U.S. patent application number 17/667699 was filed with the patent office on 2022-09-01 for diagnosis and treatment of vitiligo.
The applicant listed for this patent is University of Massachusetts. Invention is credited to John E. Harris, Jillian M. Richmond, James Pennock Strassner.
Application Number | 20220273589 17/667699 |
Document ID | / |
Family ID | 1000006336982 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220273589 |
Kind Code |
A1 |
Richmond; Jillian M. ; et
al. |
September 1, 2022 |
Diagnosis and Treatment of Vitiligo
Abstract
Methods of diagnosing and treating vitiligo.
Inventors: |
Richmond; Jillian M.;
(Dayville, CT) ; Harris; John E.; (Sterling,
MA) ; Strassner; James Pennock; (Worcester,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
|
Family ID: |
1000006336982 |
Appl. No.: |
17/667699 |
Filed: |
February 9, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16608116 |
Oct 24, 2019 |
11278505 |
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PCT/US2018/029185 |
Apr 24, 2018 |
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17667699 |
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62489191 |
Apr 24, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/137 20130101; A61P 17/00 20180101 |
International
Class: |
A61K 31/137 20060101
A61K031/137; A61P 17/00 20060101 A61P017/00 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under Grant
Nos. AR061437 and AR069114 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1.-5. (canceled)
6. A method of treating a subject who has a vitiligo lesion, the
method comprising: identifying a subject in need of treatment; and
administering a therapeutically effective amount of an inhibitor of
Sphingosine-1-phosphate receptor 1 (S1P1) to the subject.
7. The method of claim 6, wherein the inhibitor of S1P1 is
Fingolimod.
8.-19. (canceled)
20. The method of claim 6, wherein the inhibitor is administered to
the lesion.
21. The method of claim 20, wherein the inhibitor is administered
to the epidermis within the lesion.
22. The method of claim 20, wherein the inhibitor is administered
to the epidermis within the lesion by subcutaneous or intradermal
administration, or a microneedle array.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of and claims
priority to U.S. application Ser. No. 16/608,116, filed on Oct. 24,
2019, which is a 371 of International Application No.
PCT/US2018/029185, filed on Apr. 24, 2018, which claims the benefit
of U.S. Provisional Application Ser. No. 62/489,191, filed on Apr.
24, 2017. The entire contents of the foregoing are hereby
incorporated by reference.
TECHNICAL FIELD
[0003] Described herein are methods of diagnosing and treating
vitiligo.
BACKGROUND
[0004] Vitiligo is caused by CD8+ T cells that target melanocytes
for destruction (van den Boom et al., 2009), resulting in patchy
depigmentation that is disfiguring and distressing to patients
(Alikhan et al., 2011) Depigmentation typically recurs rapidly at
the same location after therapy is stopped (Cavalie et al., 2015),
indicating that autoimmune memory persists in the skin and permits
disease reactivation after cessation of treatment. It affects
approximately 1% of the population worldwide, yet there are
currently no FDA approved treatments.
SUMMARY
[0005] Skin lesions in vitiligo are maintained over time, resisting
conventional treatments by returning after they are discontinued.
Tissue resident memory T cells (Trm) provide rapid, localized
protection against reinfection from skin and mucosal-tropic
viruses. A similar memory response is observed in autoimmune
diseases as rapid relapse occurs at sites of improvement after
discontinuing therapy. As shown herein, antigen-specific Trm are
present in both mouse and human vitiligo skin lesions, an
autoimmune disease mediated by CD8+ T cells that kill melanocytes
and create white spots. Functional analysis of Trm cells indicated
that they sense autoantigen in the skin and secrete alarm signals
to recruit recirculating T cells to kill melanocytes. In addition,
Trm are not sufficient for autoimmunity: treatment with the
Sphingosine-1-phosphate 1 (S1P1) inhibitor FTY720 (fingolimod)
resulted in rapid repigmentation, yet preserved Trm in the skin
while preventing recirculating memory T cell recruitment. However,
treatment with an IL-15R.beta. blocking antibody effectively
depleted autoreactive Trm, inhibited their function, and resulted
in durable reversal of disease in mice. Based on these data and
clinical observations, depleting Trm or inhibiting their function
is a highly effective and durable treatment for vitiligo and other
autoimmune diseases.
[0006] Thus, provided herein are methods for treating a subject who
has a vitiligo lesion. The methods include identifying a subject in
need of treatment; and administering a therapeutically effective
amount of an inhibitor of Sphingosine-1-phosphate receptor 1
(S1P1), Interleukin 15 (IL-15) or the IL-15 receptor to the lesion,
and the use of inhibitors of S1P1, IL-15 or the IL-15 receptor to
treat vitiligo. In some embodiments, the inhibitor is a small
molecule inhibitor, an antibody, a peptide inhibitor, or an
inhibitory nucleic acid targeting S1P1, IL-15 or the IL-15
receptor.
[0007] In some embodiments, the inhibitor is administered to the
epidermis within the lesion, e.g., by subcutaneous or intradermal
administration, or a microneedle array.
[0008] In some embodiments, the inhibitor is administered
systemically, e.g., orally or parenterally.
[0009] Also provided herein are devices for the delivery of S1P1 or
IL-15 inhibitor to a subject with vitiligo, e.g., a microneedle
array comprising an inhibitor of S1P1, IL-15 or the IL-15
receptor.
[0010] Further, provided herein are methods for determining a score
for a subject who has vitiligo. The methods include obtaining a
sample comprising lesional interstitial skin fluid and epidermis
from a subject; evaluating CD8+ T cell infiltrate, e.g., by flow
cytometry, and elevated chemokine protein, e.g., by ELISA, in
lesional fluid, and levels of a panel of 20 genes (shown in Table
A) in the lesional skin; and using the levels to calculate a
score.
[0011] In some embodiments, the methods include comparing the score
to a reference score, and selecting a subject who has a score above
the reference score. In some embodiments, the reference score is a
control reference score that represents a normal score in an
unaffected subject, or a disease reference that represents a score
in a subject with active IFN-.gamma.-induced inflammation.
[0012] In some embodiments, the methods include selecting and
optionally administering to the subject a treatment for vitiligo,
e.g., a treatment that includes administering a therapeutically
effective amount of an inhibitor of S1P1, IL-15 or the IL-15
receptor to a vitiligo lesion. In some embodiments, the treatment
includes one or more of topical or systemic treatment with a
corticosteroid, topical treatment with an immunomodulatory agent,
topical treatment with calcipotriol, dexamethasone, phototherapy,
or targeted laser therapy.
[0013] Unless otherwise defined, 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. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0014] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0015] FIGS. 1A-1F. Melanocyte-specific CD8+ Trm cells are present
in vitiligo patient skin and express the CD122 chain of the IL-15
receptor, whereas keratinocytes express the CD215 chain. (A) Sample
photo of a vitiligo skin blister biopsy. (B) Flow cytometry
staining of blister fluid in a vitiligo patient pregated on live
single CD45+CD8+ T cells. MART-1, gp100, and tyrosinase pentamers
were pooled to identify melanocyte antigen reactive cells, and Trm
phenotype markers were assessed using CD69 and CD103 antibodies.
(C) Quantification of the frequency of melanocyte-specific cells
with a Trm phenotype in PBMCs, lesional, and nonlesional skin.
Melanocyte-specific Trm were significantly enriched in lesional
skin. (Each symbol represents one patient; open squares were
stained with MART-1 pentamer alone, closed circles were stained
with all three pentamers MART-1, gp100, and tyrosinase; one-way
ANOVA with Tukey's post tests significant as indicated). (D) Flow
cytometry staining of CD122 on melanocyte-specific Trm, and
quantification in lesional and nonlesional skin. Cells were
pregated on live single CD45+CD8+pentamer+ T cells. There was no
significant difference in CD122 expression on melanocyte-specific
Trm in lesional versus nonlesional skin. (E) Flow cytometry
staining of CD215 on keratinocytes from blister roofs, and
quantification in lesional and nonlesional skin. Cells were
pregated on live single CD45- cells. CD215 expression was higher in
lesional than nonlesional skin (Student's t tests significant as
indicated). (F) Only 1 patient's T cells were found to express
CD215.
[0016] FIGS. 2A-2G Only melanocyte-specific T cells form Trm in the
epidermis. (A) Schematic of the mouse model of vitiligo. (B)
Epidermal PMEL from vitiligo mice are CD69+CD103+. (C) Mice were
given VV-OVA and OT-1 T cells to compare another model antigen to
(D) the PMEL/vitiligo system. (E) Mice that received OVA-specific
cells and virus did not develop vitiligo. (F) OT-1s and PMEL
engrafted equally in the draining lymph nodes. (G) Only PMEL
established long-lived epidermal populations. Each dot represents
one animal; representative experiment of two shown.
[0017] FIGS. 3A-3D. Characterization of PMEL Trm in the vitiligo
mouse model reveals they are long-lived and produce IFN.gamma.. (A)
Sample flow plots pre-gated on live single epidermal PMEL at the
indicated times. (B) Quantification of epidermal PMEL numbers and
frequency of CD69+CD103+ PMEL over time in the vitiligo model. (C)
Frequency of epidermal PMEL producing IFN.gamma. in the model, and
mouse disease scores over time. (D) Representative confocal image
of ear skin reveals sparsely populated PMEL that produce IFN.gamma.
(week 10; 10.times. z stack maxima image; auto-fluorescent hair
visible as green lines in GFP channel; white arrows indicate
IFN.gamma.-GFP+ PMEL, yellow arrows indicate IFN.gamma.-GFP- PMEL;
scale bar 25 .mu.m). (n=2-8 mice per timepoint pooled from 4
separate experiments)
[0018] FIGS. 4A-4G FTY720 treatment reverses disease in mice with
established vitiligo. (A) Timing of treatments in the
repigmentation model. (B) Sample photos of vehicle control and
FTY720 treated animals at baseline and week 4. Percent of tail
pigmentation before and after treatment in (C) control and (D)
FTY720 treated animals. (E) Comparison of the final percent change
in pigmentation in PBS and FTY720 treated animals. (F) Total number
and (G) frequency of CD69+CD103+ epidermal PMEL in treated animals.
(each dot represents one animal pooled from 3 separate
experiments)
[0019] FIG. 5. Expression of IL-15Ra on PMEL Trm. Mouse epidermal
PMEL were pre-gated on live single CD69+CD103+ and then assessed
for IL-15R.alpha. (CD215, top) and IL-15R.beta. (CD122, bottom)
expression.
[0020] FIGS. 6A-6I. Treatment with IL-154 Ab durably reverses
disease in mice with established vitiligo. (A) Timing of treatments
in the efficacy/repigmentation model. (B) Sample photos of vehicle
control (PBS or isotype) and IL-15R.beta. Ab treated animals at
baseline and week 8. (C) Comparison of the final percent change in
pigmentation in vehicle and IL-15R.beta. Ab treated animals. (D)
Quantification of PMEL numbers in treated animals in the indicated
tissues. (E) Quantification of host CD8+ T cell numbers in treated
animals in the indicated tissues. (each dot represents one animal
pooled from 2 separate experiments; T tests significant as
indicated). (F) Timing of treatments in the durability study. (G)
Sample photos of vehicle control (PBS or isotype) and IL-15R.beta.
Ab treated animals at baseline and week 8. (H) Comparison of the
final percent change in pigmentation in vehicle and IL-15R.beta. Ab
treated animals (T test significant as indicated). (I) Analysis of
the percent of tail with pigmentation over time (two-way ANOVA
p=0.0015 for treatment, p<0.0001 for time, and ns for
interaction with Dunnett's comparisons to baseline pigmentation
significant as indicated by stars). (Each dot represents one animal
pooled from 3 separate experiments)
[0021] FIGS. 7A-7C. Short-term treatment with IL-15R.beta. Ab
reduces IFN.gamma. secretion by PMEL. (A) Timing of treatments in
the systemic functionality study. (B) Sample flow plots of
epidermal PMEL from vehicle or CD122 antibody treated mice showing
granzyme B and IFN.gamma. production. (C) Frequency of IFN.gamma.+
PMELs from the lymph nodes, epidermis, and dermis of mice in the
systemic functionality study (each dot represents one animal pooled
from 4 separate experiments; T tests significant as indicated).
[0022] FIGS. 8A-8E. Local intradermal injection of CD122 antibody
treatment durably reverses disease in mice. (A) Timing of
treatments in the durability study. (B) Sample photos of vehicle
control (PBS or isotype) and CD122 antibody treated animals at
treatment baseline and week 12. (C) Comparison of the final percent
change in pigmentation in control and CD122 antibody treated
animals (Each dot represents one animal pooled from 2 separate
experiments, n=7 control and 9 CD122 antibody treated mice; T test
significant as indicated). (D) Analysis of the percent of tail with
pigmentation over time (two-way ANOVA ns for treatment and time,
p=0.0017 for interaction with Dunnett's comparisons to baseline
pigmentation with simple effects within treatment groups
significant as indicated by stars, baseline versus week 10
p=0.0197, and baseline versus week 12 p=0.0002). (E) Quantification
of PMEL numbers in treated animals within the indicated tissues.
(Each symbol represents 1 animal pooled from 2 separate
experiments; all t tests ns).
