be identified by detecting the presence of one or
more markers of Schwann cell such as glial fibrillar acidic protein
(GFAP), protein S100, laminin, or nerve growth factor (NGF)
receptor, e.g., using antibodies against these markers.
Furthermore, Schwann cells have a characteristic morphology that
can be detected by microscopic examination of cultures thereof.
[0040] "Immortalized cell line" as used herein means a cell line
that can replicate and be maintained indefinitely in in vitro
cultures under conditions that promote growth, preferably at least
over a period of a year or years.
[0041] "Cell line" as used herein is a population or mixture of
cells of common origin growing together after several passages in
vitro. By growing together in the same medium and culture
conditions, the cells of the cell line share the characteristics of
generally similar growth rates, temperature, gas phase, nutritional
and surface requirements. The cell line can become more homogenous
with successive passages and selection for specific traits. Clonal
cells are those which are descended from a single cell. A cloned
cell culture is a cell culture derived from a single cell.
Immortalized cell lines are cells that have been modified to
undergo indefinite numbers of successive passages.
[0042] A SV40 Large T Antigen (SV-40 LTA) oncogene is intended to
encompass any nucleotide sequence which encodes a protein having
the function of polyoma (or SV-40) LTa and which is capable of
being expressed in the host cell, e.g., a neuronal cell.
[0043] The term "hTERT" as used herein is an abbreviation for the
human telomerase reverse transcriptase, i.e., the catalytic protein
component of human telomerase. hTERT is described in Cong, Y. S.,
et al. (1999) Hum. Mol. Genet. 8 (1), 137-142 and can be found in
GenBank as Accession Number AAD12057. Although human TERT is
exemplified herein, one of skill in the art will recognize that
TERT molecules from other species, or variants of human TERT that
maintain the biological function of hTERT are useful in the methods
of the invention.
[0044] The human telomerase catalytic subunit has been cloned (see
Nakamura, et al. (1997) Science 277: 955; Mayerson, et al. (1997)
Cell 90: 78; and Kilian, et al. (1997) Hum Mol Genet 6: 2011; U.S.
Pat. No. 6,166,178). Sources of the coding sequence for the human
telomerase subunit include any cells that demonstrate telomerase
activity such as immortal cell lines, tumor tissues, germ cells,
proliferating stem or progenitor cells, and activated lymphocytes.
The nucleic acid can be obtained using methods known in the
art.
[0045] As used herein, the term "pain" is art recognized and
includes a bodily sensation elicited by noxious chemical,
mechanical, or thermal stimuli, in a subject, e.g., a mammal such
as a human. The term "pain" includes chronic pain, such as lower
back pain; pain due to arthritis, e.g., osteoarthritis; joint pain,
e.g., knee pain or carpal tunnel syndrome; myofascial pain, and
neuropathic pain. The term "pain" further includes acute pain, such
as pain associated with muscle strains and sprains; tooth pain;
headaches; pain associated with surgery; or pain associated with
various forms of tissue injury, e.g., inflammation, infection, and
ischemia.
[0046] As used herein, the term "pain disorder" includes a disease,
disorder or condition associated with or caused by pain. Examples
of pain disorders include arthritis, allodynia, a typical
trigeminal neuralgia, trigeminal neuralgia, somatoform disorder,
hypoesthesis, hypealgesia, neuralgia, heuritis, neurogenic pain,
analgesia, anesthesia dolorosa, causlagia, sciatic nerve pain
disorder, degenerative joint disorder, fibromyalgia, visceral
disease, chronic pain disorders, migraine/headache pain, chronic
fatigue syndrome, complex regional pain syndrome, neurodystrophy,
plantar fasciitis or pain associated with cancer.
[0047] The term pain disorder, as used herein, also includes
conditions or disorders which are secondary to disorders such as
chronic pain and/or neuropathic pain, i.e., are influenced or
caused by a disorder such as chronic pain and/or neuropathic pain.
Examples of such conditions include, vasodialation, and
hypotension; conditions which are behavioral, e.g., alcohol
dependence (see, e.g., Hungund and Basavarajappa, (2000) Alcohol
and Alcoholism 35:126-133); or conditions in which detrimental
effect(s) are the result of separate disorders or injuries, e.g.,
spinal cord injuries.
[0048] The methods of the instant invention rely on the
introduction of two DNA segments, i.e., genes, into a cell which
results in the formation of an immortalized cell. These cells can
be further differentiated by the addition of a differentiating
agent. Importantly, the methods of the invention rely on the
incorporation of an oncogene and hTERT into a cell, but the method
of introducing these genes into the cell are of secondary
importance.
[0049] The manner in which the oncogene or hTERT coding region is
introduced into the cells of interest is not critical, as long as a
functional polypeptide is expressed. Expression can be
extrachromosomal or following integration into the cellular genome.
