U.S. patent application number 15/108653 was filed with the patent office on 2016-11-10 for methods and compositions relating to p62/sqstm1 for the treatment and prevention of inflammation-associated diseases.
The applicant listed for this patent is Dimitrios AGAS, Antonio CONCETTI, CURELAB ONCOLOGY, INC., Vladimir GABAI, Maria Giovanna SABBIETI, Michael SHERMAN, Victor SHIFRIN, Alexander SHNEIDER, Franco VENANZI. Invention is credited to Dimitrios Agas, Antonio Concetti, Vladimir Gabai, Maria Giovanna Sabbieti, Michael Sherman, Victor Shifrin, Alexander Shneider, Franco Venanzi.
Application Number | 20160324944 15/108653 |
Document ID | / |
Family ID | 53479722 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160324944 |
Kind Code |
A1 |
Shneider; Alexander ; et
al. |
November 10, 2016 |
Methods and compositions relating to p62/SQSTM1 for the treatment
and prevention of inflammation-associated diseases
Abstract
Provided herein are novel p62 compositions for the modulation of
expression of a proinflammatory cytokines, osteogenic transcription
factors, a bone resorptive factors and endogenous p62.
Consequently, such p62 compositions are useful for prophylaxis and
treatment of inflammatory diseases and related methods. In certain
embodiments the inflammatory diseases are not cancer-related. In
various embodiments, the inflammatory diseases include, but are not
limited to osteoporosis, obesity, metabolic syndrome, type 2
diabetes, fat liver, inflammatory bowel disease, chronic
pancreatitis, asthma, chronic obstructive pulmonary disease (COPD),
rheumatoid arthritis (RA), osteoarthritis, multiple sclerosis (MS),
psoriasis, congestive heart failure (CHF), atherosclerosis,
neurodegenerative diseases (ALS, Parkinson, Alzheimer's, Huntington
disease), depression, schizophrenia, gout, asbestosis and
silicosis.
Inventors: |
Shneider; Alexander;
(Needham, MA) ; Venanzi; Franco; (Camerino,
IT) ; Agas; Dimitrios; (Camerino, MC, IT) ;
Concetti; Antonio; (Camerino, IT) ; Sabbieti; Maria
Giovanna; (Camerino, IT) ; Gabai; Vladimir;
(Brighton, MA) ; Sherman; Michael; (Newton,
MA) ; Shifrin; Victor; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VENANZI; Franco
CONCETTI; Antonio
SABBIETI; Maria Giovanna
AGAS; Dimitrios
GABAI; Vladimir
SHIFRIN; Victor
SHERMAN; Michael
SHNEIDER; Alexander
CURELAB ONCOLOGY, INC. |
Camerino, MC
Falerone, FM
Camerino, MC
Camerino,MC
Brighton
Newton
Newton
Needham
Oedham |
MA
MA
MA
MA
MA |
IT
IT
IT
IT
US
US
US
US
US |
|
|
Family ID: |
53479722 |
Appl. No.: |
15/108653 |
Filed: |
December 29, 2014 |
PCT Filed: |
December 29, 2014 |
PCT NO: |
PCT/US14/72484 |
371 Date: |
June 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61921504 |
Dec 29, 2013 |
|
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61949597 |
Mar 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/573 20130101;
A61P 25/28 20180101; A61K 39/0008 20130101; C07K 14/47 20130101;
A61K 31/436 20130101; A61P 29/00 20180101; C07K 14/545 20130101;
C07K 16/245 20130101; C07K 14/525 20130101; A61K 2039/55566
20130101; C07K 14/5412 20130101; C07K 14/523 20130101; A61K 2039/53
20130101; C07K 16/248 20130101; A61K 38/1709 20130101; A61P 37/02
20180101; C12N 15/62 20130101; A61P 37/06 20180101; A61K 39/0005
20130101; A61K 45/06 20130101; A61K 31/519 20130101; A61K 38/1709
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06 |
Claims
1-27. (canceled)
28. A method to treat, alleviate, ameliorate, relieve, delay onset
of, inhibit progression of, reduce severity of, or reduce incidence
of one or more symptoms of a non-cancer-related chronic
inflammatory disease in a subject comprising administering to said
subject an agent comprising: a. at least 30 consecutive amino acids
of a p62/SQSTM1 polypeptide; b. a p62/SQSTM1 encoding nucleic acid,
wherein said p62/SQSTM1 encoding nucleic acid encodes at least 30
consecutive amino acids of a p62/SQSTM1 polypeptide; c. a
p62/SQSTM1 polypeptide at least 90% identical to SEQ ID NO. 2; d. a
p62/SQSTM1 polypeptide with at least one or more domain deletions;
e. a p62/SQSTM1 nucleic acid that encodes a polypeptide at least
90% identical to SEQ ID NO: 2; or f. a p62/SQSTM1 nucleic acid that
encodes a polypeptide with at least one or more domain
deletions;
29. The method according to claim 28, wherein said agent comprises
a p62/SQSTM1 polypeptide that is at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% homologous to SEQ. ID. NO. 2.
30. The method according to claim 28, wherein said agent comprises
a p62/SQSTM1 nucleic acid that encodes a polypeptide that is at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
homology to SEQ. ID. NO. 2.
31. The method according to claim 28, wherein said p62/SQSTM1
nucleic acid comprises the sequence of SEQ ID NO:1.
32. The method according to claim 28, wherein said p62/SQSTM1
nucleic acid that encodes a polypeptide with at least one or more
domain deletions comprises a domain deletion selected from the
group consisting of: PB1, ZZ, NLS2, TB, NLS1, NES, LIR, KIR, and
UBA.
33. The method according to claim 28, wherein said agent comprises
a p62/SQSTM1 nucleic acid and further comprises a plasmid, RNA or a
viral vector.
34. The method according to claim 28, wherein said p62/SQSTM1
polypeptide or p62/SQSTM1 encoding nucleic acid further comprises a
fusion polypeptide or nucleic acid encoding for a fusion
polypeptide, respectively.
35. The method according to claim 28, wherein said agent comprises
a vaccine.
36. The method according to claim 35, further comprising
administering an adjuvant to said subject.
37. The method according to claim 36, wherein said adjuvant is
selected from the group consisting of: gel-type, microbial,
particulate, oil-emulsion, surfactant-based, and synthetic
adjuvant.
38. The method according to claim 28, wherein said non-cancer
chronic inflammatory disease is selected from the group consisting
of: obesity, metabolic syndrome, type 2 diabetes, fat liver,
Crohn's Disease, pancreatitis, asthma, chronic obstructive
pulmonary disease, arthritis, osteoporosis, osteoarthritis,
multiple sclerosis, psoriasis, congestive heart failure,
atherosclerosis, neurodegenerative disease, depression,
schizophrenia, gout, asbestosis, and silicosis.
39. The method according to claim 38, wherein said
neurodegenerative disease is selected from the group consisting of:
amyotrophic lateral sclerosis, Parkinson's disease, Huntington's
disease, and Alzheimer's disease.
40. The method according to claim 28, further comprising
administering an anti-inflammatory therapy to said subject.
41. The method according to claim 28, wherein said agent comprises
a p62/SQSTM1 encoding nucleic acid, further comprising a strategy
for improving the efficiency of nucleic acid-based expression of
p62/SQSTM1 in subjects.
42. The method according to claim 41, wherein said strategy is
selected from the group consisting of: including a self-replicating
viral replicon, codon optimization, in vivo electroporation,
incorporation of a CpG stimulatory motif, including a sequence for
targeting of the endocytic or ubiquitin-processing pathways,
including a Marek's disease virus type 1 VP22 sequence, a
prime-boost regimen, a mucosal delivery vector, and a nucleic acid
delivery system.
43. The method according to claim 42, wherein said nucleic acid
delivery system is selected from the group consisting of: a polymer
gene delivery system, a liposomal delivery system, and a
cell-penetrating peptide gene delivery system.
44. The method according to claim 28, further comprising
administering an anti-inflammatory chemotherapeutic or biological
agent.
45. The method according to claim 44, wherein said chemotherapeutic
agent is selected from the group consisting of: a nonsteroidal
anti-inflammatory drug, a glucocorticoid, methotrexate,
cyclosporine, and rapamycin.
46. The method according to claim 45, wherein said
anti-inflammatory biological agent is selected from the group
consisting of: an anti-TNF antibody, an anti-IL1 antibody, an
anti-IL6 antibody, an anti-IL6 receptor antibody, an anti-IL12/23
antibody, an anti-IL17 antibody, an anti-IL1R antibody, an anti-IL1
receptor antagonist, and a soluble IL-1 receptor.
47. A method to modulate in a subject the expression of: a. a
proinflammatory cytokine; b. an osteogenic transcription factor; or
c. a bone resorptive factor comprising administering to said
subject an agent comprising: a. at least 30 consecutive amino acids
of a p62/SQSTM1 polypeptide; b. a p62/SQSTM1 encoding nucleic acid,
wherein said p62/SQSTM1 encoding nucleic acid encodes at least 30
consecutive amino acids of a p62/SQSTM1 polypeptide; c. a
p62/SQSTM1 polypeptide at least 90% identical to SEQ ID NO. 2; d. a
p62/SQSTM1 polypeptide with at least one or more domain deletions;
e. a p62/SQSTM1 nucleic acid that encodes a polypeptide at least
90% identical to SEQ ID NO: 2; or f. a p62/SQSTM1 nucleic acid that
encodes a polypeptide with at least one or more domain
deletions;
48. The method according to claim 47, wherein: a. said
proinflammatory cytokine is selected from the group consisting of:
TNF.alpha., IL-6, IL-1b, RANTES, IL-17, IL-23, CCL-1, MCP-5, and
CXCL2; b. said osteogenic transcription factor is selected from the
group of osterix and runx2; or, c. said bone resorptive factor is
selected from the group of TNF.alpha. and RANKL.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 61/921,504, filed Dec. 29,
2013 and entitled "Method of Use of Vectors, Encoding Products of
the SQSTM1 Gene as Therapeutic and Preventive Agents for
Osteoporosis" designated by Attorney Docket No. 151-00104.PRV0 and
to U.S. Provisional Patent Application Ser. No. 61/949,597, filed
Mar. 7, 2014 and entitled "Methods and Compositions Relating to p62
for the Treatment and Prevention of Inflammation-Associated
Disease" designated by Attorney Docket No. 151-00104.PRV. The
entire content of the provisional patent applications are
incorporated herein by reference, including all text, tables and
drawings.
FIELD OF THE INVENTION
[0002] This invention relates generally to prevention and treatment
of inflammatory diseases. More specifically, the invention relates
to prevention and treatment of inflammatory diseases by
administration of p62 compositions.
BACKGROUND OF THE INVENTION
[0003] Inflammation is an essential immune response that enables
survival during infection or injury and maintains tissue
homeostasis under a variety of noxious conditions. It can be
divided in acute and chronic inflammation. Acute inflammation is a
protective response to pathogens like bacteria and viruses, or to
tissue damage. In response to infection or tissue damage,
macrophages induce production of inflammatory cytokines (e.g., TNF,
IL-1, IL-6) and chemokines (e.g., CCL2 and CXCL8), as well as
prostaglandins.
[0004] These inflammatory mediators then act on target tissues,
including local blood vessels, to induce vasodilation,
extravasation of neutrophils, and leakage of plasma into the
infected tissue. In addition, IL-1, TNF, and IL-6 can have systemic
effects when secreted in sufficient amounts. They induce liver
cells (hepatocytes) to produce acute phase proteins such as
C-reactive protein and coagulation factors, and they activate brain
endothelium to produce prostaglandins, including the major
proinflammatory prostaglandin, PGE2. Locally produced PGE2, in
turn, induces specific populations of neurons in the central
nervous system to promote so-called sickness behavior: fever,
anorexia, fatigue, sleepiness, and social withdrawal (Pecchi et al.
2009. Prostaglandins and sickness behavior: old story, new
insights. Physiol Behav 97:279-292). In the case of sterile tissue
injury in the absence of infection, acute inflammation promotes
tissue repair and helps to prevent colonization of the damaged
tissues by opportunistic pathogens. The usual result of acute
inflammation is protection from the spread of infection, followed
by resolution--the restoration of affected tissues to their normal
structural and functional state. The major transcription factors
involved in inflammation are NF-kappa-B and Stat-3.
[0005] If the inflammatory trigger is not eliminated by the acute
inflammatory response or persists for any other reason, the
resolution phase may not be appropriately induced and a chronic
inflammatory state may ensue. This state can be caused by chronic
infections, unrepaired tissue damage, persistent allergens,
undigestable foreign particles, or endogenous crystals, such as
monosodium urate (Majno 2004. Cell, Tissues, and Disease; Kumar
2003. Robbins Basic Pathology.) The chronic inflammatory response
in these cases is typically localized to the site where the
inflammatory inducer is present and often results in different
types of local tissue remodeling.
[0006] In addition, a growing number of chronic inflammatory
conditions have been described where the initiating trigger is not
well defined but does not seem to involve infection or tissue
damage. These inflammatory conditions are of particular interest
because they accompany many diseases of industrialized countries,
including obesity and type 2 diabetes, atherosclerosis,
neurodegenerative diseases, and cancer. In these cases of chronic
inflammation there appear to be vicious cycles connecting
inflammation and the pathological process it accompanies.
[0007] Thus, obesity can lead to inflammation, whereas chronic
inflammation can promote obesity-associated diabetes in part by
inducing insulin resistance (Hotamisligil 2006. Inflammation and
metabolic disorders. Nature 444:860-867). Similar positive feedback
loops are present in atherosclerosis, cancer, and other chronic
inflammatory diseases. An excessive inflammatory response is
detrimental due to its negative effect on tissue function and, when
extreme, results in overt tissue damage. Frequently, acute and
chronic inflammation coexist over long periods, implying continual
reinitiation. Examples are found in rheumatoid arthritis, asthma,
chronic obstructive pulmonary disease (COPD), multiple sclerosis,
Crohn's disease, ulcerative colitis, and cancers whose stroma is
infiltrated both by macrophages and immature myeloid cells
(Mantovani et al. 2008. Cancer-related inflammation. Nature
454:436-444) No single phenomenon contributes more to the medical
burden in industrialized societies than chronic inflammation.
Chronic inflammation contributes significantly to pathogenesis of
atherosclerosis, obesity, cancer, chronic obstructive pulmonary
disease, asthma, inflammatory bowel disease, neurodegenerative
disease, multiple sclerosis, or rheumatoid arthritis and other
diseases.
[0008] Osteoporosis is the most common disease of the bone
associated with bone loss and affecting mostly women after onset of
menopause. Menopause leads to decrease in estrogen levels, thus
ovariectomy in rodents leading to cessation of estrogen generation
is the most common model for osteoporosis. Postmenopausal period is
marked by elevation of cytokines such us IL-6, TNF-alpha and
IL-1beta, and the same cytokines are elevated under ovariectomy.
TNF and IL-1 have potent antiapoptotic effects in OCs, prolonging
OC lifespan, accelerating bone resorption and inhibiting bone
formation, and blockade of TNF-alpha and IL-1beta prevents
osteoporosis due to estrogen deficiency (Mundy 2007. Osteoporosis
and Inflammation. Nutrition Reviews 65:S147-S151; Lencel and Magne
2011. Inflammaging: The driving force in osteoporosis? Medical
Hypotheses 76:317-321).
[0009] Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's
disease, is a progressive fatal neurodegenerative disease that
affects motor neurons in the brainstem, spinal cord, and motor
cortex. ALS is universally fatal, with a median age of onset of 55
years and a survival of 2-5 years after the onset of symptoms.
Prominent neuroinflammation can be easily observed in
pathologically affected areas of the CNS and in spinal cords from
both human ALS patients and mouse models of the disease (Smith et
al. 2012. Role of pro-inflammatory cytokines released from
microglia in neurodegenerative diseases. Brain Res Bull 87:10-20).
Typically, inflammation in ALS is characterized by gliosis and the
accumulation of large numbers of activated microglia and
astrocytes. Activation of glia in ALS has been extensively
characterized and is marked by elevated production of potentially
cytotoxic molecules such as ROS, inflammatory mediators such as
COX-2, and proinflammatory cytokines such as IL-1 beta, TNF-alpha,
and IL-6 (Smith et al. 2012. Role of pro-inflammatory cytokines
released from microglia in neurodegenerative diseases. Brain Res
Bull 87:10-20). The most common mouse model of ALS is transgenic
mouse expressing mutant form of superoxide dismutase, the same
mutant form as seen in some ALS patients.