[0023] FIGS. 9A-9F. Skin blistering set up and tissue analysis. (A)
Clinical setup, subject during blistering procedure. (B) Vitiligo
confetti-like depigmentation on foot. (C) Placement of orifice
plate for negative pressure chamber. (D,E) Formed blisters on (D)
lesional skin and (E) non-lesional skin. (F) Blister tissue
analysis: blister fluid (skin interstitial fluid) was withdrawn
through a needle and analyzed by flow cytometry and ELISA. Blister
roof (epidermis) was processed for RNA extraction for gene
expression analysis.
[0024] FIGS. 10A-10C. Blister fluid CD8+ T cells are elevated in
lesional skin. Blister fluid from lesional, non-lesional, and
healthy control skin was analyzed by flow cytometry for CD8+ T
cells and normalized to CD45+ cells. (A) Normalized CD8+ T cells
from multiple blisters in 8 patients and 7 controls. (B) Paired
analysis in lesional vs. non-lesional skin. (C) ROC curve comparing
normalized CD8+ T cells in lesional to non-lesional blisters and
healthy controls, which reveals decent sensitivity and specificity
of this assay to detect active lesional skin
[0025] FIGS. 11A-11C. Blister fluid CXCL9 elevated in lesional
skin. Blister fluid from lesional, non-lesional, and healthy
control skin was analyzed by ELISA for CXCL9. (ACXCL9 from multiple
blisters in 7 patients and 6 controls. (B) Paired analysis of CXCL9
average concentrations in lesional vs. non-lesional skin. (C) ROC
curve comparing lesional blisters to non-lesional and healthy
control blisters, which reveals good sensitivity and specificity of
this assay to detect active lesional skin.
[0026] FIGS. 12A-12B. Using a panel of vitiligo-specific genes to
understand disease activity. Whole genome expression was analyzed
in vitiligo lesions compared to healthy control skin. Principal
component analysis (12A) of a panel of 20 differentially expressed
genes (12B) reflect the presence of CD8+ T cells, IFN-.gamma.
signaling, and absence of melanocyte markers.
DETAILED DESCRIPTION
[0027] Immune memory is mediated by long-lived, antigen-experienced
lymphocytes that protect against reinfection. Recent studies have
defined CD8+ resident memory T cells (Trm) that remain in
non-lymphoid tissues to provide tissue surveillance against
pathogens (Gebhardt et al., 2009; Zhu et al., 2013). These Trm are
phenotypically distinct from other memory T cell populations in
that they express specific developmental markers that bolster their
function (Mackay et al., 2013; Skon et al., 2013). Upon entering
tissues such as the mucosa and skin epidermis, differentiating Trm
upregulate CD69 and CD103, downregulate the chemokine receptors
S1P1 and CCR7 to prevent recirculation, and set up residence. Most
studies have focused on anti-viral Trm responses, with the goal of
enhancing their function for vaccine development. Two key questions
that remain are: (1) what roles do Trm play in autoimmunity? and
(2) can Trm responses be targeted as a strategy to treat
autoimmunity?
[0028] To answer these questions, the present inventors sought to
define autoreactive Trm using vitiligo as a model autoimmune
disease because of its dependence on CD8+ T cells, known
autoantigens, and accessibility of target tissue. As shown herein,
lesional skin biopsies from patients contained antigen-specific
CD8+ Trm, supporting a role for these cells in human vitiligo.
[0029] A mouse model of vitiligo was developed through the adoptive
transfer of TCR transgenic T cells recognizing the human melanocyte
antigen pre-melanosome protein (Harris et al., 2012). These T
cells, called PMEL, target mouse melanocytes and induce patchy
epidermal depigmentation that mirrors human disease (Rashighi et
al., 2014). Using this model, autoreactive Trm within the skin were
characterized and their role in disease determined. The present
data indicate that once established, melanocyte-specific Trm remain
activated in skin in the absence of inflammation, and serve an
alarm/recruit function to attract recirculating memory T cells
capable of killing the target cells.
[0030] The role of Trm has been reported to vary in the context of
different skin and mucosal viral infections. In one study, Trm
alone were able to provide rapid immunity to reinfection with
vaccinia virus (Jiang et al., 2012) while others have shown that
Trm send out an alarm signal to recruit recirculating memory T
cells, which provided the key effector functions against herpes
virus (Schenkel et al., 2013) and other viruses (Ariotti, 2014).
Another study demonstrated that the recirculating memory T cells
alone are unable to provide efficient responses to reinfection with
herpes (Mackay et al., 2012), indicating that a complex interplay
between Trm and recirculating memory T cells is extant. Cooperation
of Trm with other recruited T cell populations has also been
indicated in cutaneous T cell lymphomas (Watanabe, 2015). The
present data support the role of autoreactive Trm as sentinel/alarm
cells that work together with recirculating memory cell populations
to maintain depigmentation during vitiligo.
[0031] IL-15R.beta. is a shared receptor for both IL-2 and IL-15,
and so a role for IL-2 in the maintenance of Trm is possible.
However, others have shown that IL-15 is much more potent at
generating CD8+ Trm pools than residual IL-2 signaling in skin
tissue (Adachi et al., 2015), and IL-15 is required for the
generation of CD8+ Trm in viral models in mice (Mackay et al.,
2013). Further, CD8+ T cells often express IL-15R.beta. without
CD25 (Zhang et al., 1998). Mice lacking IL-2 or CD25 develop
autoimmunity, whereas mice lacking IL-15 are protected from
autoimmunity (reviewed in ((Nelson, 2004))). Another recent study
found that IL-15R.beta. antibody administration in rhesus macaques
greatly reduced tissue effector memory T cell populations, while
recirculating populations bounced back (DeGottardi et al., 2016).
As shown herein, targeting IL-15 signaling through antibody
treatment even after the generation of Trm in the tissue was
effective at clearing these cells from the epidermis, indicating
that IL-15 signaling is required for maintenance of these
cells.
[0032] The present data support targeting this cytokine as a
strategy to clear autoreactive memory cells from the tissue,
resulting in a long-lasting, durable response to treatment. This is
in contrast to existing therapies for inflammation in skin and
other tissues, which result in rapid relapse after they are
discontinued. Further, targeting IL-15 preferentially affects
autoreactive T cells while leaving most endogenous T cell
populations intact.
Methods of Treatment
[0033] The methods described herein include methods for the
treatment of disorders associated with T cell-mediated
autoimmunity. In some embodiments, the disorder is vitiligo.
Vitiligo is a chronic, progressive autoimmune skin disease
characterized by white or depigmented patches of skin, most
commonly on the hands, forearms, feet and face. Depigmentation
results from destruction of melanocytes by T cells. This disease
affects 1% of the world population and can cause psychological
distress to affected patients. There are several clinical
presentations of active disease, including: trichrome vitiligo,
inflammatory vitiligo, confetti vitiligo, and others; Koebner
phenomenon is the development of vitiligo at the site of trauma to
the skin. Vitiligo can be further classified as localized,
generalized, or universal, depending on the distribution and extent
of depigmentation (Yaghoobi et al., 2011). Diagnosis is typically
made based on clinical findings, occasionally aided by histological
findings from skin biopsies at the sites of lesions, including
abnormalities in keratinocytes, melanocytes, and Langerhans cells;
epidermal vacuolization; and thickening of the basement membrane
(Moellmann et al., 1982). Alternatively a diagnosis can be made,
and/or a subject selected, using the Vitiligo Disease Activity
Score (VDAS), Vitiligo Area Scoring Index (VAST), or the Vitiligo
European Task Force (VETF) assessment. The VDAS is estimated by
asking the patient when they last saw a new vitiligo lesion appear,
within the last 6 weeks (4 points), 6-12 weeks (3 points), 3-6
months (2 points), 6-12 months (1 point), stable for over 1 year (0
points), or repigmenting (-1 point) (Bhatnagar et al., 2007). In
some embodiments, the subject can be one who has trichrome
(3-color) or confetti-like (multiple 1-2 mm macules) appearance of
depigmented macules in vitiligo lesions, or who has a VDAS score of
2-4. There is no known cure for vitiligo, but a variety of medical
and surgical interventions are available to improve the appearance
of the lesions. Medical interventions include topical or systemic
treatment with corticosteroids such as prednisone or clobetasol,
topical treatment with immunomodulatory agents such as tacrolimus,
topical treatment with the vitamin D3 analog calcipotriol,
pulse-dose therapy with dexamethasone (e.g., 4 mg), phototherapy
with UV light (nbUVB), targeted laser therapy on stable patches of
localized vitiligo, tattooing the depigmented skin, and
depigmentation of unaffected skin in cases where vitiligo is
widespread and repigmentation therapy is ineffective (Grimes, 2005)
(Lotti et al., 2008). Surgical interventions include
suction-blister grafts, punch grafts, autologous melanocyte
cultures, cultured epidermal suspensions, noncultured epidermal
suspensions, epidermal blister grafts, and split thickness grafts
(Grimes, 2005).
[0034] In some embodiments, the disorder to be treated is another
autoimmune disease, such as lupus, psoriasis, alopecia areata,
lichen planus, scleroderma, graft vs host disease, diabetes,
autoimmune thyroid disease, multiple sclerosis, or autoimmune
uveitis.
[0035] Generally, the methods include systemic or (more preferably)
local delivery of a therapeutically effective amount of an
inhibitor of IL-15, the IL-15 receptor, a Spingosine-1-receptor
inhibitor, and/or the downstream signaling molecules JAK1 and 3 as
described herein, to a subject who is in need of, or who has been
determined to be in need of, such treatment. As used in this
context, to "treat" means to ameliorate at least one symptom of the
disorder associated with autoimmunity. Vitiligo is characterized by
depigmentation of the skin; thus, a treatment can result in a
reduction in appearance of lesions and a return or approach to
normal pigmentation. Administration of a therapeutically effective
amount of a compound described herein for the treatment of vitiligo
will result in decreased depigmentation.
Inhibitors of IL-15 and the IL-15 Receptor
[0036] The methods and compositions described herein can include
the use of inhibitors of IL-15 and the IL-15 receptor. In some
embodiments, the therapeutic molecule to be administered comprises
a small molecule inhibitor of the IL-15 receptor alpha chain, e.g.,
benzoic acid derivatives as described in WO2014191822 A1 and amine
derivatives as described in WO2014191823 A1. In some embodiments,
the therapeutic molecule to be administered comprises a small
molecule inhibitor of IL-15-induced cellular responses, e.g.,
cefazolin as described in US20160235762 A1 and Y-320 (Ushio, 2008).
In some embodiments, the therapeutic molecule to be administered
comprises a soluble IL-15 receptor alpha chain which binds IL-15
and antagonizes the IL-15 receptor (Ruchatz et al., 1998). In some
embodiments, the therapeutic molecule to be administered comprises
an antagonistic mutant IL-15/Fc-gamma 2a fusion protein which
competitively inhibits binding of IL-15 to the IL-15 receptor (Kim
et al., 1998). In some embodiments, the therapeutic molecule to be
administered comprises a peptide that binds the IL-15 receptor
alpha chain and inhibits IL-15 activity, as described in U.S. Pat.
No. 7,736,638 B2. In some embodiments, the therapeutic molecule to
be administered comprises an antibody which specifically binds the
IL-2/IL-15 receptor beta subunit, e.g. as described in U.S. Pat.
No. 9,028,830B2. A number of inhibitors of CD122 (also known as
IL-2R13 and p'75) are known in the art, including antibodies (see,
e.g., U.S. Pat. No. 9,028,830) and small molecules (e.g.,
Ro26-4550, SP4206, ABT-737, Nutlin-2, and Compound 3; see, e.g.,
Wilson and Arkin, Curr Top Microbiol Immunol. 2011; 348:25-59; Laio
et al., Immunity. 2013 Jan. 24; 38(1): 13-25. In some embodiments,
the therapeutic molecule to be administered comprises an antibody
that specifically binds the IL-2/IL-15 receptor gamma subunit
(Hechinger et al., 2015). In some embodiments, the therapeutic
molecule to be administered comprises an antibody that specifically
binds the IL-15 receptor alpha subunit (Epron et al., 2012). In
some embodiments, the therapeutic molecule to be administered
comprises an antibody that specifically binds IL-15, e.g., as
described in WO03017935.
Inhibitors of Jak1 and Jak3
[0037] The methods and compositions described herein can include
inhibitors of Jak1 and Jak3. In some embodiments, the therapeutic
molecule to be administered comprises a small molecule inhibitor of
Jak1 and/or Jak3, for example Tofacitinib, Ruxolitinib,
Perficitinib, Baricitinib, Decernotinib, R348, AT9283, Oclacitinib,
Momelotinib, WHI-P154, ZM 39923 HCl, Filgotinib, Cerdulatinib, or
Upadacitinib. In some embodiments, the therapeutic molecule to be
administered comprises an antibody that binds Jak1 and/or Jak3.
(Craiglow and King, 2015; Harris et al., 2016) (Damsky and King,
2017).
Inhibitors of SIP1
[0038] Sphingosine-1-phosphate receptor inhibitors are
immunomodulators that include Myriocin, Fingolimod, Ozanimod,
Ponesimod, and Laquinimod. See Park et al., Biomolecules &
Therapeutics. 25 (1): 80-90.