Any of a variety of techniques can be used to introduce the
oncogene or the hTERT gene into the desired cells, including
electroporation, liposomes, or viral vectors. See Molecular
Cloning, 3.sup.rd Edition, 2001, by Sambrook and Russell. In one
embodiment, the coding sequence is introduced using a viral vector,
for example SV40, adenovirus, Herpes simplex virus,
adeno-associated virus, and the like. See Blomer et al., Human
Molecular Genetics 5 Spec No: 1397-404, 1996; Zern et al., Gene
Ther. 6: 114-120, 1999; and Robbins et al., Trends in Biotechnology
16: 35-40, 1998.
[0050] A specific means for incorporating the hTERT coding region
into the cells of interest is to use a recombinant retrovirus that
provides for integratration of the DNA segment efficiently and
stably into the genome of the target cell.
[0051] The retroviral vectors generally include as operatively
linked components, retroviral long terminal repeats, packaging
sequences and cloning site(s) for insertion of heterologous
sequences. Other operatively linked components may include a
nonretroviral promoter/enhancer and a selectable marker gene.
Examples of retrovirus expression vectors which can be used include
DC-T5T (Sullenger et al. 1990. Mol. Cell Biol. 10: 6512-65230), kat
(Blood. 1994 83: 43-50), BOSC (Proc. Natl. Acad. Sci. (USA) (1993)
90: 8392-8396), pBabe (Proc. Natl. Acad. Sci. (USA) (1995) 92:
9146-9150) and RetroXpreSS..TM.. (Clontech, Palo Alto, Calif.). An
expression vector is available that includes the hTERT gene, for
example pBabe-puro-hTERT (Morgenstern and Land 1990). In some
instances, it may be desirable to increase expression of the hTERT
gene by utilizing other promoters and/or enhancers in place of the
promoter and/or enhancers provided in the expression vector. These
promoters in combination with enhancers can be constitutive or
regulatable. Any promoter/enhancer system functional in the target
cell can be used. (See for example, Molecular Virology pp. 176-177;
Hofmann, et al. 1996. Proc. Natl. Acad. Sci. (USA) 93: 5185-5190;
Coffin and Varmus, 1996. Retroviruses. Cold Spring Harbor Press,
NY; Ausubel et al. 1994. Current Protocols in Molecular Biology.
Greene Publishing Associates, Inc. & Wiley and Sons, Inc.).
Examples include: CMV immediate-early promoter, SV40, thymidine
kinase promoter, metalothionine promoter, and tetracycline operator
(Hofman et al., (1996) Proc. Natl. Acad. Sci (USA) 93:
5185-5190).
[0052] For transfection, the neuronal cells or other cells to be
transfected are suspended in a suitable culture medium containing
recombinant retrovirus vector particles. Many different suitable
culture media are commercially available. They include DMEM, IMDM,
and .alpha.-MEM, with 5-30% serum and often further supplemented
with, e.g., BSA, one or more antibiotics and optionally growth
factors suitable for stimulating cell division. Recombinant
retrovirus vector particles are harvested into this medium by
incubating the virus-producing cells in this medium. To enhance
gene transfer, compounds such as polybrene, protamine sulphate, or
protamine HCl generally are added. Usually, the cultures are
maintained for 24 days and the recombinant retrovirus vector
containing medium is refreshed daily. Optionally, the cells to be
transfected are precultured in medium with growth factors but
without recombinant retrovirus vector particles for up to 2 days,
before adding the recombinant retrovirus vector containing medium.
For successful gene transfer it is essential that the target cells
undergo replication in culture. It is often beneficial to
transform, transfect, or electroporate a number of times to obtain
a higher number of cells containing the desired DNA segment. To
maximize the number of cells containing the desired DNA segment,
the cells are transformed, transfected, or electroporated and
allowed to recover for a number of hours or days and then
transformed, transfected, or electroporated again. This process may
be repeated 2, 3, 4, 5, 6 or more times in order to maximize the
number of cells containing the desired DNA. After the final cycle
is performed, cells containing the desired DNA are selected using
methods that are routine in the art.
[0053] In exemplified embodiments, the oncogene and hTERT are
introduced into the cell by electroporation. After one or more
rounds of electroporation, cells containing the oncogene are
selected using an antibiotic resistance marker introduced into the
cell along with the oncogene. Once cells containing the oncogene
are selected, hTERT is introduced by electroporation. After one or
more rounds of electroporation, cells containing hTERT are
selected. The selected cells contain both the oncogene and
hTERT.
[0054] Once cells containing an oncogene and hTERT are selected,
the cells can be differentiated by exposing the cells to
differentiation agent. In the case of neuronal cells, this agent
can be cAMP, a cyclic AMP analog, or a substance that increases
intracellular levels of cAMP. Exemplary cAMP analogs are
8-pCPT-2'-O-Me-cAMP, 8Br-cAMP, Sp-cAMPS, and forskolin. In one
exemplified embodiment, the agent is forskolin.
[0055] The immortalized cells produced by the methods described
herein are particularly useful in screening assays. Specifically,
the cells produced by the methods of the instant invention are
ideal for high throughput screening. In a specific embodiments, the
immortalized nociceptive DRG sensory neurons produced by the
methods of the invention are ideal for identifying modulators of
neuropathic pain. Previous attempts at identifying modulators of
neuropathic pain have focused on identifying TRPV1 antagonists.
