U.S. patent application number 14/258895 was filed with the patent office on 2014-09-11 for peripherally delivered glutamic acid decarboxylase gene therapy for spinal cord injury pain.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is The US Government as represented by the Department of Veterans Affairs, University of Pittsburgh - Of the Commonwealth System of Higher Education, The US Government as represented by the Department of Veterans Affairs. Invention is credited to David J. Fink, Joseph C. Glorioso, David Krisky, Darren Wolfe.
Application Number | 20140256801 14/258895 |
Document ID | / |
Family ID | 36319715 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256801 |
Kind Code |
A1 |
Glorioso; Joseph C. ; et
al. |
September 11, 2014 |
PERIPHERALLY DELIVERED GLUTAMIC ACID DECARBOXYLASE GENE THERAPY FOR
SPINAL CORD INJURY PAIN
Abstract
The invention provides a method of treating spinal cord injury
pain or peripheral neuropathic pain in a mammal comprising
administering to a mammal a vector comprising a nucleotide sequence
encoding a glutamic acid decarboxylase (GAD) protein in an amount
effective to treat spinal cord injury pain or peripheral
neuropathic pain.
Inventors: |
Glorioso; Joseph C.;
(Pittsburgh, PA) ; Fink; David J.; (Ann Arbor,
MI) ; Wolfe; Darren; (Pittsburgh, PA) ;
Krisky; David; (Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education
The US Government as represented by the Department of Veterans
Affairs |
Pittsburgh
Washington |
PA
DC |
US
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
The US Government as represented by the Department of Veterans
Affairs
Washington
DC
|
Family ID: |
36319715 |
Appl. No.: |
14/258895 |
Filed: |
April 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11261389 |
Oct 28, 2005 |
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14258895 |
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60622889 |
Oct 28, 2004 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 25/04 20180101;
C12N 2710/16643 20130101; C12Y 401/01015 20130101; C12N 15/86
20130101; C12N 2840/20 20130101; C12N 9/88 20130101 |
Class at
Publication: |
514/44.R |
International
Class: |
C12N 15/86 20060101
C12N015/86 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0001] This invention was made in part with Government support
under Grant Numbers NS044507, NS38850 and NS43247, awarded by the
United States National Institute of Neurological Disorders and
Stroke and a Research Grant from the Department of Veterans
Affairs. The Government may have certain rights in this invention.
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. A method of treating spinal cord injury pain or peripheral
neuropathic pain in a mammal comprising: administering to a mammal
experiencing spinal cord injury pain or peripheral neuropathic pain
a vector comprising a nucleotide sequence encoding a glutamic acid
decarboxylase (GAD) protein in an amount effective to treat spinal
cord injury pain or peripheral neuropathic pain.
14. The method of claim 13, wherein the vector is a viral
vector.
15. (canceled)
16. (canceled)
17. The method of claim 13, wherein the vector is a non-viral
vector.
18. The method of claim 13, wherein the GAD protein is GAD67.
19. The method of claim 13, wherein the nucleotide sequence
encoding the GAD protein is operably linked to a promoter.
20. The method of claim 19, wherein the promoter is a human
cytomegalovirus immediate early promoter (HCMV IEp).
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. The method of claim 13, wherein the vector is peripherally
administered.
27. The method of claim 26, wherein the vector is administered to
an appendage.
28. The method of claim 27, wherein the appendage is below a level
of spinal cord injury.
29. The method of claim 13, wherein the mammal is a human.
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. The method of claim 13, wherein the GAD protein is GAD25.
40. The method of claim 13, wherein the GAD protein is GAD65.
41. The method of claim 14, wherein the viral vector is a
retroviral vector.
42. The method of claim 14, wherein the viral vector is a
parvovirus based vector.
43. The method of claim 14, wherein the viral vector is an
adeno-associated virus (AAV) based vector.
44. The method of claim 14, wherein the viral vector is an
adenovirus-based vector.
45. The method of claim 14, wherein the viral vector is a chimeric
viral vector.
46. The method of claim 14, wherein the viral vector is a herpes
virus based vector.
47. The method of claim 46, wherein the herpes virus based vector
is a non-amplicon herpes simplex virus (HSV) based vector.
48. The method of claim 47, wherein the vector is deficient for at
least one essential HSV gene.
49. The method of claim 48, wherein the essential HSV gene is an
early, immediate-early, or late HSV gene.
50. The method of claim 49, wherein the vector is deficient for an
immediate early gene is selected from the group consisting of:
ICP4, ICP22, ICP27, ICP47, and a combination thereof.
51. The method of claim 47, wherein the vector comprises extended
deletions of ICP4, ICP27, and the deletion of UL55.
52. The method of claim 47, wherein the vector is replication
deficient.
Description
FIELD OF THE INVENTION
[0002] This invention pertains to a method and composition for
treating pain.
BACKGROUND OF THE INVENTION
[0003] Pain following spinal cord injury is an important problem
that adversely impacts quality of life and impedes effective
rehabilitation. Almost every patient that suffers from a spinal
cord injury (SCI) suffers from SCI pain. SCI pain can be at the
level of injury or below the level of injury. With each person, the
pain varies in intensity, frequency, duration of episodes, and the
type of pain experienced. The type of pain experienced has been
described by patients as a tingling, numbness, aching, throbbing,
burning, or squeezing. It is often described in specific body
regions and occurring on either side of the body or both. It may be
constant or intermittent, and is unrelated to body position or
activity.
[0004] Chronic SCI pain may begin at the time of spinal cord injury
or develop slowly over months or years after SCI. Chronic SCI pain
persists for long periods of time and often does not respond well
to conventional pain treatment. According to some reports, as many
as 90% of persons with SCI have also experienced chronic SCI pain.
The level of SCI pain can range from a mere annoyance to being
unbearable for the patient. Two categories of chronic pain after
SCI have been described. At-level neuropathic SCI pain refers to
dermatomes near the spinal cord injury site. Below-level
neuropathic SCI pain refers to dermatomes below the level of the
spinal cord injury site. The former is often present during the
early stages of spinal cord injury and resolves over time, while
the latter develops gradually and is generally refractory to
medical treatment. Chronic SCI pain following spinal cord injury is
an important problem that adversely impacts quality of life and
impedes effective rehabilitation.
[0005] SCI pain can be categorized as musculoskeletal or
neuropathic pain. Musculoskeletal SCI pain results from overuse of
tissues in the body, such as bones, joints, and muscles. It often
becomes worse with movement and eases with rest.
[0006] Neuropathic SCI pain can be due to the abnormal processing
of normal sensations as pain by the nervous system. For example,
the spinal cord and the brain may interpret otherwise normal
sensations as pain. Neuropathic SCI pain at the level of injury can
be due to damage to the actual nerve roots or to the spinal cord
itself. Doctors and other health care professionals may refer to
this as "segmental deafferentiation" or "girdle zone pain". This
type of neuropathic SCI pain is usually bilateral and follows a
circumferential pattern, for example, from your stomach around to
your back. Like other types of chronic pain, this can develop
during the first few weeks after initial spinal cord injury, or may
develop more slowly over time. Diffuse neuropathic SCI pain below
the level of injury may be due to actual changes that have occurred
to the central nervous system. This SCI neuropathic pain is often
associated with allodynia (pain from something that is not usually
painful, e.g., a light touch) or hyperalgesia (extreme pain caused
by something that normally causes a little pain, e.g., a pin
prick).
[0007] The establishment of animal models that recapitulate
essential features of the clinical condition has allowed for the
identification of neurochemical alterations in injured spinal
cords. In one model, the SCI model, a laboratory rat is subjected
to lateral hemisection of the spinal cord at T13. The lateral
hemisection of the laboratory rat's spinal cord at T13 produces
bilateral pain-related behavior below the hemisection in both hind
limbs. The SCI model provides a unique animal model to test novel
treatments for SCI pain.
[0008] Hypofunction of .gamma.-amino butyric acid (GABA)-ergic
inhibitory mechanisms and increases in calcitonin gene-related
peptide (CGRP) in spinal cords have both been reported after SCI.
GABA-mediated inhibition, acting both presynaptically and
postsynaptically, exerts a tonic modulation of nociceptive
neurotransmission between primary afferents and second-order
spinothalamic tract neurons. The pharmacologic antagonism of spinal
GABA-ergic neurotransmission results in mechanical hypersensitivity
similar to that found in neuropathic SCI pain. GABA agonist drugs
(e.g. baclofen) are approved for treatment of selected neuropathic
pain syndromes, but the ubiquitous distribution of GABA receptors
in the central nervous system results in side effects that impose
severe restrictions on administering baclofen, even when
administered intrathecally.
[0009] It has been previously demonstrated that recombinant herpes
simplex virus (HSV) based vectors delivered by subcutaneous
inoculation can be used to express neurotransmitters in neurons of
selected dorsal root ganglia (DRG). HSV vectors constructed to
express proenkephalin produce a local pain-relieving effect in
rodent models of inflammatory pain, neuropathic pain, and pain
resulting from cancer in bone. SCI pain is generally observed to be
resistant to opioid therapy, but it may respond to intrathecal
baclofen.
[0010] Other studies have demonstrated that adenoassociated virus
or herpes amplicon vectors engineered to contain the gene coding
for GAD can be used to express GABA in transduced cells. However,
the vectors in these studies were injected directly into neuronal
nuclei of the brain but not used in pain models or
experimentation.
[0011] Currently, there are no uniformly successful medical or
surgical treatments for SCI pain. Current SCI pain treatments
include opioids and neuropathic medications. The response rates to
these treatments are often limited by side effects. Unfortunately,
neuropathic SCI pain does not usually respond to opioids (e.g.,
morphine). In some cases, SCI pain does not respond to neuropathic
medications (e.g., certain anticonvulsants or antidepressants)
either. Some physicians recommend nerve root blockers that can make
the painful area numb. This numbing effect only lasts a short time.
Another approach sometimes recommended is to have a surgical
procedure such as DREZ (dorsal root entry zone), rhizotomy, or
cordotomy. These procedures ultimately raise the level of SCI pain
and may worsen neuropathic SCI pain over the long term.
[0012] Peripheral neuropathic pain is another common and difficult
to treat concomitant of polyneuropathy or structural nerve injury.
Opioids are relatively ineffective, and their use is limited by
side effects. Antidepressants and anticonvulsants have demonstrated
efficacy in randomized controlled trials but provide only 50%
relief in less than half of patients treated. Among the complex
mechanisms underlying neuropathic pain, partial nerve injury
results in a selective loss of GABAergic inhibitory synaptic
currents in spinal cord that contribute to abnormal pain
sensitivity and the phenotypic features of the neuropathic pain
syndrome. GABAergic agents have not been widely used in the
treatment of neuropathic pain because the therapeutic window of
these agents is modest and the dose is limited by side effects.
[0013] Therefore, there exists a need for a therapeutic approach to
treat SCI and neuropathic pain.
BRIEF SUMMARY OF THE INVENTION
[0014] The invention provides a vector, preferably a herpes simplex
virus (HSV) vector, comprising a polynucleotide sequence encoding a
glutamic acid decarboxylase (GAD) protein. The invention also
provides a stock of such vectors and a pharmaceutical composition
comprising such vectors. The invention further provides a method of
treating pain, such as spinal cord injury pain or peripheral
neuropathic pain, in a mammal comprising administering to a mammal
a vector comprising a nucleotide sequence encoding a glutamic acid
decarboxylase (GAD) protein in an amount effective to treat spinal
cord injury pain. These advantages, and additional inventive
features, will be apparent from the following description of the
invention and the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a schematic representation of exemplary vector
constructs that can be used in the invention.
[0016] FIG. 2 is a representation of QHGAD67 transduction by
footpad inoculation increased GAD67 mRNA in lumbar dorsal root
ganglia (DRG). One week after subcutaneous inoculation of 30 .mu.l
of 1.times.10.sup.9 pfu/ml QHGAD67 or Q0ZHG into one hind paw total
RNA was extracted from the pooled L4-L6 DRG (500 ng), amplified by
real-time PCR, and quantitated using GAPDH as a standard. The
amount relative to Q0ZHG-transduced ganglia is represented.
Means.+-.Standard Error Mean (SEM), N=6.
