U.S. patent application number 16/493242 was filed with the patent office on 2020-04-16 for methods of treatment of hla-b27 related inflammatory diseases and compositions related to same.
The applicant listed for this patent is ALMA BIO THERAPEUTICS. Invention is credited to Binah Baum, Irun R. Cohen, Raanan Margalit, Denis Ravel.
Application Number | 20200114022 16/493242 |
Document ID | / |
Family ID | 62148415 |
Filed Date | 2020-04-16 |
United States Patent
Application |
20200114022 |
Kind Code |
A1 |
Cohen; Irun R. ; et
al. |
April 16, 2020 |
METHODS OF TREATMENT OF HLA-B27 RELATED INFLAMMATORY DISEASES AND
COMPOSITIONS RELATED TO SAME
Abstract
The present disclosure provides methods and compositions for
treatment of an HLA-B27-associated autoimmune inflammatory disorder
by administration of nucleic acid encoding HSP90 or an active
fragment thereof.
Inventors: |
Cohen; Irun R.; (Rehovot,
IL) ; Ravel; Denis; (Paris, FR) ; Margalit;
Raanan; (Rehovot, IL) ; Baum; Binah; (Lyon,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALMA BIO THERAPEUTICS |
Lyon |
|
FR |
|
|
Family ID: |
62148415 |
Appl. No.: |
16/493242 |
Filed: |
March 28, 2018 |
PCT Filed: |
March 28, 2018 |
PCT NO: |
PCT/IB2018/000408 |
371 Date: |
September 11, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62480171 |
Mar 31, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
19/02 20180101; A61K 31/711 20130101; A61K 48/005 20130101; A61P
29/00 20180101; A61P 9/00 20180101; A61K 39/0008 20130101; A61P
37/00 20180101; A61K 48/00 20130101; A61P 27/02 20180101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61P 1/04 20060101 A61P001/04; A61P 19/02 20060101
A61P019/02; A61P 29/00 20060101 A61P029/00 |
Claims
1. A method of treating or preventing an autoimmune inflammatory
disease in an HLA-B27 positive subject, the method comprising:
administering to a subject having or at risk of an autoimmune
inflammatory disease and who is positive for HLA-B27 a composition
comprising a nucleic acid molecule encoding a mammalian heat shock
protein 90 (HSP90), or an active fragment thereof, wherein the
nucleic acid molecule is operatively linked to one or more
transcription control sequences, wherein the administering treats
the inflammatory disease in the subject.
2. The method of claim 1, wherein the autoimmune inflammatory
disease is an inflammatory joint disease.
3. The method of claim 2, wherein the inflammatory joint disease is
a spondyloarthropathy.
4. The method of claim 3, wherein the spondyloarthropathy is
anklyosing spondylitis, reactive arthritis, psoriatic arthritis or
enteropathic arthritis.
5. The method of claim 2, wherein the inflammatory joint disease is
juvenile spondarthritis, undifferentiated spondarthritis or
isolated peripheral enthesitis.
6. The method of claim 1, wherein the autoimmune inflammatory
disease is an ocular disorder.
7. The method of claim 6, wherein the ocular disorder is uveitis or
age-related macular degeneration.
8. The method of claim 7, wherein the uveitis is acute anterior
uveitis or posterior uveitis.
9. The method of claim 1, wherein the autoimmune inflammatory
disease is an inflammatory disease of the gastrointestinal
tract.
10. The method of claim 9, wherein the autoimmune inflammatory
disease is inflammatory bowel disease, ulcerative colitis, or
Crohn's disease.
11. The method of claim 1, wherein the autoimmune inflammatory
disease is Behcet disease.
12. The method of claim 1, wherein the autoimmune inflammatory
disease is a cardiovascular disease.
13. The method of any one of claims 1-12, wherein the subject is at
risk of the autoimmune inflammatory disorder.
14. The method of any one of claims 1-12, wherein the subject has
the autoimmune inflammatory disorder.
15. The method of any one of claims 1-14, wherein the mammalian
HSP90 is human HSP90.
16. The method of claim 15, wherein the subject is a human.
17. The method of any one of claims 1-16, wherein said
administering is by injection.
18. The method of claim 17, wherein administering is by
intramuscular injection.
19. The method of any one of claims 1-18, wherein the composition
is administered in an amount of from about 0.005 mg/kg to about 1
mg/kg of the nucleic acid molecule encoding the mammalian HSP90, or
an active fragment thereof, to the body weight of the subject.
20. The method of claim 19, wherein the composition is administered
in an amount of from about 0.01 mg/kg to about 0.5 mg/kg of the
nucleic acid molecule encoding the mammalian HSP90, or an active
fragment thereof, to the body weight of the subject.
21. The method of claim 19, wherein the composition is administered
in an amount of from about 0.10 mg/kg to about 0.30 mg/kg of the
nucleic acid molecule encoding the mammalian HSP90, or an active
fragment thereof, to the body weight of the subject.
22. The method of any one of claims 1-21, wherein the composition
is administered weekly.
23. The method of any one of claims 1-22, wherein the composition
is administered for a period of at least 4 weeks.
24. The method of any one of claims 1-23, wherein the nucleic acid
molecule is not encapsulated.
25. The method of any one of claims 1-24, wherein the nucleic acid
molecule is not contained in a virus.
26. The method of any one of claims 1-25, wherein the composition
comprises a pharmaceutically acceptable alkali metal salt.
27. The method of claim 26, wherein the pharmaceutically acceptable
alkali metal salt is sodium chloride.
28. The method of any one of claims 1-27, wherein the composition
comprises a delivery vehicle selected from the group consisting of
a liposome, a micelle, and an emulsion.
29. The method of any one of claims 1-27, wherein the composition
comprises a lipid particle.
30. The method of any one of claims 1-27, wherein the composition
comprises a catioinic lipid, polyethylene glycol, polylysine,
poloxamer, chitosan, polyL glutamate, or poly(lactide-co-glycolide)
(PLG).