However, TRPV1 has been expressed in non-neuronal cell lines and
the calcium flux induced by capsaicin was used as an outcome
measure for high throughput screening (HTS) (Garcia-Martinez C et
al. (2002) Proc Natl Acad Sci USA 99:2374-2379; Gunthorpe M J et
al. (2004) Neuropharmacology 46:133-149; Masip I et al. (2004) J
Comb Chem 6:135-141). Although these cells expressing recombinant
TRPV1 may be useful, a nociceptive sensory neuronal cell expressing
TRPV1 will be more relevant because the non-neuronal cell lines may
lack the appropriate intracellular signaling pathways associated
with and downstream of TRPV1 in nociceptive sensory neurons.
Likewise, neuronal cells from other areas of the body can be
immortalized and would be useful for high throughput screening to
identify modulators for these cells. For example, sensory, motor or
cortical neurons can be immortalized and used to identify
modulators of, for example, Alzheimer's disease, Parkinson's
disease, Huntington's disease, multiple sclerosis, or amyotrophic
lateral sclerosis (ALS).
[0056] Accordingly, the instant invention provides methods of
screening for modulators of human cells, e.g., human neuronal
cells, by contacting an immortalized cell of the invention with a
candidate modulator and determining if the modulator has a desired
biological effect, e.g., binding to and/or modulating the activity
of TRPV1.
[0057] In a specific embodiment, the invention provides screening
methods using the immortalized nociceptive DRG neurons produced by
the methods of the invention to identify modulators of pain, e.g.,
neuropathic pain. Immortalized nociceptive DRG neurons are
contacted with candidate modulators and the ability to modulate,
for example, capsaicin induced toxicity can be monitored to
determine if a candidate modulator is a modulator of neuropathic
pain.
[0058] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules, ribozymes, or antisense molecules) which bind to bind to
and/or modulate the activity of the immortalized cells of the
invention. Compounds identified using the assays described herein
may be useful for treating pain disorders.
[0059] Candidate/test compounds include, for example, 1) peptides
such as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam, K. S. et al.
(1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature
354:84-86) and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids; 2) phosphopeptides
(e.g., members of random and partially degenerate, directed
phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993)
Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0060] Candidate modulators can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the `one-bead one-compound`
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to polypeptide libraries, while the other four approaches
are applicable to polypeptide, non-peptide oligomer or small
molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug
Des. 12:145).
[0061] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233. Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310.
[0062] Alternatively, test compounds can be designed based on the
structure of known modulators of pain, or compounds that are know
to bind to receptors expressed by neurons.
[0063] The ability of a given modulating agent to modulate pain can
be quantitated by using any one of the following tests: tight
ligation of L6 and L7, as a model of neuropathic pain; complete
Freund's adjuvant into knee joint or hind paw as a model of Long
term inflammatory pain (Palecek, J. (1992) Neurophysiol
68:1951-66); nerve ligation (CCI); thermal hyperalgesia, tactile
allodynia and cold allodynia (Carlton, S. M. et al. (1994) Pain
56:155-66); thermal paw withdrawal latency (Hargreaves test); von
Frey mechanical withdrawal threshold; the hot-plate latency test;
the tail flick test (Stone, L. S., et al. (1997) NeruroReport
8:3131-3135); the warm-water immersion tail flick assay (Stone, L.
S., et al. (1997) NeruroReport 8:3131-3135); the crush injury to
the sciatic nerve test (De Konig, et al. (1986) J. Neurol. Sci.
74:237-246); the cold water allodynia test (Hunter, et al. (1997)
Pain 69:317-322; the paw pressure latency assay (Hakki-Onen, S., et
al. (2001) Brain Research 900(2):261-7; or the radiant heat test
(Yoshimura, M., (2001) Pharm. Research 44(2):105-11).
[0064] Briefly, the tail flick latency test involves projecting a
beam of light to the tail of an animal. The time is measured from
the onset of the tail heating and stops at the moment of the tail
flick. Typically, five tail flick latency (TFL) measurements are
made per rat per session with 5-10 minutes between trials.
[0065] The preceding paragraphs set forth high throughput screening
methods using immortalized nociceptive DRG neurons to identify
modulators of neuropathic pain. However, one of skill in the art
could adapt these assays to identify modulators of other conditions
and immortalized cell lines made using the methods of the
invention.
EXAMPLES
[0066] It should be appreciated that the invention should not be
construed to be limited to the examples that are now described;
rather, the invention should be construed to include any and all
applications provided herein and all equivalent variations within
the skill of the ordinary artisan.
Example 1
Generation and Characterization of DRG Neuronal Cell Line
[0067] Materials and Methods
[0068] Unless noted otherwise all reagents and materials were
purchased from Invitrogen (Carlsbad, Calif.). Animal studies were
conducted according the protocols approved by the institutional
Animal Care and Use Committee.