Inhibitory Nucleic Acids Targeting IL-15, IL-15 Receptor Subunits,
Jak1 or Jak3
[0039] The methods and compositions described herein can include
nucleic acids that target (specifically binds, or are complementary
to) S1P1, IL-15, IL-15 receptor alpha subunit, IL-15 receptor beta
subunit (CD122), IL-15 gamma subunit, Jak1, or Jak3 mRNAs.
Inhibitory nucleic acids useful in the present methods and
compositions include antisense oligonucleotides, small interfering
RNA (siRNA), small hairpin RNA (shRNA), molecules comprising
modified bases, locked nucleic acid molecules (LNA molecules),
peptide nucleic acid molecules (PNA molecules), and other
oligomeric compounds or oligonucleotide mimetics that hybridize to
at least a portion of the target nucleic acid and inhibit its
function.
[0040] In some embodiments, the inhibitory nucleic acids are 7 to
50, 7 to 20, 7 to 25, 10 or 13 to 50, or 10 or 13 to 30 nucleotides
(nts) in length, or as short as 7 or 8 up to the entire length of
the target sequence (e.g., 7 to 22, 7 to 77, or 7 to 84 nts for SEQ
ID NOs. 1, 2, and 3 respectively). One having ordinary skill in the
art will appreciate that this embodies inhibitory nucleic acids
having complementary portions of 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,
or 50 or more nucleotides in length, or any range therewithin. In
some embodiments, the inhibitory nucleic acids are 15 nucleotides
in length. In some embodiments, the inhibitory nucleic acids are 12
or 13 to 20, 25, or 30 nucleotides in length. One having ordinary
skill in the art will appreciate that this embodies inhibitory
nucleic acids having complementary portions of 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
nucleotides in length, or any range therewithin (complementary
portions refers to those portions of the inhibitory nucleic acids
that are complementary to the target sequence).
[0041] The inhibitory nucleic acids useful in the present methods
are sufficiently complementary to the target RNA, i.e., hybridize
sufficiently well and with sufficient specificity, to give the
desired effect. "Complementary" refers to the capacity for pairing,
through hydrogen bonding, between two sequences comprising
naturally or non-naturally occurring bases or analogs thereof. For
example, if a base at one position of an inhibitory nucleic acid is
capable of hydrogen bonding with a base at the corresponding
position of a RNA, then the bases are considered to be
complementary to each other at that position. 100% complementarity
is not required.
[0042] Routine methods can be used to design an inhibitory nucleic
acid that binds to the target sequence with sufficient specificity.
In some embodiments, the methods include using bioinformatics
methods known in the art to identify regions of secondary
structure, e.g., one, two, or more stem-loop structures, or
pseudoknots, and selecting those regions to target with an
inhibitory nucleic acid. For example, "gene walk" methods can be
used to optimize the inhibitory activity of the nucleic acid; for
example, a series of oligonucleotides of 10-30 nucleotides spanning
the length of a target RNA can be prepared, followed by testing for
activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can
be left between the target sequences to reduce the number of
oligonucleotides synthesized and tested. GC content is preferably
between about 30 60%. Contiguous runs of three or more Gs or Cs
should be avoided where possible (for example, it may not be
possible with very short (e.g., about 9-10 nt)
oligonucleotides).
[0043] In some embodiments, the inhibitory nucleic acid molecules
can be designed to target a specific region of the RNA sequence.
For example, a specific functional region can be targeted, e.g., a
region comprising a known RNA localization motif (i.e., a region
complementary to the target nucleic acid on which the RNA acts).
Alternatively or in addition, highly conserved regions can be
targeted, e.g., regions identified by aligning sequences from
disparate species such as primate (e.g., human) and rodent (e.g.,
mouse) and looking for regions with high degrees of identity.
Percent identity can be determined routinely using basic local
alignment search tools (BLAST programs) (Altschul et al., 1990;
Zhang and Madden, 1997), e.g., using the default parameters.
[0044] Once one or more target regions, segments or sites have been
identified, e.g., within a sequence known in the art or provided
herein, inhibitory nucleic acid compounds are chosen that are
sufficiently complementary to the target, i.e., that hybridize
sufficiently well and with sufficient specificity (i.e., do not
substantially bind to other non-target RNAs), to give the desired
effect.
[0045] In the context of this invention, hybridization means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases that pair through the formation of
hydrogen bonds. Complementary, as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a RNA molecule, then the inhibitory nucleic acid and the RNA are
considered to be complementary to each other at that position. The
inhibitory nucleic acids and the RNA are complementary to each
other when a sufficient number of corresponding positions in each
molecule are occupied by nucleotides that can hydrogen bond with
each other. Thus, "specifically hybridisable" and "complementary"
are terms which are used to indicate a sufficient degree of
complementarity or precise pairing such that stable and specific
binding occurs between the inhibitory nucleic acid and the RNA
target. For example, if a base at one position of an inhibitory
nucleic acid is capable of hydrogen bonding with a base at the
corresponding position of an RNA molecule, then the bases are
considered to be complementary to each other at that position. 100%
complementarity is not required.
[0046] It is understood in the art that a complementary nucleic
acid sequence need not be 100% complementary to that of its target
nucleic acid to be specifically hybridisable. A complementary
nucleic acid sequence for purposes of the present methods is
specifically hybridisable when binding of the sequence to the
target RNA molecule interferes with the normal function of the
target RNA to cause a loss of activity, and there is a sufficient
degree of complementarity to avoid non-specific binding of the
sequence to non-target RNA sequences under conditions in which
specific binding is desired, e.g., under physiological conditions
in the case of in vivo assays or therapeutic treatment, and in the
case of in vitro assays, under conditions in which the assays are
performed under suitable conditions of stringency. For example,
stringent salt concentration will ordinarily be less than about 750
mM NaCl and 75 mM trisodium citrate, preferably less than about 500
mM NaCl and 50 mM trisodium citrate, and more preferably less than
about 250 mM NaCl and 25 mM trisodium citrate. Low stringency
hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while high stringency hybridization can be
obtained in the presence of at least about 35% formamide, and more
preferably at least about 50% formamide. Stringent temperature
conditions will ordinarily include temperatures of at least about
30.degree. C., more preferably of at least about 37.degree. C., and
most preferably of at least about 42.degree. C. Varying additional
parameters, such as hybridization time, the concentration of
detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or
exclusion of carrier DNA, are well known to those skilled in the
art. Various levels of stringency are accomplished by combining
these various conditions as needed. In a preferred embodiment,
hybridization will occur at 30.degree. C. in 750 mM NaCl, 75 mM
trisodium citrate, and 1% SDS. In a more preferred embodiment,
hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM
trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml
denatured salmon sperm DNA (ssDNA). In a most preferred embodiment,
hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM
trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA.
Useful variations on these conditions will be readily apparent to
those skilled in the art.
[0047] For most applications, washing steps that follow
hybridization will also vary in stringency. Wash stringency
conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt
concentration or by increasing temperature. For example, stringent
salt concentration for the wash steps will preferably be less than
about 30 mM NaCl and 3 mM trisodium citrate, and most preferably
less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent
temperature conditions for the wash steps will ordinarily include a
temperature of at least about 25.degree. C., more preferably of at
least about 42.degree. C., and even more preferably of at least
about 68.degree. C. In a preferred embodiment, wash steps will
occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and
0.1% SDS. In a more preferred embodiment, wash steps will occur at
42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%
SDS. In a more preferred embodiment, wash steps will occur at
68.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%
SDS. Additional variations on these conditions will be readily
apparent to those skilled in the art. Hybridization techniques are
well known to those skilled in the art and are described (Benton
and Davis, 1977; Grunstein and Hogness, 1975), (Ausubel et al.
Current Protocols in Molecular Biology, Wiley Interscience, New
York, 2001); (Berger and Kimmel, Guide to Molecular Cloning
Techniques, 1987, Academic Press, New York; and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York).
[0048] In general, the inhibitory nucleic acids useful in the
methods described herein have at least 80% sequence complementarity
to a target region within the target nucleic acid, e.g., 90%, 95%,
or 100% sequence complementarity to the target region within an
RNA. For example, an antisense compound in which 18 of 20
nucleobases of the antisense oligonucleotide are complementary, and
would therefore specifically hybridize, to a target region would
represent 90 percent complementarity. Percent complementarity of an
inhibitory nucleic acid with a region of a target nucleic acid can
be determined routinely using basic local alignment search tools
(BLAST programs) (Altschul et al., 1990; Zhang and Madden, 1997).
Inhibitory nucleic acids that hybridize to an RNA can be identified
through routine experimentation. In general the inhibitory nucleic
acids must retain specificity for their target, i.e., must not
directly bind to, or directly significantly affect expression
levels of, transcripts other than the intended target.
[0049] For further disclosure regarding inhibitory nucleic acids,
please see US2010/0317718 (antisense oligos); US2010/0249052
(double-stranded ribonucleic acid (dsRNA)); US2009/0181914 and
US2010/0234451 (LNAs); US2007/0191294 (siRNA analogues);
US2008/0249039 (modified siRNA); and WO2010/129746 and
WO2010/040112 (inhibitory nucleic acids), as well as WO2012/065143,
WO2012/087983, and WO2014/025887 (inhibitory nucleic acids
targeting non-coding RNAs/supRNAss), all of which are incorporated
herein by reference in their entirety.
siRNA/shRNA
[0050] In some embodiments, the nucleic acid sequence that is
complementary to a target RNA can be an interfering RNA, including
but not limited to a small interfering RNA ("siRNA") or a small
hairpin RNA ("shRNA"). Methods for constructing interfering RNAs
are well known in the art. For example, the interfering RNA can be
assembled from two separate oligonucleotides, where one strand is
the sense strand and the other is the antisense strand, wherein the
antisense and sense strands are self-complementary (i.e., each
strand comprises nucleotide sequence that is complementary to
nucleotide sequence in the other strand; such as where the
antisense strand and sense strand form a duplex or double stranded
structure); the antisense strand comprises nucleotide sequence that
is complementary to a nucleotide sequence in a target nucleic acid
molecule or a portion thereof (i.e., an undesired gene) and the
sense strand comprises nucleotide sequence corresponding to the
target nucleic acid sequence or a portion thereof. Alternatively,
interfering RNA is assembled from a single oligonucleotide, where
the self-complementary sense and antisense regions are linked by
means of nucleic acid based or non-nucleic acid-based linker(s).
The interfering RNA can be a polynucleotide with a duplex,
asymmetric duplex, hairpin or asymmetric hairpin secondary
structure, having self-complementary sense and antisense regions,
wherein the antisense region comprises a nucleotide sequence that
is complementary to nucleotide sequence in a separate target
nucleic acid molecule or a portion thereof and the sense region
having nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The interfering can be a circular
single-stranded polynucleotide having two or more loop structures
and a stem comprising self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof, and wherein the circular
polynucleotide can be processed either in vivo or in vitro to
generate an active siRNA molecule capable of mediating RNA
interference.
[0051] In some embodiments, the interfering RNA coding region
encodes a self-complementary RNA molecule having a sense region, an
antisense region and a loop region. Such an RNA molecule when
expressed desirably forms a "hairpin" structure, and is referred to
herein as an "shRNA." The loop region is generally between about 2
and about 10 nucleotides in length. In some embodiments, the loop
region is from about 6 to about 9 nucleotides in length. In some
embodiments, the sense region and the antisense region are between
about 15 and about 20 nucleotides in length. Following
post-transcriptional processing, the small hairpin RNA is converted
into a siRNA by a cleavage event mediated by the enzyme Dicer,
which is a member of the RNase III family. The siRNA is then
capable of inhibiting the expression of a gene with which it shares
homology (Brummelkamp et al., 2002; Lee et al., 2002; Miyagishi and
Taira, 2002; Paddison et al., 2002; Paul et al., 2002; Sui et al.,
2002; Yu et al., 2002).
[0052] The target RNA cleavage reaction guided by siRNAs is highly
sequence specific. In general, siRNA containing a nucleotide
sequences identical to a portion of the target nucleic acid are
preferred for inhibition. However, 100% sequence identity between
the siRNA and the target gene is not required to practice the
present invention. Thus the invention has the advantage of being
able to tolerate sequence variations that might be expected due to
genetic mutation, strain polymorphism, or evolutionary divergence.
For example, siRNA sequences with insertions, deletions, and single
point mutations relative to the target sequence have also been
found to be effective for inhibition. Alternatively, siRNA
sequences with nucleotide analog substitutions or insertions can be
effective for inhibition. In general the siRNAs must retain
specificity for their target, i.e., must not directly bind to, or
directly significantly affect expression levels of, transcripts
other than the intended target.
Ribozymes
[0053] Trans-cleaving enzymatic nucleic acid molecules can also be
used; they have shown promise as therapeutic agents for human
disease (Christoffersen and Marr, 1995) (Usman & McSwiggen,
1995 Ann. Rep. Med. Chem. 30, 285-294). Enzymatic nucleic acid
molecules can be designed to cleave specific RNA targets within the
background of cellular RNA. Such a cleavage event renders the RNA
non-functional.
