U.S. patent application number 10/151702 was filed with the patent office on 2003-05-08 for transcriptional regulation of target genes.
Invention is credited to During, Matthew J., Kaplitt, Michael G., Lozano, Andres M..
Application Number | 20030087264 10/151702 |
Document ID | / |
Family ID | 26848885 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087264 |
Kind Code |
A1 |
Kaplitt, Michael G. ; et
al. |
May 8, 2003 |
Transcriptional regulation of target genes
Abstract
The present invention describes a method of identifying
inducible genetic regulatory sequences that can control the
transcription of specific gene transcripts. Methods of using
inducible genetic regulatory sequences are also discussed. In
particular, the genetic regulatory sequences of the present
invention can modulate the transcription of a nucleic acid
transcript in vivo.
Inventors: |
Kaplitt, Michael G.; (New
York, NY) ; During, Matthew J.; (Philadelphia,
PA) ; Lozano, Andres M.; (Toronto, CA) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
26848885 |
Appl. No.: |
10/151702 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60292604 |
May 21, 2001 |
|
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Current U.S.
Class: |
435/6.16 ;
205/777.5; 435/287.2 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 15/00 20180101; C12Q 1/686 20130101; A61P 35/00 20180101; C12Q
1/686 20130101; A61P 7/06 20180101; C12Q 2521/501 20130101; C12Q
2525/155 20130101; A61P 25/16 20180101; A61P 3/10 20180101; A61P
37/04 20180101 |
Class at
Publication: |
435/6 ;
205/777.5; 435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed is:
1. A method of identifying a genetic regulatory sequence responsive
to a pulsatile electromagnetic stimulus, comprising: (a) inserting
a stimulator into a tissue of an animal subject; (b) applying a
pulsatile electromagnetic signal with the stimulator; wherein at
least a part of the tissue is stimulated; (c) identifying a gene
that has either enhanced or diminished transcription in the part of
the tissue stimulated; and (d) selecting a genetic regulatory
sequence involved in the enhanced or diminished transcription of
the gene; wherein the genetic regulatory sequence is identified as
being responsive to the pulsatile electromagnetic stimulus.
2. The method of claim 1, wherein the pulsatile electromagnetic
signal is provided by a bipolar stimulator.
3. The method of claim 1, wherein the pulsatile electromagnetic
signal is provided by a monopolar stimulator.
4. The method of claim 1, wherein at least a part of the tissue is
not stimulated by the stimulator; and wherein the identifying of
step (c) is performed by comparing the transcription of the genes
in the part of the tissue stimulated with that of a part of the
tissue that is not stimulated.
5. The method of claim 4, wherein a second stimulator is placed
into the part of the tissue that is not stimulated; and wherein no
pulsatile signal is applied by the second stimulator.
6. The method of claim 1, wherein the tissue is neural tissue.
7. The method of claim 6, wherein the neural tissue is brain
tissue.
8. The method of claim 1, wherein the pulsatile electromagnetic
signal is a magnetic stimulation.
9. An isolated genetic regulatory sequence identified by performing
the method of claim 1.
10. A vector for in vivo expression of a gene of interest in a
mammalian host cell, wherein the vector comprises the gene of
interest and a transcriptional control region comprising a genetic
regulatory sequence, and wherein the gene of interest is
operatively linked to the transcriptional control region, and the
genetic regulatory sequence is responsive to a pulsatile
electromagnetic stimulus.
11. The vector of claim 10, wherein the transcriptional control
region exhibits tissue specificity and is from a gene encoding a
protein expressed by the host cell.
12. The vector of claim 10, that is a replication defective
virus.
13. The vector of claim 10, wherein the host cell is a neural
tissue cell.
14. The vector of claim 13, wherein the neural tissue cell is a
brain cell.
15. A non-human mammalian host transformed with the vector of claim
10, wherein the expression of the gene of interest in the non-human
mammalian host can be modulated by applying a pulsatile signal with
a stimulator.
16. A cell transformed with the vector of claim 10, wherein the
expression of the gene of interest in the transformed cell can be
modulated by applying a pulsatile signal with a stimulator.
17. The cell of claim 16, wherein the transformed cell may be
transplanted into a recipient mammalian host, and wherein the
expression of the gene of interest in the recipient mammalian host
can be modulated by applying a pulsatile signal with a
stimulator.
18. The replication defective viral vector of claim 10 selected
from the group consisting of a replication defective herpes simplex
virus (HSV), a replication defective papillomavirus, a replication
defective Epstein Barr virus (EBV), a replication defective
adenovirus, a gutless adenovirus, a replication defective
adeno-associated virus (AAV), and a replication defective
lentivirus.
19. A method of delivering the vector of claim 10, to a target
tissue of an animal subject comprising administering the vector to
the tissue of the animal subject, wherein the expression of the
gene of interest in the target tissue can be modulated by applying
a pulsatile signal with a stimulator.
20. The method of claim 19, wherein the transcription of the gene
of interest in the target tissue is stimulated by a stimulator that
is present in the target tissue.
21. A method of regulating the expression of a gene of interest in
a tissue of an animal subject in which the vector of claim 10 has
been administered, comprising applying a pulsatile signal with a
stimulator which modulates the transcription of the gene of
interest in the tissue.
22. The method of claim 21, wherein the response to the pulsatile
signal by the genetic regulatory sequence stimulates the
transcription of the gene of interest.
23. The method of claim 21, wherein the response to the pulsatile
signal by said genetic regulatory sequence hinders the
transcription of the gene of interest.
25. A method of identifying a genetic regulatory sequence that is
responsive to a peptide comprising: (a) contacting a peptide with a
cell; (b) identifying a gene that exhibits altered transcription
relative to a cell not contacted with the peptide; and (c)
selecting a genetic regulatory sequence involved in the altered
transcription of the gene; wherein the genetic regulatory sequence
is identified as being responsive to the peptide.
26. The method of claim 25, wherein the altered transcription is an
enhancement or diminishment of transcription.
27. An isolated genetic regulatory sequence identified by
performing the method of claim 25.
28. The method of claim 25, wherein identifying a gene exhibiting
altered transcription is by 2-dimensional gel electrophoresis or
Northern blot analysis.
29. A vector for the in vivo expression of a gene of interest in a
mammalian host cell, wherein the vector comprises the gene of
interest and a transcriptional control region comprising the
genetic regulatory sequence of claim 27, and wherein the gene of
interest is operatively linked to said transcriptional control
region and the genetic regulatory sequence is responsive to the
peptide.
30. A method of ameliorating symptoms due to Parkinson's disease
comprising: (a) administering a vector to the subthalamic nucleus
of a patient having a symptom of Parkinson's disease; wherein the
vector comprises a nucleic acid encoding glutamic acid
decarboxylase operatively under the control of a genetic regulatory
sequence that is stimulated by a pulsatile electromagnetic signal;
(b) inserting a stimulator into the subthalamic nucleus of the
patient; and (c) applying a pulsatile electromagnetic signal with
the stimulator, wherein the glutamic acid decarboxylase is
expressed and leads to the amelioration of a symptom of Parkinson's
disease.
31. The method of claim 30, wherein the vector is a replication
defective viral vector.
32. The method of claim 30, wherein the vector is a CBA-GAD65 viral
vector.