[0069] Construction of SV40 Large T-Antigen and hTERT Expression
Vectors
[0070] For cloning of the SV40 large T-antigen, plasmid construct
pZipSV776-1 was used as the template for PCR amplification of the
gene fragment coding for SV40 large T-antigen. PCR reaction was
primed by oligonucleotides 5'-CACCGCTTTGCAAAGATGGATAAAG (sense) and
5'-AATTGCATTCATTTTATGTTTCA (anti-sense). Amplification was
performed using the Expend High Fidelity PCR System (Roche,
Indianapolis, Ind.). The PCR product was cloned into the
pENTR/D-TOPO vector by directional TA-cloning. After confirmation
of the sequence, the target SV40 large T-antigen gene was
transferred into pLenti6/V5-Dest vector using Gateway technology.
In the destination vector, the SV-40 large T-antigen was under the
control of P.sub.cmv, and the selection marker, blasticidin
resistance gene, was under the control of P.sub.sv40. The hTERT
expression construct pBabe-hygro-hTERT carrying hygromycin
resistant gene (also a kind gift of William C. Hahn at Harvard
University), was used to transfer the hTERT gene into the
pLenti6/V5-Dest vector using Gateway technology. In the destination
vector, the hTERT was under the control of P.sub.cmv, and the
selection marker, hygromycin resistance gene, was under the control
of P.sub.sv40. The expression plasmids were prepared and purified
using Plasmid MIDI Kit (Qiagen, Valencia, Calif.). Endotoxin-free
plasmid was suspended in distilled water for electroporation.
[0071] Electroporation into Dissociated DRG Neurons and Selection
of Clones
[0072] Dissociated primary DRG neuronal cells were prepared as
previously described (Hoke A et al. (2003) J Neurosci 23:561-567;
Keswani S C et al. (2003) Ann Neurol 53:57-64) and the plasmid was
electroporated. Approximately 5.times.10.sup.4 cells in 90 ml
Opti-MEM media were mixed with 10 ml plasmid (1 mg/ml) and
transferred into a 0.2 cm Gene Pulser cuvette (Bio-Rad, Hercules,
Calif.). After 10 minutes of incubation at room temperature, a
single square-wave pulse (100 V, 950 mF, .about.40 ms) was
delivered by a Gene Pulser II with a Capacitance Extender Plus
(Bio-Rad, Hercules, Calif.). Culture medium at 4.degree. C. was
immediately added to the cells and the cuvette was kept on ice for
10 minutes. Cells were plated in T75 flasks in culture medium
without antibiotics (Neurobasal medium, 10% FBS, 0.5 mM glutamine,
1.times.B-27 supplement, 0.2% glucose). In order to increase the
efficiency of electroporation and incorporation of large T-antigen
into terminally differentiated sensory neurons, the process of
electroporation was repeated 3-4 times before addition of
antibiotic selection media. About 60-70% of the cells survived the
electroporation process. Twenty-four hours after the last
electroporation, culture medium was replaced by selection medium
containing blasticidin (5 .mu.g/ml) and cells were maintained in
this medium for 1-2 weeks until isolated colonies with 200-300
cells formed. Colonies were picked and expanded using standard
culture methods when reached 80-90% confluence. For hTERT
transduction, SV40 transfected and blasticidin resistant cells were
trypsinized and electroporated with the hTERT plasmid as above for
the large T-antigen. The electroporation was repeated 34 times.
About 60-70% of the cells survived the electroporation process.
Twenty-four hours after the last electroporation, culture medium
was replaced by selection medium containing hygromycin (50 mg/ml)
and cells were maintained in this medium for 1-2 weeks until
isolated colonies with 200-300 cells formed. Colonies were picked
and expanded using standard culture methods when reached 80-90%
confluence.
[0073] Induction of Neuronal Differentiation and Characterization
of the Immortalized Neuronal Clone
[0074] One of the immortalized DRG neuronal cell lines (50B11)
maintained self-replication capability over many cell divisions
(>300) and was used in further analysis of neuronal properties.
Differentiation and axonal elongation was induced in these cells by
addition of forskolin (50 .mu.M) into the culture medium. Within
hours, more than 90% cells stopped dividing and extended long
neurites. These cells were grown in 24-well plates on glass
coverslips, fixed with 4% paraformaldehyde and immunostained for
presence of neurofilament (SMI-32 antibody from Sternberger
Monoclonals Inc., Lutherville, Md.), .beta.III-tubulin (Promega,
Madison Wis.), transient receptor potential channel, vanilloid
subfamily member-1 (TRPV-1) (Abcam, Cambridge, Mass.), calcitonin
gene related protein (CGRP) (source, city, state) or isolectin B4
(IB4) (Vector Laboratories, Burlingame, Calif.) using standard
methods (Keswani et al., supra). Slides were counterstained with
4',6-Diamidino-2-phenylindole (DAPI) and mounted with Vectashield
(Vector Laboratories, Burlingame, Calif.). Specificity of all
primary and secondary antibodies was confirmed using appropriate
positive and negative control cultures.
