U.S. patent application number 11/215721 was filed with the patent office on 2006-01-12 for methods for treating pain.
This patent application is currently assigned to The Regents of the University of Colorado. Invention is credited to Linda May Rothblum Watkins.
Application Number | 20060008446 11/215721 |
Document ID | / |
Family ID | 33519855 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060008446 |
Kind Code |
A1 |
Watkins; Linda May
Rothblum |
January 12, 2006 |
Methods for treating pain
Abstract
Methods of treating pain by delivery of anti-inflammatory
cytokines, proinflammatory cytokine antagonists, and agents that
act to reduce or prevent proinflammatory cytokine actions, to the
nervous system are described. These agents can be delivered using
gene therapy techniques. Alternatively, the agents can be delivered
in protein compositions.
Inventors: |
Watkins; Linda May Rothblum;
(Boulder, CO) |
Correspondence
Address: |
ROBINS & PASTERNAK LLP
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
The Regents of the University of
Colorado
Boulder
CO
|
Family ID: |
33519855 |
Appl. No.: |
11/215721 |
Filed: |
August 29, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10742641 |
Dec 18, 2003 |
|
|
|
11215721 |
Aug 29, 2005 |
|
|
|
60504175 |
Sep 18, 2003 |
|
|
|
60480886 |
Jun 23, 2003 |
|
|
|
Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 38/1841 20130101;
A61P 25/04 20180101; A61K 48/0075 20130101; A61K 48/0083 20130101;
A61K 38/2066 20130101; A61K 38/20 20130101; A61K 48/005 20130101;
C07K 2319/75 20130101; A61K 38/1793 20130101; C12N 2750/14143
20130101; A61P 29/00 20180101; A61P 25/00 20180101; A61K 38/34
20130101; C12N 2710/10343 20130101; A61K 38/2026 20130101; C07K
2319/32 20130101; C12N 15/86 20130101; A61K 48/00 20130101; A61K
38/2086 20130101; C07K 2319/30 20130101 |
Class at
Publication: |
424/085.2 |
International
Class: |
A61K 38/20 20060101
A61K038/20 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with support under NIH Grants
NS38020 and NS40696, from the National Institute of Neurological
Diseases and Stroke, and DA15642 DA15656, from the National
Institute of Drug Abuse. Accordingly, the United States Government
may have certain rights in this invention
Claims
1. A method of treating pain in a vertebrate subject comprising
administering to the nervous system of said subject a recombinant
vector comprising a polynucleotide encoding an agent selected from
the group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions, operably linked to
expression control elements, under conditions that result in
expression of said polynucleotide in vivo to reduce pain.
2. The method of claim 1, wherein the subject is administered a
polynucleotide encoding an anti-inflammatory cytokine.
3. The method of claim 1, wherein said agent is selected from the
group consisting of interleukin-10 (IL-10), interleukin-1 receptor
antagonist (IL-1ra), interleukin-4 (IL-4), interleukin-13 (IL-13),
tumor necrosis factor soluble receptor (TNFsr), alpha-MSH, and
transforming growth factor-beta 1 (TGF-.beta.1).
4. The method of claim 2, wherein said anti-inflammatory cytokine
is IL-10.
5. The method of claim 4, wherein the IL-10 is fused to the Fc
portion of an IgG.
6. The method of claim 2, wherein said vertebrate subject is a
human and said anti-inflammatory cytokine is human IL-10.
7. The method of claim 1, wherein said recombinant vector is a
recombinant virus.
8. The method of claim 7, wherein said recombinant virus is a
recombinant adenovirus.
9. The method of claim 7, wherein said recombinant virus is a
recombinant adeno-associated virion.
10. The method of claim 1, wherein said recombinant vector is
plasmid DNA.
11. The method of claim 1, wherein said administering is by
intraparenchymal delivery.
12. The method of claim 1, wherein said administering is by
intrathecal delivery.
13. The method of claim 1, wherein said administering is by
epidural delivery.
14. The method of claim 1, wherein the pain is neuropathic
pain.
15. A method of treating pain in a mammalian subject comprising
intrathecally administering to the central nervous system of said
subject a recombinant virus or plasmid comprising a polynucleotide
encoding IL-10, operably linked to expression control elements,
under conditions that result in expression of said polynucleotide
in vivo to reduce pain.
16. The method of claim 15, wherein the IL-10 is fused to the Fc
portion of an IgG.
17. The method of claim 15, wherein said vertebrate subject is a
human and said IL-10 is human IL-10.
18. The method of claim 15, wherein said subject is administered a
recombinant virus.
19. The method of claim 18, wherein said recombinant virus is a
recombinant adenovirus.
20. The method of claim 18, wherein said recombinant virus and is a
recombinant adeno-associated virion.
21. The method of claim 1, wherein said agent is a fragment of
IL-10 comprising the sequence of SEQ ID NO:4.
22. The method of claim 21, wherein said agent consists of the
sequence of SEQ ID NO:4.
23. The method of claim 15, wherein said agent is a fragment of
IL-10 comprising the sequence of SEQ ID NO:4.
24. The method of claim 23, wherein said agent consists of the
sequence of SEQ ID NO:4.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 10/742,641, filed Dec. 18, 2003, from which priority is claimed
under 35 U.S.C. .sctn.120, which is related to U.S. Application
Nos. 60/480,886, filed Jun. 23, 2003, and 60/504,285, filed Sep.
18, 2003, from which applications the benefit is claimed under 35
USC .sctn. 19(e)(1). All of the foregoing applications are
incorporated herein by reference in their entireties.
TECHNICAL FIELD
[0003] The present invention relates generally to gene delivery
methods. In particular, the present invention pertains to methods
of treating or preventing pain by delivery of anti-inflammatory
molecules that act on proinflammatory cytokines, or nucleic acid
encoding the same, to neural tissue.
BACKGROUND
[0004] Gene therapy using genetically engineered cells and viruses
has undergone impressive development over the past 40 years. Gene
therapy techniques have been applied to diverse medical problems
and have been used in over 350 clinical trials (Wu et al., Meth.
Strat. Anesthes. (2001) 94:1119-1132). However, gene therapy has
only recently been used in attempts to control pathological pain.
Several approaches have been explored. For example, spinal
implantation of genetically engineered cells has been used to
increase inhibitory transmitters, including GABA (Eaton, M., J.
Peripheral Nerv. Sys. (2000) 5:59-74), galanin (Eaton et al., J.
Peripheral Nerv. Sys. (1999) 4:245-257), and beta-endorphin (Ishii
et al., Exp. Neurol. (2000) 166:90-98). Herpes viruses have been
utilized for their ability to be retrogradely transported from
peripheral nerve terminals to dorsal root ganglion somas. In this
way, elevations in preproenkephalin (Antunes Bras et al., J.
Neurochem. (1998) 70:1299-1303; Wilson et al., Proc. Natl. Acad.
Sci. USA (1999) 96:3211-3216) and decreases in CGRP via induced
production of CGRP antisense (Lu et al., Soc. Neurosci. Abs. (1998)
24:1625) have been produced in sensory neurons. Lastly,
adenoviruses have been injected into CSF to achieve virally driven
beta-endorphin release from meningeal cells (Finegold et al., Hum.
Gene Ther. (1999) 10:1251-1257. These gene therapy approaches focus
on decreasing the excitability of spinal cord pain transmission
neurons to incoming pain signals.
[0005] Activated spinal cord microglia and astrocytes appear to
contribute to the creation and maintenance of pathological pain. In
particular, activated glia appear to do so, at least in part, via
their release of the proinflammatory cytokines interleukin-1 (IL1),
tumor necrosis factor (TNF), and IL6 (for review, see Watkins et
al., Trends in Neurosci. (2001) 24:450-455). These proinflammatory
cytokines amplify pain by enhancing the release of "pain"
neurotransmitters from incoming sensory nerve terminals and by
enhancing the excitability of spinal cord dorsal horn pain
transmission neurons (Reeve et al., Eur. J. Pain (2000) 4:247-257;
Watkins et al., Trends in Neurosci. (2001) 24:450-455).
[0006] Astrocytes and microglia express receptors for IL-10 (Mizuno
et al., Biochem. Biophys. Res. Commun. (1994) 205:1907-1915) while
spinal cord neurons do not (Ledeboer et al., J. Neuroimmunol.
(2003) 136:94-103). In vitro studies have shown that IL-10 can
selectively suppress proinflammatory cytokine production and
signaling in these glial cells (Moore et al., Ann. Rev. Immunol.
(2001) 19:683-765). In fact, IL-10 is an especially powerful member
of the anti-inflammatory cytokine family in that it can suppress
all proinflammatory cytokines implicated in pathological pain (IL1,
TNF and IL6). IL-10 exerts this effect by inhibiting p38 MAP kinase
activation (Strie et al., Crit. Rev. Immunol. (2001) 21:427-449);
inhibiting NFkappaB activation, translocation and DNA binding
(Strie et al., Crit. Rev. Immunol. (2001) 21:427-449); inhibiting
proinflammatory cytokine transcription (Donnelly et al., J.
Interferon Cytokine Res. (1999) 19:563-573; inhibiting
proinflammatory cytokine mRNA stability and translation (Hamilton
et al., Pathobiology (1999) 67:241-244; Kontoyiannis et al., EMBO
J. (2001) 20:3760-3770); and inhibiting proinflammatory cytokine
release (Moore et al., Ann. Rev. Immunol. (2001) 19:683-765). In
addition, IL-10 stabilizes mRNAs of Suppressors of Cytokine
Signaling, thereby increasing the production of a family of
proteins that further inhibit proinflammatory cytokine production
(Strie et al., Crit. Rev. Immunol. (2001) 21:427-449). IL-10 also
interrupts proinflammatory cytokine signaling by downregulating
proinflammatory cytokine receptor expression (Sawada et al., J.
Neurochem. (1999) 72:1466-1471. Lastly, it upregulates endogenous
antagonists of proinflammatory cytokines, including IL1 receptor
antagonist and TNF decoy receptors (Foey et al., J. Immunol. (1998)
160:920-928; Huber et al., Shock (2000) 13:425-434).
[0007] The known effects of IL-10 are restricted to suppression of
proinflammatory functions of activated immune and glial cells,
leaving non-inflammatory aspects of cellular functions unaffected
(Moore et al., Ann. Rev. Immunol. (2001) 19:683-765). While some
neurons express IL-10 receptors, the only known action of IL-10 on
neurons is inhibition of cell death (apoptosis) (Bachis et al., J.
Neurosci. (2001) 21:3104-3112). Laughlin et al. (Laughlin et al.,
Pain (2000) 84:159-167) reported that intrathecal IL-10 blocks the
onset of intrathecal dynorphin-induced, IL1-mediated mechanical
allodynia. These investigators then tested the effect of IL-10 on
pathological pain induced by excitotoxic spinal cord injury, a
manipulation that activates astrocytes and microglia at the site of
injury (Brewer et al., Exp. Neurol. (1999) 159:484-493). IL-10
decreased pathological pain behaviors when given 30 minutes
following injury (Plunkett et al., Exper. Neurol. (2001)
168:144-154; Yu et al., J. Pain (2003) 4:129-140). This is in
keeping with the fact that systemic IL-10 can reduce spinal cord
proinflammatory cytokine production in response to excitotoxic
injury, a manipulation that allows systemic IL-10 to reach the
injured spinal cord due to disruption of the blood-brain barrier
(Crisi et al., Eur. J. Immunol. (1995) 2:3033-3040; Bethea et al.,
Neurotrauma (1999) 16:851-863).
[0008] However, delivery of IL-10 systemically to treat CNS
disorders is problematic. IL-10 does not cross the intact blood
brain barrier in appreciable amounts (Banks, W. A., J. Neurovirol.
(1999) 5:538-555), has a short half life such that sustainable
delivery for prolonged periods would be difficult (Radwanski et
al., Pharm. Res. (1998) 15: 1895-1901), has not been successfully
delivered orally, so presents problems for systemic administration,
and would disrupt the normal functions of the body's immune system
and would be expected to be detrimental to the health of the
patient (Xing et al., Gene Ther. (1997) 4:140-149; Fedorak et al.,
Gastroenterol. (2000) 119:1473-1482; Tilg et al., J. Immunol.
(2002) 169:2204-2209). Moreover, previous experimentors found that
delivery of IL-10 24 hours after dynorphin-induced allodynia did
not reduce the allodynia (Laughlin et al., Pain (2000)
84:159-167).
[0009] Previous reports have documented that IL-10 gene therapy
reduced pneumonia-induced lung injury (Morrison et al., Infect.
Immun. (2000) 68:4752-4758), decreased the severity of rheumatoid
arthritis (Ghivizzani et al., Clin. Orthop. (2000) 379
Suppl.:S288-299), decreased inflammatory lung fibrosis (Boehler et
al., Hum. Gene Ther. (1998) 9:541-551), inhibited cardiac allograft
rejection (Brauner et al., J. Thoracic Cardiovasc. Surg. (1997)
114:923-933), suppressed endotoxemia (Xing et al., Gene Ther.
(1997) 4:140-149), prevented and treated colitis (Lindsay et al.,
J. Immunol. (2001) 166:7625-7633), and reduced contact
hypersensitivity (Meng et al., J. Clin. Invest. (1998)
101:1462-1467).
[0010] However, the ability of IL-10 gene therapy to reverse
ongoing pain has not been documented prior to the present
invention.
SUMMARY OF THE INVENTION
[0011] The present invention is based on the surprising discovery
that pain can be successfully treated by delivering
anti-inflammatory cytokines, such as IL-10 and IL-1ra, using gene
therapy techniques, such as by targeting cells and tissues of the
nervous system, including the spinal cord glia. In particular, the
inventors herein have shown in acceptable pain models that gene
delivery of anti-inflammatory cytokines and proinflammatory
cytokine antagonists, such as IL-10 and IL-1ra, prevents and
reverses pain, such as pathological and neuropathic pain, including
thermal hyperalgesia and mechanical allodynia, without affecting
basal pain responsivity to thermal or mechanical stimuli. Because
these agents appear to selectively inhibit products of glial
activation that lead to pathology while leaving basal glial and
neuronal functions unaltered, this novel gene therapy approach for
the control of pain provides a highly desirable alternative to
neuronally focused gene therapies. Moreover, IL-10 and other agents
that act on proinflammatory cytokines can be delivered either alone
or in conjunction with gene therapy in order to treat existing
pain.
[0012] Accordingly, in one embodiment, the invention is directed to
a method of treating pain, such as neuropathic pain, in a
vertebrate subject comprising administering to the nervous system
of the subject a recombinant vector comprising a polynucleotide
encoding an agent selected from the group consisting of an
anti-inflammatory cytokine, a proinflammatory cytokine antagonist,
and an agent that acts to reduce or prevent proinflammatory
cytokine actions, operably linked to expression control elements,
under conditions that result in expression of the polynucleotide in
vivo to reduce pain.
[0013] In certain embodiments, the agent is one or more agents
selected from the group consisting of interleukin-10 (IL-10),
interleukin-1 receptor antagonist (IL-1ra), interleukin-4 (IL-4),
interleukin-13 (IL-13), tumor necrosis factor soluble receptor
(TNFsr), alpha-MSH, and transforming growth factor-beta 1
(TGF-.beta.1).
[0014] In yet further embodiments, the vertebrate subject is a
human and the anti-inflammatory cytokine is human IL-10.
[0015] In any of the above embodiments, the recombinant vector can
be a recombinant virus, such as a recombinant adenovirus or a
recombinant adeno-associated virion, or plasmid DNA. Moreover, if
IL-10 is used, the IL-10 can be stabilized by providing the
molecule as a fusion with the Fc portion of an IgG, as described
more fully below.
[0016] In additional embodiments, the administering is by
intraparenchymal, intrathecal or epidural delivery.
[0017] In further embodiments, the method further comprises
subsequently administering at five days or less, such as three days
or less, after the first administration, a recombinant vector
comprising a polynucleotide encoding an agent selected from the
group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions, operably linked to
expression control elements, under conditions that result in
expression of the polynucleotide in vivo to maintain reduced
pain.
[0018] In additional embodiments, the method further comprises
subsequently administering at five days or less, such as at three
days or less, after the first administration, a therapeutically
effective amount of a composition comprising an agent selected from
the group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions to maintain reduced
pain.
[0019] In yet another embodiment, the invention is directed to a
method of treating pain in a mammalian subject comprising
intrathecally administering to the central nervous system of the
subject a recombinant virus or plasmid comprising a polynucleotide
encoding IL-10, operably linked to expression control elements,
under conditions that result in expression of the polynucleotide in
vivo to reduce pain.
[0020] In certain embodiments, the vertebrate subject is a human
and the IL-10 is human IL-10. The IL-10 can be stabilized by
providing the molecule as a fusion with the Fc portion of an IgG,
as described more fully below.
[0021] In additional embodiments, the subject is administered a
recombinant virus, such as a recombinant adenovirus or a
recombinant adeno-associated virion. In other embodiments, the
subject is administered plasmid DNA.
[0022] In still further embodiments, the method further comprises
subsequently administering at five days or less, such as at three
days or less after the first administration, a recombinant vector
comprising a polynucleotide encoding IL-10, operably linked to
expression control elements, under conditions that result in
expression of said polynucleotide in vivo to maintain reduced
pain.
[0023] In additional embodiments, a therapeutically effective
amount of a composition comprising IL-10 is subsequently
administered at five days or less, such as three days or less after
the first administration, to maintain reduced pain.
[0024] In still further embodiments, the invention is directed to a
method of treating existing pain in a vertebrate subject comprising
intrathecally administering to the subject a therapeutically
effective amount of an agent selected from the group consisting of
an anti-inflammatory cytokine, a proinflammatory cytokine
antagonist, and an agent that acts to reduce or prevent cytokine
actions.
[0025] In certain embodiments, the agent is one or more agents
selected from the group consisting of interleukin-10 (IL-10),
interleukin-1 receptor antagonist (IL-1ra), interleukin-4 (IL-4),
interleukin-13 (IL-13), tumor necrosis factor soluble receptor
(TNFsr), alpha-MSH, and transforming growth factor-beta 1
(TGF-.beta.1).
[0026] In additional embodiments, the vertebrate subject is a human
and the anti-inflammatory cytokine is human IL-10. The IL-10 can be
stabilized by providing the molecule as a fusion with the Fc
portion of an IgG, as described more fully below.
[0027] In yet additional embodiments, the method further comprises
subsequently administering at five days or less after the first
administration, such as at three days or less, a recombinant vector
comprising a polynucleotide encoding an agent selected from the
group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions, operably linked to
expression control elements, under conditions that result in
expression of said polynucleotide in vivo to maintain reduced
pain.
[0028] In additional embodiments, the method further comprises
subsequently administering at five days or less, such as at three
days or less after the first administration, a therapeutically
effective amount of a composition comprising an agent selected from
the group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions to maintain reduced
pain.
[0029] In further embodiments, the invention is directed to a
method of treating existing pain in a vertebrate subject
comprising: [0030] (a) administering to the nervous system of the
subject a first composition comprising a therapeutically effective
amount of interleukin-10 (IL-10); and [0031] (b) administering to
the nervous system of the subject a second composition comprising a
therapeutically effective amount of IL-10 at five days or less,
such as at three days or less, after the first administration.
[0032] In certain embodiments, the first composition and the second
composition are the same. In other embodiments, the first
composition and the second composition are different. The IL-10 in
the first composition and/or in the second composition can be fused
to the Fc portion of an IgG. Additionally, in certain embodiments,
the vertebrate subject is a human and the IL-10 in the first
composition and/or the second composition is human IL-10.
[0033] In yet further embodiments, the invention is directed to a
method of treating existing pain in a vertebrate subject
comprising: [0034] (a) administering to the nervous system of the
subject a first composition comprising a therapeutically effective
amount of interleukin-10 (IL-10); and [0035] (b) administering to
the nervous system of the subject a second composition comprising a
recombinant vector comprising a polynucleotide encoding IL-10,
operably linked to expression control elements, under conditions
that result in expression of said polynucleotide in vivo, wherein
the second composition is administered at five days or less, such
as at three days or less, after the first composition is
administered.
[0036] In certain embodiments of the invention, the IL-10 in the
first composition and/or in the second composition is fused to the
Fc portion of an IgG. In additional embodiments, the vertebrate
subject is a human and the IL-10 in the first compositions and/or
the second composition is human IL-10.
[0037] In additional embodiments, the invention is directed to a
method of treating existing pain in a vertebrate subject, such as
neuropathic pain, comprising administering to the subject a
therapeutically effective amount of a composition comprising an
IL-10 polypeptide. In certain embodiments, the IL-10 polypeptide is
fused to the Fc portion of an IgG. In additional embodiments, the
vertebrate subject is a human and the anti-inflammatory cytokine is
human IL-10.
[0038] In yet further embodiments, administering is by
intraparenchymal, intrathecal or epidural delivery.
[0039] In additional embodiments, the method further comprises
subsequently administering at five days or less, such as at three
days or less, after the first administration, a recombinant vector
comprising a polynucleotide encoding an agent selected from the
group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions, operably linked to
expression control elements, under conditions that result in
expression of the polynucleotide in vivo to maintain reduced
pain.
[0040] In further embodiments, the method further comprises
subsequently administering at five days or less, such as at three
days or less, after the first administration, a therapeutically
effective amount of a composition comprising an agent selected from
the group consisting of an anti-inflammatory cytokine, a
proinflammatory cytokine antagonist, and an agent that acts to
reduce or prevent inflammatory cytokine actions to maintain reduced
pain.
[0041] These and other embodiments of the subject invention will
readily occur to those of skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 shows the effect of increasing doses of intrathecal
adenovirus on response threshold to calibrated touch/pressure
stimuli. Lower doses of adenovirus had no detectable effect on
response thresholds to calibrated touch/pressure stimuli as
assessed by the von Frey test. The highest doses lowered the
response threshold. The asterisks indicate the doses used in the
remaining experiments. In this and all subsequent figures of data
generated by the von Frey test, the y-axis represents the log
transformation used for data analysis. The log transformation
(followed by its mg force in parenthesis) of the stimuli used in
the test were as follows: 3.61 (407 mg), 3.84 (692 mg), 4.08 (1,202
mg), 4.17 (1,479 mg), 4.31 (2,041 mg), 4.56 (3,630 mg), 4.74 (5,495
mg), 4.93 (8,511 mg), 5.07 (11,749 mg), and 5.18 (15,136 mg).
[0043] FIG. 2 shows that adenoviral-delivered IL-10 prevents
intrathecal HIV-I gp120-induced mechanical allodynia. After predrug
(baseline; BL) assessment on the von Frey test, animals were
injected intrathecally with either adenovirus encoding for IL-10
(AD-IL10) or a control adenovirus that encoded for
beta-galactosidase (AD-Control). Response thresholds were
reassessed 4 and 5 days later to test whether either the presence
of virus and/or the presence of virally generated human IL-10
affected basal response thresholds. As seen, the Day 4 and Day 5
thresholds were not affected, compared to predrug BL. At this time,
animals were injected intrathecally with HIV-1 gp120 at a dose (3
.mu.g) previously shown to produce mechanical allodynia (Milligan
et al., Brain Res. (2000) 861:105-116; Milligan et al., J.
Neurosci. (2001) 21:2808-2819). Animals receiving intrathecal
AD-Control followed by intrathecal gp120 developed mechanical
allodynia as in previous experiments. In contrast, mechanical
allodynia was prevented in animals receiving intrathecal
AD-IL10.
[0044] FIG. 3 shows human IL-10 levels in lumbosacral and cervical
cerebrospinal fluid following lumbosacral adenovirus
administration. Upon completion of behavioral testing (Day 5 after
AD administration), lumbosacral and cervical CSF were collected
from animals in Examples 2 and 3. These samples were assayed by
ELISA for human IL-10. Since this ELISA does not detect rat IL-10,
this allows virally induced IL-10 to be detected independent of rat
IL-10. The 10.times.10.sup.7 plaque forming units (PFU) injected in
Experiment 2 for prevention of gp120-induced allodynia (see FIG. 2)
produced far greater levels of human IL-10 in lumbosacral CSF than
cervical CSF, indicating site specific effects of the virus.
Comparable doses of AD-Control produced low values on this
commercial ELISA test. The 5.times.10.sup.7 PFU injected in Example
3 for prevention of sciatic inflammatory neuropathy (see FIG. 4)
appears to have produced lower levels of human IL-10.
[0045] FIGS. 4A-4D show that adenoviral-delivered IL-I0 prevents
sciatic inflammatory neuropathy (SIN)-induced mechanical
allodynias. After predrug (baseline; BL) assessment on the von Frey
test, animals were injected intrathecally with either adenovirus
encoding for IL-10 (AD-IL10) (FIGS. 4B and 4D) or a control
adenovirus that encoded for beta-galactosidase (AD-Control) (FIGS.
4A and 4C). Response thresholds were reassessed 4 days later to
test whether either the presence of virus and/or the presence of
virally generated human IL-10 affected basal response thresholds.