33. A method of modulating the release of a stored compound by a
cell comprising: (a) administering a vector to the subthalamic
nucleus of an animal subject; wherein the vector comprises a
nucleic acid encoding a protein operatively under the control of a
genetic regulatory sequence that is stimulated by a pulsatile
electromagnetic signal, and wherein the expression of the protein
stimulates the production of a compound that is stored by a cell of
the animal subject; (b) inserting a stimulator into the subthalamic
nucleus of the animal subject; (c) applying a pulsatile
electromagnetic signal with the stimulator, wherein the protein is
expressed and the compound is produced; and wherein the cell stores
the compound; and (d) applying a second pulsatile electromagnetic
signal with the stimulator, wherein the release of the stored
compound from the cell is modulated.
34. The method of claim 33, wherein the compound is GABA.
35. The method of claim 34, wherein applying of a second pulsatile
electromagnetic signal is at a low frequency and the release of
GABA from the cell is increased.
36. The method of claim 34, wherein applying of a second pulsatile
electromagnetic signal is at a high frequency and the release of
GABA from the cell is decreased.
Description
RELATED PATENT APPLICATIONS
[0001] This application claims priority under 35 USC .sctn. 119(e)
to U.S. Ser. No. provisional patent application No. 60/292,604
filed May 22, 2001, which application is herein specifically
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention discloses a method of providing
transcriptional control of specific gene transcripts. One
particular use for such transcriptional control is in gene therapy.
Specific genetic regulatory sequences, signaling devices, and
peptides that emulate transcription factors, as well as the methods
of using the same are also provided.
BACKGROUND OF THE INVENTION
[0003] During the past decade researchers have begun to lay a solid
foundation for performing gene therapy related procedures. For
example, long-term expression of heterologous genes in mammals was
demonstrated using viral vectors engineered to contain tissue
specific promoters (U.S. Pat. No. 6,040,172, herein specifically
incorporated by reference in its entirety). In addition, a
modification of behavior was demonstrated in an animal model for
human Parkinson's Disease (U.S. Pat. No. 6,180,613, herein
specifically incorporated by reference in its entirety). However,
before gene therapy becomes a general medical practice, other
factors must be addressed. One such factor concerns the regulation
of the expression of the genetic transcript and/or the resulting
gene product.
[0004] For example, many gene therapy strategies involve the
expression of genes which are likely to cause adverse effects if
they are expressed continuously. Thus, administering a gene
transcript that is incapable of being regulated will oftentimes be
unacceptable. In such cases, performing gene therapy with a
regulatable gene expression system is necessary to control the
expression of the otherwise therapeutic gene and/or gene product.
Presently, regulatable promoters such as the metallothionein
promoter, the tetracycline-on, and the tetracycline-off promoters
are available. Unfortunately, such promoters are not well-suited
for gene therapy because they are controlled by inducer compounds
that are either toxic or can become toxic with long-term use. Still
others are not practical for specific in vivo applications, e.g.,
they cannot cross the blood-brain barrier. Generally, the
regulatable promoters that are currently employed in gene therapy
strategies have been identified/selected for very different
purposes and have therefore not been optimized for the particular
role they must play in gene therapy.
[0005] Therefore, there is a need for developing methods for
identifying genetic regulatory sequences (e.g., a promoter) that
will be responsive to a stimulus in a particular tissue. In
addition, there is a need for identifying stimulants of such
genetic regulatory sequences that are both non-toxic and can
readily gain access to the target tissue. There is a need for
developing vectors that employ such genetic regulatory sequences
for use in gene therapy. Further, there is a need to provide
methods of performing gene therapy which employ the novel
stimulants and genetic regulatory sequences of the present
invention.
SUMMARY OF THE INVENTION
[0006] The present invention provides specific genetic regulatory
sequences that can be controlled by specific stimulants in order to
regulate the expression of exogenous genes in vivo. The present
invention further provides methods for selecting such genetic
regulatory sequences. In one embodiment the genetic regulatory
sequence responds to a stimulus in a particular place and/or at a
particular time. Thus, such a genetic regulatory sequence may be
induced in a particular tissue and/or by the administration of a
particular stimulus. For any given gene therapy strategy the type
of tissue and stimulant is preferably pre-determined prior to its
application.
[0007] In one embodiment, the genetic regulatory sequence responds
to a pulsatile electromagnetic signal (e.g., stimulation). In a
particular embodiment, the pulsatile signal can be turned on and
off for at least 1 minute at a pulse frequency of 1 to 1000 hertz.
In a specific embodiment, the pulse frequency is 10 to 400 hertz.
In a more specific embodiment, the pulse frequency is 20 to 200
hertz. In an even more specific embodiment, the pulse frequency is
30 to 150 hertz. In one embodiment, the current is between 1
microamp and 50 milliamps. In a more specific embodiment, the
current is between 10 microamps and 5 milliamps. In one embodiment,
the voltage intensity is between 1 millivolt and 30 volts. In a
more specific embodiment, the voltage intensity is between 100
millivolts and 10 volts.
[0008] In one embodiment, the individual pulse is 1 microsecond to
10 seconds. In a specific embodiment, the pulse is 20 microseconds
to 1 second. In a more specific embodiment, the pulse width is 50
microseconds to 0.5 second. In an even more specific embodiment,
the pulse width is 90 to 180 microseconds.
[0009] Thus, in one aspect of the present invention methods are
provided for identifying a genetic regulatory sequence that is
responsive to a pulsatile electromagnetic stimulus. One such method
comprises inserting a stimulator into a tissue of an animal subject
and applying a pulsatile signal with the stimulator in which at
least a part of the tissue is stimulated. A gene is then identified
that has either enhanced or diminished transcription in the part of
the tissue stimulated, and a genetic regulatory sequence involved
in the enhanced or diminished transcription of the gene is
selected. This genetic regulatory sequence is thus identified as
being responsive to the pulsatile electromagnetic stimulus. In a
particular embodiment, the pulsatile electromagnetic signal is
provided by a bipolar stimulator. In a related embodiment the
pulsatile electromagnetic signal is provided by a monopolar
stimulator.
[0010] In a particular embodiment, at least a part of the tissue is
not stimulated by the signal of the stimulator and identifying a
gene that has either enhanced or diminished transcription in the
part of the tissue stimulated is performed by comparing the
transcription of the genes in the part of the tissue stimulated
with that of a part of the tissue that is not stimulated. In
another embodiment, a second stimulator is placed into the part of
the tissue that is not stimulated. In a specific embodiment of this
type, no pulsatile signal is applied by the second stimulator. In
an alternative embodiment, the entire tissue is stimulated.
[0011] In one embodiment of the invention, the tissue is neural
tissue. In a specific embodiment, the neural tissue is brain
tissue. In other embodiments, the tissue is heart tissue, liver
tissue, or pancreatic tissue. In a particular embodiment of this
embodiment, the pancreatic tissue contains the insulin producing
beta cells from the islets of Langerhans.
[0012] In one embodiment, the pulsatile electromagnetic signal is
magnetic. In a particular embodiment of this type the pulsatile
electromagnetic signal is a transcranial magnetic stimulation. In
another embodiment, the pulsatile electromagnetic signal is
electrical.