[0075] The electrophysiological recording techniques employed were
similar to those described by Hamill et al. (Hamill O P et al.
(1981) Pflugers Arch 391:85-100). The external solution contained
(mM) 145 NaCl, 5 KCl, 2 CaCl.sub.2, 1 MgCl.sub.2, 10 D-glucose and
10 N-2-hydroxyethylpiperazine-N.rho.-2-ethanesulfonic acid (HEPES)
(pH 7.4; 310-320 mOsmol.) Cells were continuously superfused at 2-3
ml/min. Using the whole-cell patch-clamp technique, data were
obtained with borosilicate thin-walled micropipettes (BORO,
BF150-110-10, Sutter, Novato, Calif.) made with a Flaming-Brown
Puller (P-87, Sutter Instruments, Novato, Calif.). Micropipettes
were filled with (in mM) 140 KCL, 1 CaCl.sub.2, 1 MgCl.sub.2, 10
HEPES, 10 ethyl glycol-bis(3-aminoethyl
ether)-N,N,N.rho.,N.rho.-tetraacetic acid (EGTA), 4 Mg-ATP adjusted
to a pH of 7.3 with Tris buffer. Pipette resistances measured 3 to
6 M.SIGMA.. Current-clamp recordings were obtained with an Axopatch
200B amplifier (Axon Instruments Inc. Foster City, Calif.) and data
was filtered on-line at 2 kHz. Recordings were made at a holding
potential (V.sub.H) of -60 mV. For statistical evaluation we used
ANOVA (Origin version 6, Microcal Software Inc., Northampton,
Mass.)
[0076] For analysis of changes in gene expression, 50B11 cells were
grown in media containing forskolin (50 .mu.M), NGF (10 ng/ml),
GDNF (10 ng/ml) or vehicle control for 24 hours. Total RNA was
isolated using the TRIzol Reagent according to the manufacturer's
recommendation. Two .mu.g total RNA was reverse-transcribed using
Ready-To-Go You-Prime First-Strand Bead (Amersham Biosciences,
Piscataway, N.J.) according to manufacturer's protocols. Real-time
PCR was carried out in a DNA Engine Opticon Continuous Fluorescence
Detection System using DyNAmo SYBR Green Polymerase (MJ Research,
Waltham, Mass.). All primers were designed according to the
individual gene sequence in the GenBank/EMBL nucleotide sequence
database (primer sequences are available upon request). The binding
positions of all primers were chosen to produce amplicons of 150 to
200 base pairs and to achieve maximum efficiency and specificity.
All primer sequences were checked for specificity by a BLAST search
in the GenBank/EMBL nucleotide sequence database. The primers were
synthesized by Integrated DNA Technologies, Inc. (Coralville,
Iowa). The amplification of internal control housekeeping gene,
GAPDH, was carried out using a commercial kit (Applied Biosystems,
Foster City, Calif.) according to the protocol supplied by the
manufacturer. The expression levels of individual genes before and
after treatment were calculated using the comparative C.sub.T
method.
[0077] Ca.sup.2+ microfluorimetry and imaging in
forskolin-differentiated 50HB cells were performed by ratiometric
imaging of the Ca2+-sensitive fluorescent dye fura-2. Cells were
grown on glass coverslips in 12-well dishes and calcium imaging was
done with and without differentiation with 50-.mu.M forskolin. Cell
were loaded for 15 min at 37.degree. C. with 2 M fura-2
acetoxymethyl ester (Molecular Probes, Carlsbad, Calif.) in
Krebs-HEPES buffer (100 mM NaCl, 2.0 mM KCl, 1.0 mM CaCl2, 1.0 mM
MgCl2, 1.0 mM NaH2PO4, 4.2 mM NaHCO3, 12.5 mM HEPES and 10.0 mM
glucose), then washed for 3 times in buffer to remove remaining
fura-2 ester. The coverslip with loaded cells was then mounted on
an inverted epifluorescence microscope (Zeiss, Axiovert 200) and
covered with 60 .mu.l Krebs-HEPES buffer or buffer with capsaicin.
In capsazepin pretreatment experiments, capsazepin was added Images
were acquired every .about.3 seconds with an extended Hamamatsu
Digital Camera C4742-95 (Hamamatsu Photonics, Barcelona, Spain)
using a dual filter wheel (Sutter Instruments, Novato, Calif., USA)
equipped with 340 and 380 nm, 10-nm-bandpass filters (Omega Optics,
Madrid, Spain). Data was acquired using InCyt Im2 software
(Intracellular Imaging, Inc.). Fluorescence changes are expressed
as the ratio of fluorescence at 340 and 380 nm
(F.sub.340/F.sub.380).