31. A composition comprising an HSP90-encoding nucleic acid for use
in treatment of an autoimmune inflammatory disorder in an HLA-B27
positive subject.
32. Use of an HSP90-encoding nucleic acid in the manufacture of a
medicament for in treatment of an autoimmune inflammatory disorder
in an HLA-B27 positive subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit to U.S. Provisional
Application No. 62/480,171, filed Mar. 31, 2017, the disclosure of
which is incorporated herein by reference in its entirety.
INTRODUCTION
[0002] The first human leukocyte antigen (HLA) haplotype
association with inflammatory disease was discovered in 1972 in
which the HLA type HLA-B27 was correlated with ankylosing
spondylitis. This remains one of the strongest known associations
of disease with HLA-B27. Since then, more than 100 disease
associations have been made, including spondyloarthropathies (e.g.,
ankylosing spondylitis, reactive arthritis (also known as Reiter
syndrome), psoriatic arthritis, and enteropathic arthritis), many
ocular diseases and systemic diseases with specific ocular
manifestations, and as well as inflammatory diseases of the
gastrointestinal tract (e.g., inflammatory bowel disease).
[0003] There is a need for effective therapies for relief of one or
more symptoms of autoimmune inflammatory disorders in HLA-B27
positive subjects.
SUMMARY
[0004] The present disclosure provides methods and compositions for
treatment of an HLA-B27-associated autoimmune inflammatory disorder
by administration of nucleic acid encoding HSP90 or an active
fragment thereof.
[0005] Accordingly, the present disclosure provides methods for
treating or preventing an autoimmune inflammatory disease in an
HLA-B27 positive subject, the method comprising administering to a
subject having or at risk of an autoimmune inflammatory disease and
who is positive for HLA-B27 a composition comprising a nucleic acid
molecule encoding a mammalian heat shock protein 90 (HSP90), or an
active fragment thereof, wherein the nucleic acid molecule is
operatively linked to one or more transcription control sequences,
wherein the administering treats the inflammatory disease in the
subject.
[0006] In some embodiments, the autoimmune inflammatory disease is
an inflammatory joint disease, e.g., a spondyloarthropathy, such
as, for example, anklyosing spondylitis, reactive arthritis,
psoriatic arthritis or enteropathic arthritis. In some embodiments
the inflammatory joint disease is juvenile spondarthritis,
undifferentiated spondarthritis or isolated peripheral
enthesitis.
[0007] In some embodiments, the autoimmune inflammatory disease is
an ocular disorder, such as uveitis or age-related macular
degeneration. In some embodiments, the uveitis is acute anterior
uveitis or posterior uveitis.
[0008] In some embodiments, the autoimmune inflammatory disease is
an inflammatory disease of the gastrointestinal tract, e.g.,
inflammatory bowel disease, ulcerative colitis, or Crohn's
disease.
[0009] In some embodiments, the autoimmune inflammatory disease is
Behcet disease. In some embodiments the autoimmune inflammatory
disease is a cardiovascular disease.
[0010] In any of the above embodiments, the subject can be a
subject that is at risk of the autoimmune inflammatory
disorder.
[0011] In any of the above embodiments, the subject can be a
subject has been diagnosed as having the autoimmune inflammatory
disorder.
[0012] In any of the above embodiments, the mammalian HSP90 can be
human HSP90.
[0013] In any of the above embodiments, the subject can be a
human.
[0014] In any of the above embodiments, administration can be by
injection, e.g., intramuscular injection.
[0015] In any of the above embodiments, the composition can be
administered in an amount of from about 0.005 mg/kg to about 1
mg/kg of the nucleic acid molecule encoding the mammalian HSP90, or
an active fragment thereof, to the body weight of the subject. In
related embodiments, the composition is administered in an amount
of from about 0.01 mg/kg to about 0.5 mg/kg of the nucleic acid
molecule encoding the mammalian HSP90, or an active fragment
thereof, to the body weight of the subject. In further related
embodiments, the composition is administered in an amount of from
about 0.10 mg/kg to about 0.30 mg/kg of the nucleic acid molecule
encoding the mammalian HSP90, or an active fragment thereof, to the
body weight of the subject.
[0016] In any of the above embodiments, the composition can be
administered weekly.
[0017] In any of the above embodiments, the composition can be
administered for a period of at least 4 weeks.
[0018] In some embodiments, the nucleic acid molecule is not
encapsulated.
[0019] In some embodiments, the nucleic acid molecule is not
contained in a virus.
[0020] In any of the above embodiments, the composition can
comprise a pharmaceutically acceptable alkali metal salt, such as
sodium chloride.
[0021] In some embodiments, the composition comprises a delivery
vehicle selected from the group consisting of a liposome, a
micelle, and an emulsion.
[0022] In some embodiments, the composition comprises a lipid
particle.
[0023] In some embodiments, the composition comprises a catioinic
lipid, polyethylene glycol, polylysine, poloxamer, chitosan, polyL
glutamate, or poly(lactide-co-glycolide) (PLG).
[0024] The present disclosure also provides compositions comprising
an HSP90-encoding nucleic acid for use in treatment of an
autoimmune inflammatory disorder in an HLA-B27 positive
subject.
[0025] The present disclosure also provides for use of an
HSP90-encoding nucleic acid in the manufacture of a medicament for
in treatment of an autoimmune inflammatory disorder in an HLA-B27
positive subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 schematically illustrates the protocol for a
five-week treatment of HLA-B27 rats with HSP90. Eight-week old rats
received IM injections once a week for five weeks. Animals were
euthanized on week 5, three days after receiving their last
injection.
[0027] FIG. 2 shows the results of a study to evaluate body weight
of HLA-B27 rats treated with 0.25 mg/kg of HSP90-encoding DNA
compared to HLA-B27 rats treated with the saline control.
[0028] FIGS. 3A-3B shows the results of a study to evaluate the
consistency of stool collected from HLA-B27 rats treated with 0.25
mg/kg of HSP-90 as compared to that of HLA-B27 rats treated with
saline control. At each time point in FIG. 3A, the control value is
shown by the left hand bar and the treatment value (HSP90 0.25
mg/kg) is shown by the right hand bar.