[0013] In a second aspect, the invention features an isolated
genetic regulatory sequence that has been identified by performing
a method of the present invention. In one embodiment, the genetic
regulatory sequence is responsive to a pulsatile electromagnetic
signal. In a particular embodiment the pulsatile electromagnetic
signal that the genetic regulatory sequence is responsive to is
magnetic. In an alternative embodiment, the pulsatile
electromagnetic signal that the genetic regulatory sequence is
responsive to is electrical. In a related embodiment, the genetic
regulatory sequence is responsive to a specific peptide identified
by a method of the present invention.
[0014] The present invention further provides a vector for the in
vivo expression of a gene of interest in a mammalian host cell
which contains a gene of interest operatively under control of a
genetic regulatory sequence identified by a method of the present
invention. In a particular embodiment, the vector comprises a gene
of interest operatively linked to a transcriptional control region
comprising a genetic regulatory sequence that is responsive to a
pulsatile electromagnetic stimulus. In one embodiment, the
pulsatile electromagnetic signal is magnetic. In another
embodiment, the pulsatile electromagnetic signal is electrical. In
a particular embodiment, the genetic regulatory sequence of the
transcriptional control region of the vector exhibits tissue
specificity. In a specific embodiment, the genetic regulatory
sequence of the transcriptional control region of the vector is
obtained from a gene encoding a protein expressed by the cell which
is selected to be the target cell for the vector.
[0015] In one embodiment, the vector is a replication defective
viral vector. In a particular embodiment of this type, the vector
is a replication defective herpes simplex virus (HSV). In another
embodiment, the vector is a replication defective papillomavirus.
In yet another embodiment, the vector is a replication defective
Epstein Barr virus (EBV). In still another embodiment, the vector
is a replication defective adenovirus and/or gutless adenovirus. In
yet another embodiment, the vector is a replication defective
adeno-associated virus (AAV). In still another embodiment, the
vector is a replication defective lentivirus. In yet another
embodiment, the vector is a replication defective retrovirus. In a
particular embodiment of this type, the replication defective
retrovirus vector is prepared for use in an ex vivo gene therapy
protocol.
[0016] In one embodiment, the genetic regulatory sequence of the
transcriptional control region of the vector is obtained from
neural tissue. In a more specific embodiment of this type, the
neural tissue is brain tissue. In other specific embodiment, the
genetic regulatory sequence is obtained from heart tissue, liver
tissue, or pancreatic tissue. In a particular embodiment of this
type, the pancreatic tissue contains the insulin producing beta
cells from the islets of Langerhans.
[0017] In a second aspect, the invention provides a non-human
mammalian host transformed with a vector of the present invention.
Preferably the expression of the gene of interest encoded by the
vector can be modulated by applying a pulsatile electromagnetic
signal with a stimulator. In a particular embodiment the pulsatile
electromagnetic signal is magnetic. In an alternative embodiment
the pulsatile electromagnetic signal is electrical. In a preferred
embodiment, the stimulator is also present in the non-human
mammalian host.
[0018] In a third aspect, the invention features methods of
delivering the vectors of the present invention to a target tissue
of an animal subject by administering the vector to the target
tissue of the animal subject. Preferably the expression of the gene
of interest encoded by the vector can be modulated by applying a
pulsatile electromagnetic signal with a stimulator. In one
embodiment, the pulsatile electromagnetic signal is magnetic. In an
alternative embodiment, the pulsatile electromagnetic signal is
electrical. In a particular embodiment, the transcription of the
gene of interest in the target tissue is stimulated by the
electromagnetic signal. In an alternative embodiment, the
transcription of the gene of interest in the target tissue is
hindered and/or inhibited by the electromagnetic signal. In a
particular embodiment, the pulsatile electromagnetic signal is
provided by a bipolar stimulator. In a related embodiment, the
pulsatile electromagnetic signal is provided by a monopolar
stimulator.
[0019] In a fourth aspect, the invention features methods of
identifying a genetic regulatory sequence that is responsive to a
peptide. One such method comprises contacting a peptide with a cell
and identifying a gene that has either enhanced or diminished
transcription. A genetic regulatory sequence involved in the
enhanced or diminished transcription of the gene is selected. This
genetic regulatory sequence is identified as being responsive to
the peptide. In a preferred embodiment this peptide is a random
generated peptide. In a particular embodiment, the peptide has the
amino acid sequence of SEQ ID NO:1. In a more specific embodiment,
the peptide is between 3 and 40 amino acids long. More
specifically, the peptide is between 5 and 15 amino acids long.
Randomly generated peptides that can either enhance or diminish
transcription of the gene through binding to a genetic regulatory
sequence are also included in the invention. Furthermore, genetic
regulatory sequences that respond to these peptides are also part
of the present invention, as are the vectors that comprise such
genetic regulatory sequences.
[0020] In a fifth aspect, the invention features methods of
regulating the expression of a gene of interest in a target tissue
of an animal subject in which a vector of the present invention has
been administered. One such method comprises applying a pulsatile
signal with a stimulator to modulate the transcription of a gene of
interest in the target tissue. In a particular embodiment of this
aspect, the response to the pulsatile signal by the genetic
regulatory sequence of the vector stimulates the transcription of
the gene of interest in the target tissue. In an alternative
embodiment, the response to the pulsatile signal by the genetic
regulatory sequence reduces the transcription of the gene of
interest in the target tissue. In a preferred embodiment of this
type, the response to the pulsatile signal by the genetic
regulatory sequence stops and/or prevents the transcription of the
gene of interest in the target tissue.
[0021] In a sixth aspect, the invention features methods of
ameliorating symptoms due to Parkinson's disease. One such method
comprises administering glutamic acid decarboxylase to the
subthalamic nucleus of a patient having a symptom of Parkinson's
disease. In one such embodiment, the glutamic acid decarboxylase is
administered to the subthalamic nucleus of the patient via a
vector. In one particular embodiment, the vector is constructed to
comprise a nucleic acid encoding glutamic acid decarboxylase
operatively under the control of a genetic regulatory sequence that
is stimulated by a pulsatile electromagnetic signal. A pulsatile
electromagnetic signal is applied with a stimulator that had been
placed into the subthalamic nucleus of the patient. The pulsatile
electromagnetic signal stimulates the transcription of the glutamic
acid decarboxylase in the subthalamic nucleus which leads to the
amelioration of the symptom due to Parkinson's disease. In a
preferred embodiment the vector is a replication defective viral
vector. In a particular embodiment the vector comprises a chicken
beta-actin promoter that is operatively linked to a nucleic acid
encoding human glutamic acid decarboxylase (the CBA-GAD65 viral
vector).
[0022] In a seventh aspect, the invention features a method of
modulating the release of a stored compound by a cell. In a
preferred embodiment the compound is a small organic molecule. In
one such embodiment, the compound is a hormone and/or a
neurotransmitter such as epinephrine, norepinephrine, dopamine,
dopa, serotonin and GABA.