[0054] In general, enzymatic nucleic acids with RNA cleaving
activity act by first binding to a target RNA. Such binding occurs
through the target binding portion of a enzymatic nucleic acid
which is held in close proximity to an enzymatic portion of the
molecule that acts to cleave the target RNA. Thus, the enzymatic
nucleic acid first recognizes and then binds a target RNA through
complementary base pairing, and once bound to the correct site,
acts enzymatically to cut the target RNA. Strategic cleavage of
such a target RNA will destroy its ability to direct synthesis of
an encoded protein. After an enzymatic nucleic acid has bound and
cleaved its RNA target, it is released from that RNA to search for
another target and can repeatedly bind and cleave new targets.
[0055] Several approaches such as in vitro selection (evolution)
strategies (Orgel, 1979) have been used to evolve new nucleic acid
catalysts capable of catalyzing a variety of reactions, such as
cleavage and ligation of phosphodiester linkages and amide linkages
(Bartel and Szostak, 1993; Beaudry and Joyce, 1992; Breaker, 1996;
Breaker and Joyce, 1994; Joyce, 1989, 1992; Kumar and Ellington,
1995; Szostak, 1992). The development of ribozymes that are optimal
for catalytic activity would contribute significantly to any
strategy that employs RNA-cleaving ribozymes for the purpose of
regulating gene expression. The hammerhead ribozyme, for example,
functions with a catalytic rate (kcat) of about 1 min-1 in the
presence of saturating (10 rnM) concentrations of Mg2+ cofactor. An
artificial "RNA ligase" ribozyme has been shown to catalyze the
corresponding self-modification reaction with a rate of about 100
min-1. In addition, it is known that certain modified hammerhead
ribozymes that have substrate binding arms made of DNA catalyze RNA
cleavage with multiple turn-over rates that approach 100 min-1.
Modified Inhibitory Nucleic Acids
[0056] In some embodiments, the inhibitory nucleic acids used in
the methods described herein are modified, e.g., comprise one or
more modified bonds or bases. A number of modified bases include
phosphorothioate, methylphosphonate, peptide nucleic acids, or
locked nucleic acid (LNA) molecules. Some inhibitory nucleic acids
are fully modified, while others are chimeric and contain two or
more chemically distinct regions, each made up of at least one
nucleotide. These inhibitory nucleic acids typically contain at
least one region of modified nucleotides that confers one or more
beneficial properties (such as, for example, increased nuclease
resistance, increased uptake into cells, increased binding affinity
for the target) and a region that is a substrate for enzymes
capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory
nucleic acids of the invention may be formed as composite
structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures comprise, but
are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, each of which is herein
incorporated by reference. In some embodiments, the modifications
are 2' sugar modifications including 2'-O-methyl ribose-modified
RNA (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-fluoro (2'-F) and LNA
modifications (Bernardo et al., 2015).
[0057] In some embodiments, the inhibitory nucleic acid comprises
at least one nucleotide modified at the 2' position of the sugar,
most preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or
2'-fluoro-modified nucleotide. In other preferred embodiments, RNA
modifications include 2'-fluoro, 2'-amino and 2' O-methyl
modifications on the ribose of pyrimidines, abasic residues or an
inverted base at the 3' end of the RNA. Such modifications are
routinely incorporated into oligonucleotides and these
oligonucleotides have been shown to have a higher Tm (i.e., higher
target binding affinity) than; 2'-deoxyoligonucleotides against a
given target.
[0058] A number of nucleotide and nucleoside modifications have
been shown to make the inhibitory nucleic acid into which they are
incorporated more resistant to nuclease digestion than the native
oligodeoxynucleotide; these modified oligos survive intact for a
longer time than unmodified inhibitory nucleic acids. Specific
examples of modified inhibitory nucleic acids include those
comprising modified backbones, for example, phosphorothioates,
phosphotriesters, methyl phosphonates, short chain alkyl or
cycloalkyl intersugar linkages or short chain heteroatomic or
heterocyclic intersugar linkages. Most preferred are inhibitory
nucleic acids with phosphorothioate backbones and those with
heteroatom backbones, particularly CH2-NH--O--CH2, CH,
.about.N(CH3)-O--CH2 (known as a methylene(methylimino) or MMI
backbone], CH2-O--N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and
O--N(CH3)-CH2-CH2 backbones, wherein the native phosphodiester
backbone is represented as O--P--O--CH); amide backbones (see De
Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino
backbone structures (see Summerton and Weller, U.S. Pat. No.
5,034,506); peptide nucleic acid (PNA) backbone (i.e., peptide
bonds, wherein the phosphodiester backbone of the inhibitory
nucleic acid is replaced with a polyamide backbone, the nucleotides
being bound directly or indirectly to the aza nitrogen atoms of the
polyamide backbone (Nielsen, 1991). Phosphorus-containing linkages
include, but are not limited to, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
comprising 3'alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates comprising 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563, 253; 5,571,799; 5,587,361; and 5,625,050.
[0059] Morpholino-based oligomeric compounds are described in
(Braasch and Corey, 2002) (Genesis, volume 30, issue 3, 2001;
Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al.,
Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad.
Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul.
23, 1991).
[0060] Cyclohexenyl nucleic acid inhibitory nucleic acid mimetics
are described in Wang et al., J. Am. Chem. Soc., 2000, 122,
8595-8602.
[0061] Modified inhibitory nucleic acid backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These comprise those having morpholino linkages (formed
in part from the sugar portion of a nucleoside); siloxane
backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506;
5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562;
5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;
5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; and 5,677,439, each of which is herein incorporated by
reference.
[0062] One or more substituted sugar moieties can also be included,
e.g., one of the following at the 2' position: OH, SH, SCH3, F,
OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where
n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy,
substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3;
O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2;
NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving
group; a reporter group; an intercalator; a group for improving the
pharmacokinetic properties of an inhibitory nucleic acid; or a
group for improving the pharmacodynamic properties of an inhibitory
nucleic acid and other substituents having similar properties. A
preferred modification includes 2'-methoxyethoxy [2'-O--CH2CH2OCH3,
also known as 2'-O-(2-methoxyethyl)] (Martin et al, Helv. Chim.
Acta, 1995, 78, 486). Other preferred modifications include
2'-methoxy (2'-O--CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro
(2'-F). Similar modifications may also be made at other positions
on the inhibitory nucleic acid, particularly the 3' position of the
sugar on the 3' terminal nucleotide and the 5' position of 5'
terminal nucleotide. Inhibitory nucleic acids may also have sugar
mimetics such as cyclobutyls in place of the pentofuranosyl
group.
[0063] Alternatively or in addition, the modification can be
inclusion of N,N-diethyl-4-(4-nitronaphthalene-1-ylazo)-phenylamine
(`ZEN`), which when placed near the ends of a 2'-OMe modified
oligonucleotide (ZEN-AMO) increased binding affinity and blocked
exonuclease degradation as compared to unmodified 2'-OMe
oligonucleotides (Lennox et al., Mol. Ther. Nucleic Acids 2, e117
(2013)).
[0064] Inhibitory nucleic acids can also include, additionally or
alternatively, nucleobase (often referred to in the art simply as
"base") modifications or substitutions. As used herein,
"unmodified" or "natural" nucleobases include adenine (A), guanine
(G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include nucleobases found only infrequently or transiently in
natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me
pyrimidines, particularly 5-methylcytosine (also referred to as
5-methyl-2' deoxycytosine and often referred to in the art as
5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and
gentobiosyl HMC, as well as synthetic nucleobases, e.g.,
2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,
2-(aminoalklyamino)adenine or other heterosubstituted
alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil,
5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6
(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA
Replication, W. H. Freeman & Co., San Francisco, 1980, pp
75-'7'7; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A
"universal" base known in the art, e.g., inosine, can also be
included. 5-Me-C substitutions have been shown to increase nucleic
acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., in
Crooke, S. T. and Lebleu, B., eds., Antisense Research and
Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are
presently preferred base substitutions.
[0065] It is not necessary for all positions in a given inhibitory
nucleic acid to be uniformly modified, and in fact more than one of
the aforementioned modifications may be incorporated in a single
inhibitory nucleic acid or even at within a single nucleoside
within an inhibitory nucleic acid.
[0066] In some embodiments, both a sugar and an internucleoside
linkage, i.e., the backbone, of the nucleotide units are replaced
with novel groups. The base units are maintained for hybridization
with an appropriate nucleic acid target compound. One such
oligomeric compound, an inhibitory nucleic acid mimetic that has
been shown to have excellent hybridization properties, is referred
to as a peptide nucleic acid (PNA). In PNA compounds, the
sugar-backbone of an inhibitory nucleic acid is replaced with an
amide containing backbone, for example, an aminoethylglycine
backbone. The nucleobases are retained and are bound directly or
indirectly to aza nitrogen atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds comprise, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Further teaching of PNA compounds
can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
[0067] Inhibitory nucleic acids can also include one or more
nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases comprise the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases comprise other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and
3-deazaadenine.
[0068] Further, nucleobases comprise those disclosed in U.S. Pat.
No. 3,687,808, those disclosed in `The Concise Encyclopedia of
Polymer Science And Engineering`, pages 858-859, Kroschwitz, J. I.,
ed. John Wiley & Sons, 1990, those disclosed by Englisch et
al., Angewandle Chemie, International Edition`, 1991, 30, page 613,
and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense
Research and Applications', pages 289-302, Crooke, S. T. and
Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the
oligomeric compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, comprising 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2<0>C
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, `Antisense
Research and Applications`, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications. Modified nucleobases are described in U.S. Pat. No.
3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;
5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255;
5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091;
5,614,617; 5,750,692, and 5,681,941, each of which is herein
incorporated by reference.
[0069] In some embodiments, the inhibitory nucleic acids are
chemically linked to one or more moieties or conjugates that
enhance the activity, cellular distribution, or cellular uptake of
the inhibitory nucleic acid. Such moieties comprise but are not
limited to, lipid moieties such as a cholesterol moiety (Letsinger
et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic
acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,
1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et
al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,
Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an
aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et
al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,
1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol
or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et
al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a
polyethylene glycol chain (Mancharan et al., Nucleosides &
Nucleotides, 1995, 14, 969-973), or adamantane acetic acid
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a
palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264,
229-237), or an octadecylamine or hexylamino-carbonyl-t
oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.,
1996, 277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882;
5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717,
5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;
5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;
4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263;
4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136;
5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552;
5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599,923; 5,599, 928 and 5,688,941, each of which is herein
incorporated by reference.
[0070] These moieties or conjugates can include conjugate groups
covalently bound to functional groups such as primary or secondary
hydroxyl groups. Conjugate groups of the invention include
intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the
pharmacodynamic properties of oligomers, and groups that enhance
the pharmacokinetic properties of oligomers. Typical conjugate
groups include cholesterols, lipids, phospholipids, biotin,
phenazine, folate, phenanthridine, anthraquinone, acridine,
fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance
the pharmacodynamic properties, in the context of this invention,
include groups that improve uptake, enhance resistance to
degradation, and/or strengthen sequence-specific hybridization with
the target nucleic acid. Groups that enhance the pharmacokinetic
properties, in the context of this invention, include groups that
improve uptake, distribution, metabolism or excretion of the
compounds of the present invention. Representative conjugate groups
are disclosed in International Patent Application No.
PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860,
which are incorporated herein by reference. Conjugate moieties
include, but are not limited to, lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, e.g.,
hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,
dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol
moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941.
Locked Nucleic Acids (LNAs)
[0071] In some embodiments, the modified inhibitory nucleic acids
(including ASOs) used in the methods described herein comprise
locked nucleic acid (LNA) molecules, e.g., including
[alpha]-L-LNAs. LNAs comprise ribonucleic acid analogues wherein
the ribose ring is "locked" by a methylene bridge between the
2'-oxygen and the 4'-carbon--i.e., inhibitory nucleic acids
containing at least one LNA monomer, that is, one
2'-0,4'-C-methylene-.beta.-D-ribofuranosyl nucleotide. LNA bases
form standard Watson-Crick base pairs but the locked configuration
increases the rate and stability of the basepairing reaction
(Jepsen et al., 2004). LNAs also have increased affinity to base
pair with RNA as compared to DNA. These properties render LNAs
especially useful as probes for fluorescence in situ hybridization
(FISH) and comparative genomic hybridization, as knockdown tools
for miRNAs, and as antisense oligonucleotides to target mRNAs or
other RNAs, e.g., RNAs as described herein.
[0072] The LNA molecules can include molecules comprising 10-30,
e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein
one of the strands is substantially identical, e.g., at least 80%
(or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3,
2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA.
The LNA molecules can be chemically synthesized using methods known
in the art.
[0073] The LNA molecules can be designed using any method known in
the art; a number of algorithms are known, and are commercially
available (e.g., on the internet, for example at exiqon.com). See
(Levin et al., 2006), (You et al., Nuc. Acids. Res. 34:e60 (2006);
McTigue et al., Biochemistry 43:5388-405 (2004). For example, "gene
walk" methods, similar to those used to design antisense oligos,
can be used to optimize the inhibitory activity of the LNA; for
example, a series of inhibitory nucleic acids of 10-30 nucleotides
spanning the length of a target RNA can be prepared, followed by
testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides
or more, can be left between the LNAs to reduce the number of
inhibitory nucleic acids synthesized and tested. GC content is
preferably between about 30 60%. General guidelines for designing
LNAs are known in the art; for example, LNA sequences will bind
very tightly to other LNA sequences, so it is preferable to avoid
significant complementarity within an LNA. Contiguous runs of more
than four LNA residues, should be avoided where possible (for
example, it may not be possible with very short (e.g., about 9-10
nt) inhibitory nucleic acids). In some embodiments, the LNAs are
xylo-LNAs.