[0029] FIGS. 4A-4D depicts scores for the parameters of colonic
hypervasculaization (FIG. 4A), thickness of the colonic wall (FIG.
4B), mesentery thickness in colon sample (FIG. 4C), and the global
macropscopic score (FIG. 4D) in treated and control animals.
[0030] FIGS. 5A-5B depict the results of monitoring the clinical
signs of arthritis in the tarsal joints of HLA-B27 rats over the
course of the five-week treatment with HSP90 or a control saline
solution. At each time point in FIG. 5A, the control value is shown
by the left hand bar and the treatment value (HSP90 0.25 mg/kg) is
shown by the right hand bar.
[0031] Before the present invention is described further, it is to
be understood that this invention is not limited to particular
embodiments described, and as such may, of course, vary. It is also
to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0032] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention.
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0034] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a nucleic acid molecule" includes a
plurality of nucleic acid molecules and reference to "an injection"
includes reference to one or more injections and equivalents
thereof known to those skilled in the art, and so forth. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely",
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0035] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0036] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0037] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
Definitions
[0038] "HLA-B27" refers to the human leukocyte antigen (HLA) B27
(including subtypes B*2701-2759), is a class I surface antigen
encoded by the B locus in the major histocompatibility complex
(MHC) on chromosome 6.
[0039] The terms "polypeptide," "peptide," and "protein", used
interchangeably herein, refer to a polymeric form of amino acids of
any length, which can include genetically coded and non-genetically
coded amino acids, chemically or biochemically modified or
derivatized amino acids, and polypeptides having modified peptide
backbones. The term includes fusion proteins, including, but not
limited to, fusion proteins with a heterologous amino acid
sequence, fusions with heterologous and homologous leader
sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; and the like.
[0040] A "control element" or "control sequence" refers to a
nucleotide sequence involved in an interaction of molecules that
contributes to the functional regulation of a polynucleotide,
including replication, transcription, translation, or degradation
of the polynucleotide in a host cell (e.g., in a eukaryotic host
cell, e.g., a human host cell). The regulation may affect the
frequency, speed, or specificity of the process, and may be
enhancing or inhibitory in nature. Control elements known in the
art include, for example, transcriptional regulatory sequences such
as promoters and enhancers. A promoter is a DNA region capable
under certain conditions of binding RNA polymerase and initiating
transcription of a coding region usually located downstream (in the
3' direction) from the promoter.
[0041] "Operatively linked" or "operably linked" refers to covalent
connection of genetic elements, wherein the genetic elements are
provided in a relationship permitting them to operate in a desired
manner. For instance, a promoter is operatively linked to a coding
region if the promoter facilitates initiation of transcription of
the coding sequence. There may be intervening residues between the
promoter and coding region so long as this functional relationship
is maintained.
[0042] An "expression vector" refers to a vector comprising a
region which encodes a polypeptide of interest, and is used for
effecting the expression of the protein in an intended target cell.
An expression vector also comprises control elements operatively
linked to the encoding region to facilitate expression of the
protein in the target. The combination of control elements and a
gene or genes to which they are operably linked for expression is
sometimes referred to as an "expression cassette," a large number
of which are known and available in the art or can be readily
constructed from components that are available in the art.
[0043] The terms "patient" or "subject" are used interchangeably to
refer to a human or a non-human animal (e.g., a mammal). In the
context of an HLA-B27 positive subject, such subjects are generally
human subjects or non-human animals genetically modified to express
HLA-B27.
[0044] As used herein, the terms "treatment," "treating," "treat"
and the like, refer to obtaining a desired pharmacologic and/or
physiologic effect. The effect can be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or can be therapeutic in terms of a partial or complete cure
for a disease and/or adverse effect attributable to the disease.
"Treatment," as used herein, covers any treatment of a disease in a
mammal, particularly in a human, and includes: (a) preventing the
disease from occurring in a subject which can be predisposed to the
disease but has not yet been diagnosed as having it; (b) inhibiting
the disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0045] The term "in need of treatment" as used herein refers to a
judgment made by a physician or other caregiver that a subject
requires or will benefit from treatment. This judgment is made
based on a variety of factors that are in the realm of the
physician's or caregiver's expertise.
[0046] The terms "prevent", "preventing", "prevention" and the like
refer to a course of action (such as administering IL-10 or a
pharmaceutical composition comprising IL-10) initiated in a manner
(e.g., prior to the onset of a disease, disorder, condition or
symptom thereof) so as to prevent, suppress, inhibit or reduce,
either temporarily or permanently, a subject's risk of developing a
disease, disorder, condition or the like (as determined by, for
example, the absence of clinical symptoms) or delaying the onset
thereof, generally in the context of a subject predisposed to
having a particular disease, disorder or condition. In certain
instances, the terms also refer to slowing the progression of the
disease, disorder or condition or inhibiting progression thereof to
a harmful or otherwise undesired state.
[0047] The term "in need of prevention" as used herein refers to a
judgment made by a physician or other caregiver that a subject
requires or will benefit from preventative care. This judgment is
made based on a variety of factors that are in the realm of a
physician's or caregiver's expertise.
[0048] A "therapeutically effective amount" or "efficacious amount"
refers to the amount of an agent (e.g., an HSP90-encoding nucleic
acid) that, when administered to a mammal or other subject for
treating a disease, is sufficient to effect such treatment for the
disease. The "therapeutically effective amount" will vary depending
on the agent (e.g., the HSP90-encoding nucleic acid and/or the
expression construct used to delivery the HSP90-encoding nucleic
acid), as well as patient factors such as the disease and its
severity and the age, weight, etc., of the subject to be
treated.
[0049] The phrase "therapeutically effective amount" encompassed
administration of an agent (e.g., an HSP90-encoding nucleic acid)
to a subject, either alone or as part of a pharmaceutical
composition and either in a single dose or as part of a series of
doses, in an amount capable of having any detectable, positive
effect on any symptom, aspect, or characteristic of a disease,
disorder or condition when administered to the subject. The
therapeutically effective amount can be ascertained by measuring
relevant physiological effects, and it can be adjusted in
connection with the dosing regimen and diagnostic analysis of the
subject's condition, and the like.