As seen, Day 5 thresholds were not affected, compared to predrug
BL. At this time, animals were unilaterally injected
peri-sciatically with either 4 (FIGS. 4A and 4B) or 160 (FIGS. 4C
and 4D) .mu.g zymosan (yeast cell walls). These doses have
previously been shown to produce unilateral (ipsilateral to zymosan
injection) and bilateral mechanical allodynia, respectively
(Milligan et al., J. Neurosci. (2003) 23:1026-1040). Animals
receiving intrathecal AD-Control followed by perisciatic zymosan
developed unilateral (FIG. 4A) and bilateral (FIG. 4C) mechanical
allodynia as in previous studies. In contrast, both unilateral and
bilateral mechanical allodynias were prevented in animals receiving
intrathecal AD-IL10 (FIGS. 4B and 4D).
[0046] FIG. 5 shows that adenoviral IL-10 reverses chronic sciatic
inflammatory neuropathy (SIN)-induced mechanical allodynias. After
predrug (baseline; BL) assessment on the von Frey test, animals
were repeatedly injected with either 4 or 160 .mu.g zymosan to
chronically induce unilateral (Panels A & B) or bilateral
(Panels C and D) allodynia, respectively. These peri-sciatic
zymosan injections continued throughout the behavioral testing
time-course. After behavioral verification of allodynia on Day 8,
all rats were injected intrathecally with either AD-Control (Panels
A and C) or AD-IL10 (Panels B and D). Rats receiving AD-Control
remained unilaterally (Panel A) or bilaterally (Panel C) allodynic
throughout the assessment time-course. In contrast, both unilateral
(Panel B) and bilateral (Panel D) allodynia were reversed by
intrathecal AD-IL10.
[0047] FIG. 6 shows that adenoviral IL-10 reverses both ipsilateral
and mirror-image territorial and extra-territorial allodynias.
Sciatic (territorial; Panels A and B) and saphenous
(extra-territorial; Panels C and D) nerve innervation areas were
separately tested prior to (baseline; BL) and after (Days 8, 12 and
14) chronic peri-sciatic 160 .mu.g zymosan to induce bilateral
mechanical allodynia. After the Day 8 assessment, AD-Control
(Panels A and C) or AD-IL10 (Panels B and D) was administered.
Comparable reversal by AD-IL10 was observed on Days 12 and 14 (4
and 6 days after AD, respectively) for ipsilateral and mirror image
allodynias in both sciatic (Panel B) and saphenous innervation
areas (Panel D) compared to AD-Control.
[0048] FIG. 7 shows adenoviral IL-10 attenuates sciatic chronic
constriction injury (CCI)-induced mechanical allodynias. After
predrug (baseline; BL) assessment on the von Frey test, sham
(Panels A and B) or CCI (Panels C and D) surgery was performed.
Behavioral assessments were recorded on Days 3, 5, 7 and 10 to
document the lack of allodynia in sham-operated rats and
progressive development of bilateral allodynia in CCI groups. After
the Day 10 assessment, rats received intrathecal injections of
either AD-Control (Panels A and C) or AD-IL10 (Panels B and D).
Behavioral assessments were again recorded on Days 13, 15, 17, 24,
28, and 31; that is, Days 3, 5, 7, 14, 18, and 21 days after AD.
While neither AD-Control nor AD-IL10 exerted marked effects in
sham-operated animals, AD-IL10 transiently attenuated bilateral CCI
allodynia (Panel D) compared to CCI operated AD-Control treated
animals (Panel C).
[0049] FIG. 8 shows that adenoviral IL-10 attenuates chronic
constriction injury (CCI)-induced thermal hyperalgesia. After
predrug (baseline; BL) assessment on the Hargreaves test, sham
(Panels A and B) or CCI (Panels C and D) surgery was performed.
Behavioral assessments were recorded on Days 3, 5, 7 and 10 to
document the lack of thermal hyperalgesia in sham operated rats and
progressive development of ipsilateral (unilateral) thermal
hyperalgesia in CCI groups. After the Day 10 assessment, rats
received intrathecal injections of either AD-Control (Panels A and
C) or AD-IL10 (Panels B and D). Behavioral assessments were again
recorded on Days 13, 15, 17, 24, 28, and 31; that is, Days 3, 5, 7,
14, 18, and 21 days after AD. While neither AD-Control nor AD-ILI0
exerted marked effects in sham operated animals, AD-IL10
transiently attenuated ipsilateral CCI thermal hyperalgesia (Panel
D) compared to CCI operated AD-Control treated animals (Panel
C).
[0050] FIG. 9 shows the effect of AAV-delivered IL-10 on chronic
SIN-induced allodynia. After baseline (BL) assessment, rats were
injected intrathecally with either AAV-Control or AAV-IL10. After
allowing the AAV to infect, rats were then chronically injected
over the left sciatic nerve with zymosan (yeast cell walls) to
create an inflammatory neuropathy. Profound neuropathic pain was
demonstrated by SIN in rats receiving intrathecal control virus
(open squares). Intrathecal AAV-IL10 blunted this neuropathic pain
(open circles). Filled squares and filled circles show normal pain
responses of the uninvolved hindleg (right).
[0051] FIG. 10 shows the effect of AAV-delivered IL-10 on
mechanical allodynia induced by CCI. After baseline (BL)
assessment, rats were given either sham surgery or CCI of the left
sciatic nerve to induce traumatic neuropathy. After behavioral
assessment on Day 10, rats were injected intrathecally with either
AAV-Control or AAV-IL 10. Profound neuropathic pain was
demonstrated by CCI in rats receiving intrathecal control virus
(filled squares). Intrathecal AAV-IL10 blunted this neuropathic
pain (open squares). Filled circles and open circles show normal
pain responses of sham operated rats administered either
AAV-Control or AAV-IL10.
[0052] FIG. 11 shows the effect of AAV-delivered IL-10 on chronic
thermal hyperalgesia induced by CCI. After baseline (BL)
assessment, rats were given either sham surgery or CCI of the left
sciatic nerve to induce traumatic neuropathy. After behavioral
assessment on Day 10, rats were injected intrathecally with either
AAV-Control or AAV-IL10. Profound neuropathic pain was demonstrated
by CCI in rats receiving intrathecal control virus (open circles).
Intrathecal AAV-IL10 blunted this neuropathic pain (filled
circles). Filled squares and open squares show normal pain
responses of sham operated rats administered either AAV-Control or
AAV-IL10.
[0053] FIGS. 12A and 12B show the effects of AAV-IL10 on chronic
thermal hyperalgesia induced by CCI (FIG. 12A) and chronic
mechanical allodynia induced by CCI (FIG. 12B). These were partial
timecourses as the experiments were stopped at the point of
complete pain reversal so that tissues could be collected for
analyses. After baseline (BL) assessments, rats were given either
sham surgery or CCI of the left sciatic nerve to induce traumatic
neuropathy. After behavioral assessment on Day 10, rats were
injected intrathecally with either AAV-Control or AAV-IL10.
Behavior was reassessed 3, 5 and 7 days later (corresponding to
Days 13, 15 and 17 after CCI or sham surgery). Profound neuropathic
pain was demonstrated in CCI rats receiving intrathecal control
virus. Intrathecal AAV-IL10 blunted this neuropathic pain. Normal
pain responses were observed for sham operated rats administered
either AAV-Control or AAV-IL10.
[0054] FIG. 13 shows that non-viral vector (NVV) plasmid DNA-driven
IL-10 completely reverses CCI induced mechanical allodynia and that
repeated intrathecal administration of plasmid IL-10 induces
progressively longer pain-relieving effects. After baseline (BL),
CCI was induced and rats were given intrathecal injections of
either plasmid IL-10 or plasmid GFP as a control at the time-points
indicated in the figure by arrows. Filled squares indicate CCI rats
administered plasmid IL-10; open squares indicate CCI rats
administered the GFP control plasmid; filled circles indicate sham
operated rats given plasmid IL-10; open circles indicate sham
operated rats administered GFP control plasmid.
[0055] FIG. 14 shows that AD-IL10 potentiates the analgesic effects
of acute morphine. To test whether IL-10 would affect the
pain-relieving effects of opiates such as morphine, rats were
pretreated 5 days prior to morphine with either AD-Control (open
diamonds) or AD-IL10 (filled square). A single animal received no
virus. As can be seen, rats expressing AD-IL10 (filled squares)
showed a more prolonged analgesia than rats with AD-Control
(diamonds).
[0056] FIG. 15 shows that AD-IL10 delays development of morphine
tolerance. Rats were given 10 .mu.g intrathecal morphine daily.
Even by the third day of morphine administration, it was obvious
that AD-IL10 was delaying the development of morphine
tolerance.
[0057] FIG. 16 shows that AD-IL10 delays development of morphine
tolerance. Rats were given 10 .mu.g intrathecal morphine daily.
Again, on the fifth day of morphine administration, it was obvious
that AD-IL10 was delaying the development of morphine
tolerance.
[0058] FIG. 17 shows that AD-IL10 prevents exaggerated pain which
develops as a consequence of repeated opiate administration. Prior
to morphine (day 1), all rats responded normally to the von Frey
test for mechanical pain sensitivity. Afterwards, rats got 10 .mu.g
intrathecal morphine daily. 24 hr after the last dose of morphine,
increased sensitivity (i.e. pain facilitation) was seen in rats
receiving AD-Control (open diamond). AD-IL10 (filled squares)
completely prevented the exaggerated pain responses created by
chronic morphine.
[0059] FIG. 18 shows the results of interleukin-1 receptor
antagonist (IL-1ra) on morphine analgesia in rats. To test for
generality of the concept, repeated injections of an endogenous
proinflammatory cytokine antagonist, IL-1ra, were used instead of
gene delivery of IL-10. A single injection of IL1ra, which blocks
proinflammatory cytokine function, potentiated the analgesic effect
of morphine (filled squares) compared to vehicle+morphine (open
squares).
[0060] FIG. 19 shows the results of IL-1ra on the development of
morphine tolerance. IL-1ra injected daily along with daily morphine
injections (filled squares) delayed the development of morphine
tolerance compared to rats receiving daily vehicle+morphine (open
squares).
[0061] FIG. 20 shows the continued effect of IL-1ra on morphine
tolerance. The continuing effect was still observed on the last day
of testing, that is after 5 days of morphine.
[0062] FIGS. 21A-21C show the effect of morphine administration of
pain and IL-1 production. FIG. 21A shows that after repeated
injections of morphine, exaggerated pain occurs upon
discontinuation of the opiate. Animals given repeated morphine
showed exaggerated response sensitivity to touch/pressure stimuli
24 hr after their last of 5 daily doses of intrathecal morphine
(black bar, left) compared to rats receiving intrathecal saline
instead of morphine (white bar, left). In contrast, when rats
received daily IL-1ra, this increase in pain sensitivity was
alleviated (black bar, right). FIGS. 21B and 21C show that chronic
intrathecal morphine, but not equivolume chronic intrathecal
vehicle, increases the production and release of the
proinflammatory cytokine interleukin-1 in spinal cord. Rats were
either given 5 days of once daily intrathecal injections of 10
.mu.g morphine or equivolume vehicle. Two hours after the last
injection, CSF and spinal cord were harvested and analyzed for IL-1
protein content by ELISA. As can be seen for both spinal cord CSF
(FIG. 21B) and tissue (FIG. 21C), proinflammatory cytokine content
was enhanced by chronic morphine.
[0063] FIGS. 22A and 22B show that intrathecal injection of rat
recombinant IL-10 (no plasmid; simply injection of the IL10
protein) only very briefly reverses mechanical allodynia even at
very high doses. FIG. 22A shows the hindpaw on the same side (left
side) of the CCI and FIG. 22B shows the hindpaw of the healthy
hindleg (right side). After baseline (BL) testing, rats received
either CCI or sham surgery. They were retested 3 and 10 days later
to verify that CCI (but not sham surgery) induced profound
neuropathic pain as measured by mechanical allodynia. On Days 10,
11 and 12 (relative to CCI surgery), rats received an i.t.
injection of either IL-10 protein or vehicle. The first injection
(on Day 10) was 50 ng IL-10; the second (on Day 11) and third (on
Day 12) injections were 500 ng IL-10. As seen in FIGS. 22A and 22B,
50 ng only partially reversed allodynia, a bit larger reversal was
seen with 10 times that amount. Strikingly, the reversals were very
short lived (less than 24 hr) and no increasing effectiveness was
observed with repeated injections.
[0064] FIGS. 23A and 23B show that intrathecal injection of rat
recombinant IL-10 (no plasmid; simply injection of the IL-10
protein) only very briefly reverses thermal hyperalgesia even at
very high doses. FIG. 23A shows the hindpaw on the same side (left
side) of the CCI and FIG. 23B shows the hindpaw of the healthy
hindleg (right side). It should be noted that CCI only produces
pathological pain changes in the leg on the side of the nerve
damage (that is, the left paw). The data from the right paw are
included for completeness and to show that IL-10 and vehicle
injections had no effect on the behaviors elicited from this
control paw. After baseline (BL) testing, rats received either CCI
or sham surgery. They were retested 3 and 10 days later to verify
that CCI (but not sham surgery) induced profound neuropathic pain
as measured by mechanical allodynia. On Days 10, 11 and 12
(relative to CCI surgery), rats received an i.t. injection of
either IL-10 protein or vehicle. The first injection (on Day 10)
was 50 ng IL-10; the second (on Day 11) and third (on Day 12)
injections were 500 ng IL-10. As seen in FIG. 23A, 50 ng had no
effect. A transient reversal was seen with 10 times that amount.
The reversals were very short lived (less than 24 hr) and no
increasing effectiveness was observed with repeated injections.
[0065] FIG. 24 shows that two doses of non-viral vector (NVV)
plasmid DNA-driven IL-10 delivery three days apart induces
prolonged attenuation of CCI induced mechanical allodynia. Plasmid
IL-10 was injected intrathecally at Day 10 after CCI and three days
later. Filled squares indicate the results using plasmid IL-10
while open squares show the results of control plasmid.
[0066] FIG. 25 shows that when the IL-10 plasmid from the
experiment described in FIG. 25 is linearized, it is no longer
effective in attenuating CCI induced mechanical allodynia.
[0067] FIG. 26 shows that intrathecal administration of recombinant
IL-10 protein blocked mechanical allodynia induced by peri-sciatic
injection of phospholipase A2 (PLA2). Open ovals indicate results
from intrathecal administration of IL-10 and peri-sciatic delivery
of vehicle only. Open rectangles indicate results from intrathecal
administration of vehicle only and peri-sciatic delivery of vehicle
only. Filled ovals indicate results from intrathecal administration
of IL-10 and peri-sciatic delivery of PLA2. Filled rectangles
indicate results from intrathecal administration of vehicle only
and peri-sciatic delivery of vehicle only.
[0068] FIG. 27 shows that FcIL-10, delivered intrathecally, is
effective in reversing mechanical allodynia induced by CCI. After
baseline (BL) testing, rats received CCI surgery. They were
re-tested 3 and 10 days later to verify that CCI induced profound
neuropathic pain on both measures. After the Day 10 test, rats were
injected i.t. with a stabilized variant of IL-10 (FcIL-10) plus a
plasmid encoding for IL-10. Since plasmid has no effect on behavior
until one day later, effects observed shortly after this injection
procedure reflect actions by FcIL-10 itself. As can be seen in FIG.
27 mechanical allodynia was transiently reversed by FcIL10
treatment.
[0069] Figure shows that FcIL-10 is effective in enhancing reversal
of mechanical allodynia when co-administered with a gene therapy
vector, here shown with a plasmid encoding for IL-10. After
baseline (BL) testing, rats received CCI surgery. They were
re-tested 3 and 10 days later to verify that CCI induced profound
neuropathic pain on both measures. After the Day 10 test, rats were
injected i.t. with a control plasmid that did not encode IL-10;
rather, it encoded for an inert intracellular protein (GFP). The
presence of inert plasmid DNA did not affect behaviors tested the
subsequent days. After the Day 13 test, rats were injected with
either: (a) only plasmid encoding for IL-10 or (b) an equal amount
of plasmid encoding for IL-10 plus a stabilized variant of IL-10
(FcIL-10) to test whether the presence of FcIL-10 would enhance
vector efficacy. Indeed it does. Mechanical allodynia was reversed
by plasmid-IL-10 alone for approximately 4 days. In contrast, the
co-treatment with FcIL-10 remarkably enhanced both the onset and
duration of plasmid-IL-10 efficacy on mechanical allodynia.
[0070] FIGS. 29A, 29B and 29C show that lower doses and dose
combinations of plasmid IL-10 gene therapy effectively reverse
CCI-induced mechanical allodynia. After baseline (BL) testing, rats
received CCI surgery. They were re-tested 3 and 10 days later to
verify that CCI induced profound neuropathic pain. Rats were then
injected with either: (a) 100 .mu.g plasmid encoding IL-10 (Day 10)
followed by 50 .mu.g plasmid encoding IL-10 (Day 13) (FIG. 29A);
(b) 100 .mu.g plasmid encoding IL-10 (Day 10) followed by 25 .mu.g
plasmid encoding IL-10 (Day 13) (FIG. 29B); or (c) 50 .mu.g plasmid
encoding IL-10 (Day 10) followed by 50 .mu.g plasmid encoding IL-10
(Day 13) (FIG. 29C). Each led to reversal of mechanical allodynia
over time.
[0071] FIGS. 30A and 30B show that gene therapy with IL-10 is
likely reversing CCI, because CCI is mediated by proinflammatory
cytokines. After baseline assessment on the von Frey test (BL), CCI
or sham surgery was performed, and behavior reassessed 3 and 10
days later to verify surgical efficacy. In FIG. 30A, rats then
received either intrathecal interleukin-1 receptor antagonist or
equivolume vehicle and were tested over time. In FIG. 30B, the
identical procedure was carried out except that the drug injections
were administered 2 months after surgery. As can be seen, IL-1ra
transiently reversed CCI-induced enhanced pain at both times
tested, supporting that proinflammatory cytokine are enduring
mediators of neuropathic pain in particular, and pathological pain
more generally enduring mediators of neuropathic pain in
particular, and pathological pain more generally.
[0072] FIG. 31 shows a comparison of the amino acid sequences of
mature secreted forms of human IL-10 (hIL-10) (SEQ ID NO:1), mouse
IL-10 (mIL-10) (SEQ ID NO:2) and a viral form of IL-10 (vIL-10)
(SEQ ID NO:3). Amino acid residues differing from the human
sequence are boxed.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, recombinant DNA techniques and immunology, within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Fundamental Virology, 2nd Edition, vol. I
& II (B. N. Fields and D. M. Knipe, eds.); Handbook of
Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell
eds., Blackwell Scientific Publications); T. E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and
Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,
Inc., current addition); Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.
Colowick and N. Kaplan eds., Academic Press, Inc.).
[0074] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0075] 1. Definitions
[0076] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0077] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "an anti-inflammatory cytokine"
includes a mixture of two or more such cytokines, and the like.
[0078] By "pathological pain" is meant any pain resulting from a
pathology, such as from functional disturbances and/or pathological
changes, injuries, lesions, burns and the like. One form of
pathological pain is "neuropathic pain." The term "neuropathic
pain" as used herein refers to pain caused by, but not limited to,
a neuropathy, an encephalopathy and/or a myelopathy (i.e.,
functional disturbances or pathological states of the peripheral
nervous system, brain and spinal cord, respectively). Neuropathic
pain can be caused by nerve damage, injury such as spinal cord
injury, neuritis, inflammation, noninflammatory lesions, electrical
injuries, headaches, and the like. Neuropathic pain can also be
caused by complications of various diseases, including without
limitation, demyelinating diseases, diabetes, amyloid diseases,
porphyric diseases, Lyme disease, leprosy, acromegaly, rheumatoid
arthritis, autoimmune diseases, metabolic diseases, cancer, and
viral infection. Such pain can also be caused by toxic states, such
as but not limited to, toxic states caused by arsenic, isoniazid,
lead and nitrofurantoin. Examples of neuropathic pain include, but
are not limited to, thermal or mechanical hyperalgesia, thermal or
mechanical allodynia, diabetic pain, pain arising from irritable
bowel or other internal organ disorders, endometriosis pain,
phantom limb pain, complex regional pain syndromes, fibromyalgia,
low back pain, cancer pain, pain arising from infection,
inflammation or trauma to peripheral nerves or the central nervous
system, multiple sclerosis pain, entrapment pain, pain from HIV
infection, herpesvirus infection, and the like.
[0079] "Hyperalgesia" means an abnormally increased pain sense,
such as pain that results from an excessive sensitiveness or
sensitivity.
[0080] "Hypalgesia" (or "hypoalgesia") means the decreased pain
sense.
[0081] "Allodynia" means pain that results from a non-noxious
stimulus to the skin. Examples of allodynia include, but are not
limited to, cold allodynia, tactile allodynia, and the like.
[0082] "Nociception" is defined herein as pain sense. "Nociceptor"
herein refers to a structure that mediates nociception. The
nociception may be the result of a physical stimulus, such as,
mechanical, electrical, thermal, or a chemical stimulus.
Nociceptors are present in virtually all tissues of the body.
[0083] "Analgesia" is defined herein as the relief of pain without
the loss of consciousness. An "analgesic" is an agent or drug
useful for relieving pain, again, without the loss of
consciousness.
[0084] The term "nervous system" includes both the central nervous
system and the peripheral nervous system." The term "central
nervous system" or "CNS" includes all cells and tissue of the brain
and spinal cord of a vertebrate. The term "peripheral nervous
system" refers to all cells and tissue of the portion of the
nervous system outside the brain and spinal cord. Thus, the term
"nervous system" includes, but is not limited to, neuronal cells,
glial cells, astrocytes, cells in the cerebrospinal fluid (CSF),
cells in the interstitial spaces, cells in the protective coverings
of the spinal cord, epidural cells (i.e., cells outside of the dura
mater), cells in non-neural tissues adjacent to or in contact with
or innervated by neural tissue, cells in the epineurium,
perineurium, endoneurium, funiculi, fasciculi, and the like.
[0085] The term "anti-inflammatory cytokine" as used herein refers
to a protein that decreases the action or production of one or more
proinflammatory cytokines or proteins produced by nerves, neurons,
glial cells, endothelial cells, fibroblasts, muscle, immune cells
or other cell types. Such inflammatory cytokines and proteins
include, without limitation, interleukin-1 beta (IL-1.beta.), tumor
necrosis factor-alpha (TNF-.alpha.), interleukin-6 (IL-6),
inducible nitric oxide synthetase (iNOS) and the like. Non-limiting
examples of anti-inflammatory cytokines include interleukin-10
(IL-10) including viral IL-10, interleukin-4 (IL-4), interleukin-13
(IL-13), alpha-MSH, transforming growth factor-beta 1
(TGF-.beta.1), and the like. All of these anti-inflammatory
cytokines, as well as active fragments, and active analogs thereof,
which retain the ability to decrease pain as measured in any of the
known pain models including those described further herein, are
intended for use with the present invention.
[0086] Thus, the full-length proteins and fragments thereof, as
well as proteins with modifications, such as deletions, additions
and substitutions (either conservative or non-conservative in
nature), to the native sequence, are intended for use herein, so
long as the protein maintains the desired activity. These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification. Accordingly, active proteins substantially
homologous to the parent sequence, e.g., proteins with 70 . . . 80
. . . 85 . . . 90 . . . 95 . . . 98 . . . 99% etc. identity that
retain the ability to reduce pain, are contemplated for use
herein.
[0087] By "proinflammatory cytokine antagonist" is meant any
molecule that blocks or antagonizes the biologic action of a
proinflammatory cytokine, such as by binding or interacting with a
proinflammatory cytokine receptor thereby reducing or inhibiting
the production of the proinflammatory cytokine. The terms
"antagonist", "inhibitor", and "blocker" are used interchangeably
herein. Non-limiting examples of such antagonists include
interleukin-1 receptor antagonist (IL-1ra); KINERET (recombinant
IL-1ra, Amgen); tumor necrosis factor soluble receptor (TNFsr);
soluble TNF receptor Type I (Amgen); pegylated soluble TNF receptor
Type I (PEGs TNF-R1) (Amgen); TNF decoy receptors; ETANERCEPT
(ENBREL, Amgen); INFLIXIMAB (REMICADE, Johnson & Johnson);
D2E7, a human anti-TNF monoclonal antibody (Knoll Pharmaceuticals,
Abbott Laboratories); CDP 571 (a humanized anti-TNF IgG4 antibody);
CDP 870 (an anti-TNF alpha humanized monoclonal antibody fragment),
both from Celltech; ONERCEPT, a recombinant TNF binding protein
(r-TBP-1) (Serono); IL1-Receptor Type 2 (Amgen), AMG719 (Amgen) and
IL-1 Trap (Regeneron).