Multiple Sclerosis
[0010] Multiple sclerosis (MS) is a heterogeneous and complex
autoimmune disease that is characterized by inflammation,
demyelination, and axon degeneration in the CNS. This pathology
results from a primary defect in the immune system that targets
components of the myelin sheath, resulting in secondary effects on
neurons. MS is considered an immune-mediated disease characterized
by the presence of inflammatory demyelinating lesions in the CNS.
Infection by bacteria or viruses or other environmental stimuli
trigger the activation of microglia and astrocytes in multiple
sclerosis (MS), leading to the production of proinflammatory
cytokines through activation of the transcription factors
NF-kappa-B and AP-1 (Luessi et al. 2012. Neurodegeneration in
multiple sclerosis: novel treatment strategies. Expert Rev
Neurother 12:1061-1076). Experimental autoimmune encephalomyelitis
(EAE), in which rodents are immunized with a myelin-derived antigen
and adjuvant, is the most common animal model of MS. By varying the
genetic background and immunization protocol, EAE can reproduce the
symptoms of the major forms of human MS.
[0011] There are two major classes of anti-inflammatory drugs,
chemicals and biologicals. The first class includes such well-known
drugs as aspirin, glucocorticoids, non-steroidal anti-inflammatory
agents (celecoxib) and other agents (e.g., methotrexate,
cyclosporine, rapamycin etc.). The second class includes agents
that reduce activity of specific cytokines or their receptors,
e.g., antibodies to TNF (see scheme below). Despite a variety of
drugs, there is no treatment to cure chronic inflammation. In many
instances existing drugs are not quite effective, very expensive
and have numerous side effects. For instance, major drawbacks of
anti-cytokine therapy is a decreased host immune defense against
infection and expense.
[0012] p62 is a multifunctional protein that binds ubiquitin and
regulates autophagy, activity of the nuclear factor kappa-B and
some other signaling pathways. The protein functions as a
scaffolding/adaptor protein in concert with TNF receptor-associated
factor 6 (TRF6) to mediate activation of NF-kappa-B in response to
upstream signals. Alternatively spliced transcript variants
encoding either the same or different isoforms have been identified
for this gene.
[0013] p62 was identified as 62-kDa protein that binds the src
homology 2 (SH2) domain of tyrosine kinase Lckp56 in a
phosphotyrosine-independent manner (Moscat et al. 2007. Signal
integration and diversification through the p62 scaffold protein.
Trends Biochem Sci 32:95-100). The primary sequence of p62 is
known, and p62 was shown to bind ubiquitin. (Moscat et al. 2007.
Signal integration and diversification through the p62 scaffold
protein. Trends Biochem Sci 32:95-100). FIG. 1 shows the nucleic
acid sequence of the cDNA and FIG. 2 the amino acid sequence.
SUMMARY OF THE INVENTION
[0014] Provided herein are methods to modulate the expression of a
proinflammatory cytokine in a subject by administering to the
subject an agent that includes: (a) at least 30 amino acids of a
p62/SQSTM1 polypeptide or a variant thereof; or, (b) a p62/SQSTM1
encoding nucleic acid, wherein said p62/SQSTM1 encoding nucleic
acid encodes at least 30 amino acids of a p62/SQSTM1 polypeptide or
a variant thereof. The proinflammatory cytokine can be TNF.alpha.,
IL-6, IL-1b, RANTES, IL-17, IL-23, CCL-1, MCP-5, or CXCL2.
[0015] Also provided herein are methods to modulate the expression
of an osteogenic transcription factor in a subject by administering
to the subject an agent that includes: (a) at least 30 amino acids
of a p62/SQSTM1 polypeptide or a variant thereof; or, (b) a
p62/SQSTM1 encoding nucleic acid, wherein said p62/SQSTM1 encoding
nucleic acid encodes at least 30 amino acids of a p62/SQSTM1
polypeptide or a variant thereof. The osteogenic transcription
factor can be osterix or runx2.
[0016] Also provided herein are methods to modulate the expression
of a bone resorptive factor in a subject by administering to the
subject an agent that includes: (a) at least 30 amino acids of a
p62/SQSTM1 polypeptide or a variant thereof; or, (b) a p62/SQSTM1
encoding nucleic acid, wherein said p62/SQSTM1 encoding nucleic
acid encodes at least 30 amino acids of a p62/SQSTM1 polypeptide or
a variant thereof. The bone resorptive factor can be TNF.alpha. or
RANKL.
[0017] Also provided herein are methods to modulate the expression
of endogenous p62/SQSTM1 in a subject by administering to the
subject an agent comprising that includes: (a) at least 30 amino
acids of a p62/SQSTM1 polypeptide or a variant thereof; or, (b) a
p62/SQSTM1 encoding nucleic acid, wherein said p62/SQSTM1 encoding
nucleic acid encodes at least 30 amino acids of a p62/SQSTM1
polypeptide or a variant thereof.
[0018] Provided herein are methods to treat, alleviate, ameliorate,
relieve, delay onset of, inhibit progression of, reduce severity
of, or reduce incidence of one or more symptoms of a
non-cancer-related chronic inflammatory disease in a subject by
administering to the subject an agent comprising that includes: (a)
at least 30 amino acids of a p62/SQSTM1 polypeptide or a variant
thereof; or, (b) a p62/SQSTM1 encoding nucleic acid, wherein said
p62/SQSTM1 encoding nucleic acid encodes at least 30 amino acids of
a p62/SQSTM1 polypeptide or a variant thereof.
[0019] Any of the above methods can include administration of a
variant p62/SQSTM1, wherein the variant is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% homologous to any sequence selected from the group
consisting of SEQ. ID. NO. 2-35 10. The variant can have at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% sequence homology to any sequence
selected from the group consisting of SEQ. ID. NO. 2-35, or
identical thereto.
[0020] Any of the above methods can include administration of
p62-encoding nucleic acid comprises the sequence of SEQ ID
NO:1.
[0021] Any of the above methods can include administration of p62
polypeptide or variant thereof having at least one domain deletion.
The deleted domain can be PB1, ZZ, NLS2, TB, NLS1, NES, LIR, KIR,
and UBA.
[0022] Any of the above methods can include administration of an
agent including a p62 encoding nucleic acid, wherein said p62
encoding nucleic acid encodes a polypeptide, which is at least 95%
identical to SEQ ID NO. 2, and wherein said p62 encoding nucleic
acid further comprises a plasmid, RNA or a viral vector.
[0023] Any of the above methods can include p62/SQSTM1 polypeptide
or p62/SQSTM1 encoding nucleic acid further including a fusion
polypeptide or nucleic acid encoding for a fusion polypeptide,
respectively.
[0024] Any of the above methods can include p62/SQSTM1 polypeptide
or p62/SQSTM1 encoding nucleic acid in the form of a vaccine and
further include administering an adjuvant to said subject. The
adjuvant can be gel-type, microbial, particulate, oil-emulsion,
surfactant-based, and synthetic adjuvant.
[0025] The non-cancer-related chronic inflammatory disease can be
obesity, metabolic syndrome, type 2 diabetes, fat liver, Crohn's
Disease, pancreatitis, asthma, chronic obstructive pulmonary
disease, arthritis, osteoporosis, osteoarthritis, multiple
sclerosis, psoriasis, congestive heart failure atherosclerosis,
neurodegenerative diseases, gout, asbestosis, and silicosis. The
neurodegenerative disease can be amyotrophic lateral sclerosis,
Parkinson's disease, Huntington's disease, or Alzheimer's
disease.
[0026] The methods to treat, alleviate, ameliorate, relieve, delay
onset of, inhibit progression of, reduce severity of, or reduce
incidence of one or more symptoms of a non-cancer-related chronic
inflammatory disease in a subject can further include administering
an anti-inflammatory therapy to said subject.
[0027] Any of the above methods can be applied to a subject that is
a subject diagnosed with an inflammatory disease, a subject
previously treated for an inflammatory disease, a subject with a
family history of inflammatory disease, or a subject predisposed to
an inflammatory disease.
[0028] The methods to treat, alleviate, ameliorate, relieve, delay
onset of, inhibit progression of, reduce severity of, or reduce
incidence of one or more symptoms of a non-cancer-related chronic
inflammatory disease in a subject can further include a strategy
for improving the efficiency of nucleic acid-based expression of
p62 in subjects. The strategy can include a self-replicating viral
replicon, codon optimization, in vivo electroporation,
incorporation of a CpG stimulatory motif, including a sequence for
targeting of the endocytic or ubiquitin-processing pathways,
including a Marek's disease virus type 1 VP22 sequence, a
prime-boost regimen, a mucosal delivery vector, and a nucleic acid
delivery system. The nucleic acid delivery system can be a polymer
gene delivery system, a liposomal delivery system, and a
cell-penetrating peptide gene delivery system.
[0029] Any of the above methods can further include administering
an anti-inflammatory chemotherapeutic or biological agent. The
chemotherapeutic agent can be a nonsteroidal anti-inflammatory
drug, a glucocorticoid, methotrexate, cyclosporine, or rapamycin.
The anti-inflammatory biological agent can be an anti-TNF antibody,
an anti-IL1 antibody, an anti-IL6 antibody, an anti-IL6 receptor
antibody, an anti-IL12/23 antibody, an anti-IL17 antibody, an
anti-IL1R antibody, an anti-IL1 receptor antagonist, and a soluble
IL-1 receptor.
[0030] Certain aspects and embodiments are described further in the
following description, examples, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWING
[0031] The drawings illustrate embodiments of the technology and
are not limiting.
[0032] FIG. 1 shows a wild type nucleic acid sequence of human p62
(SEQ ID NO: 1);
[0033] FIG. 2 shows a wild type amino acid sequence of the human
p62/SQSTM1 encoded by the nucleic acid sequence (SEQ ID NO: 2);
[0034] FIG. 3 shows a cartoon of the domain structure of human
p62/SQSTM1;
[0035] FIG. 4 shows the effect of p62/SQSTM1 DNA introduction on
the prevention of osteoporosis in a mouse model for
osteoporosis;
[0036] FIG. 5 shows the effect of p62/SQSTM1 DNA introduction on
osteogenic markers;
[0037] FIG. 6 shows the effect of p62/SQSTM1 DNA introduction on
bone mineral density (BMD) and content (BMC);
[0038] FIG. 7: Panel A shows the effect of p62/SQSTM1 DNA
introduction on expression of osteogenic factors; Panel B shows the
effect of p62/SQSTM1 DNA introduction on bone resorptive factors
and NF-kappa-B expression;
[0039] FIG. 8: Panel A shows the effect of exogenous p62 on p62
expression; Panel B shows increase p62-immune labelling at the
epiphyseal region of femurs of p62-OVX mice; Panel C Western Blots
demonstrate that the increased level of p62 is from endogenous
p62.
[0040] FIG. 9 shows the effect of p62/SQSTM1 DNA introduction on a
mouse model of ALS;
[0041] FIG. 10 shows the effect of p62/SQSTM1 DNA introduction on a
mouse model of MS;
[0042] FIG. 11 shows the effect of p62/SQSTM1 DNA introduction on
growth of S37 sarcoma;
[0043] FIG. 12 shows the effect of p62/SQSTM1 DNA introduction on
survival of mice with breast carcinoma;
[0044] FIG. 13 shows the effect of p62/SQSTM1 DNA introduction on
metastasis in LLC; and,
[0045] FIG. 14 shows the effect of p62/SQSTM1 DNA introduction on
metastasis in B16 melanoma.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Provided herein are p62 compositions and methods for
treatment of chronic inflammation. The inventors have found that
administering p62, such as a p62 encoding nucleic acid, to a
subject suppresses generation of inflammatory cytokines.
Consequently polynucleotides encoding a p62 polypeptide or, p62
polypeptides administered to a subject can be used to prevent
and/or mitigate development of inflammation-associated diseases
(the list of such diseases includes but is not limited to,
osteoporosis, obesity, metabolic syndrome, type 2 diabetes, fat
liver, inflammatory bowel disease, gastritis, chronic pancreatitis,
asthma, chronic obstructive pulmonary disease (COPD), rheumatoid
arthritis (RA), osteoarthritis, multiple sclerosis (MS), psoriasis,
congestive heart failure (CHF), atherosclerosis, neurodegenerative
diseases (ALS, Parkinson, Alzheimer's, Huntington disease), gout,
asbestosis and silicosis.
[0047] As used herein, "p62 polypeptide" means a polypeptide
corresponding to the full length p62/SQSTM1 protein. The term also
includes all homologs, analogs, fragments or derivatives of the
p62/SQSTM1 protein. In one embodiment, the isolated p62 polypeptide
has an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2). A
"p62 encoding nucleic acid" means a DNA or RNA that encodes at
least a portion of a p62 polypeptide or variant.
[0048] In some embodiments, the subject is a human. In other
embodiments, the subject is a non-human mammal including, but not
limited to, a horse, cow, sheep, pig, deer, dog, cat, rat, or a
mouse.
TABLE-US-00001 TABLE 1 p62/SQSTM1 of Various Species Species SEQ ID
NO Polypeptide Accession No Human 2 NP_003891.1 Domestic cow 3
AAI08088.1 Common marmoset 4 XP_002744508.1 Domestic dog 5
XP_005626405.1 White rhinoceros 6 XP_004428485.1 Star-noised mole 7
XP_004693731.1 Nine-banded armadillo 8 XP_004478189.1 Lesser
hedgehog tenrec 9 XP_004696907.1 Elephant shrew 10 XP_006898178.1
Domestic cat 11 XP_003980695.1 Gorilla 12 XP_004043182.1 Naked mole
rat 13 XP_004836787.1 Thirteen-lined ground 14 XP_005339398.1
squirrel Lesser Egyptian jerboa 15 XP_004666668.1 West Indian Ocean
16 XP_005995405.1 coelacanth Crab-eating macaque 17 XP_005558842.1
Rhesus macaque 18 AFE80687.1 Golden hamster 19 XP_005071915.1
Prairie vole 20 XP_005350213.1 House mouse 21 NP_035148.1 American
pika 22 XP_004599535.1 Degu 23 XP_004629477.1 Walrus 24
XP_004412727.1 King cobra 25 ETE69498.1 Killer whale 26
XP_004284096.1 Northern greater galago 27 XP_003799205.1 Sheep 28
ACR56704.1 Bonobo 29 XP_003809255.1 Common chimpanzee 30
XP_001153075.1 Olive baboon 31 XP_003900678.1 Sumatran orangutan 32
NP_001125548.1 Common rat 33 NP_787037.2 Bolivian squirrel monkey
34 XP_003943999.1 Florida manatee 35 XP_004387215.1
[0049] In addition to the full length amino acid sequence or the
polypeptide encoding nucleic acid thereof, the polypeptides of the
present invention may also include fragments or truncations,
analogs, and homologs of the p62 polypeptide and truncations
thereof as described herein. Fragments can include peptides (or
encode peptides) of at least 5, at least 10, at least 15, at least
20, at least 25, at least 30, at least 50, at least 100, at least
200 or at least 300 amino acid residues of the full length
polypeptide.
[0050] Deletions of one or more amino acids, or discrete portions
from the amino acid sequence of the p62/SQSTM1 protein are also
included. The deleted amino acids may or may not be contiguous. The
lower limit length of the resulting analog with a deletion mutation
is about 10, about 20, about 50, or about 100 amino acids.
[0051] In some embodiments, the p62 polypeptide (or a nucleic acid
encoding for the polypeptide) has one or more deleted domains.
While not wishing to be held by theory, the inventors hold that the
deletion of one or more domains of the p62 polypeptide provide a
more compact and manipulable polypeptide for directing an immune
response. For example, by disrupting or eliminating one or more of
the domains of a p62 polypeptide, anti-inflammatory effect can be
retained (or improved if the deleted or disrupted domain does not
contribute to this effect) in a more compact molecule and
potentially increase per weight basis.