[0078] Neuronal Toxicity Assays
[0079] Conditions for culturing the 50B11 cells and measuring the
ATP levels were optimized for the 96-well plate format. Initially
500 cells/well were plated in 96-well plates for 24 hours and then
differentiated with forskolin (50 .mu.M) in culture medium with
reduced serum (0.2%). Varying concentrations of ddC with or without
immunophilin ligand GPI-1046 were added to the wells for another 24
hours. Cellular ATP levels were measured using the ViaLight Plus
kit (Cambrex, city, state) according to manufacturer's
instructions. This luciferase-based assay allows measurement of ATP
levels on a luminometer with minimal manipulation of the well
contents.
[0080] Measurements of axonal lengths to determine axonal
degeneration induced by ddC were done as described before (Keswani
et al., supra). Briefly, DRG cultures were prepared from embryonic
E14.5 rats, plated onto collagen coated glass coverslips and
allowed to extend axons for 48 hours. Then ddC, GPI-1046 or vehicle
controls were added to the media for another 24 hours. Cells were
fixed and stained with anti-.beta.III-tubulin antibody to delineate
the axons. Axon length measurements were done in multiple fields
using a random sampling method. Each experiment was done in
triplicates and repeated at least twice. Statistical analysis was
done using ANOVA with correction for multiple comparisons.
[0081] Results
[0082] Immortalized DRG Neuronal Cells Extend Neurites Express
Neuronal Markers and Generate Action Potentials after
Differentiation
[0083] One of the clones generated after immortalization was
further studied after evaluation using the initial screening of
neurite extension in response to forskolin. This clone, 50B11,
stopped dividing immediately after addition of forskolin and within
4 hours extended neurites at least twice as long as the neuronal
body diameter (FIGS. 1A and 1B). Within 24 hours of
differentiation, the cells were positive for neuronal markers
.beta.III-tubulin and neurofilament (FIGS. 1C and 1D). We studied
these cells before and after differentiation using patch clamping.
Data were obtained from a total of 14 cells (8 undifferentiated
cells and 6 differentiated cells). Collectively these cells
displayed a mean resting membrane potential of -57.9.+-.2.1 mV.
There were no statistically significant differences between the
mean resting potential values of the undifferentiated and
differentiated groups (-57.4.+-.1.9 mV versus -58.3.+-.2.7 mV,
mean.+-.SEM; ANOVA, P>0.05). No spontaneous activity, either
synaptic or action potential discharge, was observed when
differentiated or undifferentiated cells were held at their resting
membrane potential for periods up to 10 min. Electrical stimulation
of undifferentiated cells with depolarizing current steps did not
induce an action potential (n=0/8). On the other hand, when
differentiated cells were stimulated, action potentials could be
elicited (n=5/6; FIG. 1E).
[0084] 50B11 Neuronal Line Express Markers of Small Diameter
Sensory Neurons
[0085] Small diameter DRG sensory neurons are generally divided
into two categories; peptidergic ones with dependence on NGF, and
non-peptidergic ones with dependence on GDNF (Bennett D L et al.
(1998) J Neurosci 18:3059-3072). Markers such as CGRP for
peptidergic neurons and IB4 for non-peptidergic neurons can
identify these subgroups of small diameter sensory neurons. The
50B11 line expressed both markers when differentiated in the
presence of forskolin (FIGS. 2A and 2B). Furthermore, 50B11 cells
expressed receptors for NGF (low affinity NGF receptor p75 and high
affinity Trk-A) and GDNF (c-ret and GDNF family receptor alpha-1,
GFR.alpha.-1) and upregulated these receptors when differentiated
with forskolin (FIG. 2C). Interestingly, the upregulation of
receptors was neurotrophic factor specific; NGF receptor, Trk-A was
more upregulated in the presence of NGF compared to GDNF and
similarly GDNF receptor, GFR.alpha.-1 was more upregulated in the
presence of GDNF compared to NGF.
[0086] 50B11 Neuronal Line Express Nociceptive Markers and Respond
to Capsaicin
[0087] Once we determined that the 50B11 line had small diameter
sensory neuronal markers, we explored the possibility that it was a
nociceptive neuron. The cells expressed capsaicin receptor TRPV-1
and upregulated their expression when differentiated with forskolin
with and without neurotrophic factor treatment (FIG. 3).
Furthermore, the 50B11 cells responded to capsaicin with a rapid
rise in intracellular calcium levels. This effect of capsaicin on
the 50B11 cells was preventable by pretreatment of the cells with
capsazepin, a specific blocker of TRPV-1, suggesting that the
effect of capsaicin was mediated through TRPV-1. A graph
representative of multiple intracellular calcium measurements is
shown FIG. 3C.
[0088] Next, we tested whether we can evaluate the neurotoxicity of
capsaicin in an assay suitable for 96-well plate format. Capsaicin
causes axonal degeneration and death of nociceptive DRG sensory
neurons [ref here]. In order to evaluate cell survival we used a
luciferase-based assay to measure cellular ATP levels. We first
optimized the assay by measuring ATP levels in different numbers of
cells grown in the 96-well plates and found that the optimum number
of cells for neurotoxicity assays was between 500 and 1000 cells
per well. We also optimized the culture conditions and found that
low serum levels of 0.2% fetal bovine serum provided the most
reliable results (data not shown). We then examined the
dose-response curve of capsaicin and found that 10 .mu.M of
capsaicin caused about 50% reduction in ATP levels (FIG. 4A);
similar to the dose required for neurotoxicity of capsaicin in
primary DRG neurons. The capsaicin-induced neurotoxicity was
preventable by co-administration of TRPV-1 blocker capsazepin in a
dose-dependent manner (FIG. 4B).