[0023] One embodiment of the method comprises administering to an
animal subject (preferably a human) a vector comprising a nucleic
acid encoding a protein (preferably an enzyme) operatively under
the control of a genetic regulatory sequence. The genetic
regulatory sequence is specifically chosen for its ability to be
stimulated by a pulsatile electromagnetic signal, whereas the
protein is specifically chosen for its ability, when expressed, to
stimulate the production of a compound that is subsequently stored
by a cell of the animal subject. A stimulator is then inserted into
a tissue of the animal subject such that when it is used to apply a
specific signal, the genetic regulatory sequence responds. In a
particular embodiment exemplified below, the vector and the
stimulator are administered to the subthalamic nucleus. A pulsatile
electromagnetic signal is then applied with the stimulator, causing
the protein to be expressed which thereby stimulates the production
of the compound, which in turn is stored by a cell. At a subsequent
time, a second pulsatile electromagnetic signal is applied with the
stimulator which then modulates the release of the stored compound
from the cell. In the Example 4 below, the compound is GABA. When
the second pulsatile electromagnetic signal is performed at a low
frequency, the cell is stimulated to increase the release of GABA,
whereas when the second pulsatile electromagnetic signal is
performed at a high frequency, the cell is stimulated to decrease
(and/or block) the release of GABA.
[0024] Accordingly, the present invention provides genetic
regulatory sequences that respond to pulsatile electromagnetic
stimulation and are stimulated by pulsatile electromagnetic signals
to facilitate the transcription of a nucleic acid operatively under
their control. Further provided is a genetic regulatory sequence
that responds to a pulsatile electromagnetic signal by hindering
the transcription of a nucleic acid operatively under its control
or by preventing the transcription of a nucleic acid operatively
under its control.
[0025] The present invention also provides vectors and replication
defective vectors that contain nucleic acids that are operatively
under the control of a genetic regulatory sequence identified by a
method of the present invention.
[0026] Further, the invention provides a method of modulating the
transcription of a selected nuclei acid in vivo, including turning
off transcription, by placing it operatively under the control of a
genetic regulatory sequence identified by a method of the present
invention.
[0027] Further, the present invention provides a method of
performing gene therapy with a defective viral vector comprising a
therapeutic nucleic acid that is operatively under the control of a
genetic regulatory sequence of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1-2 show the response of a neuron in the substantia
nigra to electrical stimulation of the subthalamic nucleus. A
histogram (20 ms bins) of spike counts after an electrical
stimulation at t=0 is shown. Each trial of the stimulation is used
to create the histogram and as a labeled sweep of the graph. FIG. 1
shows that in normal rats there is a large increase in impulse
activity due to subthalamic nucleus stimulation. FIG. 2 shows an
inhibition of spontaneous firing of the neuron in the substantia
nigra due to subthalamic nucleus stimulation.
[0029] FIGS. 3-4 show the change of extracellular GABA
concentration (FIG. 3) and glutamate concentration (FIG. 4) of the
substantia nigra and the subthalamic nucleus during subthalamic
nucleus stimulation in naive rats and rats transduced with a
CBA-GAD65 viral vector. The stimulation was applied for two
different time periods, two minutes and five minutes for two
different groups of rats as shown:
[0030] (i) 10 Hz, at 500 .mu.A for 2 minutes, (labeled ST1);
and
[0031] (ii) 10 Hz, at 500 .mu.A for 5 minutes (labeled ST2).
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides methods for controlling the
transcription of heterologous genes in vivo by employing specific
genetic regulatory sequences that can be up and/or down regulated,
and preferably turned off and/or on by specific stimulators.
Further provided are specific expression cassettes that include
genes of interest operably under the control these genetic
regulatory sequences, and methods of delivering these expression
cassettes to the target cells. The methods of delivery include
using standard transgenic techniques such as transfection and
transduction. The expression cassettes can be included in both
viral and non-viral vectors. The genetic regulatory sequences can
be characterized based on screening gene expression patterns in a
particular tissue following a defined stimulus. In a particular
embodiment, the delivery of the expression cassette is to the
nervous system and the genetic regulatory sequences are selected
for their ability to be regulated in the nervous system.
[0033] The invention features a method for identifying genetic
regulatory sequences for controlling expression of genes for gene
therapy which respond to a physiological stimulus. In one such
embodiment, a physiological stimulus is administered, and messenger
RNA is extracted from the cells or tissue. Microarray analysis (see
e.g., U.S. Pat. No. 6,215,894 and U.S. Pat. No. 6,004,755 herein
specifically incorporated by reference in their entirety) can then
be performed to identify genes having increased expression in
response to the physiological stimulus. The genetic regulatory
sequences from these genes can then be identified and/or isolated
(e.g., by PCR or genomic library screening). The genetic regulatory
sequences can then be inserted into expression cassettes to control
the expression of a gene of interest. The physiological stimulus
can then be used to control the expression of the gene of
interest.
[0034] Methods other than microarray analysis may include, but are
not limited to, differential display (see e.g., U.S. Pat. No.
6,045,998 and U.S. Pat. No. 5,599,672, herein specifically
incorporated by reference in their entiretiy), subtractive mRNA
hybridization (see e.g., U.S. Pat. No. 5,958,738 and U.S. Pat. No.
5,525,471, herein specifically incorporated by reference in their
entirety), peptide arrays, two-dimensional protein gel
electrophoresis and microsequencing of relevant peptides.
[0035] Stimuli may include, but are not limited to, electrical
stimulation, magnetic fields, heat or cold stimuli, peptide or
protein infusion, chemical or drug infusion, ionizing radiation,
microwave or ultrasound.
[0036] A genetic regulatory sequence (e.g., a promoter or genetic
response sequence/element) can be selected based upon the response
of an endogenous cellular gene in a target tissue to a particular
stimulus. Once identified, the genetic regulatory sequence can then
be inserted into a gene therapy vector to control expression of any
gene of interest in that target tissue.
[0037] The mammalian nervous system responds to environmental
conditions including housing and enrichment paradigms (Young et al.
(1999) Nat. Med. 5:448-53 and Rampon et al. (2000) Proc. Natl.
Acad. Sci. U.S.A. 97:12880-12884), transcranial magnetic
stimulation (Fujiki et al. (1997) Brain Res. Mol. Brain Res.
44:301-308; Muller et al. (2000) Neuropsychopharm. 2:205-15; and
Hausmann et al. (2000) Brain Res. Mol. Brain Res. 76:355-62) and
peripheral administration of exogenous compounds and peptides by
inducing transcription of specific genes.
[0038] The expression of a number of proteins have been found to be
responsive to high frequency TMS in rats, including glial acidic
fibrillary protein (GFAP) expression (Fujiki et al. (1997) supra),
c-fos, brain-derived neurotropic factor (BDNF) and cholecystokinin
(CCK) (Muller et al. (2000) supra, and Hausmann et al. (2000)
supra). The genetic regulatory equences of these genes may be
employed in expression cassettes by the method of the present
invention.
[0039] One particular aspect of the present invention is the use of
essentially any peptide e.g., preferably 3 to 40 amino acid
residues, and more preferably 5 to 15 amino acid residues, to
selectively up and/or down regulate, and preferably turn on and/or
turn off specific genes. Therefore, the present invention provides
methods of identifying/mapping genes that are up and/or down
regulated, and preferably turned on and/or off by any given
peptide.
[0040] In particular, in Example 3 below, genes encoding the NMDA
receptor, F3 contactin, and MAP-2 are shown to be upregulated in
response to a novel peptide. Therefore,
[0041] genetic regulatory sequences from the 5' promoter region of
these genes are provided as a component of the gene switch.