[0088] All of these proinflammatory cytokine antagonists, as well
as active fragments, and active analogs thereof, which retain the
ability to decrease pain as measured in any of the known pain
models including those described further herein, are intended for
use with the present invention.
[0089] Thus, the full-length molecules and fragments thereof, as
well as proteins with modifications, such as deletions, additions
and substitutions (either conservative or non-conservative in
nature), to the native sequence, are intended for use herein, so
long as the protein maintains the desired activity. These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification. Accordingly, active proteins substantially
homologous to the parent sequence, e.g., proteins with 70 . . . 80
. . . 85 . . . 90 . . . 95 . . . 98 . . . 99% etc. identity that
retain the ability to reduce pain, are contemplated for use
herein.
[0090] By "an agent that acts to reduce inflammatory cytokine
actions" is meant an agent that induces anti-inflammatory cytokine
production. Such agents include, without limitation, IL-9, Hsp27
(see, U.S. Patent Publication No. 2001/0049357).
[0091] All of these agents, as well as active fragments, and active
analogs thereof, which retain the ability to decrease pain as
measured in any of the known pain models including those described
further herein, are intended for use with the present
invention.
[0092] Thus, the full-length molecules and fragments thereof, as
well as proteins with modifications, such as deletions, additions
and substitutions (either conservative or non-conservative in
nature), to the native sequence, are intended for use herein, so
long as the protein maintains the desired activity. These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification. Accordingly, active proteins substantially
homologous to the parent sequence, e.g., proteins with 70 . . . 80
. . . 85 . . . 90.95 . . . 98 . . . 99% etc. identity that retain
the ability to reduce pain, are contemplated for use herein.
[0093] The term "analog" refers to biologically active derivatives
of the reference molecule, or fragments of such derivatives, that
retain the ability to reduce pain. In general, the term "analog"
refers to compounds having a native polypeptide sequence and
structure with one or more amino acid additions, substitutions
and/or deletions, relative to the native molecule. Particularly
preferred analogs include substitutions that are conservative in
nature, i.e., those substitutions that take place within a family
of amino acids that are related in their side chains. Specifically,
amino acids are generally divided into four families: (1)
acidic--aspartate and glutamate; (2) basic--lysine, arginine,
histidine; (3) non-polar--alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan; and (4) uncharged
polar--glycine, asparagine, glutamine, cysteine, serine threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified as aromatic amino acids. For example, it is reasonably
predictable that an isolated replacement of leucine with isoleucine
or valine, an aspartate with a glutamate, a threonine with a
serine, or a similar conservative replacement of an amino acid with
a structurally related amino acid, will not have a major effect on
the biological activity. For example, the polypeptide of interest
may include up to about 5-10 conservative or non-conservative amino
acid substitutions, or even up to about 15-25 or 50 conservative or
non-conservative amino acid substitutions, or any number between
5-50, so long as the desired function of the molecule remains
intact.
[0094] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 50%, preferably at least
about 75%, more preferably at least about 80%-85%, preferably at
least about 90%, and most preferably at least about 95%-98%
sequence identity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
[0095] In general, "identity" refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively.
Percent identity can be determined by a direct comparison of the
sequence information between two molecules by aligning the
sequences, counting the exact number of matches between the two
aligned sequences, dividing by the length of the shorter sequence,
and multiplying the result by 100. Readily available computer
programs can be used to aid in the analysis, such as ALIGN,
Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.
Dayhoff ed., 5 Suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., which adapts the local homology
algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489,
1981 for peptide analysis. Programs for determining nucleotide
sequence identity are available in the Wisconsin Sequence Analysis
Package, Version 8 (available from Genetics Computer Group,
Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,
which also rely on the Smith and Waterman algorithm. These programs
are readily utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package referred to above. For example, percent identity of a
particular nucleotide sequence to a reference sequence can be
determined using the homology algorithm of Smith and Waterman with
a default scoring table and a gap penalty of six nucleotide
positions.
[0096] Another method of establishing percent identity in the
context of the present invention is to use the MPSRCH package of
programs copyrighted by the University of Edinburgh, developed by
John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of
packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six).
From the data generated the "Match" value reflects "sequence
identity." Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
are well known in the art.
[0097] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0098] By the term "degenerate variant" is intended a
polynucleotide containing changes in the nucleic acid sequence
thereof, that encodes a polypeptide having the same amino acid
sequence as the polypeptide encoded by the polynucleotide from
which the degenerate variant is derived.
[0099] A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus. A
transcription termination sequence may be located 3' to the coding
sequence.
[0100] By "vector" is meant any genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, virus, virion, etc., which
is capable of replication when associated with the proper control
elements and which can transfer gene sequences to cells. Thus, the
term includes cloning and expression vehicles, as well as viral
vectors.
[0101] By "recombinant vector" is meant a vector that includes a
heterologous nucleic acid sequence which is capable of expression
in vivo.
[0102] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0103] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell, and a cell has been "transfected" when
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are generally known in the art.
See, e.g., Graham et al. (1973) Virology, 52 :456, Sambrook et al.
(1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous DNA
moieties into suitable host cells.
[0104] The term "heterologous" as it relates to nucleic acid
sequences such as coding sequences and control sequences, denotes
sequences that are not normally joined together, and/or are not
normally associated with a particular cell. Thus, a "heterologous"
region of a nucleic acid construct or a vector is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a nucleic acid construct
could include a coding sequence flanked by sequences not found in
association with the coding sequence in nature. Another example of
a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., synthetic sequences
having codons different from the native gene). Similarly, a cell
transformed with a construct which is not normally present in the
cell would be considered heterologous for purposes of this
invention. Allelic variation or naturally occurring mutational
events do not give rise to heterologous DNA, as used herein.
[0105] A "nucleic acid" sequence refers to a DNA or RNA sequence.
The term captures sequences that include any of the known base
analogues of DNA and RNA such as, but not limited to
4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,
aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N6-methyladenine, 7methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0106] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites ("IRES"), enhancers, and
the like, which collectively provide for the replication,
transcription and translation of a coding sequence in a recipient
cell. Not all of these control sequences need always be present so
long as the selected coding sequence is capable of being
replicated, transcribed and translated in an appropriate host
cell.
[0107] The term "promoter" is used herein in its ordinary sense to
refer to a nucleotide region comprising a DNA regulatory sequence,
wherein the regulatory sequence is derived from a gene which is
capable of binding RNA polymerase and initiating transcription of a
downstream (3'-direction) coding sequence. Transcription promoters
can include "inducible promoters" (where expression of a
polynucleotide sequence operably linked to the promoter is induced
by an analyte, cofactor, regulatory protein, etc.), "repressible
promoters" (where expression of a polynucleotide sequence operably
linked to the promoter is induced by an analyte, cofactor,
regulatory protein, etc.), and "constitutive promoters".
[0108] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0109] By "isolated" when referring to a nucleotide sequence, is
meant that the indicated molecule is present in the substantial
absence of other biological macromolecules of the same type. Thus,
an "isolated nucleic acid molecule which encodes a particular
polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not
encode the subject polypeptide; however, the molecule may include
some additional bases or moieties which do not deleteriously affect
the basic characteristics of the composition.
[0110] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "3 prime (3')" or "5 prime (5')" relative to another
sequence, it is to be understood that it is the position of the
sequences in the "sense" or "coding" strand of a DNA molecule that
is being referred to as is conventional in the art.
[0111] The terms "subject", "individual" or "patient" are used
interchangeably herein and refer to a vertebrate, preferably a
mammal. Mammals include, but are not limited to, murines, rodents,
simians, humans, farm animals, sport animals and pets.
[0112] The terms "effective amount" or "therapeutically effective
amount" of a composition or agent, as provided herein, refer to a
nontoxic but sufficient amount of the composition or agent to
provide the desired response, such as a reduction or reversal of
pain. The exact amount required will vary from subject to subject,
depending on the species, age, and general condition of the
subject, the severity of the condition being treated, and the
particular macromolecule of interest, mode of administration, and
the like. An appropriate "effective" amount in any individual case
may be determined by one of ordinary skill in the art using routine
experimentation.
[0113] "Treatment" or "treating" pain includes: (1) preventing
pain, i.e. causing pain not to develop or to occur with less
intensity in a subject that may be exposed to or predisposed to
pain but does not yet experience or display pain, (2) inhibiting
pain, i.e., arresting the development or reversing pain, or (3)
relieving pain, i.e., decreasing the amount of pain experienced by
the subject.
[0114] By "treating existing pain" is meant relieving or reversing
pain in a subject that has been experiencing pain for at least 24
hours, such as for 24-96 hours or more, such as 25 . . . 30 . . .
35 . . . 40 . . . 45 . . . 48 . . . 50 . . . 55 . . . 65 . . . 72 .
. . 80 . . . 90 . . . 96 . . . 100, etc. hours. The term also
intends treating pain that has been occurring long-term, such as
for weeks, months or even years.
[0115] 2. Modes of Carrying Out the Invention
[0116] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0117] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0118] Central to the present invention is the discovery that gene
therapy using genes encoding anti-inflammatory cytokines,
proinflammatory cytokine antagonists, and other agents that act to
reduce or prevent proinflammatory cytokine actions, serves to
reduce pain in vertebrate subjects. Advantages to this approach to
pain control are numerous. First, basal pain responsivity to at
least heat and mechanical stimuli is not altered. Thus, normal pain
processing does not appear to be noticeably influenced by the
presence of the effective dose of the anti-inflammatory cytokine
such as IL-10. Second, anti-inflammatory cytokines such as IL-10
appear to target a pathological aspect of glial activation,
suppressing the pronociceptive influences that activated glia exert
on pain modulatory systems. Third, the agents not only prevent
pathological pain from developing, but can also decrease and/or
reverse already established pathological pain states.
[0119] Gene therapy techniques can be used alone or in conjunction
with traditional drug and protein delivery techniques.
Alternatively, agents that act on proinflammatory cytokines, such
as any of the anti-inflammatory cytokines and proinflammatory
cytokine antagonists described herein, can be administered alone,
without gene delivery, to treat subjects with existing pain.
[0120] In order to further an understanding of the invention, a
more detailed discussion is provided below regarding
anti-inflammatory cytokines, as well as various gene delivery
methods for use with the present invention.
[0121] Anti-inflammatory Cytokines, Proinflammatory Cytokine
Antagonists and Agents that act to Reduce or Prevent Inflammatory
Cytokine Action
[0122] As explained above, the present invention makes use of
anti-inflammatory cytokines, proinflammatory cytokine antagonists
and agents that act to reduce or prevent inflammatory cytokine
action, to treat pain, such as pathological and neuropathic pain.
Particularly preferred anti-inflammatory cytokines and antagonists
for use with the present invention include, without limitation,
interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1ra),
interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor
soluble receptor (TNFsr), alpha-MSH and transforming growth
factor-beta 1 (TGF-.beta.1). The native molecules, as well as
fragments and analogs thereof, which retain the ability to decrease
pain as measured in any of the known pain models including those
described further herein, are intended for use with the present
invention. One particularly preferred IL-10 molecule for use herein
includes a fusion of IL-10 to the Fc portion of an IgG, described
in more detail below. Moreover, sequences derived from any of
numerous species can be used with the present invention, depending
on the animal to be treated.
[0123] For example, a number of sequences related to IL-10, as well
as IL-10 fragments, variants and agonists, which function to reduce
pain will also find use herein. For example, sequences related to
IL-10 are described in, e.g., International Publication Nos. WO
00/65027; WO 98/28425; WO 95/24425 (immunomodulator Trichinella
substances). International Publication No. WO 95/03411 describes
shortened IL-10 sequences, variants and agonists of IL-10 having
amino acid substitutions or deletions at the carboxyl and/or amino
terminus of mature human sequence; U.S. Pat. No. 6,428,985
describes IL-10 variants with a substitution of Ile at position 87
of the mature human IL-10 sequence with Ala or Gly; U.S. Pat. No.
6,159,937 describes an IL-10 fragment with the sequence
Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn) (SEQ ID NO:4); International
Publication No. WO 97/26778 describes IL-10 variants with the
sequence X1-X2-X3Thr-X4-Lys-X5-Arg-X6 (SEQ ID NO:5) where X1=Ala or
Gly; X2=Tyr or Phe; X3, X4 and X5 are independently selected from
Met, Ile, Leu and Val; and X6=Asp, Gln or Gly.
[0124] Nucleotide and amino acid sequences of anti-inflammatory
cytokines, proinflammatory cytokine antagonists and agents that act
to reduce or prevent inflammatory cytokine action, and variants
thereof, from several animal species are well known. For example,
IL-10 has been isolated from a number of animal and viral species.
IL-10 for use herein includes IL-10 from any of these various
species. Non-limiting examples of viral IL-10 include the IL-10
homologues isolated from the herpesviruses such as from
Epstein-Barr virus (see, e.g., Moore et al., Science (1990)
248:1230-1234; Hsu et al., Science (1990) 250:830-832; Suzuki et
al., J. Exp. Med. (1995) 182:477-486), Cytomegalovirus (see, e.g.,
Lockridge et al., Virol. (2000) 268:272-280; Kotenko et al., Proc.
Natl. Acad. Sci. USA (2000) 97:1695-1700; International Publication
No. WO 01/16153), and equine herpesvirus (see, e.g., Rode et al.,
Virus Genes (1993) 7:111-116), as well as the IL-10 homologue from
the OrF virus (see, e.g., Imlach et al., J. Gen. Virol. (2002)
83:1049-1058 and Fleming et al., Virus Genes (2000) 21:85-95). See,
also, FIG. 31 herein depicting the amino acid sequence of a mature,
secreted form of viral IL-10. Representative, non-limiting examples
of other IL-10 sequences for use with the present invention include
the sequences described in NCBI accession numbers NM000572, U63015,
AF418271, AF247603, AF247604, AF247606, AF247605, AY029171, UL16720
(all human sequences), and FIG. 31 herein depicting the amino acid
sequence of a mature secreted form of human IL-10; NM012854,
L02926, X60675 (rat); NM010548, AF307012, M37897, M84340 (all mouse
sequences), and FIG. 31 herein depicting the amino acid sequence of
a mature secreted form of mouse IL-10; U38200 (equine); U39569,
AF060520 (feline sequences); U00799 (bovine); U11421, Z29362 (ovine
sequences); L26031, L26029 (macaque sequences); AF294758 (monkey);
U33843 (canine); AF088887, AF068058 (rabbit sequences); AF012909,
AF120030 (woodchuck sequences); AF026277 (possum); AF097510 (guinea
pig); U11767 (deer); L37781 (gerbil); AB107649 (llama and
camel).
[0125] Non-limiting examples of IL-1ra sequences for use with the
present invention include the sequences described in NCBI accession
numbers NM173843, NM173842, NM173841, NM000577, AY196903, BC009745,
AJ005835, X64532, M63099, X77090, X52015, M55646 (all human
sequences); NM174357, AB005148 (bovine sequences); NM031167,
S64082, M57525, M644044 (mouse sequences); D21832, 568977, M57526
(rabbit sequences); SEG AB045625S, M63101 (rat sequences);
AF216526, AY026462 (canine sequences); U92482, D83714 (equine
sequences); AB038268 (dolphin).
[0126] Non-limiting examples of IL-4 sequences for use with the
present invention include the sequences described in NCBI accession
numbers NM172348, AF395008, AB015021, X16710, A00076, M13982,
NM000589 (all human sequences); BC027514, NM021283, AF352783,
M25892 (mouse sequences); NM173921, AH003241, M84745, M77120
(bovine sequences); AY130260 (chimp); AF097321, L26027 (monkey);
AY096800, AF172168, Z11897, M96845 (ovine sequences); AF035404,
AF305617 (equine sequences); AF239917, AF187322, AF054833, AF104245
(canine sequences); X16058 (rat); AF046213 (hamster); L07081
(cervine); U39634, X87408 (feline); X68330, L12991 (porcine
sequences); U34273 (goat); AB020732 (dolphin); L37779 (gerbil);
AF068058, AF169169 (rabbit sequences); AB107648 (llama and
camel).
[0127] Non-limiting examples of IL-13 sequences for use with the
present invention include the sequences described in NCBI accession
numbers NM002188, U10307, AF377331, X69079 (all human sequences);
NM053828, L26913 (rat sequences); AF385626, AF385625 (porcine
sequences); AF244915 (canine); NM174089 (bovine); AY244790
(monkey); NM008355 (mouse); AB107658 (camel); AB107650 (llama).
[0128] Non-limiting examples of TGF-.beta.1 sequences for use with
the present invention include the sequences described in NCBI
accession numbers NM000660, BD0097505, BD0097504, BD0097503,
BD0097502 (all human sequences); NM021578, X52498 (rat sequences);
AJ009862, NM011577, BC013738, M57902 (mouse sequences); AF461808,
X12373, M23703 (porcine sequences); AF175709, X99438 (equine
sequences); X76916 (ovine); X60296 (hamster); L34956 (canine).
[0129] Non-limiting examples of alpha-MSH sequences for use with
the present invention include the sequences described in NCBI
accession number NM 000939 (human); NM17451 (bovine); NM 008895
(mouse); and M 11346 (xenopus).
[0130] Non-limiting examples of TNF receptor sequences for use with
the present invention include the sequences described in NCBI
accession numbers X55313, M60275, M63121, NM152942, NM001242,
NM152877, NM152876, NM152875, NM152874, NM152873, NM152872,
NM152871, NM000043, NM 001065, NM001066, NM148974, NM148973,
NM148972, NM148971, NM148970, NM148969, NM148968, NM148967,
NM148966, NM148965, NM003790, NM032945, NM003823, NM001243,
NM152854, NM001250 (all human sequences); NM013091, M651122 (rat
sequences).
[0131] Non-limiting examples of IL-9 sequences for use with the
present invention include the sequences described in NCBI accession
numbers NM000590 (human) and NM008373 (mouse).
[0132] Polynucleotides encoding the desired anti-inflammatory
cytokine, proinflammatory cytokine antagonist and agents that act
to reduce or prevent inflammatory cytokine for use with the present
invention can be made using standard techniques of molecular
biology. For example, polynucleotide sequences coding for the
above-described molecules can be obtained using recombinant
methods, such as by screening cDNA and genomic libraries from cells
expressing the gene, or by deriving the gene from a vector known to
include the same. The gene of interest can also be produced
synthetically, rather than cloned, based on the known sequences.
The molecules can be designed with appropriate codons for the
particular sequence. The complete sequence is then assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge, Nature
(1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et
al., J. Biol. Chem. (1984) 259:6311.
[0133] Thus, particular nucleotide sequences can be obtained from
vectors harboring the desired sequences or synthesized completely
or in part using various oligonucleotide synthesis techniques known
in the art, such as site-directed mutagenesis and polymerase chain
reaction (PCR) techniques where appropriate. See, e.g., Sambrook,
supra. One method of obtaining nucleotide sequences encoding the
desired sequences is by annealing complementary sets of overlapping
synthetic oligonucleotides produced in a conventional, automated
polynucleotide synthesizer, followed by ligation with an
appropriate DNA ligase and amplification of the ligated nucleotide
sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad.
Sci. USA (1991) 88:4084-4088. Additionally,
oligonucleotide-directed synthesis (Jones et al., Nature (1986)
54:75-82), oligonucleotide directed mutagenesis of preexisting
nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and
Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic
filling-in of gapped oligonucleotides using T.sub.4 DNA polymerase
(Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033)
can be used to provide molecules for use in the subject
methods.
[0134] Gene Delivery Techniques
[0135] Anti-inflammatory genes as described above, are delivered to
the subject in question using any of several gene-delivery
techniques. Several methods for gene delivery are known in the art.
As described further below, genes can be delivered either directly
to the mammalian subject or, alternatively, delivered ex vivo, to
cells derived from the subject and the cells reimplanted in the
subject.
[0136] A number of viral based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene delivery systems. A selected gene can
be inserted into a vector and packaged in retroviral particles
using techniques known in the art. The recombinant virus can then
be isolated and delivered to cells of the subject either in vivo or
ex vivo. A number of retroviral systems have been described. See,
e.g., U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques
(1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14;
Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc.
Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and
Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.
Replication-defective murine retroviral vectors are widely utilized
gene transfer vectors. Murine leukemia retroviruses include a
single strand RNA complexed with a nuclear core protein and
polymerase (pol) enzymes encased by a protein core (gag) and
surrounded by a glycoprotein envelope (env) that determines host
range. The genomic structure of retroviruses include gag, pol, and
env genes enclosed at the 5' and 3' long terminal repeats (LTRs).
Retroviral vector systems exploit the fact that a minimal vector
containing the 5' and 3' LTRs and the packaging signal are
sufficient to allow vector packaging and infection and integration
into target cells provided that the viral structural proteins are
supplied in trans in the packaging cell line. Fundamental
advantages of retroviral vectors for gene transfer include
efficient infection and gene expression in most cell types, precise
single copy vector integration into target cell chromosomal DNA and
ease of manipulation of the retroviral genome.
[0137] A number of adenovirus vectors have also been described.
Unlike retroviruses which integrate into the host genome,
adenoviruses persist extrachromosomally thus minimizing the risks
associated with insertional mutagenesis (Haj-Ahmad and Graham, J.
Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993)
67:5911-5921; Mittereder et al., Human Gene Therapy (1994)
5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al.,
Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)
6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).
Adenovirus vectors for use in the subject methods are described in
more detail below.
[0138] Additionally, various adeno-associated virus (AAV) vector
systems have been developed for gene delivery. AAV vectors can be
readily constructed using techniques well known in the art. See,
e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International
Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO
93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell.
Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold
Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in
Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in
Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene
Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)
1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875. AAV
vector systems are also described in further detail below.
[0139] Additional viral vectors which will find use for delivering
the nucleic acid molecules of interest include those derived from
the pox family of viruses, including vaccinia virus and avian
poxvirus. By way of example, vaccinia virus recombinants expressing
the genes can be constructed as follows. The DNA encoding the
particular polypeptide is first inserted into an appropriate vector
so that it is adjacent to a vaccinia promoter and flanking vaccinia
DNA sequences, such as the sequence encoding thymidine kinase (TK).
This vector is then used to transfect cells which are
simultaneously infected with vaccinia. Homologous recombination
serves to insert the vaccinia promoter plus the gene encoding the
protein into the viral genome. The resulting TK-recombinant can be
selected by culturing the cells in the presence of
5-bromodeoxyuridine and picking viral plaques resistant
thereto.
[0140] Alternatively, avipoxviruses, such as the fowlpox and
canarypox viruses, can also be used to deliver the genes.
Recombinant avipox viruses, expressing immunogens from mammalian
pathogens, are known to confer protective immunity when
administered to non-avian species. The use of an avipox vector is
particularly desirable in human and other mammalian species since
members of the avipox genus can only productively replicate in
susceptible avian species and therefore are not infective in
mammalian cells. Methods for producing recombinant avipoxviruses
are known in the art and employ genetic recombination, as described
above with respect to the production of vaccinia viruses. See,
e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
[0141] Molecular conjugate vectors, such as the adenovirus chimeric
vectors described in Michael et al., J. Biol. Chem. (1993)
268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992)
89:6099-6103, can also be used for gene delivery.
[0142] Members of the Alphavirus genus, such as but not limited to
vectors derived from the Sindbis and Semliki Forest viruses, will
also find use as viral vectors for delivering the anti-inflammatory
cytokine gene. For a description of Sinbus-virus derived vectors
useful for the practice of the instant methods, see, Dubensky et
al., J. Virol. (1996) 70:508-519; and International Publication
Nos. WO 95/07995 and WO 96/17072.
[0143] Alternatively, the anti-inflammatory cytokines can be
delivered without the use of viral vectors, such as by using
plasmid-based nucleic acid delivery systems as described in U.S.
Pat. Nos. 6,413,942; 6,214,804; 5,580,859; 5,589,466; 5,763,270;
and 5,693,622, all incorporated herein by reference in their
entireties. Plasmids will include the gene of interest operably
linked to control elements that direct the expression of the
protein product in vivo. Such control elements are well known in
the art.
[0144] Plasmid Gene Delivery Systems
[0145] As explained above, the gene of interest can be introduced
into the subject or cells of the subject using non-viral vectors,
such as plasmids, and any of the several plasmid delivery
techniques well-known in the art. For example, vectors can be
introduced without delivery agents, as described in, e.g., U.S.
Pat. Nos. 6,413,942, 6,214,804 and 5,580,859, all incorporated by
reference herein in their entireties.
[0146] Alternatively, the vectors encoding the gene of interest can
be packaged in liposomes prior to delivery to the subject or to
cells derived therefrom, such as described in U.S. Pat. Nos.
5,580,859; 5,549,127; 5,264,618; 5,703,055, all incorporated herein
by reference in their entireties. Lipid encapsulation is generally
accomplished using liposomes which are able to stably bind or
entrap and retain nucleic acid. The ratio of condensed DNA to lipid
preparation can vary but will generally be around 1:1 (mg
DNA:micromoles lipid), or more of lipid. For a review of the use of
liposomes as carriers for delivery of nucleic acids, see, Hug and
Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17; Straubinger et
al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527. The
DNA can also be delivered in cochleate lipid compositions similar
to those described by Papahadjopoulos et al., Biochem. Biophys.
Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and
4,871,488, incorporated herein by reference in their
entireties.
[0147] The vectors may also be encapsulated, adsorbed to, or
associated with, particulate carriers, well known in the art. Such
carriers present multiple copies of a selected molecule to the
immune system and promote trapping and retention of molecules in
local lymph nodes. The particles can be phagocytosed by macrophages
and can enhance antigen presentation through cytokine release.
Examples of particulate carriers include those derived from
polymethyl methacrylate polymers, as well as microparticles derived
from poly(lactides) and poly(lactide-co-glycolides), known as PLG.
See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee
et al., J. Microencap. (1996).
[0148] Moreover, plasmid DNA can be guided by a nuclear
localization signal or like modification.
[0149] Additionally, biolistic delivery systems employing
particulate carriers such as gold and tungsten, are useful for
delivering genes of interest. The particles are coated with the
gene to be delivered and accelerated to high velocity, generally
under a reduced atmosphere, using a gun powder discharge from a
"gene gun." For a description of such techniques, and apparatuses
useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,478,744, all incorporated
herein by reference in their entireties.
[0150] A wide variety of other methods can be used to deliver the
vectors. Such methods include DEAE dextran-mediated transfection,
calcium phosphate precipitation, polylysine- or
polyornithine-mediated transfection, or precipitation using other
insoluble inorganic salts, such as strontium phosphate, aluminum
silicates including bentonite and kaolin, chromic oxide, magnesium
silicate, talc, and the like. Other useful methods of transfection
include electroporation, sonoporation, protoplast fusion, peptoid
delivery, or microinjection. See, e.g., Sambrook et al., supra, for
a discussion of techniques for transforming cells of interest; and
Felgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187,
for a review of delivery systems useful for gene transfer. Methods
of delivering DNA using electroporation are described in, e.g.,
U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6233,483, U.S.
Patent Publication No. 2002/0146831; and International Publication
No. WO/0045823, all of which are incorporated herein by reference
in their entireties.
[0151] It may also be desirable to fuse the plasmid encoding the
gene of interest to immunoglobulin molecules in order to provide
for sustained expression. One convenient technique is to fuse the
plasmid encoding the agent of interest to the Fc portion of a mouse
IgG2a with a noncytolytic mutation. Moreover, the IL-10 gene can be
present in the form of a fusion protein, fused to the Fc portion of
an IgG. Such a technique has been shown to provide for sustained
expression of cytokines, such as IL-10, especially when combined
with electroporation. See, e.g., Jiang et al., J. Biochem. (2003)
133:423-427; and Adachi et al., Gene Ther. (2002) 9:577-583.
[0152] Adenovirus Gene Delivery Systems
[0153] In a preferred embodiment of the subject invention, a
nucleotide sequence encoding the anti-inflammatory cytokine is
inserted into an adenovirus-based expression vector. The adenovirus
genome is a linear double-stranded DNA molecule of approximately
36,000 base pairs with the 55-kDa terminal protein covalently bound
to the 5' terminus of each strand. Adenoviral ("Ad") DNA contains
identical Inverted Terminal Repeats ("ITRs") of about 100 base
pairs with the exact length depending on the serotype. The viral
origins of replication are located within the ITRs exactly at the
genome ends. DNA synthesis occurs in two stages. First, replication
proceeds by strand displacement, generating a daughter duplex
molecule and a parental displaced strand. The displaced strand is
single-stranded and can form a "panhandle" intermediate, which
allows replication initiation and generation of a daughter duplex
molecule. Alternatively, replication can proceed from both ends of
the genome simultaneously, obviating the requirement to form the
panhandle structure.
[0154] During the productive infection cycle, the viral genes are
expressed in two phases: the early phase, which is the period up to
viral DNA replication, and the late phase, which coincides with the
initiation of viral DNA replication. During the early phase only
the early gene products, encoded by regions E1, E2, E3 and E4, are
expressed, which carry out a number of functions that prepare the
cell for synthesis of viral structural proteins. During the late
phase, late viral gene products are expressed in addition to the
early gene products and host cell DNA and protein synthesis are
shut off. Consequently, the cell becomes dedicated to the
production of viral DNA and of viral structural proteins.
[0155] The E1 region of adenovirus is the first region expressed
after infection of the target cell. This region consists of two
transcriptional units, the E1A and E1B genes. The main functions of
the E1A gene products are to induce quiescent cells to enter the
cell cycle and resume cellular DNA synthesis, and to
transcriptionally activate the E1B gene and the other early regions
(E2, E3, E4). Transfection of primary cells with the E1A gene alone
can induce unlimited proliferation (immortalization), but does not
result in complete transformation. However, expression of E1A in
most cases results in induction of programmed cell death
(apoptosis), and only occasionally immortalization. Coexpression of
the E1B gene is required to prevent induction of apoptosis and for
complete morphological transformation to occur. In established
immortal cell lines, high level expression of E1A can cause
complete transformation in the absence of E1B.
[0156] The E1B-encoded proteins assist E1A in redirecting the
cellular functions to allow viral replication. The E1B 55 kD and E4
33 kD proteins, which form a complex that is essentially localized
in the nucleus, function in inhibiting the synthesis of host
proteins and in facilitating the expression of viral genes. Their
main influence is to establish selective transport of viral mRNAs
from the nucleus to the cytoplasm, concomittantly with the onset of
the late phase of infection. The E1B 21 kD protein is important for
correct temporal control of the productive infection cycle, thereby
preventing premature death of the host cell before the virus life
cycle has been completed.
[0157] Adenoviral-based vectors express gene product peptides at
high levels. Adenoviral vectors have high efficiencies of
infectivity, even with low titers of virus. Additionally, the virus
is fully infective as a cell-free virion so injection of producer
cell lines are not necessary. Adenoviral vectors achieve long-term
expression of heterologous genes in vivo. Adenovirus is not
associated with severe human pathology, the virus can infect a wide
variety of cells and has a broad host-range, the virus can be
produced in large quantities with relative ease, and the virus can
be rendered replication defective by deletions in the early-region
1 ("E1") of the viral genome. Thus, vectors derived from human
adenoviruses, in which at least the E1 region has been deleted and
replaced by a gene of interest, have been used extensively for gene
therapy experiments in the pre-clinical and clinical phase.
[0158] Adenoviral vectors for use with the present invention are
derived from any of the various adenoviral serotypes, including,
without limitation, any of the over 40 serotype strains of
adenovirus, such as serotypes 2, 5, 12, 40, and 41. The adenoviral
vectors used herein are replication-deficient and contain the gene
of interest under the control of a suitable promoter, such as any
of the promoters discussed below with reference to adeno-associated
virus. For example, U.S. Pat. No. 6,048,551, incorporated herein by
reference in its entirety, describes replication-deficient
adenoviral vectors that include the hum an gene for the
anti-inflammatory cytokine IL-10, as well as vectors that include
the gene for the anti-inflammatory cytokine IL-1ra, under the
control of the Rous Sarcoma Virus (RSV) promoter, termed
Ad.RSVIL-10 and Ad.RSVIL-1ra, respectively.
[0159] Other recombinant adenoviruses, derived from any of the
adenoviral serotypes, and with different promoter systems, can be
used by those skilled in the art. For example, U.S. Pat. No.
6,306,652, incorporated herein by reference in its entirety,
describes adenovirus vectors with E2A sequences, containing the hr
mutation and the ts125 mutation, termed ts400, to prevent cell
death by E2A overexpression, as well as vectors with E2A sequences,
containing only the hr mutation, under the control of an inducible
promoter, and vectors with E2A sequences, containing the hr
mutation and the ts125 mutation (ts400), under the control of an
inducible promoter.
[0160] Moreover, "minimal" adenovirus vectors as described in U.S.
Pat. No. 6,306,652 will find use with the present invention. Such
vectors retain at least a portion of the viral genome that is
required for encapsidation of the genome into virus particles (the
encapsidation signal), as well as at least one copy of at least a
functional part or a derivative of the ITR. Packaging of the
minimal adenovirus vector can be achieved by co-infection with a
helper virus or, alternatively, with a packaging-deficient
replicating helper system as described in U.S. Pat. No.
6,306,652.
[0161] Other useful adenovirus-based vectors for delivery of
anti-inflammatory cytokines include the "gutless"
(helper-dependent) adenovirus in which the vast majority of the
viral genome has been removed (Wu et al., Anesthes. (2001)
94:1119-1132). Such "gutless" adenoviral vectors essentially create
no viral proteins, thus allowing virally driven gene therapy to
successfully ensue for over a year after a single administration
(Parks, R. J., Clin. Genet. (2000) 58:1-11; Tsai et al., Curr.
Opin. Mol. Ther. (2000) 2:515-523) and eliminates interference by
the immune system. In addition, removal of the viral genome creates
space for insertion of control sequences that provide expression
regulation by systemically administered drugs (Burcin et al., Proc.
Natl. Acad. Sci. USA (1999) 96:355-360), adding both safety and
control of virally driven protein expression. These and other
recombinant adenoviruses will find use with the present
methods.
[0162] Adeno-Associated Virus Gene Delivery Systems
[0163] Adeno-associated virus (AAV) has been used with success to
deliver genes for gene therapy. The AAV genome is a linear,
single-stranded DNA molecule containing about 4681 nucleotides. The
AAV genome generally comprises an internal, nonrepeating genome
flanked on each end by inverted terminal repeats (ITRs). The ITRs
are approximately 145 base pairs (bp) in length. The ITRs have
multiple functions, including providing origins of DNA replication,
and packaging signals for the viral genome. The internal
nonrepeated portion of the genome includes two large open reading
frames, known as the AAV replication (rep) and capsid (cap) genes.
The rep and cap genes code for viral proteins that allow the virus
to replicate and package into a virion. In particular, a family of
at least four viral proteins are expressed from the AAV rep region,
Rep 78, Rep 68, Rep 52, and Rep 40, named according to their
apparent molecular weight. The AAV cap region encodes at least
three proteins, VPI, VP2, and VP3.
[0164] AAV has been engineered to deliver genes of interest by
deleting the internal nonrepeating portion of the AAV genome (i.e.,
the rep and cap genes) and inserting a heterologous gene (in this
case, the gene encoding the anti-inflammatory cytokine) between the
ITRs. The heterologous gene is typically functionally linked to a
heterologous promoter (constitutive, cell-specific, or inducible)
capable of driving gene expression in the patient's target cells
under appropriate conditions. Termination signals, such as
polyadenylation sites, can also be included.
[0165] AAV is a helper-dependent virus; that is, it requires
coinfection with a helper virus (e.g., adenovirus, herpesvirus or
vaccinia), in order to form AAV virions. In the absence of
coinfection with a helper virus, AAV establishes a latent state in
which the viral genome inserts into a host cell chromosome, but
infectious virions are not produced. Subsequent infection by a
helper virus "rescues" the integrated genome, allowing it to
replicate and package its genome into an infectious AAV virion.
While AAV can infect cells from different species, the helper virus
must be of the same species as the host cell. Thus, for example,
human AAV will replicate in canine cells coinfected with a canine
adenovirus.
[0166] Recombinant AAV virions comprising the anti-inflammatory
cytokine coding sequence may be produced using a variety of
art-recognized techniques described more fully below. Wild-type AAV
and helper viruses may be used to provide the necessary replicative
functions for producing rAAV virions (see, e.g., U.S. Pat. No.
5,139,941, incorporated herein by reference in its entirety).
Alternatively, a plasmid, containing helper function genes, in
combination with infection by one of the well-known helper viruses
can be used as the source of replicative functions (see e.g., U.S.
Pat. No. 5,622,856 and U.S. Pat. No. 5,139,941, both incorporated
herein by reference in their entireties). Similarly, a plasmid,
containing accessory function genes can be used in combination with
infection by wild-type AAV, to provide the necessary replicative
functions. These three approaches, when used in combination with a
rAAV vector, are each sufficient to produce rAAV virions. Other
approaches, well known in the art, can also be employed by the
skilled artisan to produce rAAV virions.
[0167] In a preferred embodiment of the present invention, a triple
transfection method (described in detail in U.S. Pat. No.
6,001,650, incorporated by reference herein in its entirety) is
used to produce rAAV virions because this method does not require
the use of an infectious helper virus, enabling rAAV virions to be
produced without any detectable helper virus present. This is
accomplished by use of three vectors for rAAV virion production: an
AAV helper function vector, an accessory function vector, and a
rAAV expression vector. One of skill in the art will appreciate,
however, that the nucleic acid sequences encoded by these vectors
can be provided on two or more vectors in various combinations.
[0168] As explained herein, the AAV helper function vector encodes
the "AAV helper function" sequences (i.e., rep and cap), which
function in trans for productive AAV replication and encapsidation.
Preferably, the AAV helper function vector supports efficient AAV
vector production without generating any detectable wt AAV virions
(i.e., AAV virions containing functional rep and cap genes). An
example of such a vector, pHLP19, is described in U.S. Pat. No.
6,001,650, incorporated herein by reference in its entirety. The
rep and cap genes of the AAV helper function vector can be derived
from any of the known AAV serotypes, as explained above. For
example, the AAV helper function vector may have a rep gene derived
from AAV-2 and a cap gene derived from AAV-6; one of skill in the
art will recognize that other rep and cap gene combinations are
possible, the defining feature being the ability to support rAAV
virion production.
[0169] The accessory function vector encodes nucleotide sequences
for non-AAV--derived viral and/or cellular functions upon which AAV
is dependent for replication (i.e., "accessory functions"). The
accessory functions include those functions required for AAV
replication, including, without limitation, those moieties involved
in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV DNA replication, synthesis of cap expression
products, and AAV capsid assembly. Viral-based accessory functions
can be derived from any of the well-known helper viruses such as
adenovirus, herpesvirus (other than herpes simplex virus type-1),
and vaccinia virus. In a preferred embodiment, the accessory
function plasmid pLadeno5 is used (details regarding pLadeno5 are
described in U.S. Pat. No. 6,004,797, incorporated herein by
reference in its entirety). This plasmid provides a complete set of
adenovirus accessory functions for AAV vector production, but lacks
the components necessary to form replication-competent
adenovirus.
[0170] In order to further an understanding of AAV, a more detailed
discussion is provided below regarding recombinant AAV expression
vectors and AAV helper and accessory functions
[0171] Recombinant AAV Expression Vectors
[0172] Recombinant AAV (rAAV) expression vectors are constructed
using known techniques to at least provide as operatively linked
components in the direction of transcription, control elements
including a transcriptional initiation region, the
anti-inflammatory polynucleotide of interest and a transcriptional
termination region. The control elements are selected to be
functional in a mammalian muscle cell. The resulting construct
which contains the operatively linked components is bounded (5' and
3') with functional AAV ITR sequences.
[0173] The nucleotide sequences of AAV ITR regions are known. See,
e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Bems, K. I.
"Parvoviridae and their Replication" in Fundamental Virology, 2nd
Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2
sequence. AAV ITRs used in the vectors of the invention need not
have a wild-type nucleotide sequence, and may be altered, e.g., by
the insertion, deletion or substitution of nucleotides.
Additionally, AAV ITRs may be derived from any of several AAV
serotypes, including without limitation, AAV-1, AAV-2, AAV-3,
AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8, etc. Furthermore, 5' and 3'
ITRs which flank a selected nucleotide sequence in an AAV
expression vector need not necessarily be identical or derived from
the same AAV serotype or isolate, so long as they function as
intended, i.e., to allow for excision and rescue of the sequence of
interest from a host cell genome or vector, and to allow
integration of the DNA molecule into the recipient cell genome when
AAV Rep gene products are present in the cell.
[0174] Suitable polynucleotide molecules for use in AAV vectors
will be less than about 5 kilobases (kb) in size. The selected
polynucleotide sequence is operably linked to control elements that
direct the transcription or expression thereof in the subject in
vivo. Such control elements can comprise control sequences normally
associated with the selected gene. Alternatively, heterologous
control sequences can be employed. Useful heterologous control
sequences generally include those derived from sequences encoding
mammalian or viral genes. Examples include, but are not limited to,
neuron-specific enolase promoter, a GFAP promoter, the SV40 early
promoter, mouse mammary tumor virus LTR promoter; adenovirus major
late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a
cytomegalovirus (CMV) promoter such as the CMV immediate early
promoter region (CMVIE), a rous sarcoma virus (RSV) promoter,
synthetic promoters, hybrid promoters, and the like. In addition,
sequences derived from nonviral genes, such as the murine
metallothionein gene, will also find use herein. Such promoter
sequences are commercially available from, e.g., Stratagene (San
Diego, Calif.).
[0175] The AAV expression vector which harbors the polynucleotide
molecule of interest bounded by AAV ITRs, can be constructed by
directly inserting the selected sequence(s) into an AAV genome
which has had the major AAV open reading frames ("ORFs") excised
therefrom. Other portions of the AAV genome can also be deleted, so
long as a sufficient portion of the ITRs remain to allow for
replication and packaging functions. Such constructs can be
designed using techniques well known in the art. See, e.g., U.S.
Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos.
WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4
Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;
Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory
Press); Carter (1992) Current Opinion in Biotechnology 3:533-539;
Muzyczka (1992) Current Topics in Microbiol. and Immunol.
158:97-129; Kotin (1994) Human Gene Therapy 5:793-801; Shelling and
Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.
Med. 179:1867-1875.
[0176] Alternatively, AAV ITRs can be excised from the viral genome
or from an AAV vector containing the same and fused 5' and 3' of a
selected nucleic acid construct that is present in another vector
using standard ligation techniques, such as those described in
Sambrook et al., supra. For example, ligations can be accomplished
in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 .mu.g/ml BSA,
10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss) units
T4 DNA ligase at 0.degree. C. (for "sticky end" ligation) or 1 mM
ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for
"blunt end" ligation). Intermolecular "sticky end" ligations are
usually performed at 30-100 .mu.g/ml total DNA concentrations
(5-100 nM total end concentration). AAV vectors which contain ITRs
have been described in, e.g., U.S. Pat. No. 5,139,941. In
particular, several AAV vectors are described therein which are
available from the American Type Culture Collection ("ATCC") under
Accession Numbers 53222, 53223, 53224, 53225 and 53226.
[0177] For the purposes of the invention, suitable host cells for
producing rAAV virions from the AAV expression vectors include
microorganisms, yeast cells, insect cells, and mammalian cells,
that can be, or have been, used as recipients of a heterologous DNA
molecule and that are capable of growth in, for example, suspension
culture, a bioreactor, or the like. The term includes the progeny
of the original cell which has been transfected. Thus, a "host
cell" as used herein generally refers to a cell which has been
transfected with an exogenous DNA sequence. Cells from the stable
human cell line, 293 (readily available through, e.g., the American
Type Culture Collection under Accession Number ATCC CRL1573) are
preferred in the practice of the present invention. Particularly,
the human cell line 293 is a human embryonic kidney cell line that
has been transformed with adenovirus type-5 DNA fragments (Graham
et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral
E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293
cell line is readily transfected, and provides a particularly
convenient platform in which to produce rAAV virions.
[0178] AAV Helper Functions
[0179] Host cells containing the above-described AAV expression
vectors must be rendered capable of providing AAV helper functions
in order to replicate and encapsidate the nucleotide sequences
flanked by the AAV ITRs to produce rAAV virions. AAV helper
functions are generally AAV-derived coding sequences which can be
expressed to provide AAV gene products that, in turn, function in
trans for productive AAV replication. AAV helper functions are used
herein to complement necessary AAV functions that are missing from
the AAV expression vectors. Thus, AAV helper functions include one,
or both of the major AAV ORFs, namely the rep and cap coding
regions, or functional homologues thereof.
[0180] By "AAV rep coding region" is meant the art-recognized
region of the AAV genome which encodes the replication proteins Rep
78, Rep 68, Rep 52 and Rep 40. These Rep expression products have
been shown to possess many functions, including recognition,
binding and nicking of the AAV origin of DNA replication, DNA
helicase activity and modulation of transcription from AAV (or
other heterologous) promoters. The Rep expression products are
collectively required for replicating the AAV genome. For a
description of the AAV rep coding region, see, e.g., Muzyczka, N.
(1992) Current Topics in Microbiol. and Immunol. 158:97-129; and
Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Suitable
homologues of the AAV rep coding region include the human
herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2
DNA replication (Thomson et al. (1994) Virology 204:304-311).
[0181] By "AAV cap coding region" is meant the art-recognized
region of the AAV genome which encodes the capsid proteins VP1,
VP2, and VP3, or functional homologues thereof. These Cap
expression products supply the packaging functions which are
collectively required for packaging the viral genome. For a
description of the AAV cap coding region, see, e.g., Muzyczka, N.
and Kotin, R. M. (supra).
[0182] AAV helper functions are introduced into the host cell by
transfecting the host cell with an AAV helper construct either
prior to, or concurrently with, the transfection of the AAV
expression vector. AAV helper constructs are thus used to provide
at least transient expression of AAV rep and/or cap genes to
complement missing AAV functions that are necessary for productive
AAV infection. AAV helper constructs lack AAV ITRs and can neither
replicate nor package themselves.
[0183] These constructs can be in the form of a plasmid, phage,
transposon, cosmid, virus, or virion. A number of AAV helper
constructs have been described, such as the commonly used plasmids
pAAV/Ad and pIM29+45 which encode both Rep and Cap expression
products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828;
and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other
vectors have been described which encode Rep and/or Cap expression
products. See, e.g., U.S. Pat. No. 5,139,941.
[0184] AAV Accessory Functions
[0185] The host cell (or packaging cell) must also be rendered
capable of providing nonAAV-derived functions, or "accessory
functions," in order to produce rAAV virions. Accessory functions
are nonAAV-derived viral and/or cellular functions upon which AAV
is dependent for its replication. Thus, accessory functions include
at least those nonAAV proteins and RNAs that are required in AAV
replication, including those involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of Cap expression products and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses.
[0186] In particular, accessory functions can be introduced into
and then expressed in host cells using methods known to those of
skill in the art. Typically, accessory functions are provided by
infection of the host cells with an unrelated helper virus. A
number of suitable helper viruses are known, including
adenoviruses; herpesviruses such as herpes simplex virus types 1
and 2; and vaccinia viruses. Nonviral accessory functions will also
find use herein, such as those provided by cell synchronization
using any of various known agents. See, e.g., Buller et al. (1981)
J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222;
Schlehofer et al. (1986) Virology 152:110-117.
[0187] Alternatively, accessory functions can be provided using an
accessory function vector as defined above. See, e.g., U.S. Pat.
No. 6,004,797 and International Publication No. WO 01/83797,
incorporated herein by reference in its entirety. Nucleic acid
sequences providing the accessory functions can be obtained from
natural sources, such as from the genome of an adenovirus particle,
or constructed using recombinant or synthetic methods known in the
art. As explained above, it has been demonstrated that the
full-complement of adenovirus genes are not required for accessory
helper functions. In particular, adenovirus mutants incapable of
DNA replication and late gene synthesis have been shown to be
permissive for AAV replication. Ito et al., (1970) J. Gen. Virol.
9:243; Ishibashi et al, (1971) Virology 45:317. Similarly, mutants
within the E2B and E3 regions have been shown to support AAV
replication, indicating that the E2B and E3 regions are probably
not involved in providing accessory functions. Carter et al.,
(1983) Virology 126:505. However, adenoviruses defective in the E1
region, or having a deleted E4 region, are unable to support AAV
replication. Thus, EIA and E4 regions are likely required for AAV
replication, either directly or indirectly. Laughlin et al., (1982)
J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA
78:1925; Carter et al., (1983) Virology 126:505. Other
characterized Ad mutants include: E1B (Laughlin et al. (1982),
supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology
104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss
et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol.
35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927;
Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter,
Adeno-Associated Virus Helper Functions, in I CRC Handbook of
Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983),
supra); and E4 (Carter et al. (1983), supra; Carter (1995)).
Although studies of the accessory functions provided by
adenoviruses having mutations in the E1B coding region have
produced conflicting results, Samulski et al., (1988) J. Virol.
62:206-210, recently reported that E1B55k is required for AAV
virion production, while EIB19k is not. In addition, International
Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy
5:938-945, describe accessory function vectors encoding various Ad
genes. Particularly preferred accessory function vectors comprise
an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding
region, an adenovirus E2A 72 kD coding region, an adenovirus E1A
coding region, and an adenovirus E1B region lacking an intact
E1B55k coding region. Such vectors are described in International
Publication No. WO 01/83797.
[0188] As a consequence of the infection of the host cell with a
helper virus, or transfection of the host cell with an accessory
function vector, accessory functions are expressed which
transactivate the AAV helper construct to produce AAV Rep and/or
Cap proteins. The Rep expression products excise the recombinant
DNA (including the DNA of interest) from the AAV expression vector.