[0052] The p62 polypeptide has a domain structure as provided in
Table 2 below and as shown in FIG. 3:
TABLE-US-00002 TABLE 2 p62 Polypeptide Domain Structure Domain/site
Full name Location Description PB1 Phox/Bem1p 20-102 PB1-domain is
conserved among eukaryotes domain (protista, plants, fungi and
animals). PB1- domain (=OPR has specific - ubiquitin-like
beta-grasp fold. There domain) are 3 types of PB1-domains: type I
domains contains acid OPCA-motif, type II domains contain
conservative Lys residue in the first beta-sheet, and I/II type
domains contain both of the above. OPCA- motif can bind to basic
amino acids (e.g., lysine) via salt bridges, enabling ability of
PB1-domains to form heteromeric structures (Sumimoto et al., 2007).
PB1- domain of p62 is type I/II (Lamark et al., 2003). PB1-domain
is responsible for di- and multimerization of p62, as well as
interaction with other proteins: MEKK3, MEK5, PKC.zeta.,
PKC-lambda/ (protein kinases containing PB1-domain), NBR1 (Next to
BRCA1, contains PB1-domain) (Nezis, Stenmark, 2011). ZZ Zn2+-finger
122-167 ZZ- domain is Zn.sup.2+-finger of C2H2 type. ZZ-domain ZZ
type of p62 binds to RIP1 (receptor interacting protein 1). RIP1 is
a regulatory protein kinase which integrates signaling pathways
activated by bacterial or viral infection (via PAMP), death
receptors, or genotoxins; it takes part in determination of cell
fate (survival, apoptosis, or necrosis) (Festjens et al., 2007).
NLS2 Nuclear 183-194 Tentative nuclear localization signal (Pankiv
et al., localization 2009) signal 2 TBS TRAF6- 228-233 p62 binds
via TB domain to E3-ubiquitin protein binding ligase TRAF6. TRAF6
activates kinase TAK1, domain polyubiqitinating it via K63). TRAF6
participates in signaling from RANK-L, IL-1R, TCR, BCR and TGFbeta
receptors (Landstrom, 2010). Interaction of p62 with TRAF6
stimulates autoubiqitination of TRAF6 nu E3- ligase activity. This
process requires PB1- and UBA-domains (Moscat et al., 2006). NLS1
Nuclear 261-273 Tentative nuclear localization signal (Pankiv et
al., localization 2009) signal 1 NES Nuclear 303-321 Tentative
nuclear export signal (Pankiv et al., 2009) export signal LIR LC3
321-342 LIR-domain is required for binding of p62 to LC3
interaction protein (wild-type human microtubule-associated region
protein 1 light chain 3, Light Chain 3) (Pankiv et al., 2007). LC3
-ubiquitin-like protein, conjugating with phosphatidyl ethanolamine
of autophagosome membrane (Tanida, 2011). P62 via interaction of
with LC3, p62 is recruited to autophagosomes (Shvets et al., 2011),
apparently transporting ubiquitinated proteins associated with UBA
domain. KIR Keap1 343-357 KIR domain is required for interaction
with DC interaction domain of Keap1 protein, containing Kelch
repeats region (Komatsu et al., 2010). Keap1 (Kelch-like ECH-
associated protein 1) is a regulator of activity of transcription
factor Nrf2 (NF-E2-related factor 2). Nrf2 regulates expression of
genes involved in glutathione synthesis, ROS detoxification,
metabolism of xenobiotics and drug transport (Taguchi et al.,
2011). Overexpression of p62 displaces Nrf2 from Keap1, Nrf2 is
stabilized which lead to stimulation of expression of
Nrf2-dependent genes. Paradoxically, hyperactivation of Nrf2 and
overexpression of genes considered "cytoprotective" causes severe
pathology (Komatsu et al., 2010). PEST 267-283 Targets of
proteosomal degradation (Okazaki et al. 346-380 1999. Cloning,
Expression Profile, and Genomic Organization of the Mouse STAP/A170
Gene. Genomics 60: 87-95) UBA Ubiquitin- 389-434 UBA-domain is one
of the domains which can bind associated to polyubiquitinated
labels (along with CUE, UIM, domain NZF etc.). UBA-domains can be
divided in four classes depending on their ability to bind
polyubiquitin labels of different structures (K6, K29, K48, K63).
UBA-domain of p62 belongs to class 4, which consists of domains
with equal affinity for binding to K6, K29, K48, K63 (Raasi et al.,
2005). UBA domain also participates in p62 dimerization (Garner et
al., 2011). Most of the mutations associated with Paget disease are
localized in UBA domain (Yan Jenny Chung, Van Hul, 2011). However,
p62 mutations are not enough for osteoblasts to acquire the
specific Paget phenotype: The expression of nucleocapsid protein of
measles virus is also required (Singer, 2011). The structure of the
UBA domain is known (Isogai et al., 2011). p62 NCBI reference
sequence: NP_003891 (sequestosome-1 isoform 1 [Homo sapiens]).
[0053] In some embodiments, one or more of the above domains are
deleted from a human p62 polypeptide at corresponding codons for
the nucleic acid regions of the p62 nucleic acid (in-frame
deletions), as presented below.
TABLE-US-00003 TABLE 3 Deletions in p62 Start of the deletion, End
of the deletion, Deleted domain between nts between nts PB1 1 and
20 102 and 122 ZZ 102 and 122 167 and 183 NLS2 167 and 183 194 and
228 TB 194 and 228 233 and 261 NLS1 233 and 261 273 and 303
NES-LIR-KIR 273 and 303 357 and 389 UBA Stop codon between Not
applicable 357 and 389 Nucleotide numbers refer to p62 NCBI
reference sequence NP_003891 (sequestosome-1 isoform 1 [Homo
sapiens]).
[0054] For example, any deletion of the encoding nucleic acid
sequence that starts at nucleotide 102 up to nucleotide 122 and
ends at 167 up to 183 is considered a ZZ deletion. Therefore, e.g.
a deletion of nucleotides 110-175 is a ZZ deletion. Techniques for
creating in-frame deletions are well known to those skilled in the
art.
[0055] As used herein, "biologically active" refers to polypeptides
according to the present invention having a similar structural
function (but not necessarily to the same degree), and/or similar
regulatory function (but not necessarily to the same degree),
and/or similar biochemical function (but not necessarily to the
same degree) as the individual wild type polypeptides.
[0056] As used herein, a "deletion" is defined as a change in the
nucleotide or amino acid sequence in which one or more nucleotide
or amino acid residues are absent as compared to the wild-type
polynucleotide or polypeptide, respectively.
[0057] As used herein an "insertion" or "addition" is a change in
the nucleotide or amino acid sequence that has resulted in the
addition of one or more nucleotide or amino acid residues as
compared to the wild-type polynucleotide or polypeptide,
respectively.
[0058] As used herein "substitution" results from the replacement
of one or more nucleotides or amino acids by different nucleotides
or amino acids, respectively, as compared to the wild-type
polynucleotide or polypeptide, respectively. In some embodiments,
the amino acid substitution mutation is C145R or Q418R.
[0059] As used herein, the term "variant" means any polypeptide
(including polypeptides encoded by the corresponding nucleic acid)
having a substitution of, deletion of or addition of one (or more)
amino acid from or to the sequence (or any combination of these),
including allelic variations, as compared with the wild-type
polypeptide. In some embodiments, the resultant polypeptide retains
at least 75%, 80%, 85%, 90%, 95%, 99% or more of the biological
activity as compared to the wild-type polypeptides as used in the
present invention. Variants of the p62 polypeptides (including
polypeptides encoded by the corresponding nucleic acid) can have at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% sequence identity to any of the
amino acid sequences listed in Table 1.
[0060] Sequence identity or homology can be determined using
standard techniques known in the art, such as the Best Fit sequence
program described by Devereux et al., Nucl. Acid Res. 12:387-395
(1984) or the BLASTX program (Altschul et al., J Mol. Biol.
215:403-410). The alignment may include the introduction of gaps in
the sequences to be aligned. In addition, for sequences which
contain either more or fewer amino acids than the proteins
disclosed herein, it is understood that the percentage of homology
will be determined based on the number of homologous amino acids in
relation to the total number of amino acids. Consequently, variants
of the p62 polypeptides (including polypeptides encoded by the
corresponding nucleic acid) can have at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence homology to any of the polypeptide sequences listed in
Table 1
[0061] In some embodiments, variants or derivatives of the
polypeptides of the present invention maintain the
hydrophobicity/hydrophilicity of the amino acid sequence.
Conservative amino acid substitutions are known in the art and may
be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions.
Amino acid substitutions may include the use of non-naturally
occurring analogues, for example to increase blood plasma
half-life.
[0062] The term "derivative" as used herein in relation to the
amino acid sequence means chemical modification of a polypeptide of
the invention.
[0063] Non-limiting examples of such modifications may include but
are not limited to aliphatic esters or amides of the carboxyl
terminus or of residues containing carboxyl side chains, O-acyl
derivatives of hydroxyl group-containing residues, and N-acyl
derivatives of the amino-terminal amino acid or amino-group
containing residues, e.g., lysine or arginine.
[0064] Additional modifications can include, for example,
production of a polypeptide conjugated with a polymer such as
polyethylene glycol (PEG), or addition of PEG during chemical
synthesis of a polypeptide of the invention.
[0065] Modifications of polypeptides or portions thereof can also
include reduction/alkylation, chemical coupling to an appropriate
carrier, or mild formalin treatment.
[0066] The term "post-translationally modified" or "modified"
refers to any modification of an amino acid after its incorporation
into a polypeptide chain. The term encompasses, but is not limited
to, co-translational in vivo modifications, post-translational in
vivo modifications, and post-translational in vitro
modifications.
[0067] Other derivatives of the polypeptides of the present
invention include incorporation of unnatural amino acid residues,
or phosphorylated amino acid residues such as phosphotyrosine,
phosphoserine or phosphothreonine residues. Other potential
modifications include sulfonation, biotinylation, or the addition
of other moieties, particularly those which have molecular shapes
similar to phosphate groups.
[0068] Derivatives also include polypeptides modified by
glycosylation. These can be made by modifying glycosylation
patterns during synthesis and processing in various alternative
eukaryotic host expression systems, or during further processing
steps. Methods for producing glycosylation modifications include
exposing the fusion proteins to glycosylating enzymes derived from
cells that normally carry out such processing, such as mammalian
glycosylation enzymes. Alternatively, deglycosylation enzymes can
be used to remove carbohydrates attached during production in
eukaryotic expression systems. Additionally, one can also modify
the coding sequence so that glycosylations site(s) are added or
glycosylation sites are deleted or disabled. Furthermore, if no
glycosylation is desired, the proteins can be produced in a
prokaryotic host expression system.
[0069] Variants and/or derivatives of the polypeptides of the
invention can be prepared by chemical synthesis or by using
site-directed mutagenesis (Gillman et al., Gene 8:81 (1979);
Roberts et at, Nature 328:731 (1987) or Innis (Ed.), 1990, PCR
Protocols: A Guide to Methods and Applications, Academic Press, New
York, N.Y.) or the polymerase chain reaction (PCR) method (Saiki et
al, Science 239:487 (1988)), as exemplified by Daugherty et at
(Nucleic Acids Res. 19:2471 (1991)) to modify nucleic acids
encoding the p62 polypeptides of invention.
[0070] In another embodiment, polypeptides of the present invention
may contain a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of the fusion protein can be increased through use of a
heterologous signal sequence. Signal sequences are typically
characterized by a core of hydrophobic amino acids, which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal peptides contain processing
sites that allow cleavage of the signal sequence from the mature
proteins as they pass through the secretory pathway. Thus, the
invention pertains to the described polypeptides having a signal
sequence, as well as to polypeptides from which the signal sequence
has been proteolytically cleaved (i.e., the cleavage products). In
order to enhance stability and/or reactivity, the polypeptides of
the present invention can also be modified to incorporate one or
more polymorphisms in the amino acid sequence resulting from
natural allelic variation. Additionally, D-amino acids, non-natural
amino acids or non-amino acid analogues can be substituted or added
to produce a modified p62 polypeptide within the scope of this
invention.
[0071] The polypeptides of the present invention may be produced by
expression of a nucleotide sequence coding for same in a suitable
expression system.
[0072] In addition, or in the alternative, the polypeptides can be
produced using chemical methods to synthesize the desired amino
acid sequence, in whole or in part. For example, polypeptides can
be synthesized by solid phase techniques, cleaved from the resin,
and purified by preparative high performance liquid chromatography
(e.g., Creighton (1983) Proteins Structures And Molecular
Principles, WH Freeman and Co, New York N.Y.). The composition of
the synthetic polypeptides may be confirmed by amino acid analysis
or sequencing (e.g., the Edman degradation procedure).
Additionally, the amino acid sequence of a p62 polypeptide, or any
part thereof, may be altered during direct synthesis and/or
combined using chemical methods with a sequence from other
subunits, or any part thereof, to produce a variant
polypeptide.
[0073] As used herein, the term "fusion proteins" refers to
chimeric proteins comprising amino acid sequences of two or more
different proteins. Typically, fusion proteins result from in vitro
recombinant techniques well known in the art.
[0074] In additional embodiments, the fusion proteins of the
present invention may further comprise one or more additional
polypeptide domains added to facilitate protein purification, to
increase expression of the recombinant protein, or to increase the
solubility of the recombinant protein. Such
purification/expression/solubility facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals (Porath J (1992) Protein Expr Purif 3-.26328 1), protein A
domains that allow purification on immobilized immunoglobulin, and
the domain utilized in the FLAGS extension/affinity purification
system (Immunex Corp, Seattle, Wash.). The inclusion of a cleavable
linker sequence such as Factor Xa or enterokinase (Invitrogen, San
Diego, Calif.) between the purification domain and the protein is
useful to facilitate purification.
[0075] Additional fusion expression vectors include pGEX
(Pharmacia, a Piscataway, N.J.), pMAL (New England Biolabs,
Beverly, Mass.) and pRITS (GE Healthcare Biosciences, Piscataway,
N.J.) which fuse glutathione S transferase (GST), maltose B binding
protein, or protein A, respectively, to the target recombinant
protein. EBV, BKV, and other episomal expression vectors
(ThermoFisher Scientific) can also be used.
[0076] In another aspect of the invention, p62 polypeptides can be
non-covalently linked to a transport moiety or transfection agent.
An example of a non-covalently linked peptide transfection agent is
the Chariot protein delivery system (See U.S. Pat. No. 6,841,535;
Morris et al. (1999) J. Biol. Chem. 274(35):24941-24946; and Morris
et al. (2001) Nature Biotech. 19:1173-1176).
[0077] In certain embodiments, a nucleic acid molecule encoding p62
polypeptide is utilized. The nucleic acid molecule may comprise or
consist of a nucleotide sequence encoding one or more p62
polypeptides, or fragments (including fragments that code for
domains in any order or polypeptides wherein one or more domains
are deleted or disrupted) or derivatives thereof, such as that
contained in a DNA insert in an ATCC Deposit. Variants of the p62 a
nucleic acid molecules encoding p62 polypeptide can have at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% sequence identity to SEQ ID NO:
1.
[0078] The term "nucleic acid sequence" or "nucleic acid molecule"
refers to a DNA or RNA sequence. The term encompasses molecules
formed from any of the known base analogs of DNA and RNA such as,
but not limited- to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,
N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5' methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic
acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine,
2-thiocytosine, and 2,6-diaminopurine, among others.
[0079] In certain embodiments of the present invention, vectors are
used to transfer a polynucleotide encoding a polypeptide to a cell.
A vector is any molecule used to transfer a nucleic acid sequence
to a host cell. In certain cases, an expression vector is utilized.
An expression vector is a nucleic acid molecule that is suitable
for introduction to and/or propagation in a host cell and contains
nucleic acid sequences that direct and/or control the expression of
the transferred nucleic acid sequences. Expression includes, but is
not limited to, processes such as transcription, translation, and
splicing, if introns are present. Expression vectors typically
comprise one or more flanking sequences operably linked to a
heterologous nucleic acid sequence encoding a polypeptide. Flanking
sequences may be homologous (i.e., from the same species and/or
strain as the host cell), heterologous (i.e., from a species other
than the host cell species or strain), hybrid (i.e., a combination
of flanking sequences from more than one source), or synthetic, for
example.
[0080] A flanking sequence is capable of effecting the replication,
transcription and/or translation of the coding sequence and is
operably linked to a coding sequence. As used herein, the term
operably linked refers to a linkage of polynucleotide elements in a
functional relationship. For instance, a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the coding sequence. However, a flanking sequence
need not necessarily be contiguous with the coding sequence, so
long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
may still be considered operably linked to the coding sequence.