DETAILED DESCRIPTION OF EXAMPLES OF EMBODIMENTS
[0050] The present invention is based on the discovery that
DNA-encoding HSP90 is effective as a therapeutic in the treatment
of subjects who are HLA-B27 positive and have, or at risk of, an
autoimminue inflammatory disease associated with HLA-B27 positive
status.
Heat Shock Protein 90 (HSP90)
[0051] In general, the methods of the present disclosure involve
administration of a nucleic acid encoding a heat shock protein 90
(HSP90) to a subject so that the HSP90-encoding nucleic acid
provides for production of HSP90 protein in the host. Such
constructs can be a DNA construct in which the HSP90-encoding DNA
is operably linked to a promoter that facilitates expression in the
subject.
[0052] The HSP90 encoded by the nucleic acid of the construct can
be any suitable HSP90, or an active fragment thereof, e.g., a
mammalian HSP90 or active fragment thereof, e.g., a human HSP90,
rat HSP90, or mouse HSP90, or an active fragment thereof. The
nucleic acid sequence encoding the mammalian heat shock proteins
may include DNA, RNA, or derivatives of either DNA or RNA with the
proviso such are amenable to production of the encoded HSP90
protein gene product in a host cell, e.g., a human cell. The
nucleic acid sequence encoding the HSP90 can be obtained from a
natural source, either as encoding a full-length HSP90 or a portion
thereof. A nucleic acid molecule can also be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Nucleic acids
encoding HSP90 include naturally-occurring HSP90-encoding nucleic
acids and homologues thereof, including, but not limited to,
natural allelic variants and modified nucleic acid sequences in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications do not
substantially interfere with the nucleic acid molecule's ability to
provide for production a functional HSP90 or an active fragment
thereof in a cell of the subject to whom it is administered.
[0053] Where the subject is human, a nucleic acid encoding a human
HSP90 (either full-length or an active fragment thereof) is of
particular interest. The human HSP90 can be either the alpha
isoform or the beta isoform. The amino acid sequences of examples
of human HSP0 proteins suitable for use in the present methods
include, for example, NCBI reference sequence NP_001017963 (heat
shock protein HSP 90-alpha isoform 1) and NCBI reference sequence
NP_005339.3 (heat shock protein HSP 90-alpha isoform 2). The amino
acid sequence of an example of a full-length human HSP90 protein is
provided below:
TABLE-US-00001 (SEQ ID NO: 1)
MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELIS
NSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTK
ADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTV
ITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEE
RRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEEKEDKEEEKEKEEK
ESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRN
PDDITNEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFVPRRAPFD
LFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISR
EMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIH
EDSQNRKKLSELLRYYTSASGDEMVSLKDYCTRMKENQKHIYYITGETKD
QVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLE
LPEDEEEKKKQEEKKTKFENLCKIMKDILEKKVEKVVVSNRLVTSPCCIV
TSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKA
EADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDE
DDPTADDTSAAVTEEMPPLEGDDDTSRMEEVD.
[0054] Any suitable constructs to facilitate delivery of the
HSP-encoding nucleic acid into a host cell, particularly a human
host cell, to facilitate expression in the host cell can be used.
Suitable constructs include plasmids.
[0055] In some embodiments, the HSP90-enocoding construct does not
contain a CpG motif, which may be defined by the generic formula
5'-X.sub.1CGX.sub.2-3', wherein X1 and X2 represent any nucleotide,
and the the central CG dinucleotide is unmethylated. Examples of
CpG motif-containing sequences include nucleic acid having a
sequence of the formulae 5'-RRCGYY-3', 5'-RTCGYY-3',
5'-RRCGYYCG-3', 5'-RTCGYYCG-3', wherein the "CG" are unmethylated
CpG dinucleotides, R represents a purine (A or G) and Y represents
a pyrimidine (C or T).
[0056] The HSP90-encoding constructs generally include a nucleic
acid sequence encoding an HSP90 (or active fragment thereof), e.g.,
a human HSP90, operatively linked to one or more transcription
control elements in an expression construct. Transcription control
elements include nucleic acids having sequences that facilitate
control of initiation, elongation, and termination of
transcription. Transcription control elements include promoters,
enhancers, operators and repressors. Of particular interest are
transcription control elements that facilaite expression in a
mammalian cell, particularly a human cell. Promoters of interest in
the present constructs include constitutive promoters, inducible,
promoters, and tissue-specific promotoers, with constitutive
promoters being of particular interest. Examples of promoters that
can find use in the constructs of the present of disclosure to
provide for expression of HSP90 in a subject include, but at not
necessarily limed to, viral promoters, such as CMV promoters, RSV
promoters, retroviral promoters, and SV-40 promoters.
[0057] Examples of eukaryotic expression constructs for use in the
methods of the present disclosure include: pcDNA3, pcDNA3.1(+/-),
pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto,
pCR3.1, pCI, pBK-RSV, pBK-CMV, pTRES or their derivatives.
Formulations
[0058] The HSP90-encoding constructs can be provided in any
formulation suitable for administration to a subject, e.g.,
suitable for administration of a human subject.
[0059] Compositions suitable for administration to a subject can
include the HSP90-encoding construct and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" includes any material which, when combined with an
HSP-encoding construct of the present disclosure, retains the
biological activity and improves stability of the construct without
causing significant adverse reactions. Examples include, but are
not limited to, any of the standard carriers such as an isotonic
solution of a pharmaceutically acceptable alkali metal salt (e.g.,
sodium chloride), a phosphate buffered saline (PBS) solution,
water, Ringer's solution, dextrose solution, emulsions such as
oil/water emulsion, and various types of wetting agents. Aqueous
carriers can contain suitable auxiliary substances required to
approximate the physiological conditions of the recipient, for
example, by enhancing chemical stability and isotonicity. Examples
of additives of interest in formulation of the present disclosure
include, but are not limited to, sodium citrate, malic acid,
ethanol, and PLURONIC F-68.RTM.. Additional examples of additives
include, for example, pharmaceutically acceptable salts (e.g.,
sodium acetate, sodium lactate, potassium chloride, calcium
chloride), and preservatives. In general formulations are
sterile.
[0060] Compositions of interest a include a sterile formulation
comprising HSP90-encoding nucleic acid (e.g., HSP90-encoding DNA,
e.g., in an expression construct operably linked to a promoter,
such as a strong constitutive promoter (e.g., CMV)) formulated with
0.9% pharmaceutically acceptable alkali metal salt (e.g., sodium
salt, e.g., NaCl).
[0061] HSP90-encoding nucleic acid can optionally formulated with a
lipid carrier, e.g., so as to be provided as a payload in a
stabilized lipid particle, as in a complex with a catioinic lipid,
e.g., in a liposome or micelle. In such embodiments, the lipid
carrier can be modified (e.g., by manipulating the chemical formula
of the lipid portion of the delivery vehicle and/or providing a
compound capable of targeting the lipid to a target site, for
example, a target cell type so as to provide for interaction of
lipid carrier (or, for example, other liposome component) with a
molecule on the surface of the cell. Suitable targeting compounds
include ligands capable of selectively binding another molecule at
a particular site. Examples of such ligands include antibodies,
antigens, receptors and receptor ligands.
[0062] HSP90-encoding nucleic acids can optionally be formulated
with a suitable polymer, such as polyethylene glycol, polylysine,
poloxamer, Chitosan, polyL glutamate, poly(lactide-co-glycolide)
(PLG).
[0063] The components of the formulations of the HSP90-encoding
nucleic acid can be selected according to a variety of factors,
such as the route of administration. For example, where the
formulation is to be administered transdermally or topically, the
formulation can include absorption enhancers (e.g., DMSO) or other
components to promote transport of the nucleic acid across the
skin.
[0064] In some embodiments, the HSP90-encoding nucleic acid is not
encapsulated (e.g., within a liposome). In some the HSP90-encoding
nucleic acid is not contained in a viral particle. In some
embodiments, the nucleic acid is "naked", i.e., is not encapsulated
(e.g., within a liposome) and is not contained in a viral
particle.
[0065] The HSP90-encoding nucleic acid construct may be provided in
a sterile container (e.g., a syringe) and, optionally, may be
lyophilized and reconstituted (e.g., with sterile PBS) prior to
administration.
Subjects for Treatment
[0066] The methods of the present disclosure generally involve
treatment of HLA-B27 positive human subjects who have, or who are
at risk of, an autoimmune inflammatory disease. "HLA-B27-associated
disease" refers to the observation that bearing the HLA-B27 allele
carries a statistically significant increased risk of developing
the disease compared to persons who do not bear the HLA-B27 allele.
HLA-B27 positive subjects include those who express any subtype of
HLA-B27, e.g., any of HLA-B27 subtypes B*2701-2759, with the
subtype HLA-B*2705 being of particular interest.
[0067] HLA-B*27:05 (specifically the HLA-B*27:0502 allele) is the
most widely distributed disease-associated subtype in the world.
Other common disease-associated subtypes include HLA-B*27:02
(Mediterranean populations) and HLA-B*27:04 (Chinese and other
Asian populations). HLA-B*27:01, HLA-B*27:03, HLA-B*27:07,
HLA-B*27:08, HLA-B*27:10, HLA-B*27:13, HLA-B*27:14, HLA-B*27:15,
HLA-B*27:19, HLA-B*27:23, HLA-B*27:24, HLA-B*27:25 and HLA-B*27:49
have also been implicated as associated with disease. It will be
understood that ancestry can influence the strength of association
of an HLA-B27 subtype and disease incidence. It should be noted
that HLA-B*27:05 and HLA-B*27:02 confer about equal susceptibility
to ankylosing spondylitis (AS) in Caucasian populations. Among
Chinese populations, HLA-B*27:04 has been demonstrated to show a
stronger association with AS than HLA-B*27:05.
[0068] HLA-B27 status (i.e., HLA-B27 positive or HLA-B27 negative)
can be determined by any suitable method accepted in the art for
determining HLA status. For example, HLA-B27 status can be
determined by detection of the HLA-B27 antigen in a sample (e.g.,
blood sample) obtained from the subject, e.g., using a suitable
commercially available detection reagent and method (e.g., an
anti-HLA-B27 antibody, and in an ELISA or flow cytometry assay). An
example of a suitable assay is the ELISA sandwich assay available
from LSBio (LifeSpan BioSciences, Inc.) (Human HLA-B27 ELISA Kit,
Catalog No. LS-F21903) for antibody-based detection of HLA-B27 in a
plasma or serum sample of a human subject.
[0069] A HLA-B27 positive status has been associated with a number
of autoimmune inflammatory diseases, and thus the methods of the
present disclosure contemplates treatment of individuals having, or
at risk of having, such HLA-B27-associated autoimmune inflammatory
diseases. HLA-B27 positive subjects may be either male or female,
and may be of any age, including an age at which risk of onset of
an HLA-B27-associated autoimmune inflammatory disorder is increased
in an HLA-B27 subject as compared to an HLA-B27 negative
subject.
[0070] Examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include inflammatory joint diseases, such as
spondyloarthropathies (e.g., seronegative spondyloarthropathies
(where the subject is seronegative for rheumatoid factor). Examples
of HLA-B27-associated spondyloarthropathies include anklyosing
spondylitis, reactive arthritis, psoriatic arthritis, enteropathic
arthritis, undifferentiated spondyloarthropathy, and juvenile
idiopathic arthritis (JIA, including late-onset oligoarticular
JIA). Other examples of HLA-B27-associated inflammatory joint
diseases include juvenile spondarthritis, undifferentiated
spondarthritis or isolated peripheral enthesitis. Notably, not all
subjects who have such autoimmune inflammatory joint diseases are
HLA-B27 positive. In other words not all subjects having, for
example, axial spondylitis (e.g., anklyosing spondylitis), reactive
arthritis, psoriatic arthritis and enteropathic are HLA-B27
positive, and thus HLA-B27 positive subjects are a subset of
subjects having these diseases.
[0071] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include ocular disorders, such as uveitis, e.g.,
anterior uveitis (e.g., acute anterior uveitis (AAU)), posterior
uvetitis, age-related macular degeneration (ARMD), conjunctivitis,
keratitis, vitritis, retinal vasculitis, dry eye, keratitis,
episcleritis/scleritis and peripheral ulcerative keratitis).
Uvetitis may be present in a subject having or at risk of
ankylosing spondylitis, reactive arthritis (also known as Reiter's
syndrome), undifferentiated spondyloarthropathy, or psoriatic
arthropathy.
[0072] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include inflammatory disease of the
gastrointestinal tract such as inflammatory bowel disease,
ulcerative colitis, or Crohn's disease. Notably, not all subjects
who have such autoimmune inflammatory gastrointestinal diseases are
HLA-B27 positive. In other words not all subjects having
inflammatory bowel disease, ulcerative colitis, or Crohn's disease
are HLA-B27 positive, and thus HLA-B27 positive subjects are a
subset of subjects having these diseases.
[0073] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include HLA-B27-associated autoimmune
inflammatory skin disease, such as psoriasis associated with
psoriatic arthritis, which may be characterized by psoriasiform
skin lesions and/or nail lesions.
[0074] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include Behcet disease.
[0075] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the methods of the
present disclosure include cardiovascular disease, e.g., a cardiac
syndrome that involves severe conduction system abnormalities plus
aortic regurgitation. See, e.g., Bergfeldt (1997) Ann Intern Med.
127:621-629.
[0076] Other examples of HLA-B27-associated autoimmune inflammatory
diseases amenable to treatment according to the present methods
include fibrosis, e.g., pulmonary fibrosis, e.g., upper lobe
pulmonary fibrosis.
[0077] Subjects amenable to therapy according to the methods of the
present disclosure include those who present with one or more
HLA-B27-associated autoimmune inflammatory diseases, e.g., such
that the subject presents symptoms of both an autoimmune
inflammatory joint disease and an autoimmune inflammatory
gastrointestinal disease. For examples, subjects having have an
enteropathic arthropathy or spondylitis have an autoimmune
inflammatory joint disease that is associated with, e.g., Crohn's
disease or ulcerative colitis. In another example, subjects may
have an autoimmune inflammatory ocular disease, such as uveitis,
which is associated with an autoimmune inflammatory joint disease.
Examples of HLA-B27 autoimmune inflammatory diseases that can be
associated, e.g., occur in the same subject where a second disease
may occur on or after onset of a first disease, are provided in the
table below.
TABLE-US-00002 TABLE Examples of Associations of HLA-B27-Associated
Autoimmune Inflammatory Diseases Condition Ocular Involvement
Ankylosing Spondylitis Acute anterior uveitis (AAU) Conjunctivitis
Reiter's Syndrome and Reactive Conjunctivitis Arthritis Anterior
uveitis Keratitis Undifferentiated Spondyloarthropathy Anterior
uveitis (acute or chronic) Vitritis Retinal Vasculitis Psoriatic
Arthropathy Anterior uveitis Conjunctivitis Dry eye Keratitis
Inflammatory Bowel Disease Conjunctivitis Episcleritis/Scleritis
Anterior Uveitis Vitritis Retinal vasculitis Peripheral ulcerative
keratitis
Treatment Regimen
[0078] The HSP90-encoding constructs of the present disclosure are
generally administered to an HLA-B27 positive subject having, or at
risk of, an autoimmune inflammatory disorder in an amount and for a
period of time to provide for improvement in at least one disease
symptom. As will be appreciated, the dosage regiment will vary
according to a number of factors including, but not limited to, the
HLA-B27-associated disease to be treated, severity of the disease,
the age and health of the patient, and the like.
[0079] The HSP90-encoding constructs may be administered according
to any suitable conventional and pharmaceutically acceptable route
of administration. Such routes include any suitable parenteral
route, including by injection or transdermal delivery, e.g.,
intramuscular injection, subcutaneous injection, intradermal
injection, topical administration, and intravenous administration.
Enteral routes of administration are also contemplated (e.g.,
intranasal and other routes of effective inhalation routes).
Administration by injection, particularly by intramuscular
injection is of particular interest.
[0080] The HSP90-encoding construct can be administered in a single
dose, or in multiple doses, and may encompass administration of
additional doses, to elicit and/or maintain the desired effect. The
amount administered (e.g., by injection, e.g., by intramuscular or
subcutaneous injection) can be from about 0.001 mg/kg body weight
to about 1 mg/kg body weight, from about 0.005 mg/kg body weight to
about 0.5 mg/kg body weight, from about 0.01 mg/kg body weight to
about 0.4 mg/kg body weight, from about from about 0.20 mg/kg body
weight to about 0.30 mg/kg body weight, of HSP90-encoding nucleic
acid. Doses of HSP90-encoding nucleic acid can be from about 0.1 mg
to about 30 mg, from about 0.2 mg to about 25 mg, from about 0.3 mg
to about 20 mg, or from about 0.5 mg to about 15 mg per subject
(e.g., where subject is from about 65 kg to about 70 kg). In some
embodiments the dose of HSP90-encoding nucleic acid administered is
about 0.20 mg/kg to about 0.30 mg/kg, or 0.25 mg/kg of
HSP90-encoding nucleic acid per body weight of the subject. In some
embodiments, the dose of HSP90-encoding nucleic acid is from about
0.2 mg to about 20 mg, or from about 0.5 mg to about 15 mg per
subject. For example, the dose can be from about 0.001 to 1 mg/kg
corresponding to 0.06 to 65 mg per subject for a body weight of 65
kg subject.
[0081] The HSP90-encoding nucleic acid can be administered daily,
every other day, twice per week, once per week, every other week,
or once per month, with weekly doses being of particular interest.