[0017] FIG. 3 represents protein from the dorsal quadrant of lumbar
spinal cord determined by Western blot using 0-actin as an internal
standard and quantitated by relative optical density. Means.+-.SEM,
N=6, *P<0.05. increased GAD67-like immunoreactivity after
transduction with QHGAD67.
[0018] FIG. 4A is a representation of the amount of gamma amino
butyric acid (GABA) released from primary DRG neurons transduced in
vitro at an m.o.i. of 1 increased substantially in QHGAD67-infected
compared to control or Q0ZHG-infected cells. GABA released over 5
min was determined by HPLC as described under Materials and
Methods. The measurement of GABA concentration/well was performed
three times and triplicate samples were used for each condition.
Means.+-.SEM, *P<0.01 vs. Q0HG or vehicle. FIG. 4B is a
representation of the amount of GABA released from nerve terminals
in spinal cord in vivo was determined by HPLC in microdialysate of
dorsal horn. One week after subcutaneous inoculation of 30 .mu.l of
1.times.10.sup.9 pfu/ml QHGAD67 into one hind paw the amount of
GABA (pmol/10 .mu.l fraction of microdialysate) was substantially
increased in QHGAD67-inoculated compared to control animals.
Means.+-.SEM, N=6, *P<0.05.
[0019] FIGS. 5A-D are representations of the mechanical allodynia
and thermal hyperalgesia were significantly reduced by QHGAD67
inoculation. FIGS. 5A and 5B demonstrate that one week after
hemisection there was a decrease in paw withdrawal threshold
(mechanical allodynia), which persisted for 15 weeks as shown in
vehicle-treated animals (x). Inoculation with QHGAD67 produced an
antialiodynic effect reflected in an increased threshold value
(open circle). Seven weeks after initial inoculation the
antialiodnyic effect of QHGAD67 decreased, but reinoculation of
QHGAD67 into the same animals reestablished the antinociceptive
effect (*P<0.05, **P<0.01 vs. Q0ZHG-inoculated, N=6). (A)
Ipsilateral and (B) contralateral to hemisection. FIGS. 5C and 5D
demonstrate that one week after hemisection there was a significant
decrease in paw withdrawal latency (thermal hyperalgesia) and
injection of the vector resulted in a significant increase in paw
withdrawal latency (open circles). By 7 weeks after initial
inoculation both the antihyperalgesic effects of QHGAD67 had
decreased, but reinoculation of QHGAD67 reestablished the
antihyperalgesic effects (*P<0.05, **P<0.01 vs.
Q0ZHG-inoculated, N=6. (C) Ipsilateral and (D) contralateral to
hemisection. Q0ZHG-inoculated animals (open triangles) were
indistinguishable from vehicle-treated controls in all cases
(A-D).
[0020] FIGS. 6A and 6B demonstrate that bicuculline (0.5 .mu.g) or
phaclofen (0.8 .mu.g) administrated intrathecally 3 weeks after
hemisection and 2 weeks after footpad inoculation partially
reversed the (A) antialiodynic and (B) antihyperalgesic effects of
vector inoculation. The dotted line represents the mean threshold
(A) and latency (B) in animals after SCI inoculated with control
vector or vehicle. Means.+-.SEM, N=6, *P<0.05, **P<0.01 vs
vehicle-treated.
[0021] FIG. 7 is a histogram of the relative optical density
measurements of CGRP-like immunoreactivity in dorsal horn. The
relative optical density measurements were taken from a series of
six continuous sections in the L5 segment of each animal. The
dotted line indicates that the density of CGRP-IR in normal spinal
cord is increased substantially both ipsilateral and contralateral
to T13 hemisection in animals inoculated with vehicle or Q0ZHG, and
inoculation with QHGAD67 significantly attenuates this increase.
Means.+-.SEM, n=6, *P<0.051 compared to vehicle or Q0ZHG.
[0022] FIG. 8 depicts SEQ ID NO:1 discussed herein.
[0023] FIG. 9 depicts SEQ ID NO:2 discussed herein.
[0024] FIG. 10 depicts SEQ ID NOs:3-8 discussed herein.
[0025] FIGS. 11(a) and 11(b) depict data demonstrating
Antinociceptive effect of QOGAD67 in neuropathic pain. (A) L5
spinal nerve ligation (SNL) caused a significant decrease in the
threshold to tactile stimulation, which persisted for more than 4
months. Subcutaneous inoculation of QHGAD67 (arrow) produced an
antiallodynic effect reflected in an increase in the mechanical
threshold. Reinoculation of QHGAD67 7 weeks after the initial
inoculation (arrow) reestablished the antiallodynic effect. Results
are expressed as mean.+-.standard error of the mean. (open circles)
QHGAD67; (closed circles) QOZHG; *p<0.05; **p<0.01; n=8
animals per group. (B) L5 SNL also caused a significant thermal
hyperalgesia, which persisted for 6 weeks. Inoculation with QHGAD67
(arrow), but not QOZHG, reversed the thermal hyperalgesia induced
by spinal nerve injury. *p<0.05; **p<0.01 versus
QOZHG-inoculated; n=8 animals per group. The statistical
significances of the differences were determined by analysis of
variance (StatView 5.2; SAS Institute, Cary, N.C.) corrected for
the number of post hoc comparisons using Scheffe's F test.
[0026] FIG. 12 is a histogram depicting data concerning the effect
of QHGAD67 on Fos-LI in dorsal horn. Fos-LI in dorsal horn induced
by 10 minutes of gentle tactile stimulation was markedly increased
in rats inoculated with QOZHG 1 week after spinal nerve ligation
(SNL) and tested 2 weeks later (3 weeks after SNL). This increase
was blocked in rats with SNL that had been inoculated with QHGAD67
1 week after SNL and tested 2 weeks later (3 weeks after SNL), and
it was found in laminae I-VI of dorsal horn. Results are expressed
as mean.+-.standard error of the mean. **p<0.01; n=5 animals per
group. The difference between sham-operated and SNL animals
inoculated with QOZHG was also statistically significant
(p<0.01).
[0027] FIG. 13 graphically depicts data concerning the effect of
QHGAD67 on the phosphorylated extracellular signal-regulated kinase
1 and 2 (p-ERK1/2) expression in dorsal horn. Results are expressed
as mean.+-.standard error of the mean. **p<0.01; ***p<0.001;
n=5 animals per group.
[0028] FIG. 14 depicts the construction of an HSV vector having
extended deletions of the ICP4 and ICP27 loci and a deletion of
UL55.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The invention provides a vector comprising a polynucleotide
sequence encoding a glutamic acid decarboxylase protein. The vector
can be any suitable gene transfer vector. Examples of suitable
vectors include plasmids, liposomes, molecular conjugates (e.g.,
transferrin), and viruses. Preferably, the vector is a viral
vector. Suitable viral vectors include, for example, retroviral
vectors, herpes virus based vectors and parvovirus based vectors
(e.g., adeno-associated virus (AAV) based vectors, AAV-adenoviral
chimeric vectors, and adenovirus-based vectors). One of ordinary
skill in the art has the requisite understanding to determine the
appropriate vector for a particular situation.
[0030] In a preferred embodiment, the vector is a herpesviral based
vector, such as based on HSV. An HSV based viral vector is suitable
for use as a vector to introduce a nucleic acid sequence into
numerous cell types. The mature HSV virion consists of an enveloped
icosahedral capsid with a viral genome consisting of a linear
double-stranded DNA molecule that is 152 kb. In a preferred
embodiment, the HSV based viral vector is deficient in at least one
essential HSV gene. Of course, the vector can alternatively or in
addition be deleted for non-essential genes. Preferably, the HSV
based viral vector that is deficient in at least one essential HSV
gene is replication deficient. Most replication deficient HSV
vectors contain a deletion to remove one or more
intermediate-early, early, or late HSV genes to prevent
replication. For example, the HSV vector may be deficient in an
immediate early gene selected from the group consisting of: ICP 4,
ICP22, ICP27, ICP47, and a combination thereof. Advantages of the
HSV vector are its ability to enter a latent stage that can result
in long-term DNA expression and its large viral DNA genome that can
accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors
are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782,
5,849,572, and 5,804,413, and International Patent Applications WO
91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, which are
incorporated herein by reference. Preferably, the HSV vector is
"multiply deficient," meaning that the HSV vector is deficient in
more than one gene function required for viral replication. The
sequence of HSV is available on the internet at
www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list-
_uids=9629378&d opt=GenBank&term=hsv-1&qty=1, which may
facilitate the generation of desired mutations in designing
vectors.
[0031] The HSV vector can be deficient in replication-essential
gene functions of only the early regions of the HSV genome, only
the immediate-early regions of the HSV genome, only the late
regions of the HSV genome, or both the early and late regions of
the HSV genome. The HSV vector also can have essentially the entire
HSV genome removed, in which case it is preferred that at least
either the viral inverted terminal repeats (ITRs) and one or more
promoters or the viral ITRs and a packaging signal are left intact
(i.e., an HSV amplicon). The larger the region of the HSV genome
that is removed, the larger the piece of exogenous nucleic acid
sequence that can be inserted into the genome. However, it is
preferred that the vector of the present invention be a
non-amplicon HSV vector.
[0032] It should be appreciated that the deletion of different
regions of the HSV vector can alter the immune response of the
mammal. In particular, the deletion of different regions can reduce
the inflammatory response generated by the HSV vector. Furthermore,
the HSV vector's protein coat can be modified so as to decrease the
HSV vector's ability or inability to be recognized by a
neutralizing antibody directed against the wild-type protein
coat.
[0033] The HSV vector, when multiply replication deficient,
preferably includes a spacer element to provide viral growth in a
complementing cell line similar to that achieved by singly
replication deficient HSV vectors. The spacer element can contain
any nucleic acid sequence or sequences which are of the desired
length. The spacer element sequence can be coding or non-coding and
native or non-native with respect to the HSV genome, but does not
restore the replication essential function(s) to the deficient
region. In addition, the inclusion of a spacer element in any or
all of the deficient HSV regions will decrease the capacity of the
HSV vector for large inserts. The production of HSV vectors
involves using standard molecular biological techniques well known
in the art.
[0034] Replication deficient HSV vectors are typically produced in
complementing cell lines that provide gene functions not present in
the replication deficient HSV vectors, but required for viral
propagation, at appropriate levels in order to generate high titers
of viral vector stock. A preferred cell line complements for at
least one and preferably all replication essential gene functions
not present in a replication deficient HSV vector. The cell line
also can complement non-essential genes that, when missing, reduce
growth or replication efficiency (e.g., UL55). The complementing
cell line can complement for a deficiency in at least one
replication essential gene function encoded by the early regions,
immediate-early regions, late regions, viral packaging regions,
virus-associated regions, or combinations thereof, including all
HSV functions (e.g., to enable propagation of HSV amplicons, which
comprise minimal HSV sequences, such as only inverted terminal
repeats and the packaging signal or only ITRs and an HSV promoter).
The cell line preferably is further characterized in that it
contains the complementing genes in a non-overlapping fashion with
the HSV vector, which minimizes, and practically eliminates, the
possibility of the HSV vector genome recombining with the cellular
DNA. Accordingly, the presence of replication competent HSV is
minimized, if not avoided in the vector stock, which, therefore, is
suitable for certain therapeutic purposes, especially gene therapy
purposes. The construction of complementing cell lines involves
standard molecular biology and cell culture techniques well known
in the art.
[0035] When the vector is a replication deficient HSV, the nucleic
acid sequence encoding the protein (e.g., GAD protein) is
preferably located in the locus of an essential HSV gene, most
preferably either the ICP4 or the ICP27 gene locus of the HSV
genome. The insertion of a nucleic acid sequence into the HSV
genome (e.g., the ICP4 or the ICP27 gene locus of the HSV genome)
can be facilitated by known methods, for example, by the
introduction of a unique restriction site at a given position of
the HSV genome.
[0036] A preferred HSV vector for use in the context of the
invention contains expanded ICP4, or ICP27 deletions, and
preferably both. By "expanded" deletions in this context, it is
meant that the preferred vectors have no homologous sequences at
either or both of these loci relative to the complementing cell
line used for their production. Desirably, the virus has no
remaining ICP4 or ICP27 (or both) coding or promoter sequences.