Similarly, an enhancer sequence can be located upstream or
downstream from the coding sequence and affect transcription of the
sequence. In certain embodiments, the flanking sequence is a
transcriptional regulatory region that drives high-level gene
expression in the target cell. The transcriptional regulatory
region can include, for example, a promoter, enhancer, silencer,
repressor element, or combinations thereof. The transcriptional
regulatory region can be constitutive, tissue-specific, cell-type
specific (i.e., the region is drives higher levels of transcription
in a one type of tissue or cell as compared to another), or
regulatable (i.e., responsive to interaction with a molecule). The
source of a transcriptional regulatory region may be any
prokaryotic or eukaryotic organism, any vertebrate or invertebrate
organism, or any plant, provided that the flanking sequence
functions in a cell by causing transcription of a nucleic acid
within that cell. A wide variety of transcriptional regulatory
regions can be utilized.
[0081] Suitable transcriptional regulatory regions include, for
example, the CMV promoter (i.e., the CMV-immediate early promoter);
promoters from eukaryotic genes (i.e., the estrogen-inducible
chicken ovalbumin gene, the interferon genes, the
gluco-corticoid-inducible tyrosine aminotransferase gene, and the
thymidine kinase gene); and the major early and late adenovirus
gene promoters; the SV40 early promoter region (Bernoist and
Chambon, 1981, Nature 290:304-10); the promoter contained in the 3'
long terminal repeat (LTR) of Rous sarcoma virus (RSV) (Yamamoto,
et al., 1980, Cell 22:787-97); the herpes simplex virus thymidine
kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:1444-45); the regulatory sequences of the
metallothionine gene (Brinster et al., 1982, Nature 296:39-42);
prokaryotic expression vectors such as the beta-lactamase promoter
(VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,
75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl.
Acad. Sci. U.S.A., 80:21-25). Tissue- and/or cell-type specific
transcriptional control regions include, for example, the elastase
I gene control region which is active in pancreatic acinar cells
(Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold
Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,
1987, Hepalology 7:425-515); the insulin gene control region which
is active in pancreatic beta cells (Hanahan, 1985, Nature
315:115-22); the immunoglobulin gene control region which is active
in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; Adames
et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell.
Biol., 7:1436-44); the mouse mammary tumor virus control region
intesticular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45:485-95); the albumin gene control region, in liver (Pinkert
et al., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein
gene control region in liver (Krumlauf et al., 1985, Mol. Cell.
Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); the
alpha 1-antitrypsin gene control region in liver (Kelsey et al.,
1987, Genes and Devel. 1:161-71); the beta-globin gene control
region in myeloid cells (Mogram et al., 1985, Nature 315:338-40;
Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene
control region in oligodendrocyte cells in the brain (Readhead et
al., 1987, Cell 48:703-12); the myosin light chain-2 gene control
region in skeletal muscle (Sani, 1985, Nature 314:283-86); the
gonadotropic releasing hormone gene control region in the
hypothalamus (Mason et al., 1986, Science 234:1372-78), and the
tyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996
February; 23(1):154-8; Siders, et al. Cancer Gene Ther 1998
September-October; 5(5):281-91), among others. Inducible promoters
that are activated in the presence of a certain molecule or
condition such as light, heat, radiation, tetracycline, or heat
shock proteins, for example, can also be used (see, for example, WO
00/10612). Other suitable promoters are known in the art.
[0082] As described above, enhancers may also be suitable flanking
sequences. Enhancers are cis-acting elements of DNA, usually about
10-300 bp in length, that act on the promoter to increase
transcription. Enhancers are typically orientation- and
position-independent, having been identified both 5' and 3' to
controlled coding sequences. Several enhancer sequences available
from mammalian genes are known (i.e., globin, elastase, albumin,
alpha-feto-protein and insulin). Similarly, the SV40 enhancer, the
cytomegalovirus early promoter enhancer, the polyoma enhancer, and
adenovirus enhancers are useful with eukaryotic promoter sequences.
While an enhancer may be spliced into the vector at a position 5'
or 3' to nucleic acid coding sequence, it is typically located at a
site 5' from the promoter. Other suitable enhancers are known in
the art, and would be applicable to the present invention.
[0083] In some embodiments of the invention, provided herein are
vaccines that include a p62 polynucleotide or p62 polypeptide. Such
vaccines can further include an adjuvant. Any of a variety of
adjuvants can be employed in the vaccines of this invention to
enhance the immune response. Most adjuvants contain a substance
designed to protect the antigen from rapid catabolism, such as
aluminum hydroxide or mineral oil, and a nonspecific stimulator of
immune responses. Suitable adjuvants are commercially available and
include, for example, Freund's Incomplete Adjuvant and Freund's
Complete Adjuvant (Difco Laboratories) and Merck Adjuvant 65 (Merck
and Company, Inc., Rahway, N.J.). Suitable adjuvant types include,
but are not limited to, gel-type, microbial, particulate,
oil-emulsion, surfactant-based, and synthetic adjuvants.
[0084] In certain embodiments, it may be advantageous to combine a
p62 polypeptide or polynucleotide encoding a p62 polypeptide, or
derivative thereof, with one or more co-stimulatory component(s)
such as cell surface proteins, cytokines, chemokines, or signaling
molecules in a composition of the present invention. The
co-stimulatory component may be included in the composition as a
polypeptide or as a nucleic acid encoding the polypeptide, for
example. Suitable co-stimulatory molecules include, for instance,
polypeptides that bind members of the CD28 family (i.e., CD28,
ICOS; Hutloff, et al. Nature 1999, 397: 263-265; Peach, et al. J
Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides
B7.1 (CD80; Schwartz, 1992; Chen et al, 1992; Ellis, et al. J.
Immunol., 156(8): 2700-9) and B7.2 (CD86; Ellis, et al. J.
Immunol., 156(8): 2700-9); polypeptides which bind members of the
integrin family (i.e., LFA-1 (CD11a/CD18); Sedwick, et al. J
Immunol 1999, 162: 1367-1375; Wulfing, et al. Science 1998, 282:
2266-2269; Lub, et al. Immunol Today 1995, 16: 479-483) including
members of the ICAM family (i.e., ICAM-1, -2 or -3); polypeptides
which bind CD2 family members (i.e., CD2, signaling lymphocyte
activation molecule (CDw150 or "SLAM"; Aversa, et al. J Immunol
1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis, et
al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al.
Nature 1998, 395: 462-469); polypeptides which bind heat stable
antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27:
2524-2528); polypeptides which bind to members of the TNF receptor
(TNFR) family (i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol
1998, 10: 481-489), OX40 (CD134; Weinberg, et al. Semin Immunol
1998, 10: 471-480; Higgins, et al. J Immunol 1999, 162: 486-493),
and CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499)) such as
4-1 BBL (4-1 BB ligand; Vinay, et al. Semin Immunol 1998, 10:
481-48; DeBenedette, et al. J Immunol 1997, 158: 551-559), TNFR
associated factor-1 (TRAF-1; 4-1BB ligand; Saoulli, et al. J Exp
Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18:
558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med
1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526,
Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB
and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565;
Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620;
Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40
ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5
(OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565;
Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27,
ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD154
(CD40 ligand or "CD40L"; Gurunathan, et al. J. Immunol., 1998, 161:
4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) may
also be suitable.
[0085] Additional strategies for improving the efficiency of
nucleic acid-based immunization may also be used including, for
example, the use of self-replicating viral replicons (Caley, et al.
1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6:
723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon
optimization (Liu, et al. 2000. Mol. Ther., 1: 497-500; Dubensky,
supra; Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo
electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640),
incorporation of CpG stimulatory motifs (Gurunathan, et al. Ann.
Rev. Immunol., 2000, 18: 927-974; Leitner, supra; Cho, et al. J.
Immunol. 168(10):4907-13), sequences for targeting of the endocytic
or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol.
72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373),
Marek's disease virus type 1 VP22 sequences (J. Virol.
76(6):2676-82, 2002), prime-boost regimens (Gurunathan, supra;
Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998.
Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), and
the use of mucosal delivery vectors such as Salmonella (Darji, et
al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19:
2945-2954). Other methods are known in the art, some of which are
described below.
[0086] Nucleic acids encoding p62 polypeptides can be administered
to subjects by any of several available techniques. Various viral
vectors that have been successfully utilized for introducing a
nucleic acid to a host include retrovirus, adenovirus,
adeno-associated virus (AAV), herpes virus, and poxvirus, among
others. It is understood in the art that many such viral vectors
are available in the art. The vectors can be constructed using
standard recombinant techniques widely available to one skilled in
the art. Many such techniques are disclosed in Molecular Cloning: A
Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor
Laboratory Press), Gene Expression Technology (Methods in
Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press,
San Diego, Calif.), and PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego,
Calif.).
[0087] Suitable retroviral vectors include derivatives of
lentivirus as well as derivatives of murine or avian retroviruses.
Examples of suitable retroviral vectors include, for example,
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and
Rous Sarcoma Virus (RSV). A number of retroviral vectors can
incorporate multiple exogenous nucleic acid sequences. As
recombinant retroviruses are defective, they require assistance in
order to produce infectious vector particles. This assistance can
be provided by, for example, helper cell lines encoding retrovirus
structural genes. Suitable helper cell lines include. PSI.2, PA317
and PA12, among others. The vector virions produced using such cell
lines can be used to infect a tissue cell line, such as NIH 3T3
cells, to produce large quantities of chimeric retroviral virions.
Retroviral vectors may be administered by traditional methods
(i.e., injection) or by implantation of a "producer cell line" in
proximity to the target cell population (Culver, K., et al., 1994,
Hum. Gene Ther., 5 (3): 343-79; Culver, K., et al., Cold Spring
Harb. Symp; Quant. Biol., 59: 685-90); Oldfield, E., 1993, Hum.
Gene Ther., 4 (1): 39-69). The producer cell line is engineered to
produce a viral vector and releases viral particles in the vicinity
of the target cell. A portion of the released viral particles
contact the target cells and infect those cells, thus delivering a
nucleic acid of the present invention to the target cell. Following
infection of the target cell, expression of the nucleic acid of the
vector occurs.
[0088] Adenoviral vectors are useful for gene transfer into
eukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004):
431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51).
Routes for administrating recombinant adenovirus to different
tissues in vivo have included intratracheal instillation
(Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection into
muscle (Quantin, B., et al., 1992, Proc. Natl. Acad. Sci. U.S.A.,
89 (7): 2581-4), peripheral intravenous injection (Herz, J., and
Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6)
and stereotactic inoculation to brain (Le Gal La Salle, G., et al.,
1993, Science, 259 (5097): 988-90), among others.
[0089] Adeno-associated virus (AAV) demonstrates high-level
infectivity, broad host range and specificity in integrating into
the host cell genome (Hermonat, P., et al., 1984, Proc. Natl. Acad.
Sci. U.S.A., 81 (20): 6466-70). Herpes Simplex Virus type-1 (HSV-1)
is yet another attractive vector system, especially for use in the
nervous system because of its neurotropic property (Geller, A., et
al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso, et al.,
1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, et al., 1995, Annu.
Rev. Microbiol., 49: 675-710).
[0090] Poxvirus is another useful expression vector (Smith, et al.
1983, Gene, 25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20:
345-62; Moss, et al, 1992, Curr. Top. Microbiol. Immunol., 158:
25-38; Moss, et al. 1991. Science, 252: 1662-1667). Poxviruses
shown to be useful include vaccinia, NYVAC, avipox, fowlpox,
canarypox, ALVAC, and ALVAC(2), among others.
[0091] NYVAC (vP866) was derived from the Copenhagen vaccine strain
of vaccinia virus by deleting six nonessential regions of the
genome encoding known or potential virulence factors (see, for
example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci
were also engineered as recipient loci for the insertion of foreign
genes. NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4L reverse,
pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under
the terms of the Budapest Treaty, accession numbers VR-2559,
VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914,
respectively
[0092] ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2)
are also suitable vectors (see, for example, U.S. Pat. No.
5,756,103). ALVAC(2) is identical to ALVAC(1) except that ALVAC(2)
genome comprises the vaccinia E3L and K3L genes under the control
of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al.,
1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). ALVAC
was deposited under the terms of the Budapest Treaty with the
American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, USA, ATCC accession number
VR-2547.
[0093] Another useful poxvirus vector is TROVAC. TROVAC refers to
an attenuated fowlpox that was a plaque-cloned isolate derived from
the FP-1 vaccine strain of fowlpoxvirus which is licensed for
vaccination of 1 day old chicks. TROVAC was likewise deposited
under the terms of the Budapest Treaty with the ATCC, accession
number 2553.
[0094] "Non-viral" plasmid vectors may also be suitable in
practicing the present invention. Suitable plasmid vectors are
compatible with bacterial, insect, and/or mammalian host cells.
Such vectors include, for example, PCR-II, pCR3, and pcDNA3.1
(ThermoFisher), pBSII (Agilent Technology Santa Clara CApET15 (EMD
Millipore, Bullerica, Mass.) pGEX (GE HealthCare Bioscience,
Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL
(BlueBacII, Thermofisher), pDSR-alpha (PCT pub. No. WO 90/14363)
and pFastBacDual (ThermoFisher)) as well as Bluescript.RTM. plasmid
derivatives (a high copy number COLE1-based phagemid, (Agilent
Technology Santa Clara, Calif.), PCR cloning plasmids designed for
cloning Taq-amplified PCR products (e.g., TOPO.TM. TA Cloning.RTM.
kit, PCR2.1.RTM. plasmid derivatives, Thermofisher Bacterial
vectors may also be used with the current invention. These vectors
include, for example, Shigella, Salmonella, Vibrio cholerae,
Laclobacillus, Bacille calmette guerin (BCG), and Streptococcus
(see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796;
and WO 92/21376). Many other non-viral plasmid expression vectors
and systems are known in the art and could be used with the current
invention.
[0095] Suitable nucleic acid delivery techniques include DNA-ligand
complexes, adenovirus-ligand-DNA complexes, direct injection of
DNA, CaPO4 precipitation, gene gun techniques, electroporation
polymer gene delivery system, cell-penetrating peptide gene
delivery system, and colloidal dispersion systems, among others.
Polymer gene delivery system includes polyetherimide- and
pluronic-based delivery systems. Cell-penetrating peptide-based
systems include 9-35 mer cationic and/or amphipathic peptides
capable of mediating translocation of DNA across plasma membrane.
Colloidal dispersion systems include macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. Liposomes are artificial membrane vesicles useful as
delivery vehicles in vitro and in vivo. RNA, DNA and intact virions
can be encapsulated within the aqueous interior and be delivered to
cells in a biologically active form (Fraley, R., et al., 1981,
Trends Biochem. Sci., 6: 77). The composition of the liposome is
usually a combination of phospholipids, particularly
high-phase-transition-temperature phospholipids, usually in
combination with steroids, especially cholesterol. Other
phospholipids or other lipids may also be used. The physical
characteristics of liposomes depend on pH, ionic strength, and the
presence of divalent cations. Examples of lipids useful in liposome
production include phosphatidyl compounds, such as
phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, sphingolipids, cerebrosides, and
gangliosides. The lipid can be a diacylphosphatidylglycerol. The,
lipid moiety of diacylphosphatidylglycerol can have 14-18 carbon
atoms. The lipid moiety of diacylphosphatidylglycerol can have
16-18 carbon atoms. The lipid moiety of diacylphosphatidylglycerol
can be saturated. Illustrative phospholipids include egg
phosphatidylcholine, phosphatidylethanolamine, sphingolipids,
cerebrosides, and gangliosides.
[0096] In an embodiment, a method for the treatment of inflammatory
diseases is provided. In various embodiments, the method for the
treatment of inflammatory diseases that are not related to cancer
is provided. In some embodiments, the treatment of inflammatory
diseases comprises administering a therapeutically effective amount
of p62 polypeptides or p62 encoding nucleic acids to a subject in
need thereof, in such amounts and for such time as is necessary to
achieve the desired result. In certain embodiments of the present
invention a "therapeutically effective amount" of an inventive
targeted particle is that amount effective for treating,
alleviating, ameliorating, relieving, delaying onset of, inhibiting
progression of, reducing severity of, and/or reducing incidence of
one or more symptoms or features of inflammatory diseases.
