U.S. patent application number 16/130774 was filed with the patent office on 2019-03-21 for aquaporin tolerizing vaccines and methods of use thereof.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Peggy Pui-Kay Ho, Lawrence Steinman.
Application Number | 20190083594 16/130774 |
Document ID | / |
Family ID | 57204415 |
Filed Date | 2019-03-21 |
United States Patent
Application |
20190083594 |
Kind Code |
A1 |
Steinman; Lawrence ; et
al. |
March 21, 2019 |
AQUAPORIN TOLERIZING VACCINES AND METHODS OF USE THEREOF
Abstract
The instant disclosure provides aquaporin DNA tolerizing
vaccines and methods of using such vaccines for treating
individuals having neuromyelitis optica (NMO) and NMO spectrum
disorders. Aspects of the methods include administering to the
individual, in need thereof, an effective amount of an aquaporin
DNA tolerizing vaccine to reduce one or more symptoms of NMO or an
NMO spectrum disorder. Compositions and kits for practicing the
methods of the disclosure are also provided.
Inventors: |
Steinman; Lawrence;
(Stanford, CA) ; Ho; Peggy Pui-Kay; (Cupertino,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
57204415 |
Appl. No.: |
16/130774 |
Filed: |
September 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15134931 |
Apr 21, 2016 |
10098935 |
|
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16130774 |
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62195625 |
Jul 22, 2015 |
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62155947 |
May 1, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61K 2039/53 20130101; A61K 2039/54 20130101; A61K 39/001
20130101; A61K 39/001102 20180801; A61K 39/0008 20130101; A61K
2039/55561 20130101; A61K 39/39 20130101; A61K 2039/577
20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/39 20060101 A61K039/39 |
Claims
1. An aquaporin tolerizing vaccine comprising a DNA vector
comprising: a) an aquaporin cDNA; b) a promoter sequence operably
linked the aquaporin cDNA; and c) a DNA backbone, linked to the
promoter sequence and the aquaporin cDNA, comprising 4 or fewer
immunostimulatory CpG motifs.
2. The aquaporin tolerizing vaccine according to claim 1, wherein
the aquaporin cDNA is an Aquaporin-4 (AQP4) cDNA.
3. The aquaporin tolerizing vaccine according to claim 2, wherein
the aquaporin cDNA is a mammalian AQP4 cDNA
4. The aquaporin tolerizing vaccine according to claim 3, wherein
the mammalian aquaporin cDNA is a human AQP4 cDNA.
5. The aquaporin tolerizing vaccine according to claim 4, wherein
the human AQP4 cDNA is full length.
6. The aquaporin tolerizing vaccine according to claim 4, wherein
the human AQP4 cDNA is not full length and comprises cDNA encoding
amino acids 61-80 of human AQP4.
7. The aquaporin tolerizing vaccine according to claim 1, wherein
the aquaporin cDNA encodes a modified aquaporin polypeptide that is
non-naturally occurring.
8. The aquaporin tolerizing vaccine according to claim 1, wherein
the DNA vector, excluding the aquaporin cDNA, comprises 29 or fewer
immunostimulatory CpG motifs.
9-19. (canceled)
Description
CROSS-REFERENCE
[0001] This application claims the benefit of and is a Continuation
of application Ser. No. 15/134,931 filed Apr. 21, 2016, which
claims benefit of U.S. Provisional Patent Application No.
62/155,947, filed May 1, 2015 and U.S. Provisional Patent
Application No. 62/195,25, filed Jul. 22, 2015, which applications
are incorporated herein by reference in its entirety.
INTRODUCTION
[0002] Neuromyelitis optica (NMO) or Devic's disease is a severe,
demyelinating disease of the central nervous system that
preferentially affects the optic nerve and spinal cord. NMO and NMO
spectrum disorder attacks are characterized mainly by uni- or
bilateral inflammation of the optic nerve, optic neuritis, and
acute, traverse myelitis. Most patients have relapsing attacks
(separated by months or years with partial recovery) and most
relapsing patients are female. A less common form of NMO results in
a single attack lasting one to two months. In some rare instances,
patients suffer other neurological disorders including, intractable
vomiting, nausea, endocrine disorders, sleep disorders, confusion,
and coma. Untreated or unrecognized, NMO can be fatal within 5
years of onset in nearly a third of patients. Many NMO patients may
become legally blind in one or both eyes and may have significant
partial paralysis. The etiology of NMO remains unknown.
[0003] NMO has an estimated prevalence of about 0.5 to 4.4 per
100,000 people and incidence per 100,000 people of about 0.05 to
0.4 worldwide. Diagnosis of NMO is typically clinical and generally
based on the co-presence of primary symptomatic optic neuritis and
myelitis with other supporting criteria, typically normal brain MRI
(i.e., imaging that is not diagnostic of multiple sclerosis), MRI
evidence of extended myelitis in the spinal cord and/or the
presence of biomarker NMO-IgG antibody (i.e., anti-aquaporin-4
(AQP4) antibody).
[0004] Data from randomized clinical treatment studies of NMO
remains limited, thus, treatment modalities for NMO are varied.
Current palliative and preventative treatments include
corticosteroids, plasma exchange, immunomodulatory therapy with
purified immunoglobulins from healthy donors, interferon therapy
and various conventional immunosuppressive therapies.
PUBLICATIONS
[0005] Collongues & de Seze (2011) Ther Adv Neurol Disord.
4(2): 111-121. [0006] Marrie & Gryba (2013) Int J MS Care.
15(3): 113-118. [0007] Wingerchuk et al. (2006) Neurology.
66(10):1485-9.
SUMMARY
[0008] Methods are provided for treating individuals having
neuromyelitis optica (NMO) and NMO spectrum disorders. Aspects of
the instant disclosure include aquaporin DNA tolerizing vaccines
and methods of using such vaccines. Aspects of the subject methods
further include administering to the individual, in need thereof,
an effective amount of an aquaporin DNA tolerizing vaccine to at
least reduce one or more symptoms of NMO or an NMO spectrum
disorder and/or prevent the onset of one or more symptoms of NMO or
an NMO spectrum disorder as described herein. Also provided are
compositions and kits for practicing the methods of the
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. The patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee. It is
emphasized that, according to common practice, the various features
of the drawings are not to-scale. On the contrary, the dimensions
of the various features are arbitrarily expanded or reduced for
clarity. Included in the drawings are the following figures.
[0010] FIG. 1 depicts a schematic of the pBHT1 vector showing the
relative positions of the antigen sequence, the polyadenylation
signal, the Kanamycin resistance sequence, the pUC origin of
replication and the CMV promoter.
[0011] FIG. 2 depicts the antigen portion of the pBHT1-murine AQP4
p21-40 construct. The construct was constructed by digesting double
stranded AQP4 p21-40 oligonucleotide and pBHT1 with HindIII and
EcoRI restriction enzymes, removing the 5' phosphates from pBHT1
and performing a ligation reaction. The ligated vector was then
transformed into bacteria and the transformed bacteria were plated
(SEQ ID NO:1).
[0012] FIG. 3 depicts confirmation by restriction enzyme digest of
the successful cloning of the AQP4 p21-40 insert as described in
FIG. 2.
[0013] FIG. 4 depicts confirmation by DNA sequencing and BLAST
search of the successful cloning of the AQP4 p21-40 insert as
described in FIG. 2 (SEQ ID NO:2).
[0014] FIG. 5 depicts a schematic of NMO mouse model DNA
vaccination experiment #1. Arrows along the timeline represent when
each action of the experiment was performed. 10 C57BL6 mice were
used in 3 groups (2 immunized mice that did not receive treatment,
4 immunized mice that received treatment with empty DNA vaccine
vector and 4 immunized mice that received treatment with the
AQP4p21-40 DNA vaccine vector).
[0015] FIG. 6 depicts that the AQP4 p21-40 DNA vaccination is
effective in suppressing AQP4 p21-40 specific T cell proliferation.
Proliferation assay was performed using the spleens of untreated
mice, mice treated with empty vector, and mice treated with
AQP4p21-40 DNA vaccine and T cell stimulation was performed at
various concentrations of antigen (x-axis). Statistically
significant differences at p<0.05 are indicated (*).
[0016] FIG. 7 depicts that AQP4 p21-40 IgM antibodies decreased
following DNA vaccination in C57BL/6 mice. B6 IgM levels were
measured in mice that were not immunized and did not receive
treatment (B6 naive), mice that were immunized but did not receive
treatment (B6 immunized), mice that were not immunized and treated
with empty vehicle (B6 naive vehicle), mice that were immunized and
treated with empty vehicle (B6 immunized vehicle), mice that were
not immunized and treated with AQP4 DNA vaccine (B6 naive AQP4) and
mice that were immunized and treated with AQP4 DNA vaccine (B6
immunized AQP4).
[0017] FIG. 8 depicts that AQP4 p21-40 IgG antibodies decreased
following DNA Vaccination in C57BL/6 mice. B6 IgG levels were
measured in mice that were not immunized and did not receive
treatment (B6 naive), mice that were immunized but did not receive
treatment (B6 immunized), mice that were not immunized and treated
with empty vehicle (B6 naive vehicle), mice that were immunized and
treated with empty vehicle (B6 immunized vehicle), mice that were
not immunized and treated with AQP4 DNA vaccine (B6 naive AQP4) and
mice that were immunized and treated with AQP4 DNA vaccine (B6
immunized AQP4).
[0018] FIG. 9 depicts that AQP4 p21-40 DNA vaccination is effective
in suppressing IL-17A. IL-17A production was measured at various
concentrations of antigen (x-axis) in untreated mice, mice treated
with empty vector, and mice treated with AQP4p21-40 DNA vaccine.
Statistically significant differences are indicated (*).
[0019] FIG. 10 depicts that AQP4 p21-40 DNA vaccination is
effective in suppressing IL-6. IL-6 production was measured at
various concentrations of antigen (x-axis) in untreated mice, mice
treated with empty vector, and mice treated with AQP4p21-40 DNA
vaccine. Statistically significant differences are indicated
(*).
[0020] FIG. 11 depicts a schematic of NMO mouse model DNA
vaccination experiment #2. Arrows along the timeline represent when
each action of the experiment was performed. 15 C57BL6 mice were
used in 3 groups (5 immunized mice that did not receive treatment,
5 immunized mice that received treatment with empty DNA vaccine
vector and 5 immunized mice that received treatment with the
AQP4p21-40 DNA vaccine vector).
[0021] FIG. 12 depicts that AQP4 p21-40 DNA vaccination is
effective in suppressing AQP4 p21-40 specific T cell proliferation.
Proliferation assay was performed using untreated mice, mice
treated with empty pBHT1 vector, and mice treated with
pBHT1-AQP4p21-40 DNA vaccine and T cell stimulation was performed
at various concentrations of antigen (x-axis). Statistically
significant differences at are indicated (*).
[0022] FIG. 13 depicts that AQP4 p21-40 DNA vaccination is
effective in suppressing IL-17A. IL-17A production was measured at
various concentrations of antigen (x-axis) in mice that received
only delivery vehicle (vehicle), mice treated with empty vector
(pBHT1), and mice treated with p-BHT1-AQP4p21-40 DNA vaccine
(p-BHT1-AQP4p21-40 DNA). Statistically significant differences are
indicated (*).
[0023] FIG. 14 depicts that AQP4 p21-40 DNA vaccination is
effective in suppressing IFN-gamma. IFN-gamma production was
measured at various concentrations of antigen (x-axis) in mice that
received only delivery vehicle (vehicle), mice treated with empty
vector (pBHT1), and mice treated with p-BHT1-AQP4p21-40 DNA vaccine
(p-BHT1-AQP4p21-40 DNA). Statistically significant differences are
indicated (*).
[0024] FIG. 15 depicts that AQP4 p21-40 DNA vaccination is
effective in suppressing IL-6. IL-6 production was measured at
various concentrations of antigen (x-axis) in mice that received
only delivery vehicle (vehicle), mice treated with empty vector
(pBHT1), and mice treated with p-BHT1-AQP4p21-40 DNA vaccine
(p-BHT1-AQP4p21-40 DNA). Statistically significant differences are
indicated (*).
[0025] FIG. 16 depicts that AQP4 p21-40 DNA vaccination maintains
AQP4 expression on astrocytes in AQP4 p21-40-immunized spinal cord.
Astrocytes from mice that received only delivery vehicle (vehicle,
top row), mice treated with empty vector (pBHT1, middle row), and
mice treated with p-BHT1-AQP4p21-40 DNA vaccine (p-BHT1-AQP4p21-40
DNA, bottom row) were stained for GFAP (left column) and AQP4
(middle column) and DNA (DAPI) and imaged (merged images (right
column) showing both GFAP and AQP4 staining are also provided).
Staining and imaging displayed that AQP4 expression is maintained
in astrocytes from AQP4p21-40 immunized spinal cord.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] The instant disclosure provides aquaporin DNA tolerizing
vaccines and methods of using such vaccines for treating
individuals having neuromyelitis optica (NMO) and NMO spectrum
disorders. Aspects of the methods include administering to the
individual, in need thereof, an effective amount of an aquaporin
DNA tolerizing vaccine to reduce one or more symptoms of NMO or an
NMO spectrum disorder. Compositions and kits for practicing the
methods of the disclosure are also provided.
[0027] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0028] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0030] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0031] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g. polypeptides, known to those
skilled in the art, and so forth.
[0032] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed
Definitions
[0033] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0034] A "vector" is capable of transferring nucleic acid sequences
to target cells. For example, a vector may comprise a coding
sequence capable of being expressed in a target cell. For the
purposes of the present invention, "vector construct," "expression
vector," and "gene transfer vector," generally refer to any nucleic
acid construct capable of directing the expression of a gene of
interest and which is useful in transferring the gene of interest
into target cells. Thus, the term includes cloning and expression
vehicles, as well as integrating vectors, however, in some
instances a vector may be configured to prevent, eliminate or
inhibit the integration of the vector into a host cell genome.
[0035] The term "plasmid" is encompassed within the term "vector"
and refers to any genetic element that is capable of replication by
comprising proper control and regulatory elements when present in a
host cell. Plasmids may be designated by a lower case p followed by
letters and/or numbers. Starting plasmids are commercially
available, publicly available on an unrestricted basis, can be
constructed from available plasmids in accord with published
procedures, can be isolated from organisms harboring the plasmid
(e.g., naturally occurring organisms or laboratory stocks (e.g.,
bacterial stocks, etc.), or synthesized, in whole or in part, on a
standard or custom basis, e.g., as provided by commercial suppliers
such as DNA2.0, Inc. (Menlo Park, Calif.)). In addition, where
equivalent plasmids to those described are known in the art such
plasmids will be readily apparent to the ordinarily skilled artisan
and the nucleic acid sequences of such plasmids may be readily
available.
[0036] Vectors are capable of transferring nucleic acid sequences
to target cells and, in some instances, are used to manipulate
nucleic acid sequence, e.g., recombine nucleic acid sequences (i.e.
to make recombinant nucleic acid sequences). For purposes of this
invention examples of vectors include, but are not limited to,
plasmids, phage, transposons, cosmids, virus, and the like.
[0037] "Naked nucleic acid" as used herein refers to a nucleic acid
molecule that is not encapsulated (such as, e.g., within a viral
particle, bacterial cell, or liposome) and not complexed with a
molecule that binds to the nucleic acid (such as, e.g.,
DEAE-dextran) nor otherwise conjugated to the nucleic acid (e.g.,
gold particles or polysaccharide-based supports).
[0038] An "expression cassette" comprises any nucleic acid
construct capable of directing the expression of any RNA transcript
including gene/coding sequence of interest as well as
non-translated RNAs. Such cassettes can be constructed into a
"vector," "vector construct," "expression vector," or "gene
transfer vector," in order to transfer the expression cassette into
target cells. Thus, the term includes cloning and expression
vehicles, as well as viral vectors.
[0039] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues linked
by peptide bonds, and for the purposes of the instant disclosure,
have a minimum length of at least 5 amino acids. Oligopeptides,
oligomers multimers, and the like, typically refer to longer chains
of amino acids and are also composed of linearly arranged amino
acids linked by peptide bonds, whether produced biologically,
recombinantly, or synthetically and whether composed of naturally
occurring or non-naturally occurring amino acids, are included
within this definition. Both full-length proteins and fragments
thereof greater than 5 amino acids are encompassed by the
definition. The terms also include polypeptides that have
co-translational (e.g., signal peptide cleavage) and
post-translational modifications of the polypeptide, such as, for
example, disulfide-bond formation, glycosylation, acetylation,
phosphorylation, proteolytic cleavage (e.g., cleavage by furins or
metalloproteases), and the like. Furthermore, as used herein, a
"polypeptide" refers to a protein that includes modifications, such
as deletions, additions, and substitutions (generally conservative
in nature as would be known to a person in the art) to the native
sequence, as long as the protein maintains the desired activity
relevant to the purposes of the described methods. These
modifications can be deliberate, as through site-directed
mutagenesis, or can be accidental, such as through mutations of
hosts that produce the proteins, or errors due to PCR amplification
or other recombinant DNA methods.
[0040] Modified polypeptides may also include, e.g., those
polypeptides that have been modified to improve their use as a
therapeutic. Such polypeptide modification may include any
combination of N- and/or C-terminal truncation (e.g., to achieve
the minimal active sequence (MAS)), deletion of one or more
consecutive amino acid(s) to achieve the MAS, combinatorial
deletion with two or more positions omitted independently to
achieve the MAS, structure simplification (e.g., following alanine
or D amino acid scanning to identify non-active sites), cleave site
elimination, cyclization between side chains, cyclization between
terminal ends, cyclization between the backbone, cyclization
between a terminal end and a side chain, cyclization between a
terminal end and the backbone, cyclization between a side chain and
the backbone, cyclization through disulfide bonding, modification
to reduce polypeptide flexibility (e.g., through peptide bridging,
e.g., lanthionine bridging, dicarba bridging, hydrazine bridging,
lactam bridging), modification to reduce hydrogen bonding,
modification to increase membrane permeability (e.g., by modifying
the overall or regional (e.g., surface) charge of a polypeptide),
unnatural amino acid (e.g., a D-amino acid) substitution,
N-methyl-.alpha.-amino acid substitution, .beta.-amino acid
substitution, amide bond replacement, terminal end blocking (e.g.,
through N-acylation, N-pyroglutamate, C-amidation, etc.), addition
of carbohydrate chains, N-terminal esterification, pegylation, and
the like. Polypeptide modifications have been described, e.g., by
Vlieghe et al. (2010) Drug Discovery Today. 15:(1/2) 40-56, the
disclosure of which is incorporated herein by reference. The
ordinary skilled artisan will readily understand where a
polypeptide modification may be encoded (e.g., an amino acid
substitution, amino acid addition, amino acid truncation, etc.) in
a nucleic acid. The ordinary skilled artisan will also readily
understand that where a polypeptide modification is initially
synthetically produced (e.g., through enzymatic truncation of a
polypeptide) such modification may, in some instances, also be
achieved by modifying a nucleic acid that encodes the
polypeptide.
[0041] The term "gene" refers to a particular unit of heredity
present at a particular locus within the genetic component of an
organism. A gene may be a nucleic acid sequence, e.g., a DNA or RNA
sequence, present in a nucleic acid genome, a DNA or RNA genome, of
an organism and, in some instances, may be present on a chromosome.
Typically a gene will be a DNA sequence that encodes for an mRNA
that encodes a protein. A gene may be comprised of a single exon
and no introns or multiple exons and one or more introns. One of
two or more identical or alternative forms of a gene present at a
particular locus is referred to as an "allele" and, for example, a
diploid organism will typically have two alleles of a particular
gene. New alleles of a particular gene may be generated either
naturally or artificially through natural or induced mutation and
propagated through breeding or cloning.
[0042] The terms "specific binding," "specifically binds," and the
like, refer to non-covalent or covalent preferential binding to a
molecule relative to other molecules or moieties in a solution or
reaction mixture (e.g., an antibody specifically binds to a
particular polypeptide (e.g., antigen) or epitope relative to other
available polypeptides). In some embodiments, the affinity of one
molecule for another molecule to which it specifically binds is
characterized by a K.sub.D (dissociation constant) of 10.sup.-5 M
or less (e.g., 10.sup.-6 M or less, 10.sup.-7 M or less, 10.sup.-8
M or less, 10.sup.-9 M or less, 10.sup.-10 M or less, 10.sup.-11 M
or less, 10.sup.-12 M or less, 10.sup.-13 M or less, 10.sup.-14 M
or less, 10.sup.-15 M or less, or 10.sup.-16 M or less). "Affinity"
refers to the strength of binding, increased binding affinity being
correlated with a lower K.sub.D.
[0043] General methods in molecular and cellular biochemistry can
be found in such standard textbooks as Molecular Cloning: A
Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press
2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et
al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et
al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy
(Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift
& Loewy eds., Academic Press 1995); Immunology Methods Manual
(I. Lefkovits ed., Academic Press 1997); and Cell and Tissue
Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which
are incorporated herein by reference. Reagents, cloning vectors,
and kits for genetic manipulation referred to in this disclosure
are available from commercial vendors such as BioRad, Stratagene,
Life Technologies, Inc., Sigma-Aldrich, and ClonTech.
Aquaporin Polypeptides and Nucleic Acids
[0044] Aspects of the disclosure include aquaporin polypeptides and
nucleic acids encoding for aquaporin polypeptides for use in
treating neuromyelitis optica. By "aquaporin polypeptide" is meant
a polypeptide having homology with the product of one or more
aquaporin genes encoding an aquaporin protein. Aquaporin proteins
form water specific channels that provide plasma membranes with
permeability to water and serve to regulate cellular, tissue, organ
and organismal water balance by mediating water flow along osmotic
gradients and/or functioning as osmoreceptors. DNA tolerizing
vectors, described in more detail below, may include one or more
nucleic acid sequences encoding for one or more aquaporin
polypeptides or portion(s) thereof.
[0045] Aquaporin polypeptides may be recombinantly or synthetically
produced and may vary in their homology with naturally occurring
aquaporin polypeptides. As such, an aquaporin polypeptide of the
instant disclosure may share 100% or less sequence identity with a
naturally occurring aquaporin polypeptide. An aquaporin polypeptide
having less than 100% sequence identity with a naturally occurring
aquaporin polypeptide may be a modified polypeptide, e.g.,
recombinantly modified, such that one or more amino acid residues
of a naturally occurring aquaporin polypeptide sequence have been
modified such that the recombinant aquaporin polypeptide is a
non-naturally occurring aquaporin polypeptide.
[0046] In some instances, a recombinant aquaporin polypeptide may
be encoded from a recombinant aquaporin nucleic acid. Such
recombinant aquaporin polypeptides may contain one or more amino
acid residue mutations relative to a naturally occurring aquaporin
polypeptide. By "mutations" is meant any amino acid reside
substitution, deletion or insertion in the primary amino acid
sequence relative to a starting aquaporin polypeptide, e.g., a
naturally occurring aquaporin polypeptide or other reference
aquaporin polypeptide sequence. Amino acid mutations may be
generated through synthetic means, e.g., through mutation of a
naturally occurring or reference nucleic acid sequence encoding an
aquaporin polypeptide. In some instances, a recombinant aquaporin
nucleic acid excludes one or more non-coding sequences included in
a naturally occurring aquaporin gene or genetic locus. For example,
in some instances, a recombinant aquaporin nucleic acid may be an
aquaporin cDNA that excludes one or more introns of an aquaporin
gene or genetic locus. The number of excluded non-coding sequences
in an aquaporin cDNA may vary depending on, e.g., the overall
length of the cDNA, the particular aquaporin gene from which the
cDNA may be derived, the length of the particular aquaporin from
which the cDNA may be derived, the particular isoform from which
the cDNA may be derived, the particular aquaporin allele or mutant
allele from which the cDNA may be derived, etc., and may range from
1 to 3 or more, including 1 or more, 2 or more, 3 or more. In some
instances, an aquaporin cDNA may comprise the entire coding region
of the aquaporin gene and exclude all non-coding sequences, e.g.,
all introns and untranslated regions, of the reference gene locus
and/or transcript.
[0047] In some instances, an aquaporin polypeptide or aquaporin
nucleic acid may be derived, in part or in whole, from a mammalian
aquaporin gene. Mammalian aquaporin genes include placentalia
aquaporins, monotremata aquaporins and marsupialia aquaporins.
Placentalia aquaporins include those of primates, rodents,
even-toes ungulates, carnivores, bats, odd-toed ungulates,
insectivores, rabbits and hares, cingulata, macroscelidea,
tenrecidae, scandentia, dermoptera, proboscidea, tubulidentata,
chrysochloridae, and sirenia. Primate aquaporins include but are
not limited to those of baboons (e.g., Papio papio, Papio Anubis,
Papio cynocephalus, Papio hamadryas, Papio ursinus and the like),
macaques (e.g., Macaca fascicularis, Macaca nemestrina, Macaca
mulatta, and the like), green monkeys (e.g., Chlorocebus genus),
mangabey (e.g., Cercocebus agilis, Cercocebus galeritus, Cercocebus
torquatus, Cercocebus atys, Cercocebus lunulatus, and the like),
patas monkeys (e.g., Erythrocebus patas), squirrel monkeys (e.g.,
Saimiri sciureus), species of the family Hominidae (e.g.,
chimpanzees, gorillas, orangutans, and humans). Rodent aquaporins
include but are not limited to those of mouse, rat, squirrel,
gopher, vole, hamster, gerbil, guinea pig and the like.
[0048] In some instances, an aquaporin protein, e.g., as encoded by
an aquaporin gene, may be described in terms of sequence similarity
and/or sequence identity in relationship to a described amino acid
sequence. As such, an aquaporin polypeptide may share up to 100%
sequence identity with a particular amino acid sequence, e.g., one
or more of the aquaporin amino acid sequences described herein. In
some instances, an aquaporin polypeptide may share less than 100%
sequence identity to a particular amino acid sequence, e.g., one or
more of the aquaporin amino acid sequences described herein,
including but not limited to, e.g., at least 99%, at least 98%, at
least 97%, at least 96%, at least 95%, at least 94%, at least 93%,
at least 92%, at least 91%, at least 90%, at least 89%, at least
88%, at least 87%, at least 86%, at least 85%, at least 84%, at
least 83%, at least 82%, at least 81%, at least 80%, at least 79%,
at least 78%, at least 77%, at least 76%, at least 75%, at least
74%, at least 73%, at least 72%, at least 71% or at least 70%
sequence identity with an aquaporin amino acid sequence described
herein. In some instances, an aquaporin polypeptide may share no
less than 60% sequence identity to one or more of the aquaporin
sequences described herein.
[0049] In some instances, an aquaporin protein, e.g., as encoded by
an aquaporin gene, may share 100% sequence identity with an
aquaporin 4 (AQP4) polypeptide including but not limited to a
mammalian AQP4 polypeptide, a rodent AQP4 polypeptide, a human AQP4
polypeptide, a mouse AQP4 polypeptide, and the like. In some
instances, an aquaporin polypeptide may share less than 100%
sequence identity to a AQP4 polypeptide, e.g., one or more of the
AQP4 polypeptide amino acid sequences described herein, including
but not limited to, e.g., at least 99%, at least 98%, at least 97%,
at least 96%, at least 95%, at least 94%, at least 93%, at least
92%, at least 91%, at least 90%, at least 89%, at least 88%, at
least 87%, at least 86%, at least 85%, at least 84%, at least 83%,
at least 82%, at least 81%, at least 80%, at least 79%, at least
78%, at least 77%, at least 76%, at least 75%, at least 74%, at
least 73%, at least 72%, at least 71% or at least 70% sequence
identity with an AQP4 amino acid sequence described herein. In some
instances, an aquaporin polypeptide may share no less than 60%
sequence identity to one or more of the AQP4 sequences described
herein.
[0050] In some instances, an aquaporin polypeptide may, in whole or
in part, share 100% sequence identity or less than 100% sequence
identity (including but not limited to, e.g., 99%, 95%, 90%, 85%,
80%, etc.) with one or more proteins of UniProt ID Nos: P47863,
Q5I4F9, Q923J4, H0Y2J1, U5L133, U5L0U2, U5L1A8, U5L0F3, U5L1A7,
U5L0K2, U5L0T9, A0A096NGN8, Q866S4, Q6XVT6, U5L134, H2NW35, H0V364,
A0A091E3Y2, O77750, F7IQC1, H2QED7, U5L0K6, G3SXM7, G1SVZ6, P55087,
F1DSG4, F6NF8, G3R4L3, G7PWJ7, P55088, L8HYR7, H0WNR2, G3WQD1,
G3WQD2, F6SPF1, U5L0F5, M3XDP6, G1M6L3, Q53H97, G1R432, U5L1A9,
D2GUV5, I3MKS2, F7I6W3, Q9H3V7, G5BJS5, F7FSF9, A8V978, L5LSC4,
F1PEB5, M3Y5D3, U6CQ82, L5K2V7, L9KGP3, G3HDY9, Q8K4M1. In some
instances, an aquaporin polypeptide may share at least 90%
(including, e.g., 95%, 96%, 97%, 98% 99%, etc.) amino acid sequence
similarity over at least 80% (including, e.g., 85%, 90%, 95%, 96%,
97%, 98% 99%, etc.) of its length with one or more of of UniProt ID
Nos: P47863, Q5I4F9, Q923J4, H0Y2J1, U5L133, U5L0U2, U5L1A8,
U5L0F3, U5L1A7, U5L0K2, U5L0T9, A0A096NGN8, Q866S4, Q6XVT6, U5L134,
H2NW35, H0V364, A0A091E3Y2, O77750, F7IQC1, H2QED7, U5L0K6, G3SXM7,
G1SVZ6, P55087, F1DSG4, F6TNF8, G3R4L3, G7PWJ7, P55088, L8HYR7,
H0WNR2, G3WQD1, G3QD2, F6SPF1, U5L0F5, M3XDP6, G1M6L3, Q53H97,
G1R432, U5L1A9, D2GUV5, I3MKS2, F7I6W3, Q9H3V7, G5BJS5, F7FSF9,
A8V978, L5LSC4, F1PEB5, M3Y5D3, U6CQ82, L5K2V7, L9KGP3, G3HDY9,
Q8K4M1.
[0051] In some embodiments of the invention, the aquaporin of
interest is human aquaporin, including without limitation human
aquaporin 4. The sequences of human aquaporins are publicly
available, e.g. the reference sequence of aquaporin-4 isoform b
GenBank accession number NP_004019.1; and the reference sequence of
human aquaporin-4 isoform a GenBank accession number
NP_001641.1.
[0052] In some instances, an aquaporin protein, e.g., as encoded by
an aquaporin gene, may share 100% sequence identity or less
(including but not limited to, e.g., at least 99%, at least 98%, at
least 97%, at least 96%, at least 95%, at least 94%, at least 93%,
at least 92%, at least 91%, at least 90%, at least 89%, at least
88%, at least 87%, at least 86%, at least 85%, at least 84%, at
least 83%, at least 82%, at least 81%, at least 80%, at least 79%,
at least 78%, at least 77%, at least 76%, at least 75%, at least
74%, at least 73%, at least 72%, at least 71% or at least 70%) with
a human aquaporin sequence, including but not limited to, e.g.,
human AQP4 (UniProtID: P55087) the amino acid sequence of which
is:
TABLE-US-00001 (SEQ ID NO: 3)
MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVL
LSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAV
TVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMV
HGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIG
HLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEY
VFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVH
VIDVDRGEEKKGKDQSGEVLSSV.
[0053] In some instances, an aquaporin protein, e.g., as encoded by
an aquaporin gene, may share 100% sequence identity or less
(including but not limited to, e.g., at least 99%, at least 98%, at
least 97%, at least 96%, at least 95%, at least 94%, at least 93%,
at least 92%, at least 91%, at least 90%, at least 89%, at least
88%, at least 87%, at least 86%, at least 85%, at least 84%, at
least 83%, at least 82%, at least 81%, at least 80%, at least 79%,
at least 78%, at least 77%, at least 76%, at least 75%, at least
74%, at least 73%, at least 72%, at least 71% or at least 70%) with
a mouse aquaporin sequence, including but not limited to, e.g.,
mouse AQP4 (UniProtlD: P55088) the amino acid sequence of which
is:
TABLE-US-00002 (SEQ ID NO: 4)
MSDGAAARRWGKCGHSCSRESIMVAFKGVWTQAFWKAVSAEFLATLIFVL
LGVGSTINWGGSENPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAV
TVAMVCTRKISIAKSVFYIIAQCLGAIIGAGILYLVTPPSVVGGLGVTTV
HGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIG
HLFAINYTGASMNPARSFGPAVIMGNWANHWIYWVGPIMGAVLAGALYEY
VFCPDVELKRRLKEAFSKAAQQTKGSYMEVEDNRSQVETEDLILKPGVVH
VIDIDRGEEKKGKDSSGEVLSSV.
[0054] In some instances, an AQP4 polypeptide or a nucleic acid
encoding an AQP4 polypeptide may include or exclude all or a
portion of a domain of the APQ4 protein. Domains of APQ4 include
but are not limited to topological, transmembrane, and/or
functional domains. Such domains include but are not limited to,
e.g., Cytoplasmic Domain from amino acids 1 to 36 of P55087,
Helical Domain from amino acids 37 to 57 of P55087, Extracellular
Domain from amino acids 58 to 64 of P55087, Helical Domain from
amino acids 65 to 85 of P55087, Cytoplasmic Domain from amino acids
86 to 115 of P55087, Helical Domain from amino acids 116 to 136 of
P55087, Extracellular Domain from amino acids 137 to 155 of P55087,
Helical Domain from amino acids 156 to 176 of P55087, Cytoplasmic
Domain from amino acids 177 to 184 of P55087, Helical Domain from
amino acids 185 to 205 of P55087, Extracellular Domain from amino
acids 206 to 231 of P55087, Helical Domain from amino acids 232 to
252 of P55087, Cytoplasmic Domain from amino acids 253 to 323 of
P55087. In some instances, an AQP4 polypeptide or a nucleic acid
encoding an AQP4 polypeptide may include or exclude one or more
APQ4 motifs, including but not limited to one or more (Asn-Pro-Ala)
(i.e., NPA) motifs.
[0055] In some instances, an AQP4 polypeptide or a nucleic acid
encoding an AQP4 polypeptide may include or exclude one or more
modification sites of the APQ4 protein. Modification sites may
include but are not limited to, e.g., phosphorylation sites,
glycosylation sites. Such sites may include but are not limited to
the following phosphoserine sites relative to the sequence of
P55087: residue 111, residue 180, residue 285 and residue 321. Such
sites include but are not limited to the following glycosylation
sites relative to the sequence of P55087: residue 153 and residue
206. In some instances, an AQP4 polypeptide may include or exclude
one or more post-translational modifications.
[0056] In some instances, an aquaporin polypeptide, e.g., as
encoded from a nucleic acid of the subject disclosure, may include
a portion of a full length aquaporin amino acid sequence, e.g., an
aquaporin amino acid sequence disclosed herein. The length of such
portions of aquaporin amino acid sequence may vary and may range,
e.g., from 5 to 322 amino acids in length, including, e.g., 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,
250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,
276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,
302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,
315, 316, 317, 318, 319, 320, 321 or 322 residues in length.
[0057] In some instances, such polypeptide portions of an aquaporin
may include specific amino acid residues relative to the human AQP4
sequence (UniProt ID P55087) where the first amino acid of the
polypeptide portion may range from residues 1-318 of P55087,
including but not limited to, e.g., residue 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,
134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,
199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,
212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,
225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,
251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,
264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,
277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,
290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,
303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,
316, 317 and 318.
[0058] In some instances, such polypeptide portions of an aquaporin
may include specific amino acid residues relative to the human AQP4
sequence (UniProt ID P55087) where the last amino acid of the
polypeptide portion may range from residues 5-323 of P55087,
including but not limited to, e.g., residue 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,
176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,
189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,
215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,
228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,
241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,
254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,
267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,
280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,
293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,
306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,
319, 320, 321, 322, and 323.
[0059] In some instances, a polypeptide portion of an aquaporin may
include amino acid residues 61-80 of the human AQP4 sequence
(UniProt ID P55087).
[0060] In some instances, modification of an AQP4 polypeptide,
including e.g., selection of amino acid mutations, amino acid
deletions, and/or amino acid insertion sites, may be performed
based on the three-dimensional structure of the AQP4 polypeptide.
For example, in some instances, a modified AQP4 polypeptide and/or
a nucleic acid encoding a modified AQP4 polypeptide may be based on
rational design of the modified AQP4 polypeptide three-dimensional
structure. Rational design of modified APQ4 polypeptides may be
achieved through use of one or more three-dimensional APQ4 protein
structures including but not limited to, e.g., RCSP Protein Data
Bank (PDB) structure 3GD8 (see Ho et al., (2009) Proc. Natl. Acad.
Sci. USA 106: 7437-7442, the disclosure of which is incorporated
herein by reference in its entirety).
[0061] In some instances, an aquaporin nucleic acid, e.g., as
included in a DNA tolerizing vaccine, may encode for an aquaporin
polypeptide described herein, e.g., a mammalian AQP4 polypeptide, a
human AQP4 polypeptide, a mouse AQP4 polypeptide. In some
instances, an aquaporin nucleic acid may share 100% sequence
identity over its entire length with all or a portion of an nucleic
acid encoding for an aquaporin polypeptide described herein, e.g.,
a mammalian AQP4 polypeptide, a human AQP4 polypeptide, a mouse
AQP4 polypeptide, etc.
[0062] In some instances, an aquaporin nucleic acid, e.g., as
included in a DNA tolerizing vaccine, may be derived from human
AQP4 nucleotide sequence. Human AQP4 nucleotide sequences include
but are not limited to, e.g., those DNA sequences included in human
genome locations chr18:24,432,008-24,442,575 and
chr18:24,436,175-24,442,526. In some instances, an aquaporin
nucleic acid may include, in part or in whole, or be derived from
one or more sequences having the following Accession Numbers:
U34846, U34845, D63412, U63622, U63623, AC018371, BCO22286, RefSeq
NM_001650.4, RefSeq NM_004028.3. In some instances, an aquaporin
nucleic acid may include, in part or in whole, or be derived from
CCDS11889.1 having the sequence:
TABLE-US-00003 (SEQ ID NO: 5)
ATGAGTGACAGACCCACAGCAAGGCGGTGGGGTAAGTGTGGACCTTTGTG
TACCAGAGAGAACATCATGGTGGCTTTCAAAGGGGTCTGGACTCAAGCTT
TCTGGAAAGCAGTCACAGCGGAATTTCTGGCCATGCTTATTTTTGTTCTC
CTCAGCCTGGGATCCACCATCAACTGGGGTGGAACAGAAAAGCCTTTACC
GGTCGACATGGTTCTCATCTCCCTTTGCTTTGGACTCAGCATTGCAACCA
TGGTGCAGTGCTTTGGCCATATCAGCGGTGGCCACATCAACCCTGCAGTG
ACTGTGGCCATGGTGTGCACCAGGAAGATCAGCATCGCCAAGTCTGTCTT
CTACATCGCAGCCCAGTGCCTGGGGGCCATCATTGGAGCAGGAATCCTCT
ATCTGGTCACACCTCCCAGTGTGGTGGGAGGCCTGGGAGTCACCATGGTT
CATGGAAATCTTACCGCTGGTCATGGTCTCCTGGTTGAGTTGATAATCAC
ATTTCAATTGGTGTTTACTATCTTTGCCAGCTGTGATTCCAAACGGACTG
ATGTCACTGGCTCAATAGCTTTAGCAATTGGATTTTCTGTTGCAATTGGA
CATTTATTTGCAATCAATTATACTGGTGCCAGCATGAATCCCGCCCGATC
CTTTGGACCTGCAGTTATCATGGGAAATTGGGAAAACCATTGGATATATT
GGGTTGGGCCCATCATAGGAGCTGTCCTCGCTGGTGGCCTTTATGAGTAT
GTCTTCTGTCCAGATGTTGAATTCAAACGTCGTTTTAAAGAAGCCTTCAG
CAAAGCTGCCCAGCAAACAAAAGGAAGCTACATGGAGGTGGAGGACAACA
GGAGTCAGGTAGAGACGGATGACCTGATTCTAAAACCTGGAGTGGTGCAT
GTGATTGACGTTGACCGGGGAGAGGAGAAGAAGGGGAAAGACCAATCTGG
AGAGGTATTGTCTTCAGTATGA.
[0063] In some instances, an aquaporin nucleic acid, e.g., as
included in a DNA tolerizing vaccine, may be derived from mouse
AQP4 nucleotide sequence. In some instances, an aquaporin nucleic
acid may include, in part or in whole, or be derived from one or
more sequences having the following Accession Numbers: U48398,
U48397, U33012, U48400, U48399, U88623, AF469168, AF469169,
AF219992, CT010362, BCO24526, RefSeq NM_009700.2, RefSeq
XM_006525540.1. In some instances, an aquaporin nucleic acid may
include, in part or in whole, or be derived from CCDS29073.1 having
the sequence:
TABLE-US-00004 (SEQ ID NO: 6)
ATGAGTGACAGAGCTGCGGCAAGGCGGTGGGGTAAGTGTGGACATTCCTG
CAGTAGAGAGAGCATCATGGTGGCTTTCAAAGGAGTCTGGACTCAGGCTT
TCTGGAAGGCAGTCTCAGCAGAATTTCTGGCCACGCTTATCTTTGTTTTG
CTCGGTGTGGGATCCACCATAAACTGGGGTGGCTCAGAAAACCCCTTACC
TGTGGACATGGTCCTCATCTCCCTTTGCTTTGGACTCAGCATTGCTACCA
TGGTGCAGTGCTTTGGCCACATCAGTGGTGGCCACATCAATCCCGCTGTG
ACTGTAGCCATGGTGTGCACACGAAAGATCAGCATCGCTAAGTCCGTCTT
CTACATCATTGCACAGTGCCTGGGGGCCATCATTGGAGCCGGCATCCTCT
ACCTGGTCACACCTCCCAGTGTGGTTGGAGGATTGGGAGTCACCACGGTT
CATGGAAACCTCACCGCTGGCCATGCCCTCCTGGTGGAGTTAATAATCAC
TTTCCAGTTGGTGTTCACTATTTTTGCCAGCTGTGATTCCAAACGAACTG
ATGTTACTGGTTCAATAGCTTTAGCAATTGGATTTTCCGTTGCAATTGGA
CATTTGTTTGCAATCAATTATACTGGAGCCAGCATGAATCCAGCTCGATC
TTTTGGACCCGCAGTTATCATGGGAAACTGGGCAAACCACTGGATATATT
GGGTTGGACCAATCATGGGCGCTGTGCTGGCAGGTGCCCTTTATGAGTAT
GTCTTCTGTCCTGATGTGGAGCTCAAACGTCGCCTTAAGGAAGCCTTCAG
CAAAGCCGCGCAGCAGACAAAAGGGAGCTACATGGAGGTGGAGGACAACC
GGAGCCAAGTGGAGACGGAAGACTTGATCCTGAAGCCCGGAGTGGTGCAT
GTGATTGACATTGACCGTGGAGAAGAGAAGAAGGGGAAAGACTCTTCGGG
AGAGGTATTGTCTTCCGTATGA.
[0064] In some instances, a nucleic acid as described herein may be
appended with one or more additional nucleic acids or one or more
additional nucleotides. Additional nucleic acid may be appended to
the described nucleic acids for a variety of purposes including but
not limited to, e.g., cloning purposes (e.g., to facilitate
homologous recombination, to facilitate ligation, etc.). As such,
in some instances a nucleic acid as described herein may be
appended with one or more additional nucleic acids to attach one or
more nucleic acid spacers, one or more homologous sequences (e.g.,
a sequence homologous with a vector into which the subject nucleic
acid may be cloned), one or more restriction enzyme recognition
sites, and the like. Additional sequences appended to a subject
nucleic acid may be added through any convenient method including
but not limited to, e.g., ligation-based methods, PCR-based
methods, de novo polynucleotide synthesis, etc.
DNA Tolerizing Vaccines
[0065] Aspects of the disclosure include DNA tolerizing vaccines
comprising nucleic acid encoding for an aquaporin polypeptide. In
some instances, an aquaporin polypeptide may be referred herein to
as an antigen. Methods are provided, described in more detail
below, for treating a subject having an adverse immune response to
an aquaporin autoantigen. Accordingly, as used herein, the term
"DNA tolerizing vaccine" may refer to a composition containing a
nucleic acid containing one or more aquaporin nucleic acid
sequences and encoding for one or more aquaporin polypeptides. In
some instances, an encoded aquaporin polypeptide of a DNA
tolerizing vaccine may be essentially the same as an aquaporin
autoantigen. In some instances, an encoded aquaporin polypeptide of
a DNA tolerizing vaccine may be different from an aquaporin
autoantigen. Encompassed differences between an encoded aquaporin
polypeptide of a DNA tolerizing vaccine and an aquaporin
autoantigen include but are not limited to those polypeptide
modifications described herein.
[0066] The components of an aquaporin DNA tolerizing vaccine will
vary and will include, at a minimum, a vector that contains a
nucleic acid sequence encoding an aquaporin polypeptide and the
necessary components for expression of the aquaporin polypeptide
from the vector. Nucleic acid sequence encoding for any aquaporin
polypeptide, including those described herein, may find use in a
DNA tolerizing vaccine.
[0067] In some instances, a DNA tolerizing vaccine includes a
minigene that includes nucleic acid encoding for one or more
aquaporin polypeptides. As used herein the term "minigene" refers
to a minimal gene fragment that excludes one or more components of
a native gene locus but includes the necessary elements for
expression of the gene product or some portion of the gene product
or a synthetic construct. In some instances, an aquaporin minigene
may exclude at least one aquaporin intron, or portion thereof,
including but not limited to 1 or more introns, 2 or more intron,
or all the introns of the native aquaporin genetic locus. In some
instances, an aquaporin minigene may include at least one aquaporin
intron, or portion thereof, including but not limited to 1 or more
introns, 2 or more intron, or all the introns of the native
aquaporin genetic locus. In some instances, an aquaporin minigene
may exclude at least one aquaporin exon, or portion thereof,
including but not limited to 1 or more exons, 2 or more exons, 3
exons or all but a portion of one exon of the native aquaporin
genetic locus. In some instances, an aquaporin minigene may include
at least one aquaporin exon, or portion thereof, including but not
limited to 1 or more exons, 2 or more exons, 3 exons or all the
exons of the native aquaporin genetic locus.
[0068] In some instances, a minigene may include nucleic acid
encoding for one antigenic epitope of an aquaporin gene. In some
instances, a minigene may include two or more antigenic epitopes of
an aquaporin gene, e.g., arranged in series and joined with or
without one or more optional linkers. An antigenic epitope may
consist of an exon or one or more portions thereof or portions of
two or more exons. In some instances, multiple antigenic epitopes
from the same exon and/or from multiple different exons are
arranged in series and contained within a minigene. Methods of
generating such strings of antigenic epitopes include but are not
limited to, e.g., those described in Whitton et al. J Virol. 1993
January; 67(1): 348-352, the disclosure of which is incorporated
herein by reference in its entirety.
[0069] A minigene will also include at least some regulatory
sequence that controls or enhances the expression of the minigene
transcript. In some instances, a minigene regulatory sequence will
include a promoter. Promoters useful in an aquaporin minigene will
vary and selection of such a minigene promoter will depend on
various factors including the desired expression level of the
minigene transcript, the desired control of minigene expression,
the desired size of the overall minigene, the intended use of the
minigene, including the subject to which the minigene may be
delivered. Such minigene promoters may include but are not limited
to a native aquaporin promoter, a native non-aquaporin (i.e., a
promoter native to organism from which the aquaporin sequence was
derived but not associated with the native aquaporin locus), a
heterologous promoter (i.e., a promoter derived from an organism
other than the organism from which the aquaporin sequence was
derived (e.g., a non-human promoter, a non-mammalian promoter,
etc.)), a minimal promoter, a minipromter, a constitutive promoter,
a tissue specific promoter, an inducible promoter, a synthetic
promoter and the like.
[0070] In some instances, a DNA tolerizing vaccine will include a
vector backbone, e.g., a plasmid polynucleotide backbone. Vector
backbones useful in a DNA tolerizing vaccine will vary and may be
selected based on a number of factors. For example, in some
instances, a vector backbone may be selected based on the absence
or minimal presence of nucleotide sequence that is homologous with
one or more desired host organisms of the DNA tolerizing vaccine
(i.e., an organism that will ultimately receive the DNA tolerizing
vaccine) in order to prevent or minimize the likelihood of
homologous recombination between the vector and the host organism
genome. The amount of homologous sequence between the vector
backbone and the host organism may vary and, in some instances, the
vector backbone may not contain any sequence homologous to the host
organism that is longer than 200 nucleotides, including but not
limited to, e.g., longer than 150 nucleotides, longer than 100
nucleotides, longer than 90 nucleotides, longer than 80
nucleotides, longer than 70 nucleotides, longer than 60
nucleotides, longer than 50 nucleotides, longer than 40
nucleotides, longer than 30 nucleotides or longer than 25
nucleotides. In some instances, the vector may have further
features that prevent integration into a host genome, e.g., the
vector may be a closed- circular plasmid.
[0071] A vector of a DNA tolerizing vaccine may include one or more
vector specific elements. By "vector specific elements" is meant
elements that are used in making, constructing, propagating,
maintaining and/or assaying the vector before, during or after its
construction and/or before its use in a DNA tolerizing vaccine.
Such vector specific elements include but are not limited to, e.g.,
vector elements necessary for the propagation, cloning and
selection of the vector during its use and may include but are not
limited to, e.g., an origin of replication, a multiple cloning
site, a prokaryotic promoter, a phage promoter, a selectable marker
(e.g., an antibiotic resistance gene, an encoded enzymatic protein,
an encoded fluorescent or chromogenic protein, etc.), and the like.
Any convenient vector specific elements may find use, as
appropriate, in the vectors as described herein.
[0072] In some instances, a vector backbone or one or more vector
specific elements of a DNA tolerizing vaccine is configured to
reduce the number of immunostimulatory motifs present in the
vector. For example, a nucleic acid vector may be modified where a
non-CpG dinucleotide is substituted for one or more CpG
dinucleotides of the formula
5'-purine-pyrimidine-C-G-pyrimidine-pyrimidine-3' or
5'-purine-purine-C-G-pyrimidine-pyrimidine-3', thereby producing a
vector in which immunostimulatory activity is reduced. Such vectors
are useful, for example, in methods for administering immune
modulatory nucleic acids and/or for administering a DNA tolerizing
vaccine encoding one or more aquaporin polypeptides.
[0073] For example, the cytosine of the CpG dinucleotide can be
substituted with guanine, thereby yielding a region having a GpG
motif of the formula
5'-purine-pyrimidine-G-G-pyrimidine-pyrimidine-3' or
5'-purine-purine-G-G-pyrimidine-pyrimidine-3'. The cytosine can
also be substituted with any other non-cytosine nucleotide. The
substitution can be accomplished, for example, using site-directed
mutagenesis. Typically, the substituted CpG motifs are those CpGs
that are not located in one or more regulatory regions of the
vector (e.g., promoter regions) and/or vector specific element, as
described herein. In addition, where the CpG is located within a
coding region of an expression vector (e.g., the coding region of a
vector specific element, such as a selectable marker), the
non-cytosine substitution is typically selected to yield a silent
mutation or a codon corresponding to a conservative substitution of
the encoded amino acid.
[0074] For example, in certain embodiments, a modified pVAX1 vector
is utilized in which one or more CpG dinucleotides of the formula
5'-purine-pyrimidine-C-G-pyrimidine-pyrimidine-3' is mutated by
substituting the cytosine of the CpG dinucleotide with a
non-cytosine nucleotide. The pVAX1 vector is known in the art and
is commercially available from Life Technologies, Inc. (Grand
Island, N.Y.). In one exemplary embodiment, the modified pVAX1
vector has the following cytosine to non-cytosine substitutions
within a CpG motif: cytosine to guanine at nucleotides 784, 1161,
1218, and 1966; cytosine to adenine at nucleotides 1264, 1337,
1829, 1874, 1940, and 1997; and cytosine to thymine at nucleotides
1963 and 1987; with additional cytosine to guanine mutations at
nucleotides 1831, 1876, 1942, and 1999 where the nucleotide number
designations as set forth above are according to the numbering
system for pVAX1 provided by Life Technologies, Inc. (Grand Island,
N.Y.). In some instances, a modified pVAX1 vector is pBHT1 (SEQ ID
NO:7).
[0075] In some instances, vectors, including vector specific
elements, include or exclude certain immune modulatory sequences,
e.g., exclude immunostimulatory sequences and/or include
immunoinhibitory sequences, or have increased numbers of
immunoinhibitory sequences and/or decreased numbers of
immunostimulatory sequences. The use of immune modulatory
sequences, including but not limited to, e.g.: GTGGTT, ATGGTT,
GCGGTT, ACGGTT, GTGGCT, ATGGCT, GCGGCT, ACGGCT, GTGGTC, ATGGTC,
GCGGTC, ACGGTC, GTGCTT, ATGCTT, GCGCTT, ACGCTT, GTGCCT, ATGCCT,
GCGCCT, ACGCCT, GTGCTC, ATGCTC, GCGCTC, ACGCTC, GGGGTT, AGGGTT,
GAGGTT, AAGGTT, GGGGCT, AGGGCT, GAGGCT, AAGGCT, GGGGTC, AGGGTC,
GAGGTC, AAGGTC, GGGCTT, AGGCTT, GAGCTT, AAGCTT, GGGCCT, AGGCCT,
GAGCCT, AAGCCT, GGGCTC, AGGCTC, GAGCTC and AAGCTC; and the
generation and use of vectors containing reduced numbers of
immunostimulatory sequences and increased numbers of
immunoinhibitory sequences have been described, e.g., in U.S. Pat.
No. 7,811,813, the disclosure of which is incorporated herein by
reference in its entirety.
[0076] In some instances, the vector backbone (i.e., the nucleotide
sequence of the vector excluding any regulatory elements,
replication sequences and/or coding sequences), e.g., the resulting
vector backbone after modification to remove immunostimulatory CpG
motifs as described herein, contains 4 or fewer immunostimulatory
CpG motifs, including but not limited to, e.g., 3 or fewer
immunostimulatory CpG motifs, 2 or fewer immunostimulatory CpG
motifs, 1 or fewer immunostimulatory CpG motifs. In some instances,
a vector backbone as described herein of a DNA tolerizing vaccine
may be modified to contain no immunostimulatory CpG motifs.
[0077] In some instances, the vector (i.e., the nucleotide sequence
of the vector excluding only any inserted aquaporin coding
sequence), e.g., the resulting vector after modification to remove
immunostimulatory CpG motifs as described herein, contains 30 or
fewer immunostimulatory CpG motifs, including but not limited to,
e.g., 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or
fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or
fewer, 19 or fewer, 18 or fewer immunostimulatory CpG motifs.
[0078] A vector of a DNA tolerizing vaccine, whether or not
configured to contain a minigene, will further include one or more
regulatory elements. Such regulatory elements will vary and may
include but are not limited to, e.g., a promoter, an enhancer, an
intron, a polyadenylation signal, an initiation sequence (e.g., a
Kozak sequence), and the like. Promoters useful in the expression
of an aquaporin polynucleotide include but are not limited to,
e.g., a native aquaporin promoter, a native non-aquaporin promoter
(i.e., a promoter native to organism from which the aquaporin
sequence was derived but not associated with the native aquaporin
locus), a heterologous promoter (i.e., a promoter derived from an
organism other than the organism from which the aquaporin sequence
was derived (e.g., a non-human promoter, a non-mammalian promoter,
etc.)), a minimal promoter, a minipromter, a constitutive promoter,
a tissue specific promoter, an inducible promoter, a synthetic
promoter and the like.
[0079] Promoters may be operably linked to an aquaporin polypeptide
encoding nucleic acid to control production of encoded transcript
either in vitro or in vivo. Such promoters may be constitutively
active or controllable through the introduction of a stimulus,
e.g., an environmental stimulus (e.g., change in temperature, pH,
light exposure, and the like), a chemical or biological stimulus
(e.g., a small molecule, a chemical, a polypeptide that binds to
the promoter, and the like). In some instances, a vector of a DNA
tolerizing vaccine may include a cytomegalovirus promoter.
[0080] A DNA tolerizing vaccine regulatory element may also include
more or more enhancer elements. Enhancers may be operably linked to
an aquaporin polypeptide encoding nucleic acid to control
production of encoded transcript either in vitro or in vivo. Such
enhancers may be constitutively active or controllable through the
introduction of a stimulus, e.g., an environmental stimulus (e.g.,
change in temperature, pH, light exposure, and the like), a
chemical or biological stimulus (e.g., a small molecule, a
chemical, a polypeptide that binds to the enhancer, and the like).
In some instances, a vector of a DNA tolerizing vaccine may include
a cytomegalovirus enhancer.
[0081] Suitable promoter and enhancer elements are known in the
art. For expression in a bacterial cell, suitable promoters
include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P
and trc. For expression in a eukaryotic cell, suitable promoters
include, but are not limited to, light and/or heavy chain
immunoglobulin gene promoter and enhancer elements; cytomegalovirus
immediate early promoter; herpes simplex virus thymidine kinase
promoter; early and late SV40 promoters; promoter present in long
terminal repeats from a retrovirus; mouse metallothionein-I
promoter; and various art-known tissue specific promoters.
[0082] Suitable inducible promoters, including reversible inducible
promoters are known in the art. Such inducible promoters may be
isolated and derived from many organisms, e.g., eukaryotes and
prokaryotes. Modification of inducible promoters derived from a
first organism for use in a second organism, e.g., a first
prokaryote and a second a eukaryote, a first eukaryote and a second
a prokaryote, etc., is well known in the art. Such inducible
promoters, and systems based on such inducible promoters but also
comprising additional control proteins, include, but are not
limited to, e.g., tetracycline regulated promoters, (e.g., promoter
systems including TetActivators, TetON, TetOFF, etc.), steroid
regulated promoters (e.g., rat glucocorticoid receptor promoter
systems, human estrogen receptor promoter systems, retinoid
promoter systems, thyroid promoter systems, ecdysone promoter
systems, mifepristone promoter systems, etc.), metal regulated
promoters (e.g., metallothionein promoter systems, etc.),
temperature regulated promoters (e.g., heat shock inducible
promoters (e.g., HSP-70, HSP-90, etc.), synthetic inducible
promoters, and the like.
[0083] Transcriptional control elements, e.g., promoters,
enhancers, etc., may be bound to an aquaporin polypeptide encoding
nucleic acid singly or in arrays containing multiple
transcriptional control elements, e.g., about 2, about 3, about 4,
about 5, or more than 5 transcriptional control elements. In
certain embodiments, transcriptional control elements are operably
linked, directly or indirectly to the 5' end of an aquaporin
polypeptide encoding nucleic acid with or without intervening
"spacer" nucleic acid(s). Transcriptional control elements, methods
of making and/or arranging and/or modifying transcription control
elements (e.g., in expression cassettes) useful in the nucleic
acids described herein may, in some instances, include those
described in Liu et al., Gene Therapy (2004) 11:52-60; Zheng &
Baum, Methods Mol Biol. 2008, 434:205-19; Papadakis et al., Curr
Gene Ther. 2004, 4(1):89-113; the disclosures of which are
incorporated herein by reference in their entirety.
[0084] A DNA tolerizing vaccine regulatory element may also include
more or more introns wherein inclusion of the intron in the vector
and/or minigene enhances the expression of the encoded aquaporin
polypeptide of the DNA tolerizing vector. Such introns may be
aquaporin gene introns (i.e., introns or portions thereof derived
from a native aquaporin genetic locus) or may be native
non-aquaporin introns (i.e., introns derived from the intended host
genome but from a non-aquaporin locus) or may be heterologous
introns (i.e., introns derived from a genome or organism other than
the intended host genome or host organism). For example, in some
instances, a DNA tolerizing vaccine may include an intron, or
fragment thereof, from a cytomegalovirus including but not limited
to the first intron of the cytomegalovirus or a minimal intron,
including a minimal first intron, of the cytomegalovirus and/or
those introns described in Quilici et al. Biotechnol Lett. 2013,
35(1):21-7 and Xu et al. Gene. 2001, 272(1-2): 149-56; the
disclosures of which are incorporated herein by reference in their
entirety.
[0085] In some instances, a DNA tolerizing vaccine includes an
appropriate diluent, e.g., a suitable solution or liquid for
dissolving a vector as described herein. Such diluents may vary and
may depend upon, e.g., the concentration of vector to be suspended,
the pharmaceutical formulation, of the DNA tolerizing vaccine, the
mode of delivery of the DNA tolerizing vaccine, the method of
storage of the DNA tolerizing vaccine, and the like. In some
instances, a suitable solution or liquid may include but is not
limited to, e.g., aqueous solutions, water (e.g., nuclease-free
water, water for injection (WFI), etc.), saline, phosphate buffered
saline (PBS), tris buffer saline (TBS), tris-EDTA (TE) buffer,
combinations thereof, and the like. Pharmaceutical formulations of
DNA tolerizing vaccines are discussed in more detail below.
Methods and Compositions
[0086] Aspects of the disclosure include methods and compositions
for repressing an immune response to an aquaporin autoantigen in a
subject. Because such methods can be used to treat a subject, such
methods can also be referred to as methods of treating an
individual. Aspects of the subject methods generally involve the
administration of a therapeutically effective amount of a nucleic
acid and/or DNA tolerizing vaccine, as described herein, to a
subject in need thereof.
[0087] A "therapeutically effective amount" or "therapeutically
effective dose" or "therapeutic dose" is an amount sufficient to
effect desired clinical results (i.e., achieve therapeutic
efficacy). A therapeutically effective dose can be administered in
one or more administrations. For purposes of this disclosure, a
therapeutically effective dose of DNA tolerizing vaccine (e.g.,
APQ4 DNA tolerizing vaccine, and the like) and/or compositions
(e.g., DNA tolerizing vaccine compositions) is an amount that is
sufficient, when administered to (e.g., injected into, delivered
intravenously, etc.) the individual, to palliate, ameliorate,
stabilize, reverse, prevent, slow or delay the progression of the
disease state (e.g., autoimmune disease, NMO spectrum disorder,
NMO, etc.) by, for example, reducing the subject's immune activity,
reducing the subject's immune response, reducing the subject's
immune response to an aquaporin, reducing the subject's immune
response to APQ4, reducing the subject's immune response to
self-APQ4. In some instances, an effective amount reduces one or
more symptoms of NMO and/or an NMO spectrum disorder (including but
not limited to, e.g., vision impairment, vision loss, eye pain, eye
strain, muscle weakness (e.g., in the arms and/or legs), numbness
(e.g., in the arms and/or legs), partial paralysis (e.g., of the
arms and/or legs), incontinence (e.g., urinary incontinence, fecal
incontinence, etc.), intractable vomiting, intractable hiccups,
nausea, endocrine disorders, sleep disorders, confusion, coma,
etc.).
[0088] In some instances, a therapeutically effective dose, whether
delivered in a single administration or multiple administrations,
of a DNA tolerizing vaccine may remain effective for an extended
period of time, e.g., by nature of the extended transient
expression of the encoded polypeptide. The extended time period
during which an administered therapeutically effective dose of a
DNA tolerizing vaccine may remain effective will vary and may range
from days to weeks including but not limited to, e.g., 2-3 days,
3-4 days, 4-5 days, 5-6 days, 6-7 days, 2-5 days, 3-6 days, 4-7
days, 1 week to 2 weeks, 2 weeks to 3 weeks, 3 weeks to 4 weeks, 1
week to 3 weeks, 2 weeks to 4 weeks, 1 week to 4 weeks, etc.
[0089] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect can be prophylactic in terms
of completely or partially preventing a disease or symptom(s)
thereof and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. The term "treatment" encompasses any
treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease and/or symptom(s) from
occurring in a subject who may be predisposed to the disease or
symptom(s) but has not yet been diagnosed as having it; (b)
inhibiting the disease and/or symptom(s), i.e., arresting
development of a disease and/or the associated symptoms; or (c)
relieving the disease and the associated symptom(s), i.e., causing
regression of the disease and/or symptom(s). Those in need of
treatment can include those already inflicted (e.g., those with NMO
or an NMO spectrum disorder, e.g. those having NMO or an NMO
spectrum disorder) as well as those in which prevention is desired
(e.g., those with increased susceptibility to NMO or an NMO
spectrum disorder; those with relapsing NMO; those suspected of
having NMO or an NMO spectrum disorder; those having one or more
risk factors for NMO or an NMO spectrum disorder or an NMO relapse,
etc.).
[0090] A therapeutic treatment is one in which the subject is
inflicted prior to administration and a prophylactic treatment is
one in which the subject is not inflicted prior to administration.
With respect to relapsing conditions, a prophylactic treatment may
include a treatment administered to a subject with a diagnosed
condition in a remitting state, e.g., to prevent a relapse of the
condition or to prevent the reoccurrence of one or more symptoms of
the condition. In some embodiments, the subject has an increased
likelihood of becoming inflicted or is suspected of having an
increased likelihood of becoming inflicted (e.g., relative to a
standard, e.g., relative to the average individual, e.g., a subject
may have a genetic predisposition to autoimmune disease and/or a
family history indicating increased risk of NMO spectrum disorders
and/or autoimmune disease), in which case the treatment can be a
prophylactic treatment.
[0091] In some embodiments, the individual to be treated is an
individual with NMO or an NMO spectrum disorder. As used herein
"NMO" includes any form of NMO spectrum disorder, including
relapsing NMO, acute NMO; unilateral NMO, bilateral NMO, and the
like. In some cases, the individual has recently undergone
treatment for NMO (e.g., corticosteroid therapy, immunomodulatory
therapy, immunosuppressive therapy, etc.) and may therefore be at
risk for recurrence and/or relapse. Those subjects in NMO remission
may, in some instances, be treated according to the methods
described herein, e.g., to prevent or delay relapse. In some
instances, an NMO spectrum disorder suitable to be treated by the
subject methods, compositions, and kits as described herein is a
NMO diagnosed, at least in part, as based on the presence of
NMO-IgG antibody in a sample from the subject.
[0092] The terms "individual", "subject", "recipient", "host", and
"patient", are used interchangeably herein and refer to any
mammalian subject for whom treatment or therapy is desired,
particularly humans. "Mammal" for purposes of treatment refers to
any animal classified as a mammal, including humans, domestic and
farm animals, and zoo, sports, or pet animals, such as dogs,
horses, cats, cows, sheep, goats, pigs, camels, etc. In some
embodiments, the mammal is human. In some instances, a subject may
also be a research subject, including but not limited to, e.g., a
human research subject (e.g., a clinical trial participant), a
pre-clinical research subject (e.g., a mammalian research subject,
a laboratory animal, etc.), an animal model (e.g., a rodent animal
model, a mouse model, a rat model, etc.).
[0093] Animal models as used herein include but are not limited to
animal models of multiple sclerosis (MS), animal models of MS
related disorders, animal models of NMO, animal models of NMO
spectrum disorders which may include but are not limited to, e.g.,
those described in Jones et al. (2012) Mult Scler Relat Disord.
1(4):174-179 and Denic et al. (2010) Pathophysiology 18: 21-29; the
disclosures of which are incorporated herein by reference in their
entirety. In some instances, a method of treatment and/or a DNA
tolerizing vaccine and/or nucleic acid, as described herein, may be
evaluated, tested, or developed through the use of one or more
animal models. In such instances, a treated animal model or group
thereof may be compared to one or more controls, including positive
controls and/or negative controls, and/or control groups.
[0094] In some instances, a nucleic acid and/or DNA tolerizing
vaccine, as described herein may be co-administered with one or
more agents of one or more additional therapies. For example, a
nucleic acid and/or DNA tolerizing vaccine may be co-administered
with one or more conventional NMO therapies, e.g., steroid therapy,
anti-inflammatory therapy, immunomodulatory therapy,
immunosuppressive therapy, etc. In some instances, two or more
nucleic acids and/or DNA tolerizing vaccines, as described herein
may be administered in combination, e.g., as part of a nucleic acid
and/or DNA tolerizing vaccine "cocktail".
[0095] The terms "co-administration" and "in combination with"
include the administration of two or more therapeutic agents either
simultaneously, concurrently or sequentially within no specific
time limits. In one embodiment, the agents are present in the cell
or in the subject's body at the same time or exert their biological
or therapeutic effect at the same time. In one embodiment, the
therapeutic agents are in the same composition or unit dosage form.
In other embodiments, the therapeutic agents are in separate
compositions or unit dosage forms. In certain embodiments, a first
agent can be administered prior to (e.g., minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second therapeutic agent.
[0096] Whether administered alone or as part of a combination
therapy, any convenient and appropriate method of delivery of the
nucleic acids and/or DNA tolerizing vaccines described herein may
be utilized. Nucleic acid constructs can be delivered with cationic
lipids (Goddard, et al, Gene Therapy, 4:1231-1236, 1997; Gorman, et
al, Gene Therapy 4:983-992, 1997; Chadwick, et al, Gene Therapy
4:937-942, 1997; Gokhale, et al, Gene Therapy 4:1289-1299, 1997;
Gao, and Huang, Gene Therapy 2:710-722, 1995, the disclosures of
which are incorporated herein by reference in their entirety), by
uptake of "naked DNA", and the like. In some instances, a method of
delivery of the nucleic acids and/or DNA tolerizing vaccines may
include or may be enhanced by electroporation, particle bombardment
(i.e., biolistics), sonoporation, magnetofection, hydrodynamic
delivery and the like. In some instances, a method of delivery of
the nucleic acids and/or DNA tolerizing vaccines may include or may
be enhanced by the use of one or more chemical methods to enhance
delivery including but not limited to, e.g., the use of nucleic
acid specifically modified to enhance delivery, lipoplexes,
polymersomes, polyplexes, dendrimers, nanoparticles (e.g.,
inorganic nanoparticles), cell-penetrating peptides,
cell-penetrating proteins (e.g., supercharged proteins), and the
like. In some instances, the exact formulation, route of
administration and dosage can be chosen empirically. Methods of
nucleic acid and/or DNA vaccine delivery include but are not
limited to, e.g., those described in U.S. Pat. Nos. 9,018,187,
8,877,729, 8,785,202, 8,759,499, 8,754,062, 8,747,903, 8,697,667,
8,591,862, 8,466,122, 8,338,584, 8,268,796, 8,242,089, 8,178,128,
7,922,709, 7,915,230, 7,829,657, 7,795,380, 7,795,017, 7,767,456,
7,655,467, 7,604,803, 7,534,424, 7,294,511, 7,015,040, the
disclosures of which are incorporated herein by reference in their
entirety.
[0097] Methods of interest for the delivery of nucleic acids and
DNA vaccines, as described herein, include but are not limited to
injection delivery, oral delivery, inhalation delivery, topical
delivery (e.g., transdermal delivery, transmucosal delivery, etc.),
and the like. Such delivery methods may or may not make use of
methods for enhancing nucleic acid delivery, e.g., as described
above, where appropriate. Of interest are injection delivery
methods, including but not limited to needle and needleless
injection methods. As such, in many instances, nucleic acids may be
delivered in a suitable diluent by intramuscular injection and, in
some instances, a course of therapy may include multiple
intramuscular injections, e.g., according to a pre-determined
treatment schedule. In some instances, methods of intramuscular
injection of nucleic acids and DNA vaccines, as described herein,
may include formulating the subject nucleic acid or DNA vaccine in
phosphate buffered saline (PBS) containing 0.9 mM calcium
(Ca.sup.2+) as a sterile solution.
[0098] A pharmaceutical composition (e.g., a DNA vaccine
composition) of the instant disclosure is formulated to be
compatible with its intended route of administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0099] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Kolliphor EL or phosphate buffered saline
(PBS). In all cases, the composition must be sterile and should be
fluid to the extent that easy syringeability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0100] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0101] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0102] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0103] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0104] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0105] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0106] In some instances, methods as described herein my include
methods for assaying for the effectiveness of a DNA tolerizing
vaccine therapy. Methods of evaluating DNA tolerizing therapy
effectiveness may include measuring the level of one or more
biomarkers of NMO in a biological sample from the subject. In some
instances, the level of an NMO biomarker may include the use of one
or more specific binding agents of an aquaporin or an aquaporin
antibody, e.g., for the detection and or measurement of an NMO
biomarker. For example, in some instances, the level of one or more
aquaporin antibodies (i.e., anti-APQ4-IgG, anti-APQ4-IgM, etc.)
present in a biological sample from a subject may be measured
through an assay involving binding of a detectable aquaporin
antibody specific binding member to an aquaporin antibody of the
sample.
[0107] In other instances, the level of an indirect biomarker,
e.g., of aquaporin levels, of aquaporin antibody levels, of immune
system activation, etc. may be measured or detected as a means of
assessing a DNA tolerizing vaccine treatment as described herein.
For example, in some instances the level of one or more immune
system activation markers (e.g., one or more cytokines, IL-6,
IL-17a, INF-gamma, etc.) may be measured in a biological sample
from a subject as a means of determining the subject's response to
treatment with a DNA tolerizing vaccine. In some instances,
immunoglobulin levels may be assessed in a sample from a subject,
including but not limited to, e.g., IgG levels, IgM levels, total
immunoglobulin, etc., as a means of evaluating a subject's response
to DNA tolerizing vaccine treatment. In some instances, the number,
relative amounts, and/or activity of immune cells, e.g., collected
from a biological sample from a subject, may be assessed as a means
of determining a subject's response to DNA tolerizing vaccine
treatment. Any convenient immune system evaluation assay may find
use in assessing the immune system of a subject undergoing or
having had treatment with a DNA tolerizing vaccine as described
herein.
[0108] In some instances, a subject's response to therapy may be
determined by measuring the response of one or more immune cell
populations of the subject to the therapy. Immune cell populations
that may be measured as a means of determining a subject's immune
response may include but are not limited to, e.g., granulocytes and
their progeny (e.g., basophils, eosinophils, and neutrophils), mast
cells, monocytes and their progeny (e.g., macrophages, dendritic
cells), natural killer cells, T cells (e.g., CD8+ T cells, CD4+ T
cells (e.g., TH1 CD4+ T cells, TH2 CD4+ T cells, TH17 CD4+ T cells,
and Treg CD4+ T cells), B cells, and the like. In some instances, a
subject's response to treatment may be evaluated based on a
subject's T cell response. In some instances, a subject's response
to treatment may be evaluated based on a subject's B cell response.
Methods of measuring a subject's immune system activity including
response to therapy or autoimmune response include but are not
limited to, e.g., T-cell proliferation assay, immunoblot assay,
autoantibody detection, flow cytometric methods, etc. and those
methods described in Seyfert-Margolis et al., Diabetes. 2006 55(9):
2588-2594; Bercovici et al., Clin Vaccine Immunol. 2000 7(6):
859-864; Gratama et al. Cytometry A. 2008 73(11): 971-974; the
disclosures of which are incorporated herein by reference in their
entirety. The ordinary artisan will readily recognize where a
particular immune assay, e.g., an assay for a particular autoimmune
disease, may be adapted for use in the methods as described herein,
e.g., adaption of an existing autoimmune assay for evaluation of a
treatment response as described herein and/or for the evaluation of
NMO and/or a NMO spectrum disorder.
[0109] In some instances, assessments of a subject's immune system
and/or immune response to a particular antigen may be performed
prior to DNA tolerizing vaccine administration, e.g., to establish
a baseline. In some instances, assessments of a subject's immune
system and/or immune response to a particular antigen may be
performed during therapy, e.g., at a pre-determined time point
after the first administration and before the final administration,
to assess a subject's response to DNA tolerizing vaccine therapy.
In some instances, assessments of a subject's immune system and/or
immune response to a particular antigen may be performed after
therapy, e.g., at a pre-determined time point after administration
of the final dose, to assess a subject's response to the course of
DNA tolerizing vaccine treatment. In some instances, the results of
such assessments may inform the therapeutic regimen and therapy may
be adjusted, e.g., extended or terminated or modified (e.g., dose
modification), based on the results of one or more of the
assessments described herein.
Kits
[0110] Also provided are kits for use in the subject methods. The
subject kits include any combination of components and compositions
for performing the subject methods. In some embodiments, a kit can
include the following: a DNA tolerizing vaccine, a vaccine delivery
device, a suitable buffer and any combination thereof.
[0111] In some embodiments, a subject kit includes lyophilized DNA
tolerizing vaccine and a suitable diluent for resuspending the
lyophilized DNA tolerizing vaccine before use where the DNA
tolerizing vaccine and the diluent are present in separate
containers. In some instances, a subject kit may include one or
more pre-formulated doses of DNA tolerizing vaccine in
"ready-to-use" format. In instances where a dosing regimen is
desired that includes multiple administrations of one or more DNA
tolerizing vaccines, a subject kit may include two or more doses of
DNA tolerizing vaccine, in a pre-formulated or an unformulated
configuration, and may, optionally, include instructions (e.g.,
instructions as to when each dose should be administered,
instruction for preparing unformulated doses, instructions for dose
delivery, etc.). In some instances, a subject kit may include one
or more testing reagents or testing devices or combinations thereof
for assaying a subject's need for therapy (e.g., before or after
therapy), assaying the effectiveness of therapy (e.g., during or
after therapy), etc.
[0112] In addition to the above components, the subject kits may
further include (in certain embodiments) instructions for
practicing the subject methods. These instructions may be present
in the subject kits in a variety of forms, one or more of which may
be present in the kit. One form in which these instructions may be
present is as printed information on a suitable medium or
substrate, e.g., a piece or pieces of paper on which the
information is printed, in the packaging of the kit, in a package
insert, and the like. Yet another form of these instructions is a
computer readable medium, e.g., diskette, compact disk (CD), flash
drive, and the like, on which the information has been recorded.
Yet another form of these instructions that may be present is a
website address which may be used via the internet to access the
information at a removed site.
EXAMPLES
[0113] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., room temperature (RT); base pairs (bp); kilobases (kb);
picoliters (ph; seconds (s or sec); minutes (m or min); hours (h or
hr); days (d); weeks (wk or wks); nanoliters (nl); microliters
(ul); milliliters (ml); liters (L); nanograms (ng); micrograms
(ug); milligrams (mg); grams ((g), in the context of mass);
kilograms (kg); equivalents of the force of gravity ((g), in the
context of centrifugation); nanomolar (nM); micromolar (uM),
millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base
pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal
(i.p.); subcutaneous (s.c.); and the like.
Example 1
[0114] A tolerizing DNA vaccine minigene was developed using AQP4
peptide 21-40 inserted into a modified pBHT1 expression vector
(FIG. 1). The modified pBHT1 expression vector has been modified to
reduce the CpG sequences in the noncoding backbone. Modified pBHT1
and AQP4p21-40 encoding oligos were digested with HindIII and EcoRI
(FIG. 2), 5' phosphates from pBHT1 were removed, and the resultant
polynucleotides were ligated. Newly ligated plasmids were
transformed, plated, and screened. Presence of the AQP4p21-40
insert (100 bp) was verified by restriction enzyme digest of the
newly ligated plasmid (FIG. 3) and DNA sequencing/BLAST (FIG.
4).
[0115] The 21-40 AQP4 peptide sequence is the dominant T cell
epitope in both SJLJ and C57BL6 mice. In comparison, the dominate T
cell epitope in humans is AQP4 peptide 61-80 (see, e.g., Nelson et
al. (2010) PLOS one, 5(11):e15050 and Varrin-Doyer et al. (2012)
AnnNeurol, 72(1):53-64, the disclosures of which are incorporated
herein by reference in their entirety).
[0116] In two separate trials, C57BL6 mice were immunized with AQP4
peptide 21-40 in complete Freund's adjuvant. On days 7, 14, and 21,
the mice were given intramuscular injections of vehicle (1xPBS,
with calcium and magnesium), empty pBHT1 vector, or
pBHT1-AQP4p21-40 tolerizing DNA vaccine (see FIG. 5 and FIG. 11 for
experimental timelines). On day 23, spleens and serum or spleens,
serum, spinal cord and optic nerve were harvested from the mice
(see FIG. 5 and FIG. 11). 72 hour T cell proliferation assays in
the presence of AQP4 p21-40 showed a statistically significant
reduction in T cell proliferation from the mice treated with
AQP4p21-40 tolerizing DNA vaccine compared to the two control
groups (FIG. 6 and FIG. 12). ELISAs performed on the cellular
supernatants showed a decrease in IL-6, IL-17, and IFN-gamma
production in the AQP4p21-40 tolerizing DNA vaccine treated mice as
compared to the two control groups (FIG. 9, FIG. 10, FIG. 13, FIG.
14 and FIG. 15). ELISA analysis of the serum indicated that
AQP4p21-40 specific IgM and IgG antibodies were also significantly
reduced in the AQP4p21-40 tolerizing DNA vaccine treated mice as
compared to the two control groups (FIG. 7 and FIG. 8).
Furthermore, Glial fibrillary acidic protein (GFAP) staining (a
marker for astrocytes) and AQP4 staining in astrocytes from AQP4
p21-40-immunized spinal cord indicates that AQP4 p21-40 DNA
vaccination results in the maintenance of astrocyte AQP4 expression
(FIG. 16).
[0117] These experiments conclusively show that pBHT1-AQP4p21-40
tolerizing DNA vaccine is effective in suppressing both T cell and
B cell responses to AQP4 p21-40 in mice.
Example 2
[0118] Human specific AQP4 tolerizing DNA vaccines are generated,
including AQP4p61-80 minigene DNA vaccine and full-length AQP4 cDNA
vaccine. Human specific AQP sequences, including AQP4p61-80 and
full-length AQP4 cDNA, are cloned into modified pBHT1 expression
vector.
[0119] Mice, optionally humanized mice (e.g., humanized-NSG mice
(e.g., CD34 humanized mice, BLT humanized mice, PBMC humanized
mice, and the like) available from The Jackson Laboratory (Bar
Harbor, Me.)), are immunized with either human AQP4 peptide or
full-length human AQP4 or a combination thereof. Following
immunization, mice are given intramuscular injections of vehicle,
empty vector, or one or more human specific AQP4 tolerizing DNA
vaccines according to a predetermined injection schedule. At a
predetermined time-point spleens and serum are harvested from the
mice. Assays are performed to evaluate immune system response in
the mice, e.g. T cell and B cell responses, IL-6, IL-7 and
IFN-gamma production, and the like. The results of the assays for
the experimental and control groups are compared to evaluate the
effect of human specific AQP4 tolerizing DNA vaccines on immune
response in the AQP4 immunized mice.
[0120] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of the present invention is embodied by the
appended claims.