Initially, the isolated promoters can be cloned upstream of a
marker gene such as green fluorescent protein (see e.g., U.S. Pat.
No. 5,625,048, WO 97/26333, and WO 99/64592, herein specifically
incorporated by reference in their entirety) or luciferase, which
can then be used to further characterize the responsiveness to the
peripheral administration of the peptide. Subsequently, important
neural proteins (e.g., tyrosine hydroxylase, DARP-32 etc.) can be
inserted in place of the marker proteins for gene therapy, for
example.
[0042] Similarly, synthetic promoter constructs including multiple
genetic regulatory sequences from the genes encoding CRE, p53,
AP-1, SRE, NF-kappaB, SRF, Spl (using e.g., 3 to 10 copies of the
response element) can be initially placed upstream of a luciferase
and GFP cDNA. These expression cassettes can be cloned into an AAV
cis plasmid and used to generate rAAV-RE (generic response
element)-luc/GFP, for example. One such expression vector, the
rAAV-Cre-luc has already been packaged and successfully injected
into a group of rats. The present invention further provides
"cocktails" of response elements which can be used to generate
synthetic response elements that can either upregulate a given
transgene or downregulate the transgene, depending on the stimulus
applied.
[0043] Vectors may include, but are not limited to, adenovirus
(recombinant and "gutless" vectors), herpes simplex virus
(recombinant vectors and amplicons), adeno-associated virus,
lentivirus, retrovirus, synthetic or non-viral vectors (including
liposome and plasmid-based systems). Preferably the viral vectors
are reproduction defective viral vectors.
[0044] It should also be noted that chimeric promoters or genetic
response elements which contain some part of an endogenous promoter
identified to be responsive to an external stimulus are also part
of the present invention.
[0045] Definitions
[0046] As used herein, the term "gene" refers to an assembly of
nucleotides that encodes a polypeptide and includes cDNA and
genomic DNA nucleic acids. A gene is a nucleic acid that does not
necessarily correspond to the naturally occurring gene which
contains all of the introns and regulatory sequences, e.g.,
promoters, present in the natural genomic DNA. Rather, a gene
encoding a particular protein can minimally contain just the
corresponding coding sequence for the protein.
[0047] As used herein a "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded at its 3' terminus by the transcription initiation site
and extends upstream (5' direction) to include the minimum number
of bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence will be
found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0048] As used herein transcriptional and translational control
sequences are DNA regulatory sequences, such as promoters,
enhancers, terminators, and the like, that provide for the
expression of a coding sequence in a host cell. In eukaryotic
cells, polyadenylation signals are control sequences.
[0049] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "operatively under the control"
of transcriptional and translational control sequences in a cell
when RNA polymerase transcribes the coding sequence into a
precursor RNA, which is then trans-RNA spliced to yield mRNA and
translated into the protein encoded by the coding sequence.
[0050] A nucleotide sequence is "operatively under the control" of
a genetic regulatory sequence when the genetic regulatory sequence
controls and/or regulates the transcription of that nucleotide
sequence. That genetic regulatory sequence can also be referred to
as being "operatively linked" to that nucleotide sequence.
[0051] As used herein, a "genetic regulatory sequence" is a nucleic
acid that: (a) acts in cis to control and/or regulate the
transcription of a nucleotide sequence, and (b) can be acted upon
in trans by a regulatory stimulus to promote and/or inhibit the
transcription of the nucleotide sequence. Therefore, an inducible
promoter is a genetic regulatory sequence. In addition, a portion
of a promoter (e.g., fragment/element) that retains and/or
possesses the ability to control and/or regulate the transcription
of a nucleotide sequence either alone or in conjunction with an
alternative promoter or fragment thereof (e.g., a chimeric
promoter) is also a genetic regulatory sequence. Such fragments
include response elements (genetic response elements) and promoter
elements.
[0052] As used herein, an "expression cassette" is a nucleic acid
that minimally comprises a nucleotide sequence to be transcribed
(e.g., a coding sequence) that is operatively under the control of
a genetic regulatory sequence.
[0053] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0054] As used herein, a "defective viral vector", abbreviated
"dvv" is a viral vector that requires the expression and/or
transcription of at least one nucleic acid that it lacks in order
to be replicated and/or packaged. Preferably the dvv is a
replication defective viral vector.
[0055] As used herein, a "heterologous gene" is a gene that has
been placed into a vector or cell that does not naturally occur in
that vector or cell.
[0056] As used herein, a gene is an "exogenous gene" when the gene
is not encoded by the particular vector or cell.
[0057] A "vector" as used herein is a genetic construct that
facilitates the efficient transfer of a nucleic acid (e.g., a gene)
to a cell. The use of a vector can also facilitate the
transcription and/or expression of that nucleic acid in that cell.
Examples of vectors include plasmids, phages, amplicons, viruses
and cosmids, to which another DNA segment may be attached so as to
bring about the replication of the attached segment.
[0058] As used herein, "pulsatile" stimulation is a stimulation in
which more than one pulse per unit time (and preferably a series of
pulses) is generated at a defined pulse width. The number of pulses
per unit time is termed its frequency which can be denoted in
Hertz.
[0059] As used herein, a "pulse width" is the length of time a
single pulse lasts.
[0060] As used herein, a "small organic molecule" is an organic
compound, or organic compound complexed with an inorganic compound
(e.g., metal) that has a molecular weight of less than 3
kilodaltons, and preferably less than 1.5 kilodaltons.
[0061] Gene Therapy
[0062] The genetic regulatory sequences of the present invention
can be used to modulate gene transcription in any cell, including
human cells. However, the genetic regulatory sequences of the
present invention can be used to modulate gene transcription in
cells of other mammals, such as rodents, e.g., mice, rats, rabbits,
hamsters and guinea pigs; farm animals e.g., sheep, goats, pigs,
horses and cows; domestic pets such as cats and dogs, higher
primates such as monkeys, and the great apes such baboons,
chimpanzees and gorillas.
[0063] The genetic regulatory sequences of the present invention
can be operatively linked to any heterologous nucleic acid of
interest, preferably those encoding proteins. Indeed, any protein
can be encoded by the heterologous nucleic acid operatively under
the control of a genetic regulatory sequence of the present
invention. A short list of a few of these proteins and their roles
in particular conditions and/diseases is included in Table 3 below.
However, this listing should in no way limit the general
methodology of the present invention which provides the ability to
modulate the expression of any nucleic acid of choice. In addition,
the expression cassettes of the present invention can be
constructed to comprise multiple nucleic acids each encoding a
different protein and all under the control of the same genetic
regulatory sequence. Alternatively, different nucleic acids can be
placed under the control of different genetic regulatory sequences.
For example, the use of two genetic regulatory sequences, one of
which stimulates transcription when treated with a pulsatile
electromagnetic signal and the other which hinders transcription
under the same conditions can be used to control the expression of
two different genes at the same time by operatively linking one
coding sequence to one genetic regulatory sequence and the other
coding sequence to the other genetic regulatory sequence. For
example, insulin and glucagon expression could be controlled in
this manner. Alternatively, multiple expression cassettes can be
employed encoding multiple different proteins.