[0089] The Immortalized 50B11 Neuronal Line is Suitable for
High-Throughout Drug Screening
[0090] One of the potential uses of 50B11 sensory neuronal line
will be their use in high-throughput screening assays. Our
laboratory had developed in vitro model of antiretroviral toxic
neuropathy using primary DRG sensory neurons (Keswani et al.,
supra). We adapted this assay to the 50B11 cells and measured
cellular ATP levels after varying concentrations of ddC (FIG. 5A).
There was a dose-dependent toxicity of ddC at concentrations
similar to therapeutic plasma levels in HIV patients. This
neurotoxicity was preventable by co-administration of a
neuroprotective compound, GPI-1046, a non-immunosuppressive
immunophilin ligand. We also validated this toxicity using a more
standard measure of axonal degeneration where we differentiated the
50B11 cells, let them extend neurites and then treated them with
ddC with and without GPI-1046 for 24 hours and then measured their
total neuritic lengths (FIG. 5B).
[0091] Discussion
[0092] Cellular tools for drug development for peripheral
neuropathies and neuropathic pain are limited. We developed a novel
method to immortalize nociceptive dorsal root ganglion (DRG)
neurons and show that the immortalized DRG neuronal line (50B11)
extend neurites when differentiated, express receptors
characteristic of small sensory neurons and nociceptive receptor
TRPV-1, generate action potentials when depolarized and respond to
capsaicin. Furthermore, the cells are easy to grow in large
quantities and suitable for high throughput drug screening using in
vitro assays for neuropathic pain and peripheral neuropathies.
[0093] DRG neurons are terminally differentiated cells that extend
long axons to their target tissues. More than half of all DRG
neurons are unmyelinated, extend axons to the skin and have
nociceptive properties. In many studies on peripheral neuropathies
and neuropathic pain, rat or mouse primary DRG neurons are used.
However, obtaining these cells in sufficient quantities to perform
high-throughput drug screening is nearly impossible. An alternative
is to use neuronal cell lines derived from neuroblastomas or
immortalized neural crest precursor/stem cell lines (Rao M S and
Anderson D J (1997) J Neurobiol 32:722-746). However, these
approaches have caveats for neuropathic pain research and drug
screening. These cells are often heterogeneous and retain the
potential to differentiate into multiple neuronal and non-neuronal
cell types in a mixed environment. Furthermore, they are not likely
to respond to drugs in a consistent manner because of this
heterogeneity in culture. In contrast, the nociceptive DRG neuronal
cell line that we developed is clonal; therefore, all of the cells
are similar to each other and in biological assays behave in a
predictable manner. These cells can be grown in large quantities,
differentiated into nociceptive neurons that express proper markers
and ion channels necessary for nociception and generation of action
potentials, and used in high-throughput drug screening.
[0094] The immortalized DRG neuronal line, 50B11, was likely to be
generated from a nociceptive neuron with potential to differentiate
into either a peptidergic neuron with NGF dependency or
non-peptidergic neuron with GDNF dependency. During development,
all future nociceptive neurons are dependent on NGF and express
markers for NGF-dependent neurons (p75 and TrkA) (McMahon S B et
al. (1994) Neuron 12:1161-1171), but a subgroup switch dependency
and express markers of GDNF-dependent neurons such as IB4 (Molliver
D C et al. (1997) Neuron 19:849-861). The 50B11 line retained this
bi-potentiality because in the presence of a given neurotrophic
factor, it upregulated the appropriate receptors. Furthermore, the
cells extended longer neurites in the presence of either NGF or
GDNF.
[0095] In order to immortalize rat DRG neurons, we used a two-step
transformation process. Although combination of SV-40 large T
antigen and hTERT had been used before to immortalize primary
airway epithelial cells (Lundberg A S et al. (2002) Oncogene
21:4577-4586), this approach had not been applied to generation of
immortalized cell lines from terminally differentiated cells such
as neurons. In order to increase our transfection efficiency and
generation of transformed neurons, we used a different approach and
performed multiple electroporations before adding the selection
antibiotic to the media. We obtained multiple clones and
characterized one in detail. We were able to grow this cell line,
50B11, through multiple doublings (well over 300) without loss of
differentiation potential. Stocks of cells from early and late
passages had similar properties in terms of their differentiation
potential.
[0096] Differentiation into neurons with nociceptive properties was
accomplished by using forskolin. During development DRG neurons
express high levels of cAMP but downregulate it after they are
mature and their axons reach the target tissues. Elevated cAMP
levels in the DRG line could be accomplished by methods other than
forskolin, but we chose forskolin mainly because of ease of use and
relatively lower cost compared to other choices such as membrane
permeable dibutryl-cAMP (db-cAMP). This is an important issue to
consider in designing assays for high-throughput screening.