Sequence CWU 1
1
71100DNAArtificial sequencesynthetic polynucleotide sequence
1actgactgaa gcttgccgcc atgagcatca tggtggcttt caaaggagtc tggactcagg
60ctttctggaa ggcagtctca gcatgagaat tcactgactg 100272DNAArtificial
sequencesynthetic polynucleotide sequence 2gagcatcatg gtggctttca
aaggagtctg gactcaggct ttctggaagg cagtctcagc 60atgagaattc tg
723323PRTHomo sapiens 3Met Ser Asp Arg Pro Thr Ala Arg Arg Trp Gly
Lys Cys Gly Pro Leu 1 5 10 15 Cys Thr Arg Glu Asn Ile Met Val Ala
Phe Lys Gly Val Trp Thr Gln 20 25 30 Ala Phe Trp Lys Ala Val Thr
Ala Glu Phe Leu Ala Met Leu Ile Phe 35 40 45 Val Leu Leu Ser Leu
Gly Ser Thr Ile Asn Trp Gly Gly Thr Glu Lys 50 55 60 Pro Leu Pro
Val Asp Met Val Leu Ile Ser Leu Cys Phe Gly Leu Ser 65 70 75 80 Ile
Ala Thr Met Val Gln Cys Phe Gly His Ile Ser Gly Gly His Ile 85 90
95 Asn Pro Ala Val Thr Val Ala Met Val Cys Thr Arg Lys Ile Ser Ile
100 105 110 Ala Lys Ser Val Phe Tyr Ile Ala Ala Gln Cys Leu Gly Ala
Ile Ile 115 120 125 Gly Ala Gly Ile Leu Tyr Leu Val Thr Pro Pro Ser
Val Val Gly Gly 130 135 140 Leu Gly Val Thr Met Val His Gly Asn Leu
Thr Ala Gly His Gly Leu 145 150 155 160 Leu Val Glu Leu Ile Ile Thr
Phe Gln Leu Val Phe Thr Ile Phe Ala 165 170 175 Ser Cys Asp Ser Lys
Arg Thr Asp Val Thr Gly Ser Ile Ala Leu Ala 180 185 190 Ile Gly Phe
Ser Val Ala Ile Gly His Leu Phe Ala Ile Asn Tyr Thr 195 200 205 Gly
Ala Ser Met Asn Pro Ala Arg Ser Phe Gly Pro Ala Val Ile Met 210 215
220 Gly Asn Trp Glu Asn His Trp Ile Tyr Trp Val Gly Pro Ile Ile Gly
225 230 235 240 Ala Val Leu Ala Gly Gly Leu Tyr Glu Tyr Val Phe Cys
Pro Asp Val 245 250 255 Glu Phe Lys Arg Arg Phe Lys Glu Ala Phe Ser
Lys Ala Ala Gln Gln 260 265 270 Thr Lys Gly Ser Tyr Met Glu Val Glu
Asp Asn Arg Ser Gln Val Glu 275 280 285 Thr Asp Asp Leu Ile Leu Lys
Pro Gly Val Val His Val Ile Asp Val 290 295 300 Asp Arg Gly Glu Glu
Lys Lys Gly Lys Asp Gln Ser Gly Glu Val Leu 305 310 315 320 Ser Ser
Val 4323PRTMus musculus 4Met Ser Asp Gly Ala Ala Ala Arg Arg Trp
Gly Lys Cys Gly His Ser 1 5 10 15 Cys Ser Arg Glu Ser Ile Met Val
Ala Phe Lys Gly Val Trp Thr Gln 20 25 30 Ala Phe Trp Lys Ala Val
Ser Ala Glu Phe Leu Ala Thr Leu Ile Phe 35 40 45 Val Leu Leu Gly
Val Gly Ser Thr Ile Asn Trp Gly Gly Ser Glu Asn 50 55 60 Pro Leu
Pro Val Asp Met Val Leu Ile Ser Leu Cys Phe Gly Leu Ser 65 70 75 80
Ile Ala Thr Met Val Gln Cys Phe Gly His Ile Ser Gly Gly His Ile 85
90 95 Asn Pro Ala Val Thr Val Ala Met Val Cys Thr Arg Lys Ile Ser
Ile 100 105 110 Ala Lys Ser Val Phe Tyr Ile Ile Ala Gln Cys Leu Gly
Ala Ile Ile 115 120 125 Gly Ala Gly Ile Leu Tyr Leu Val Thr Pro Pro
Ser Val Val Gly Gly 130 135 140 Leu Gly Val Thr Thr Val His Gly Asn
Leu Thr Ala Gly His Gly Leu 145 150 155 160 Leu Val Glu Leu Ile Ile
Thr Phe Gln Leu Val Phe Thr Ile Phe Ala 165 170 175 Ser Cys Asp Ser
Lys Arg Thr Asp Val Thr Gly Ser Ile Ala Leu Ala 180 185 190 Ile Gly
Phe Ser Val Ala Ile Gly His Leu Phe Ala Ile Asn Tyr Thr 195 200 205
Gly Ala Ser Met Asn Pro Ala Arg Ser Phe Gly Pro Ala Val Ile Met 210
215 220 Gly Asn Trp Ala Asn His Trp Ile Tyr Trp Val Gly Pro Ile Met
Gly 225 230 235 240 Ala Val Leu Ala Gly Ala Leu Tyr Glu Tyr Val Phe
Cys Pro Asp Val 245 250 255 Glu Leu Lys Arg Arg Leu Lys Glu Ala Phe
Ser Lys Ala Ala Gln Gln 260 265 270 Thr Lys Gly Ser Tyr Met Glu Val
Glu Asp Asn Arg Ser Gln Val Glu 275 280 285 Thr Glu Asp Leu Ile Leu
Lys Pro Gly Val Val His Val Ile Asp Ile 290 295 300 Asp Arg Gly Glu
Glu Lys Lys Gly Lys Asp Ser Ser Gly Glu Val Leu 305 310 315 320 Ser
Ser Val 5972DNAHomo sapiens 5atgagtgaca gacccacagc aaggcggtgg
ggtaagtgtg gacctttgtg taccagagag 60aacatcatgg tggctttcaa aggggtctgg
actcaagctt tctggaaagc agtcacagcg 120gaatttctgg ccatgcttat
ttttgttctc ctcagcctgg gatccaccat caactggggt 180ggaacagaaa
agcctttacc ggtcgacatg gttctcatct ccctttgctt tggactcagc
240attgcaacca tggtgcagtg ctttggccat atcagcggtg gccacatcaa
ccctgcagtg 300actgtggcca tggtgtgcac caggaagatc agcatcgcca
agtctgtctt ctacatcgca 360gcccagtgcc tgggggccat cattggagca
ggaatcctct atctggtcac acctcccagt 420gtggtgggag gcctgggagt
caccatggtt catggaaatc ttaccgctgg tcatggtctc 480ctggttgagt
tgataatcac atttcaattg gtgtttacta tctttgccag ctgtgattcc
540aaacggactg atgtcactgg ctcaatagct ttagcaattg gattttctgt
tgcaattgga 600catttatttg caatcaatta tactggtgcc agcatgaatc
ccgcccgatc ctttggacct 660gcagttatca tgggaaattg ggaaaaccat
tggatatatt gggttgggcc catcatagga 720gctgtcctcg ctggtggcct
ttatgagtat gtcttctgtc cagatgttga attcaaacgt 780cgttttaaag
aagccttcag caaagctgcc cagcaaacaa aaggaagcta catggaggtg
840gaggacaaca ggagtcaggt agagacggat gacctgattc taaaacctgg
agtggtgcat 900gtgattgacg ttgaccgggg agaggagaag aaggggaaag
accaatctgg agaggtattg 960tcttcagtat ga 9726972DNAMus musculus
6atgagtgaca gagctgcggc aaggcggtgg ggtaagtgtg gacattcctg cagtagagag
60agcatcatgg tggctttcaa aggagtctgg actcaggctt tctggaaggc agtctcagca
120gaatttctgg ccacgcttat ctttgttttg ctcggtgtgg gatccaccat
aaactggggt 180ggctcagaaa accccttacc tgtggacatg gtcctcatct
ccctttgctt tggactcagc 240attgctacca tggtgcagtg ctttggccac
atcagtggtg gccacatcaa tcccgctgtg 300actgtagcca tggtgtgcac
acgaaagatc agcatcgcta agtccgtctt ctacatcatt 360gcacagtgcc
tgggggccat cattggagcc ggcatcctct acctggtcac acctcccagt
420gtggttggag gattgggagt caccacggtt catggaaacc tcaccgctgg
ccatgggctc 480ctggtggagt taataatcac tttccagttg gtgttcacta
tttttgccag ctgtgattcc 540aaacgaactg atgttactgg ttcaatagct
ttagcaattg gattttccgt tgcaattgga 600catttgtttg caatcaatta
tactggagcc agcatgaatc cagctcgatc ttttggaccc 660gcagttatca
tgggaaactg ggcaaaccac tggatatatt gggttggacc aatcatgggc
720gctgtgctgg caggtgccct ttatgagtat gtcttctgtc ctgatgtgga
gctcaaacgt 780cgccttaagg aagccttcag caaagccgcg cagcagacaa
aagggagcta catggaggtg 840gaggacaacc ggagccaagt ggagacggaa
gacttgatcc tgaagcccgg agtggtgcat 900gtgattgaca ttgaccgtgg
agaagagaag aaggggaaag actcttcggg agaggtattg 960tcttccgtat ga
97272999DNAArtificial sequencesynthetic polynucleotide sequence
7gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
60atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc
cattgacgtc aatgggtgga 240ctatttacgg taaactgccc acttggcagt
acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg
gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt
cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac
caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac
gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc
tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg
actcactata gggagaccca agctggctag cgtttaaact taagcttggt
720accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca
gcacagtggc 780ggcggctcga gtctagaggg cccgtttaaa cccgctgatc
agcctcgact gtgccttcta 840gttgccagcc atctgttgtt tgcccctccc
ccgtgccttc cttgaccctg gaaggtgcca 900ctcccactgt cctttcctaa
taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 960attctattct
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
1020gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt
tatggacagc 1080aagcgaaccg gaattgccag ctggggcgcc ctctggtaag
gttgggaagc cctgcaaagt 1140aaactggatg gctttctcgc ggccaaggat
ctgatggcgc aggggatcaa gctctgatca 1200agagacagga tgaggatggt
ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1260ggcagcttgg
gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
1320tgatgccgcc gtgttcaggc tgtcagcgca ggggcgcccg gttctttttg
tcaagaccga 1380cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
cggctatcgt ggctggccac 1440gacgggcgtt ccttgcgcag ctgtgctcga
cgttgtcact gaagcgggaa gggactggct 1500gctattgggc gaagtgccgg
ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1560agtatccatc
atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
1620attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
aagccggtct 1680tgtcgatcag gatgatctgg acgaagagca tcaggggctc
gcgccagccg aactgttcgc 1740caggctcaag gcgagcatgc ccgacggcga
ggatctcgtc gtgacccatg gcgatgcctg 1800cttgccgaat atcatggtgg
aaaatggcag gttttctgga ttcatcgact gtggccggct 1860gggtgtggcg
gacaggtatc aggacatagc gttggctacc cgtgatattg ctgaagagct
1920tggcggcgaa tgggctgaca ggttcctcgt gctttacggt attgcggctc
ccgattcgca 1980gcgcattgcc ttctataggc ttcttgacga gttcttctga
attattaacg cttacaattt 2040cctgatgcgg tattttctcc ttacgcatct
gtgcggtatt tcacaccgca tacaggtggc 2100acttttcggg gaaatgtgcg
cggaacccct atttgtttat ttttctaaat acattcaaat 2160atgtatccgc
tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa
2220cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
catgaccaaa 2280atcccttaac gtgagttttc gttccactga gcgtcagacc
ccgtagaaaa gatcaaagga 2340tcttcttgag atcctttttt tctgcgcgta
atctgctgct tgcaaacaaa aaaaccaccg 2400ctaccagcgg tggtttgttt
gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460ggcttcagca
gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
2520cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
gttaccagtg 2580gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
actcaagacg atagttaccg 2640gataaggcgc agcggtcggg ctgaacgggg
ggttcgtgca cacagcccag cttggagcga 2700acgacctaca ccgaactgag
atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760gaagggagaa
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
2820agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
tcgccacctc 2880tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
ggagcctatg gaaaaacgcc 2940agcaacgcgg cctttttacg gttcctgggc
ttttgctggc cttttgctca catgttctt 2999
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