The Rep proteins also serve to duplicate the AAV genome. The
expressed Cap proteins assemble into capsids, and the recombinant
AAV genome is packaged into the capsids. Thus, productive AAV
replication ensues, and the DNA is packaged into rAAV virions. A
"recombinant AAV virion," or "rAAV virion" is defined herein as an
infectious, replication-defective virus including an AAV protein
shell, encapsidating a heterologous nucleotide sequence of interest
which is flanked on both sides by AAV ITRs.
[0189] Following recombinant AAV replication, rAAV virions can be
purified from the host cell using a variety of conventional
purification methods, such as column chromatography, CsCl
gradients, and the like. For example, a plurality of column
purification steps can be used, such as purification over an anion
exchange column, an affinity column and/or a cation exchange
column. See, for example, International Publication No. WO
02/12455. Further, if infection is employed to express the
accessory functions, residual helper virus can be inactivated,
using known methods. For example, adenovirus can be inactivated by
heating to temperatures of approximately 60*C for, e.g., 20 minutes
or more. This treatment effectively inactivates only the helper
virus since AAV is extremely heat stable while the helper
adenovirus is heat labile.
[0190] The resulting rAAV virions containing the nucleotide
sequence of interest can then be used for gene delivery using the
techniques described below.
[0191] Compositions and Delivery
[0192] A. Compositions
[0193] Once produced, the vectors (or virions) encoding the
anti-inflammatory cytokine, will be formulated into compositions
suitable for delivery. Compositions will comprise sufficient
genetic material to produce a therapeutically effective amount of
the anti-inflammatory cytokine of interest, i.e., an amount
sufficient to reduce or ameliorate pain. The compositions will also
contain a pharmaceutically acceptable excipient. Such excipients
include any pharmaceutical agent that does not itself induce the
production of antibodies harmful to the individual receiving the
composition, and which may be administered without undue toxicity.
Pharmaceutically acceptable excipients include, but are not limited
to, sorbitol, any of the various TWEEN compounds, and liquids such
as water, saline, glycerol and ethanol. Pharmaceutically acceptable
salts can be included therein, for example, mineral acid salts such
as hydrochlorides, hydrobromides, phosphates, sulfates, and the
like; and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. Additionally, auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, may be present in such vehicles. A
thorough discussion of pharmaceutically acceptable excipients is
available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co.,
N.J. 1991).
[0194] One particularly useful formulation comprises the vector or
virion of interest in combination with one or more dihydric or
polyhydric alcohols, and, optionally, a detergent, such as a
sorbitan ester. See, for example, International Publication No. WO
00/32233.
[0195] As is apparent to those skilled in the art in view of the
teachings of this specification, an effective amount can be
empirically determined. Representative doses are detailed below.
Administration can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of
determining the most effective means and dosages of administration
are well known to those of skill in the art and will vary with the
vector, the composition of the therapy, the target cells, and the
subject being treated. Single and multiple administrations can be
carried out with the dose level and pattern being selected by the
treating physician.
[0196] As shown in the examples below, one particularly effective
way to produce long-term alleviation of pain involves administering
two or more doses of IL-10 at close intervals, e.g., at less than
10 days apart, preferably less than 5 days apart, more preferably
less than 4 days apart, such as at 3 . . . 2 . . . 1 . . . etc. and
any amount of time within the stated ranges.
[0197] If multiple doses are administered, the first formulation
administered can be the same or different than the subsequent
formulations. Thus, for example, the first administration can be in
the form of an adenovirus vector and the second administration in
the form of an adenovirus vector, plasmid DNA, an AAV virion, a
subunit vaccine composition, or the like. Moreover, subsequent
delivery can also be the same or different than the second mode of
delivery.
[0198] It should be understood that more than one transgene can be
expressed by the delivered recombinant vector. For example, the
recombinant vectors can encode more than one anti-inflammatory
cytokine. Alternatively, separate vectors, each expressing one or
more different transgenes, can also be delivered to the nervous
system as described herein. Thus, multiple anti-inflammatory
cytokines can be delivered concurrently or sequentially.
Furthermore, it is also intended that the vectors delivered by the
methods of the present invention be combined with other suitable
compositions and therapies. For instance, other pain alleviators
and analgesics, such as anti-prostaglandins, including, without
limitation, cyclooxygenase-2 (COX-2) inhibitors, 5-lipoxygenase
(5-LOX) inhibitors, and the like, can be coadministered with the
compositions of the invention. Other compounds for delivery include
agents used in the treatment of neuropathic pain such as, but not
limited to, tricyclic antidepressants (e.g., amitriptyline,
imipramine, desipramine), anti-convulsants (e.g., gabapentin,
carbamazepine, phenyloin) and local anesthetics (e.g., mexiletine,
lidocaine); and agents used in the treatment of inflammatory pain
including, but not limited to, NSAIDs (e.g., ibuprofen, naprosyn
sodium, aspirin, diclofenac sodium, indomethacin, toletin),
steroids (e.g., methylprednisone, prednisone), analgesics (e.g.,
acetaminophen), and opiates (e.g., tramadol, demerol, darvon,
vicodin, fentanyl).
[0199] B. Delivery
[0200] The recombinant vectors may be introduced into the nervous
system, including into any cell or tissue of the CNS or peripheral
nervous system, or cells or tissues in close proximity thereto.
Thus, delivery can be, for example, into any neural tissue
including, without limitation, peripheral nerves, the retina,
dorsal root ganglia, neuromuscular junction, as well as the CNS,
e.g., to target spinal cord glial cells, cells in the cerebrospinal
fluid (CSF), cells in the interstitial spaces, cells in the
protective coverings of the spinal cord, epidural cells (i.e.,
cells outside of the dura mater), cells in non-neural tissues
adjacent to or in contact with or innervated by neural tissue. The
recombinant vectors be introduced either in vivo or in vitro (also
termed ex vivo) to treat preexisting neuronal damage, neuropathies
and other causes of neuropathic pain as defined above. If
transduced in vitro, the desired recipient cell will be removed
from the subject, transduced with rAAV virions and reintroduced
into the subject. Alternatively, syngeneic or xenogeneic cells can
be used where those cells will not generate an inappropriate immune
response in the subject. Additionally, neural progenitor cells can
be transduced in vitro and then delivered to the CNS.
[0201] Suitable methods for the delivery and introduction of
transduced cells into a subject have been described. For example,
cells can be transduced in vitro by combining recombinant vectors
with cells to be transduced in appropriate media, and those cells
harboring the DNA of interest can be screened using conventional
techniques such as Southern blots and/or PCR, or by using
selectable markers. Transduced cells can then be formulated into
pharmaceutical compositions, as described above, and the
composition introduced into the subject by various techniques as
described below, in one or more doses.
[0202] For in vivo delivery, the recombinant vectors will be
formulated into pharmaceutical compositions and one or more dosages
may be administered directly in the indicated manner.
Therapeutically effective doses can be readily determined by one of
skill in the art and will depend on the particular delivery system
used. For AAV-delivered anti-inflammatory cytokines, a
therapeutically effective dose will include on the order of from
about 10.sup.6 to 10.sup.15 of the rAAV virions, more preferably
10.sup.7 to 10.sup.12, and even more preferably about 10.sup.8 to
10.sup.10 of the rAAV virions (or viral genomes, also termed "vg"),
or any value within these ranges. For adenovirus-delivered
anti-inflammatory cytokines, a therapeutically effective dose will
include about 1.times.10.sup.6 plaque forming units (PFU) to
1.times.10.sup.12 PFU, preferably about 1.times.10.sup.7 PFU to
about 1.times.10.sup.10 PFU, or any dose within these ranges which
is sufficient to alleviate pain.
[0203] Generally, from 1 .mu.l to 1 ml of composition will be
delivered, such as from 0.01 to about 0.5 ml, for example about
0.05 to about 0.3 ml, such as 0.08, 0.09, 0.1, 0.2, etc. and any
number within these ranges, of composition will be delivered.
[0204] Recombinant vectors, or cells transduced in vitro, may be
delivered directly to neural tissue such as peripheral nerves, the
retina, dorsal root ganglia, neuromuscular junction, as well as the
CNS, e.g., to target spinal cord glial cells, cells in the
cerebrospinal fluid (CSF), cells in the interstitial spaces, cells
in the protective coverings of the spinal cord, epidural cells
(i.e., cells outside of the dura mater), cells in non-neural
tissues adjacent to or in contact with or innervated by neural
tissue, and the like, by injection into, e.g., the ventricular
region, as well as to the striatum (e.g., the caudate nucleus or
putamen of the striatum), spinal cord and neuromuscular junction,
into the interstitial space, with a needle, catheter or related
device, using techniques known in the art, such as by stereotactic
injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999;
Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.
Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.
11:2315-2329, 2000), epidural delivery, etc.
[0205] A particularly preferred method for targeting the nervous
system, such as spinal cord glia, is by intrathecal delivery,
rather than into the cord tissue itself. Such delivery presents
many advantages. The targeted protein is released into the
surrounding CSF and/or tissues and unlike viruses, released
proteins can penetrate into the spinal cord parenchyma, just as
after acute intrathecal injections. Indeed, intrathecal delivery of
viral vectors can keep expression local. Moreover, in the case of
IL-10, its brief half-life also serves to keep it local following
intrathecal gene therapy; that is, its rapid degradation keeps the
active protein concentrated close to its site of release. An
additional advantage of intrathecal gene therapy is that the
intrathecal route mimics lumbar puncture administration (i.e.,
spinal tap) already in routine use in humans.
[0206] Another method for delivery is by administration into the
epidural space. The epidural space occupies the vertebral canal
between the periosteum lining the canal and the dura. The epidural
space is readily approached through the lumbar area. Generally, a
needle, catheter or the like is inserted in the midline and passes
through the skin, fascia, supraspinous and interspinous ligaments,
and the ligamentum flavum prior to reaching the extradural space.
However, administration can also be through the thoracic area.
Methods for delivering agents epidurally are well known in the art.
See, e.g., Textbook of Surgery, (D. C. Sabiston, ed.) W.B. Saunders
Company.
[0207] Another preferred method for administering the recombinant
vectors or transduced cells is by delivery to dorsal root ganglia
(DRG) neurons, e.g., by injection into the epidural space with
subsequent diffusion to DRG. For example, the recombinant vectors
or transduced cells can be delivered via intrathecal cannulation
under conditions where the protein is diffused to DRG. See, e.g.,
Chiang et al., Acta Anaesthesiol. Sin. (2000) 38:31-36; Jain, K.
K., Expert Opin. Investig. Drugs (2000) 9:2403-2410.
[0208] Yet another mode of administration to the CNS uses a
convection-enhanced delivery (CED) system. In this way, recombinant
vectors can be delivered to many cells over large areas of the CNS.
Moreover, the delivered vectors efficiently express transgenes in
CNS cells (e.g., glial cells). Any convection-enhanced delivery
device may be appropriate for delivery of recombinant vectors. In a
preferred embodiment, the device is an osmotic pump or an infusion
pump. Both osmotic and infusion pumps are commercially available
from a variety of suppliers, for example Alzet Corporation,
Hamilton Corporation, Alza, Inc., Palo Alto, Calif.). Typically, a
recombinant vector is delivered via CED devices as follows. A
catheter, cannula or other injection device is inserted into CNS
tissue in the chosen subject. Stereotactic maps and positioning
devices are available, for example from ASI Instruments, Warren,
Mich. Positioning may also be conducted by using anatomical maps
obtained by CT and/or MRI imaging to help guide the injection
device to the chosen target. Moreover, because the methods
described herein can be practiced such that relatively large areas
of the subject take up the recombinant vectors, fewer infusion
cannula are needed. Since surgical complications are related to the
number of penetrations, this mode of delivery serves to reduce the
side-effects seen with conventional delivery techniques. For a
detailed description regarding CED delivery, see U.S. Pat. No.
6,309,634, incorporated herein by reference in its entirety.
[0209] Protein Delivery Techniques
[0210] As explained above, agents that act on proinflammatory
cytokines, such as any of the anti-inflammatory cytokines and
proinflammatory cytokine antagonists described herein, can be
administered alone, without gene delivery, or in conjunction with
gene therapy, to treat or prevent pain. Thus, for example, one or
more of IL-10 (including viral IL-10), IL-1ra, IL-4, IL-13, TNFsr,
alpha-MSH, TGF-.beta.1, proinflammatory cytokine antagonists and/or
other agents that act on proinflammatory cytokines, can be
formulated into compositions and delivered to subjects prior to,
concurrent with or subsequent to gene delivery of one or more of
these agents. Alternatively, these agents can be delivered alone,
without the genes, to subjects with existing pain.
[0211] Compositions will comprise a therapeutically effective
amount of the agent such that pain is reduced or reversed. The
compositions will also contain a pharmaceutically acceptable
excipient. Such excipients include any pharmaceutical agent that
does not itself induce the production of antibodies harmful to the
individual receiving the composition, and which may be administered
without undue toxicity. Pharmaceutically acceptable excipients
include, but are not limited to, sorbitol, any of the various TWEEN
compounds, and liquids such as water, saline, glycerol and ethanol.
Pharmaceutically acceptable salts can be included therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles. A thorough discussion of pharmaceutically acceptable
excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. 1991). The pharmaceutical compositions may
comprise the compound or its pharmaceutically acceptable salt or
hydrate as the active component.
[0212] In general, the agents will be formulated into compositions
for oral (including buccal and sub-lingual), rectal, nasal,
topical, pulmonary, vaginal or parenteral (including intramuscular,
intraarterial, intrathecal, epidural, subcutaneous and intravenous)
administration or in a form suitable for administration by
inhalation or insufflation. The preferred manner of administration
is into the nervous system, for example into any neural tissue
including, without limitation, peripheral nerves, the retina,
dorsal root ganglia, neuromuscular junction, as well as the CNS,
e.g., to target spinal cord glial cells, cells in the cerebrospinal
fluid (CSF), cells in the interstitial spaces, cells in the
protective coverings of the spinal cord, epidural cells (i.e.,
cells outside of the dura mater), cells in non-neural tissues
adjacent to or in contact with or innervated by neural tissue,
using any of the techniques described above with reference to
recombinant vectors.
[0213] Preferably, the compositions are formulated in order to
improve stability and extend the half-life of the active agent. For
example, the active agent, such as IL-10, can be derivatized with
polyethlene glycol (PEG). Pegylation techniques are well known in
the art and include, for example, site-specific pegylation (see,
e.g., Yamamoto et al., Nat. Biotech. (2003) 21:546-552; Manjula et
al., Bioconjug. Chem. (2003) 14:464-472; Goodson and Katre,
Biotechnology (1990) 8:343-346; U.S. Pat. No. 6,310,180
incorporated herein by reference in its entirety), pegylation using
size exclusion reaction chromatography (see, e.g., Fee, C. J.,
Biotechnol. Bioeng. (2003) 82:200-206), and pegylation using solid
phase (see, e.g., Lu and Felix, Pept. Res. (1993) 6:140-146). For
other methods of pegylation see, e.g., International Publication
No. WO 02/26265, U.S. Patent Nos. 5,206,344 and 6,423,685, all
incorporated herein by reference in their entireties, as well as
reviews by Harris and Chess, Nat. Rev. Drug. Discov. (2003)
2:214-221; Greenwald et al., Adv. Drug. Deliv. Rev. (2003)
55:217-256; and Delgado et al., Crit. Rev. Ther. Drug Carrier Syst.
(1992) 9:249-304.
[0214] Moreover, the active agent may be fused to antibodies or
peptides, to improve stability and extend half-life, using
techniques well known in the art. For example, the active agent may
be fused to immunoglobulin molecules in order to provide for
sustained release. One convenient technique is to fuse the agent of
interest to the Fc portion of an IgG such as a human or mouse IgG2a
with a noncytolytic mutation. See, e.g., Jiang et al., J. Biochem.
(2003) 133:423-427; Adachi et al., Gene Ther. (2002) 9:577-583; and
U.S. Pat. No. 6,410,008, incorporated herein by reference in its
entirety. A non-lytic recombinant human IL-10/Fc chimera is
commercially available from Sigma Chemical Co. (St. Louis,
Mo.).
[0215] Additionally, the active agent can be fused to an
enzymatically inactive polypeptide, such as albumin, as well as
enzymes that have enzymatic activity in an organism other than the
organism to which the agent will be delivered. For example, useful
polypeptides include plant enzymes, porcine or rodent
glycosyltransferases, and .alpha.-1,3-galactosyltransferases. See,
e.g., Sandrin et al., Proc. Natl. Acad. Sci. USA (1993) 90:11391
and U.S. Pat. No. 6,403,077, incorporated herein by reference in
its entirety. Other methods for stabilizing the agent of interest
is to make the protein larger or less accessible to proteases, such
as by introducing glycosylation sites and/or removing sites
involved in activation (e.g., that target the protein for
degradation).
[0216] Additionally, the active agent may be delivered in
sustained-release formulations. Controlled or sustained-release
formulations are made by incorporating the protein into carriers or
vehicles such as liposomes, nonresorbable impermeable polymers such
as ethylenevinyl acetate copolymers and Hytrel.RTM. copolymers,
swellable polymers such as hydrogels, or resorbable polymers such
as collagen and certain polyacids or polyesters such as those used
to make resorbable sutures. Additionally, the active agent can be
encapsulated, adsorbed to, or associated with, particulate
carriers. Examples of particulate carriers include those derived
from polymethyl methacrylate polymers, as well as microparticles
derived from poly(lactides) and poly(lactide-co-glycolides), known
as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368;
and McGee et al., J. Microencap. (1996).
[0217] As explained above, administration can be effected in one
dose, continuously or intermittently throughout the course of
treatment. Methods of determining the most effective means and
dosages of administration are well known to those of skill in the
art and will vary with the formulation, the composition of the
therapy, the target cells, and the subject being treated. Single
and multiple administrations can be carried out with the dose level
and pattern being selected by the treating physician.
[0218] One particularly effective way to produce long-term
alleviation of pain involves administering two or more doses of
IL-10 at close intervals, e.g., at less than 10 days apart,
preferably less than 5 days apart, more preferably less than 4 days
apart, such as at 3 . . . 2 . . . 1 . . . etc. and any amount of
time within the stated ranges.
[0219] If multiple doses are administered, the first formulation
administered can be the same or different than the subsequent
formulations. Thus, for example, the first administration can be in
the form of a subunit vaccine composition, and the second
administration in the form of a subunit vaccine composition, an
adenovirus vector, an AAV virion, a DNA plasmid, etc. Moreover,
subsequent delivery can also be the same or different than the
second mode of delivery.
[0220] Pain Models
[0221] The ability of an anti-inflammatory cytokine to treat pain
can be evaluated by any of the accepted pain models known in the
art. Examples of such models are as follows.
[0222] Tail Flick Model: The tail-flick test (D'Amour et al., J.
Pharmacol. Exp. and Ther. (1941) 72:74-79) is a model of acute
pain. A gently-restrained rat is placed on a test stage such that a
focused light source beams on the dorsal or ventral surface of the
rat's tail. A photosensor is present on the test stage located
opposite the light source. To begin the test, the rat's tail blocks
the light, thus preventing the light reaching the photosensor.
Latency measurement begins with the activation of the light source.
When a rat moves or flicks its tail, the photosensor detects the
light source and stops the measurement. The test measures the
period of time (duration) that the rat's tail remains immobile
(latent). Rats are tested prior to administration thereto of a
compound of interest and then at various times after such
administration.
[0223] Rat Tail Immersion Model: The rat tail immersion assay is
also a model of acute pain. A rat is loosely held in hand while
covered with a small folded thin cotton towel with its tail
exposed. The tip of the tail is dipped into a, e.g., 52.degree. C.
water bath to a depth of two inches. The rat responds by either
wiggling of the tail or withdrawal of the tail from the water;
either response is scored as the behavioral end-point. Rats are
tested for a tail response latency (TRL) score prior to
administration thereto of a compound of interest and then retested
for TRL at various times after such administration.
[0224] Carrageenan-induced Paw Hyperalgesia Model: The carrageenan
paw hyperalgesia test is a model of inflammatory pain. A
subcutaneous injection of carrageenan is made into the left
hindpaws of rats. The rats are treated with a selected agent
before, e.g., 30 minutes, the carrageenan injection or after, e.g.,
two hours after, the carrageenan injection. Paw pressure
sensitivity for each animal is tested with an analgesymeter three
hours after the carrageenan injection. See, Randall et al., Arch.
Int. Pharmacodyn. (1957) 111:409-419.
[0225] The effects of selected agents on carrageenan-induced paw
edema can also be examined. This test (see, Vinegar et al., J.
Phamacol. Exp. Ther. (1969) 166:96-103) allows an assessment of the
ability of a compound to reverse or prevent the formation of edema
evoked by paw carrageenan injection. The paw edema test is carried
out using a plethysmometer for paw measurements. After
administration of a selected agent, a carrageenan solution is
injected subcutaneously into the lateral foot pad on the plantar
surface of the left hind paw. At three hours post-carrageenan
treatment, the volume of the treated paw (left) and the un-treated
paw (right) is measured using a plethysmometer.
[0226] Formalin Behavioral Response Model: The formalin test is a
model of acute, persistent pain. Response to formalin treatment is
biphasic (Dubuisson et al., Pain (1977) 4:161-174). The Phase I
response is indicative of a pure nociceptive response to the
irritant. Phase 2, typically beginning 20 to 60 minutes following
injection of formalin, is thought to reflect increased
sensitization of the spinal cord.
[0227] Von frey Filament Test: The effect of compounds on
mechanical allodynia can be determined by the von Frey filament
test in rats with a tight ligation of the L-5 spinal nerve: a model
of painful peripheral neuropathy. The surgical procedure is
performed as described by Kim et al., Pain (1992) 50 :355-363. A
calibrated series of von Frey filaments are used to assess
mechanical allodynia (Chaplan et al., J. Neurosci. Methods (1994)
53:55-63). Filaments of increasing stiffness are applied
perpendicular to the midplantar surface in the sciatic nerve
distribution of the left hindpaw. The filaments are slowly
depressed until bending occurred and are then held for 4-6 seconds.
The filament application order and number of trials were determined
by the up-down method of Dixon (Chaplan et al., supra). Flinching
and licking of the paw and paw withdrawal on the ligated side are
considered positive responses.
[0228] Chronic Constriction Injury: Heat and cold allodynia
responses can be evaluated as described below in rats having a
chronic constriction injury (CCI). A unilateral mononeuropathy is
produced in rats using the chronic constriction injury model
described in Bennett et al., Pain (1988) 33:87-107. CCI is produced
in anesthetized rats as follows. The lateral aspect of each rat's
hind limb is shaved and scrubbed with Nolvasan. Using aseptic
techniques, an incision is made on the lateral aspect of the hind
limb at the mid-thigh level. The biceps femoris is bluntly
dissected to expose the sciatic nerve. On the right hind limb of
each rat, four loosely tied ligatures (for example, Chromic gut
4.0; Ethicon, Johnson and Johnson, Somerville, N.J.) are made
around the sciatic nerve approximately 1-2 mm apart. On the left
side of each rat, an identical dissection is performed except that
the sciatic nerve is not ligated (sham). The muscle is closed with
a continuous suture pattern with, e.g., 4-0 Vicryl (Johnson and
Johnson, Somerville, N.J.) and the overlying skin is closed with
wound clips. The rats are ear-tagged for identification purposes
and returned to animal housing.
[0229] The Hargreaves Test: The Hargreaves test (Hargreaves et al.,
Pain (1998) 32:77-88) is also a radiant heat model for pain. CCI
rats are tested for thermal hyperalgesia at least 10 days post-op.
The test apparatus consists of an elevated heated (80-82.degree.
F.) glass platform. Eight rats at a time, representing all testing
groups, are confined individually in inverted plastic cages on the
glass floor of the platform at least 15 minutes before testing. A
radiant heat source placed underneath the glass is aimed at the
plantar hind paw of each rat. The application of heat is continued
until the paw is withdrawn (withdrawal latency) or the time elapsed
is 20 seconds. This trial is also applied to the sham operated leg.
Two to four trials are conducted on each paw, alternately, with at
least 5 minutes interval between trials. The average of these
values represents the withdrawal latency.
[0230] Cold Allodynia Model: The test apparatus and methods of
behavioral testing is described in Gogas et al., Analgesia (1997)
3:111-118. The apparatus for testing cold allodynia in neuropathic
(CCI) rats consists of a Plexiglass chamber with a metal plate 6 cm
from the bottom of the chamber. The chamber is filled with ice and
water to a depth of 2.5 cm above the metal plate, with the
temperature of the bath maintained at 0-4.degree. C. throughout the
test. Each rat is placed into the chamber individually, a timer
started, and the animal's response latency was measured to the
nearest tenth of a second. A "response" is defined as a rapid
withdrawal of the right ligated hindpaw completely out of the water
when the animal is stationary and not pivoting. An exaggerated limp
while the animal is walking and turning is not scored as a
response. The animals' baseline scores for withdrawal of the
ligated leg from the water typically range from 7-13 seconds. The
maximum immersion time is 20 seconds with a 20-minute interval
between trials.