Furthermore, we chose an assay that is also easy to scale up for
high-throughput screening. Measurement of cellular ATP levels using
the luciferase-based assay is suitable in multiple ways. First, one
can use it as a measure of cellular health and cell numbers as ATP
levels in cells correlate with the number of healthy cells. Second,
the assay is simple to administer with no wash steps involved. It
requires addition of reagents into the wells twice, once to lyse
the cells and second to add reagents for the luminescence. This
improves reproducibility of the assay and will reduce the number of
multiplicates needed during drug screening.
[0097] In summary we have developed an immortalized DRG sensory
neuronal line with nociceptive properties suitable for
high-throughput drug screening for peripheral neuropathies and
neuropathic pain. The transfection and selection methods we
developed can be used to generate other neuronal populations,
including neurons from human tissues such as brain, spinal cord,
dorsal root sensory ganglia or autonomic ganglia.
Example 2
Generation and Characterization of Human Fetal Astrocytes and
Schwann Cells
[0098] Human astrocytes or Schwann cells were prepared from aborted
fetal tissues using standard cell culture techniques. Cells were
cultured in Neurobasal media (Neurobasal MEM plus 10% FBS, 0.5 uM
glutamin, 2% glucose, 1.times.B27 supplement) for 3-7 days in an
incubator with 5% CO2 at 37.degree. C. Cells were detached with a
cell scraper and washed 2 times in Opti-MEM (Invitrogen, CA) and
0.8-1.0.times.10.sup.6 cells were suspended in 100 ul Opti-MEM and
dissociated with pipetting using a 200 .mu.l tip.
[0099] Five .mu.g pLenti6/V5-DEST plasmid carrying hTERT gene
(pLenti6/hTERT) in 1.7 .mu.l TE (pH8.0) and 15 .mu.g
pLenti6/V5-DEST plasmid carrying SV40 large T antigen gene
(pLenti6/SV40) in 5 .mu.l TE (pH8.0) were mixed with cells,
transferred into a 0.2 cm gene-pulser cuvette (Bio-Rad, CA) and
incubated for 5 minutes at room temperature. Gene Pulser Xcell
Electroporation System (Bio-Rad, CA) was set at 850 .mu.F.times.90V
and the cells were pulsed once (with a time constant of 3540
mini-second). 500 .mu.l ice-chilled culture media (antibiotics
free) was immediately added to the cells and the cuvette was kept
on ice for 5 minutes. Cells were then transferred into a 75
cm.sup.2 (T75) culture flask and cultured in antibiotics-free
Neurobasal media for 3-7 days until 70-80% confluence.
[0100] The next 3 electroporations were done with the same
procedure, but with different amount of pLenti6/hTERT and
pLenti6/SV40. For the second electroporation, 10 .mu.g
pLenti6/hTERT (in 3.3 .mu.l TE, pH 8.0) and 10 .mu.g pLenti6/SV40
(in 3.3 .mu.l TE, pH 8.0) were mixed with cells. For the third time
electroporation, 15 .mu.g pLenti6/hTERT (in 5 .mu.l TE, pH8.0) and
5 .mu.g pLenti6/SV40 (in 1.7 .mu.l TE, pH8.0) were mixed with
cells. For the fourth electroporation, 20 .mu.g pLenti6/hTERT (in
6.6 .mu.l TE, pH8.0) and 2 .mu.g pLenti6/SV40 (in 0.6 .mu.l TE,
pH8.0) were mixed with cells.
[0101] After the fourth electroporation, cells from each
gene-pulser cuvette were transferred into 3 T75 culture flasks and
cultured in Neurobasal media containing 5 .mu.g/ml blasticidin
(Invitrogen, CA) for 6-8 days. Blasticidin-resistant colonies were
detached with 0.05% trypsin, isolated with glass capillary pipettes
and transferred into 24 well plates. Cells were then cultured for
5-10 days (depending on the size of the original colony) in
blasticidin-containing media and a portion of the cells were plated
in a T75 culture flask for further cloning. The resulted colonies
were propagated in T75 flasks, stored frozen in freezing media
(culture media plus 10% DMSO), and characterized.
[0102] The immortalized human astrocyte and Schwann cell lines were
further characterized by RT-PCR, immunohistochemistry and Western
blotting. Cell lines expressed glial markers such as GFAP, S100,
CD44 and tenascin by RT-PCR, and GFAP and S100 by
immunohistochemistry. Furthermore, astrocyte line expressed
glutamate transporters EAAT-1 and EAAT-2 by RT-PCR and western
blotting. Further characterization, including biological assays are
ongoing.
INCORPORATION BY REFERENCE
[0103] The contents of all references, patents, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference.
EQUIVALENTS
[0104] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
2125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1caccgctttg caaagatgga taaag
25223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2aattgcattc attttatgtt tca 23
* * * * *