[0074] For additional information regarding LNAs see U.S. Pat. Nos.
6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207;
7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos.
20100267018; 20100261175; and 20100035968; (Jepsen et al., 2004;
Kauppinen et al., 2005; Ponting et al., 2009) Koshkin et al.
Tetrahedron 54, 3607-3630 (1998); Obika et al. Tetrahedron Lett.
39, 5401-5404 (1998); and references cited therein.
[0075] See also (Baigude and Rana, 2014; Bernardo et al., 2015;
Figueira et al., 2014; Stenvang et al., 2012).
Making and Using Inhibitory Nucleic Acids
[0076] The nucleic acid sequences used to practice the methods
described herein, whether RNA, cDNA, genomic DNA, vectors, viruses
or hybrids thereof, can be isolated from a variety of sources,
genetically engineered, amplified, and/or expressed/generated
recombinantly. Recombinant nucleic acid sequences can be
individually isolated or cloned and tested for a desired activity.
Any recombinant expression system can be used, including e.g. in
vitro, bacterial, fungal, mammalian, yeast, insect or plant cell
expression systems.
[0077] Nucleic acid sequences of the invention can be inserted into
delivery vectors and expressed from transcription units within the
vectors. The recombinant vectors can be DNA plasmids or viral
vectors. Generation of the vector construct can be accomplished
using any suitable genetic engineering techniques well known in the
art, including, without limitation, the standard techniques of PCR,
oligonucleotide synthesis, restriction endonuclease digestion,
ligation, transformation, plasmid purification, and DNA sequencing,
for example as described in Sambrook et al. Molecular Cloning: A
Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997))
and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford
University Press, (2000)). As will be apparent to one of ordinary
skill in the art, a variety of suitable vectors are available for
transferring nucleic acids of the invention into cells. The
selection of an appropriate vector to deliver nucleic acids and
optimization of the conditions for insertion of the selected
expression vector into the cell, are within the scope of one of
ordinary skill in the art without the need for undue
experimentation. Viral vectors comprise a nucleotide sequence
having sequences for the production of recombinant virus in a
packaging cell. Viral vectors expressing nucleic acids of the
invention can be constructed based on viral backbones including,
but not limited to, a retrovirus, lentivirus, adenovirus,
adeno-associated virus, pox virus or alphavirus. The recombinant
vectors capable of expressing the nucleic acids of the invention
can be delivered as described herein, and persist in target cells
(e.g., stable transformants).
[0078] Nucleic acid sequences used to practice this invention can
be synthesized in vitro by well-known chemical synthesis
techniques(Frenkel et al., 1995) Adams (1983) J. Am. Chem. Soc.
105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Blommers
(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol.
68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra.
Lett. 22:1859; U.S. Pat. No. 4,458,066.
[0079] Nucleic acid sequences of the invention can be stabilized
against nucleolytic degradation such as by the incorporation of a
modification, e.g., a nucleotide modification. For example, nucleic
acid sequences of the invention includes a phosphorothioate at
least the first, second, or third internucleotide linkage at the 5'
or 3' end of the nucleotide sequence. As another example, the
nucleic acid sequence can include a 2'-modified nucleotide, e.g., a
2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl
(2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA). As another example, the nucleic
acid sequence can include at least one 2'-O-methyl-modified
nucleotide, and in some embodiments, all of the nucleotides include
a 2'-O-methyl modification. In some embodiments, the nucleic acids
are "locked," i.e., comprise nucleic acid analogues in which the
ribose ring is "locked" by a methylene bridge connecting the 2'-O
atom and the 4'-C atom(Kauppinen et al., 2005) Koshkin et al., J.
Am. Chem. Soc., 120(50):13252-13253 (1998)). For additional
modifications see US 20100004320, US 20090298916, and US
20090143326.
[0080] Techniques for the manipulation of nucleic acids used to
practice this invention, such as, e.g., subcloning, labeling probes
(e.g., random-primer labeling using Klenow polymerase, nick
translation, amplification), sequencing, hybridization and the like
are well described in the scientific and patent literature, see,
e.g., Sambrook et al., Molecular Cloning; A Laboratory Manual 3d
ed. (2001); Current Protocols in Molecular Biology, Ausubel et al.,
eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); Laboratory
Techniques In Biochemistry And Molecular Biology: Hybridization
With Nucleic Acid Probes, Part I. Theory and Nucleic Acid
Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Pharmaceutical Compositions and Methods of Administration
[0081] The methods described herein include the use of
pharmaceutical compositions comprising inhibitors of S1P1, IL-15,
the IL-15 receptor, or Jak1/3.
[0082] Pharmaceutical compositions typically include a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes saline, solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Supplementary active compounds
can also be incorporated into the compositions, e.g., bioactive
molecules that promote melanocyte regeneration, growth, or
migration, such as .alpha.-MSH analogs (afamelanotide, etc), WNT
agonists, piperine or its analogs, or surgical transplantation of
melanocytes.
[0083] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration.
[0084] Methods of formulating suitable pharmaceutical compositions
are known in the art, see, e.g., Remington: The Science and
Practice of Pharmacy, 21st ed., 2005; and the books in the series
Drugs and the Pharmaceutical Sciences: a Series of Textbooks and
Monographs (Dekker, NY). For example, 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 bisulfate; 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.
[0085] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should 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 that
delays absorption, for example, aluminum monostearate and
gelatin.
[0086] 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 yield a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0087] Oral compositions generally include an inert diluent or an
edible carrier. 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, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. 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.
[0088] For administration by inhalation, the compounds can be
delivered in the form of an aerosol spray from a pressured
container or dispenser that contains a suitable propellant, e.g., a
gas such as carbon dioxide, or a nebulizer. Such methods include
those described in U.S. Pat. No. 6,468,798.
[0089] Systemic administration of a therapeutic compound as
described herein 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.
[0090] In some embodiments, compositions comprising an IL-15, IL-15
receptor, or Jak1/3 inhibitor for transdermal application can
further comprise cosmetically-acceptable carriers or vehicles and
any optional components. A number of such cosmetically acceptable
carriers, vehicles and optional components are known in the art and
include carriers and vehicles suitable for application to skin
(e.g., sunscreens, creams, milks, lotions, masks, serums, etc.),
see, e.g., U.S. Pat. Nos. 6,645,512 and 6,641,824. In particular,
optional components that may be desirable include, but are not
limited to absorbents, anti-acne actives, anti-caking agents,
anti-cellulite agents, anti-foaming agents, anti-fungal actives,
anti-inflammatory actives, anti-microbial actives, anti-oxidants,
antiperspirant/deodorant actives, anti-skin atrophy actives,
anti-viral agents, anti-wrinkle actives, artificial tanning agents
and accelerators, astringents, barrier repair agents, binders,
buffering agents, bulking agents, chelating agents, colorants,
dyes, enzymes, essential oils, film formers, flavors, fragrances,
humectants, hydrocolloids, light diffusers, nail enamels,
opacifying agents, optical brighteners, optical modifiers,
particulates, perfumes, pH adjusters, sequestering agents, skin
conditioners/moisturizers, skin feel modifiers, skin protectants,
skin sensates, skin treating agents, skin exfoliating agents, skin
lightening agents, skin soothing and/or healing agents, skin
thickeners, sunscreen actives, topical anesthetics, vitamin
compounds, and combinations thereof.
[0091] The pharmaceutical compositions can also be prepared in the
form of suppositories (e.g., with conventional suppository bases
such as cocoa butter and other glycerides) or retention enemas for
rectal delivery.
[0092] Therapeutic compounds that are or include nucleic acids can
be administered by any method suitable for administration of
nucleic acid agents, such as a DNA vaccine. These methods include
gene guns, bio injectors, and skin patches as well as needle-free
methods such as the micro-particle DNA vaccine technology disclosed
in U.S. Pat. No. 6,194,389, and the mammalian transdermal
needle-free vaccination with powder-form vaccine as disclosed in
U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is
possible (Hamajima et al., 1998). Liposomes (e.g., as described in
U.S. Pat. No. 6,472,375) and microencapsulation can also be used.
Biodegradable targetable microparticle delivery systems can also be
used (e.g., as described in U.S. Pat. No. 6,471,996).
[0093] In one embodiment, the therapeutic compounds are prepared
with carriers that will protect the therapeutic compounds against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Such formulations
can be prepared using standard techniques, or obtained
commercially, e.g., from Alza Corporation and Nova Pharmaceuticals,
Inc. Liposomal suspensions (including liposomes targeted to
selected cells with monoclonal antibodies to cellular antigens) can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0094] In some embodiments, the therapeutic agents are administered
by a microneedle array, e.g. as described in U.S. Pat. No.
6,379,324B1, U.S. Pat. No. 6,881,203B2, U.S. Pat. No. 6,256,533B1,
U.S. Pat. No. 6,790,372B2, and U.S. Pat. No. 3,964,482A. These
arrays comprise a number of either hollow or solid microneedles
which are used to pierce through the stratum corneum, the outermost
layer of skin comprising dead skin cells, to facilitate transdermal
drug delivery. Penetration of drugs delivered locally by this
method is improved as compared to topical delivery, is painless,
and causes minimal trauma to the skin. Drugs can be delivered by
passive diffusion, injection, or iontophoresis.
[0095] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Dosage
[0096] An "effective amount" is an amount sufficient to effect
beneficial or desired results. For example, a therapeutic amount is
one that achieves the desired therapeutic effect. This amount can
be the same or different from a prophylactically effective amount,
which is an amount necessary to prevent onset of disease or disease
symptoms. An effective amount can be administered in one or more
administrations, applications or dosages. A therapeutically
effective amount of a therapeutic compound (i.e., an effective
dosage) depends on the therapeutic compounds selected. The
compositions can be administered one from one or more times per day
to one or more times per week; including once every other day. The
skilled artisan will appreciate that certain factors may influence
the dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of the therapeutic
compounds described herein can include a single treatment or a
series of treatments.
[0097] Dosage, toxicity and therapeutic efficacy of the therapeutic
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
that exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0098] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0099] Methods of Diagnosis
[0100] Included herein are methods for diagnosing vitiligo. The
methods rely on determining a vitiligo score using VDAS, VASI
and/or VETF. Biomarkers CXCL9, CXCL10 and CD8+ T cells may also be
assessed using a minimally invasive, non-scarring skin biopsy
technique to reliably and accurately sample vitiligo lesions by
inducing suction blisters, which provide both fluid (interstitial
skin fluid) and roof (epidermis) for analysis, or tape-stripping
the outer layer of the skin for analysis (Strassner et al., 2017).
The methods include obtaining a sample comprising lesional
interstitial skin fluid and a sample comprising epidermis from the
lesion from a subject, and evaluating CD8+ T cell infiltrate, e.g.,
by flow cytometry, and elevated chemokine protein, e.g., by ELISA,
in lesional fluid, and levels of a panel of 20 genes (shown in
Table A) in the lesional skin.
[0101] A negative pressure instrument was used to create suction
blisters, a process that induced minimal discomfort and does not
leave a permanent scar (Babu et al., 2008; Gupta et al., 1999a;
Gupta et al., 1999b). The blister fluid, comprised of interstitial
fluid from the epidermis and superficial dermis where active
inflammation is located (Rossing and Worm, 1981), is drawn from a
broad area of the skin, providing an opportunity to sample much
more of the lesion than a conventional biopsy. We found that
blister fluid from lesional skin contains significantly elevated
numbers of CD8+ T cells and CXCL9 protein compared to non-lesional
and healthy control skin (Strassner et al., 2017). The expression
of a small panel of genes involved in CD8+ T cell activity,
IFN-.gamma. signaling, and melanocyte activity mark active vitiligo
lesions compared to healthy skin (Rashighi et al., 2014). Our
unpublished studies reveal that much of this activity is located in
the epidermis, and thus analyzing epidermal blister roofs may be an
effective, innovative way to monitor disease activity through gene
expression. Another option is through tape-stripping the epidermis
and analyzing gene expression on the cells removed in this way (Liu
et al., 2010). An innovative combined analysis of the blister fluid
and roof will be more sensitive and specific than any single
measure alone, and could become the standard tool to measure
IFN-.gamma.-specific disease activity and treatment responses in
future clinical studies.
[0102] Various methods are well known within the art for the
identification and/or isolation and/or purification of a biological
marker (e.g., cell, protein or nucleic acid) from a sample. An
"isolated" or "purified" biological marker is substantially free of
cellular material or other contaminants from the cell or tissue
source from which the biological marker is derived i.e. partially
or completely altered or removed from the natural state through
human intervention. For example, nucleic acids contained in the
sample are first isolated according to standard methods, for
example using lytic enzymes, chemical solutions, or isolated by
nucleic acid-binding resins following the manufacturer's
instructions.