[0231] 2. Experimental
[0232] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0233] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Materials and Methods
Subjects
[0234] Pathogen-free adult male Sprague-Dawley rats (300-450 g;
Harlan Labs, Madison, Wis.) were used in all experiments. Rats were
housed in temperature- and light-controlled rooms with standard
rodent chow and water available ad libitum. Behavioral testing was
performed during the light cycle.
Drugs
[0235] Sterile aliquots of recombinant gp120 (1 .mu.g/.mu.l;
product #1021; lot #8D159M2; ImmunoDiagnostics, Bedford, Mass.)
were stored at -75.degree. C. At the time of testing, gp120 was
slowly thawed and maintained on crushed ice. Each aliquot of gp120
was used within 30 min of thawing. The gp120 was diluted to a
concentration of 0.5 .mu.g/.mu.l in a 0.1% rat serum albumen
vehicle (RSA; Life Technologies, Gaithersburg, Md., in Dulbecco's
Phosphate Buffered Saline (DPBS,1.times.), 0.10 m pore-filtered, pH
7.2, cat#14190-144; Gibco, Invitrogen Corp, Grand Island, N.Y.) as
described previously (Milligan et al., J. Neurosci. (2001)
21:2808-2819).
[0236] Zymosan (yeast cell walls; Sigma Chemical Co., St. Louis,
Mo.) was made fresh daily by suspension in a vehicle of incomplete
Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.). The final
concentrations were 0, 0.08, or 3.2 .mu.g/.mu.l as described
previously (Milligan et al., J. Neurosci. (2003) 23:1026-1040).
[0237] For experiments using adenovirus, a replication-defective
adenovirus expression vector containing the cDNA encoding for human
IL-10 (AD-IL10) driven by the Rous Sarcoma Virus (RSV) promoter was
used and is described in U.S. Pat. No. 6,048,551. The control
adenovirus (AD-Control) was an analogous adenovirus expression
vector in which the RSV promoter directed the expression of the E.
coli beta-galactosidase gene. Recombinant adenoviruses were grown
by infecting 36 100-mm plates of Human Embryonic Kidney 293
(HEK293) cells (5.times.10.sup.6 cells/plate) at a multiplicity of
25 plaque-forming units/cell. The infected cells were collected
after 48 hr, concentrated by low speed centrifugation, and
resuspended in 20 ml of growth media (DMEM, 10% calf serum). After
4 freeze-thaw cycles, the cell lysates were layered on cesium
chloride step gradients (1 ml of 1.4 g/ml cesium chloride/PBS
cushion, 1.5 ml 1.25 g/ml cesium chloride/PBS step) and centrifuged
in a Beckman SW 40 rotor for 1 hr at 36 K rpm. Viral bands were
harvested and further purified in isopycnic gradients consisting of
1.35 g/ml cesium chloride/PBS in a Beckman VTi65 rotor centrifuged
for 2 hrs at 65 K rpm. Mature viral particles were isolated and
dialyzed for 1 hr against DPBS (Ix) and twice each for 2 hr against
DPBS-3% sucrose. Dialysed virus preparations were stored as 10
.mu.l aliquots at -80.degree. C. Viral titers were determined by
viral plaque assay as previously described (Schaack et al., J.
Virol. (1995) 69:3920-3923).
[0238] For experiments with AAV, an AAV expression vector was
produced (packaged and purified) as previously described
(Zolotukhin et al., Gene Ther. (1999) 6:973-985). In brief,
cotransfection of the proviral cassette with plasmid (pDG) that
provides the AAV rep and cap genes in trans as well as adenoviral
genes E2a, E4 and VA was conducted. The E1a and E1b genes were in
the complimentary cell line, HEK 293. The vector cassette
containing the cDNA encoding rat IL-10 (AAV-IL10) was driven by the
hybrid CMV enhancer/chicken beta actin promoter/hybrid intron
(pTR2-CB-rIL-10). The control AAV (AAV-Control) was an analogous
AAV expression vector in which the CMV enhancer/chicken beta actin
promoter directs the expression of the reporter gene UF11 encoding
Jellyfish green florescent protein (GFP). Viral titers were
determined by infectious center assay as previously described
(Zolotukhin et al., Gene Ther. (1999) 6:973-985). Here, viral
titers for rat IL-10 and UF11 of 2.6.times.10.sup.13 physical
particles (Dot blot)/ml and 1.32.times.10.sup.13 physical
particles/ml, respectively, were achieved. These titers correspond
to 1.7.times.10.sup.11 infectious particles/ml and
1.69.times.10.sup.11 infectious particles/ml for rat IL-10 and UF
11 (GFP), respectively.
[0239] For experiments using plasmid or "naked" DNA, free plasmid
DNA was the identical plasmid engineered for transfection of AAV
described above. In these studies, plasmid DNA encoding IL-10
(pTR2-CB-rIL-10) or GFP (pTR2-CB-GFP-TK-NEO (UF11)) was subcloned
and purified similar to procedures described previously (Sambrook,
J, Fritsch, E. R., Maniatis, T. Molecular cloning, 2.sup.nd ed.,
Cold Spring Harbor Press, pp 1.38-1.39, 1989). After isolation
procedures, plasmids (pDNA) were dialyzed for 1 hr against DPBS
(1.times.) and twice each for 2 hr against DPBS-3% sucrose.
Dialysed pDNA preparations were stored as 300 .mu.l aliquots at
-80.degree. C. The concentration of the pDNA-IL10 and pDNA-UF11
preparations were determined by 260 nm adsorption and were 4.2
.mu.g/.mu.l and 5.6 .mu.g/ul respectively. Animals were given 100
.mu.g pDNA for each injection day. There were a total of four
injections during the 77 day experiment.
Behavioral Measures
[0240] von Frey Test. The von Frey test (Chaplan et al., J.
Neurosci Meth. (1994) 53:55-63) was performed within the sciatic
and saphenous innervation area of the hindpaws as previously
described (Milligan et al., Brain Res. (2000) 861:105-116; Chacur
et al., Pain (2001) 94:231-244; Gazda et al., J. Peripheral Nerv.
Sys. (2001) 6:111-129; Milligan et al., J. Neurosci. (2001)
21:2808-2819. Briefly, a logarithmic series of 10 calibrated
Semmes-Weinstein monofilaments (von Frey hairs; Stoelting, Wood
Dale, Ill.) was applied randomly to the left and right hind paws to
determine the stimulus intensity threshold stiffness required to
elicit a paw withdrawal response. Log stiffness of the hairs is
determined by log10 (milligrams.times.10). The 10 stimuli had the
following log-stiffness values (values in milligrams are given in
parenthesis): 3.61 (407 mg), 3.84 (692 mg), 4.08 (1202 mg), 4.17
(1479 mg), 4.31 (2041 mg), 4.56 (3630 mg), 4.74 (5495 mg), 4.93
(8511 mg), 5.07 (11,749 mg), and 5.18 (15,136 mg). The range of
monofilaments used in these experiments (0.407-15.136 gm) produces
a logarithmically graded slope when interpolating a 50% response
threshold of stimulus intensity [expressed as log10
(milligrams.times.10)] (Chaplan et al., J. Neurosci Meth. (1994)
53:55-63). Assessments were made prior to (baseline) and at
specific times after peri-sciatic and intrathecal drug
administration, as detailed below for each experiment. Behavioral
testing was performed blind with respect to drug administration.
The behavioral responses were used to calculate the 50% paw
withdrawal threshold (absolute threshold), by fitting a Gaussian
integral psychometric function using a maximum-likelihood fitting
method (Harvey, Behav. Res. Meth. Instrum. Comput. (1986)
18:623-632; Treutwein and Strasburger, Percept. Psycholphys. (1999)
61:87-106), as described in detail previously (Milligan et al.,
Brain Res. (2000) 861:105-116). This fitting method allows
parametric statistical analyses (Milligan et al., Brain Res. (2000)
861:105-116).
[0241] Hargreaves Test. Thresholds for behavioral response to heat
stimuli applied to each hind paw were assessed using the Hargreaves
test (Hargreaves et al., Pain (1998) 32:77-88), as previously
described (Milligan et al., Brain Res. (2000) 861:105-116).
Briefly, baseline (BL) paw withdrawal values were calculated from
an average of 3-6 consecutive withdrawal latencies of both the left
and right hind paws measured during a 1 hr period. Voltage to the
heat source was adjusted to yield BL latencies ranging 8-12 sec and
a cut off time of 20 sec was imposed to avoid tissue damage. This
procedure was followed by intrathecal injections and a timecourse
of post-drug behavioral assessments, as described below. Behavioral
testing was performed blind with respect to drug administration.
The order of paw testing varied randomly.
Surgery and Microinjections
[0242] Chronic intrathecal catheters. Lumbosacral intrathecal
(intrathecal) catheters were constructed and implanted by lumbar
approach as previously described in detail (Milligan et al., J.
Neurosci. Meth. (1999) 90:81-86; Milligan et al., (2003) in Pain
Research Methods and Protocols: Methods of Molecular Medicine (Luo
D., ed.), in press. New York: Humana Press). The indwelling
catheters were used to microinject recombinant adenovirus,
recombinant AAV, gp120, or vehicle into the CSF space surrounding
the lumbosacral spinal cord. All intrathecal microinjections were
performed as detailed previously, using an 8 .mu.l void volume to
ensure complete drug delivery (Milligan et al., J. Neurosci. Meth.
(1999) 90:81-86). All catheter placements were verified upon
completion of behavioral testing by visual inspection. Data were
only analyzed from animals with catheters verified as having the
catheter tip within the CSF space at the lumbosacral spinal
level.
[0243] Chronic peri-sciatic catheters. Peri-sciatic catheters were
constructed and implanted at mid-thigh level of the left hindleg as
previously described (Chacur et al., Pain (2001) 94:231-244; Gazda
et al., J. Peripheral Nerv. Sys. (2001) 6:111-129; Milligan et al.,
(2003) in Pain Research Methods and Protocols: Methods of Molecular
Medicine (Luo D., ed.), in press. New York: Humana Press). This
method allowed multi-day recovery of the animal from isoflurane
anesthesia prior to unilateral microinjection of an immune
activator around the sciatic nerve. This avoids the deleterious
effects of anesthetics on the function of both immune (Lockwood et
al., Anesthes. Analg. (1993) 77 :769-774; Sato et al., Masui.
(1995) 44:971-975; Miller et al., Int. J. Microcirc. Clin. Exp.
(1996) 16:147-154) and glial cells (Feinstein et al., J. Neurosurg.
Anesthesiol. 13:99-105; Tas et al., Proc. Natl. Acad. Sci. USA
(1987) 84:5972-5975; Mantz et al., Anesthesiology (1993)
78:892-901; Miyazaki et al., Anesthesiology (1997) 86:1359-1366).
In addition, this indwelling catheter method allowed peri-sciatic
immune activation to be either acute (single injection of an immune
activator) or chronic (repeated injections across weeks) (Milligan
et al., J. Neurosci. (2003) 23:1026-1040). Both methods were used
in the present experiments in awake, unrestrained rats. These acute
and chronic peri-sciatic microinjections over the left sciatic
nerve were performed as previously described (Chacur et al., Pain
(2001) 94:231-244; Milligan et al., (2003) in Pain Research Methods
and Protocols: Methods of Molecular Medicine (Luo D., ed.), in
press. New York: Humana Press). Catheters were verified at
sacrifice by visual inspection. Data were only analyzed from
confirmed sites.
[0244] Chronic constriction injury (CCI). CCI was created at
mid-thigh level of the left hindleg as previously described
(Bennett and Xie, Pain (1988) 33:87-107). Four sterile, absorbable
surgical chromic gut sutures (cuticular 4-0, chromic gut, 27'',
cutting FS-2; Ethicon, Somerville, N.J.) were loosely tied around
the gently isolated sciatic nerve under isoflurane anesthesia
(Phoenix Pharm., St. Joseph, Mo.). The sciatic nerves of
sham-operated rats were identically exposed but not ligated. Suture
placements were verified at sacrifice by visual inspection. Data
were only analyzed from confirmed sites.
[0245] Intrathecal microinjection of AA V into lumbosacral spinal
cord. For experiments injecting either AAV or pDNA, no chronic
indwelling catheters were used. Instead, an acute catheter
application method under brief isoflurane anesthesia (2% vol in
oxygen) was employed. Here, a 25 cm PE-10 catheter, attached by a
30-gauge, 0.5-inch sterile needle to a sterile, 50 .mu.l glass
Hamilton syringe, was marked with black permanent ink at 7.7-7.8 cm
from the open end and placed in a sterile, dry container until the
time of injection. Rats were lightly anesthetized, the lower dorsal
pelvic area was shaved and lightly swabbed with 70% alcohol. An
18-gauge sterile needle with the plastic hub removed was inserted
between lumbar vertebrae L5 and L6. The open end of the PE-10
catheter was inserted into the 18-gauge needle and threaded to the
7.7 cm mark allowing for intrathecal PE-10 catheter-tip placement
at the level of the lumbosacral enlargement. Drugs were injected
with a 1 .mu.l pre- and post 0.9% sterile, isotonic saline solution
flush for 1 min. The PE-10 catheter was immediately withdrawn and
the 18-gauge needle was removed from the L5-L6 inter-vertebral
space. This acute injection method took 2-3 min to complete, and
rats showed full recovery from anesthesia within 10 min. No
abnormal motor behavior was observed in 100% of injections.
Cerebrospinal Fluid (CSF) Collection & Analysis
[0246] Immediately upon completion of behavioral testing in
Examples 2 and 3, rats were overdosed with sodium pentobarbital
(Abbot Laboratories, North Chicago, Ill.). Cervical and lumbosacral
CSF were collected as previously described (Milligan et al., J.
Neurosci. (2001) 21:2808-2819). These samples were flash frozen in
liquid nitrogen and stored at -80.degree. C. until analyzed by an
enzyme linked immunosorbant assay (ELISA) to detect IL-10. As noted
above, the IL-10 used was the human protein. This allowed virally
driven IL-10 production to be assessed unconfounded by rat IL-10 by
use of the R & D (Minneapolis, Minn.) human IL-10 ELISA kit
(Cat # D1000) that detects human IL-10 but not rat IL-10
(manufacturer's information). CSF sample preparation was as
previously described (Milligan et al., J. Neurosci. (2001)
21:2808-2819). The ELISAs were performed according to
manufacturer's instructions.
[0247] Similarly, for the for the AAV and pDNA experiments, rats
were treated with sodium pentobarbital as above, cervical and
lumbosacral CSF collected as described above and samples were flash
frozen until analysis using an ELISA to detect rat IL-10. Rat IL-10
was measured using the R & D (Minneapolis, Minn.) rat IL-10
ELISA kit. CSF sample preparation was as previously described
(Milligan et al., J. Neurosci. (2001) 21:2808-2819). The ELISAs
were performed according to manufacturer's instructions.
Dorsal Root Ganglion and Spinal Cord Tissue Collection &
Analysis.
[0248] Immediately following collection of CSF in the AAV and pDNA
experiments, L4-L6 dorsal root ganglia and lumbosacral spinal cord
were collected ipsilateral and contralateral to CCI as well as
bilateral cervical spinal cord according to methods previously
described (Milligan et al., J. Neurosci. (2001) 21:2808-2819).
These samples were quickly frozen on dry ice, transferred to
pre-cooled labeled tubes and stored at -80.degree. C. until
analyzed by real time polymerase chain reaction to detect rat IL-10
mRNA, using techniques well known in the art. See, e.g., Giulietti
et al., Methods (2001) 25:386-401.
Data Analysis
[0249] All statistical comparisons were computed using Statview
5.0.1 for the Macintosh. Data from the von Frey test were analyzed
as the interpolated 50% threshold (absolute threshold) in log base
10 of stimulus intensity (monofilament stiffness in
milligrams.times.10). Baseline measures for both the von Frey and
Hargreaves tests, and dose response effects, were analyzed by
one-way ANOVA. Timecourse measures for each behavioral test were
analyzed by repeated measures ANOVAs followed by Fisher's protected
least significant difference posthoc comparisons, where
appropriate. Cervical and lumbosacral CSF IL-10 contents were
analyzed by 2.times.2 ANOVA, followed by Fisher's protected least
significant difference posthoc comparisons, where appropriate.
EXAMPLE 1
Dose Response Characterization of Intrathecal Adenovirus Effects on
Behavioral Sensitivity to Calibrated Touch/Pressure Stimuli
[0250] The following experiment was conducted in order to define a
range of adenovirus doses that produced no apparent change in
threshold responses to calibrated touch/pressure stimuli. After
assessment of baseline von Frey responses, rats were intrathecally
injected with either 0 (n=7), 5 (n=5), 10 (n=5), 60 (n=2), 80
(n=7), 160 (n=4), 300 (n=2), or 600 (n=2).times.10.sup.7 plaque
forming units (PFU) of adenovirus. Testing of 1200.times.10.sup.7
PFU was attempted but terminated upon observing vestibulomotor
effects of this dose. Rats injected intrathecally with adenovirus
were assessed on the von Frey test 24 hr later.
[0251] While doses of adenovirus up to 300.times.10.sup.7 PFU had
no reliable effect on responses to calibrated touch/pressure
stimuli compared to vehicle controls, the highest dose
(600.times.10.sup.7 PFU) lowered the response threshold (FIG. 1).
Pre-viral-injected BL values showed no reliable differences between
groups (F.sub.7,26=1.1715, p>0.14). One-Way ANOVA revealed a
reliable effect of viral dose (F.sub.7,26=5.694, p<0.005).
Posthoc analysis revealed that only the 600.times.10.sup.7 PFU
adenovirus dose decreased response thresholds compared to controls
(p<0.002). Adenovirus doses employed in subsequent experiments
were restricted to the lower end of the dose range (see asterisks
in FIG. 1) so to minimize the chances of virally induced
alterations in pain sensitivity.
EXAMPLE 2
Prevention of Intrathecal HIV-1 gp120 Induced Mechanical Allodynia
by Intrathecal AD-ILI0
[0252] It has previously been shown that spinal immune activation
induced by intrathecal delivery of gp120, an envelope glycoprotein
of human immunodeficiency virus-I, lowers the response threshold to
touch/pressure stimuli (Milligan et al., Brain Res. (2000)
861:105-116; Milligan et al., J. Neurosci. (2001) 21:2808-2819).
This pain response is the result of spinal cord glial activation
and the release of the glial proinflammatory cytokines IL1 and TNF
(Milligan et al., Brain Res. (2000) 861:105-116; Milligan et al.,
J. Neurosci. (2001) 21:2808-2819). Hence the ability of AD-IL10 to
prevent this glially-driven mechanical allodynia was examined.
[0253] Based on pilot studies of adenoviral doses within the range
defined in Example 1, 10.times.10.sup.7 PFU of AD-ILI0 in 10 .mu.l
was chosen for study. An equal volume of AD-Control
(16.times.10.sup.7 PFU in 10 .mu.l) was administered to the control
group. Rats were first assessed for their responses to the von Frey
test prior to (baseline; BL) and on Days 4 and 5 after intrathecal
AD-IL10 or AD-Control injection (n=8/group). Based on prior studies
of this AD-IL10 vector, near maximal levels of viral ly directed
IL-10 should be induced by this time (Gudmundsson et al., Amer. J.
Resp. Cell & Molec. Biol. (1998) 19:812-818). The behavioral
tests on Days 4 and 5 were performed to verify that neither this
intrathecal adenoviral dose nor virally directed ILI0 release had
any observable confounding effect on this measure. Upon completion
of the Day 5 test, all rats were injected with 3 .mu.g gp120. This
gp120 dose has previously been shown to produce mechanical
allodynia as measured by the von Frey test (Milligan et al., Brain
Res. (2000) 861:105-116; Milligan et al., J. Neurosci. (2001)
21:2808-2819; Milligan et al., J. Pain (2001) 6:326-333).
Vehicle-injected controls were not included as it has repeatedly
been demonstrated that this procedure has no effect on this
behavioral measure (Milligan et al., Brain Res. (2000) 861:105-116;
Milligan et al., J. Pain (2001) 6:326-333; Milligan et al., J.
Neurosci. (2001) 21:2808-2819). Following gp120 injections,
responses to touch/pressure stimuli were reassessed each 20 min for
120 min, in accordance with prior publications (Milligan et al.,
Brain Res. (2000) 861:105-116; Milligan et al., J. Pain (2001)
6:326-333; Milligan et al., J. Neurosci. (2001) 21:2808-2819). Upon
completion of testing, cervical and lumbosacral CSF samples were
collected for IL-10 analyses.
[0254] Intrathecal administration of 10.times.10.sup.7 AD-IL10 and
AD-Control had no reliable effect on behavioral responses on the
von Frey test compared to BL (F.sub.1,21=1.385, p>0.25) (FIG.
2). Thus neither IL-10 released by the adenovirus nor the presence
of this dose of adenovirus itself altered basal pain responsivity.
As in previous studies (Milligan et al., Brain Res. (2000)
861:105-116; Milligan et al., J. Neurosci. (2001) 21:2808-2819),
intrathecal gp120 produced robust mechanical allodynia in
AD-Controls. In contrast, no mechanical allodynia developed in the
AD-IL10 treated animals. Repeated measures ANOVA revealed a
reliable main effect of IL-10 (F.sub.1,21=235.694,
p<0.0001).
[0255] CSF collected upon completion of behavioral testing
supported that AD-IL10 induced the release of human IL-10,
concentrated at the lumbosacral level (FIG. 3). ANOVA revealed
reliable main effects of IL-10 (F.sub.1,21=37.430, p<0.0001) and
site of CSF collection (lumbosacral vs. cervical;
F.sub.1,21=46.240, p>0.0001) and an interaction between IL10 and
site of CSF collection (F.sub.1,21=36.577, p<0.0001), supporting
that AD-ILI0 caused a greater site-specific effect of IL-10
concentrations at lumbosacral than cervical levels.
EXAMPLE 3
Prevention of Sciatic Inflammatory Neuropathy (SIN) Induced
Mechanical Allodynia by Intrathecal AD-IL10
[0256] The purpose of Examples 3 through 5 was to extend the
results of Example 2 by examining the effect of AD-IL10 on
neuropathic pain. Neuropathic pain arises as a consequence of
inflammation and/or trauma of peripheral nerves. Neuropathic pain
is poorly managed by currently available drugs developed to target
neurons (for review, see (Watkins and Maier, Physiol. Rev. (2002)
82:981-1011).
[0257] AD-IL10 was tested for its ability to prevent mechanical
allodynia induced by sciatic inflammatory neuropathy (SIN) as
follows. Based on pilot studies of adenoviral doses within the
range defined in Example 1, 5.times.10.sup.7 PFU of AD-IL 10 in 5
.mu.l was chosen for study. An equal volume of AD-Control
(8.times.10.sup.7 PFU in 5 .mu.l) was administered to the control
group. Rats were first assessed for their responses to the von Frey
test prior to (BL) and again on Day 4 after intrathecal AD-ILI0 or
AD-Control injection. As noted above, near maximal levels of
virally directed IL-10 are expected by this time (Gudmundsson et
al., Amer. J. Resp. Cell & Molec. Biol. (1998) 19:812-818). The
behavioral test on Day 4 was performed to verify that neither this
intrathecal adenoviral dose nor virally directed IL-10 release had
any observable confounding effect on this measure. Immediately upon
completion of the Day 4 test, all rats were peri-sciatically
injected with either 4 or 160 .mu.g zymosan (n=5-6/group).
Peri-sciatic vehicle injected controls were not included as it has
repeatedly been demonstrated that this procedure has no effect on
this behavioral measure (Chacur et al., Pain (2001) 94:231-244;
Gazda et al., J. Peripheral Nerv. Sys. (2001) 6:111-129; Milligan
et al., J. Neurosci. (2003) 23:1026-1040). The 4 and 160 .mu.g
zymosan doses have previously been shown to induce unilateral and
bilateral mechanical allodynia, respectively, in intrathecal
catheterized rats (Milligan et al., J. Neurosci. (2003)
23:1026-1040). Behavioral. responses on the von Frey test were
reassessed 3 and 24 hr later, in accordance with prior studies
(Chacur et al., Pain (2001) 94:231-244; Gazda et al., J. Peripheral
Nerv. Sys. (2001) 6:111-129; Milligan et al., J. Neurosci. (2003)
23:1026-1040). Upon completion of testing, cervical and lumbosacral
CSF samples were collected for IL-10 analyses.