[0097] In one aspect of the invention, a method for administering
p62 polypeptides or p62 encoding nucleic acids to a subject
suffering from inflammatory disease or relapse is provided. In some
embodiments, p62 polypeptides or p62 encoding nucleic acids are
administered to a subject in such amounts and for such time as is
necessary to achieve the desired result (i.e. treatment of
inflammatory disease). In certain embodiments of the present
invention a "therapeutically effective amount" of p62 polypeptides
and p62 encoding nucleic acids is that amount effective for
treating, alleviating, ameliorating, relieving, delaying onset of,
inhibiting progression of, reducing severity of, and/or reducing
incidence of one or more symptoms or features of inflammatory
disease. In some embodiments, the p62 polypeptides or p62 encoding
nucleic acids of the invention are administered to a subject
previously treated for inflammatory disease. In some embodiments,
the p62 polypeptides or p62 encoding nucleic acids of the invention
are administered to a subject with a family history of inflammatory
disease. In some embodiments, the p62 polypeptides or p62 encoding
nucleic acids of the invention are administered to a subject with a
predisposition for inflammatory diseases. For example, a subject
who is genetically predisposed to inflammatory diseases (ALS,
Parkinson disease, Huntington disease), or subjected to
environmental factors provoking inflammatory disease (e.g., tobacco
smoke, asbestos, silica particles). Genetic predisposition to ALS
is associated with mutations in genes of superoxidismutase or TAR
DNA-binding protein 43 (TDP-43). Genetic predisposition to
Parkinson disease is associated with the parkin and synuclein
genes. Genetic predisposition to Huntington disease is associated
with a mutation in huntingtin gene (Glass, C. K., K. Saijo, et al.
(2010). "Mechanisms Underlying Inflammation in Neurodegeneration."
Cell 140(6): 918-934). Tobacco smoke, asbestos, silica particles
are all well-known inducers of chronic inflammation as persistent
allergens and undigestable foreign particles; they lead to chronic
obstructive pulmonary disease, asbestosis and silicosis (Medzhitov
2010. Inflammation 2010: New Adventures of an Old Flame. Cell
140:771-776)
[0098] In some embodiments, p62 polypeptides or p62 encoding
nucleic acids in accordance with the present invention may be used
to treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of an
inflammatory disease, disorder, and/or condition. In some
embodiments, the inflammatory disease, disorder, and/or condition
is non-cancer-related. Such diseases include, but not limited to,
osteoporosis, obesity, metabolic syndrome, type 2 diabetes, fat
liver, inflammatory bowel disease, gastritis, chronic pancreatitis,
asthma, chronic obstructive pulmonary disease (COPD), rheumatoid
arthritis (RA), osteoarthritis, multiple sclerosis (MS), psoriasis,
congestive heart failure (CHF), atherosclerosis, neurodegenerative
diseases (ALS, Parkinson, Alzheimer's, Huntington disease), gout,
asbestosis and silicosis.
[0099] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of
osteoporosis. Osteoporosis is the most common disease of the bone
associated with bone loss and affecting mostly women after onset of
menopause. Menopause leads to decrease in estrogen levels, thus
ovariectomy in rodents leading to cessation of estrogen generation
is the most common model for osteoporosis. Postmenopausal period is
marked by elevation of cytokines such us IL-6, TNF-alpha and IL-1
beta, and the same cytokines are elevated under ovariectomy. TNF
and IL-1 have potent antiapoptotic effects in osteoclasts
prolonging OC lifespan, accelerating bone resorption and inhibiting
bone formation, and blockade of TNF-alpha and IL-1beta prevents
osteoporosis due to estrogendeficiency. (Mundy 2007. Osteoporosis
and Inflammation. Nutrition Reviews 65:S147-S151; Lencel and Magne
2011. Inflammaging: The driving force in osteoporosis? Medical
Hypotheses 76:317-321).
[0100] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of obesity,
type 2 diabetes and fat liver disease. Obesity is increasingly
prevalent in the population and strongly associated with the
development of insulin resistance, an underlying feature of both
type 2 diabetes (T2D) and metabolic syndrome. Insulin resistance
has been recognized as the integral feature of metabolic syndrome,
which includes glucose intolerance, insulin resistance, obesity,
hypertriglyceridemia, low HDL cholesterol, hypertension, and
accelerated atherosclerosis. Growing evidence links a chronic,
subacute inflammatory state to the development of obesity and the
coexisting conditions of insulin resistance, T2D and metabolic
syndrome. The proinflammatory cytokine TNF-.alpha. has been
demonstrated to mediate insulin resistance as a result of obesity
in many rodent obesity models. In addition to TNF-alpha, various
other inflammatory mediators and cytokines are also overexpressed
in adipose and other tissues in experimental mouse models of
obesity and in humans. Proinflammatory cytokines can cause insulin
resistance and anti-inflammatory medications may reverse it
suggesting that inflammation may be directly involved in its
(Hotamisligil 2006. Inflammation and metabolic disorders. Nature
444:860-867).
[0101] Non-alcoholic fatty liver disease (NAFLD) is recognized as
the hepatic manifestation of metabolic syndrome, and is
characterized by the accumulation of fatty infiltrations affecting
>5% of the liver. The clinical implications of NAFLD are derived
by its potential to progress to steatohepatitis (NASH), fibrosis,
cirrhosis, and in some cases, hepatocellular carcinoma. The
prevalence of NAFLD is rising in parallel with the increasing rate
of obesity, and this global trend is attributed to the diet of the
Western lifestyle. The pathogenesis of NAFLD is not well
understood, but is proposed to be a "two-hit" process. The first
"hit" leads to lipid accumulation and steatosis. Mechanisms of this
lipid accumulation are unclear, but likely involve dysregulated
lipid homeostasis including beta-oxidation, very low density
lipoprotein secretion, de novo lipogenesis, and lipid trafficking
and storage. This hepatic steatosis sensitizes the liver to a
"second hit" leading to inflammation, a key pathophysiologic
feature of steatohepatitis, and progressive liver disease (Renaud
et al. 2014. Effect of Diet on Expression of Genes Involved in
Lipid Metabolism, Oxidative Stress, and Inflammation in Mouse
Liver-Insights into Mechanisms of Hepatic Steatosis. PLoS ONE
9:e88584)
[0102] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of IBD,
including, but not limited to ulcerative colitis and Crohn's
disease. Inflammatory Bowel Disease (IBD) is a chronic inflammatory
disorder of gastrointestinal tract. Examples of IBD include
ulcerative colitis and Crohn's disease. Substantial evidence from
human genetic studies and from preclinical IBD models suggests that
failures by either the mucosal epithelial layer or the immune
system to properly interact with the lumen microbial community may
underlie the pathogenic processes. IBD is also an important risk
factor for development of colon carcinoma, where proinflammatory
cytokines TNFa, interleukin-1, IL-6 plays a significant role role
(Danese and Mantovani 2010. Inflammatory bowel disease and
intestinal cancer: a paradigm of the Yin-Yang interplay between
inflammation and cancer. Oncogene 29:3313-3323)
[0103] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of asthma.
Asthma is an inflammatory disease in the airway, leading to airway
hyperresponsiveness, obstruction, mucus hyper-production and airway
wall remodeling. Asthma is classically recognized as the typical
Th2 disease, with increased IgE levels and eosinophilic
inflammation in the airway. Emerging Th2 cytokines modulates the
airway inflammation, which induces airway remodeling. However, the
relatively simple paradigm has been doubted because of the
realization that strategies designed to suppress Th2 function are
not effective enough for all patients in the clinical trials. (Kudo
et al. 2013. Pathology of asthma. Frontiers in Microbiology
4:263).
[0104] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of COPD
(chronic obstructive pulmonary disease). COPD is associated with
chronic inflammation affecting predominantly lung parenchyma and
peripheral airways and results in largely irreversible and
progressive airflow limitation. This inflammation is characterized
by increased numbers of alveolar macrophages, neutrophils, and T
lymphocytes, which are recruited from the circulation. Oxidative
stress plays a key role in driving this inflammation. The pulmonary
inflammation may enhance the development and growth of lung cancer.
The peripheral inflammation extends into the circulation, resulting
in systemic inflammation with the same inflammatory proteins.
Systemic inflammation may worsen comorbidities. Treatment of
pulmonary inflammation may therefore have beneficial effects.
(Barnes 2014. Cellular and Molecular Mechanisms of Chronic
Obstructive Pulmonary Disease. Clinics in Chest Medicine
35:71-86).
[0105] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of Rheumatoid
Arthritis (RA). RA is a chronic, inflammatory, systemic autoimmune
disease that affects about 1% of the general population in Western
countries and is two to three times more common in women than in
men. Although the etiology and pathogenesis of RA is not yet fully
understood, the disease is characterized by aggressive synovial
hyperplasia (pannus formation) and inflammation (synovitis), which,
if left untreated, leads to progressive destruction of joint
cartilage and bone. The destructive lesions result from immune
responses and nonantigen-specific innate inflammatory
processes.
[0106] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of
osteoarthritis (OA) is a disease of the joints that affects several
million individuals worldwide. This disease, which involves mainly
the diarthrodial joints, is chronic and develops slowly over
decades. The role of synovial inflammation in the pathophysiology
of OA is now widely accepted. Synovitis has been considered
secondary to the cartilage changes yet findings indicate that
synovial inflammation could be a component of the early events
leading to the clinical stage of OA. Synovial inflammation leads to
the production and release of pro-inflammatory cytokines and
several other inflammatory mediators. Some of these factors,
including the pro-inflammatory cytokines, diffuse through the
synovial fluid into the cartilage, where they activate chondrocyte
production of the catabolic factors through auto- and paracrine
mechanisms.
[0107] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of Multiple
Sclerosis (MS). MS is characterized by inflammation, demyelination,
and axon degeneration in the CNS. Individuals are plagued by
MS-associated comorbidities, such as chronic pain, fatigue,
depression, sleep disorders, spasticity, gait and coordination
imbalances, migraines, sensory organ dysfunctions, and overall
cognitive impairment (Damal et al. 2013. Optimizing therapeutics in
the management of patients with multiple sclerosis: a review of
drug efficacy, dosing, and mechanisms of action. Biologics
7:247-258). This pathology results from a primary defect in the
immune system that targets components of the myelin sheath,
resulting in secondary effects on neurons. MS is considered an
immune-mediated disease characterized by the presence of
inflammatory demyelinating lesions in the CNS. Infection by
bacteria or viruses or other environmental stimuli trigger the
activation of microglia and astrocytes in multiple sclerosis (MS),
leading to the production of proinflammatory cytokines through
activation of the transcription factors NF-kappa-B and AP-1 (Luessi
et al. 2012. Neurodegeneration in multiple sclerosis: novel
treatment strategies. Expert Rev Neurother 12:1061-1076).
[0108] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of
atherosclerosis. Atherosclerosis shares features with traditional
inflammatory diseases including rheumatoid arthritis.
Atherosclerosis is the main cause of coronary artery and
cerebrovascular disease, which are the leading cause of death
worldwide. Atherogenesis is thought to begin with the development
of endothelial dysfunction caused by the exposure of the vessel
wall to systemic risk factors and local hemodynamics. The ensuing
endothelial activation promotes the accumulation of inflammatory
cells in the vessel wall. As atheroma progresses, inflammatory
cells produce cytokines and growth factors, which evoke smooth
muscle cell migration into the intima. The architecture of the
intima changes profoundly leading to the formation of two
compartment lesions, the fibrous cap and the necrotic core.
Inflammatory cells may also produce matrix degrading enzymes that
disrupt the integrity of the fibrous cap or procoagulant molecules
such as tissue factor, ultimately leading to plaque rupture and
thrombosis (Cole et al. 2011. Toll-like receptors in
atherosclerosis: a "Pandora's box" of advances and controversies.
Trends in Pharmacological Sciences 34:629-636).
[0109] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of gastritis.
A cause of gastritis is Helicobacter pylon colonizing the gastric
mucosa causing chronic inflammation, which is characterized by
enhanced expression of many inflammatory genes.
[0110] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of chronic
pancreatitis (CP). Chronic pancreatitis (CP) is a
fibro-inflammatory disease involving the pancreatic parenchyma
which is progressively destroyed and replaced by fibrotic tissues.
Histologically, acinar cell damage, mononuclear cell infiltration,
and fibrosis are observed. There are various causes that may lead
to CP, but the exact pathophysiology of the disease is still
unclear.
[0111] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of psoriasis.
Psoriasis is a chronic inflammatory skin disease, most commonly
resulting in the occurrence of red and silver scaly plaques; it
affects approximately 2-3% of the general population. Although its
pathogenesis is not fully understood, there is an underlying
interaction between numerous immune effector cells and aberrant
hyperproliferation and differentiation of epidermal keratinocytes.
It is a prototype of immune dysregulation mediated by TH1
proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and
IL-12 (Goldminz et al. 2012. NF-kappB: An essential transcription
factor in psoriasis. Journal of Dermatological Science
69:89-94).
[0112] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of congestive
heart failure (CHF). CHF is a leading cause for both
hospitalization and death in the western world. Its prevalence is
rather increasing with the broad implementation of standardized
evidence-based treatment algorithms for heart failure. The heart
failure syndrome is characterized by impaired systolic and/or
diastolic function and various clinical signs such as fatigue,
dyspnea, fluid retention, and cachexia. An inflammatory activation
in CHF patients has long been recognized. Indeed, immune mechanisms
modulate interstitial fibrosis, cardiomyocyte apoptosis, and
hypertrophy, all of which are central processes leading to
maladaptive remodeling in response to a variety of stimuli.
Especially for heart failure evolving from large myocardial
infarction there is substantial evidence for a causal contribution
of immunity early in the course of the disease. Systemic cytokines
came have been monitored in several clinical trials. The broadest
amount of data was gathered for tumor necrosis factor-alpha
(TNF-alpha) which was demonstrated to correlate well with diverse
clinical and laboratory parameters, such as exercise capacity and
neurohormonal activation in CHF patients.
[0113] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of
neurodegenerative disease. In an embodiment, p62 polypeptides or
p62 encoding nucleic acids in accordance with the present invention
may be used to treat, alleviate, ameliorate, relieve, delay onset
of (prophylaxis), inhibit progression of, reduce severity of,
and/or reduce incidence of one or more symptoms or features of
Alzheimer's disease (AD). AD is the most common form of dementia in
the elderly resulting in a progressive decline in a number of
cognitive functions including short-term memory. AD is
characterized by the formation of two characteristic lesions:
extracellular beta-amyloid deposits forming senile plaques and
intracellular neurofibrillary tangles made up of the microtubule
associated protein tau. A strong link between inflammation,
primarily mediated by pro-inflammatory cytokines, and AD has been
established both in clinical data and bench research. Recent
findings also suggest that AD may be associated with a more
widespread inflammatory state characterized by increased peripheral
blood levels of IL-1, IL-6, TNF-alpha, TGF-beta, and IL-18 (Smith
et al. 2012. Role of pro-inflammatory cytokines released from
microglia in neurodegenerative diseases. Brain Res Bull
87:10-20).
[0114] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of Parkinson's
disease (PD). PD is the most common neurodegenerative movement
disorder and is caused by the progressive loss of dopaminergic
neurons from the substantia nigra pars compacta that normally
innervate the striatum. The pathological hallmark of PD is
intracellular accumulation of alpha-synuclein leading to the
formation of Lewy bodies. PD may result in a number of different
presenting symptoms including resting tremor, bradykinesia,
cogwheel rigidity, and postural instability. Epidemiologic findings
from a number of studies suggest that inflammation may be involved
in the pathogenesis of PD. This is also supported, in part, by
post-mortem analysis of cerebrospinal fluid and brain demonstrating
elevated protein levels of pro-inflammatory cytokines in PD
patients (Smith et al. 2012. Role of pro-inflammatory cytokines
released from microglia in neurodegenerative diseases. Brain Res
Bull 87:10-20).
[0115] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of Amyotrophic
Lateral Sclerosis (ALS), or Lou Gehrig's disease. ALS is a
progressive fatal neurodegenerative disease that affects motor
neurons in the brainstem, spinal cord, and motor cortex. ALS is
universally fatal, with a median age of onset of 55 years and a
survival of 2-5 years after the onset of symptoms. Prominent
neuroinflammation can be easily observed in pathologically affected
areas of the CNS and in spinal cords from both human ALS patients
and mouse models of the disease. Typically, inflammation in ALS is
characterized by gliosis and the accumulation of large numbers of
activated microglia and astrocytes. Activation of glia in ALS has
been extensively characterized and is marked by elevated production
of potentially cytotoxic molecules such as ROS, inflammatory
mediators such as COX-2, and proinflammatory cytokines such as
IL-1beta, TNF-alpha, and IL-6 (Smith et al. 2012. Role of
pro-inflammatory cytokines released from microglia in
neurodegenerative diseases. Brain Res Bull 87:10-20).
[0116] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of Huntington
disease. Huntington's disease (HD) is characterized by a
progressive course of disease until death 15-20 years after the
first symptoms occur and is caused by a mutation with expanded CAG
repeats in the huntingtin (htt) protein. Mutant htt (mhtt) in the
striatum is assumed to be the main reason for neurodegeneration.
Both innate and adaptive immune systems may play an important role
in HD. Activation of microglia with expression of proinflammatory
cytokines, impaired migration of macrophages, and deposition of
complement factors in the striatum indicate an activation of the
innate immune system (Ellrichmann et al. 2013. The Role of the
Immune System in Huntington's Disease. Clinical and Developmental
Immunology 2013:11). Enhancement of the NFkB-mediated inflammatory
response in astrocytes contributes to HD pathogenesis (Hsiao et al.
2013. A critical role of astrocyte-mediated nuclear
factor-kB-dependent inflammation in Huntington's disease. Human
Molecular Genetics 22:1826-1842).
[0117] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of depression
or schizophrenia. Depression is associated with both a chronic
low-grade inflammatory response, activation of cell-mediated
immunity and activation of the compensatory anti-inflammatory
reflex system (CIRS), characterized by negative immunoregulatory
processes. Meta-analyses of over 100 studies provided in vivo
evidence that schizophrenia can be, in part, explained by an
inflammatory imbalance.
[0118] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of asbestosis.
Asbestosis is a chronic inflammatory and fibrotic medical condition
affecting the parenchymal tissue of the lungs caused by the
inhalation and retention of asbestos fibers. It usually occurs
after high intensity and/or long-term exposure to asbestos
(particularly in those individuals working on the production or
end-use of products containing asbestos) and is therefore regarded
as an occupational lung disease. Sufferers may experience severe
dyspnea (shortness of breath) and are at an increased risk for
certain malignancies, including lung cancer but especially
mesothelioma. Asbestosis specifically refers to interstitial
(parenchymal) fibrosis from asbestos, and not pleural fibrosis or
plaquing. The primary symptom of asbestosis is generally the slow
onset of dyspnea, especially on exertion. Clinically advanced cases
of asbestosis may lead to respiratory failure. The characteristic
pulmonary function finding in asbestosis is a restrictive
ventilatory defect. In the more severe cases, the drastic reduction
in lung function due to the stiffening of the lungs and reduced TLC
may induce right-sided heart failure (cor pulmonale). In addition
to a restrictive defect, asbestosis may produce reduction in
diffusion capacity and arterial hypoxemia.
[0119] In an embodiment, p62 polypeptides or p62 encoding nucleic
acids in accordance with the present invention may be used to
treat, alleviate, ameliorate, relieve, delay onset of
(prophylaxis), inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of silicosis.
Silicosis, (previously miner's phthisis, grinder's asthma, potter's
rot and other occupation-related names) is a form of occupational
lung disease caused by inhalation of crystalline silica dust, and
is marked by inflammation and scarring in the form of nodular
lesions in the upper lobes of the lungs. It is a type of
pneumoconiosis. Silicosis (particularly the acute form) is
characterized by shortness of breath, cough, fever, and cyanosis
(bluish skin). Signs and symptoms include: dyspnea, cough, fatigue,
tachypnea, loss of appetite and weight loss, chest pain, and fever.
In advanced cases, symptoms may include cyanosis, cor pulmonale,
and respiratory insufficiency. Subjects with silicosis are
particularly susceptible to tuberculosis (TB) infection--known as
silicotuberculosis. Pulmonary complications of silicosis also
include Chronic Bronchitis and airflow limitation
(indistinguishable from that caused by smoking), non-tuberculous
Mycobacterium infection, fungal lung infection, compensatory
emphysema, and pneumothorax. There are some data revealing an
association between silicosis and certain autoimmune diseases,
including nephritis, Scleroderma, and Systemic Lupus Erythematosus,
especially in acute or accelerated silicosis.
[0120] Methods of the invention further include administering one
or more anti-inflammatory therapies to a subject. Anti-inflammatory
chemotherapeutic agents are any chemical entity or drug that
reduces inflammation. Anti-inflammatory chemotherapeutic agents
include, but are not limited to, a nonsteroidal anti-inflammatory
drug (NSAID), a glucocorticoid, methotrexate, cyclosporine, and
rapamycin. NSAIDs are cyclooxygenase inhibitors. Examples of NSAIDs
include aspirin, ibuprofen, naproxen sodium, diclofenac, etodolac,
fenoprofen, flurbiprofen, oxaprozin. Glucocorticoids are a class of
steroid hormones that bind to the glucocorticoid receptor.
Methotrexate is a chemical analog of folic acid which inhibits the
metabolism of folic acid. Cyclosporine and rapamycin,
anti-rejection drugs, have anti-inflammatory properties.
[0121] Anti-inflammatory biologic agents are any
naturally-occurring biologic entity that reduces inflammation.
Anti-inflammatory biologic agents include, but are not limited to,
an anti-TNF antibody, an anti-IL1 antibody, an anti-IL6 antibody,
an anti-IL6 receptor antibody, an anti-IL12/23 antibody, an
anti-IL17 antibody, an anti-IL1R antibody, an anti-IL1 receptor
antagonist, and a soluble IL-1 receptor.
[0122] Compounds and compositions described herein can be
administered as a pharmaceutical or medicament formulated with a
pharmaceutically acceptable carrier. Accordingly, the compounds and
compositions may be used in the manufacture of a medicament or
pharmaceutical composition. Pharmaceutical compositions of the
invention may be formulated as solutions or lyophilized powders for
parenteral administration. Powders may be reconstituted by addition
of a suitable diluent or other pharmaceutically acceptable carrier
prior to use. Liquid formulations may be buffered, isotonic,
aqueous solutions. Powders also may be sprayed in dry form.
Examples of suitable diluents are normal isotonic saline solution,
standard 5% dextrose in water, or buffered sodium or ammonium
acetate solution. Such formulations are especially suitable for
parenteral administration, but may also be used for oral
administration or contained in a metered dose inhaler or nebulizer
for insufflation. It may be desirable to add excipients such as
polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium chloride, sodium citrate, and
the like.
[0123] Alternately, compounds may be encapsulated, tableted or
prepared in an emulsion or syrup for oral administration.
Pharmaceutically acceptable solid or liquid carriers may be added
to enhance or stabilize the composition, or to facilitate
preparation of the composition. Solid carriers include starch,
lactose, calcium sulfate dihydrate, terra alba, magnesium stearate
or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid
carriers include syrup, peanut oil, olive oil, saline and water.
The carrier may also include a sustained release material such as
glyceryl monostearate or glyceryl distearate, alone or with a wax.
The amount of solid carrier varies but, preferably, will be between
about 20 mg to about 1 g per dosage unit. The pharmaceutical
preparations are made following the conventional techniques of
pharmacy involving milling, mixing, granulating, and compressing,
when necessary, for tablet forms; or milling, mixing and filling
for hard gelatin capsule forms. When a liquid carrier is used, the
preparation may be in the form of a syrup, elixir, emulsion, or an
aqueous or non-aqueous suspension. For rectal administration, the
invention compounds may be combined with excipients such as cocoa
butter, glycerin, gelatin or polyethylene glycols and molded into a
suppository.
[0124] Compounds or compositions may be formulated to include other
medically useful drugs or biological agents. The compounds or
compositions also may be administered in conjunction with the
administration of other drugs or biological agents useful for the
disease or condition to which the invention compounds or
compositions are directed.
[0125] As employed herein, the phrase "an effective amount," refers
to a dose sufficient to provide concentrations high enough to
impart a beneficial effect on the recipient thereof. The specific
therapeutically effective dose level for any particular subject
will depend upon a variety of factors including the disorder being
treated, the severity of the disorder, the activity of the specific
compound or composition, the route of administration, the rate of
clearance of the compound or composition, the duration of
treatment, the drugs used in combination or coincident with the
compound or composition, the age, body weight, sex, diet, and
general health of the subject, and like factors well known in the
medical arts and sciences. Various general considerations taken
into account in determining the "therapeutically effective amount"
are known to those of skill in the art and are described, e.g., in
Gilman et al., eds., Goodman And Gilman's: The Pharmacological
Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and
Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co.,
Easton, Pa., 1990. Dosage levels typically fall in the range of
about 0.001 up to 100 mg/kg/day; with levels in the range of about
0.05 up to 10 mg/kg/day are generally applicable. A compound can be
administered parenterally, such as intravascularly, intravenously,
intraarterially, intramuscularly, subcutaneously, or the like.
Administration can also be orally, nasally, rectally,
transdermally, intravaginally or inhalationally via an aerosol. A
compound or composition can be administered to the
inflammation-related lesions. The compound or composition may be
administered as a bolus, or slowly infused, or be administered as
an intradermal, subcutaneous or intramuscular or intraperitoneal
injection.
[0126] A therapeutically effective dose can be estimated initially
from cell culture assays by determining p62 expression levels upon
introduction of the DNA or RNA encoding p62 into the cells. A dose
can then be confirmed in animal models to achieve suppression of
generation of inflammatory cytokines and/or alleviation of
inflammation. Such information can be used to more accurately
determine useful initial doses in humans. The exact formulation,
route of administration and dosage can be chosen by medical
professional in view of the subject's presentation.
EXAMPLES
Example 1
Materials and Methods
Vector Construction
[0127] As a source of cDNA encoding p62, total RNA was extracted
from HeLa cells. Full length cDNA encoding the longer isoform of
p62 (Transcript Variant 1, Gen Bank reference No. NP_003891) was
amplified by PCR (HotStar HiFidelity Polymerase Kit Qiagen) using
the following primers: FW: 5-CCCGCTAGCATGGCGTCGCTCACCGTG-3 and REV:
5'-CCCAAGCTTTCACAACGGCGGGGGATGCTTTG-3'. PCR products were purified
and Nhe I-Hind III digested fragments cloned into a DNA vector with
nucleic acid sequence corresponding to pcDNA3.1 resulting in p62
plasmid. A full length ovalbumin gene (pOVA) cloned in pcDNA3.1 was
taken as reference plasmid. Large scale preparations of the
endotoxin-free plasmids were routinely performed by alkaline lysis
using either Endo Free Plasmid Kit (Qiagen) or Gen Elute HPSelect
Plasmid Giga Prep columns (SIGMA # NA0800). The DNA constructs were
confirmed by sequencing.
Animals and Treatments
[0128] Three-month old female FVB and Balb/c mice (Harlan Italy
SrL, Correzzana Milano, Italy) were used. Mice were kept in
laminar-flow cage in a standardized environmental condition. In
prevention trials mice were randomly distributed in three groups
(G1-G3) and injected intramuscularly at week 0, 1, 2 with only
saline (G1, n=12), with pcDNA3.1 (G2, n=12), or with hp62 DNA (G3
n=12). At day forty-five after the last injection, mice from each
group were randomly divided in two subgroups and were sham operated
(SO; n=6) or ovariectomized (OVX n=6). After two months mice were
sacrificed by CO2 narcosis according to the recommendation of the
Italian Ethical Committee. For therapeutic trials mice were
ovariectomized (OVX) and left untreated for 2 months. Afterwards,
mice were randomized in 4 subgroups, and injected with plasmid as
described above. After 2 months mice were sacrificed for
analysis.
Histological Bone Analysis and Immuflourescence
[0129] Femurs, dissected of adhering tissue, were fixed in 4%
paraformaldehyde (PFA) for 24 h, decalcified in 14% EDTA solution
for 3 days and soaked in 30% sucrose overnight. Samples, embedded
with Tissue-Tek OCT compound, were sectioned (8 .mu.m thick
sections) by a rotatory -30.degree. C. microtome cryostat (Ames
Cryostat Miles) and stained with toluidine blue. Other sections,
after permeabilization with 0.3% Triton X-100 were incubated with
rabbit anti-p62 diluted 1:800 (Enzo Life Sciences; Vinci-Biochem
s.r.l., Firenze, Italy) diluted 1:400. After rinsing, sections were
incubated with chicken anti-rabbit IgG Alexa Fluor 488 conjugated
(Molecular Probes; Invitrogen, Milano Italy) diluted 1:100. Control
experiments were performed by omitting the appropriate primary
antibody or by complexing the primary antibodies with the relative
blocking peptide. Slides were imaged using a Leica DM 2500
fluorescent microscopy. Fluorescence analysis was performed by a
fluorimeter Tecan Infinite [29].
Ex Vivo Dual-Energy X-Ray Absorptiometry (DXA) Analyses
[0130] Femurs were dissected and fixed as above described. Bone
mineral density (BMD) and bone mineral content (BMC) were measured
using a PIXImus DEXA.
Bone Marrow Cell (BMSC) Preparation
[0131] Long bones (femurs, tibiae and humeri) from the mouse groups
were dissected free of adhering tissue. The ends were removed and
the marrow cavity was flushed and cultured in DMEM.
Cytokines and Chemokines Assay
[0132] The cytokine/chemokine profiles of BMSCs supernatants were
assessed by ELISA-based cytokine array by using Mouse Cytokine
Array Panel A kit (R&D Systems, Milano, Italy) accordingly to
the manufacturer's instructions. Immunoreactive dots were
visualized using LiteAblot Turbo luminol reagents (Euroclone,
Milano, Italy) and Hyperfilm-ECL film (Euroclone, Milano, Italy)
and quantitated densitometrically.
Western Immunoblotting
[0133] Proteins from total bone marrow cells population were
extracted in Cell Lysis Buffer (Cell Signaling Euroclone, Milano,
Italy) immediately after flushing the bone marrow cavity, and the
concentration was determined by the BCA protein assay reagent
(Pierce, Euroclone Milano, Italy). Western blotting was performed
by standard methods.
Statistical Analysis
[0134] All in vitro and in vivo experiments were repeated at least
three times. t-student was used to test for significant differences
between two groups, and differences were considered significant at
*p<0.05.
Example 2
p62 Vaccine Prevents Osteoporosis in a Mouse Model
[0135] To evaluate whether p62 vaccine was able to prevent
osteoporosis, groups of mice were first injected either with p62DNA
or reference plasmids (pcDNA 3.1, pOVA) and then ovariectomized
(OVX). For each trial a control group of sham operated (SO) mice
was included. Two months after surgery mice were sacrificed, and
the collected long bones subjected to histological examination. The
metaphyseal regions of the distal femurs from pcDNA3.1-OVX
(control) mice displayed classic osteoporotic features
characterized by significant bone loss and thinned disconnected
trabecular structure. On the other hand, p62-OVX bones (treated
mice) showed a micro-architecture essentially indistinguishable to
that seen in SO mice. Moreover, examination of cross sections femur
diaphysis from p62DNA-OVX mice revealed (at variance of those
obtained from reference plasmids treated mice) an enhanced
anabolic-osteoblastic activity as evidenced by new cortical bone
apposition suggesting an anabolic action of p62 treatment.
[0136] BMCs were flushed from the bone cavities, and cultured for 3
days. Afterwards, both supernatants and cells were collected and
analyzed respectively either for the release of inflammatory
cytokines, or for expression of osteogenic markers. As shown in
FIG. 4 the marked up-regulation and release of pro-inflammatory
cytokines by BMCs from OVX compare to SO operated mice was
drastically suppressed by p62-DNA pre-treatment. The inhibitory
effect of p62 DNA extended to an array of cytokines such as
TNF.alpha., IL-6, IL-1b IL-17, all known to be essential inducers
of inflammatory diseases and bone loss. As far as the capability of
p62DNA to induce new bone formation is concerned, western blotting
analysis of p62-OVX BMCs extracts indicated a strong and selective
increase of osteogenic markers, such as Runx2 and Osterix
transcription factors. An increase of Runx2 and Osterix, although
weaker, was also found in p62 SO mice (FIG. 5).
[0137] Consequently, administration of p62 plasmid prevented
osteoporosis in a mouse model.
Example 3
p62 Vaccine Reverses Osteoporosis in a Mouse Model
[0138] In these trials, mice were ovariectomized and, after two
months, injected either with p62-DNA or reference plasmids (see
M&M for details). Two months after last plasmids injections,
bones were collected and histologically evaluated. OVX-p62 treated
mice group (in contrast to control groups) showed a restored
trabecular microarchitecture at metaphyseal regions of the distal
femurs and a decreased porosity in cortical bone. In addition,
p62-DNA treatment proved to increase both bone mineral density
(BMD) and content (BMC) as judged by DXA analysis (FIG. 6).
Finally, coupled with marked up-regulation of osteoblastogenic
Runx2 and Osterix (FIG. 7, panel A), a strong inhibition of two
majors bone resorptive factors such as TNF.alpha. and RANKL was
also observed in BMCs from OVX-p62 mice. RANKL is a key mediator of
inflammation that, by binding to its receptor RANK on osteoclast
precursors, fosters osteoclastogenesis via intracellular NF-kB
signaling. Down-regulation of NF-kappa-B in OVX-p62 BMCs was also
observed (FIG. 7, panel B). Consequently, p62 administration
reversed osteoporosis in a mouse model.
Example 4
p62 Vaccine Upregulates Endogenous p62 in a Mouse Model
[0139] The expression levels of p62 in BMCs retrieved from plasmids
injected mice before ovariectomy were determined. Surprisingly,
although p62 expression in BMCs was down-regulated by ovariectomy,
BMCs from p62 DNA pre-injected mice demonstrated up-regulation of
p62 (FIG. 8, panel A). Consistently, an increased p62-immune
labeling was observed at the epiphyseal region of femurs of p62-OVX
mice (FIG. 8, panel B).
[0140] Western Blotting analysis that can distinguish between human
(exogenous) and mouse (endogenous) p62 was performed. As shown in
FIG. 8, panel C, p62DNA administration up-regulates endogenous p62
protein in bone marrow-resident cells. Consequently, administration
of p62 plasmid increased endogenous p62 levels in a mouse
model.
Example 5
p62 Administration Reduced Symptoms of Amyotrophic Lateral
Sclerosis (ALS) in a Mouse Model
[0141] FIG. 9 shows the effect of p62 on a mouse model of ALS.
Neuroinflammation is a prominent pathologic feature in the spinal
cord of patients with ALS, and is characterized by glial activation
and infiltrating T cells. A similar inflammatory response is
present in spinal cords of ALS mice. This is the most common mouse
model of ALS expressing G93A mutant form of SOD, the same mutation
which occurs in a humans with ALS, and this model is widely used
for testing of drugs against ALS. Mice were treated with p62
plasmid or pcDNA 3.1 plasmids (6 times weekly with 150 ug/mouse
i.m.) from day 75 after birth (6 mice per group), and a standard
test for ALS (hind limb extension reflex) was applied. This reflex
was evaluated as the ability to perform complete extension of the
hind limbs when the animal was suspended by the tail. Without ALS,
both control and p62 treated mice demonstrate strong reflex, which
started to decline in ALS mice after 60 days of age, and it
decreased from 100% to zero on 100 days. Treatment with p62,
however, markedly delayed decline in the reflex. Consequently,
administration of p62 plasmid reduced symptoms related to ALS in a
mouse model.
Example 6
p62 Administration Reduced Symptoms of Multiple Sclerosis (MS) in a
Mouse Model
[0142] MS is a chronic inflammatory disease resulting in
demyelination and axonal loss throughout the central nervous system
(CNS), with unknown cause and only limited treatment options
(Noseworthy, J. H., C. Lucchinetti, et al. (2000)"Multiple
sclerosis." N Engl J Med 343(13): 938-52; Lassmann et al.,
2001"Heterogeneity of multiple sclerosis pathogenesis: implications
for diagnosis and therapy." Trends Mol Med 7(3): 115-21). The most
commonly used animal model for MS research is murine experimental
allergic encephalomyelitis (EAE) induced by administration of MOG
polypeptides. This model resembles both the inflammatory phase,
i.e., the generation of autoreactive myelin specific T cells, as
well as the neurodegenerative phase of the disease, i.e.,
destruction of the myelin sheath around the axons and subsequent
loss of axons, as observed in human disease (Steinman, 2001,
"Multiple sclerosis: a two-stage disease." Nat Immunol 2(9):
762-4.). FIG. 10 demonstrates clinical signs of rMOG-induced EAE
showing mean clinical scores. Mice (4 per group) were examined
daily for clinical signs of EAE and were scored as followed: 0, no
disease; 1, limp tail; 2, hind limb weakness; 3, complete hind limb
paralysis; 4, hind limb paralysis plus forelimb paralysis; and 5,
moribund or dead. On day 0 MOG immunization. p62 DNA (100 .mu.g/50
.mu.l) administered at days 16 and 22 after MOG immunization as
indicated by red arrows. As one can see, whereas control mice had
higher EAE score till the end of observation (day 31), in mice
treated with p62 plasmid this score progressively declined (to
about 0.5 at day 31). Consequently, administration of p62 plasmid
alleviated MS-like symptoms in a mouse model of MS.
Example 7
p62 Administration on Tumor Growth and Mouse Survival
[0143] Chronic inflammation is involved in pathogenesis of
sarcomas, in particular, Kaposi sarcoma (Douglas J L, 2010 "Kaposi
Sarcoma Pathogenesis: A Triad of Viral Infection, Oncogenesis and
Chronic Inflammation." Transl Biomed 1(2). Transplantable sarcoma
37 in mice was used to study effect of p62 vaccination on tumor
growth. Mice (6 per group) were injected i.m. with p62 plasmid or
empty vector (250 ug per mouse) 14, 7 days before 1, 8, 14 days
after tumor inoculation and growth of tumors was monitored by
caliper. Injection of p62 plasmid almost completely prevented
growth of sarcoma 37. Consequently, administration of p62 plasmid
reduced growth of mouse S37 sarcoma
[0144] Chronic inflammation accompanies breast cancer, and
increased levels of IL-6 is a negative prognostic factor Lippitz,
B. E. (2013). "Cytokine patterns in patients with cancer: a
systematic review." The Lancet Oncology 14(6): e218-e228. There is
specific form of breast cancer, IBC (inflammatory breast cancer)
which is the most aggressive and with poor prognosis (Fernandez,
Robertson et al. 2013 "Inflammatory breast cancer (IBC): clues for
targeted therapies." Breast Cancer Research and Treatment 140(1):
23-33). To study effect of p62 vaccination on survival of mice with
Ca755 breast cancer, mice were treated with p62 polynucleotide and
their survival was monitored. Treatment with p62 increased mouse
mean survival with breast cancer by 56% (from 25 to 39 days) both
in fast and slow-growing tumors (FIG. 12).
Example 8
Effect of p62 Polynucleotide Administration on Tumor Metastases
[0145] LLC is a widely used model of metastatic lung cancer: after
subcutaneous inoculation of tumor cells in flanks of mice, within 3
weeks they form metastases in lungs which can be easily counted.
Treatment of mice with p62 polynucleotide markedly suppresses
formation of both small and big metastases thus effectively
blocking metastatic process (FIG. 13). Consequently, administration
of p62 plasmid suppresses lung cancer in a mouse model.
[0146] B16 melanoma cells were injected intravenously (in tail
vein), thereby imitating a clinical situation wherein cancer is
diagnosed when tumor cells are already in blood stream. Tumor cells
forms metastases in the lung, which can be detected and counted.
p62 polynucleotide was administered 1, 8, 15 days after tumor cell
inoculation. As seen in FIG. 14, treatment with p62 polynucleotide
significantly reduced number and size of metastatic tumors, thereby
demonstrating ant-metastatic effect.
Example 9
Obesity and Metabolic Syndrome
[0147] Three groups of newborn male mice received subcutaneous
injections of i) placebo or ii) and iii) 3 mg/kg of monosodium
glutamate (MSG) daily for 10 days. Group 3 then received 5 weekly
injections of 200 ug of p62-encoding plasmid intramuscularly. Body
weight of animas in all 3 groups was measured and compared. MSG
markedly increased body weight in both groups compared to the
control group (p<0.01 and p<0.05). However, increase of body
weight in the group receiving p62 plasmid was significantly less
than in a group receiving MSG alone. Consequently, administration
of the p62-encoding plasmid reduced obesity in the mouse model.
Example 10
Type 2 Diabetes
[0148] Two groups of Zucker Diabetic Fatty (ZDF) rats, a standard
model for Type 2 Diabetes, were fed with 6.5% fatty diet. One group
received 5 weekly injections of p62 plasmid, 200 ug/injection,
while another was used as a control. In the control group
hyperglycemia was observed between 8 and 10 weeks of age, while in
the plasmid treated group hyperglycemia was delayed an average of 3
weeks. Consequently, administration of the p62 plasmid delayed the
incidence of Type 2 Diabetes in the rat model.
Example 11
Fat Liver
[0149] Two groups of mice were fed with MCD diet (10% fat, 40%
sucrose, no choline, no methionine). Each group contained 15 mice.
Serum level of alanine aminotransferase (ALT) was monitored
starting at 10 weeks on the diet. An experimental group received 5
weekly intramuscular injections of p62-encoding plasmid, 200
ug/injection. The placebo group received MCD diet with no plasmid
intervention. Control group received normal diet and no plasmid
through the entire research period. Both groups dieting on MCD have
demonstrated elevated level of ALT compared to the control group
(p<0.01 for both groups). However, p62 treatment reduced ALT
level compared to placebo group (p<0.05). Consequently,
administration of the p62 plasmid reduced the incidence of fat
liver disease in the mouse model.
Example 12
Crohn's Disease
[0150] Mice received drinking water containing 8% dextran sulfate
sodium (DSS) for 2 weeks. Contemporarily, one group of mice
received 5 weekly in injections of 200 ug of p62-encoding plasmid
while the other group constituted the control (10 animals in each
group). p62-treated animals demonstrated reduction in bloody stool
and diarrhea. Also, the weight of each animal was monitored
individually once a week. p62 inhibited weight loss (p<0.05)
although it did not entirely stop it. Consequently, administration
of the p62 plasmid reduced symptoms related to Crohn's Disease in a
mouse model.
Example 13
Pancreatitis
[0151] Mice were treated with 4 mg/animal LPS and 1 ug per animal
caerulein for 10 weeks, twice a week, to induce pancreatitis. 3
mice were used as a control group, and 3 received 5 weekly
injections of p62 vaccine 200 ug/injection intramuscularly. After
15 weeks mice were sacrificed and subjected to histological
analysis. p62 plasmid treatment reduced the extent of observed
chronic pancreatitis. Consequently, administration of the p62
plasmid reduced pancreatitis in a mouse model.
Example 14
Asthma
[0152] Mice were subjected to three intraperitoneal injections of
1% ovalbumin solution. Injections were made with 14 days intervals.
A week after the 3 d injection animals were exposed to a 1%
ovalbumin aerosol for 30 min each day for 3 days to induce
asthma-like disease. During this 5 weeks period, one group of mice
received 5 weekly intramuscular injections of 200 ug of p62
plasmid, while another was kept as a control. Two days after the
final exposure, airway hyperresponsiveness (AHR) was measured and
compared in control and treatment group. AHR was induced either by
cold air or by hyperventilation. AHR manifestation was
significantly reduced in p62-treated group. Consequently,
administration of the p62 plasmid reduced asthma symptoms in a
mouse model.
Example 15
Arthritis Osteoarthritis
[0153] Two groups of mice (10 animals per group) were administered
collagen II (CII) with complete Freund adjuvant to induce
collagen-induced arthritis. Animals were 6 weeks of age to avoid
spontaneous arthritis, which can be observed in older animals. An
experimental group also received 5 weekly injections of 200 of of
p62-encoding plasmid. The control group manifested first signs on
arthritis 30-33 weeks after the CII challenge. The p62-treated
group demonstrated collagen-induced arthritis 38-43 weeks after CII
injections. Consequently, administration of the p62 plasmid delayed
collagen-induced arthritis in a mouse model.
Example 16
Atherosclerosis
[0154] ApoE(-/-) mice were maintained on a high-fat diet for 8 wks.
The control group was not vaccinated while the experimental group
received 5 weekly injections of 200 ug of p62-encoding plasmid. The
plasmid treatment reduced the atherosclerotic plaque area and
plaque neovessel density. It increased the plaque collagen and
elastin contents, and reduced plasma angiotensin II levels and
plaque macrophage infiltration and cathepsin S (CatS) protein. p62
administration also decreased the levels of AT1R, gp91phox, TLR2,
monocyte chemotactic protein-1 in the aortic roots. Consequently,
administration of the p62 plasmid reduced atherosclerosis in a
mouse model.
Example 17
Parkinson's Disease
[0155] Male Wistar rats were either pretreated with 5 weekly
injections of p62-encoding plasmid, 200 ug/injections, or used as a
control group. Then, 6-hydroxydopamine, 6-OHDA (10 micrograms in
0.1% ascorbic acid in normal saline) was administered to
experimental and control groups rats via unilateral intrastriatal
injection to simulate Parkinson's Disease. Three weeks after 6-OHDA
infusion, rats were tested for neurobehavioral activity (open-field
test and rotarod muscular coordination test). A control group which
received neither the plasmid nor 6-OHDA was utilized to assess the
effect of 6-OHDA. Pretreatment with p62 plasmid markedly reduced
Parkinson's-like manifestation induced by 6-OHDA in the rat
model.
Example 18
Huntington's and Alzheimer's Disease
[0156] Ovariectomy induces neurodegenerative changes analogous to
Huntington and Alzheimer's diseases in the mouse. Mice were
subjected to sham operation or ovariectomy. Both operated groups
were divided in 2 sub-groups: one subgroup received p62-plasmid
vaccination, and another did not. A battery of behavioral tests
were conducted as described below. In each test we have observed
that p62 vaccination significantly alleviated the neurodegenerative
effect of ovariectomy, which indicates it may have strong potential
in treatment and/or prevention of Alzheimer's and Huntington's
diseases.
Behavioral Tests
[0157] All experiments were performed during the light period in
accordance with the European Community Council Directive for Care
and Use of Laboratory Animals (86/609/EEC).
[0158] Behavioral testing took place during four consecutive days.
On the first day, animals were subjected to the whole battery of
sensorimotor tests. On the second day, open field was performed. On
the third day, animals were subjected to the Porsolt-test. Finally,
the plus-maze test or light/dark test was performed on the last
day.
[0159] The sequence of testing was based on previous reports by
different authors (Johansson et al., Proc Natl Acad Sci USA. 2001
Jul. 31; 98(16):9407-12 and Gimenez-Llort et al., Eur J Neurosci.
2002 August; 16(3):547-50; Baeza et al., J Neuroimmunol. 2010 Feb.
26; 219(1-2):90-9). Behavior was evaluated by three independent
observers. Mice were weighted before performing the tests, in order
to be sure that all of them were active in the same way. Olfactory
trails were removed by cleaning the surface of the apparatuses
after each test.
Sensorimotor Abilities
Visual Placing Reflex
[0160] The visual placing reflex was tested in order to evaluate
the function of the visual system. For the performance of this
placing response, the mouse was suspended by the tail and lowered
toward a solid black surface. Complete extension of the forelimbs
was considered a positive response. The mean response was rated in
three trials (Baeza et al., 2010).
Hindlimb Extensor Reflex
[0161] This reflex was evaluated during the previous test as the
ability to perform complete extension of the hindlimbs when the
animal was suspended by the tail. Such response was considered
positive. The mean response was rated in three trials (Baeza et
al., 2010).
Tightrope Test
[0162] This method is used to evaluate the vitality loss in aging
mice by testing their muscular vigor, motor coordination and
traction in two training trials of 5 s and a test trial of 60 s
(Miguel and Blasco, Ex p Gerontol. 1978; 13(6):389-96 and Baeza et
al., 2010). Mice were suspended by their forelimbs in the middle of
an elevated horizontal tightrope (40 cm height, 60 cm length and
divided in segments of 10 cm). Muscular vigor was assessed as the
percentage of mice falling off the rope and the latency to fall (in
seconds). Motor coordination included the percentage of mice that
walk at least 1 segment (criteria 1) and the percentage of mice
that complete the test (criteria 2). Traction was evaluated by
analyzing the different parts of the body that mice used to remain
suspended (forelimbs, hindlimbs and tail) and, subsequently, the
percentages of mice displaying the maximum (forelimbs, hindlimbs
and tail) and minimum (forelimbs only) traction capacities were
assessed within each group.
Exploratory and Anxiety-Like Behavioral Tests
[0163] This group includes different tests that study the
depressive-like and anxiety-like behaviors in the animals: the FST,
the open field, the light/dark and the EPM tests.
Forced Swimming Test (FST)
[0164] The forced-swimming test is the best recognized
pharmacological model for assessing antidepressant-like activity in
rodents (Porsolt et al., 1977a Arch Int Pharmacodyn Ther 229:
327-336, Porsolt et al., 1977b Nature 266: 730-732; Willner, 1990
Pharmacol Ther 45: 425-455; Al-Rahbi et al., Biomed Res Int. 2013;
2013:493643.doi: 10.1155/2013/493643). The development of learned
helplessness syndrome, when mice are placed in a cylinder filled
with water that they cannot escape from, reflects the cessation of
persistent escape-directed behavior, as seen by increased periods
of immobility (Lucki, 1997 Behav Pharmacol 8: 523-532). The
reduction in immobility is considered as a behavioral profile that
is consistent with an antidepressant-like action (Cryan et al.,
2002 Trends Pharmacol Sci 23: 238-245; Walf and Frye., 2010 Physiol
Behay. 2010 Feb. 9; 99(2):169-74; Al-Rahbi et al., 2013). Briefly,
the mice were placed individually in a glass cylinder (20 cm in
height, 14 cm in diameter) filled to a 15 cm depth with water
(23.+-.1.degree. C.). At this water depth, the mice could touch the
bottom of the jar with their tail, but they could not support
themselves with their hind limbs. Each mouse was given a 6 min
swimming test, and the duration of immobility was noted during the
final 4 min interval of the test, since the first 2 min were used
to allow the animals to familiarize them-selves with the
surroundings. All the swim-test sessions were recorded by a video
camera positioned directly above the cylinder. Two experienced
observers, who were blind to the treatment conditions, scored the
videotapes. An immobility period was regarded as the time spent by
the mouse floating in the water without struggling and while making
only the very slight movements that are necessary to keep its head
above the water. Following these swimming sessions, the mice were
towel dried and returned to their housing. Each animal was tested
only once.
Open Field Test
[0165] Locomotor activity was measured by an open-field apparatus
consisting of a square arena (43.2 cm.times.43.2 cm) equipped with
two lines of 16 photocells to measure horizontal and vertical
activity. The arena was lit by one red light lamp (25 W), and a
white noise generator in the room produced an ambient background
noise of .about.70 dB. All data were recorded on a personal
computer (MED-PC Open-Field Activity Software) in an adjacent
control room. Mice were placed in the centre of the apparatus and
the test was carried out for 5 min. A number of conventional and
ethological parameters (Choleris et al., 2001 Neurosci Biobehav Rev
25: 353-360; Perfumi and Mattioli, Phytother Res. 2007 January;
21(1):37-43) were collected during the session. The horizontal
activity (i.e. distance travelled, ambulation time, resting time)
and the vertical activity (i.e. rearing) in the central and
peripheral zone were recorded automatically. The time spent in the
central area, the ambulation time and vertical activity in this
zone, and the latency to leave the starting central point and that
to reach the periphery ("freezing behavior") were measured as
indicators of the emotional reactivity of the mouse (Baeza et al.,
2010). Moreover aging involves a decrease in the defecatory
behaviour and an increase of urine incontinence. Therefore, the
number of fecal boli and presence of urine were also considered in
the different groups of age (in an attempt to study whether
ovariectomy in mature animals caused these behaviors to be more
similar to those observed in aged animals).
Light/Dark Test
[0166] A relevant test system to detect anxiety-related behaviour
in mice is the light/dark exploration test, which uses the aversion
of rodents for brightly lit large spaces (Hascoet et al., 2001 Prog
Neuropsychopharmacol Biol Psychiatry 25: 141-166; Bourin and
Hascoet, 2003 Eur J Pharmacol 463: 55-65). The light-dark apparatus
consisted of an open-topped rectangular Plexiglas box
(45.times.30.times.30 cm; I.times.b.times.h) that was divided into
a small (18.times.30 cm) area and a large (27.times.30 cm) area
with an opening door (7.5.times.7.5 cm) located in the center of
the partition at floor level. The small compartment was painted
black and stayed dark, whereas the large compartment was painted
white and was brightly illuminated with a 60 W (400 l.times.) light
source. Briefly, each animal was placed at the center of the
illuminated compartment, facing one of the dark areas. The latency
time for their first passage from the light compartment to the dark
one, the number of entries into each compartment, the time spent in
the illuminated area, and the number of times that the mouse reared
on its hindpaws in the light space (rearing), were recorded for 5
min (Waif and Frye., 2010).
Elevated Plus Maze Test (EPM)
[0167] The elevated plus maze assesses anxiety-like behavior and
consisted of black Plexiglas with two open arms (30.times.3.5 cm)
and two enclosed arms of the same size (14 cm high walls). The four
arms were connected by a central square (6.times.6 cm square) and
were elevated approximately 74 cm from the ground. Briefly, mice
were placed in the central square facing one of the closed arms and
its behavior was scored for 5 min. The number of entries with all
four paws within the arms and the time spent in the arms were
scored separately for open and closed arms. A greater amount of raw
time and a greater proportion of time (%) spent on the brightly-lit
open arms of the elevated plus maze was considered an index of
anxiety-like behavior (Kolosova et al., Aging (Albany N.Y.). 2013
June; 5(6):474-84; Walf and Frye., 2010).
[0168] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[0169] Modifications may be made to the foregoing without departing
from the basic aspects of the technology. Although the technology
has been described in substantial detail with reference to one or
more specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, yet these modifications and
improvements are within the scope and spirit of the technology.
[0170] The technology illustratively described herein suitably may
be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising," "consisting essentially of," and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and use of such terms
and expressions do not exclude any equivalents of the features
shown and described or portions thereof, and various modifications
are possible within the scope of the technology claimed. The term
"a" or "an" can refer to one of or a plurality of the elements it
modifies (e.g., "a reagent" can mean one or more reagents) unless
it is contextually clear either one of the elements or more than
one of the elements is described. The term "about" as used herein
refers to a value within 10% of the underlying parameter (i.e.,
plus or minus 10%), and use of the term "about" at the beginning of
a string of values modifies each of the values (i.e., "about 1, 2
and 3" refers to about 1, about 2 and about 3). For example, a
weight of "about 100 grams" can include weights between 90 grams
and 110 grams. Further, when a listing of values is described
herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing
includes all intermediate and fractional values thereof (e.g., 54%,
85.4%). Thus, it should be understood that although the present
technology has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this technology.
[0171] Certain embodiments of the technology are set forth in the
claim(s) that follow(s).
Sequence CWU 1
1
212923DNAHomo sapiens 1cctctcgagg cggggcgggg cctccgcgtt cgctacaaaa
gccgcgcggc ggctgcgacc 60gggacggccc gttttccgcc agctcgccgc tcgctatggc
gtcgctcacc gtgaaggcct 120accttctggg caaggaggac gcggcgcgcg
agattcgccg cttcagcttc tgctgcagcc 180ccgagcctga ggcggaagcc
gaggctgcgg cgggtccggg accctgcgag cggctgctga 240gccgggtggc
cgccctgttc cccgcgctgc ggcctggcgg cttccaggcg cactaccgcg
300atgaggacgg ggacttggtt gccttttcca gtgacgagga attgacaatg
gccatgtcct 360acgtgaagga tgacatcttc cgaatctaca ttaaagagaa
aaaagagtgc cggcgggacc 420accgcccacc gtgtgctcag gaggcgcccc
gcaacatggt gcaccccaat gtgatctgcg 480atggctgcaa tgggcctgtg
gtaggaaccc gctacaagtg cagcgtctgc ccagactacg 540acttgtgtag
cgtctgcgag ggaaagggct tgcaccgggg gcacaccaag ctcgcattcc
600ccagcccctt cgggcacctg tctgagggct tctcgcacag ccgctggctc
cggaaggtga 660aacacggaca cttcgggtgg ccaggatggg aaatgggtcc
accaggaaac tggagcccac 720gtcctcctcg tgcaggggag gcccgccctg
gccccacggc agaatcagct tctggtccat 780cggaggatcc gagtgtgaat
ttcctgaaga acgttgggga gagtgtggca gctgccctta 840gccctctggg
cattgaagtt gatatcgatg tggagcacgg agggaaaaga agccgcctga
900cccccgtctc tccagagagt tccagcacag aggagaagag cagctcacag
ccaagcagct 960gctgctctga ccccagcaag ccgggtggga atgttgaggg
cgccacgcag tctctggcgg 1020agcagatgag gaagatcgcc ttggagtccg
aggggcgccc tgaggaacag atggagtcgg 1080ataactgttc aggaggagat
gatgactgga cccatctgtc ttcaaaagaa gtggacccgt 1140ctacaggtga
actccagtcc ctacagatgc cagaatccga agggccaagc tctctggacc
1200cctcccagga gggacccaca gggctgaagg aagctgcctt gtacccacat
ctcccgccag 1260aggctgaccc gcggctgatt gagtccctct cccagatgct
gtccatgggc ttctctgatg 1320aaggcggctg gctcaccagg ctcctgcaga
ccaagaacta tgacatcgga gcggctctgg 1380acaccatcca gtattcaaag
catcccccgc cgttgtgacc acttttgccc acctcttctg 1440cgtgcccctc
ttctgtctca tagttgtgtt aagcttgcgt agaattgcag gtctctgtac
1500gggccagttt ctctgccttc ttccaggatc aggggttagg gtgcaagaag
ccatttaggg 1560cagcaaaaca agtgacatga agggagggtc cctgtgtgtg
tgtgtgctga tgtttcctgg 1620gtgccctggc tccttgcagc agggctgggc
ctgcgagacc caaggctcac tgcagcgcgc 1680tcctgacccc tccctgcagg
ggctacgtta gcagcccagc acatagcttg cctaatggct 1740ttcactttct
cttttgtttt aaatgactca taggtccctg acatttagtt gattattttc
1800tgctacagac ctggtacact ctgattttag ataaagtaag cctaggtgtt
gtcagcaggc 1860aggctgggga ggccagtgtt gtgggcttcc tgctgggact
gagaaggctc acgaagggca 1920tccgcaatgt tggtttcact gagagctgcc
tcctggtctc ttcaccactg tagttctctc 1980atttccaaac catcagctgc
ttttaaaata agatctcttt gtagccatcc tgttaaattt 2040gtaaacaatc
taattaaatg gcatcagcac tttaaccaat gacgtttgca tagagagaaa
2100tgattgacag taagtttatt gttaatggtt cttacagagt atctttaaaa
gtgccttagg 2160ggaaccctgt ccctcctaac aagtgtatct cgattaataa
cctgccagtc ccagatcaca 2220catcatcatc gaagtcttcc ccagttataa
agaggtcaca tagtcgtgtg ggtcgaggat 2280tctgtgcctc caggaccagg
ggcccaccct ctgcccaggg agtccttgcg tcccatgagg 2340tcttcccgca
aggcctctca gacccagatg tgacggggtg tgtggcccga ggaagctgga
2400cagcggcagt gggcctgctg aggccttctc ttgaggcctg tgctctgggg
gtcccttgct 2460tagcctgtgc tggaccagct ggcctggggt ccctctgaag
agaccttggc tgctcactgt 2520ccacatgtga actttttcta ggtggcagga
caaattgcgc ccatttagag gatgtggctg 2580taacctgctg gatgggactc
catagctcct tcccaggacc cctcagctcc ccggcactgc 2640agtctgcaga
gttctcctgg aggcaggggc tgctgccttg tttcaccttc catgtcaggc
2700cagcctgtcc ctgaaagaga agatggccat gccctccatg tgtaagaaca
atgccagggc 2760ccaggaggac cgcctgccct gcctgggcct tggctgggcc
tctggttctg acactttctg 2820ctggaagctg tcaggctggg acaggctttg
attttgaggg ttagcaagac aaagcaaata 2880aatgccttcc acctcaccgc
aaaaaaaaaa aaaaaaaaaa aaa 29232440PRTHomo sapiens 2Met Ala Ser Leu
Thr Val Lys Ala Tyr Leu Leu Gly Lys Glu Asp Ala 1 5 10 15 Ala Arg
Glu Ile Arg Arg Phe Ser Phe Cys Cys Ser Pro Glu Pro Glu 20 25 30
Ala Glu Ala Glu Ala Ala Ala Gly Pro Gly Pro Cys Glu Arg Leu Leu 35
40 45 Ser Arg Val Ala Ala Leu Phe Pro Ala Leu Arg Pro Gly Gly Phe
Gln 50 55 60 Ala His Tyr Arg Asp Glu Asp Gly Asp Leu Val Ala Phe
Ser Ser Asp 65 70 75 80 Glu Glu Leu Thr Met Ala Met Ser Tyr Val Lys
Asp Asp Ile Phe Arg 85 90 95 Ile Tyr Ile Lys Glu Lys Lys Glu Cys
Arg Arg Asp His Arg Pro Pro 100 105 110 Cys Ala Gln Glu Ala Pro Arg
Asn Met Val His Pro Asn Val Ile Cys 115 120 125 Asp Gly Cys Asn Gly
Pro Val Val Gly Thr Arg Tyr Lys Cys Ser Val 130 135 140 Cys Pro Asp
Tyr Asp Leu Cys Ser Val Cys Glu Gly Lys Gly Leu His 145 150 155 160
Arg Gly His Thr Lys Leu Ala Phe Pro Ser Pro Phe Gly His Leu Ser 165
170 175 Glu Gly Phe Ser His Ser Arg Trp Leu Arg Lys Val Lys His Gly
His 180 185 190 Phe Gly Trp Pro Gly Trp Glu Met Gly Pro Pro Gly Asn
Trp Ser Pro 195 200 205 Arg Pro Pro Arg Ala Gly Glu Ala Arg Pro Gly
Pro Thr Ala Glu Ser 210 215 220 Ala Ser Gly Pro Ser Glu Asp Pro Ser
Val Asn Phe Leu Lys Asn Val 225 230 235 240 Gly Glu Ser Val Ala Ala
Ala Leu Ser Pro Leu Gly Ile Glu Val Asp 245 250 255 Ile Asp Val Glu
His Gly Gly Lys Arg Ser Arg Leu Thr Pro Val Ser 260 265 270 Pro Glu
Ser Ser Ser Thr Glu Glu Lys Ser Ser Ser Gln Pro Ser Ser 275 280 285
Cys Cys Ser Asp Pro Ser Lys Pro Gly Gly Asn Val Glu Gly Ala Thr 290
295 300 Gln Ser Leu Ala Glu Gln Met Arg Lys Ile Ala Leu Glu Ser Glu
Gly 305 310 315 320 Arg Pro Glu Glu Gln Met Glu Ser Asp Asn Cys Ser
Gly Gly Asp Asp 325 330 335 Asp Trp Thr His Leu Ser Ser Lys Glu Val
Asp Pro Ser Thr Gly Glu 340 345 350 Leu Gln Ser Leu Gln Met Pro Glu
Ser Glu Gly Pro Ser Ser Leu Asp 355 360 365 Pro Ser Gln Glu Gly Pro
Thr Gly Leu Lys Glu Ala Ala Leu Tyr Pro 370 375 380 His Leu Pro Pro
Glu Ala Asp Pro Arg Leu Ile Glu Ser Leu Ser Gln 385 390 395 400 Met
Leu Ser Met Gly Phe Ser Asp Glu Gly Gly Trp Leu Thr Arg Leu 405 410
415 Leu Gln Thr Lys Asn Tyr Asp Ile Gly Ala Ala Leu Asp Thr Ile Gln
420 425 430 Tyr Ser Lys His Pro Pro Pro Leu 435 440
* * * * *