[0103] The presence and/or level of a protein can be evaluated
using methods known in the art, e.g., using standard
electrophoretic and quantitative immunoassay methods for proteins,
including but not limited to, Western blot; enzyme linked
immunosorbent assay (ELISA); biotin/avidin type assays; protein
array detection; radio-immunoassay; immunohistochemistry (IHC);
immune-precipitation assay; FACS (fluorescent activated cell
sorting); mass spectrometry (Brody et al., 2010; Kim et al., 2010;
Pfaffe et al., 2011; Phillips et al., 2014; Yasun et al., 2012).
The methods typically include revealing labels such as fluorescent,
chemiluminescent, radioactive, and enzymatic or dye molecules that
provide a signal either directly or indirectly. As used herein, the
term "label" refers to the coupling (i.e. physically linkage) of a
detectable substance, such as a radioactive agent or fluorophore
(e.g. phycoerythrin (PE) or indocyanine (Cy5), to an antibody or
probe, as well as indirect labeling of the probe or antibody (e.g.
horseradish peroxidase, HRP) by reactivity with a detectable
substance.
[0104] In some embodiments, an ELISA method may be used, wherein
the wells of a mictrotiter plate are coated with an antibody
against which the protein is to be tested. The sample containing or
suspected of containing the biological marker is then applied to
the wells. After a sufficient amount of time, during which
antibody-antigen complexes would have formed, the plate is washed
to remove any unbound moieties, and a detectably labelled molecule
is added. Again, after a sufficient period of incubation, the plate
is washed to remove any excess, unbound molecules, and the presence
of the labeled molecule is determined using methods known in the
art. Variations of the ELISA method, such as the competitive ELISA
or competition assay, and sandwich ELISA, may also be used, as
these are well-known to those skilled in the art.
[0105] In some embodiments, an IHC method may be used. IHC provides
a method of detecting a biological marker in situ. The presence and
exact cellular location of the biological marker can be detected.
Typically, a sample is fixed with formalin or paraformaldehyde,
embedded in paraffin, and cut into sections for staining and
subsequent inspection by confocal microscopy. Current methods of
IHC use either direct or indirect labelling. The sample may also be
inspected by fluorescent microscopy when immunofluorescence (IF) is
performed, as a variation to IHC.
[0106] Mass spectrometry, and particularly matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) and
surface-enhanced laser desorption/ionization mass spectrometry
(SELDI-MS), is useful for the detection of biomarkers of this
invention. (See U.S. Pat. Nos. 5,118,937; 5,045,694; 5,719,060;
6,225,047)
[0107] The presence and/or level of a nucleic acid in Table A can
be evaluated using methods known in the art, e.g., using polymerase
chain reaction (PCR), reverse transcriptase polymerase chain
reaction (RT-PCR), quantitative or semi-quantitative real-time
RT-PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification,
Magnetics) (Diehl et al., 2006); RNAse protection assay; Northern
blot; various types of nucleic acid sequencing (Sanger,
pyrosequencing, NextGeneration Sequencing); fluorescent in-situ
hybridization (FISH); or gene array/chips) (Lehninger Biochemistry
(Worth Publishers, Inc., current addition; Sambrook, et al,
Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001);
(Bernard and Wittwer, 2002) (Bianchi et al., 2011; Miranda et al.,
2010) (Taylor and Gercel-Taylor, 2013; Yang et al., 2014)
(Nordstrom et al., 2000) (Ahmadian et al., 2000). In some
embodiments, high throughput methods, e.g., protein or gene chips
as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths
et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman and
Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218;
MacBeath and Schreiber, Science 2000, 289(5485): 1760-1763;
Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring
Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and
Applications: Nuts & Bolts, DNA Press, 2003), can be used to
detect the presence and/or level of the nucleic acids in the roof.
Measurement of the level of a biomarker can be direct or indirect.
For example, the abundance levels of a specific mRNA can be
directly quantitated. Alternatively, the amount of a biomarker can
be determined indirectly by measuring abundance levels of cDNA,
amplified RNAs or DNAs, or by measuring quantities or activities of
RNAs, or other molecules that are indicative of the expression
level of the biomarker. In some embodiments a technique suitable
for the detection of alterations in the structure or sequence of
nucleic acids, such as the presence of deletions, amplifications,
or substitutions, can be used for the detection of biomarkers of
this invention.
[0108] RT-PCR can be used to determine the expression profiles of
biomarkers (U.S. Patent No. 2005/0048542A1). The first step in
expression profiling by RT-PCR is the reverse transcription of the
RNA template into cDNA, followed by its exponential amplification
in a PCR reaction (Ausubel et al (1997) Current Protocols of
Molecular Biology, John Wiley and Sons). To minimize errors and the
effects of sample-to-sample variation, RT-PCR is usually performed
using an internal standard, which is expressed at constant level
among tissues, and is unaffected by the experimental treatment.
Housekeeping genes are most commonly used, e.g., GAPDH, TUBB, or
GUSB.
[0109] Gene arrays are prepared by selecting probes that comprise a
polynucleotide sequence, and then immobilizing such probes to a
solid support or surface. For example, the probes may comprise DNA
sequences, RNA sequences, co-polymer sequences of DNA and RNA, DNA
and/or RNA analogues, or combinations thereof. The probe sequences
can be synthesized either enzymatically in vivo, enzymatically in
vitro (e.g. by PCR), or non-enzymatically in vitro.
TABLE-US-00001 TABLE A 24-Gene VITAL Score GenBank GenBank GenBank
cytokines RefSeq ID T cell RefSeq ID melanocyte RefSeq ID CXCL9
NM_002416.2 GZMB NM_004131.5 or DCT NM_001129889.2, NM_001346011.1
NM_001322182.1, NM_001322183.1, NM_001322184.1, NM_001322185.1,
NM_001322186.1, or NM_001922.4 CXCL10 NM_001565.3 CD8A
NM_001145873.1, KIT NM_000222.2 or NM_001768.6, or NM_001093772.1
NM_171827.3 CXCL11 NM_005409.4, or CXCR3 NM_001504.1 or TYR
NM_000372.4 NM_001302123.1 NM_001142797.1 STAT1 NM_007315.3, or CD4
NM_000616.4, TRPM1 NM_001252020.1, NM_139266.2 NM_001195014.2,
NM_001252024.1, NM_001195015.2, NM_001252030.1, NM_001195016.2, or
or NM_002420.5 NM_001195017.2 CCL5 NM_002985.2, or FOXP3
NM_014009.3 or NM_001278736.1 NM_001114377.1 CCL18 NM_002988.3 IFNG
NM_000619.2 other HOUSE IL23A NM_016584.2 HLA-A GAPDH
NM_001256799.2, NM_001289745.1, NM_001289746.1, or NM_002046.5 TNF
NM_000594.3 B2M NM_004048.2 TUBB NM_001293212.1, NM_001293213.1,
NM_001293214.1, NM_001293215.1, NM_001293216.1, or NM_178014.3
CXCL2 NM_002089.3 GUSB NM_000181.3, NM_001284290.1, NM_001293104.1,
or NM_001293105.1
[0110] Reference Scores
[0111] Suitable reference score values can be determined using
methods known in the art, e.g., using standard clinical trial
methodology and statistical analysis. The reference values can have
any relevant form.
[0112] The predetermined level can be a single cut-off (threshold)
value, such as a median or mean, or a level that defines the
boundaries of an upper or lower quartile, tertile, or other segment
of a clinical trial population that is determined to be
statistically different from the other segments. It can be a range
of cut-off (or threshold) values, such as a confidence interval. It
can be established based upon comparative groups, such as where
association with risk of developing disease or presence of disease
in one defined group is a fold higher, or lower, (e.g.,
approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the
risk or presence of disease in another defined group. It can be a
range, for example, where a population of subjects (e.g., control
subjects) is divided equally (or unequally) into groups, such as a
low-risk group, a medium-risk group and a high-risk group, or into
quartiles, the lowest quartile being subjects with the lowest risk
and the highest quartile being subjects with the highest risk, or
into n-quantiles (i.e., n regularly spaced intervals) the lowest of
the n-quantiles being subjects with the lowest risk and the highest
of the n-quantiles being subjects with the highest risk.
[0113] In some embodiments, the predetermined level is a level or
occurrence in the same subject, e.g., at a different time point,
e.g., an earlier time point.
EXAMPLES
[0114] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Materials and Methods
[0115] The following materials and methods were used in the
Examples set forth below.
[0116] Study Design
[0117] The objectives of this study were to determine whether Trm
exist in human vitiligo, how they contribute to disease in a mouse
model of vitiligo, and to determine whether these cells could be
targeted therapeutically. These objectives were proposed to test
the hypothesis that vitiligo is resistant to treatment because Trm
persist in the skin and reactivate disease upon cessation of
treatment. This hypothesis was formed based on clinical
observations, as well as others reported in the literature
(Ariotti, 2014; Clark et al., 2012; Jiang et al., 2012; Mackay et
al., 2013; Schenkel et al., 2013; Skon et al., 2013; Watanabe,
2015).
[0118] Mice
[0119] All mice were housed in pathogen-free facilities at UMMS,
and procedures were approved by the UMMS Institutional Animal Care
and Use Committee and in accordance with the National Institutes of
Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice
used for these studies were on the C57BL/6J (B6) background or a
mixed 129.times.C57BL/6 background that had been backcrossed to B6
for more than 10 generations. Age and sex-matched mice were used,
and both male and female mice of all strains were tested to avoid
gender bias. Replicate experiments were performed two to five
times.
[0120] KRT14-Kitl*4XTG2Bj1 (Krt14-Kitl*) mice were a gift from B.
J. Longley (University of Wisconsin, Madison, Wis.; now available
at The Jackson Laboratory, stock no. 009687). The following strains
were bred to Krt14-Kitl* mice for use as hosts in the vitiligo
model: GREAT (The Jackson Laboratory, stock no. 017580), and REX3
(provided by A. Luster, Massachusetts General Hospital). For
consistency, the Krt14-Kitl* allele was heterozygous on all mice
used in vitiligo experiments. Thy1.1+ PMEL TCR transgenic mice were
obtained from The Jackson Laboratory (stock no. 005023) and were
used as donors in the vitiligo model. GFP-PMEL TCR transgenic mice
were produced by crossing PMEL transgenic mice with DPE-GFP mice,
which express GFP in T cells (provided by U. von Andrian, Harvard
Medical School, Boston, Mass.).
[0121] Vitiligo Induction
[0122] Vitiligo was induced as previously described (Harris et al.,
2012). Briefly, PMEL CD8+ T cells were isolated from the spleens of
PMEL TCR transgenic mice through negative selection on microbeads
(Miltenyi Biotec) according to the manufacturer's instructions.
Purified CD8+ T cells (1.times.10.sup.6) were injected
intravenously into sublethally irradiated (500 rads 1 day before
transfer) Krt14-Kitl* hosts (8 to 16 weeks of age). On the same day
of transfer, recipient mice received intraperitoneal injection of
1.times.10.sup.6 plaque-forming units of rVV-hPMEL (N. Restifo,
National Cancer Institute, NIH).
[0123] Vitiligo score was objectively quantified by an observer
blinded to the experimental groups, using a point scale based on
the extent of depigmentation at four easily visible locations,
including the ears, nose, rear footpads, and tails as described
previously (Harris et al., 2012). The extent of depigmentation was
estimated as a percentage of the anatomic site; both left and right
ears and left and right rear footpads were estimated together and
therefore evaluated as single sites. Points were awarded as
follows: no evidence of depigmentation (0%) received a score of 0,
>0 to 10%=1 point, >10 to 25%=2 points, >25 to 75%=3
points, >75 to <100%=4 points, and 100%=5 points. The
"vitiligo score" was the sum of the scores at all four sites, with
a maximum score of 20 points.
[0124] Repigmentation, Functionality and Durability Experiments
[0125] Vitiligo mice with >75% depigmentation and stable disease
(between week 12-20 post-vitiligo induction) were used for
repigmentation studies. FTY720 (Cayman chemical) treatment was
performed by i.p. injection of (1) 1 mg/kg FTY720 diluted in water
or (2) vehicle (water) three times weekly for the duration of the
observation period (4 weeks) as previously described (Chiba, 2005;
Murooka et al., 2012). IL-15R.beta. antibody treatment was
performed by i.p. injection of 100 .mu.g of (1) anti-IL-15R.beta.
antibody ChMBC7 or (2) isotype control or vehicle (PBS) three times
weekly. For long-term treatment studies, mice were treated for the
duration of the observation period (8 weeks) for repigmentation
studies. For durability studies, mice were treated for 2 weeks, and
monitored for an additional 8 weeks after cessation of treatment.
For functionality studies, mice were treated for 2 weeks,
sacrificed at week 3, and PMEL T cells were restimulated ex vivo
using 3 .mu.g/mL plate bound anti-CD3 antibody with 2 .mu.g/mL
anti-CD28 antibody in complete RPMI with brefeldin A for 6-18 hours
for cytokine production evaluation via flow cytometry analysis.
Repigmentation analysis was performed with ImageJ. Photos were
taken of each individual mouse before treatment and again after
treatment was completed. The images were converted into black and
white and the change in pigment was quantified with Image J
software as previously described (Agarwal et al., 2015).
[0126] In Situ PMEL Labeling
[0127] Ears were injected with 40 ul of an antibody cocktail
containing 0.05 mg/ml TCR-Vbeta 13-APC (BD Biosciences clone
MR12-3) and 0.35 mg/ml Fc block 2.4G2. 2 hours post injection, mice
were euthanized and ears excised. The dorsal and ventral sides were
separated and mounted on slides for confocal microscopy (Cummings
et al., 2008).