[0258] It was found that intrathecally administered AD-IL10 (a)
successfully induced the site-specific release of human IL-10 into
CSF and (b) prevented mechanical allodynia created in response to
spinal cord immune activation. ANOVA revealed that AD-ILI0 and
AD-Control had no effect on mechanical response thresholds measured
5 days after virus delivery, compared to BL (F.sub.7,88=0.686,
p>0.68) (FIG. 4). Hence, neither the presence of IL10 nor
adenovirus had measurable effects on basal pain responses. As in
our previous studies (Milligan et al., J. Neurosci. (2003)
23:1026-1040), low dose zymosan induced a unilateral allodynia
(FIG. 4A) while higher dose zyrnosan induced a bilateral allodynia
(FIG. 4C), compared to BL measures. Repeated measures ANOVA
revealed reliable main effects of peri-sciatic zymosan dose
(F.sub.1,40=12.093, p<0.002), intrathecal IL-10
(F.sub.1,40=69.829, p<0.0001), laterality (F.sub.1,40=22.315,
p<0.0001) and time after peri-sciatic zymosan application
(F.sub.1,40=13.029, p<0.001), and interactions between zymosan
dose and intrathecal IL-10 (F.sub.1,40=6.161, p<0.02) and
between intrathecal IL-10 and laterality (F.sub.1,40=15.412,
p<0.001). Post hoc means comparison revealed that 4 .mu.g
zymosan induced mechanical al lodynia in the left (ipsilateral)
hindpaw compared to the right (contralateral) hindpaw in AD-control
treated animals (p<0.0001). Mechanical response of the right
hindpaw after 4 .mu.g peri-sciatic zymosan did not differ from that
at BL, indicating that 4 .mu.g zymosan induced only a unilateral
allodynia ipsilateral to the site of injection (p>0.45). In
addition, posthoc analyses revealed that bilateral mechanical
allodynia occurred in response to 160 .mu.g peri-sciatic zymosan in
AD-Control treated animals. That is, the thresholds for both the
left and right paws were reliably different from BL measures
(p<0.0001). Both ipsilateral (p>0.05) {FIG. 4B) and bilateral
(p>0.15) (FIG. 4D) allodynias were blocked by AD-IL10 as von
Frey responses after peri-sciatic zymosan did not differ from
BL.
[0259] Lumbosacral CSF collected upon completion of behavioral
testing indicated that AD-ILI0 induced the release of human IL-10
(FIG. 3). One-way ANOVA revealed a reliable main effect of AD-ILI0
(F.sub.1,10=8.362, p<0.02). FIG. 3 suggests a dose-dependent
effect of 5.times.10.sup.7 PFU AD-IL10 (this example) compared to
10.times.10.sup.7 PFU ILI0 (Example 2). As these assays were
performed at separate times with different kits, these values were
not statistically compared.
EXAMPLE 4
Reversal of Sciatic Inflammatory Neuropathy (SIN) Induced
Mechanical Allodynia by Intrathecal AD-ILI0
[0260] Example 3 revealed that intrathecal AD-IL10 prevented
SIN-induced mechanical allodynia. In this example, the chronic SIN
method (Milligan et al., J. Neurosci. (2003) 23:1026-1040) was used
to test whether AD-ILI0 could reverse established SIN-induced
mechanical allodynia. The dose of AD-ILI0 chosen for study was
identical to that in Example 3 (5.times.10.sup.7 PFU of adenovirus
in 5 .mu.l). An equal volume of AD-Control (8.times.10.sup.7 PFU in
5 .mu.l) was administered to the control group. Rats were assessed
for their responses to the von Frey test prior to (BL) initiation
of chronic SIN. Unilateral and bilateral chronic SIN were created
as described previously (Milligan et al., J. Neurosci. (2003)
23:1026-1040). Peri-sciatic microinjection of zymosan (either 4 or
160 .mu.g) was delivered immediately after BL (Day 0) and 2, 4, 6,
8, 10, and 12 days later. Von Frey tests were again performed on
Days 1, 4, 8, 9, 10, 12 and 14. When behavioral testing and
peri-sciatic injections occurred on the same day, behavioral
testing preceded the peri-sciatic injection. The Day 8 behavioral
assessment provided verification that the 4 .mu.g and 160 .mu.g
chronic zymosan regimens produced unilateral and bilateral
allodynia, respectively. Intrathecal adenovirus (either AD-IL10 or
AD-Control) was delivered immediately after the Day 8 test
(n=5-6/group). The Day 9-14 behavioral assessments allowed
assessment of the ability of AD-IL 10 to reverse well-established
inflammatory neuropathy pain.
[0261] To examine whether ipsilateral territorial (skin innervated
by the sciatic nerve), ipsilateral extra-territorial (skin
innervated by the saphenous nerve), mirror-image territorial, and
mirror-image extra-territorial allodynias were comparably affected
by intrathecal ILI0 gene therapy, sciatic and saphenous innervation
zones were separately tested at BL, Day 8 (prior to AD
administration), and Days 12 and 14 (4 and 6 days after AD
administration) in rats chronically administered 160 .mu.g
peri-sciatic zymosan.
[0262] As previously reported (Milligan et al., J. Neurosci. (2003)
23:1026-1040), the repeated low (4 .mu.g) and high (160 .mu.g)
zymosan protocols produced chronic unilateral and bilateral
allodynia, respectively (FIG. 5). Eight days after initiation of
zymosan administration, prior to adenoviral administration, ANOVA
revealed reliable main effects of zymosan dose (F.sub.1,36=35.049,
p<0.0001) and laterality (F.sub.1,36=41.634, p<0.0001) and
time after peri-sciatic zymosan application (F.sub.2,72=7.537,
p<0.001), and interactions between zymosan dose and laterality
(F.sub.1,36=35.919, p<0.0001). Post hoc means comparison
revealed that 4 .mu.g zymosan induced mechanical allodynia in the
left (ipsilateral) hindpaw compared to the right (contralateral)
hindpaw in IL-10- and control virus-treated groups (p<0.0001).
Mechanical responses of the right hindpaw after 4 .mu.g
peri-sciatic zymosan did not differ from that at BL (p>0.66),
supporting that 4 .mu.g zymosan induced only a unilateral allodynia
ipsilateral to the site of injection. In addition, posthoc analyses
supported that bilateral mechanical allodynia occurred in response
to 160 .mu.g peri-sciatic zymosan. That is, the thresholds of the
left and right hindpaw did not differ (p>0.29) but the
thresholds for both the left and right paws were reliably different
from BL (p<0.0001).
[0263] After intrathecal adenoviral administration, AD-IL10
reversed these ongoing pathological pain states. That is, AD-IL10
reversed both ipsilateral and bilateral allodynias induced by
peri-sciatic zymosan. ANOVA revealed reliable main effects of
zymosan dose (F.sub.1,36=22.724, p<0.0001), IL10
(F.sub.1,36=50.044, p<0.0001), laterality (F.sub.1,36=35.532,
p<0.0001) and time after intrathecal adenoviral administration
(F.sub.3,108.times.6.301, p<0.001), and interactions between
IL-10 and laterality (F.sub.1,36=35.919, p<0.05). Posthoc means
comparisons supported that IL-10 attenuated the allodynic effects
of 4 .mu.g zymosan in the ipsilateral hindpaw (p<0.0001,
comparing ipsilateral hindpaw responses on day 8 vs. ipsilateral
hindpaw responses on day 14 in the AD-IL10 group), whereas
AD-control group hindpaw responses remained allodynic through day
14 (p>0.8, comparing ipsilateral hindpaw responses on day 8 vs.
ipsilateral hindpaw responses on day 14 in the AD-control
group).
[0264] IL-10 also attenuated allodynic effects of 160 .mu.g zymosan
in the contralateral hindpaw (p<0.0001, comparing contralateral
hindpaw responses on day 8 vs. contralateral hindpaw responses on
day 14 in the AD-IL10 group), whereas virus alone did not alter
ongoing mirror image allodyma (p>0.2, comparing contralateral
hindpaw responses on day 8 vs. contralateral hind paw responses on
day 14 in the AD-control group)
[0265] Differential testing of sciatic nerve (territorial) and
saphenous nerve (extra-territorial) innervation areas of the
ipsilateral and contralateral hind paws at BL (F.sub.1,50=1.352,
p>0.90) and at 8 days (F.sub.1,50=0.170, p>0.89) after
chronic peri-sciatic 160 .mu.g zymosan (prior to AD administration)
revealed no differences between groups (FIG. 6). At Day 8 (compared
to BL), territorial and extraterritorial mechanical allodynia was
observed in both the ipsilateral and mirror-image hind paws. ANOVA
revealed main effect of time (BL vs. Day 8) after peri-sciatic
zymosan (F.sub.1,38=22.398, p<0.0001). Chronic peri-sciatic 160
.mu.g zymosan produced reliable bilateral allodynia in both the
territorial and extraterritorial innervation areas of both hind
paws. ANOVA revealed no differences between the saphenous versus
sciatic territories (F.sub.1,36=0.008, p>0.92). In addition, no
differences were found between ipsilateral vs. contralateral
hindpaw responses (F.sub.1,36=0.716, p>0.40). AD-ILI0 reliably
reversed bilateral mechanical allodynia produced by peri-sciatic
zymosan in both the territorial and extra-territorial innervation
areas of both hindpaws. Repeated measures ANOVA revealed reliable
main effects of IL-10 (F.sub.1,32=45.174, p<0.0001) and time
after viral treatment (F 2.sub.,64=37.354, p<0.0001), and an
interaction between time after viral treatment and IL-10 (F
2.sub.,64=15.265, p<0.0001). Posthoc analyses revealed that
AD-IL10 reliably reversed ipsilateral territorial (p<0.05),
ipsilateral extraterritorial (p<0.001), mirror-image territorial
(p<0.01), and mirror-image extraterritorial (p<0.01)
allodynias compared to AD-Control treated animals. The degree of
reversal of each of these allodynias was comparable at both Days 12
(4 days after AD-ILI0; p<0.02 comparing AD-ILI0 ipsilateral and
contralateral saphenous and sciatic terrirotires to respective
AD-Controls) and 14 (6 days after AD-IL10; p<0.005 comparing
AD-IL10 ipsilateral and contralateral saphenous and sciatic
terrirotires to respective AD-Controls).
EXAMPLE 5
Reversal of Chronic Constriction Injury (CCI) Induced Mechanical
Allodynia and Thermal Hyperalgesia by Intrathecal AD-ILI0
[0266] Example 4 revealed that adenoviral IL-10 can fully reverse
SIN-induced pathological pain changes as measured by the von Frey
test. While approximately 50% of clinical neuropathies are
infective/inflammatory in nature, the rest involve peripheral nerve
trauma (Said and Hontebeyrie-Joskowicz, Res. Immunol (1992)
143:589-599). Hence, it was important to determine whether
adenoviral IL-10 could reverse traumatic neuropathy induced pain
changes, in addition to its effectiveness on inflammatory
neuropathy. A classic partial nerve injury model was used for
study; namely, chronic constriction injury (CCI) (Bennett and Xie,
Pain (1988) 33:87-107). The dose of AD-IL-10 chosen for study was
identical to that in Examples 3 and 4 (5.times.10.sup.7 PFU of
adenovirus in 5 .mu.l). An equal volume of AD-Control
(8.times.10.sup.7 PFU in 5 .mu.l) was administered to the control
group. Rats were assessed for their responses to the von Frey test
and Hargreaves test prior to (BL) and again on Day 10 after CCI or
sham surgery. This latter test allowed verification of the
development of mechanical allodynia and thermal hyperalgesia in CCI
rats, compared to controls. Immediately after the test on Day 10,
all rats received intrathecal AD-IL10 or AD-Control (n=6/group).
Von Frey and Hargreaves tests were again performed on Days 3, 5, 7,
14, 18, and 21 after viral administration. This corresponds to Days
13, 15, 17, 24, 28, and 31 after CCI or Sham surgery. These tests
allowed assessment of (a) the ability of AD-IL10 to reverse
well-established traumatic neuropathy pain and (b) the duration of
AD-IL10 effectiveness.
[0267] CCI produced chronic bilateral mechanical allodynia (FIG. 7)
and chronic ipsilateral thermal hyperalgesia (FIG. 8). Such a
pattern of pain changes is in accord with prior publications
(Paulson et al., Pain (2000) 84:233-245). For behavioral
assessments between Days 3-10, prior to adenoviral administration,
ANOVA for the von Frey test revealed reliable main effects of CCI
(F.sub.1,38=143.235, p<0.0001), laterality (F.sub.1,38=16.797,
p<0.001) and time after CCI surgery (F.sub.3,114=15.699,
p<0.0001), and interactions between CCI surgery and laterality
(F.sub.1,38=13.824, p<0.001) and time after CCI surgery and CCI
(F.sub.3,114=7.054, p<0.001). Post hoc means comparison revealed
that CCI induced bilateral mechanical allodynia compared to sham
operated controls (ipsilateral: p<0.0001; contralateral:
p<0.0001). In addition, prior to adenoviral administration,
ANOVA for the Hargreaves test revealed reliable main effects of CCI
(F.sub.1,38=239.135, p<0.0001) and laterality
(F.sub.1,38=150.902, p<0.0001), and interactions between CCI and
laterality (F.sub.1,38=103.228, p<0.0001). Post hoc means
comparison revealed that CCI induced unilateral thermal
hyperalgesia compared to sham operated controls (ipsilateral:
p<0.0001; contralateral: p>0.49).
[0268] After intrathecal adenoviral administration, AD-IL10
reversed these ongoing pathological pain states. That is, analyzing
data between Days 13-24, AD-IL10 reversed both bilateral allodynia
and ipsilateral thermal hyperalgesia induced by CCI. ANOVA revealed
for the von Frey test reliable main effects of CCI
(F.sub.1,38=105.832, p<0.0001), IL-10 (F.sub.1,38=8.998,
p<0.005), and time (F.sub.3,114=5.651, p<0.01), and
interactions between CCI and IL10 (F.sub.1,38=14.301, p<0.001),
time after intrathecal adenovirus and IL-I0 (F.sub.3,114=7.29,
p<0.001) and time after intrathecal adenovirus, CCI and IL-10
(F.sub.3,114=2.604, p=0.05). Posthoc means comparisons supported
that IL-10 reversed the bilateral mechanical allodynic effects of
CCI by Day 15 (p<0.0001 and p<0.02, respectively, comparing
the ipsilateral and contralateral paw of the AD-ILI0 group vs.
AD-Control on Day 15), as well as on day 17 (p<0.0001 and
p<0.01, respectively, comparing the ipsilateral and
contralateral paw of the AD-ILI0 group vs. AD-Control on Day
17).
[0269] ANOVA revealed for the Hargreaves test reliable main effects
of CCI (F.sub.1,38=48.069, p<0.0001), ILI0 (F.sub.1,38=4.727,
p<0.05) and laterality (F.sub.1,38=48.466, p<0.0001) and
interactions between CCI and laterality (F.sub.1,38=30.955,
p<0.0001), ILI0 and laterality (F.sub.1,38=6.494, p<0.01) and
time after intrathecal adenovirus, CCI and ILI0 (F.sub.3,114=3.116,
p<0.05). Posthoc means comparisons supported that ILI0 reversed
the ipsilateral thermal hyperalgesic effects of CCI by Day 15
(p<0.002, comparing the ipsilateral paw of the AD-ILI0 group vs.
AD-Control on Day 15), as well as on day 17 (p<0.01, comparing
the ipsilateral paw of the AD-ILI0 group vs. AD-Control on Day
17).
[0270] Intrathecal AD-ILI0 did not permanently reverse these
ongoing pathological pain states. This was expected, given that
cells infected by adenovirus are readily detected and deleted by
the immune system. Indeed, the literature on the adenovirus used in
this study supported that it would only temporarily reverse the
consequences of a proinflammatory challenge (Gudmundsson et al.,
Amer. J. Resp. Cell & Molec. Biol. (1998) 19:812-818). In
support of this, AD-IL10 reversal of CCI-induced pathological pain
states began dissipating by Day 24. From Day 24-31, both mechanical
allodynia and thermal hyperalgesia progressively returned. By Day
28, mechanical allodynia and thermal hyperalgesia had returned to
the preadenoviral levels observed at Day 10. This was supported by
ANOVA that mechanical allodynia (F.sub.1,38=0.450, p>0.50) and
thermal hyperalgesia (F.sub.1,38=0.612, p>0.43) did not differ
from those at preadenoviral levels.
[0271] The above examples demonstrate that lumbosacral intrathecal
delivery of replication-deficient adenovirus containing the cDNA
for human IL-10 produces site-specific release of IL-10 into CSF.
Neither the presence of 5-10.times.10.sup.7 PFU of adenovirus nor
virally driven IL-10 caused observable effects on basal response
thresholds to calibrated touch/pressure (von Frey test) or thermal
(Hargreaves test) stimuli. However, adenoviral IL10 prevented and
reversed pathological pain states. Adenoviral IL-10 prevented
mechanical allodynias induced by spinal immune activation with
intrathecal HIV-1 gp120 and by sciatic inflammatory neuropathy
(SIN). It reversed mechanical allodynias induced by SIN and sciatic
traumatic neuropathy (CCI). Lastly, it reversed thermal
hyperalgesia induced by CCI. Given that neuropathic pain is
especially difficult to treat with currently available drugs
{McQuay et al., Brit. Med. J. (1995) 311:1047-1052; McQuay et al.,
Pain (1996) 68:217-227; Collins et al., J. Pain Symptom. Manage.
(2000) 20:339-457, the success of this gene therapy is
dramatic.
EXAMPLE 6
Prevention of Sciatic Inflammatory Neuropathy (SIN) Induced
Mechanical Allodynia by Intrathecal AAV-ILI0
[0272] Given the profound results achieved with adenoviral-IL10 and
in order to test whether the results were achievable with different
vectors and molecules, the following experiments were conducted
using (a) a different viral vector (AAV) and (b) rat IL-10 instead
of human IL-10. The use of rat IL-10 eliminates potential
interference from the immune system to the foreign human IL-10
protein when delivered to rats.
[0273] The dose of AAV-IL10 chosen for study was based on
observations from Example 3 (5.times.10.sup.7 PFU of adenovirus in
5 .mu.l). Here, it was estimated that 8.5.times.10.sup.8 infectious
particles in 5 .mu.l would be efficacious. An equal volume of
AAV-Control (8.5.times.10.sup.8 PFU in 5 .mu.l) was administered to
the control group. Rats were assessed for their responses to the
von Frey test prior to (BL) intrathecal AAV (either AAV-10 or
AAV-Control) was delivered (n=5-6/group). The second BL assessment
(BL-2) was conducted 3 days after AAV injection to ensure that this
dose of AAV did not alter normal threshold responses. Unilateral
and bilateral chronic SIN was created as described previously
(Milligan et al., J. Neurosci. (2003) 23:1026-1040). Peri-sciatic
microinjection of zymosan (either 4 or 160 ug) was delivered
immediately after BL-2 (Day 0) and 2, 4, 6 and 8 days later. Von
Frey tests were again performed daily until Day 8 and on Day 10.
When behavioral testing and peri-sciatic injections occurred on the
same day, behavioral testing preceded the peri-sciatic
injection.
[0274] As shown in FIG. 9, AAV-delivered IL-10 had no effect on the
normal (right) leg pain responses but returned the neuropathic leg
(left) to normal levels of pain sensitivity. ANOVA revealed that
AAV-IL10 and AAV-Control had no effect on mechanical response
thresholds measured 3 days after virus delivery, compared to BL
(F.sub.1,48=1.069, p>0.30). Hence neither the presence of IL-10
or AAV had measurable effects on basal pain responses. Low dose
zymosan induced a unilateral allodynia while higher dose zymosan
induced a bilateral allodynia, compared to BL measures. Repeated
measures ANOVA revealed reliable main effects of peri-sciatic
zymosan dose (F.sub.2,44=237.795, p<0.0001), intrathecal
AAV-IL10 (F.sub.1,44=399.912, p<0.0001), laterality
(F.sub.1,44=125.122, p<0.0001) and time after peri-sciatic
zymosan application (F.sub.8,352=14.865, p<0.0001), and
interactions between intrathecal AAV-IL10 and zymosan dose
(F.sub.2,44=125.975, p<0.0001), intrathecal AAV-IL10 and
laterality (F.sub.1,44=24.906, p<0.0001), zymosan dose and
laterality (F.sub.2,44=69.651, p<0.0001), intrathecal AAV-IL10,
zymosan dose and laterality (F.sub.2,44=24.323, p<0.0001) and
time after peri-sciatic zymosan application, intrathecal AAV-IL10,
zyrnosan dose and laterality (F.sub.16,352=1.706, p<0.05).
EXAMPLE 7
Full Time Course of Reversal of Chronic Constriction Injury (CCI)
Induced Mechanical Allodynia and Thermal Hyperalgesia by
Intrathecal AAV-ILI0
[0275] In order to determine whether AAV-mediated IL-10 gene
delivery was effective in reversing CCI induced mechanical
allodynia and thermal hyperalgesia, the following experiments were
conducted. The dose of AAV-IL10 chosen for study was
8.5.times.10.sup.8 infectious particles of AAV in 5 .mu.l. An equal
volume of AAV-Control (8.5.times.10.sup.8 infectious particles in 5
.mu.l) was administered to the control group. Rats were assessed
for their responses to the von Frey test and Hargreaves test prior
to (BL) and again on Days 3 and 10 after CCI or sham surgery. This
latter test allowed verification of the development of chronic
mechanical allodynia and thermal hyperalgesia in CCI rats, compared
to controls. Immediately after the test on Day 10, all rats
received intrathecal AAV-IL10 or AAV-Control (n=6/group). Von Frey
and Hargreaves tests were again performed on Days 3, 5, 7, 9, 11,
14, 16 and 20 after viral administration. This corresponds to Days
13, 15, 17, 19, 21, 24, 26, and 30 after CCI or Sham surgery. These
tests allowed assessment of (a) the ability of AAV-IL10 to reverse
well-established traumatic neuropathy pain and (b) the duration of
AAV-IL10 effectiveness.
[0276] As shown in FIGS. 10 and 11, AAV-IL10 had no effect on the
sham operated rats but returned the neuropathic pain back to normal
levels of pain sensitivity. Prior to CCI surgery, all groups showed
similar BL values (F.sub.7,40=0.345, p>0.9). As observed in the
experiments above, CCI produced chronic bilateral mechanical
allodynia and chronic ipsilateral thermal hyperalgesia. For
behavioral assessments at Days 3 and 10, prior to AAV intrathecal
administration, ANOVA for the von Frey test revealed reliable main
effects of CCI (F.sub.1,40=197.446, p<0.0001) and laterality
(F.sub.1,40=6.356, p<0.05).
[0277] In addition, prior to CCI surgery, all groups showed no
behavioral BL differences for the Hargreaves test
(F.sub.7,40=2.102, p>0.05). Before AAV intrathecal
administration, ANOVA for the Hargreaves test revealed reliable
main effects of CCI (F.sub.1,40=140.740, p<0.0001) and
laterality (F.sub.1,38=48.901, p<0.0001), and an interaction
between CCI and laterality (F.sub.1,40=104.295, p<0.0001).
[0278] After intrathecal AAV administration, AAV-IL10 reversed
these ongoing pathological pain states. That is, analyzing data
between Days 13-30 (corresponding to days 3-20), AAV-IL10 reversed
both bilateral allodynia and ipsilateral thermal hyperalgesia
induced by CCI. ANOVA revealed for the von Frey test reliable main
effects of CCI (F.sub.1,40=496.336, p<0.0001), AAV-IL10
(F.sub.1,40=59.636, p<0.0001), laterality (F.sub.1,40=28.565,
p<0.0001), and time after AAV (F.sub.7,280=10.462, p<0.0001),
and interactions between CCI and AAV-IL10 (F.sub.1,40=72.988,
p<0.0001), CCI and laterality (F.sub.1,40=9.325, p<0.01),
time after AAV and CCI (F.sub.7,280=5.823, p<0.0001), time after
AAV and AAV-IL10 (F.sub.7,280=5.993, p<0.0001) and time after
AAV, CCI and AAV-IL10 (F.sub.7,280=4.840, p=0.0001).
[0279] ANOVA revealed for the Hargreaves test reliable main effects
of CCI (F.sub.1,39=134.036, p<0.0001), AAV-IL10
(F.sub.1,39=12.047, p<0.01) and laterality (F.sub.1,39=66.284,
p<0.0001) and time after intrathecal AAV administration
(F.sub.7,273=12.237, p<0.005), and interactions between CCI and
AAV-IL10 (F.sub.1,39=24.486, p<0.0001), CCI and laterality
(F.sub.1,39=91.956, p<0.0001), IL-10 and laterality
(F.sub.1,39=17.392, p<0.0001) CCI, AAV-IL10 and laterality
(F.sub.1,39=35.721, p<0.0001) and time after intrathecal AAV
administration and IL10 (F.sub.7,273=3.783, p<0.005).
EXAMPLE 8
Partial Time Course of Reversal of Chronic Constriction Injury
(CCI) Induced Mechanical Allodynia and Thermal Hyperalgesia by
Intrathecal Aav-ILI0 to Collect CSF and Tissue Samples at Time of
Full Reversal
[0280] Example 7 was repeated with one exception. That is, the time
course of intrathecal AAV-IL10 was truncated at the time of full
behavioral reversal of both thermal hyperalgesia and low threshold
allodynia to examine the mechanism of action of spinal AAV-IL10.
The dose of AAV-IL10 was 8.5.times.10.sup.8 infectious particles of
AAV in 5 .mu.l. An equal volume of AAV-Control (8.5.times.10.sup.8
infectious particles in 5 .mu.l) was administered to the control
group. Rats were assessed for their responses to the von Frey test
and Hargreaves test prior to (BL) and again on Days 3, 5, 7 and 10
after CCI or sham surgery. Immediately after the test on Day 10,
all rats received intrathecal AAV-IL10 or AAV-Control (n=6/group).
Von Frey and Hargreaves tests were again performed on Days 3, 5 and
7 after viral administration. This corresponds to Days 13, 15 and
17 after CCI or Sham surgery. These tests allowed assessment of (a)
the production and release of AAV-IL10 compared to control-AAV (b)
the action AAV-IL10 on proinflammatory cytokines (IL-1, TNF-b and
IL-6) and their respective receptors as well as IL10 receptors.
[0281] As seen in FIGS. 12A and 12B, AAV-delivered IL-10 again
reversed chronic thermal hyperalgesia induced by CCI. This is a
partial timecourse as the experiment was stopped at the point of
complete pain reversal so that tissues could be collected for
analyses. After baseline (BL) assessment, rats were given either
sham surgery or CCI of the left sciatic nerve to induce traumatic
neuropathy. After behavioral assessment on Day 10, rats were
injected intrathecally with either AAV-Control or AAV-IL10.
Behavior was reassessed 3, 5 and 7 days later (corresponding to
Days 13, 15 and 17 after CCI or sham surgery). After testing on Day
17, the animals were sacrificed and tissues collected for analyses.
Profound neuropathic pain was demonstrated in CCI rats receiving
intrathecal control virus. Intrathecal AAV-IL10 blunted this
neuropathic pain. Normal pain responses were observed for sham
operated rats administered either AAV-Control or AAV-IL10.
[0282] In particular, prior to induction of CCI, all groups
revealed similar BL values (F.sub.7,42=0.497, p>0.80). For
behavioral assessments between Days 3-10, after induction of CCI
and prior to AAV administration, ANOVA for the von Frey test
revealed reliable main effects of CCI (F.sub.1,42=282.369,
p<0.0001), laterality (F.sub.1,42=13.119, p<0.001) and an
interaction between CCI surgery and laterality (F.sub.1,42=8.076,
p<0.01).
[0283] Prior to the induction of CCI, BL values assessed from the
Hargreaves test revealed no differences (F.sub.7,42=0.957,
p>0.47). However, after induction of CCI and prior to AAV
administration, ANOVA for the Hargreaves test revealed reliable
main effects of CCI (F.sub.1,42=137.312, p<0.0001) and
laterality (F.sub.1,42=40.480, p<0.0001), and an interaction
between CCI and laterality (F.sub.1,42=156.562, p<0.0001).
[0284] After intrathecal AAV administration, AAV-IL10 reversed
these ongoing pathological pain states. That is, analyzing data
between Days 13-17, AAV-IL10 reversed both bilateral allodynia and
ipsilateral thermal hyperalgesia induced by CCI. ANOVA revealed for
the von Frey test reliable main effects of CCI (F.sub.1,42=220.489,
p<0.0001), AAV-IL10 (F.sub.1,42=38.931, p<0.0001), laterality
(F.sub.1,42=86.812, p<0.0001). ANOVA revealed for the Hargreaves
test reliable main effects of CCI (F.sub.1,42=43.169, p<0.0001),
AAV-IL10 (F.sub.1,42=14.740, p<0.001) and laterality
(F.sub.1,42=31.609, p<0.0001) and interactions between CCI and
laterality (F.sub.1,42=18.402, p<0.0001) and AAV-IL10 and
laterality (F.sub.1,42=6.494, p<0.01) and time after intrathecal
adeno-associated virus, CCI and IL10 (F.sub.3,114=5.534,
p<0.05).
EXAMPLE 9
Reversal of Chronic Constriction Injury (CCI) Neuropathic Pain with
Intrathecally Injected Plasmid DNA Encoding for IL-I0
[0285] In order to determine whether the effect of IL-10 could be
elicited by delivery using a non-viral vector (NVV), the following
experiment was conducted. 100 .mu.g of plasmid ("naked") DNA (pDNA)
encoding either rat IL-10 or GFP (as a control) was injected
intrathecally 10 days, 15 days (five days after the first
injection), 24 days (nine days after the second injection) and 67
days (43 days after the third injection) later. As shown in FIG.
13, the first injection completely but only briefly reversed
pathological pain in the rats. The second injection, given after
return to baseline, again completely reversed pain, but for a
longer time. The third injection, given after return to baseline,
again completely reversed pain but for an even longer time period.
Remarkably, the fourth injection, given after the allodynia was
fully reestablished for six days (Days 38-43 in FIG. 13), again
completely reversed pain. The control plasmid had no effect in the
CCI or sham operated rats. These results are remarkable. To the
best of the inventors' knowledge, no published report has examined
repeated plasmid injections at such short time intervals. Moreover,
given that equal doses of the control GFP plasmid had no effect on
CCI, the results appear specific for IL-10.
[0286] These data raised the question of what might happen if the
inter-injection interval for successive plasmid administrations
were further shortened. Therefore, 100 .mu.g of pDNA encoding rat
IL-10 was injected intrathecally 10 days after CCI induced
mechanical allodynia (Day 10). This induced full reversal of
allodynia by Day 12 (FIG. 24). A second intrathecal injection of
100 .mu.g of the plasmid was given on Day 13, while CCI
pain-enhancement remained fully reversed, as opposed to the
experiment shown in FIG. 13 and described above where the second
plasmid injection was given after allodynia was allowed to reoccur.
As shown in FIG. 24, when the second plasmid injection was
delivered while CCI pain-enhancement remained fully reversed, the
effectiveness of the second injection was remarkably enhanced.
[0287] As a further control, the IL-10 and GFP control plasmids
were enzymatically cut to linearize them. Linearized plasmids are
known to be far more susceptible to enzymatic degradation and show
little to no activity. As expected, an equal dose of linearized
plasmid had no effect on CCI (FIG. 25).
EXAMPLE 10
Effects of Ad-IL10 on Morphine Analgesia, Morphine Tolerance and
Exaggerated Pain Accompanying Cessation of Chronic Opiates
[0288] Morphine tolerance and pathological pain have many features
in common, leading to the concept that both have common biological
underpinnings. Thus, the ability of gene therapy to induce an
anti-inflammatory cytokine might impact this phenomenon as well.
Rats were injected intrathecally with either AD-IL10 or AD-Control
at 5 days prior to the beginning of morphine challenge. They were
behaviorally tested prior to and after intrathecal morphine (10
.mu.g) vs. saline across days. On days 1, 3 and 5, the rats were
tested for tactile sensitivity (von Frey test) and thermal pain
sensitivity (Tail flick test). After morphine, testing followed a 6
hr timecourse.
[0289] As seen in FIG. 14, IL-10 expression in spinal cord caused
even the first dose of morphine to have a more prolonged analgesic
(i.e., pain suppressive) effect as IL-10-expressing rats had longer
tailflick latencies than did controls 100-240 min later.
[0290] As seen in FIGS. 15 and 16, IL-10 expression in spinal cord
caused a delay in the development of morphine tolerance as
IL-10-expressing rats showed greater morphine analgesia than did
control rats.
[0291] As seen in FIG. 17, repeated morphine administration caused
a decrease in pain threshold (increased in pain responsivity) in
animals administered AD-Control (labeled Vehicle on the figure).
This is a classic effect of chronic morphine, wherein abstinence
from the opiate causes exaggerated pain responses. Here, it was
recorded immediately prior to the daily dose of morphine, thus 24
hr after the last dose of morphine. AD-IL10 prevented this increase
in pain sensitivity.
EXAMPLE 11
Effects of IL-1ra on Morphine Analgesia, Morphine Tolerance and
Exaggerated Pain Accompanying Cessation of Chronic Opiates
[0292] A. To test for generality, the ability of an antagonist of
proinflammatory cytokines to exert parallel effects as IL-10 was
tested. Antagonists of proinflammatory cytokines are known to block
and reverse various pathological pain states. Here, rats were
injected intrathecally with either IL-1ra (interleukin-1 receptor
antagonist) or vehicle daily, along with daily morphine or vehicle.
They were behaviorally tested prior to and after these daily
intrathecal injections. On days 1, 3 and 5, the rats were tested
for tactile sensitivity (von Frey test) and thermal pain
sensitivity (Tail flick test). After morphine, testing followed a 6
hr timecourse.
[0293] As seen in FIG. 18, IL-1ra injected into the cerebrospinal
fluid surrounding spinal cord caused even the first dose of
morphine to have a more prolonged analgesic (i.e., pain
suppressive) effect as IL-1ra-injected rats had longer tailflick
latencies than did controls 100-240 min later. Hence, effects on
morphine and pain were again parallel.
[0294] As seen in FIGS. 19 and 20, IL-1ra injected into spinal
cerebrospinal fluid caused a delay in the development of morphine
tolerance as IL-1ra-injected rats showed greater morphine analgesia
than did control rats.
[0295] As seen in FIG. 21A, repeated morphine administration caused
a decrease in pain threshold (increased in pain responsivity) in
animals administered intrathecal morphine+vehicle (left black bar).
This is a classic effect of chronic morphine, wherein abstinence
from the opiate causes exaggerated pain responses. Here, is it
recorded immediately prior to the daily dose of morphine, thus 24
hr after the last dose of morphine. Daily intrathecal IL-1ra
prevented this increase in pain sensitivity (right black bar).
[0296] B. To test whether the effects observed in Examples 10 and
11A implied that chronic morphine increased the production and
release of proinflammatory cytokines, levels of IL-1 protein were
assayed by ELISA from tissues collected after chronic intrathecal
morphine versus vehicle administration. Hence rats either received
5 days of 10 .mu.g morphine or equivolume of vehicle. Two hours
after the last intrathecal injection (at a time when chronic
morphine-induced mechanical allodynia and thermal hyperalgesia
occur), rats were overdosed with sodium pentobarbital and
lumbosacral CSF and dorsal spinal cord were collected. Samples were
immediately flash-frozen in liquid nitrogen and stored at -80C
until assayed by ELISA.
[0297] As seen in FIGS. 21B and 21C, chronic morphine treatment
increased expression of IL-1 protein in both spinal cord CSF (FIG.
21B) and in dorsal spinal cord tissue (FIG. 21C). The increase in
CSF levels is important as it shows that IL-1 was not simply
produced but rather was also released, thus enabling it to exert
effects on neurons and other glia.
EXAMPLE 12
Effects of IL-1ra on CCI Induced Mechanical Allodynia
[0298] The data presented to this point suggested that IL-10 might
be blocking/reversing pathological pain states because it was
suppressing proinflammatory cytokines. To test whether CCI was in
fact mediated by proinflammatory cytokines, the following
experiment was performed. Rats were first assessed for baseline
(BL) responsivity on the von Frey test and then subjected to either
CCI or sham surgery. Behaviors were reassessed 3 and 10 days later
to verify surgical efficacy. One group of rats was then immediately
administered either 100 .mu.g IL-1ra or equivolume (1 .mu.l)
vehicle intrathecally, then monitored for behaviors on the von Frey
test for several hours (FIG. 30A). The second group of rats was
treated identically save that these intrathecal injections occurred
2 months after surgery (FIG. 30B). As shown in FIGS. 30A and 30B,
in both cases, the proinflammatory cytokine antagonist transiently
reversed mechanical allodynia in CCI treated animals, while having
no effect on sham operated controls. These data support that
proinflammatory cytokines are key players in both creating and
maintaining pathological pain states over extended periods of
time.
EXAMPLE 13
Effect of Injected IL-10 on Chronic Constriction Injury (CCI)
Induced Mechanical Allodynia
[0299] The previous examples illustrate the therapeutic efficacy of
delivering viral and non-viral vectors encoding IL-10 in order to
treat pain. In order to compare the effect of injected IL-10
protein versus gene therapy using DNA encoding IL-10, the following
experiment was conducted. Recombinant rat IL-10 protein (Sigma
Chemical Co., St. Louis, Mo.; lot # 101K0290) was reconstituted in
sterile Dulbecco's PBS containing 0.1% rat serum albumen at a stock
concentration of 0.1 mg/mL, aliquoted in sterile eppendorf tubes
and stored at -80.degree. C. until the time of injection. Animals
received three injections of rat IL-10 protein. At the time of the
first injection, stock IL-10 protein was thawed on ice and diluted
with Dulbecco's PBS containing 0.1% rat serum albumen to a final
concentration of 0.01 mg/mL. The dose of the first injection was 50
ng in 50 .mu.l. The second and third injections of rat IL-10
protein were higher (500 ng in 5 .mu.l), thus stock solution of
IL-10 protein was thawed on ice immediately followed by an i.t.
injection. Control animals received equivolume vehicle (sterile
Dulbecco's PBS containing 5% bovine serum albumen and 0.1% rat
serum albumen) injections.
[0300] Rats were assessed for their BL responses to the von Frey
test and Hargreaves test prior to and again on Days 3 and 10 after
CCI or sham surgery. In this experiment, three separate, temporally
spaced i.t. injections were administered starting day 10 after the
induction of CCI. Behaviors on the von Frey test and Hargreaves
test were assessed at 1 and/or 2 hr and 24 hr after each injection.
The dose of rat recombinant IL-10 protein was 50 ng in 5 .mu.l for
the first i.t. injection and 500 ng in 5 .mu.l for the second and
third injection. The higher dose for the second and third injection
was to ensure that maximal effects of the IL-10 protein on both
behavioral tests could be observed.
[0301] All groups showed similar BL values (F.sub.7,26=0.510,
p>0.8) for the von Frey test prior to CCI surgery. As observed
in previous experiments and shown in FIGS. 22 and 23, CCI again
produced chronic bilateral mechanical allodynia and chronic
ipsilateral thermal hyperalgesia. For behavioral assessments at
Days 3 and 10, prior to rat recombinant IL-10 intrathecal
administration, ANOVA for the von Frey test revealed reliable main
effects of CCI (F.sub.1,26=1102.390, p<0.0001).
[0302] In addition, prior to CCI surgery, all groups showed no
behavioral BL differences for the Hargreaves test
(F.sub.7,26=0.324, p>0.9). Before rat recombinant IL-10
intrathecal administration, ANOVA for the Hargreaves test revealed
reliable main effects of CCI (F.sub.1,26=94.228, p<0.0001) and
laterality (F.sub.1,26=37.784, p<0.0001), and an interaction
between CCI and laterality (F.sub.1,26=42.128, p<0.0001).
[0303] After the first intrathecal rat recombinant IL-10
administration, rat recombinant IL-10 reversed these ongoing
pathological pain states. The lower dose of rat recombinant IL-10
(only for the first injection; 50 ng) reversed onlybilateral
allodynia, but not ipsilateral thermal hyperalgesia induced by CCI.
ANOVA revealed for the von Frey test reliable main effects of CCI
(F.sub.1,26=913.411, p<0.0001), rat recombinant IL-10
(F.sub.1,26=26.744, p<0.0001) and time after rat recombinant
IL-10 (F.sub.1,26=11.538, p<0.0001), and interactions between
CCI and rat recombinant IL-10 (F.sub.1,26=17.755, p<0.001), time
and CCI (F.sub.1,26=48.915, p<0.0001), time and rat recombinant
IL-10 (F.sub.1,26=17.344, p<0.001), time, CCI and rat
recombinant IL-10 (F.sub.1,26=23.563, p<0.0001).
[0304] ANOVA revealed for the Hargreaves test reliable main effects
of CCI (F.sub.1,26=28.492, p<0.0001) and laterality
(F.sub.1,26=25.603, p<0.0001) and an interaction between CCI and
laterality (F.sub.1,26=34.857, p<0.0001). There was no reliable
main effect of rat recombinant IL-10 at this lower dose on the
Hargreaves test.
[0305] After the second intrathecal rat recombinant IL-10
administration, which was given at the higher dose of 500 ng, rat
recombinant IL-10 reversed both bilateral allodynia and ipsilateral
thermal hyperalgesia induced by CCI. ANOVA revealed for the von
Frey test reliable main effects of CCI (F.sub.1,26=450.175,
p<0.0001), rat recombinant IL-10 (F.sub.1,26=51.815,
p<0.0001) and time after rat recombinant IL-10
(F.sub.2,52=31.983, p<0.0001), and interactions between CCI and
rat recombinant IL-10 (F.sub.1,26=60.758, p<0.0001), time and
CCI (F.sub.2,26=38.202, p<0.0001), time and rat recombinant
IL-10 (F.sub.2,26=39.030, p<0.001), and time, CCI and rat
recombinant IL-10 (F.sub.2,26=44.300, p<0.0001).
[0306] ANOVA revealed for the Hargreaves test reliable main effects
of CCI (F.sub.1,26=15.957, p<0.001), rat recombinant IL-10
(F.sub.1,26=11.337, p<0.005), and laterality (F.sub.1,26=25.278,
p<0.0001) and interactions between CCI and laterality
(F.sub.1,26=27.133, p<0.0001) and time, rat recombinant IL-10
and laterality (F.sub.2,52=2.239, p<0.05).
[0307] After the third intrathecal rat recombinant IL-10 injection,
which was also given at the higher dose of 500 ng, IL-10 again
reversed both bilateral allodynia and ipsilateral thermal
hyperalgesia induced by CCI. ANOVA revealed for the von Frey test
reliable main effects of CCI (F.sub.1,26=1130.649, p<0.0001),
rat recombinant IL-10 (F.sub.1,26=38.190, p<0.0001) and time
after rat recombinant IL-10 (F.sub.4,104=32.709, p<0.0001), and
interactions between CCI and rat recombinant IL-10
(F.sub.1,26=45.951, p<0.0001), time and CCI (F.sub.4,104=81.860,
p<0.0001), time and rat recombinant IL-10 (F.sub.4,104=37.044,
p<0.001), and time, CCI and rat recombinant IL-10
(F.sub.4,104=34.969, p<0.0001). Rats remained fully allodynic
from 24 through 72 hrs after the third injection. ANOVA revealed a
main effect of only CCI (F.sub.1,26=1506.028, p<0.0001). All
other comparisons were not reliable (p>0.10).
[0308] ANOVA revealed for the Hargreaves test reliable main effects
of CCI (F.sub.1,26=293.036, p<0.0001), and laterality
(F.sub.1,26=47.126, p<0.0001) and interactions between CCI and
laterality (F.sub.1,26=56.134, p<0.0001) and time, rat
recombinant IL-10 and (F.sub.4,104=3.396, p<0.05). Rats remained
fully allodynic from 24 through 72 hrs after the third injection.
ANOVA revealed a main effect of CCI (F.sub.1,26=37.706,
p<0.0001), laterality (F.sub.1,26=44.118, p<0.0001) and an
interaction between CCI and laterality (F.sub.1,26=72.034,
p<0.0001). All other comparisons were not reliable
(P>0.15).
EXAMPLE 14
Effect of Injected IL-10 on PLA2 Induced Mechanical Allodynia
[0309] Mechanical allodynia was induced in rats by peri-sciatic
injection of phospholipase A2 (PLA2), an inflammatory mediator
released by activated immune cells. Allodynia induced by
peri-sciatic injection of PLA2 is mediated by spinal
proinflammatory cytokines (Chacur et al., Pain (2001) 94:231-244).
10 ng of IL-10 was administered to rats intrathecally, followed by
PLA2 induced allodynia. As shown in FIG. 26, intrathecal
administration of 10 ng of IL-10 blocked development of allodynia,
at least for five hours.
EXAMPLE 15
Effect of Fc-IL10 on Chronic Constriction Injury (CCI) Induced
Mechanical Allodynia
[0310] Examples 13 and 14 showed the ability of IL-10 protein to
reverse enhanced pain states. Here, the efficacy of a stabilized
variant of IL-10 (FcIL-10) was examined to test whether it too
exerted such effects. Rats were first tested for baseline (BL)
responses on the von Frey test. All rats then underwent CCI
surgery. Behaviors were reassessed at Days 3 and 10 to verify that
CCI surgery did produce mechanical allodynia (FIG. 27). Rats were
injected i.t. with 250 ng FcIL-10 (a non-lytic recombinant human
IL-10/Fc chimera, Sigma Chemical Co., St. Louis, Mo., product
number I9404) plus a plasmid encoding for IL-10. Since plasmid has
no effect on behavior until one day later, effects observed shortly
after this injection procedure reflect actions by FcIL-10 itself.
As can be seen in FIG. 27, mechanical allodynia was transiently
reversed by FcIL-10 treatment.
EXAMPLE 16
FcIL-10 Enhances the Effectiveness of Gene Therapy
[0311] The present experiment illustrates the therapeutic efficacy
of IL-10 delivered closely in time with a gene therapy vector.
After baseline (BL) testing, rats received CCI surgery. They were
re-tested 3 and 10 days later to verify that CCI induced profound
neuropathic pain (FIG. 28). After the Day 10 test, rats were
injected i.t. with a control plasmid that did not encode IL-10;
rather, it encoded for an inert intracellular protein (GFP). It can
be seen that the presence of inert plasmid DNA did not affect
behaviors tested the subsequent days. After the Day 13 test, rats
were injected with either: (a) only plasmid encoding for IL-10 or
(b) an equal amount of plasmid encoding for IL-10 plus a stabilized
variant of IL-10 (FcIL-10) to test whether the presence of FcIL-10
would enhance vector efficacy. Indeed it did. Mechanical allodynia
was reversed by plasmid-IL10 alone for approximately 4 days (see,
the effect of the first injection of plasmid-IL10 shown in FIG.
13). In contrast, the co-treatment with FcIL-10 remarkably enhanced
both the onset and duration of plasmid-IL10 efficacy on mechanical
allodynia.
EXAMPLE 17
Effectiveness of Lower Doses and Dose Combinations of Plasmid IL10
Gene Therapy
[0312] After baseline (BL) testing, rats received CCI surgery. They
were re-tested 3 and 10 days later to verify that CCI induced
profound neuropathic pain on the von Frey test (mechanical
allodynia). Rats were then injected with either: (a) 100 .mu.g
plasmid encoding IL-10 (Day 10) followed by 50 .mu.g plasmid
encoding IL-10 (Day 13) (FIG. 29A); (b) 100 .mu.g plasmid encoding
IL-10 (Day 10) followed by 25 .mu.g plasmid encoding IL-10 (Day 13)
(FIG. 29B); or (c) 50 .mu.g plasmid encoding IL-10 (Day 10)
followed by 50 .mu.g plasmid encoding IL-10 (Day 13) (FIG. 29C). As
shown in the figures, each led to reversal of mechanical allodynia
over time.
[0313] Thus, methods for delivering anti-inflammatory cytokines to
the CNS for the treatment of pathological pain are described.
Although preferred embodiments of the subject invention have been
described in some detail, it is understood that obvious variations
can be made without departing from the spirit and the scope of the
invention as defined herein.
Sequence CWU 1
1
5 1 160 PRT Homo sapiens 1 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85
90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys
Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Met Lys Ile Arg Asn 145 150 155 160 2 160 PRT Artificial
mouse IL-10 (mIL-10) 2 Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn
Cys Thr His Phe Pro 1 5 10 15 Val Gly Gln Ser His Met Leu Leu Glu
Leu Arg Thr Ala Phe Ser Gln 20 25 30 Val Lys Thr Phe Phe Gln Thr
Lys Asp Gln Leu Asp Asn Ile Leu Leu 35 40 45 Thr Asp Ser Leu Met
Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu
Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala 65 70 75 80 Glu
Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu 85 90
95 Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser
Asp Phe 115 120 125 Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met
Asn Glu Phe Asp 130 135 140 Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met
Met Ile Lys Met Lys Ser 145 150 155 160 3 145 PRT Artificial viral
form of IL-10 (vIL-10) 3 Gln Cys Asp Asn Phe Pro Gln Met Leu Arg
Asp Leu Arg Asp Ala Phe 1 5 10 15 Ser Arg Val Lys Thr Phe Phe Gln
Thr Lys Asp Glu Val Asp Asn Leu 20 25 30 Leu Leu Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 35 40 45 Gln Ala Leu Ser
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 50 55 60 Gln Ala
Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser Leu 65 70 75 80
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 85
90 95 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys
Asn 100 105 110 Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
Met Ser Glu 115 120 125 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Ile Lys Ala 130 135 140 Arg 145 4 9 PRT Artificial IL-10
fragment 4 Ala Tyr Met Thr Met Lys Ile Arg Asn 1 5 5 9 PRT
Artificial IL-10 variants 5 Xaa Xaa Xaa Thr Xaa Lys Xaa Arg Xaa 1
5
* * * * *