1TABLE 3 Genetic Defects Disease/Symptom adenosine deaminase severe
combined immunodeficiency disease alpha, - antitrypsin pulmonary
emphysema 5-alpha reductase male pseudohemaphroditism 17-alpha
reductase male pseudohemaphroditism p53 or ARF-P19 proteins linked
to cancer insulin insulin-dependent diabetes sickle cell anemia
B-globin hypoxanthine guanine Lesh-Nyhan Syndrone
phosphoribosyl-transferase ornithine transcarbamolase Fatal to
newborn males phenylalanine hydroxylase Phenylketonuria Dralassemia
x- or B-globin AT Page 7A Menkes' syndrome AT Page 7B Wilson
Disease hexosamindase A Tay-Sachs Disease acid cholesterylester
hydrolase Wolmon Disease
[0064] In one particular example a defective viral vector
comprising a nucleic acid encoding insulin operatively under the
control of a genetic regulatory sequence of the present invention
can be employed to transduce the pancreas in vivo to treat
insulin-dependent diabetes. For example, if the genetic regulatory
sequence is induced to stimulate transcription when a pulsatile
electromagnetic signal is provided by a stimulator, expression of
insulin could be controlled and the insulin-dependent diabetes
treated. The vectors of the present invention can be delivered in
vitro, ex vivo and in vivo.
[0065] When the genetic regulatory sequence is contained in a viral
vector, the delivery can be performed by stereotaxic injection into
the brain for example, as previously exemplified (U.S. Pat. No.
6,180,613, herein specifically incorporated by reference in its
entirety); or via a guide catheter (U.S. Pat. No. 6,162,796, herein
specifically incorporated by reference in its entirety) to an
artery to treat the heart. In addition, the vectors of the present
invention may also be delivered intravenously,
intracerebroventricularly and/or intrathecally, for specific
applications. Additional routes of administration can be local
application of the vector under direct visualization, e.g.
superficial cortical application, or other non-stereotactic
applications.
[0066] For targeting a vector to a particular type of cell, it may
be necessary to associate the vector with a homing agent that binds
specifically to a surface receptor of the cell. Thus, the vector
may be conjugated to a ligand (e.g., enkephalin) for which certain
nervous system cells have receptors, or a surface specific
antibody. The conjugation may be covalent, e.g., a crosslinking
agent such as glutaraldehyde, or noncovalent, e.g., the binding of
an avidinated ligand to a biotinylated vector.
[0067] In addition, the helper-free defective viral vectors of the
present invention can be delivered ex vivo, as exemplified by
Anderson et al. (U.S. Pat. No. 5,399,346, herein specifically
incorporated by reference in its entirety).
[0068] Alternatively, a vector can be introduced by lipofection.
Liposomes can be used for encapsulation and transfection of nucleic
acids. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7417; see also Mackey et al. (1988), Proc. Natl. Acad. Sci.
U.S.A 85:8027-8031). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Felgner et al.
(1989) Science 337:387-388). The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as
pancreas, liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting (Mackey et.
al. (1988) supra).
[0069] It is also possible to introduce the vector as a naked DNA
plasmid. Naked DNA vectors for gene therapy can be introduced into
the desired host cells by methods known in the art, e.g.,
transfection, electroporation, microinjection, transduction, cell
fusion, DEAE dextran, calcium phosphate precipitation, use of a
gene gun, or use of a DNA vector transporter (see, e.g., Wu et al.
(1992) J. Biol. Chem. 267:963-967; Wu et al. (1988) J. Biol. Chem.
263:14621-14624; Hartmut et al., Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990).
[0070] In an ex vivo method of the invention, the genetic
regulatory sequences of the invention are delivered to a host cell
to be transplanted into a mammalian recipient. The host cells may
be endogenous or exogenous to the mammalian recipient. The term
"transplant cell" refers broadly to the component, e.g., tissue or
cells, being grafted, implanted, or transplanted into a recipient
subject. As used herein, the term "transplantation" refers to the
transfer or grafting of tissues or cells from one part of a subject
to another part of the same subject or to another subject.
Transplanted tissue may comprise a collection of cells of identical
composition, or derived from a donor organism, or from an in vitro
culture. Delivery of the genetic regulatory sequences of the
invention to a transplant cell may be accomplished by any of the
methods known to the art and described above, e.g., as a plasmid,
as part of a vector; by injection, lipofection, etc.
[0071] A variety of dissociated cells can be implanted, using
standard techniques for isolation and transplantation of tissue or
organs, such as livers. See, for example, U.S. Pat. No. 6,281,015,
herein specifically incorporated by reference.
[0072] Transgenic Animals
[0073] A transgenic animal model can be prepared so as to contain a
nucleic acid operatively under the control of a genetic regulatory
sequence of the present invention. For example transgenic vectors,
including viral vectors, or cosmid clones (or phage clones) can be
constructed. Cosmids may be introduced into transgenic mice using
published procedures (Jaenisch (1988) Science 240:1468-1474).
[0074] Thus the present invention further provides transgenic,
knock-in, and knockout animals that contain one or more
heterologous genes operatively under the control of a genetic
regulatory sequence of the present invention. These animals can be
used as animal models in drug screening assays. In one such
example, a drug can be added under various "controlled" expression
levels of a particular gene, or at various time points before
and/or after induced expression of the particular gene, allowing a
much more detailed investigation of the effects of that drug on a
particular condition. In a specific embodiment, the transgenic,
knock-in, or knockout animal is a mouse. Cells from the inducible
knockout, knock-in and/or transgenic animals of the present
invention are also part of the present invention. These cells can
also be used in the drug assays, for example.
[0075] Transgenic animals can be obtained through gene therapy
techniques described above or by microinjection of a nucleic acid
for example, into an embryonic stem cell or an animal zygote (such
as a bacterial artificial chromosome (BAC) comprising a nucleic
acid operatively under the control of a genetic regulatory sequence
of the present invention). Microinjection of BACs has been shown to
be successful in a number of animals including rats, rabbits, pigs,
goats, sheep, and cows (in Transgenic Animals Generation and Use
(1997) ed., L. M. Houdebine, Harwood Academic Publishers, The
Netherlands). Methods of constructing BACs or other DNAs such as
bacteriophage P1 derived artificial chromosomes (PACs) that encode
specific nucleic acids through homologous recombination have
recently been described in great detail (Heintz et al. (1998)
PCT/US98/12966, herein specifically incorporated by reference in
its entirety). Alternatively, a yeast artificial chromosome (YAC)
can be used.
[0076] Ribozymes and Antisense
[0077] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(see Weintraub (1990) Sci. Amer. 262:40-46; Marcus-Sekura (1987)
Nucl. Acid Res, 15:5749-5763; Marcus-Sekura (1988) Anal. Biochem.
172:289-295); Brysch et al. (1994) Cell Mol. Neurobiol.
14:557-568). Preferably, the antisense molecule employed is
complementary to a substantial portion of the mRNA. In the cell,
the antisense molecule hybridizes to that mRNA, forming a double
stranded molecule. The cell does not translate an mRNA in this
double-stranded form. Therefore, antisense nucleic acids interfere
with the expression of mRNA into protein. Preferably a DNA
antisense nucleic acid is employed since such an RNA/DNA duplex is
a preferred substrate for RNase H.
[0078] Oligomers of greater than about fifteen nucleotides and
molecules that hybridize to the AUG initiation codon will be
particularly efficient. Antisense methods have been used to inhibit
the expression of many genes in vitro (Marcus-Sekura (1988) supra;
Hambor et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014)
and in situ (Arima et al. (1998) Antisense Nucl. Acid Drug Dev.
8:319-327; Hou et al. (1998) Antisense Nucl. Acid Drug Dev.
8:295-308).
[0079] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single stranded RNA molecules in a manner
somewhat analogous to DNA restriction endonucleases. Ribozymes were
discovered from the observation that certain mRNAs have the ability
to excise their own introns. By modifying the nucleotide sequence
of these ribozymes, researchers have been able to engineer
molecules that recognize specific nucleotide sequences in an RNA
molecule and cleave it (Cech (1988) JAMA 260:3030-3034; Cech (1989)
Biochem. Intl. 18:7-14). Because they are sequence-specific, only
mRNAs with particular sequences are inactivated.
[0080] Investigators have identified two types of ribozymes,
Tetrahymena-type and "hammerhead"-type (Haselhoff et al. (1988)
Nature 334:585-591). Tetrahymena-type ribozymes recognize four-base
sequences, while "hammerhead"-type recognize eleven- to
eighteen-base sequences. The longer the recognition sequence, the
more likely it is to occur exclusively in the target mRNA species.
Therefore, hammerhead-type ribozymes are preferable to
Tetrahymena-type ribozymes for inactivating a specific mRNA
species, and eighteen base recognition sequences are preferable to
shorter recognition sequences.
[0081] Antisense nucleic acids and/or ribozyme can also be placed
operatively under the control of a genetic regulatory sequence of
the present invention. Such an expression cassette, when placed in
vector (e.g., a viral vector) and appropriately administered can be
used to selectively modulate the expression of a particular protein
in a cell, tissue or animal subject. This procedure would be
complementary to modulating the transcription of the gene encoding
the protein described above, since in this case the gene would be
transcribed but selectively prevented from being expressed. For
example, the administration of an antisense nucleic acid or
ribozyme that prevents the over-expression of tumor necrosis factor
alpha, which arises in septic shock, leprosy or tuberculosis, by
the methodology disclosed herein may be particularly
beneficial.
[0082] Specific Embodiments
[0083] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
[0084] One method for regulating the expression of a heterologous
gene is to place it operatively under the control of an inducible
promoter. Unfortunately, whereas promoter activity and gene
expression differ fundamentally when examined in vitro relative to
in vivo most of the inducible promoters currently being employed
have been optimized to control gene expression in cultured cells,
rather than in a living animal. For example, electrical stimulation
of astroglial cells in tissue culture recently has been shown to
induce expression of a marker gene under the control of the
inducible hsp70 promoter (hsp 70) (Pelham (1982) Cell 30:517-528;
Yanagida et al. (2000) J. Biotechnology 79:53-61). However, the
hsp70 promoter responds non-specifically to a variety of stresses
including chemical toxicity, heat stress, and ischemia and there
was no direct evidence provided that the hsp 70 promoter was
specifically responsive to the electrical stimulation applied.
Moreover, whereas astroglial cells placed in tissue culture are
required to divide, in their native state in the brain they
normally do not undergo cell division. Therefore, it was not
surprising to find that in vivo the hsp 70 promoter was not
effected by electrical stimulation of the brain (Example 1).
[0085] Prior to the experiments described below, the use of
electrical stimulation to control the expression of a heterologous
protein in vivo has not been substantiated. Therefore, a nucleic
acid encoding a heterologous protein (luciferase) was placed into
an expression cassette under the control of a genetic regulatory
sequence (i.e., a 2.2 kb 5' fragment of the human glial acidic
fibrillary protein gene) that responds to electrical stimulation.
The expression cassette, the recombinant viral vector,
rAAV-GFAP-Luc, was then delivered (i.e., injected) to an animal
subject. The electrical stimulation was applied and the luciferase
activity was determined in resected hippocampi.
[0086] As described in Example 2, an electrical stimulator can be
inserted into the brain to control expression of a heterologous
gene contained in an expression vector (e.g., a viral vector).
Whereas a variety of stimulators are currently used for nerve or
brain stimulation to alter neuronal firing activity, they have
never been used to control gene expression.
[0087] Example 3 describes the infusion of a peptide intranasally
into rats, and subsequent changes in gene expression in a variety
of brain regions were analyzed.
[0088] Parkinson's Disease is a neurodegenerative disorder
characterized by loss of the nigrostriatal pathway and is
responsive to treatments which facilitate dopaminergic transmission
in the caudate-putamen (Yahr et al. (1987) Parkinson's Disease
(Raven Press); Yahr et al. (1969) Arch. Neurol. 21:343-54).
Unilateral 6-hydroxydopamine lesions of the substantia nigra have
been used to generate an established rodent model of Parkinson's
Disease.
[0089] Electrophysiology studies have demonstrated that the
subthalamic nucleus (STN) has a prominent excitatory connection
with the substantia nigra (SN). In Parkinson's disease (PD), the
subthalamic nucleus is overactive and this overactivity may lead to
progressive degeneration of dopamine neurons in the subthalamic
nucleus as well as the common features of Parkinson's disease such
as tremor, rigidity and bradykinesia. As described in Example 4
below, changing the excitatory projection from the subthalamic
nucleus to the substantia nigra into an inhibitory projection
alleviates the symptoms associated with Parkinson's disease.
EXAMPLES
Example 1
Use of Electrical Stimulation to Regulate Genes
[0090] Materials and Methods: Rats: Male-Sprague-Dawley, Fisher,
Wistar and Lewis strains of rats were used for all experiments.
[0091] Transcranial Magnetic Stimulation: An adeno-associated viral
(AAV) vector expressing the luciferase transgene under control of a
genetic regulatory sequence from the human 2.2 kb glial acidic
fibrillary protein (GFAP) promoter was injected stereotactically
into the left dorsal hippocampus of young adult male rats. After
four weeks, groups of rats were randomized to exposure to the 5 cm
Cadwell coil without activating the stimulator (i.e., mock
stimulation), to low frequency stimulation (5 Hz) or to high
frequency (25 Hz) delivered as trans (30 trains, each 10 second
duration). Stimulation or mock stimulation was given once daily for
four days with the 5 cm coil centered over the left hippocampus. On
day 5, rats were euthanized and hippocampi resected and analyzed
for luciferase activity.
[0092] As shown in Table 1, the amount of luciferase activity
measured correlated with the amount of electrical stimulation
applied. As a further control, luciferase activity in a group of
completely naive control rats was found to be undetectable after
stimulation (<10 pg). As is apparent from Table 1, expression of
luciferase that is operatively under the control of the GFAP
promoter element increases with electrical stimulation (see
methods). Therefore, the use of 5' elements or response elements
isolated from the promoter regions of inducible genes can be used
to generate similarly regulated transcription units.
2TABLE 1 Effect of Electrical Stimulation on Luciferase Activity in
The Hippocampi of Rats Injected with an rAAV-GFAP-Luc Vector.
STIMULATION (Hz) ACTIVITY (pg/hc).sup.a FOLD INCREASE mock 109 .+-.
92.sup.b 1 5 348 .+-. 178 2 25 1754 .+-. 326 16 .sup.apg luciferase
activity/hippocampus .sup.b(mean, S.D.)
Example 2
Selection of Genetic Regulatory Sequences to Regulate an Exogenous
Gene
[0093] Bipolar stimulators were inserted bilaterally into the
caudate nuclei of the rat. Stimulation was then performed on the
left caudate for 30 minutes at high frequency (200 Hz) with a
current of 200 .mu.A, while a stimulator was inserted into the
right caudate but remained off. Following the stimulation the
animals were sacrificed, the right and left caudate nuclei were
dissected and the tissue samples were rapidly frozen. mRNA was
extracted from the tissues, and microarray analysis was performed
comparing the mRNA from the left (stimulated) caudate nucleus to
the right (unstimulated) caudate. This procedure was replicated in
four animals.
[0094] Analysis of the data revealed significant changes in
approximately 70 genes, with a high consensus among all samples and
replicates. The majority of genes were turned off, with some genes
decreasing in amount and eight genes were expressed in increasing
amounts. Promoters were next isolated by PCR from four genes which
were turned off (i.e., protein kinase B kinase, snyaptic vesicle
protein 2B, phosphatidylinositol 3-kinase p85, and calcineurin B),
and one gene, insulin-like growth factor-1 (IGF-1) that was
increased in response to stimulation.
[0095] Next promoter fragments between 2.0 and 3.5 kb were inserted
into adeno-associated virus (AAV) vectors containing the gene for
green fluorescent protein (GFP). AAV vectors were then packaged and
purified using standard techniques. In a specific example, the
calcineurin B promoter/GFP construct was packaged and 3 .mu.l of
purified vector stocks were then infused bilaterally into the rat
caudate nucleus in five animals. Brain stimulators were inserted
bilaterally into the same point in the brain and were fixed to the
skull with dental cement. After 48-72 hours of recovery, the left
stimulator was turned on for 30 minutes at high frequency (200 Hz)
with a current of 200 .mu.A, while the right stimulator remained
off. Animals were then sacrificed, and the tissue was harvested.
Protein was extracted for western blot analysis that were performed
using a monoclonal anti-GFP protein and standard techniques. The
results showed that the GFP protein levels from the left
(stimulated) caudate nucleus was decreased relative to that of the
right (unstimulated) caudate.
[0096] The construct was then modified to encode tyrosine
hydroxylase (TH) rather than green fluorescent protein. Previously
the expression of tyrosine hydroxylase from viral vectors in the
caudate nucleus of rat models of Parkinson's disease was shown to
result in therapeutic improvement (U.S. Pat. No. 6,180,613, herein
specifically incorporated by reference in its entirety).
[0097] In an analogous experiment, the construct encoding TH was
also shown to be responsive to electronic stimulation (i.e., as
described above for the corresponding GFP construct). In the case
of the TH construct, a significant decrease in tyrosine hydroxylase
mRNA levels from the left (stimulated) caudate nucleus relative to
that of the right (unstimulated) caudate was determined by
quantitative polymerase chain reaction (PCR) assays.
Example 3
Use of Peripherally Administered Peptides to Regulate Genes
[0098] Peripherally Administered Peptides: A novel peptide was
synthesized that comprised nine amino acids having the amino acid
sequence of HSEGTFTSD (SEQ ID NO:1). This peptide was administered
intranasally at a dose of 30 .mu.g to rats and was found to have no
effect on spontaneous locomotor behavior, stereotype, feeding, nor
did it influence pain threshold. Twenty minutes following the
administration, the rats were sacrificed and their hippocampi
removed. The hippocampi were subsequently dissected and the
hippocampi RNA was isolated. The isolated RNA was then used for
gene expression profiling using AFFYMETRIC rat gene chips
(containing approximately 7000 cDNA's and EST's). Additional groups
of rats received the vehicle control or amphetamine. Analysis of
the gene expression data showed that in comparison to the vehicle
treated rats, a large number of genes had expression that increased
by greater than twenty fold. These genes included the neural
adhesion molecule F3, bcl-w, MAP-2, NMDA Receptor, mGluR5 (another
glutamate receptor) each of which increased by 20-70 fold at this
twenty minute time point.
Example 4
Administrating a Viral Vector Encoding Glutamic Acid Decarboxylase
into the Subthalamic Nucleus
[0099] Electrophysiology and microdialysis were performed in the
substantia nigra of normal rats and rats treated with a CBA-GAD65
viral vector encoding human glutamic acid decarboxylase (GAD65/67).
Glutamic acid decarboxylase converts glutamate to GABA in neurons.
The CBA-GAD viral vector was injected into the subthalamic nucleus
three weeks before 6-OHDA lesions of the medial forebrain bundle.
Electrophysiology and microdialysis were performed at least 4
months after the transduction of the viral vector.
[0100] Inhibitory GABA containing connections were detected from
the subthalamic nucleus to the substantia nigra using
electrophysiology and microdialysis. In the microdialysis
experiments a much higher (.about.10.times.) increase in GABA was
detected due to low frequency electrical stimulation of the
subthalamic nucleus, compared to the increase in naive control
rats. Table 2 shows the extracellular concentration of GABA and
glutamate in the substantia nigra obtained before and after low
frequency stimulation in a rat transduced with the CBA-GAD viral
vector as compared to a naive control rat. FIGS. 3 and 4 correspond
to the data in Table 2. In contrast, high frequency stimulation
blocked GABA release in the microdialysis experiment, demonstrating
that the release of GABA, quite independent of transcriptional
regulation, can be modulated by electromagnetic (e.g., electrical)
stimulation.
3TABLE 2 SUBSTANTIA NIGRA MICRODLALYSIS DURING THE SUBTHALAMIC
NUCLEUS STIMULATION GAD65 NAIVE Sample Flow Rate (1.0 GABA GLU GABA
GLU 5 ul/15 ul ul/min) uM uM uM uM Basal 1 0.031 0.328 0.027 0.056
Basal 2 0.033 0.357 0.007 0.133 Basal 3 0.030 0.894 0.004 0.168
ST1-1 LFS-1: 10 Hz, 500 uA for 0.006 0.125 0.004 0.178 2' ST1-2
0.010 0.143 0.031 0.553 ST1-3 0.410 1.008 0.021 0.606 ST1-4 0.026
0.673 0.011 0.501 ST1-5 0.139 1.290 0.032 0.644 ST1-6 0.033 0.624
0.037 0.623 ST1-7 0.034 0.787 0.052 0.904 ST1-8 0.065 1.009 0.027
0.514 ST1-9 0.043 0.976 0.023 0.639 ST2-1 LFS-2: 10 Hz, 500 uA for
0.032 0.758 0.078 0.938 5' ST2-2 0.023 0.819 0.108 1.121 ST2-3
0.033 0.580 0.061 1.213 ST2-4 0.016 0.629 0.043 0.661 ST2-5 0.332
1.564 0.036 0.718 ST2-6 0.044 0.809 0.068 1.220 ST2-7 0.049 0.863
0.049 0.796 ST2-8 0.041 0.866 0.164 1.183 ST2-9 0.038 0.951 0.061
0.852 Note: Each sample was collected every 5-6 minutes.
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