Preferably, the deletion in ICP27 extends as well into the UL55
locus, and desirably both genes are deleted. Thus, a most preferred
virus for use in the invention contains extended deletions in ICP4,
ICP27 and UL 55 such that there is no viral homology to these genes
used in a complementing cell line. Desirably, the vector further
does not include any homologous DNA sequences to that employed in
the complementing cell line (e.g., even using different regulatory
sequences and polyadenylation sequences).
[0037] As noted above, cell lines complementing the function of
genes, particularly essential genes, deleted from an HSV vector are
desirable (and in the case of essential HSV genes, necessary) to
replicate the vector. Thus, to employ a preferred vector that lacks
both ICP4 and ICP2, a cell line engineered to complement both
essential genes should be employed. Moreover, as UL55.sup.- HSV
strains grow poorly, a cell line complementing it is desirable for
use when it is deleted from the vector backbone. Methods for
generating complementing cell lines are known to those of ordinary
skill in the art.
[0038] As noted above, the inventive vector also comprises a
nucleic acid sequence encoding a GAD protein (i.e., one or more
nucleic acid sequences encoding one or more GAD proteins). The
nucleic acid sequence encoding the GAD protein can be obtained from
any source, e.g., isolated from nature, synthetically generated,
isolated from a genetically engineered organism, and the like. An
ordinarily skilled artisan will appreciate that any type of nucleic
acid sequence (e.g., DNA, RNA, and cDNA) that can be inserted into
a vector can be used in connection with the invention.
[0039] The nucleic acid sequence of the inventive vector can encode
a secreted protein, e.g., a protein that is naturally secreted by
the infected cell. Alternatively, the nucleic acid sequence can
encode a protein, such as GAD, that generates a secreted product
(e.g., GABA) or peptide by enzymatic catalysis within the cell.
Alternatively, the nucleic acid sequence can encode a protein that
is not naturally secreted by the cell (i.e., a non-secretable
protein), but which comprises a signal peptide that facilitates
protein secretion. In this manner, for example, the nucleic acid
sequence encodes an endoplasmic reticulum (ER) localization signal
peptide and the non-secretable protein. The ER localization signal
peptide functions to direct DNA, RNA, and/or a protein to the
membrane of the endoplasmic reticulum, wherein a protein is
expressed and targeted for secretion. The ER localization signal
peptide desirably functions to increase the secretion (i.e., the
secretion potential) by a cell of (i) proteins that are not
normally secreted (i.e., secretable) by the cell and/or (ii)
proteins that are normally secreted by a cell, but in low (i.e.,
less than desired) quantities. The ER localization signal peptide
encoded by the polynucleotide can be any suitable ER localization
signal peptide or polypeptide (i.e., protein). For example, the ER
localization signal peptide encoded by the nucleic acid sequence
can be a peptide or polypeptide (i.e., protein) selected from the
group consisting of nerve growth factor (NGF), immunoglobulin (Ig)
(e.g., an Ig .kappa. chain leader sequence), and midkine (MK), or a
portion thereof. Suitable ER localization signal peptides also
include those described in Ladunga, Current Opinions in
Biotechnology, 11, 13-18 (2000).
[0040] Although the nucleic acid sequence can encode any protein,
the protein preferably is a GAD protein or an enkephalin. There are
several isoforms of mammalian GAD encoded by several different
genes, in particular, GAD25, GAD65, and GAD67. GAD65, targeted
principally to membranes and nerve terminals, is regulated by
pyridoxal-5'-phosphate and other cofactors. GAD65 is thought to be
responsible for the packaging of GABA into vesicles in preparation
of synaptic release. Another isoform of mammalian GAD, GAD67, is
predominantly cytosolic and its enzymatic activity appears to be
regulated by protein level. GAD25 is an alternate splicing variant
of GAD67. The vector preferably comprises a nucleic acid sequence
coding for GAD67. The coding sequence of the human GAD67 gene and
the amino acid sequence of the encoded gene product (i.e., the
encoded protein) are publicly available at the National Center for
Biotechnology Information (NCBI) website as GenBank Accession No.
NM.sub.--000817 (SEQ ID NO: 1) and NP.sub.--000808 (SEQ ID NO: 2),
respectively. Similarly, the coding sequence of the human
enkephalin gene and the amino acid sequence of the encoded gene
product (i.e., the encoded protein) are publicly available as
GenBank Accession No. NM.sub.--006211.
[0041] The nucleic acid sequence can encode any variant, homolog,
or functional portion of the aforementioned proteins. A variant of
the protein can include one or more mutations (e.g., point
mutations, deletions, insertions, etc.) from a corresponding
naturally occurring protein. By "naturally occurring" is meant that
the protein can be found in nature and has not been synthetically
modified. Thus, where mutations are introduced in the nucleic acid
sequence encoding the protein, such mutations desirably will effect
a substitution in the encoded protein whereby codons encoding
positively-charged residues (H, K, and R) are substituted with
codons encoding positively-charged residues, codons encoding
negatively-charged residues (D and E) are substituted with codons
encoding negatively-charged residues, codons encoding neutral polar
residues (C, G, N, Q, S, T, and Y) are substituted with codons
encoding neutral polar residues, and codons encoding neutral
non-polar residues (A, F, I, L, M, P, V, and W) are substituted
with codons encoding neutral non-polar residues. In addition, a
homolog of the protein can be any peptide, polypeptide, or portion
thereof, that is more than about 70% identical (preferably more
than about 80% identical, more preferably more than about 90%
identical, and most preferably more than about 95% identical) to
the protein at the amino acid level. The degree of amino acid
identity can be determined using any method known in the art, such
as the BLAST sequence database. A "functional portion" is any
portion of a GAD protein that retains the biological activity of
the naturally occurring, full-length GAD protein at measurable
levels. A functional portion of the GAD protein produced by
expression of the nucleic acid sequence of the vector can be
identified using standard molecular biology and cell culture
techniques, such as assaying the biological activity of the GAD
protein portion in human cells transiently transfected with a
nucleic acid sequence encoding the GAD protein portion.
[0042] The expression of the nucleic acid sequence encoding the
protein is controlled by a suitable expression control sequence
operably linked to the nucleic acid sequence. An "expression
control sequence" is any nucleic acid sequence that promotes,
enhances, or controls expression (typically and preferably
transcription) of another nucleic acid sequence. Suitable
expression control sequences include constitutive promoters,
inducible promoters, repressible promoters, and enhancers. The
nucleic acid sequence encoding the protein in the vector can be
regulated by its endogenous promoter or, preferably, by a
non-native promoter sequence. Examples of suitable non-native
promoters include the human cytomegalovirus (HCMV) promoters, such
as the HCMV immediate-early promoter (HCMV IEp), promoters derived
from human immunodeficiency virus (HIV), such as the HIV long
terminal repeat promoter, the phosphoglycerate kinase (PGK)
promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, the
Lap2 promoter, or the herpes thymidine kinase promoter (Wagner et
al., Proc. Natl. Acad. Sci., 78, 144-145 (1981)), promoters derived
from SV40 or Epstein Barr virus, and the like. In a preferred
embodiment, the promoter is HCMV IEp. The HCMV IEp promoter can be
inserted into the ICP4 locus of the recombinant HSV. Alternatively,
expression of the nucleic acid sequence encoding the protein can be
controlled by a chimeric promoter sequence. A promoter sequence is
"chimeric" if it comprises at least two nucleic acid sequence
portions obtained from, derived from, or based upon at least two
different sources (e.g., two different regions of an organism's
genome, two different organisms, or an organism combined with a
synthetic sequence). Techniques for operably linking sequences
together are well known in the art.
[0043] The promoter can be an inducible promoter, i.e., a promoter
that is up- and/or down-regulated in response to an appropriate
signal. For example, an expression control sequence up-regulated by
a pharmaceutical agent is particularly useful in pain management
applications. For example, the promoter can be a
pharmaceutically-inducible promoter (e.g., responsive to
tetracycline). Examples of such promoters are marketed by Ariad.
The promoter can be introduced into the genome of the vector by
methods known in the art, for example, by the introduction of a
unique restriction site at a given region of the genome.
Alternatively, the promoter can be inserted as part of the
expression cassette comprising the nucleic acid sequence coding for
the protein, such as GAD. In a preferred embodiment, the inducible
promoter is operably linked to the polynucleotide sequence encoding
for the GAD protein.
[0044] Preferably, the nucleic acid sequence encoding the protein
further comprises a transcription-terminating region such as a
polyadenylation sequence located 3' of the region encoding the
protein. Any suitable polyadenylation sequence can be used,
including a synthetic optimized sequence, as well as the
polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma
virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus).
[0045] In addition to the nucleic acid encoding the protein
(including the promoter and transcription-terminating region), the
vector can comprise at least one additional nucleic acid sequence
encoding at least one other gene product, e.g., which itself
performs a prophylactic or therapeutic function, or augments or
enhances a prophylactic or therapeutic potential of the protein.
The gene product encoded by the additional nucleic acid sequence
can be an RNA, peptide, or polypeptide with a desired activity. If
the additional nucleic acid sequence confers a prophylactic or
therapeutic benefit, the nucleic acid sequence can exert its effect
at the level of RNA or protein. Alternatively, the additional
nucleic acid sequence can encode an antisense molecule, a ribozyme,
a protein that affects splicing or 3' processing (e.g.,
polyadenylation), or a protein that affects the level of expression
of another gene within the cell (i.e., where gene expression is
broadly considered to include all steps from initiation of
transcription through production of a process protein), such as by
mediating an altered rate of mRNA accumulation or transport or an
alteration in post-transcriptional regulation. The additional
nucleic acid sequence can encode any one of a variety of gene
products that confers a prophylactic or therapeutic benefit,
depending on the intended end-use of the composition. The
additional nucleic acid sequence also can encode a factor that acts
upon a different target than the protein encoded by the nucleic
acid sequence of the vector, thereby providing multifactorial
treatment. The additional nucleic acid sequence can encode a
chimeric protein for combination therapy. The additional gene
product can be secreted, or remain within the cell in which it is
produced unless or until the cell is lysed. A variety of gene
products can enhance the therapeutic potential of the vector.
[0046] The additional nucleic acid sequence can encode one gene
product or multiple gene products. Alternatively, multiple
additional nucleic acid sequences, each encoding one or more gene
products, can be inserted into the vector. In either case,
expression of the gene product(s) can be separately regulated by
individual expression control sequences, or coordinately regulated
by one common expression control sequence. Alternatively,
expression of the additional nucleic acid(s) can be regulated by
the same expression control sequence that regulates expression of
the protein encoded by the nucleic acid sequence of the vector;
however, any transcription terminating regions present in the
nucleic acid encoding the protein would be eliminated to allow for
transcriptional read-through of the additional nucleic acid
sequence(s). The additional nucleic acid sequence(s) can comprise
any suitable expression control sequence(s) and any suitable
transcription-termination region(s) discussed herein in connection
with expression of the protein produced by expression of the
nucleic acid sequence of the vector.
[0047] After the vector has been created, the vector is purified.
Vector purification to enhance the concentration of the vector in
the composition can be accomplished by any suitable method, such as
by density gradient purification, by chromatography techniques, or
limiting dilution purification. The vector, preferably a
replication deficient HSV vector, is desirably purified from cells
infected with the replication deficient HSV vector using a method
that comprises lysing cells infected with the HSV vector and
collecting a fraction containing the HSV vector.
[0048] The cells can be lysed using any suitable method, such as
exposure to detergents, freeze-thawing, and cell membrane rupture
(e.g., via French press or microfluidization). The cell lysate then
optionally can be clarified to remove large pieces of cell debris
using any suitable method, such as gentle centrifugation,
filtration, or tangential flow filtration (TFF). The clarified cell
lysate then optionally can be treated with an enzyme capable of
digesting DNA and RNA (a "DNase/RNase") to remove any DNA or RNA in
the clarified cell lysate not contained within the vector
particles.
[0049] Generally, the inventive recombinant HSV is most useful when
enough of the virus can be delivered to a cell population to ensure
that the cells are confronted with a predefined number of viruses.
Thus, the present invention provides a stock, preferably a
homogeneous stock, comprising the inventive HSV vector. The
preparation and analysis of HSV stocks is well known in the art.
For example, a viral stock can be manufactured in roller bottles
containing cells transduced with the HSV vector. The viral stock
can then be purified on a continuous nycodenze gradient, and
aliquotted and stored until needed. Viral stocks vary considerably
in titer, depending largely on viral genotype and the protocol and
cell lines used to prepare them. Preferably, such a stock has a
viral titer of at least about 10.sup.5 plaque-forming units (pfu),
such as at least about 10.sup.6 pfu or even more preferably at
least about 10.sup.7 pfu. In still more preferred embodiments, the
titer can be at least about 10.sup.8 pfu, or at least about
10.sup.9 pfu, and high titer stocks of at least about 10.sup.10 pfu
or at least about 10.sup.11 pfu are most preferred.
[0050] The invention additionally provides a composition comprising
the HSV vector and a carrier, preferably a
physiologically-acceptable carrier. The carrier of the composition
can be any suitable carrier for the vector. The carrier typically
will be liquid, but also can be solid, or a combination of liquid
and solid components. The carrier desirably is a pharmaceutically
acceptable (e.g., a physiologically or pharmacologically
acceptable) carrier (e.g., excipient or diluent). Pharmaceutically
acceptable carriers are well known and are readily available. The
choice of carrier will be determined, at least in part, by the
particular vector and the particular method used to administer the
composition. The composition can further comprise any other
suitable components, especially for enhancing the stability of the
composition and/or its end-use. Accordingly, there is a wide
variety of suitable formulations of the composition of the
invention. The following formulations and methods are merely
exemplary and are in no way limiting.
[0051] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of a sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0052] In addition, the composition can comprise additional
therapeutic or biologically-active agents. For example, therapeutic
factors useful in the treatment of a particular indication can be
present. Factors that control inflammation, such as ibuprofen or
steroids, can be part of the composition to reduce swelling and
inflammation associated with in vivo administration of the vector
and physiological distress. Immune system suppressors can be
administered with the composition method to reduce any immune
response to the vector itself or associated with a disorder.
Alternatively, immune enhancers can be included in the composition
to upregulate the body's natural defenses against disease.
[0053] Antibiotics, i.e., microbicides and fungicides, can be
present to reduce the risk of infection associated with gene
transfer procedures and other disorders.
[0054] The invention also provides a method of treating spinal cord
injury pain or peripheral neuropathic pain in a mammal comprising
administering to a mammal a vector or composition of the present
invention comprising a nucleotide sequence encoding a glutamic acid
decarboxylase (GAD) protein in an amount effective to treat spinal
cord injury pain or peripheral neuropathic pain. In a preferred
embodiment, the administered vector is a viral vector. In a
preferred embodiment, the mammal is a human.
[0055] The method of treating spinal cord injury pain or peripheral
neuropathic pain further can comprise the administration (i.e.,
pre-administration, co-administration, and/or post-administration)
of other treatments and/or agents to modify (e.g., enhance) the
effectiveness of the method. The method of the invention can
further comprise the administration of other substances which
locally or systemically alter (i.e., diminish or enhance) the
effect of the composition on a host. For example, substances that
diminish any systemic effect of the protein produced through
expression of the nucleic acid sequence of the vector in a host can
be used to control the level of systemic toxicity in the host.
Likewise, substances that enhance the local effect of the protein
produced through expression of the nucleic acid sequence of the
vector in a host can be used to reduce the level of the protein
required to produce a prophylactic or therapeutic effect in the
host. Such substances include antagonists, for example, soluble
receptors or antibodies directed against the protein produced
through expression of the nucleic acid sequence of the vector, and
agonists of the protein.
[0056] One skilled in the art will appreciate that suitable methods
of administering the inventive vector and composition of the
invention to an animal (especially a human) for therapeutic or
prophylactic purposes, e.g., gene therapy, vaccination, and the
like (see, for example, Rosenfeld et al., Science, 252, 431-434
(1991), Jaffe et al., Clin. Res., 39(2), 302A (1991), Rosenfeld et
al., Clin. Res., 39(2), 311A (1991), Berkner, BioTechniques, 6,
616-629 (1988)), are available, and, although more than one route
can be used to administer the composition, a particular route can
provide a more immediate and more effective reaction than another
route. A preferred route of administration involves transduction of
DRG neurons through peripheral inoculation to release GABA in the
dorsal horn. In many embodiments, this can be accomplished by
delivering the GAD vector by subcutaneous inoculation, which is an
attractive feature of the inventive approach to treat SCI pain or
peripheral neuropathic pain.
[0057] The dose administered to an animal, particularly a human, in
the context of the invention will vary with the particular vector,
the composition containing the vector and the carrier therefor (as
discussed above), the method of administration, and the particular
site and organism being treated. The dose should be sufficient to
effect a desirable response, e.g., therapeutic or prophylactic
response, within a desirable time frame. Thus, the dose of the
vector of the inventive composition typically will be about
1.times.10.sup.5 or more particle units (e.g., about
1.times.10.sup.6 or more particle units, about 1.times.10.sup.7 or
more particle units, 1.times.10.sup.8 or more particle units,
1.times.10.sup.9 or more particle units, 1.times.10.sup.10 or more
particle units, 1.times.10.sup.11 or more particle units, or about
1.times.10.sup.12 or more particle units). The dose of the vector
typically will not be 1.times.10.sup.13 or less particle units
(e.g., 4.times.10.sup.12 or less particle units, 1.times.10.sup.12
or less particle units, 1.times.10.sup.11 or less particle units,
or even 1.times.10.sup.10 or less particle units).
[0058] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope. In these examples, several measurements were recorded and
statistically analyzed. The statistical significance of the
difference between vector treated and control animals was
determined using multivariate analysis of variance or the
Kruskal-Wallis test for non-parametric measures. Single comparison
was performed with Student's t test, using a P value of <0.05 as
significant. All data are expressed as means.+-.the standard error
of the mean (SEM).
Example 1
[0059] This example demonstrates the construction of the GAD
vector.
[0060] The nonreplicating HSV vector QHGAD67 is defective in
expression of the HSV immediate early (IE) genes ICP4, ICP22,
ICP27, and ICP47, and contains the human GAD67 gene under the
control of the human cytomegalovirus immediate early promoter (HCMV
IEp) in the U.sub.L41 locus (FIG. 1). Control vector Q0ZHG
(constructed according to the method described in Chen et al. J.
Virol, 74(21), 10132-41 (2000)) is defective in the same genes, but
contains the Escherichia coli lacZ reporter gene in the same
position (FIG. 1).
[0061] GAD67 cDNA (constructed according to the method described in
Bu, D. F., et al., Proc. Natl. Acad. Sci. USA, 89, 2115-2119
(1992)) was individually sub-cloned as a ClaI/Xbal fragment
downstream of the human cytomegalovirus immediate early promoter in
the shuttle plasmid p41H, containing the promoter and adequate HSV
flanking DNA sequence in order to enable efficient homologous
recombination at the U.sub.L41 gene locus of the vector. The
expression/targeting cassette was recombined into the U.sub.L41
locus of vector Q0ZHG by cotransfection of complementing 7b cells
with PmeI-digested viral and targeting plasmid DNA to replace the
LacZ marker gene with the GAD expression construct. The recombinant
QHGAD67 was purified by three rounds of limiting dilution
purification and the genetic structure confirmed by Southern blot.
Vector stocks were produced in 7b cells in roller bottles, purified
on a continuous nycodenze gradient, and aliquotted and stored at
-80.degree. C. until thawed for use. The titer of the final vector
product was determined as described in Krisky, D., et al., In
Methods in Molecular Medicine, Human Press, Totowa, N.J.
(1996))
Example 2
[0062] This example demonstrates the construction of an HSV vector
having extended deletions of the ICP4 and ICP27 loci and a deletion
of UL55.
[0063] The schematic for constructing this vector is set forth in
FIG. 14. Specifically, plasmid d106 (Hadjipanayis and DeLuca, Can
Res 65(12): 5310-6 (2005)) was virally crossed with plasmid TOZ.1
(Arafat et al., Clin Can Res 6: 4442-8 (2000)) to produce QOZHG.1.
The details of vector QOZHG.1 are described in Example 1 and its
construction described in Chen et al., J Virol 74(21), 10132-41
(2000).
[0064] Plasmid pPXE (Niranjan et al., Mol Ther 8(4):530-42 (2003))
was recombined into the ICP27 locus of QOZHG.1 to rescue the
previous ICP27 deletion and to remove HCMV-eGFP gene. A single
recombinant was then isolated, purified and verified by selecting a
plaque that did not exhibit green fluorescence under a fluorescent
microscope. The recombinant was termed E1. E1 was negative for the
GFP gene and positive for the LacZ gene.
[0065] Plasmid 41HN was produced by cloning the Hind III to Not I
fragment (HSV-1 genomic nucleotides 90145 to 93858) containing the
UL41 coding sequence into the Hind III to Not I sites of pBSSK
(Stratagene). Plasmid 41HN was then recombined into the UL41 locus
of E1 to rescue the wildtype UL41 gene and remove LacZ. The
resulting vector, named E1-1 was isolated, purified, and verified
by standard methods. This vector was negative for both gfp and lacZ
genes.
[0066] Plasmid pSASB3 was constructed by cloning the Sph I to Afl
III (Sal I linkered) fragment (1928 bp) of the HSV-1 KOS strain
genome (nucleotides 124485-126413) into Sph I/Sal I digested pSP72
followed by insertion of a the 695 bp BglII to BamH I fragment
(nucleotides 131931 to 132626) containing regions upstream of the
ICP4 promoter including the viral origin contained within the short
inverted repeat regions into the Bgl II to BamH I sites of the
vector plasmid.
[0067] Plasmid pSASB3gfp was contructed by cloning a HCMV-eGFP
fragment in the BamHI site of pSASB3. Plasmid pSASB3gfp was then
recombined into the ICP4 locus of E1-1 to expand the ICP4 deletion.
The resulting vector, named E1 G6/d106-4HG was then isolated,
purified, and verified by standard methods.
[0068] The plasmid pSASB3-HPPE was created by cloning a EcoR I to
Sal I fragment of the plasmid pCMV-hPPE from Dr. Steven Wilson,
University of South Carolina (Liu F, Housley P R, Wilson S P. [J
Neurochem. 1996 October; 67(4):1457-62.) containing the HCMV
immediate early promoter, SV40 intron (180 bp XhoI-PstI) from the
16 s/19 s RNA of SV40, the entire hPPE coding sequence, and the
SV40 polyadenylation signal (SV40 bases 2533 to 2729 into plasmid
pSASB3 at the unique Sal I site. Sal I release of the hPPE
expression construct was made possible by previous EcoR I digestion
followed by Klenow fragment blunting of the EcoR I site and
ligation with a Sal I linker. The product of this ligation was then
digested with Sal I to purify the Sal I flanked expression
construct. pCMV-hPPE was subcloned from a cDNA clone pUR292 from
Dr. Barbara Spruce as 946 bp BamHI-HindIII fragment from pUR292
that was blunt-ended, NotI linkers added, and cloned into the
unique NotI site of the expression plasmid pCMV.beta..
[0069] The final enkephalin expression/ICP4 targeting construct
(pSASB3-HPPE) contains the following elements; 1) bases 131931 to
132626 of the HSV genome to provide a 5' recombination flanking
sequence targeting the ICP4 locus, 2) the human cytomegalovirus
(HCMV) immediate early promoter (IEp), 3) the SV40 16 s/19 s intron
splice donor and acceptor sites, 4) the preproenkephalin cDNA, 5)
the SV40 late polyadenylation signal, 6) bases 124485 to 126413 of
the HSV genome to provide a 3' recombination flank targeting the
ICP4 locus.
[0070] Plasmid pSASB3-hppe was then recombined into the ICP4 locus
of E1G6/d106-4HG to produce the NurelP1 (NP1) vector, which is
generically known as 6221.
[0071] Plasmid PS-UB6R-6 was created by cloning a Bgl II-BamH I
flanked ubiquitin promoter driven Red2 (Invitrogen) in the BamH I
site of PSP4. The BamH I site of PSP.4 is located in between the 5'
ICP27 flank fragment and the UL56 coding sequence.
[0072] Plasmid PS-UB6R-6 was recombined into the ICP27 locus of NP1
to expand the ICP27 deletion to include all of ICP27 and UL55 and
to insert the UB6-Red gene. The resulting vector, termed HPPE6221R,
was isolated, purified, and verified by selecting red plaques.
[0073] Plasmid PSP4 was created by cloning the EcoR I to BamH I
(HSV-1 genomic 110095 to 113322) sequence 5' to the ICP27 coding
sequence into the plasmid PS.2. PS.2 contains the Dde I to Sma I
(HSV-1 genomic fragment 116156 to 117119) blunt end ligated into
the Not I site of pBSSK (Stratagene).
[0074] Plasmid PSP4 was recombined into the ICP27 locus of
HPPE6221R vector to remove UB6-Red and to leave the ICP27/UL55
deletion. The resulting vector, termed NurelP2 (NP2); was isolated,
purified, and verified by standard methods.
[0075] Plasmid pSASB3GFP was recombined into the ICP4 locus of NP2
to replace HCMV-hppe with HCMV-eGFP. The resulting vector, termed
SAS2, was then isolated, purified, and verified by standard
methods.
[0076] Plasmid pRC2 (Invitrogen) was modified sequentially by
converting the 1) HinDIII site into a ClaI site, 2) the BbvII site
into a HinDIII site, 3) the resulting HinDIII site into a BglII
site to make plasmid pRC2HB2. The BglII fragment of pRC2HB2, about
1200 base pairs, was cloned into the BamHI site of plasmid pSASB3
creating plasmid pSHB3. Separately the GAD67 clone was modified by
converting the HinDIII site into a BamHI site creating pGADHB2. The
resulting 2.8 kb BamHI fragment from pGADHB2 was cloned into the
BamHI site of pSHB3 to make pGADL1.
[0077] Plasmid pGAD-L1 was recombined into the ICP4 locus of SAS2
to produce the vector NurelG2 (NG2).
[0078] The vectors NP2, SAS2 and NG2 are vectors that have no
homology with the cell line, such that no homologous recombination
can take place between the cell line and the vector. Therefore,
these vectors are ideal for using in combination with the cell
ICP4, ICP27, IL55 complementing line for vector production.
Example 3
[0079] This example demonstrates the expression of GAD protein by
GAD vector transduced cells.
[0080] One week after subcutaneous inoculation of QHGAD67 into the
plantar surface of the hind paw of a laboratory rat the amount of
GAD67 mRNA in the pooled L4-L6 DRG detected by real-time RT-PCR was
fivefold greater than in contralateral DRG transduced with Q0ZHG
(FIG. 2). Also, GAD67 immunoreactivity in transduced DRG was
present in neurons in a broad spectrum of DRG neurons of all sizes
compared to the contralateral (vehicle-injected) DRG. GAD67
protein, determined by Western blot, was significantly increased in
both the lumbar DRG (0.048.+-.0.009 OD units) compared to
sham-inoculated controls (0.025.+-.0.006 OD units, P<0.01.
[0081] One week after subcutaneous inoculation of 30 .mu.l of
11.times.10.sup.9 pfu/ml QHGAD67 increased immunoreactivity was
seen predominantly in laminae II and III compared to the
contralateral (vehicle-injected) dorsal horn. In the superficial
dorsal horn of control rats GAD67 immunoreactivity was located
predominantly in small round cells of lamina III with few neuritic
extensions that appeared to be endogenous GABA-ergic interneurons
in the superficial dorsal horn and some smaller, punctuate, densely
staining, irregular profiles that appeared to be varicosities or
small axonal terminals. The intensity of immunostaining was
increased substantially in the dorsal horn containing central
terminals of the axons from DRG transduced with QHGAD67 and the
increased immunoreactivity appeared to be located in the irregular
profiles representing axonal terminals. By Western blot the amount
of GAD in the dorsal spinal cord of the lumbar segments containing
the central terminal of those axons (0.041.+-.0.008 OD units) was
significantly increased compared to the sham-inoculated controls
(0.027.+-.0.004 OD units) (P<0.01, FIG. 3).
[0082] The analysis of GAD RNA by real-time RT-PCR was performed as
follows: L4-6 DRGs were rapidly removed and total RNA extracted
from the pooled L4-6 ganglia using TriReagent (Sigma). After DNase
I digestion, first-strand cDNA was produced using Omniscript
reverse transcriptase (Qiagen, Valencia, Calif., USA). Primers and
probes for GAD67 and GAPDH (Synthegen, Houston, Tex., USA) were
designed using Primer Express (Applied Biosystems, Foster City,
Calif., USA). GAD67 forward primer SEQ ID NO: 3; reverse primer SEQ
ID NO: 4; probe, SEQ ID NO: 5 (Synthegen); and CAPDH forward primer
SEQ ID NO: 6; reverse primer SEQ ID NO: 7; probe SEQ ID NO: 8
(Invitrogen). PCRs were performed in an ABI Prism 7700 sequence
detection system (Applied Biosystems) in a total volume of 50
.mu.l. The amount of RNA was determined using GAPDH as an internal
control and calculated relative to DRG transduced with Q0ZHG. Each
PCR amplification was performed in triplicate wells, using the
following conditions: 2 minutes at 50.degree. C. and 10 minutes at
95.degree. C., followed by 40 cycles of 15 s at 95.degree. C. and 1
minute at 60.degree. C.
[0083] The amount of GAD67 protein was determined by Western blot
according to the following protocol: The L4-L6 DRG or the dorsal
quadrant of the lumbar enlargement (L4 to L6 segments) of spinal
cord was sonicated in homogenization buffer (100 mg tissue/ml)
consisting of 60 mM phosphate buffer, pH 7.4, 1 mM
phenylmethylsulfonyl fluoride and 0.5% TritonX-100. The homogenate
was centrifuged for 15 minutes at 100,000 g, using a TL100
ultracentrifuge ultracentrifuge (Beckman), and the total protein in
the supernatant was measured by Bradford assay (BioRad, Hercules,
Calif., USA). Proteins were separated on a 4-15% SDS gradient
polyacrylamide gel, transferred to nitrocellulose membrane
(Immobilon-P, Millipore, Billerica, Ma, USA), incubated with rabbit
anti-GAD67 (1:4000, Chemicon, Temecula, Calif., USA) followed by
horseradish peroxidase-conjugated goat anti-mouse (1:10,000,
Jackson Laboratories, Bar Harbor, Me., USA), and detected by
enhanced chemiluminescence (NEN, Boston, Mass., USA). The membranes
were stripped and reprobed with rabbit anti-.beta.-actin as a
loading control. The intensity of each band was determined by
quantitative densitometry using a PC-based image analysis system
(MCID, Imaging Research, Brock, Ontario, Canada).
Example 4
[0084] This example demonstrates the increased release of GABA in
GAD vector transduced cells.
[0085] Primary DRG neurons transduced in vitro with QHGAD67 at a
multiplicity of infection (m.o.i.) of 1 released GABA into the
medium (9.53.+-.2.15 pmol/10 .mu.l) in amounts substantially
greater than those released from control (2.34.+-.0.22 pmmol/10
.mu.l, P<0.01) or Q0ZHG-transduced DRG neurons (2.56.+-.0.54
pmol/10 .mu.l, P<0.01) (FIG. 4A). The in vivo amount of GABA
released into the dorsal spinal cord from the central terminals of
DRG transduced by subcutaneous inoculation into the foot 1 week
earlier was determined by microdialysis from a catheter implanted
in the ipsilateral lumbar dorsal horn. GABA contained in the
dialysate collected from animals inoculated with Q0ZHG contained
0.74.+-.0.24 pmol/10 .mu.l compared to the dialysate from animals
inoculated with QHGAD67 which contained 1.46.+-.0.25 pmol/10 .mu.l
(P<0.05) (FIG. 4B).
[0086] Dissociated DRG neurons from 17-day-old rat embryos were
plated on poly-D-lysine-treated coverslips at a density of 10.sup.5
cells/well in a 24-well plate. Each well contained 500 .mu.l
Neurobasal medium containing B27, Glutamax I, Albumax II, and
penicillin/streptomycin (Gibco-BRL, Carlsbad, Calif.), supplemented
with 100 ng/ml of 7.0 S NGF (Sigma, St. Louis, Mo.). At 14 days in
culture, the cells were infected with either QHGAD67 or Q0ZHG at a
m.o.i. of 1 for 1 hour, after which the virus was removed.
Forty-eight hours later the medium was changed to 100 .mu.l of
artificial cerebrospinal fluid and after 5 minutes collection time
the bathing solution was centrifuged 5 minutes at 10,000 g and the
supernatant taken for determination of GABA by HPLC. The DRG cells
were examined for expression of GAD67 protein by
immunocytochemistry.
[0087] The amount of GABA released from nerve terminals in the
dorsal horn in vivo was determined by HPLC of a microdialysate
using the following protocol: Rats were anesthetized with chloral
hydrate (400 mg/kg), the laminae of T11 and T12 vertebrae that
overlie the lumbar segments of the spinal cord were removed,
leaving the dura intact, and the animals were fixed in a
stereotaxic apparatus. A heating lamp was used to prevent heat loss
and the body temperature was kept at 37.5.degree. C. using a
feedback sensor. A small dural incision lateral to midline was made
with a sharp needle, and a microdialysis probe (CMA/11, cuprophane
dialySis membrane, length 1 mm, diameter 0.24 mm, molecular cut-off
6 kDa, CMA/Microdialysis, Stockholm, Sweden) was inserted into the
dorsal horn through the dural incision and perfused with artificial
cerebrospinal fluid (CMA/Microdialysis) at a rate of 1 .mu.l/min.
After 1 h to allow for equilibration with the extracellular fluid,
samples were collected for 1 h. At the end of the experiment, the
probe was checked for the presence of air bubbles and the position
in the dorsal horn of the spinal cord verified by microscopy of
perfusion-fixed sections.
[0088] Next, the amount of GABA released from transduced cells in
vitro, or collected by microdialysis in vivo, was determined using
HPLC with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
derivatization (AccQ.Fluor Reagent Kit; Waters, USA). Twenty
microliters of culture solution was mixed with 20 .mu.l of
derivatizing reagent in 60 .mu.l of borate buffer; 10 .mu.l of the
sample was allowed to react for 10 minutes at 55.degree. C. and
separated by gradient HPLC (Waters 2695 Separations Module) on an
AccQ.Taq column (3.9.times.150 mm; Waters) with a mobile phase
consisting of Eluent A (Waters) and acetonitrile and a flow rate of
1 ml/min. The peaks were detected by fluorescence at 37.degree. C.
(Waters 2475 Detector) using an excitation wavelength of 250 nm and
emission wavelength of 395 nm.
[0089] The intrathecal catheter was surgically implanted in the
laboratory rat according to the following protocol: An intrathecal
catheter was placed 1 week after T13 left spinal hemisection using
a modification of the method of Storkson. Briefly, the animals were
reanesthetized with chloral hydrate (400 mg/kg), a longitudinal
incision was made from L2 to L6 a few millimeters left of the
midline, and a polyethylene catheter (PE-10, Clay Adams,
Parsippany, N.J., USA) was introduced from the L4-L5 intervertebral
space into the lumbar subarachnoid space so that the tip of the
catheter was located near the lumbar enlargement of the spinal
cord. The distal end of the catheter was tunneled subcutaneously to
emerge at the neck, leaving 7 .mu.l of dead space. After
implantation of the intrathecal catheter, the rats were housed in
individual cages and those animals showing evidence of motor
dysfunction were sacrificed. The location of the catheter tip was
confirmed by infusion of 15 .mu.l of lidocaine (20 mg/ml) followed
by 8 .mu.l of saline to produce a motor paralysis lasting for 20-30
minutes. Intrathecal drug administration was performed using a
microinjection syringe (Hamilton Co., Reno, Nev., USA) connected to
the intrathecal catheter in awake, briefly restrained rats.
Example 5
[0090] This example demonstrates that subcutaneous inoculation of a
GAD vector in a laboratory rat reduces mechanical allodynia and
thermal hyperalgesia after SCI.
[0091] One day after T13 left spinal cord hemisection, all animals
showed ipsilateral hind-limb paralysis with no motor dysfunction in
the hind limb contralateral to spinal cord hemisection. Two weeks
after spinal cord hemisection, there was considerable return of
motor function (BBB score of 12-13, data not shown). A BBB score of
12, corresponding to frequent-to-consistent weight-supporting
phantom steps and occasional front leg-hind leg coordination is
sufficient to allow full behavioral testing of
somatosensory-induced paw withdrawal. At one week after spinal cord
hemisection, mechanical allodynia, manifested by a significant
decrease in hind-paw withdrawal threshold to a graded series of von
Frey filament stimuli (1.71.+-.0.35 g ipsilateral, 1.9.+-.0.52 g
contralateral) as compared to the preoperative threshold
(11.2.+-.1.68 g ipsilateral, 11.6.+-.1.71 g contralateral), in both
hind paws was observed. Inoculation of QHGAD67 (1.times.10.sup.9
pfu/ml, 30 .mu.l/paw) subcutaneously in the plantar surface of the
hind paws bilaterally one week after spinal cord hemisection
significantly increased the hind-paw withdrawal threshold
(4.4.+-.1.12 g ipsilateral, 4.1.+-.0.75 g contralateral) measured
one week after inoculation (two weeks after injury). Control
animals inoculated with Q0ZHG showed no change in their mechanical
threshold (1.8.+-.0.28 g ipsilateral, 1.6.+-.0.34 g contralateral,
P<0.01 compared to QHGAD67). The maximal antiallodynic effect
(i.e., increase in paw withdrawal threshold) occurred two weeks
after QHGAD67 inoculation (5.19.+-.0.82 g ipsilateral and
5.8.+-.1.14 g contralateral) and the antiallodynic effect persisted
for five weeks, decreasing to 2.86.+-.0.63 g ipsilateral and
3.2.+-.0.47 g contralateral at six weeks after inoculation (FIGS.
5A and 5B). Reinoculation with the same dose of QHGAD67 into the
footpads bilaterally at six weeks after initial inoculation
reestablished the antiallodynic effect. The magnitude of the effect
obtained by reinoculation was at least as great as that Produced by
the initial injection of the vector, and the duration of the effect
produced by reinoculation was slightly longer (6-7 weeks) than that
which resulted from the initial inoculation. There was no
significant difference in paw withdrawal thresholds between
Q0ZHG-inoculated and vehicle-treated rats at all time points (FIGS.
5A and 5B).
[0092] After spinal cord hemisection, animals also demonstrated
thermal hyperalgesia manifested by a decrease in Withdrawal latency
in response to noxious thermal stimuli (6.7.+-.0.51 s ipsilateral,
6.9.+-.0.6 s contralateral) compared to the preoperative values
(12.6.+-.1.23 s ipsilateral, 12.1.+-.1.25 s contralateral). One
week after inoculation of QHGAD67 (two weeks after spinal cord
hemisection) there was a statistically significant increase in
thermal latency (8.8.+-.0.48 s ipsilateral, 8.7.+-.0.71 s
contralateral) compared to Q0ZHG-inoculated controls (6.5.+-.0.43 s
ipsilateral, 7.1.+-.0.42 sec contralateral). The time courses of
antihyperalgesic effect were similar to those of antiallodynic
effect of the vector (FIGS. 5C and 5D), except that the peak effect
occurred four weeks after inoculation (9.7.+-.0.71 s ipsilateral,
9.62.+-.0.78 s contralateral). Reinoculation of QHGAD67 vector at
six weeks also reestablished the antihyperalgesic effect. The
duration and the magnitude of the antihyperalgesic effect after the
second inoculation was longer and greater than those which followed
the initial inoculation. There was no significant difference
between vehicle-treated and Q0ZHG-inoculated animals in both
hind-paw withdrawal latencies at any time period (FIGS. 5C and
5D).
[0093] For these tests, male Sprague-Dawley rats, weighing 175-200
g were used. Housing conditions and experimental procedures were
approved by the University of Pittsburgh Institutional Animal Care
and Use Committee. With the rat under chloral hydrate anesthesia
(400 mg/kg) the T11-T12 spinal laminae were located by palpating
the last rib (attached to T13). A longitudinal incision was made
exposing several segments, and a laminectomy was performed at two
vertebral segments (T11-T12). The lumbar enlargement was identified
by accompanying dorsal vessels, and the spinal cord was hemisected
at T13 using a No. 11 scalpel blade with care taken not to damage
the major dorsal vessels and vascular branches. A tuberculin
syringe with a 28-gauge needle was placed dorsoventrally at the
midline of the cord and pulled laterally to ensure that the spinal
cord hemisection was complete. Muscle and fascia were sutured
closed, and the skin was closed with autoclips. Following surgery,
animals were maintained under the same preoperative conditions. All
the animals were eating and drinking within 3 hours after surgery.
Locomotor function was observed and recorded using the BBB
Locomotor Rating Scale to ensure that motor recovery of the limb
ipsilateral to the spinal cord hemisection was sufficient to allow
for somatosensory behavioral testing. Animals that demonstrated a
loss of locomotion in both hind limbs, indicating bilateral
corticospinal tract transaction, were excluded from the study at
that time. Animals that met the criteria were then inoculated with
a vector. Six animals in each group were inoculated with a vector
and tested by reinoculation.
[0094] Behavioral testing for mechanical allodynia and thermal
hyperalgesia was performed during the day portion of the circadian
cycle (8:00 AM to 5:00 PM). Mechanical allodynia was assessed by
measuring the threshold of paw-withdrawal response to graded
mechanical stimuli using a series of von Frey filaments (0.4, 0.7,
1.2, 1.5, 2.0, 3.6, 5.5, 8.5, 11.8, and 15.1 g). Rats were placed
in transparent plastic cubicles on a mesh floor for a period of at
least 30 minutes for acclimatization, after which von Frey
filaments were applied to the plantar surface of the foot serially
in ascending order of strength with sufficient force to cause
slight bending against the paw and held for 6 s. A brisk foot
withdrawal to von Frey filament application was regarded as a
positive response and cause to present the next weaker stimulus.
Thermal hyperalgesia was assessed by measuring the latency of paw
withdrawal from a radiant heat source. In this test, the rats were
placed on a glass plate over a light box. After a ten minute
habituation period the plantar surface of the paw was exposed to a
beam of radiant heat applied through the glass floor. The light
beam was turned off automatically by a photocell when the rat
lifted the limb, allowing the measurement of time between the start
of the light beam and the paw withdrawal. This time was defined as
the paw withdrawal time. Testing was always performed in triplicate
at five minute intervals and twenty seconds was used as the cut-off
time.
Example 6
[0095] This example demonstrates that the behavioral effects of GAD
vector inoculation are reversed by bicuculline and phaclofen.
[0096] The pharmacologic basis of the QHGAD67-mediated
antinociceptive effect using the GABA.sub.A receptor-selective
antagonist bicuculline and the GABA.sub.B receptor-selective
antagonist phaclofen was examined. Intrathecal administration of
bicuculline (0.5 .mu.g; Sigma) or phaclofen (0.8 .mu.g; Sigma) to
sham-operated animals two weeks after surgery did not alter the
mechanical threshold or thermal latency. Administration of the same
dose of bicuculline to rats with spinal cord hemisection inoculated
with QHGAD67 reduced the mechanical threshold from 4.87.+-.1.13 g
to 3.5.+-.0.7 g (P<0.05) ipsilateral to the spinal cord
hemisection and from 5.75.+-.1.41 g to 3.38.+-.0.9 g (P<0.01)
contralateral to the spinal cord hemisection (FIG. 6A) measured
10-15 minutes after drug administration. Intrathecal phaclofen
reduced the mechanical threshold to 3.6.+-.0.78 g (P<0.05)
ipsilateral to the spinal cord hemisection and 4.05.+-.0.75 g
(P<0.05) contralateral to the spinal cord hemisection (FIG. 6A).
The thermal withdrawal latency was reduced from 9.28.+-.1.39 s to
7.23.+-.1.21 s (P<0.05) ipsilateral, and from 9.56.+-.1.5 s to
7.41.+-.1.29 s P<0.05) contralateral by administration of
bicuculline and to 7.54.+-.1.16 s (P<0.05) ipsilateral,
7.66.+-.1.24 s (P<0.05) contralateral by administration of
phaclofen (FIG. 6B). In each case the effect of the drug was
measured at the peak effect 30 minutes after drug administration.
One hour after inoculation there was no longer any detectable drug
effect. There were no significant changes in either mechanical
threshold or thermal latency in spinal cord hemisected rats
inoculated with Q0ZHG and treated with bicuculline or phaclofen at
the same doses.
Example 7
[0097] This example demonstrates that transduction of cells with a
GAD vector reduces CGRP immunoreactivity in the dorsal horn.
[0098] In sham-operated animals, CGRP immunoreactivity at the L5
segment was weak, confined largely to laminae I and II within the
superficial dorsal horn of the spinal cord bilaterally. One week
after left T13 hemisection, CGRP immunoreactivity was increased and
staining could be detected extending into laminae III and IV of
dorsal spinal cord bilaterally. In spinal cord hemisected rats 1
week after inoculation of QHGAD67 in both hindpaws, CGRP-like
immunoreactivity was reduced (76.5.+-.13.3 OD unit ipsilateral,
63.6.+-.12.4 OD unit contralateral) compared to spinal cord
hemisected rats inoculated with Q0ZHG (107.3.+-.22.4 OD unit
ipsilateral, 86.+-.23.5 unit contralateral, P<0.0-5) or
vehicle-treated animals (96.7.+-.21.8 OD unit ipsilateral,
104.6.+-.22.6 OD unit contralateral, P<0.05). There was no
significant difference in staining between Q0ZHG-inoculated and
vehicle-treated animals (FIG. 7).
[0099] The distribution of GAD protein in unlesioned transduced
animals and CGRP peptide in lesioned transduced animals was
determined by immunohistochemistry. Rats were perfused
intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer,
the L5 segment of spinal cord and attached roots removed, postfixed
in the same solution for two hours, and cryoprotected with 30%
sucrose in PBS for two days. Twenty-micrometer cryostat sections
were thaw mounted onto cold Superfrost microscope slides (Fisher,
Pittsburgh, Pa., USA) and incubated overnight at 4.degree. C. with
rabbit anti-GAD67 (1:2000, Chemicon) or rabbit anti-CGRP (1:500,
PLI, San Carlos, Calif., USA) followed by fluorescent anti-rabbit
IgG (Alexa Fluor 594, 1:500, Molecular Probes, Eugene, Oreg., USA)
for two hours at room temperature. Fluorescent images were captured
by confocal microscopy (Diagnostic Instruments, Sterling Heights,
Mich., USA).
Example 8
[0100] This example demonstrates that transfer of the gene encoding
GAD to dorsal root ganglion using a herpes simplex virus vector
attenuates peripheral neuropathic pain.
[0101] Male Sprague-Dawley rats weighing 225 to 250 gm underwent
selective L5 SNL (as described in Hao et al., Pain; 102:135-42
(2003)). One week after SNL, 30 .mu.l of vector (either QHGAD67 or
QOZHG, 4.times.10.sup.8 plaque-forming units per milliliter) was
injected subcutaneously in the plantar surface of the left hind
paw, ipsilateral to the ligation. Mechanical allodynia induced by
SNL was determined by assessing the response of paw withdrawal to
von Frey hairs of graded tensile strength (see Hao et al., supra,
and Chaplan et al., J Neurosci Methods, 53:55-63 (1994)) with a
tactile stimulus producing a 50% likelihood of withdrawal
determined using the up-down method (see Dixon et al., Annu Rev
Pharmacol Toxicol; 20:441-62 (1980)) Thermal hyperalgesia was
determined using a Hargreaves apparatus (as described in Hargreaves
et al., Pain; 32:77-88 (1988)) recording the time to withdrawal
from a radiant thermal stimulus positioned directly under the hind
paw.