[0128] Study Subjects
[0129] Patient shave skin biopsies and suction blister biopsies
were collected under IRB-approved protocols at UMMS by
board-certified dermatologists, and all samples were de-identified
before use in experiments. For suction blister skin biopsies,
lesional sites were chosen based on the presence of depigmentation.
Active patients were defined as having changes in their lesions
over the previous 6 months, as well as the presence of confetti
depigmentation, a recently described clinical sign of active
vitiligo (Sosa et al., 2015). Stable patients were defined as
lacking confetti depigmentation, and having no changes in their
lesions over the previous 6 months. Non-lesional sites were
selected as normal-appearing, non-depigmented skin when examined by
Wood's lamp, at least 2 cm from the nearest depigmented macule.
Patients were excluded from the study if they had received
treatment within the previous three months.
[0130] Blister Induction and Processing
[0131] Suction blisters were induced on the skin using the Negative
Pressure Instrument Model NP-4 (Electronic Diversities, Finksburg
Md.) as previously described (Strassner et al., 2017). Briefly, the
suction chambers were applied to the patient skin with 10-12 mm Hg
of negative pressure and a constant temperature of 40.degree. C.;
blisters formed between 30 minutes and one hour after initiation of
the procedure. After blister formation, the blister fluid was
aspirated using 1 mL insulin syringes. Cells within the blister
fluid were pelleted at 330.times.g for 10 minutes for cell staining
and the supernatant was collected for ELISA.
[0132] Flow Cytometry & Cell Sorting
[0133] Tail skin and draining lymph nodes were harvested at the
indicated times. Lymph nodes were disrupted and tail skin was
incubated with 5 U/mL Dispase II (Roche) for 1 h at 37.degree. C.
Epidermis was removed and mechanically dissociated using 70 .mu.m
filters. Dermis was incubated with 1 mg/mL collagenase IV and 2
mg/mL DNAse I (Sigma Aldrich) for 1 h at 37.degree. C. before
mechanical dissociation. Samples were filtered prior to staining
and analysis, and UltraComp eBeads (eBiosciences) were used for
compensation controls. All murine flow cytometry samples were
blocked with Fc block 2.4G2 (Bio X Cell) and stained with LiveDead
Blue (Invitrogen, 1:1000). The following antibodies were used at a
1:200 dilution: CD45, Thy1.1, CD3, CD8.beta., CD69, CD44, CD103,
and CD62L (Biolegend). IL-15R.alpha. (R&D Systems) was used at
a 1:10 dilution (10 uL per 100 uL). For intracellular cytokine
staining with IFN.gamma. and Granzyme B antibodies (Biolegend), a
Cytofix/Cytoperm kit (BD Biosciences) was used per the
manufacturer's instructions.
[0134] All human flow cytometry samples were blocked with Human
TruStain FcX (Biolegend) and LiveDead Blue (Invitrogen 1:1000). The
following antibodies were used at a 1:20 dilution: CD45, CD4 (Tonbo
Biosciences), and CD8, HLA-A2, CD69 and CD103 (Biolegend). CD3
(Biolegend) was used at a 1:200 dilution. Human blood was screened
for HLA-A2 expressing cells by flow cytometry, and HLA-A2 positive
patient samples were treated with 50 nM dasatinib (Axon Medchem BV)
for 30 minutes prior to labeling with a MART-1 loaded class I
pentamer (Proimmune) per the manufacturer's protocol. Additional
surface staining was performed to identify phenotypes of
antigen-specific cells. Peripheral blood was used to make FMOs to
assist in gating, and samples were stained, then fixed and lysed
using RBC Fixation/Lysis Buffer (Biolegend) per the manufacturer's
instructions. Data were collected with an LSR II and were analyzed
with FlowJo software.
[0135] Statistics
[0136] All statistical analyses were performed with GraphPad Prism
software. Dual comparisons were made with unpaired Student's t
test, and groups of three or more were analyzed by ANOVA with
Tukey's or Dunnett's post-tests. P values <0.05 were considered
significant.
Example 1. Autoreactive T Cells within Lesions of Vitiligo Patients
Express a Trm Phenotype, and Express IL-15R
[0137] Viral infections of the skin generate both central memory T
cells (Tcm) that localize to lymph nodes and recirculate, as well
as resident memory T cells (Trm) that remain in the skin for very
long periods, and both populations help prevent reinfection. We
hypothesized that melanocyte-specific Trm cells form long-lived
memory cells within the vitiligo lesions, similar to those in viral
infections, and make them refractory to treatment. We performed
blister biopsies on vitiligo patients with active and stable
disease (FIG. 1A) (Strassner et al., 2017). We identified
antigen-specific T cells in both blood and skin fluid from these
patients. In patients with active disease, the majority of
antigen-specific T cells were CD69 single positive, whereas in
stable disease they were CD69+CD103+(FIG. 1B). There was an
enrichment of antigen-specific T cells in lesional fluid with a Trm
phenotype (FIG. 1C). Antigen-specific cells Trm expressed the CD122
chain of the IL-15R (FIG. 1D). Keratinocytes express the CD215
chain of the IL-15 receptor to present IL-15 in trans to T cells,
and keratinocytes in lesional skin express higher CD215 than in
nonlesional skin (FIG. 1E). Taken together, these data indicate
that human vitiligo patients possess antigen-specific Trm cells in
lesional skin that express IL-15Rf.beta./CD122.
Example 2. Autoreactive T Cells in the Epidermis of a Mouse Model
of Vitiligo Possess a Resident Memory Phenotype
[0138] To address the functional roles of Trm in vitiligo, we
employed our mouse model, which uses the adoptive transfer of CD8+
T cells that recognize the autoantigen Pre-melanosome protein
(PMEL) physiologically expressed in melanocytes (FIG. 2A). Previous
studies using this model revealed that adoptively transferred,
autoreactive T cells accumulate within the epidermis of the skin
during the progression of vitiligo (Agarwal et al., 2015; Harris et
al., 2012; Rashighi et al., 2014) (8, 9, 12). We found that these
cells accumulate in the epidermis where melanocytes reside, and
that a large fraction of these cells expressed both CD69 and CD103,
markers of Trm formation (FIG. 2B).
[0139] To determine the role of self antigens in the recruitment
and retention of Trm within the epidermis of mice with
autoimmunity, we compared the generation of skin Trm that recognize
PMEL physiologically expressed in melanocytes to T cells that
recognize the irrelevant foreign OVA antigen (OT-1). We induced
immune responses with recombinant vaccinia virus (VV) expressing
pre-melanosome protein (VV-PMEL) and PMEL T cells, or expressing
ovalbumin (VV-OVA) and OT-1 T cells. Only melanocyte-specific T
cells established Trm in the epidermis, whereas OT-1s did not (FIG.
2C-G). These data indicate that autoreactive Trm are generated
directly in the skin where autoantigen is expressed during vitiligo
in our mouse model.
Example 3. PMEL Trm are Long-Lived, Produce IFN-.gamma., and
Chronically Encounter Self-Antigen
[0140] Self-reactive Trm in vitiligo differ from viral-reactive Trm
in that they are frequently re-exposed to antigen as target cells
regenerate, in contrast to viral antigen that is cleared. Thus, we
measured the kinetics of transformation of autoreactive T cells
into Trm, their persistence over time, activation status, and
functional status. We found that PMEL upregulate CD69 and CD103
maximally by 7-8 weeks post vitiligo induction (FIGS. 3A and 3B).
In accordance with previous studies, PMEL Trm also downregulated
CCR7 and CD62L (Table 1). PMEL infiltration in the epidermis was
maximal between weeks 5-7, indicating ongoing inflammation and
active disease. After this time, the autoreactive T cell pool in
the epidermis contracted, and remaining cells were mostly of the
Trm phenotype; therefore, we considered any time point after 7
weeks the stable/Trm phase of disease. Mice maintained a pool of
approximately 25-50 PMEL per 10,000 live single epidermal cells
that were 60-80% CD69+CD103+ past 30 weeks. Total PMEL numbers in
the epidermis waned one-year post vitiligo induction, though the
proportion of Trm PMEL in that pool was maintained. We also found
distinct populations of melanocyte-specific Tcm/Tem (CD44+CD62L+)
in lymph nodes and spleen (summarized in Table 1).
[0141] We previously reported that vitiligo is dependent on
IFN.gamma. and IFN.gamma.-dependent chemokines (Agarwal et al.,
2015; Harris et al., 2012; Rashighi et al., 2014). Therefore, we
evaluated the effector function of autoreactive Trm in our model.
We bred PMEL mice to GREAT mice, which report IFN.gamma. expression
(Reinhardt et al., 2009). We found that PMEL Trm maintain
IFN.gamma. reporter expression up to 30 weeks post-vitiligo
induction (FIGS. 3C and 3D). By 60 weeks, IFN.gamma. reporter
expression was reduced to basal levels, possibly due to exhaustion
of the T cells, or depletion of the melanocyte stem cell reservoirs
resulting in reduced self-antigen load in the skin. Taken together,
these data indicate that PMEL Trm are long-lived and maintain
effector function for long periods.
TABLE-US-00002 TABLE 1 Phenotype characteristics of PMEL in
different tissues in the vitiligo mouse model. Marker Epidermis
Dermis Spleen Lymph Node Blood CD93 70% 60% 5% 25% 2% CD44low/+ 20%
50% 2.5% 12% 2.5% (55% CD93+) (54% CD93+) (60% CD93+) (75% CD93+)
(31% CD93+) KLRG1 4% 12% 34% 5% 58% CD52L 0% 0% 4% 13% 5% CD126 0%
2% 3% 5% 0% CCR5 23% 28% 14% 14% 2% PD-1 5% 3% 0% 1% 0% (all CCR5+)
CXCR3 1% 7% 52% 68% 85%
Example 4. Persistence of Depigmentation in Vitiligo Requires
Circulating Memory T Cells
[0142] Previous studies in virus models are conflicted as to the
function of Trm within tissues. Some studies report enhanced
effector function of Trm(Jiang et al., 2012), while others describe
primarily an alarm function that serves to efficiently recruit
effectors to sites of reinfection(Ariotti, 2014; Schenkel et al.,
2013). We previously reported that blocking CXCL10 not only
prevented the progression of vitiligo, but also reversed stable
disease, demonstrating that the chemokine continued to play an
important role even after melanocytes were destroyed and Trm became
established in the epidermis(Rashighi et al., 2014). This suggested
that Trm may not be sufficient for the memory observed in lesions
after treatment is discontinued. In addition, it suggested that
continued chemokine production is required to maintain
depigmentation in the lesions.
[0143] We evaluated the role of circulating PMEL populations in
maintenance of vitiligo using the S1P1 inhibitor FTY720, which
inhibits circulation of T cells from the lymph nodes, to determine
whether T cell recirculation is required during the maintenance of
vitiligo. We found that treatment with FTY720 resulted in rapid
reversal of disease (FIGS. 4A-G). In accordance with other studies
(Pinschewer et al., 2000), PMEL Trm were still present in the skin
after treatment. These data indicate that recirculating memory T
cells contribute to sustained melanocyte killing during the
maintenance of vitiligo, and that Trm are not sufficient effectors
for this function.
Example 5. IL-15143 is Required for the Function and Maintenance of
Trm in the Skin, and IL-15R11 Blockade Serves as a Durable
Treatment
[0144] Existing treatments for vitiligo are not durable, as the
disease returns at the site of treatment after discontinuation. The
persistence of Trm following FTY720 treatment indicates this also
will not be a durable therapy, as they remain in the tissue and
would likely regain their function after stopping the treatment.
Previous studies reported that IL-15 signaling was important for
the generation of skin Trm that reside in hair follicles (Adachi et
al., 2015), and we hypothesized that targeting this cytokine might
deplete Trm in the skin and result in long-lasting repigmentation.
We first confirmed that the majority of PMEL expressed the IL-15R
(CD122 & CD215 antibodies; FIG. 5). To test the impact of IL-15
on the survival and maintenance of the Trm population in the
epidermis, mice with longstanding, stable vitiligo (>12 weeks
after disease initiation) were treated with an anti-mouse
IL-15R.beta. antibody, which blocks IL-15 signaling. The antibody
used, ChMBC7, is a chimeric rat/mouse antibody with diminished
Fc-mediated effector functions. After 8 weeks of systemic
treatment, we saw significant repigmentation in treated mice
compared to controls (FIG. 6A-C). We found that PMEL were depleted
from the epidermis in longstanding lesions, and within the PMEL
population, Trm (CD69+, CD103+) were largely affected. There were
also fewer PMEL in the dermis, lymph nodes, spleen, and blood of
treated mice compared to controls (FIG. 6D). However, IL-15R.beta.
antibody treatment had small effects on host CD8+ T cell
populations, indicating that targeting this pathway may
preferentially have an effect on autoreactive cells (FIG. 6E).