The HSP90-encoding nucleic acid can be administered for a period of
at least 3 days, at least 5 days, at least 1 week, at least 2
weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or at
least 6 weeks or more.
EXAMPLES
[0082] The following examples are provided in order to demonstrate
and further illustrate certain embodiments and aspects of the
present invention and are not to be construed as limiting the scope
thereof.
Methods and Materials
Animals
[0083] Human leukocyte antigen B27 (HLA-B27) transgenic rats, which
are transgenic for HLA-B*2705 and human 32-microglobulin, have
previously been described, for example by Taurog et al. (J. Exp Med
1994 180:2359-64) and Hammer et al. (Cell 1990 63:1099-1112).
Transgenic rats, overexpressing the human HLA-B27 and
.beta.2-microglobulin proteins, develop lesions of the
gastrointestinal system, the joints, the skin, and the gonads,
which seem similar to the spondyloarthropathies in humans that have
been associated with the HLA-B27 and .beta.2-microglobulin genes.
The gastrointestinal inflammation is mostly limited to the mucosa
and submucosa, exhibiting histological features similar to those
present in inflammatory bowel disease (IBD) (Taurog et al. J. Exp
Med 1994 180:2359-64; Hammer et al. Cell 1990 63:1099-1112; Sartor
et al. Baillieres Clin Rheumatol 1996 10:55-76; Breban et al. Ann
Med Interne (Paris) 1998 149:139-141). Chronic intestinal
inflammation is the first to occur, with clinical signs of diarrhea
apparent after 12 weeks of age.
[0084] The HLA-B27 transgenic rats uniformly develop a progressive
inflammatory disease similar to the HLA B27-associated human
spondyloarthropathies (e.g., anklyosing spondylitis, reactive
arthritis, and arthritis associated with inflammatory bowel
disease), including colitis, enteritis, arthritis, male genital
inflammation, and psoriasiform skin and nail lesions. HLA-B27
transgenic rats developed inflammatory disease involving the
gastrointestinal tract, peripheral and vertebral joints, male
genital tract, skin, nails, and heart. Accordingly, the HLA-B27
transgenic rat is an art-accepted model of human HLA-B27-associated
inflammatory diseases.
[0085] Male and female HLA-B27 rats were raised under pathogen-free
conditions in the accredited facilities at Institut Pasteur in
Lille and those of the Nord-Pas de Calais Ethical Committee for
animal use. The animals were maintained at an ambient temperature
of 20.+-.5.degree. C. with relative humidity of 50.+-.20%. The
animal housing were maintained at a light and dark cycle of 12
hours each and the ventilation was set to approximately 12
cycles/hour using filtered, non-recycled air.
[0086] Five animals were housed per cage with free access to
standard mice chow and tap water. The experiments were performed
using animals that were eight-week old at the beginning of the
experiments. Representative eight week old animals weighted 100 g
and representative twelve week old animals weighed 280 g. The
experiments were performed and the animals were maintained
according to governmental guidelines (articles R214-87-R214-137
code rural update 13 Feb. 2013 according to European directive
2010/63/UE).
[0087] The animals were randomized in two groups. At the beginning
of the study, each animal received a unique identity number. The
animals were individually identified by an ear tattoo at the
breeder's facility. Eight animals were randomly assigned to group 1
and another eight animals were randomly assigned to group 2. Group
1, the control group, was treated with saline while Group 2, the
experimental group, was treated with DNA-encoding human HSP90 (FIG.
1).
[0088] Animals were euthanized by cervical dislocation at day 3
following the last injection upon completion of the treatment (FIG.
1).
DNA Plasmids and Buffers, Injection Protocol
[0089] DNA-encoding HSP90 was provided as a construct encoding
human HSP90 (alpha isoform) in the vector pCDNA3 (obtained from
Delphi Genetics SA (batch number DE 160043-160044)) and stored at
4.degree. C. in buffer until ready for use.
[0090] Plasmid DNA was prepared by diluting in DNAse/RNAse free PBS
buffer pH 7.5 to a concentration of 6 mg/ml and kept at 4.degree.
C. during the whole treatment period. Sampling of the solution were
made in aseptic conditions using sterile and pyrogen-free material.
Briefly, as illustrated in FIG. 1, eight-week old rats were treated
weekly over the course of five weeks by intramuscular injection of
200 uL of 0.9% NaCl (Group 1) or 200 uL of 0.25 mg/kg of HSP90
(Group 2). Intramuscular injections were performed on the right and
left legs, alternatively following skin preparation with ethyl
alcohol swabs to reduce skin bacterial counts.
Clinical Examination
[0091] Each animal was checked for clinical signs once a week over
the course of the five week treatment period. Each animal's body
weight was recorded twice a week starting at the first week of the
treatment period until euthanasia.
Example 1: Effect of HSP90-Encoding DNA on Colonic Inflammation
[0092] As discussed above, eight-week old rats were treated weekly
over the course of five weeks by intramuscular injection of 200 uL
of 0.9% NaCl (Group 1, saline control) or 200 uL of 0.25 mg/kg of
HSP90 (Group 2). Animals were euthanized at day 3 following the
last injection upon completion of the treatment.
[0093] To evaluate the effect of HSP90 on colonic inflammation,
several parameters were monitored daily during the five-week
treatment period. The parameters were assigned scores and the
scores were used to calculate a disease activity index (DAI)
ranging between 0 and 5. The parameters used included weight loss,
stool consistency, and blood occurrence in the animals' stool. No
weight loss was assigned a score of 0, less than 10% weight loss
was assigned a score of 1 and 10% or more weight loss was assigned
a score of 2. A score of 0 was assigned for regular stool
consistency, a score of 1 for soft stool consistency and a score of
2 for diarrhea. Blood occurrence observed in the animal's feces was
assigned a score of 1 while the absence of blood was assigned a
score of 0. DAI was calculated by adding the scores for the
parameters for weight loss, stool consistency and blood occurrence.
Table 1 provides a summary of the parameters in calculating
DAI.
TABLE-US-00003 TABLE 1 Scoring Disease Activity Index Parameters
Scores Disease Weight loss 0: no; 1: <10%; 2: .gtoreq.10%
Activity Index Stool consistency 0: regular; 1: soft; 2: diarrhea
(DAI) Blood occurrence 0: absence; 1: presence (0-5)
[0094] FIG. 2 provides the results of the effect of HSP90-encoding
DNA on weight. As shown in FIG. 2, there was no significant
difference for the body weight evolution in the group of rats
treated with DNA encoding HSP90 as compared to control rats.
[0095] FIGS. 3A and 3B provide the results of analysis of stool
consistency. As expected in HLA-B27 rats, a high score for the
stool consistency (diarrhea) was observed since the beginning of
the study. No significant difference in this parameter was observed
in HLA-B27 treated rats compared to control HLA-B27 rats. In
HLA-B27 rats treated with HSP-90 at 0.25 mg/kg, no improvement of
this parameter was observed even in the end of the study.
Example 2: Evaluation of Effect of HSP90-Encoding DNA on Intensity
of Colonic Lesions at the Macroscopic Level
[0096] To determine the intensity of inflammation in the colon,
several parameters were scored after euthanasia of the animals, 3
days after the last week of treatment (week 12+3 days). The
parameters were assigned scores and the scores were used to
calculate a score ranging between 0 and 11 to represent macroscopic
evaluation of the colonic lesions. The parameters included mucosal
lesions, colonic hypervascularization, colon wall thickness, and
mesentery thickness. Mesentery thickness refers to the region of
the mesentery to which the sigmoid colon is attached at the
gastrointestinal mesenteric margin. The scoring strategies for the
visual assessment of these parameters are summarized in the table
below.
TABLE-US-00004 TABLE 2 Scoring Macroscopic Evaluation of Colonic
Lesions Parameters Score Macroscopic Mucosal lesions 0: Absence
evaluation of 1: Congestive leiong, erythema the colonic 2:
Ulceration <20%, no stenosis lesions 3: Ulceration .gtoreq.20%
no stenosis (0-11) 4: Stenosis Colonic 0: Absence
hypervascularization 1: Moderate 2: Diffuse Colon wall thickness 0:
Normal 1: Weak, 1-2 mm 2: Moderate, 2-3 mm 3: Important, >3 mm
Thickness of the 0: Absence mesocolon and 1: Moderate
intra-abdominal 2: Diffuse mesenteric lymph nodes
[0097] The results are summarized in the table below.
TABLE-US-00005 TABLE 3 Analysis of Parameters for Global
Macroscopic Score ID hyper- colonic num- Mucosal vascular- wall
mesentery Group Sex ber lesions ization thickness thickness Control
M 208 2 1 2 1 1M PBS M 209 0 1 2 2 M 211 0 0 0 0 F 210 0 0 2 1 F
212 0 1 1 1 F 213 0 0 1 0 M 215 0 1 2 2 F 220 0 2 2 1 HSP90 M 198 0
0 0 0 0.25 M 200 0 0 1 0 mg/kg F 201 0 0 1 1 1M 1X/ F 204 0 0 1 0
week F 205 2 0 1 0 F 207 0 0 1 0 M 218 0 0 1 0 F 222 0 0 1 0
[0098] As shown in FIG. 4A, HSP90 treatment significantly decreased
the hypervascularization as compared to control. In addition, HSP90
treatment significantly decreased the thickness of mesentery as
compared to control (FIG. 4C). In addition, a strong trend to
decrease the thickness of the colonic wall was observed in HSP90
treated rats compared to control rats (FIG. 4B). As reflected by
the global macroscopic score in shown in FIG. 4D, animal treated
with HSP90-encoding DNA exhibited significantly improvement in
inflammation as compared to controls.
Example 3: Evaluation of Effect of HSP90-Encoding DNA on Arthritis
in the HLA-B27 Transgenic Rats
[0099] To determine the intensity of arthritis, clinical signs of
arthritis in the tarsal joins were monitored daily in all the
animals. The clinical signs monitored were swelling and erythema of
the hindpaws. Swelling was assessed visually and each animal
assigned a score ranging from 0 to 3, a score of 0 was considered
normal, a score of 1 was assigned for mild swelling, a score of 2
assigned for moderate swelling, and a score of 3 assigned for
severe swelling. The same scoring strategy was followed for
erythema of the hindpaws (normal=0, mild=1, moderate=2,
severe=3).
[0100] The results of assessment of swelling are shown in FIGS. 5A
and 5B. As shown in FIGS. 5A and 8B, a trend toward a decrease in
the signs of arthritis was observed in HLA-B27 treated rats as
compared to control HLA-B27 rats at day 24 and day 31. In HLA-B27
treated rats an improvement in these signs of arthritis was also
observed was observed at day 17 and day 21 of treated animals as
compared to the beginning of the study.
Example 4: Evaluation of Effect of HSP90-Encoding DNA on
Hematology
[0101] For hematology, blood was collected from each animal before
the first IM injection (d-1) and at the time of euthanasia (d30)
and the following hematology parameters assessed: haematocrit, mean
glomerular volume, mean corpuscular hemoglobin content, mean
corpuscular hemoglobin concentration, and variability of the size
of red cells.
[0102] Prior to the first injection, no significant differences
regarding the different analyzed parameters between the treatment
group of HLA-B27 rats and the control group of HLA-B27 rats were
observed, indicating a good homogeneity among the animals in the
test and control groups. At day 30, following the 5 injections of
HSP90-encoding DNA or saline control, no significant differences
were observed, indicating that HSP90 administration did not have a
significant effect on the different studied parameters compared to
injection of saline control.
[0103] In the control HLA-B27 rats, a significant difference was
observed for the IDR % at day 30 as compared to prior to injection.
In the HLA-B27 rats treated with HSP90-encoding DNA, a significant
difference was observed in the number of red corpuscules, the level
of hemoglobin, the hematocrit, the CCMH and IDR % at day 30
compared to the levels observed prior to the first injection.
* * * * *