[0102] After L5 SNL, rats displayed a significant decrease in the
magnitude of the mechanical stimulus necessary to evoke a brisk
withdrawal response to von Frey hair stimulation (FIG. 11A) and a
significant reduction in la-tency to withdraw from a heat stimulus
(thermal hyperalgesia; see FIG. 11B). Rats inoculated with QH-GAD67
showed a statistically significant increase in mechanical threshold
beginning 1 week after inoculation. The antiallodynic effect of
QHGAD67-mediated GABA expression was sustained and continuous,
lasting 5 to 6 weeks and peaking at 2 weeks after inoculation (see
FIG. 11A). The peak value of mechanical threshold, 8.6 gm, was
close to the preoperative value. By 7 weeks after inoculation, the
antiallodynic effect of vector transduction disappeared, and the
mechanical threshold of QHGAD67-injected rats was identical to that
of control rats. Reinoculation into the same paw with the same dose
of QHGAD67 reestablished the antiallodynic effect (see FIG. 11A).
SNL induced a decrease in the thermal latency from 10.7 to 6.5
seconds, which lasted 3 weeks before gradually recovery. Rats
inoculated with QHGAD67 showed a statistically significant increase
in thermal latency in the ipsilateral paw beginning 1 week after
inoculation (see FIG. 11B), an effect that was sustained and
continuous, lasting 3 to 4 weeks (see FIG. 11B). Sham-operated
animals had no change in mechanical thresh-old or thermal
latency.
[0103] Expression of c-Fos and phosphorylated extracellular
signal-regulated kinase 1 and 2 (p-ERK1/2) induced by gentle touch
is one indirect biological marker of nociceptive processes
(Catheline et al., Pain; 92:389-98 (2001)). Three weeks after SNL,
gentle touch was applied once every 4 seconds for 10 minutes, with
the flat surface of the experimenter's thumb to the rat's paw, and
the number of immunoreactive cells (anti-c-Fos or anti-p-ERK1/2
antibodies; Santa Cruz Biotechnology, Santa Cruz, Calif.) detected
avidin-biotin horseradish peroxidase followed by nickel-enhanced
diaminobenzidine (Vector Laboratories, Bur-lingame, CA).12 The
number of Fos-LI-positive neurons was substantially increased
ipsilateral to SNL compared with sham-operated control rats, and
inoculation of vector QHGAD67 significantly reduced the number
Fos-LI-positive neurons in laminae I-VI (see FIG. 12). p-ERK1/2
expression in laminae I and II was also increased in rats after
gentle touch stimulation with SNL, and that increase was blocked in
animals inoculated with QHGAD67. p-ERK was not induced by 10
minutes of gentle tactile stimulation in sham-operated animals but
it was substantially induced after spinal nerve ligation (SNL) in
animals inoculated with QOZHG. Touch-induced expression of p-ERK1/2
was suppressed in animals inoculated with QHGAD67, confirmed by
counts of p-ERK1/2-positive neurons in the dorsal horn (FIG.
13).
[0104] These results demonstrate that subcutaneous inoculation of
an HSV vector expressing GAD to transduce DRG in vivo attenuated
the behavioral manifestations of mechanical allodynia and thermal
hyperalgesia in a model of peripheral neuropathic pain; the effect
on behavior was confirmed by histological measures showing a block
in the induction of expression of c-Fos and p-ERK1/2 in the
ipsilateral spinal dorsal horn.
DISCUSSION
[0105] The lateral hemisection of a laboratory rat's spinal cord at
T13 produces bilateral SCI pain related behavior below the lesion
in both hind limbs ("the SCI model"). The SCI pain related behavior
is manifested as mechanical allodynia and thermal hyperalgesia.
This phenomenon is accompanied by bilateral spinal cord
reorganization. In the above mentioned experiments, this SCI model
was used to examine the effects of the local production and release
of GABA due to a GAD vector mediated gene transfer to the lumbar
DRG in alleviating some of the manifestations of SCI pain.
[0106] DRG neurons were transduced with a HSV vector encoding for
GAD ("the GAD vector"). These transduced DRG neurons expressed GAD
in vitro and in vivo. The expression of GAD in these cells resulted
in the release of GABA. In the laboratory rats subjected to lateral
hemisection of the spinal cord at T13 followed by subcutaneous
inoculation with the GAD vector, regional GABA release from
transduced DRG neurons reduced mechanical allodynia and thermal
hyperalgesia in the hind limbs. This effect could be reversed by
either GABA.sub.A or GABA.sub.B receptor antagonists administered
at doses that did not alter nociception in normal animals or in
laboratory rats subjected to lateral hemisection of the spinal cord
at T13 without subcutaneous inoculation with the GAD vector.
Moreover, GAD vector mediated GABA release also attenuated the
increase in CGRP immunoreactivity in the lumbar dorsal horn that
occurs after SCI. Therefore, the inventive method involving GAD
vector mediated gene transfer to DRG can effectively be used to
treat below level neuropathic SCI pain.
[0107] Release of GABA from primary DRG neurons transduced in vitro
with the GAD vector was not increased in a medium containing 60 mM
K.sup.+ and was unaffected by removal of Ca.sup.2+ from the medium
suggesting that GABA release is not vesicular, but occurs
constitutively, perhaps through reversal of the GABA transporter.
Although the amount of GABA released from nerve terminals in vivo
was sufficient to elevate GABA levels in microdialysate of the
dorsal horn significantly, there was no evidence of motor weakness
in these animals, suggesting that transgene mediated GABA release
was limited to the dorsal horn. This would be consistent with the
observation that animals transduced by subcutaneous inoculation of
the proenkephalin expressing HSV vector acquired an analgesic
effect that was limited to the limb ipsilateral to the
inoculation.
[0108] In laboratory rats with uninjured spinal cords, tonic
GABA-ergic inhibition of low threshold afferent inputs modulates
sensory processing, and bicuculline block of GABA receptor function
produces pain-related behavior. Electrophysiologic studies suggest
that both GABA.sub.A and GABA.sub.B receptors contribute to the
tonic modulation of nociceptive neurotransmission at the spinal
cord level. Peripheral nerve injury results in a decrease in GABA
levels in the dorsal horn and a reduction in the primary afferent
evoked inhibitory post synaptic currents in the dorsal horn after
partial nerve injury. However, whether these phenomena result from
a loss of GABA-ergic interneurons or a desensitization of GABA
receptors as found in central sensitization has not been fully
established. A transient reduction in GABA immunoreactivity has
been reported in the lumbar spinal cord after photochemical
induction of spinal ischemia, but previous studies have not
examined GABA immunoreactivity below the level of hemisection.
Nonetheless, constitutive delivery of GABA to the dorsal horn below
the level of the hemisection as a result of GAD vector transduction
of DRG neurons reduced behavioral measures of mechanical allodynia
and thermal hyperalgesia after SCI. This indicates that SCI pain is
susceptible to modulation by GABA.
[0109] The effect of baclofen on central neurogenic pain has been
assessed in several trials with generally positive results in brief
trials, but with less remarkable long-term relief. Intrathecal
administration of baclofen partially alleviates chronic mechanical
and cold allodynia in an ischemia model of SCI. In a chronic
constriction injury model of neuropathic pain, intrathecal
transplantation of GABA-releasing cells reverses some of the
manifestations of neuropathic pain. It has been discovered that the
pain-relieving effects of GAD vector mediated GABA release were
continuous over the course of several weeks.
[0110] The amount of GABA release over time was not measured.
However, it was observed that the reinoculation of the GAD vector
after the analgesic effect had waned reestablished the reduction in
mechanical allodynia and thermal hyperalgesia. This observation
demonstrates that the loss of therapeutic effect was not due to the
development of tolerance, but to a reduction in gene expression.
This is consistent with the findings of previous studies that
examined vectors in which transgene expression was driven by
transiently active human cytomegalovirus immediate early promoter
(HCMV IEp).
[0111] There are no "objective" measures of pain and pain relief.
However, measurements of the increase in the amount of CGRP
immunoreactivity in spinal cord segments below the level of injury
in the SCI model have been used to measure pain and pain relief in
the laboratory. Unfortunately, little is known about the phenomena
of increased spinal cord CGRP in the SCI model. The mechanisms
responsible for the increase in below-level CGRP have not been
defined. It is not known whether the increase in immunoreactivity
correlates with increased release on turnover of CGRP. It is also
not known whether CGRP plays any role in the SCI pain phenomenon or
occurs as an epiphenomenon of SCI. Nonetheless, the increase in
CGRP that occurs after SCI in these studies serves as a histologic
correlate to the behavioral measures of pain analogous to the
increase in c-fos immunoreactivity induced by nonnoxious touch in
the spinal cord nerve ligation model of peripheral neuropathic
pain. Therefore, measurements of CGRP can be used to determine the
effect of GAD vectors in treating SCI pain. While not wishing to be
bound to any particular theory, because CGRP is located in the
unmyelinated and thinly myelinated afferents that project
principally to laminae I, II, and V, of the dorsal horn, the
increase in CGRP is believed to result from the sprouting of
primary afferents. The mechanism through which GABA release from
GAD transduced cells prevents the increase in CGRP expression is
not known.
[0112] From the foregoing, in accordance with the invention, a
nonreplicating HSV vector designed to express human glutamic acid
decarboxylase (GAD) was successfully constructed, and this vector
was used to treat SCI pain and also peripheral neuropathic pain.
These examples demonstrate that the inventive method involving the
use of a gene transfer approach can be used to transduce DRG
neurons through peripheral inoculation to release GABA in the
dorsal horn. These examples also demonstrate that the inventive
method involving gene transfer using the GAD vector reduces
below-level mechanical allodynia and thermal hyperalgesia after
SCI. The ability to deliver the GAD vector by subcutaneous
inoculation is an attractive feature of the inventive approach to
treat SCI pain and also peripheral neuropathic pain.
[0113] The practice of the present invention employs, unless
otherwise indicated, conventional methods of virology,
microbiology, molecular biology and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in
the literature. (See, e.g., Sambrook, et al. Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook ofParvoviruses, Vol. I & II (P.
Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II
(B. N. Fields and D. M. Knipe, eds.))
[0114] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0115] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0116] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
913610DNAHomo Sapiens 1gaattcttcg taggaattat cttttccctc ctctcacccg
acagcctgcc tatttccaaa 60ggaaaaaaaa aaagcgtgtt gagtacgttc tggattactc
ataagacctt ttttttttcc 120ttccgggcgc aaaaccgtga gctggattta
taatcgccct ataaagctcc agaggcggtc 180aggcacctgc agaggagccc
cgccgctccg ccgactagct gcccccgcga gcaacggcct 240cgtgatttcc
ccgccgatcc ggtccccgcc tccccactct gcccccgcct accccggagc
300cgtgcagccg cctctccgaa tctctctctt ctcctggcgc tcgcgtgcga
gagggaacta 360gcgagaacga ggaagcagct ggaggtgacg ccgggcagat
tacgcctgtc agggccgagc 420cgagcggatc gctgggcgct gtgcagagga
aaggcgggag tgcccggctc gctgtcgcag 480agccgagcct gtttctgcgc
cggaccagtc gaggactctg gacagtagag gccccgggac 540gaccgagctg
atggcgtctt cgaccccatc ttcgtccgca acctcctcga acgcgggagc
600ggaccccaat accactaacc tgcgccccac aacgtacgat acctggtgcg
gcgtggccca 660tggatgcacc agaaaactgg ggctcaagat ctgcggcttc
ttgcaaagga ccaacagcct 720ggaagagaag agtcgccttg tgagtgcctt
cagggagagg caatcctcca agaacctgct 780ttcctgtgaa aacagcgacc
gggatgcccg cttccggcgc acagagactg acttctctaa 840tctgtttgct
agagatctgc ttccggctaa gaacggtgag gagcaaaccg tgcaattcct
900cctggaagtg gtggacatac tcctcaacta tgtccgcaag acatttgatc
gctccaccaa 960ggtgctggac tttcatcacc cacaccagtt gctggaaggc
atggagggct tcaacttgga 1020gctctctgac caccccgagt ccctggagca
gatcctggtt gactgcagag acaccttgaa 1080gtatggggtt cgcacaggtc
atcctcgatt tttcaaccag ctctccactg gattggatat 1140tattggccta
gctggagaat ggctgacatc aacggccaat accaacatgt ttacatatga
1200aattgcacca gtgtttgtcc tcatggaaca aataacactt aagaagatga
gagagatagt 1260tggatggtca agtaaagatg gtgatgggat attttctcct
gggggcgcca tatccaacat 1320gtacagcatc atggctgctc gctacaagta
cttcccggaa gttaagacaa agggcatggc 1380ggctgtgcct aaactggtcc
tcttcacctc agaacagagt cactattcca taaagaaagc 1440tggggctgca
cttggctttg gaactgacaa tgtgattttg ataaagtgca atgaaagggg
1500gaaaataatt ccagctgatt ttgaggcaaa aattcttgaa gccaaacaga
agggatatgt 1560tcccttttat gtcaatgcaa ctgctggcac gactgtttat
ggagcttttg atccgataca 1620agagattgca gatatatgtg agaaatataa
cctttggttg catgtcgatg ctgcctgggg 1680aggtgggctg ctcatgtcca
ggaagcaccg ccataaactc aacggcatag aaagggccaa 1740ctcagtcacc
tggaaccctc acaagatgat gggcgtgctg ttgcagtgct ctgccattct
1800cgtcaaggaa aagggtatac tccaaggatg caaccagatg tgtgcaggat
atctcttcca 1860gccagacaag cagtatgatg tctcctacga caccggggac
aaggcaattc agtgtggccg 1920ccacgtggat atcttcaagt tctggctgat
gtggaaagca aagggcacag tgggatttga 1980aaaccagatc aacaaatgcc
tggaactggc tgaatacctc tatgccaaga ttaaaaacag 2040agaagaattt
gagatggttt tcaatggcga gcctgagcac acaaacgtct gtttttggta
2100tattccacaa agcctcaggg gtgtgccaga cagccctcaa cgacgggaaa
agctacacaa 2160ggtggctcca aaaatcaaag ccctgatgat ggagtcaggt
acgaccatgg ttggctacca 2220gccccaaggg gacaaggcca acttcttccg
gatggtcatc tccaacccag ccgctaccca 2280gtctgacatt gacttcctca
ttgaggagat agaaagactg ggccaggatc tgtaatcatc 2340cttcgcagaa
catgagttta tgggaatgcc ttttccctct ggcactccag aacaaacctc
2400tatatgttgc tgaaacacac aggccatttc attgagggaa aacataatat
cttgaagaat 2460attgttaaaa ccttacttaa agcttgtttg ttctagttag
caggaaatag tgttcttttt 2520aaaaagttgc acattaggaa cagagtatat
atgtacagtt atacatacct ctctctatat 2580atacatgtat agtgagtgtg
gcttagtaat agatcacggc atgtttcccg ctccaagaga 2640attcacttta
ccttcagcag ttaccgagga gctaaacatg ctgccaacca gcttgtccaa
2700caactccagg aaaactgttt ttcaaaacgc catgtcctag gggccaaggg
aaatgctgtt 2760ggtgagaatc gacctcactg tcagcgtttc tccacctgaa
gtgatgatgg atgagaaaaa 2820acaccaccaa atgacaagtc acaccctccc
cattagtatc ctgttagggg aaaatagtag 2880cagagtcatt gttacaggtg
tactatggct gtattttaga gattaatttg tgtagattgt 2940gtaaattcct
gttgtctgac cttggtggtg ggaggggaga ctatgtgtca tgatttcaat
3000gattgtttaa ttgtaggtca atgaaatatt tgcttattta tattcagaga
tgtaccatgt 3060taaagaggcg tcttgtattt tcttcccatt tgtaatgtat
cttatttata tatgaagtaa 3120gttctgaaaa ctgtttatgg tattttcgtg
catttgtgag ccaaagagaa aagattaaaa 3180ttagtgagat ttgtatttat
attagagtgc ccttaaaata atgatttaag cattttactg 3240tctgtaagag
aattctaaga ttgtacatga cataagttat agtaatcatg gcaaatcctg
3300ttacttaaat agcatctgct cttctcttac gctctctgtc tggctgtacg
tctggtgttc 3360tcaatgcttt tctagcaact gttggataat aactagatct
cctgtaattt tgtagtagtt 3420gatgaccaat ctctgtgact cgcttagctg
aaacctaagg caacatttcc gaagaccttc 3480tgaagatctc agataaagtg
accaggctca caactgtttt tgaagaaggg aaattcacac 3540tgtgcgtttt
gagtatgcaa gaagaatata aataaataaa atatctcatg gagattgaca
3600aaaaaaaaaa 36102594PRTHomo sapiens 2Met Ala Ser Ser Thr Pro Ser
Ser Ser Ala Thr Ser Ser Asn Ala Gly 1 5 10 15 Ala Asp Pro Asn Thr
Thr Asn Leu Arg Pro Thr Thr Tyr Asp Thr Trp 20 25 30 Cys Gly Val
Ala His Gly Cys Thr Arg Lys Leu Gly Leu Lys Ile Cys 35 40 45 Gly
Phe Leu Gln Arg Thr Asn Ser Leu Glu Glu Lys Ser Arg Leu Val 50 55
60 Ser Ala Phe Arg Glu Arg Gln Ser Ser Lys Asn Leu Leu Ser Cys Glu
65 70 75 80 Asn Ser Asp Arg Asp Ala Arg Phe Arg Arg Thr Glu Thr Asp
Phe Ser 85 90 95 Asn Leu Phe Ala Arg Asp Leu Leu Pro Ala Lys Asn
Gly Glu Glu Gln 100 105 110 Thr Val Gln Phe Leu Leu Glu Val Val Asp
Ile Leu Leu Asn Tyr Val 115 120 125 Arg Lys Thr Phe Asp Arg Ser Thr
Lys Val Leu Asp Phe His His Pro 130 135 140 His Gln Leu Leu Glu Gly
Met Glu Gly Phe Asn Leu Glu Leu Ser Asp 145 150 155 160 His Pro Glu
Ser Leu Glu Gln Ile Leu Val Asp Cys Arg Asp Thr Leu 165 170 175 Lys
Tyr Gly Val Arg Thr Gly His Pro Arg Phe Phe Asn Gln Leu Ser 180 185
190 Thr Gly Leu Asp Ile Ile Gly Leu Ala Gly Glu Trp Leu Thr Ser Thr
195 200 205 Ala Asn Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe
Val Leu 210 215 220 Met Glu Gln Ile Thr Leu Lys Lys Met Arg Glu Ile
Val Gly Trp Ser 225 230 235 240 Ser Lys Asp Gly Asp Gly Ile Phe Ser
Pro Gly Gly Ala Ile Ser Asn 245 250 255 Met Tyr Ser Ile Met Ala Ala
Arg Tyr Lys Tyr Phe Pro Glu Val Lys 260 265 270 Thr Lys Gly Met Ala
Ala Val Pro Lys Leu Val Leu Phe Thr Ser Glu 275 280 285 Gln Ser His
Tyr Ser Ile Lys Lys Ala Gly Ala Ala Leu Gly Phe Gly 290 295 300 Thr
Asp Asn Val Ile Leu Ile Lys Cys Asn Glu Arg Gly Lys Ile Ile 305 310
315 320 Pro Ala Asp Phe Glu Ala Lys Ile Leu Glu Ala Lys Gln Lys Gly
Tyr 325 330 335 Val Pro Phe Tyr Val Asn Ala Thr Ala Gly Thr Thr Val
Tyr Gly Ala 340 345 350 Phe Asp Pro Ile Gln Glu Ile Ala Asp Ile Cys
Glu Lys Tyr Asn Leu 355 360 365 Trp Leu His Val Asp Ala Ala Trp Gly
Gly Gly Leu Leu Met Ser Arg 370 375 380 Lys His Arg His Lys Leu Asn
Gly Ile Glu Arg Ala Asn Ser Val Thr 385 390 395 400 Trp Asn Pro His
Lys Met Met Gly Val Leu Leu Gln Cys Ser Ala Ile 405 410 415 Leu Val
Lys Glu Lys Gly Ile Leu Gln Gly Cys Asn Gln Met Cys Ala 420 425 430
Gly Tyr Leu Phe Gln Pro Asp Lys Gln Tyr Asp Val Ser Tyr Asp Thr 435
440 445 Gly Asp Lys Ala Ile Gln Cys Gly Arg His Val Asp Ile Phe Lys
Phe 450 455 460 Trp Leu Met Trp Lys Ala Lys Gly Thr Val Gly Phe Glu
Asn Gln Ile 465 470 475 480 Asn Lys Cys Leu Glu Leu Ala Glu Tyr Leu
Tyr Ala Lys Ile Lys Asn 485 490 495 Arg Glu Glu Phe Glu Met Val Phe
Asn Gly Glu Pro Glu His Thr Asn 500 505 510 Val Cys Phe Trp Tyr Ile
Pro Gln Ser Leu Arg Gly Val Pro Asp Ser 515 520 525 Pro Gln Arg Arg
Glu Lys Leu His Lys Val Ala Pro Lys Ile Lys Ala 530 535 540 Leu Met
Met Glu Ser Gly Thr Thr Met Val Gly Tyr Gln Pro Gln Gly 545 550 555
560 Asp Lys Ala Asn Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr
565 570 575 Gln Ser Asp Ile Asp Phe Leu Ile Glu Glu Ile Glu Arg Leu
Gly Gln 580 585 590 Asp Leu 319DNAArtificialForward primer
3gcgggagcgg atcctaata 19419DNAArtificialReverse primer 4tggtgcatcc
atgggctac 19528DNAArtificialprobe 5cgtcctacaa catatgatac ttggtgtg
28618DNAArtificialForward primer 6ccgagggccc actaaagg
18721DNAArtificialReverse primer 7tgctgttgaa gtcacaggag a
21824DNAArtificialProbe 8catcctgggc tacactgagg acca 2491239DNAHomo
sapiens 9cggcgagggt cctgccgagg gacccgttct gcgcccaggc aggctcgaag
cacgcgtccc 60tctctcctcg cagtccatgg cgcggttcct gacactttgc acttggctgc
tgttgctcgg 120ccccgggctc ctggcgaccg tgcgggccga atgcagccag
gattgcgcga cgtgcagcta 180ccgcctagtg cgcccggccg acatcaactt
cctggcttgc gtaatggaat gtgaaggtaa 240actgccttct ctgaaaattt
gggaaacctg caaggagctc ctgcagctgt ccaaaccaga 300gcttcctcaa
gatggcacca gcaccctcag agaaaatagc aaaccggaag aaagccattt
360gctagccaaa aggtatgggg gcttcatgaa aaggtatgga ggcttcatga
agaaaatgga 420tgagctttat cccatggagc cagaagaaga ggccaatgga
agtgagatcc tcgccaagcg 480gtatgggggc ttcatgaaga aggatgcaga
ggaggacgac tcgctggcca attcctcaga 540cctgctaaaa gagcttctgg
aaacagggga caaccgagag cgtagccacc accaggatgg 600cagtgataat
gaggaagaag tgagcaagag atatgggggc ttcatgagag gcttaaagag
660aagcccccaa ctggaagatg aagccaaaga gctgcagaag cgatatgggg
gcttcatgag 720aagagtaggt cgcccagagt ggtggatgga ctaccagaaa
cggtatggag gtttcctgaa 780gcgctttgcc gaggctctgc cctccgacga
agaaggcgaa agttactcca aagaagttcc 840tgaaatggaa aaaagatacg
gaggatttat gagattttaa tatttttccc actagtggcc 900ccaggcccca
gcaagcctcc ctccatcctc cagtgggaaa ctgttgatgg tgttttattg
960tcatgtgttg cttgccttgt atagttgact tcattgtctg gataactata
caacctgaaa 1020actgtcattt caggttctgt gctctttttg gagtctttaa
gctcagtatt agtctattgc 1080agctatctcg ttttcatgct aaaatagttt
ttgttatctt gtctcttatt tttgacaaac 1140atcaataaat gcttacttgt
atatagagat aataaaccta ttaccccaag tgcaaaaaaa 1200aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1239
* * * * *
References