[0145] To determine whether IL-15R.beta. blockade provided a
durable treatment option, we treated mice with IL-15R.beta.
antibody for only 2 weeks, then stopped treatment and observed
repigmentation over a full 8-week period. Treatment with this
short-term approach resulted in significant repigmentation over the
total 10-week period (FIG. 6F-I). Analysis of PMEL depletion
following short-term IL-15R.beta. antibody treatment revealed
variable effects in the lymph node, spleen, blood and skin (Table
2). Therefore, we performed a systemic functionality study in which
mice with vitiligo were dosed for 2 weeks, but PMEL were
restimulated in vitro with 3 .mu.g/mL plate bound anti-CD3 with 2
.mu.g/mL soluble anti-CD28. Cytokine production was assessed via
flow cytometry and we found that PMEL from the skin of mice treated
with anti-CD122 antibody made significantly less IFN.gamma. than
PMEL from control animals, indicating that short-term treatment
affects T cell function (FIG. 7A-C). To evaluate whether local
injection of antibody could induce a durable treatment response
similar to systemic administration, another durability study was
conducted using intradermal injection of IL-15R.beta. antibody. We
treated mice with established vitiligo with CD122 antibody
intradermally for 4 weeks (2 weeks at 5 .mu.g for loading dose, and
2 weeks at lug for maintenance dose), then stopped treatment and
observed repigmentation over a 10-week period. Similar to the
systemic durability study, experimental mice treated with
intradermal CD122 antibody had significant repigmentation compared
to control mice without depletion of PMEL T cells (FIG. 8). This
suggests that targeting IL-15 in the skin in vitiligo, unlike
existing therapies, could provide a durable treatment option for
patients, with longstanding effects after a limited treatment
course.
TABLE-US-00003 TABLE 2 PMEL analysis in IL-15R.beta. antibody
durability studies. IL-15R.beta. Average # P value antibody PMEL
normalized to 9{circumflex over ( )}4 summary Significant
lot/experiment population live singlets .+-. SEM (t test)
depletion? Lot A - Trm IL-15R.beta. Ab: 0.4067 No experiment 1
105.5 .+-. 34.28 Veh: 162.5 + 55.87 Tcm spleen IL-15R.beta. Ab:
*0.0283 Yes 6.53 .+-. 1.575 Veh: 34.31 + 9.31 Tcm lymph
IL-15R.beta. Ab: *0.0228 Yes node 21.38 .+-. 5.585 Veh: 68.73 +
18.88 Lot A - Trm IL-15R.beta. Ab: *0.0457 Yes experiment 2 54.63
.+-. 14.7 Veh: 164 + 44.08 Tcm spleen IL-15R.beta. Ab: 0.8065 No
9.83 .+-. 2.582 Veh: 9.35 + 4.018 Tcm lymph IL-15R.beta. Ab: 0.5602
No node 15.78 .+-. 5.838 Veh: 12.58 + 2.676 Summary lot A Trm
IL-15R.beta. Ab: *0.0308 Yes experiments 1 & 2 76.64 .+-. 17.43
Veh: 163.4 + 32.1 Tcm spleen IL-15R.beta. Ab: 0.1057 No 8.418 .+-.
1.575 Veh: 18.34 + 5.032 Tcm lymph IL-15R.beta. Ab: 0.1745 No node
17.34 .+-. 4.44 Veh: 36.7 + 14.03 Lot B - Trm IL-15R.beta. Ab:
0.1566 No experiment 3 218.1 .+-. 95.5 Veh: 54.18 + 21.71 Tcm
spleen IL-15R.beta. Ab: 0.3045 No 20.76 .+-. 5.577 Veh: 30.77 +
5.763 Tcm lymph IL-15R.beta. Ab: 0.391 No node 31.15 .+-. 5.635
Veh: 43.78 + 8.855 Summary of all Trm IL-15R.beta. Ab: 0.7767 No 3
experiments 134.6 .+-. 41.05 Veh: 126.5 + 25.56 Tcm spleen
IL-15R.beta. Ab: 0.0521 No 12.84 .+-. 2.465 Veh: 23.17 + 4.635 Tcm
lymph IL-15R.beta. Ab: 0.0851 No node 22.27 .+-. 3.714 Veh: 38.73 +
8.55
Example 6. Vitiligo Activity Assessment
[0146] The present inventors discovered that the
IFN-.gamma.-chemokine axis plays a critical role in the progression
and maintenance of vitiligo, and hypothesize that targeting this
axis will be an effective, new targeted treatment strategy (Harris
et al., 2012; Rashighi et al., 2014; Rashighi and Harris, 2015).
Serum levels of the IFN-.gamma.-dependent chemokine CXCL10 were
elevated in vitiligo patients compared to healthy controls
(Rashighi et al., 2014). Others have validated these findings in
additional vitiligo patients, and one study reported that serum
CXCL9 and CXCL10 levels correlated with disease activity and
decreased after systemic immunosuppression (Regazzetti et al.,
2015; Wang et al., 2016). Two vitiligo patients rapidly repigmented
following treatment with two different JAK inhibitors that both
interfere with IFN-.gamma. signaling (Craiglow and King, 2015;
Harris et al., 2016), and a case series reported that topical
ruxolitinib was effective for vitiligo patients (Rothstein et al.,
2017). One patient had elevated serum CXCL10 for over a year off of
treatment that corrected 2-3 months after starting treatment with
ruxolitinib, suggesting that targeted therapies could result in
detectable changes in components of IFN-.gamma.-induced
inflammatory markers.
[0147] However, a caveat of using serum cytokines as markers of
disease activity is that while a large group of vitiligo patients
(n=30 or more in existing studies) have average chemokine levels
statistically higher than healthy controls, many subjects have
undetectable levels and the standard deviation is high, precluding
identification of a "normal" vs. "abnormal" value. In addition,
serum cytokines are not organ- or disease-specific, so patients
with other sources of inflammation (multiple autoimmune diseases
are common in vitiligo patients) have elevated levels that are
unrelated to vitiligo disease activity (Antonelli et al., 2011;
Rotondi et al., 2005). Thus, serum chemokines are not sensitive or
specific for disease activity, and are therefore unlikely to be
reliable markers of treatment response. A reliable method to sample
lesional skin and reliably capture markers of inflammation is
needed.
[0148] Suction blister biopsies: To induce blisters in vitiligo
patient skin as a minimally invasive method to sample vitiligo
lesions, we used the NP-4 negative pressure instrument (Electronic
Diversities, Finksburg, Md.), which creates an adjustable suction
through a warmed aluminum orifice plate. Blisters form with minimal
discomfort in 30-45 minutes at the site of 1 cm openings in the
plate. Up to 4 chambers can be used at a time, each with up to 5
openings, for up to 20 blisters. Blister fluid is extracted through
a 30-gauge needle, and the blister roof is removed with iris
scissors and gradle forceps (FIGS. 9A-F). The roof was stored at
-80.degree. C., fluid centrifuged at 330.times.g for 5 minutes, and
the supernatant removed and stored at -80.degree. C. The cellular
pellet was resuspended in FACS buffer, cells stained with live/dead
blue (viability) and fluorescent antibodies against CD45 (immune
cells), CD3 (T cells), and CD8 (cytotoxic T cells), and analyzed on
a FACSCalibur flow cytometer. Stored blister fluid supernatant was
analyzed for IFN-.gamma., CXCL9, CXCL10, and CXCL11 by ELISA
(R&D Systems, Minneapolis, Minn.). The stored blister roof was
processed for RNA isolation (RNeasy, Qiagen).
[0149] CD8.sup.+ T cells and CXCL9 protein are elevated in lesional
skin: Eight subjects with clinical evidence of active vitiligo
lesions (confetti depigmentation, see (Sosa et al., 2015)) and off
all treatment participated (Strassner et al., 2017). Six blisters
were induced per patient: 2 in one lesion, 2 in a separate lesion,
and 2 in non-lesional skin. Seven healthy subjects contributed 3
blisters each in normal-appearing skin. Blister fluid CD8.sup.+ T
cells and total CD45.sup.+ cells were averaged between blisters. We
calculated the ratio of CD8.sup.+ T cells to 10.sup.4 total
CD45.sup.+ cells (CD8:CD45), which was elevated in lesional blister
fluid vs. non-lesional fluid in all subjects tested (p=0.005,
paired, 2-tailed T test), ranging from 2-fold to 25-fold. The
Receiver Operating Characteristic (ROC) curve is a method to
determine the sensitivity and specificity of an assay for
separating disease and control subjects. It can be used to set a
threshold value that best represents "normal" vs. "disease". The
ROC curve for CD8:CD45 in blister fluid was highly significant
(p=0.0002) and revealed that this test alone had decent sensitivity
(lesions consistently positive) and specificity (non-lesions
consistently negative) to separate vitiligo lesions from
non-lesional and healthy control skin: A threshold value of >912
CD8.sup.+ T cells had 83% sensitivity and 61% specificity for
detecting active lesional skin (FIGS. 10A-C). We also quantified T
cells without normalization, or normalized to total single cells or
blister fluid volume, but these were not significant, and lesional
blister roof T cells were not significantly different from
non-lesional in the subjects tested (not shown). This suggests that
the fluid CD8.sup.+ T cell:CD45.sup.+ cell ratio is the best method
for comparison.
[0150] Cytokine proteins in blister fluid were quantified by
enzyme-linked immunosorbent assay (ELISA). We found that
IFN-.gamma. and CXCL11 were undetectable in all lesions tested.
CXCL10 was inconsistently elevated in a small number of blisters,
but this was not statistically significant in the subjects tested
(not shown). However, CXCL9 protein was consistently elevated in
the majority of lesional blisters tested, and there was minimal
detectable CXCL9 protein in non-lesional or healthy control
blisters, suggesting that this could be a more sensitive and
specific marker for disease activity(Strassner et al., 2017).
Paired lesional:non-lesional comparison was statistically
significant in the subjects tested (p=0.03, paired, 2-tailed T
test), with increases ranging from 2-fold to many thousand-fold.
The ROC curve for CXCL9 blister fluid protein was highly
significant (p<0.0001) and revealed that this test alone had
good sensitivity and specificity to separate vitiligo lesions from
both non-lesional and healthy control skin: A threshold value of
>48 pg/ml CXCL9 had 83% sensitivity and 78% specificity for
detecting active lesional skin, while a threshold of >429 pg/ml
had 71% sensitivity and 97% specificity (FIGS. 11A-C). This
suggests that CXCL9 protein is the most sensitive and specific
cytokine in blister fluid for comparison.
[0151] In addition to obtaining blister roofs for gene expression
analysis as described above, samples could also be obtained by tape
stripping the skin and performing expression analysis on the
removed skin cells.
[0152] Gene expression in active lesional skin: We previously
published the gene expression profile of 5 vitiligo patients/5
controls (whole genome Illumina DASL), which we then validated in
an additional 8 patients/3 controls (Nanostring, inflammation code
set). This revealed an IFN-.gamma. signature in active lesional
skin that included the chemokines CXCL9, 10, and 11 as the genes
with most highly elevated expression (Rashighi et al., 2014).
Subsequent principal component (PC) analysis revealed a panel of 20
genes that efficiently separate diseased from control subjects.
This panel is comprised of 3 distinct gene sets: 1) markers of a
CD8.sup.+ T cell response, 2) markers of IFN-.gamma.-induced
inflammation, and 3) melanocyte-specific markers. Active lesional
skin reflects increased expression of CD8.sup.+ T cell genes,
increased expression of IFN-.gamma.-induced genes, and decreased
expression of melanocyte-specific genes, and each set is reflected
by a different axis (FIGS. 12A-B). We hypothesized that CD8.sup.+ T
cell genes and IFN-.gamma.-induced genes will decrease while
melanocyte-specific genes increase following treatment. We have
isolated RNA from blister roofs and demonstrated that it is of high
integrity (high RNA integrity number on Bioanalyzer analysis) and
provides good material for RT-PCR analysis of housekeeping genes
(not shown).
[0153] Vitiligo biomarkers: The vitiligo biomarkers to be used to
assess patient disease activity are comprised of 3 parameters: 1)
CD8.sup.+ T cell numbers normalized to CD45.sup.+ immune cells in
blister fluid; 2) CXCL9 protein concentration (pg/ml) in blister
fluid; and 3) 20 differentially expressed genes in the epidermal
blister roof, although either of these parameters could be used as
markers of disease activity alone, as well(Strassner et al., 2017).
The panel of differentially expressed genes can be quantified into
a single score by calculating the Mahalanobis distance across the
expression data, an approach that not only takes into account the
direction of the expression (so that a positive expression does not
cancel out a negative expression) but also the covariance of the
gene expressions. The Mahalanobis distance accounts for the
variance of each variable and the covariance between variables by
transforming the data into standardized uncorrelated data and
computing the ordinary Euclidean distance for the transformed data.
In this way, the Mahalanobis distance is like a univariate z-score:
it provides a way to measure distances that takes into account the
scale of the data. We use an approach such as Stouffer's Z-score
method (essentially an average of the z-scores) to combine the
z-scores into a single z-score for the gene expression panel. Each
parameter (CD8+ T cell numbers and CXCL9 protein concentration) in
lesional skin will be divided by the parameter in non-lesional
skin, resulting in a fold change in lesional vs. non-lesional skin.
All three parameters can be compared to assess disease activity by
transforming the fold-change results into z-scores and then again
using Stouffer's z-score method to combine them into a single
z-score for each subject.
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OTHER EMBODIMENTS
[0251] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *