U.S. patent application number 14/063844 was filed with the patent office on 2014-09-25 for antisense compositions and methods for modulating contact hypersensitivity or contact dermatitis.
This patent application is currently assigned to SAREPTA THERAPEUTICS, INC.. The applicant listed for this patent is SAREPTA THERAPEUTICS, INC.. Invention is credited to Patrick L. Iversen, Nikki B. Marshall, Dan V. Mourich.
Application Number | 20140287983 14/063844 |
Document ID | / |
Family ID | 42124650 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287983 |
Kind Code |
A1 |
Mourich; Dan V. ; et
al. |
September 25, 2014 |
ANTISENSE COMPOSITIONS AND METHODS FOR MODULATING CONTACT
HYPERSENSITIVITY OR CONTACT DERMATITIS
Abstract
Provided are methods and compositions, including topical
compositions, for inducing tolerance to a sensitizing agent known
to provoke contact hypersensitivity in a subject. Included are
methods of topically applying to the subject an effective amount of
an antisense composition targeting the start site or splice site of
a CFLAR mRNA.
Inventors: |
Mourich; Dan V.; (Albany,
OR) ; Marshall; Nikki B.; (Corvallis, OR) ;
Iversen; Patrick L.; (Corvallis, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAREPTA THERAPEUTICS, INC. |
CORVALLIS |
OR |
US |
|
|
Assignee: |
SAREPTA THERAPEUTICS, INC.
CORVALLIS
OR
|
Family ID: |
42124650 |
Appl. No.: |
14/063844 |
Filed: |
October 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12641159 |
Dec 17, 2009 |
8592386 |
|
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14063844 |
|
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61138460 |
Dec 17, 2008 |
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Current U.S.
Class: |
514/1.2 ;
514/44A |
Current CPC
Class: |
A61K 38/19 20130101;
C12N 2310/11 20130101; C12N 2310/3513 20130101; A61K 45/06
20130101; C12N 2320/32 20130101; C12N 15/1135 20130101; A61K
31/7125 20130101; C12N 15/1136 20130101; A61P 17/00 20180101 |
Class at
Publication: |
514/1.2 ;
514/44.A |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 45/06 20060101 A61K045/06; A61K 38/19 20060101
A61K038/19; A61K 31/7125 20060101 A61K031/7125 |
Claims
1. A method of inducing tolerance to a sensitizing agent,
comprising topically applying to a subject, an effective amount of
an antisense composition containing an antisense oligonucleotide,
wherein the oligonucleotide contains morpholino subunits and
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit, between 12-40 nucleotide bases, and a base sequence
effective to hybridize to at least 12 contiguous bases of a target
sequence contained within SEQ ID NO:11, wherein the oligonucleotide
binding to the target sequence is effective to reduce expression of
a functional human CFLAR in CFLAR-expressing lymphocytes.
2. The method of claim 1, wherein the antisense oligonucleotide
comprises a cell-penetrating peptide capable of enhancing uptake of
the oligonucleotide into activated T cells.
3. The method of claim 1, comprising continuing said applying step
on a periodic basis to reduce skin or mucous membrane inflammation
resulting from contact with the agent.
4. The method of claim 1, wherein the composition is applied to a
skin area of the subject prior to contact or after contact with the
sensitizing agent.
5. (canceled)
6. The method of claim 1, wherein the sensitizing agent is i) skin
or mucous membrane irritant selected from the group consisting of
an acid, an alkali, a solvent, a heavy metal, rubber, latex, a
cosmetic, and a fragrance, or ii) a skin allergen from a plant
containing urushiol oil.
7. (canceled)
8. The method of claim 6, wherein the plant is selected from the
group consisting poison oak, poison ivy, and poison sumac.
9. The method of claim 1, wherein the composition comprises a
carrier or delivery vehicle for topical administration.
10. (canceled)
11. The method of claim 1, wherein the oligonucleotide in the
conjugate has a base sequence effective to hybridize to at least 12
contiguous bases of a target sequence contained within SEQ ID
NO:12.
12. The method of claim 2, wherein the cell-penetrating peptide is
an arginine-rich peptide.
13. (canceled)
14. The method of claim 1, wherein the morpholino subunits in the
oligonucleotide are joined by phosphorodiamidate linkages, in
accordance with the structure: ##STR00006## where Y.sub.1.dbd.,
Z.dbd.O, Pj is a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino
e.g., wherein X.dbd.NR.sub.2, where each R is independently
hydrogen or methyl.
15. The method of claim 14, wherein the intersubunit linkages,
which are uncharged, are interspersed with linkages that are
positively charged at physiological pH, where the total number of
positively charged linkages is between 2 and no more than half of
the total number of linkages.
16. The method of claim 15, wherein the positively charged linkages
have a phosphorodiamidate structure in which X is 1-piperazine.
17. A composition adapted for topical administration, comprising an
antisense oligonucleotide, wherein the oligonucleotide contains
morpholino subunits and phosphorus-containing intersubunit linkages
joining a morpholino nitrogen of one subunit to a 5' exocyclic
carbon of an adjacent subunit, between 12-40 nucleotide bases, and
a base sequence effective to hybridize to at least 12 contiguous
bases of a target sequence contained within SEQ ID NO:11, and a
delivery vehicle or carrier for topical uptake of the
composition.
18. The composition of claim 17, wherein the antisense
oligonucleotide comprises a cell-penetrating peptide capable of
enhancing uptake of the oligonucleotide into activated T cells.
19. (canceled)
20. The composition of claim 17, wherein the oligonucleotide has a
base sequence effective to hybridize to at least 12 contiguous
bases of a target sequence contained within SEQ ID NO:12.
21. The composition of claim 18, wherein the cell-penetrating
peptide is an arginine-rich peptide.
22. (canceled)
23. The composition claim 17, wherein the morpholino subunits in
the oligonucleotide are joined by phosphorodiamidate linkages, in
accordance with the structure: ##STR00007## where Y.sub.1.dbd.,
Z.dbd.O, Pj is a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino
e.g., wherein X.dbd.NR.sub.2, where each R is independently
hydrogen or methyl.
24. The composition of claim 23, wherein the intersubunit linkages,
which are uncharged, are interspersed with linkages that are
positively charged at physiological pH, where the total number of
positively charged linkages is between 2 and no more than half of
the total number of linkages.
25. The composition of claim 24, wherein the positively charged
linkages have a phosphorodiamidate structure in which X is
1-piperazine.
26. The composition of claim 17, further comprising a sensitizing
agent.
27-36. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 12/641,159, filed Dec. 17, 2009 (now allowed); which claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application No. 61/138,460 filed Dec. 17, 2008; both of these
applications are incorporated herein by reference in their
entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120178.sub.--409C1_SEQUENCE_LISTING.txt. The text file is 10 KB,
was created on Oct. 23, 2013, and is being submitted electronically
via EFS-Web, concurrent with the filing of the specification.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and antisense
compounds for modulating contact hypersensitivity responses induced
by exposure to antigens, including haptens or metal ions complexed
with cellular proteins.
REFERENCES
[0004] The following references are cited in the Background or
Methods sections of this application. [0005] Brand, R. M. (2001).
"Topical and transdermal delivery of antisense oligonucleotides."
Curr Opin Mol Ther 3(3): 244-8. [0006] Brand, R. M. and P. L.
Iversen (2000). "Transdermal delivery of antisense compounds." Adv
Drug Deliv Rev 44(1): 51-7. [0007] Isomura, I., K. Tsujimura, et
al. (2006). "Antigen-specific peripheral tolerance induced by
topical application of NF-kappaB decoy oligodeoxynucleotide." J
Invest Dermatol 126(1): 97-104. [0008] Kirchhoff, S., W. W. Muller,
et al. (2000). "TCR-mediated up-regulation of c-FLIPshort
correlates with resistance toward CD95-mediated apoptosis by
blocking death-inducing signaling complex activity." J Immunol
165(11): 6293-300. [0009] Lazou, K., N. S. Sadick, et al. (2007).
"The use of antisense strategy to modulate human melanogenesis." J
Drugs Dermatol 6(6 Suppl): s2-7. [0010] Leung, D. Y., L. A. Diaz,
et al. (1997). "Allergic and immunologic skin disorders." Jama
278(22): 1914-23. [0011] Marshall, N. B., S. K. Oda, et al. (2007).
"Arginine-rich cell-penetrating peptides facilitate delivery of
antisense oligomers into murine leukocytes and alter pre-mRNA
splicing." J Immunol Methods 325(1-2): 114-26. [0012] Merk, H. F.,
J. M. Baron, et al. (2006). "Concepts in molecular
dermatotoxicology." Exp Dermatol 15(9): 692-704. [0013] Mourich, D.
V., S. K. Oda, et al. (2007). "Ligand independent form of CTLA-4
induced by antisense EXON skipping in NOD mouse inhibits autoimmune
diabetes." J Exp Med(In Press). [0014] Perlman, H., L. J. Pagliari,
et al. (1999). "FLICE-inhibitory protein expression during
macrophage differentiation confers resistance to fas-mediated
apoptosis." J Exp Med 190(11): 1679-88. [0015] Regnier, V., T. Le
Doan, et al. (1998). "Parameters controlling topical delivery of
oligonucleotides by electroporation." J Drug Target 5(4): 275-89.
[0016] Saint-Mezard, P., F. Berard, et al. (2004). "The role of
CD4+ and CD8+ T cells in contact hypersensitivity and allergic
contact dermatitis." Eur J Dermatol 14(3): 131-8. [0017]
Saint-Mezard, P., M. Krasteva, et al. (2003). "Afferent and
efferent phases of allergic contact dermatitis (ACD) can be induced
after a single skin contact with haptens: evidence using a mouse
model of primary ACD." J Invest Dermatol 120(4): 641-7. [0018]
Saint-Mezard, P., A. Rosieres, et al. (2004). "Allergic contact
dermatitis." Eur J Dermatol 14(5): 284-95. [0019] Schmitz, I., H.
Weyd, et al. (2004). "Resistance of short term activated T cells to
CD95-mediated apoptosis correlates with de novo protein synthesis
of c-FLIPshort." J Immunol 172(4): 2194-200. [0020] Stein, C. A.
and J. S. Cohen (1989). Phosphorothioate Oligodeoxynucleotide
Analogues. Boca Raton, Fla., CRC Press. [0021] Thorburn, A. (2004).
"Death receptor-induced cell killing." Cell Signal 16(2): 139-44.
[0022] Wang, J., A. A. Lobito, et al. (2000). "Inhibition of
Fas-mediated apoptosis by the B cell antigen receptor through
c-FLIP." Eur J Immunol 30(1): 155-63.
BACKGROUND OF THE INVENTION
[0023] Contact dermatitis is responsible for over 5.6 million
doctor visits each year in the United States and accounts for
15-20% of all occupational diseases. Including lost workdays and
loss of productivity, the estimated total annual costs associated
with occupational skin diseases approach $1 billion annually in the
United States (CDC National Institute of Occupational Health,
Update July 1997) and up to $3 billion annually in Germany (Merk,
Baron et al. 2006). Eighty percent of contact dermatitis instances
are due to irritants while in the other 20% the compound induces an
immunologic cascade and are classified as allergic (Leung, Diaz et
al. 1997).
[0024] Contact dermatitis and many hypersensitivity reactions of
the skin are produced by haptens, in the form of low molecular
weight molecules or metal ions, complexing with cellular proteins.
Subsequently these are processed into peptides and presented on the
surface of antigen-presenting cells (APCs), typically Langerhans
cells, the principle APC of the skin, residing in the epidermis
(Saint-Mezard, Krasteva et al. 2003). Once Langerhans undergo
maturation they migrate to the regional lymph node and present
hapten-modified peptides in the context of major histocompatibility
class I and II molecules to hapten-specific CD8+ and CD4+ T cells,
respectively (Saint-Mezard, Berard et al. 2004). Antigen-specific
activation of T cells constitutes the sensitization phase of
contact sensitivity responses. Upon subsequent exposure to hapten,
the challenge phase, effector memory T cells migrate to the
peripheral tissues harboring hapten-presenting APCs. Here antigen
recognition induces the T cells to express various mediators of
inflammation and cytotoxicity, ultimately causing dermatitis and
tissue damage (Saint-Mezard, Rosieres et al. 2004).
[0025] One of the anti-apoptotic proteins that is upregulated in T
cells following T-cell activation is CFLAR (Kirchhoff, Muller et
al. 2000). T cells that upregulate CFLAR as a consequence of T-cell
receptor (TCR) engagement are resistant to Fas-mediated apoptosis.
It has been clearly established that this CFLAR associated
resistance correlates with de novo protein synthesis of CFLAR.sub.S
(Schmitz, Weyd et al. 2004). In these studies, it was also shown
that CFLAR exerted its anti-apoptotic effect by blocking DISC
activity. Increased expression of CFLAR is also seen following
cross-linking of the B-cell receptor for antigen. In this case the
upregulation was seen in the levels of CFLAR.sub.L and was also
associated with inhibition of Fas-mediated apoptosis (Wang, Lobito
et al. 2000). CFLAR expression levels are also associated with the
resistance to apoptosis that is seen following monocyte to
macrophage differentiation (Perlman, Pagliari et al. 1999). It
appears that CFLAR is commonly upregulated as a first step to
prevent Fas-mediated apoptosis following signals for subsequent
cell differentiation.
[0026] Although the signaling pathways associated with apoptosis
and immunoregulation are complex and incompletely understood, CFLAR
is one anti-apoptotic molecule that appears to play an important
role in cell survival especially following death receptor ligation
(Thorburn 2004).
[0027] A disclosure of antisense targeting of CFLAR, as shown in
Mourich, et al (US20050203041 and WO2005030799), describes the use
of such compounds to treat transplantation rejection and autoimmune
conditions. The circulating T cells targeted by the methods and
compositions of US20050203041 are activated by alloantigens that
induce a graft versus host response in the case of transplantation
and hyper-activated T cells responding to self antigens in the case
of autoimmune conditions.
[0028] Accordingly, given the absence of a sufficient number of
interventions for combating contact hypersensitivity, the present
invention solves this deficiency while providing other related
advantages.
SUMMARY OF THE INVENTION
[0029] The present invention is based in part on the discoveries
that targeting CFLAR expression in cells circulating in the
epidermal region of a subject is effective to produce tolerance to
a sensitizing agent, including an agent known to provoke contact
hypersensitivity such as contact dermatitis; and that such
targeting can be achieved by topical delivery to the subject of an
antisense oligonucleotide such as a morpholino oligonucleotide. In
certain embodiments, the oligonucleotide may be conjugated to
cell-penetrating peptide, such as an arginine-rich peptide, and
delivered in a suitable topical delivery vehicle.
[0030] The invention includes, in one aspect, a method of inducing
tolerance to a sensitizing agent known to provoke contact
hypersensitivity in a subject. The method includes topically
applying to the subject, an effective amount of an antisense
composition containing, in a suitable topical delivery vehicle, an
antisense oligonucleotide containing between 12-40 nucleotide bases
and having a base sequence effective to hybridize to at least 12
contiguous bases of a target sequence contained within SEQ ID
NO:11, wherein the oligonucleotide binding to the target sequence
is effective to block normal expression of a functional human CFLAR
in CFLAR-expressing lymphocytes.
[0031] In certain embodiments, the antisense oligonucleotide is
composed of morpholino subunits and phosphorus-containing
intersubunit linkages joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. In certain
embodiments, the oligonucleotide comprises an cell-penetrating
peptide such as an arginine-rich peptide that enhances uptake of
the oligonucleotide into activated T cells in culture.
[0032] The method may further include continuing the applying step
on a periodic basis as long as needed to reduce skin or mucous
membrane inflammation resulting from contact with the agent.
[0033] Where the sensitizing agent to which the subject is exposed
is a skin irritant, such as an acid, an alkali such as a soap or
detergent, a solvent, a heavy metal, rubber, a cosmetic, or a
fragrance, the composition in certain embodiments may be applied to
the skin area of the subject expected to come into contact with the
irritant.
[0034] Where the sensitizing agent to which the subject is exposed
is a skin allergen, such as poison oak, poison ivy, poison sumac,
or other plants containing urushiol oil, the composition in certain
embodiments may be applied to the skin area of the subject expected
to come into contact with the allergen.
[0035] In certain embodiments, the delivery vehicle in the
composition may include propylene glycol and an acyl-chain lipid,
such as a fatty acid or phospholipids. One exemplary acyl-chain
lipid is linoleic acid.
[0036] In certain embodiments, the oligonucleotide in the conjugate
may have a base sequence effective to hybridize to at least 12
contiguous bases of a target sequence contained within SEQ ID
NO:12. The arginine-rich peptide in the conjugate may have a
sequence identified by any one of SEQ ID NO:1-10.
[0037] In certain embodiments, the morpholino subunits in the
compound may be joined by phosphorodiamidate linkages, in
accordance with the structure:
##STR00001##
where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, or alkyl amino e.g., wherein X.dbd.NR.sub.2, where each
R is independently hydrogen or methyl. The intersubunit linkages,
which are uncharged, may be interspersed with linkages that are
positively charged at physiological pH, where the total number of
positively charged linkages is between 2 and no more than half of
the total number of linkages. An exemplary positively charged
linkage is the above phosphorodiamidate structure in which X is
1-piperazine.
[0038] Certain embodiments include compositions for use in treating
contact hypersensitivity in a subject, by topical application of
the composition to an effected skin area of the subject. In certain
embodiments, the composition includes an antisense oligonucleotide
containing between 12-40 nucleotide bases and having a base
sequence effective to hybridize to at least 6 contiguous bases of a
target sequence contained within SEQ ID NO:11, where
oligonucleotide binding to the target sequence is effective to
block normal expression of a functional human CFLAR in
CFLAR-expressing lymphocytes exposed to the oligonucleotide.
[0039] In certain embodiments, the antisense oligonucleotide is
composed of morpholino subunits and phosphorus-containing
intersubunit linkages joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. In certain
embodiments, the oligonucleotide is a conjugate that comprises an
arginine-rich peptide capable of enhancing uptake of the
oligonucleotide into activated T cells in culture.
[0040] In still another aspect, the invention includes a method of
achieving transdermal uptake of an antisense oligonucleotide into
target cells in the epidermis, by applying to the skin or mucous
membrane of a subject, an antisense composition comprising an
antisense oligonucleotide that is conjugated to a cell-penetrating
peptide as described herein. Exemplary embodiments of the method
are as noted above.
[0041] Certain embodiments include methods of treating contact
hypersensitivity, comprising contacting the skin or mucous membrane
of a subject with an effective amount of an antisense composition
containing an antisense oligonucleotide as described herein,
wherein the oligonucleotide reduces expression of a functional
human CFLAR in CFLAR-expressing lymphocytes. In certain
embodiments, the oligonucleotide is a peptide nucleic acid (PNA), a
locked nucleic acid (LNA), an RNA interference agent with a duplex
region, or a morpholino oligomer.
[0042] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIGS. 1A-1E show exemplary backbone linkages in a morpholino
oligomer;
[0044] FIGS. 2A and 2B show a conjugate of an arginine-rich peptide
and an uncharged PMO oligomer (2A), and a conjugate of an
arginine-rich peptide and a PMO have uncharged linkages and two
different types of positively charged linkages (2B);
[0045] FIG. 3 shows inhibition of CFLAR protein expression and
detection of a higher molecular weight stress-induced insoluble
aggregate of GAPDH in activated T cells treated with CFLAR PPMO
(SEQ ID NO: 28);
[0046] FIGS. 4A and 4B show inhibition of FITC-induced dermatitis
in mice with topically applied CFLAR PPMO. Topical application of
CFLAR PPMO (SEQ ID NO: 28) caused a dose-dependent inhibition of
initial FITC-induced DTH (FIG. 4A) and FITC-induced memory response
at 15 days post initial FITC challenge and topical application of
CFLAR PPMO (FIG. 4B).
[0047] FIGS. 5A-5E show reduction of leukocyte numbers as
illustrated by a plot (FIG. 5A) and as seen by histological
examination in (FIGS. 5B-5E) under various treatment
conditions;
[0048] FIGS. 6A-6D show by immunohistochemical examination, the
reduction in CFLAR positive cells under various treatment
conditions; and
[0049] FIGS. 7A and 7B show inhibition of oxazolone-induced
dermatitis in mice with topically applied CFLAR PPMO (SEQ ID NO:
28). Topical application of CFLAR PPMO caused inhibition of initial
oxazolone-induced delayed-type hypersensitivity (FIG. 7A), and
oxazolone-induced memory response at 15 days post initial oxazolone
challenge and topical application of CFLAR PPMO (FIG. 7B).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0051] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0052] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0053] By "coding sequence" is meant any nucleic acid sequence that
contributes to the code for the polypeptide product of a gene. By
contrast, the term "non-coding sequence" refers to any nucleic acid
sequence that does not contribute to the code for the polypeptide
product of a gene.
[0054] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises," and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0055] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of:" Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they materially
affect the activity or action of the listed elements.
[0056] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing rules. For example, the sequence "A-G-T," is
complementary to the sequence "T-C-A." Polynucleotides are
described as "complementary" to one another when hybridization
occurs in an antiparallel configuration between two single-stranded
polynucleotides. Complementarity (the degree that one
polynucleotide is complementary with another) is quantifiable in
terms of the proportion of bases in opposing strands that are
expected to form hydrogen bonds with each other, according to
generally accepted base-pairing rules.
[0057] Complementarity may be "partial," in which only some of the
nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. While
perfect complementarity is often desired, some embodiments can
include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches
with respect to the target RNA. Variations at any location within
the oligomer are included. In certain embodiments, variations in
sequence near the termini of an oligomer are generally preferable
to variations in the interior, and if present are typically within
about 6, 5, 4, 3, 2, or 1 nucleotides of the 5' and/or 3'
terminus.
[0058] The terms "cell penetrating peptide" or "CPP" are used
interchangeably and refer to cationic cell penetrating peptides,
also called transport peptides, carrier peptides, or peptide
transduction domains. The peptides, as illustrated, have the
capability of inducing cell penetration within 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 100% of cells of a given cell culture population,
including all integers in between, and allow macromolecular
translocation within multiple tissues in vivo upon systemic
administration. A cell-penetrating peptide may enhance uptake of
the oligonucleotide into T-cells, including activated T-cells,
quiescent T-cells, or both. A peptide may be an arginine-rich
peptide, including the peptides in SEQ ID NOS:1-10.
[0059] The terms "antisense oligomer" or "antisense
oligonucleotide" or "antisense compound" are used interchangeably
and refer to a sequence of subunits, each having a base carried on
a backbone subunit composed of ribose or other pentose sugar or
morpholino group, and where the backbone groups are linked by
intersubunit linkages that allow the bases in the compound to
hybridize to a "target sequence" in a nucleic acid (typically an
RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer
heteroduplex within the target sequence. The cyclic subunits may be
based on ribose or another pentose sugar or, in certain
embodiments, a morpholino group (see description of morpholino
oligomers below). Included are single-stranded antisense oligomers,
and antisense oligomers having at least one duplex or
double-stranded region. Also included are peptide nucleic acids
(PNAs), locked nucleic acids (LNAs), RNA interference agents (e.g.,
siRNA agents), and other antisense agents known in the art.
[0060] The oligomer may have exact sequence complementarity to the
target sequence or near complementarity. Such antisense compounds
are designed to block or inhibit translation of the mRNA containing
the target sequence or designed to block pre-mRNA processing (i.e.,
splicing) and may be said to be "directed to" a sequence with which
it hybridizes. Antisense oligonucleotides and oligonucleotide
analogs may contain between about 8 and 40 subunits, typically
about 8-25 subunits, and preferably about 12 to 25 subunits.
[0061] Antisense oligomers can be designed to block or inhibit
translation of mRNA or to inhibit natural pre-mRNA splice
processing, or induce degradation of targeted mRNAs, and may be
said to be "directed to" or "targeted against" a target sequence
with which it hybridizes. In certain embodiments, the target
sequence includes a region including an AUG start codon of a CFLAR
mRNA, a 3' or 5' splice site of a pre-processed CFLAR mRNA, or a
branch point of a pre-processed CFLAR mRNA. The target sequence may
be within an exon or within an intron. The target sequence for a
splice site may include an mRNA sequence having its 5' end 1 to
about 25 base pairs downstream of a normal splice acceptor junction
in a preprocessed mRNA. A preferred target sequence for a splice is
any region of a preprocessed mRNA that includes a splice site or is
contained entirely within an exon coding sequence or spans a splice
acceptor or donor site.
[0062] Included are antisense oligonucleotides that comprise,
consist essentially of, or consist of one or more of SEQ ID
NOS:23-33. Also included are variants of these antisense oligomers,
including variant oligomers having 80%, 85%, 90%, 95%, 97%, 98%, or
99% (including all integers in between) sequence identity or
sequence homology to any one of SEQ ID NOS: 23-33, and/or variants
that differ from these sequences by about 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 nucleotides, preferably those variants that reduce CFLAR
expression in a cell such as a T-cell. Also included are
oligonucleotides of any one or more of SEQ ID NOS: 23-33, which
comprise a suitable number of charged linkages, as described
herein, e.g., up to about 1 per every 2-5 uncharged linkages, such
as about 4-5 per every 10 uncharged linkages, and/or which comprise
an Arg-rich peptide attached thereto, as also described herein.
[0063] A "morpholino oligomer" refers to a polymeric molecule
having a backbone which supports bases capable of hydrogen bonding
to typical polynucleotides, wherein the polymer lacks a pentose
sugar backbone moiety, and more specifically a ribose backbone
linked by phosphodiester bonds which is typical of nucleotides and
nucleosides, but instead contains a ring nitrogen with coupling
through the ring nitrogen. A preferred "morpholino" oligomer is
composed of morpholino subunit structures linked together by
phosphoramidate or phosphorodiamidate linkages, joining the
morpholino nitrogen of one subunit to the 5' exocyclic carbon of an
adjacent subunit, each subunit including a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide. Morpholino oligomers
(including antisense oligomers) are detailed, for example, in
co-owned U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,
5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are
expressly incorporated by reference herein.
[0064] A "phosphoramidate" group comprises phosphorus having three
attached oxygen atoms and one attached nitrogen atom, while a
"phosphorodiamidate" group (see e.g. FIGS. 1A-B) comprises
phosphorus having two attached oxygen atoms and two attached
nitrogen atoms. In the uncharged or the cationic intersubunit
linkages of the oligomers described herein, one nitrogen is always
pendant to the backbone chain. The second nitrogen in a
phosphorodiamidate linkage is typically the ring nitrogen in a
morpholino ring structure (again, see FIGS. 1A-B). A
phosphoramidate or phosphorodiamidate linkage may include a
thiophosphoramidate or thiophosphorodiamidate linkage,
respectively, in which one oxygen atom, typically the oxygen
pendant to the backbone in the oligomers described herein, is
replaced with sulfur.
[0065] The terms "uncharged" and "cationic" are used herein to
refer to the predominant charge state of a backbone linking groups
in an antisense compound at near-neutral pH, e.g. about 6 to 8.
Preferably, the term refers to the predominant state of the
chemical moiety at physiological pH, that is, about 7.4. A
"substantially uncharged," phosphorus containing backbone in an
oligonucleotide analog is one in which a majority of the subunit
linkages, e.g., between 50-100%, typically at least 60% to 100% or
75% or 80% of its linkages, are uncharged at physiological pH, and
contain a single phosphorous atom.
[0066] A "subunit" of an oligonucleotide refers to one nucleotide
(or nucleotide analog) unit. The term may refer to the nucleotide
unit with or without the attached intersubunit linkage, although,
when referring to a "charged subunit", the charge typically resides
within the intersubunit linkage (e.g., a phosphate or
phosphorothioate linkage or a cationic linkage).
[0067] The purine or pyrimidine base pairing moiety is typically
adenine, cytosine, guanine, uracil, thymine or inosine. Also
included are bases such as pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2,4,6-trime115thoxy benzene, 3-methyl uracil,
dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,
5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or
6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine,
2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,
4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,
5''-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, .beta.-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonylmethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine,
.beta.-D-mannosylqueosine, uridine-5-oxyacetic acid,
2-thiocytidine, threonine derivatives and others (Burgin et al.,
1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By
"modified bases" in this aspect is meant nucleotide bases other
than adenine (A), guanine (G), cytosine (C), thymine (T), and
uracil (U), as illustrated above; such bases can be used at any
position in the antisense molecule. Persons skilled in the art will
appreciate that depending on the uses of the oligomers, Ts and Us
are interchangeable. For instance, with other antisense chemistries
such as 2'-O-methyl antisense oligonucleotides that are more
RNA-like, the T bases may be shown as U (see, e.g., Sequence
Listing).
[0068] An "amino acid subunit" or "amino acid residue" can refer to
an .alpha.-amino acid residue (--CO--CHR--NH--) or a .beta.- or
other amino acid residue (e.g., --CO--(CH.sub.2).sub.nCHR--NH--),
where R is a side chain (which may include hydrogen) and n is 1 to
6, preferably 1 to 4.
[0069] The term "naturally occurring amino acid" refers to an amino
acid present in proteins found in nature, such as the 20 (L)-amino
acids utilized during protein biosynthesis as well as others such
as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,
homocysteine, citrulline and ornithine. The term "non-natural amino
acids" refers to those amino acids not present in proteins found in
nature, examples include beta-alanine (.beta.-Ala), 6-aminohexanoic
acid (Ahx) and 6-aminopentanoic acid. Additional examples of
"non-natural amino acids" include, without limitation, (D)-amino
acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and
the like, which are known to a person skilled in the art.
[0070] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide" or
"isolated oligonucleotide," as used herein, may refer to a
polynucleotide that has been purified or removed from the sequences
that flank it in a naturally-occurring state, e.g., a DNA fragment
that has been removed from the sequences that are normally adjacent
to the fragment.
[0071] A first sequence is an "antisense sequence" with respect to
a second sequence if a polynucleotide whose sequence is the first
sequence specifically binds to, or specifically hybridizes with,
the second polynucleotide sequence under physiological
conditions.
[0072] The term "target sequence" refers to a portion of the target
RNA against which the oligonucleotide or antisense agent is
directed, that is, the sequence to which the oligonucleotide will
hybridize by Watson-Crick base pairing of a complementary
sequence.
[0073] The term "targeting sequence" is the sequence in the
oligonucleotide analog that is complementary (meaning, in addition,
substantially complementary) to the "target sequence" in either the
mature CFLAR mRNA or a pre-processed mRNA transcript, and
specifically the pre-processed mRNA transcript of the human CFLAR
gene. The entire targeting sequence, or only a portion, of the
compound may be complementary to the target sequence. For example,
in an antisense compound having about 10-40 bases, about 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40
may be targeting sequences. Typically, the targeting sequence is
formed of contiguous bases in the compound, but may alternatively
be formed of non-contiguous sequences that when placed together,
e.g., from opposite ends of the compound, constitute sequence that
spans the target sequence.
[0074] Target and targeting sequences are described as
"complementary" to one another when hybridization occurs in an
antiparallel configuration. A targeting sequence may have "near" or
"substantial" complementarity to the target sequence and still
function for the purpose of the presently described methods, that
is, still be "complementary." Preferably, the oligonucleotide
analog compounds employed in the presently described methods have
at most one mismatch with the target sequence out of 10
nucleotides, and preferably at most one mismatch out of 20.
Alternatively, the antisense compounds employed have at least 90%
sequence homology, and preferably at least 95% sequence homology,
with the exemplary targeting sequences as designated herein. For
purposes of complementary binding to an RNA target, and as
discussed below, a guanine base may be complementary to either an
adenine or uracil RNA base.
[0075] An oligonucleotide "specifically hybridizes" to a target
polynucleotide if the oligomer hybridizes to the target under
physiological conditions, with a T.sub.m substantially greater than
45.degree. C., preferably at least 50.degree. C., and typically
60.degree. C.-80.degree. C. or higher. Such hybridization
preferably corresponds to stringent hybridization conditions. At a
given ionic strength and pH, the T.sub.m is the temperature at
which 50% of a target sequence hybridizes to a complementary
polynucleotide. Again, such hybridization may occur with "near" or
"substantial" complementary of the antisense compound to the target
sequence, as well as with exact complementarity.
[0076] "Homology" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions.
Homology may be determined using sequence comparison programs such
as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395).
In this way sequences of a similar or substantially different
length to those cited herein could be compared by insertion of gaps
into the alignment, such gaps being determined, for example, by the
comparison algorithm used by GAP.
[0077] The recitations "sequence identity" or, for example,
comprising a "sequence 50% identical to," as used herein, refer to
the extent that sequences are identical on a
nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis
over a window of comparison. Thus, a "percentage of sequence
identity" may be calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,
Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu,
Asn, Gln, Cys and Met) occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity.
[0078] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence,"
"comparison window," "sequence identity," "percentage of sequence
identity," and "substantial identity". A "reference sequence" is at
least 8 or 10 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0079] A "nuclease-resistant" oligomeric molecule (oligomer) refers
to one whose backbone is substantially resistant to nuclease
cleavage, in non-hybridized or hybridized form; by common
extracellular and intracellular nucleases in the body; that is, the
oligomer shows little or no nuclease cleavage under normal nuclease
conditions in the body to which the oligomer is exposed.
[0080] A "heteroduplex" refers to a duplex between an
oligonucleotide analog and the complementary portion of a target
RNA. A "nuclease-resistant heteroduplex" refers to a heteroduplex
formed by the binding of an antisense compound to its complementary
target, such that the heteroduplex is substantially resistant to in
vivo degradation by intracellular and extracellular nucleases, such
as RNAse H, which are capable of cutting double-stranded RNA/RNA or
RNA/DNA complexes.
[0081] The term "relative amount" is used where a comparison is
made between a test measurement and a control measurement. The
relative amount of a reagent forming a complex in a reaction is the
amount reacting with a test specimen, compared with the amount
reacting with a control specimen. The control specimen may be run
separately in the same assay, or it may be part of the same sample
(for example, normal tissue surrounding a malignant area in a
tissue section).
[0082] "Monocytes, lymphocytes, and dendritic cells" refer to three
types of white blood cells of the immune system. The cell types
have their common, textbook definitions.
[0083] The term "activated T cells" refers to either chronically
activated T cells (i.e. autoimmunity) or naive T cells responding
to alloantigens (i.e. transplantation), or chemical modification of
self-antigens, (hapten-induced contact sensitivity).
[0084] The acronym "CFLAR" refers to the CASP8 and FADD-like
apoptosis regulator and also has several other designations
including: FLICE inhibitory protein; FADD-like anti-apoptotic
molecule; Inhibitor of FLICE; Caspase-related inducer of apoptosis;
Caspase homolog; Caspase-like apoptosis regulatory protein; and
usurpin beta. CFLAR also refers to the protein with the following
aliases: CASH; CASP8AP1; CLARP; Casper; FLAME; FLAME-1; FLAME1;
FLIP; I-FLICE; MRIT; USURPIN; cFLIP, c-FLIPL; c-FLIPR; and c-FLIPS.
The human CFLAR NCBI Gene ID is 8837 and the GenBank reference
sequence for the human CFLAR gene can be found using accession
NM.sub.--003879.
[0085] The acronym "PMO" refers to a phosphorodiamidate morpholino
oligonucleotide.
[0086] An arginine-rich peptide refers to a peptide transport
moiety effective to enhance transport of the compound into cells.
The transport moiety may be attached to a terminus of the oligomer
and in certain embodiments consists of about 6 to 16 amino acid
subunits selected from subunits with a guanidyl side chain moiety,
as in the alpha amino acid subunit arginine (Arg) and the beta
amino acid subunits defined by --CO--(CH.sub.2).sub.n--CHR--NH--,
where n is 2 to 7 and R is H. For example, when n is 5 and R is H,
the subunit is a 6-aminohexanoic acid subunit; when n is 2 and R is
H, the subunit is a .beta.-alanine subunit.
[0087] The acronym "PPMO" refers to a peptide-conjugate of an
arginine-rich peptide and a PMO.
[0088] An agent is "effective to enhance transport" or "effective
to promote uptake" of the compound into mammalian cells if the
compound is taken up by these cells by passive transport across the
cell membrane or by an active transport mechanism involving, for
example, transport across the membrane by e.g., an ATP-dependent
transport mechanism, or by "facilitated transport", referring to
transport of antisense agents across the cell membrane by a
transport mechanism that requires binding of the agent to a
transport protein, which then facilitates passage of the bound
agent across the membrane, or by cell membrane invagination. Uptake
of the compound into the target cells may be confirmed, for
example, by uptake of a fluoresceinated compound in the cells.
[0089] An "effective amount" or "therapeutically effective amount"
refers to an amount of antisense compound administered topically to
a mammalian subject, either as a single dose or as part of a series
of doses, which is effective to produce a desired therapeutic
effect, such as reduced inflammation, reduction in dermatitis,
reduced localized infiltration of lymphocytes, or any combination
thereof. For an antisense oligomer, this effect is typically
brought about by inhibiting translation or natural
splice-processing of a selected target sequence, such as CFLAR.
[0090] By "enhance" or "enhancing," or "increase" or "increasing,"
or "stimulate" or "stimulating," refers generally to the ability of
one or antisense or RNAi compounds or compositions to produce or
cause a greater physiological response (i.e., downstream effects)
in a cell or a subject, as compared to the response caused by
either no antisense compound or a control compound. Examples of a
physiological response included increased activation-induced cell
death (AICD) of T-cells, including CD4+ and CD8+ T-cells. An
"increased" or "enhanced" amount is typically a "statistically
significant" amount, and may include an increase that is 1.1, 1.2,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g.,
500, 1000 times) (including all integers and decimal points in
between and above 1), e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount
produced by no antisense compound (the absence of an agent) or a
control compound.
[0091] The term "reduce" or "inhibit" may relate generally to the
ability of one or more antisense or RNAi compounds of the invention
to "decrease" a relevant physiological or cellular response, such
as a symptom of a disease or condition described herein, as
measured according to routine techniques in the diagnostic art.
Relevant physiological or cellular responses (in vivo or in vitro)
will be apparent to persons skilled in the art, and may include
reductions in CFLAR expression, T-cell activation or infiltration,
inflammation, or the various symptoms of contact hypersensitivity.
A "decrease" in a response may be "statistically significant" as
compared to the response produced by no antisense compound or a
control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% decrease, including all integers in between.
[0092] A "topical delivery vehicle" or carrier refers generally to
a pharmaceutical composition appropriate for application to the
skin or mucous membranes. Certain illustrative delivery vehicles
incorporate propylene glycol and/or an acyl-chain lipid, e.g.,
fatty acid, fatty ester, phospholipid, and triglycerides. An
exemplary acyl-chain lipid is linoleic acid. Other exemplary
topical formulations are described below.
[0093] "Treatment" of an individual (e.g. a mammal, such as a
human) or a cell is any type of intervention used in an attempt to
alter the natural course of the individual or cell. Treatment
includes, but is not limited to, administration of a pharmaceutical
composition, and may be performed either prophylactically,
simultaneously or subsequent to the initiation of a pathologic
event or contact with an etiologic agent. Treatment includes any
desirable effect on the symptoms or pathology of a disease or
condition associated contact hypersensitivity. The related term
"improved therapeutic outcome" relative to a patient diagnosed as
having such a condition, may refer to a slowing or diminution in
the condition, or detectable symptoms associated with the
condition.
[0094] Also included are "prophylactic" treatments, which can be
directed to reducing the rate of progression of the disease or
condition being treated, delaying the onset of that disease or
condition, or reducing the severity of its onset. "Treatment" or
"prophylaxis" does not necessarily indicate complete eradication,
cure, or prevention of the disease or condition, or associated
symptoms thereof.
[0095] A "subject," as used herein, may include any animal that
exhibits a symptom, or is at risk for exhibiting a symptom, which
can be treated with an antisense compound of the invention, such as
a subject that has or is at risk for having contact
hypersensitivity, or related symptoms. Suitable subjects (patients)
include laboratory animals (such as mouse, rat, rabbit, or guinea
pig), farm animals, and domestic animals or pets (such as a cat or
dog). Non-human primates and, preferably, human patients, are
included.
[0096] A subject is "sensitized" to an etiologic agent if the
extent of contact dermatitis, e.g., inflammatory response to the
agent, is more severe than response from the initial contact with
the agent. The degree of sensitization may increase with subsequent
exposure(s) to the agent, and may decline during a prolonged period
without exposure to the agent.
[0097] "Inducing tolerance" to a sensitizing agent known to provoke
contact hypersensitivity including contact dermatitis means
reducing the extent to which a sensitized subject reacts to skin
contact with the agent, as evidenced, for example, by a reduced
inflammatory response at the skin or mucous membrane site of
contact with the agent.
[0098] "Alkyl" refers to a fully saturated monovalent radical
containing carbon and hydrogen, which may be branched, linear, or
cyclic (cycloalkyl). Examples of alkyl groups are methyl, ethyl,
n-butyl, t-butyl, n-heptyl, isopropyl, cyclopropyl, cyclopentyl,
ethylcyclopentyl, and cyclohexyl. Generally preferred are alkyl
groups having one to six carbon atoms, referred to as "lower
alkyl", and exemplified by methyl, ethyl, n-butyl, i-butyl,
t-butyl, isoamyl, n-pentyl, and isopentyl. In one embodiment, lower
alkyl refers to C.sub.1 to O.sub.4 alkyl.
[0099] "Alkenyl" refers to an unsaturated monovalent radical
containing carbon and hydrogen, which may be branched, linear, or
cyclic. The alkenyl group may be monounsaturated or
polyunsaturated. Generally preferred are alkenyl groups having one
to six carbon atoms, referred to as "lower alkenyl."
[0100] "Alkynyl" refers to an unsaturated straight or branched
chain hydrocarbon radical containing from 2 to 18 carbons
comprising at least one carbon to carbon triple bond. Examples
include without limitation ethynyl, propynyl, iso-propynyl,
butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl. The term
"lower alkynyl" refers to an alkynyl group, as defined herein,
containing between 2 and 8 carbons.
[0101] "Cycloalkyl" refers to a mono- or poly-cyclic alkyl radical.
Examples include without limitation cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl and cyclooctyl.
[0102] "Aryl" refers to a substituted or unsubstituted monovalent
aromatic radical, generally having a single ring (e.g., phenyl) or
two condensed rings (e.g., naphthyl). This term includes heteroaryl
groups, which are aromatic ring groups having one or more nitrogen,
oxygen, or sulfur atoms in the ring, such as furyl, pyrrolyl,
pyridyl, and indolyl. By "substituted" is meant that one or more
ring hydrogens in the aryl group is replaced with a halide such as
fluorine, chlorine, or bromine; with a lower alkyl group containing
one or two carbon atoms; nitro, amino, methylamino, dimethylamino,
methoxy, halomethoxy, halomethyl, or haloethyl. Preferred
substituents include halogen, methyl, ethyl, and methoxy. Generally
preferred are aryl groups having a single ring.
[0103] "Aralkyl" refers to an alkyl, preferably lower
(C.sub.1-C.sub.4, more preferably C.sub.1-C.sub.2) alkyl,
substituent which is further substituted with an aryl group;
examples are benzyl (--CH.sub.2C.sub.6H.sub.5) and phenethyl
(--CH.sub.2CH.sub.2C.sub.6H.sub.5).
[0104] "Thioalkoxy" refers to a radical of the formula --SRc where
Rc is an alkyl radical as defined herein. The term "lower
thioalkoxy" refers to an alkoxy group, as defined herein,
containing between 1 and 8 carbons.
[0105] "Alkoxy" refers to a radical of the formula --ORda where Rd
is an alkyl radical as defined herein. The term "lower alkoxy"
refers to an alkoxy group, as defined herein, containing between 1
and 8 carbons. Examples of alkoxy groups include, without
limitation, methoxy and ethoxy.
[0106] "Alkoxyalkyl" refers to an alkyl group substituted with an
alkoxy group.
[0107] "Carbonyl" refers to the --C(.dbd.O)-- radical.
[0108] "Guanidynyl" refers to the H.sub.2N(C.dbd.NH.sub.2)--NH--
radical.
[0109] "Amidinyl" refers to the H.sub.2N(C.dbd.NH.sub.2)CH--
radical.
[0110] "Amino" refers to the --NH.sub.2 radical.
[0111] "Alkylamino" refers to a radical of the formula --NHRd or
--NRdRd where each Rd is, independently, an alkyl radical as
defined herein. The term "lower alkylamino" refers to an alkylamino
group, as defined herein, containing between 1 and 8 carbons.
[0112] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 to 4
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Preferably, the ring
atoms include 3 to 6 carbon atoms. Such heterocycles include, for
example, pyrrolidine, piperidine, piperazine, and morpholine.
[0113] Heterocycles include heteroaryls as defined below. Thus, in
addition to the heteroaryls listed below, heterocycles also include
morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,
piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiopyranyl, and the like.
[0114] "Heteroaryl" means an aromatic heterocycle ring of 5- to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bicyclic ring systems. Representative heteroaryls
are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,
quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0115] The term "substituted", with respect to an alkyl, alkenyl,
alkynyl, aryl, aralkyl, or alkaryl group, refers to replacement of
a hydrogen atom with a heteroatom-containing substituent, such as,
for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino,
alkylamino, imino, oxo (keto), nitro, cyano, or various acids or
esters such as carboxylic, sulfonic, or phosphonic.
[0116] The term "substituted", particularly with respect to an
alkyl, alkoxy, thioalkoxy, or alkylamino group, refers to
replacement of a hydrogen atom on carbon with a
heteroatom-containing substituent, such as, for example, halogen,
hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo
(keto), nitro, cyano, or various acids or esters such as
carboxylic, sulfonic, or phosphonic. It may also refer to
replacement of a hydrogen atom on a heteroatom (such as an amine
hydrogen) with an alkyl, carbonyl or other carbon containing
group.
[0117] In certain embodiments, the terms "optionally substituted
alkyl", "optionally substituted alkenyl", "optionally substituted
alkoxy", "optionally substituted thioalkoxy", "optionally
substituted alkyl amino", "optionally substituted lower alkyl",
"optionally substituted lower alkenyl", "optionally substituted
lower alkoxy", "optionally substituted lower thioalkoxy",
"optionally substituted lower alkyl amino" and "optionally
substituted heterocyclyl" mean that, when substituted, at least one
hydrogen atom is replaced with a substituent. In the case of an oxo
substituent (.dbd.O) two hydrogen atoms are replaced.
[0118] In this regard, substituents include: deuterium, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted heterocycle, optionally substituted cycloalkyl, oxo,
halogen, --CN, --ORx, NRxRy, NRxC(.dbd.O)Ry, NRxSO2Ry,
--NRxC(.dbd.O)NRxRy, C(.dbd.O)Rx, C(.dbd.O)ORx, C(.dbd.O)NRxRy,
--SOmRx and --SOmNRxRy, wherein m is 0, 1 or 2, Rx and Ry are the
same or different and independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted heterocycle or optionally substituted cycloalkyl and
each of said optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
aryl, optionally substituted heterocycle and optionally substituted
cycloalkyl substituents may be further substituted with one or more
of oxo, halogen, --CN, --ORx, NRxRy, NRxC(.dbd.O)Ry, NRxSO2Ry,
--NRxC(.dbd.O)NRxRy, C(.dbd.O)Rx, C(.dbd.O)ORx, C(.dbd.O)NRxRy,
--SOmRx and --SOmNRxRy.
[0119] Target and Targeting Sequences
[0120] In certain embodiments, the antisense compound of the
invention targets the AUG start site codon of a CFLAR mRNA. In
certain embodiments, the oligonucleotide has a base sequence
effective to hybridize to at least 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 39, 39, 40 or more contiguous or non-contiguous
bases of surrounding or spanning the AUG start codon of a human
CFLAR mRNA transcript, such as the region within SEQ ID NO:11, and
block or reduce normal expression of a functional human CFLAR in
CFLAR expressing lymphocytes. Certain exemplary targeting sequences
are able to hybridize to at least 12 contiguous bases contained
within SEQ ID NO:12, and include SEQ ID NOS:23-27, and variants
thereof having at least about 80%, 85%, 90%, 95%, or 98% identity
to these sequences.
[0121] In certain embodiments, the antisense compound of the
invention may target a human CFLAR splice-site target sequence,
such as a splice acceptor site, a splice donor site, or a branch
point. Included are splice acceptor and splice donor sites at or
near the border of any one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, or 14 of the various alternatively spliced mRNAs that
derive from the CFLAR gene. Specific examples included splice
acceptor and splice donor sites at or near the border of any one of
exons 1, 3, 4, 5, 6, 8, 9, 10, 12, or 13 of C-FLIP.sub.L, exons 3,
4, 5, 6, or 7 of C-FLIP.sub.S, and exons 3, 4, 5, 6, or 7 of
C-FLIP.sub.R. Also included are branch points, which are typically
located about 20-50 bases upstream of an acceptor site. Hence, in
certain embodiments, an antisense oligomer may target about 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more bases
surrounding a splice donor or splice acceptor site or branch point
of a CFLAR mRNA transcript.
[0122] In certain embodiments, an antisense compound may target a
splice site contained within SEQ ID NOS:13-17. Human CFLAR
splice-site target sequences contained within SEQ ID NOS:13-17
include any contiguous sequence of bases, typically at least 6 to
12 to 22 to 25 to 30 or more contiguous or non-contiguous bases
(including all integers in between), at which hybridization by an
antisense oligonucleotide is effective to block or reduce normal
processing of a functional human CFLAR in CFLAR expressing
lymphocytes. Exemplary targeting sequences include SEQ ID
NOS:29-33, and variants thereof having at least about 80%, 85%,
90%, 95%, or 98% identity to these sequences.
[0123] Exemplary human (Hu) and murine (Mu) CFLAR target sequences
are shown below in Table 1.
TABLE-US-00001 TABLE 1 Exemplary CFLAR Target Sequences SEQ Target
Target Sequence (5' to 3') ID NO: Hu-AUG (+30))
CCTTGTGAGCTTCCCTAGTCTAAGAGTAGG 11 ATGTCTGCTGAAGTCATCCATCAGGTTGAA
Hu-AUG (+12) TCTAAGAGTAGGATGTCTGCTGAAG 12 Hu-Ex2SA
CAGAAAAATTCCCTTTTAACCACAG/AACT 13 CCCCCACTGGAAAGGATTCTG Hu-Ex3SA
CTAAATGAACTTGTCTGGTTTGCAG/ 14 AGTGCTGATGGCAGAGATTGGTGAG Hu-Ex4SA
TGTTTTTTGTTGGTGGTTCTCTTAG/ 15 AGTTTCTTGGACCTTGTGGTTGAGT Hu-Ex2SD
ACCCTCACCTTGTTTCGGACTATAG/ 16 GTAATTCATCAACTCTTCCTGAGGC Hu-Ex3SD
CCGAGGCAAGATAAGCAAGGAGAAG/ 17 GTGAGTTTTCTTCTTTTGGTTCATG Mu-Ex2SA
ATAAGAGGATTCTCTTTCACCACAG/ 18 AGTGTCTCTATTGCAAGAACTCTGA Mu-Ex2SD
ACCCTCACCTGGTTTCTGATTATAG/ 19 GTAAGTCATCCCCTGGGGGAGGGGA Mu-Ex3SA
CTGAAGACACTTTTATGGTTTACAG/ 20 GGTCCTGCTGATGGAGATTGGTGAG Mu-Ex3SD
CAGAGGCAAGATAGCCAAGGACAAG/ 21 GTGAGTTGTCTTTGCTCGGTGCCTG Mu-Ex4SA
CATTTCTTGTTCATGGCTTTCTTAG/ 22 AGTTTCTTGGATCTGGTGATTGAAT
[0124] Human (hu) and murine (mu) CFLAR antisense targeting
sequences that are complementary to regions contained within the
target sequences listed in Table 1 are shown below in Table 2.
TABLE-US-00002 TABLE 2 Exemplary Human and Mouse CFLAR Targeting
Sequences Oligomer Sequence (5' to 3') SEQ ID NO: CFLAR-huAUG1
CTTCAGCAGACATCCTACTC 23 CFLAR-huAUG2 GACTAGGGAAGCTCACAAGG 24
CFLAR-huAUG3 TCAACCTGATGGATGACTTC 25 CFLAR-huAUG(-5)
GATGACTTCAGCAGACATCCTAC 26 CFLAR-huAUG(-11) CTTCAGCAGACATCCTACTC 27
TTAG CFLAR-muAUG CTGGGCCATGTTCAGAACC 28 CFLAR-huSA2
GGAGTTCTGTGGTTAAAAGG 29 CFLAR-huSD2 CTATAGTCCGAAACAAGGTGAGG 30
CFLAR-huSA3 CACCAATCTCTGCCATCAGCACT 31 CFLAR-huSA4
TCAACCACAAGGTCCAAGAAACT 32 CFLAR-huSD3 CTTCTCCTTGCTTATCTTGCCT 33
R.sub.9F.sub.2- RRRRRRRRRFFC- 34 CFLARmuAUG; CTGGGCCATGTTCAGAACC
CFLAR PPMO Scrambled Control TGCGCGTCATGTACGCCAA 35
R.sub.9F.sub.2-Scr. Control; RRRRRRRRRFFC- 36 Scrambled Control
TGCGCGTCATGTACGCCAA PPMO
[0125] Additional targeting sequences may be selected by first
identifying an AUG translation start site or splice-site target
sequence within SEQ ID NOS: 11-16, and constructing a targeting
sequence complementary to at least 12 contiguous bases, and
typically 20 or more bases, of the target sequence.
[0126] Once a targeting sequence has been identified, it can be
readily tested for its ability to interfere with normal CFLAR
expression or processing, through steps described below. Briefly,
in one illustrative embodiment, a morpholino antisense compound
(PMO) or peptide-conjugated morpholino antisense compound (PPMO) be
prepared according to methods described in Sections B and C below,
and the compound can be tested for its ability to block normal
CFLAR expression or processing in CFLAR producing cells in
accordance with the methods given in Example 1. This process can be
applied to other antisense and RNAi chemistries and
methodologies.
[0127] More generally, any type of assay or determination used to
measure levels of CFLAR isoforms in culture samples may be
employed, such as, but not limited to, immunoassays, including
direct competitive, sandwich, direct and indirect cellular, and
crisscross enzyme-linked immunosorbent assays (ELISAs), enzyme
linked immunosorbent spot (ELISPOT) assays, radioimmunoassays
(RIAs), immunoprecipitation, immunohistochemistry,
immunofluorescence, immunoblotting, and the like may be employed
using polyclonal, monoclonal, polyclonal, and fusion phage
antibodies. Simple immunofluorescence using monoclonal and/or
fusion phage antibodies are especially preferred in many
embodiments. Moreover, the sequence of CFLAR is known so that
assessment of mRNA levels by RT-PCR, ribonuclease protection
assays, or Northern analysis, are feasible and in many cases
preferred.
[0128] In certain embodiments, the antisense compound can be tested
for its ability to block normal expression or processing of CFLAR
by direct screening of the compound in a test animal, e.g., murine
model, where the sequence tested is targeted against a selected AUG
translation start site or splice site target sequence of the
corresponding animal (mice) CFLAR processed or preprocessed
transcript sequence. In this approach, the test agent is
administered to the experimental animal, a biological sample is
taken from the animal and from a control animal of the same
species, and the CFLAR protein or mRNA concentration of the spliced
products are measured.
Antisense Oligonucleotides
[0129] As detailed above, the antisense oligonucleotides described
herein typically comprise a base sequence targeting a region that
includes one or more of the following; the region surrounding the
AUG start codon of a CFLAR mRNA, a region surrounding the splice
donor or acceptor sites of a CFLAR mRNA, or a region surrounding
the branch points of a CFLAR mRNA. In addition, the oligomer is
able to effectively reduce expression of CFLAR mRNA in a cell, such
as an activated T-cell. This requirement is typically met when the
oligomer compound (a) has the ability to be actively taken up by
mammalian cells, and (b) once taken up, form a duplex with the
target RNA with a Tm greater than about 45.degree. C.
[0130] A variety of antisense chemistries may be employed. Examples
include RNA interference based compounds (e.g., siRNA, shRNA, or
RNAi-inducing vectors), hybrid interfering RNA molecules, RNA
amidates and thioamidates, thio-siRNA aptamers, phosphorothioates,
shRNA without interferon and/or cytotoxicity induction (e.g.,
having one or a plurality of G(s) at the 5' end of the sense
strand), DNA and antisense RNA hybrid constructs, oligomers having
universal bases that can pair with all of the four naturally
occurring bases, alternate oligonucleotide analogue chemistries in
U.S. Application No. 2009/0149404 (herein incorporated by
reference), 2' and/or 3' prodrugs of 1', 2', 3' or 4'-branched
nucleosides, immunostimulatory oligonucleotide analogs (see, e.g.,
U.S. Application Nos. 2008/0027214 and 2006/0135454, herein
incorporated by reference), ssDNA, bicyclonucleoside
oligonucleotide analogues, circularly permuted chimeric pRNA
molecules, caged RNAs (e.g., photoactivatable caged RNAs),
self-cleaving ribozymes, oligonucleotides with alternating segments
of sugar-modified nucleosides and 2'-deoxynucleosides, polyamide
nucleic acid derivatives, oligos comprising a 5'- and/or a 3'-cap
structure, chimeric nucleic acid molecules (e.g., having a target
region and a largely double-stranded region of specific nucleotide
sequences for intracellular targeting, siDNA, and oligomers of
ribose groups linked by achiral 5' to 3' internucleoside phosphate
linkages.
[0131] In certain embodiments, the oligomer backbone may be
substantially uncharged, and, preferably, may be recognized as a
substrate for active or facilitated transport across the cell
membrane. The ability of the oligomer to form a stable duplex with
the target RNA may also relate to other features of the oligomer
backbone, including the length and degree of complementarity of the
antisense oligomer with respect to the target, the ratio of G:C to
A:T base matches, and the positions of any mismatched bases. The
ability of the antisense oligomer to resist cellular nucleases may
promote survival and ultimate delivery of the agent to the cell
cytoplasm.
[0132] Certain embodiments included peptide nucleic acids (PNAs),
analogs of DNA in which the backbone is structurally homomorphous
with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine
units to which pyrimidine or purine bases are attached. PNAs
containing natural pyrimidine and purine bases hybridize to
complementary oligonucleotides obeying Watson-Crick base-pairing
rules, and mimic DNA in terms of base pair recognition (Egholm,
Buchardt et al. 1993). The backbone of PNAs is formed by peptide
bonds rather than phosphodiester bonds, making them well-suited for
antisense applications. The backbone is uncharged, resulting in
PNA/DNA or PNA/RNA duplexes which exhibit greater than normal
thermal stability. PNAs are not recognized by nucleases or
proteases.
[0133] Certain embodiments employ morpholino-based subunits bearing
base-pairing moieties, joined by uncharged linkages, as described
above. Especially preferred is a substantially uncharged
phosphorodiamidate-linked morpholino oligomer. Morpholino
oligonucleotides, including antisense oligomers, are detailed, for
example, in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866,
5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and
5,506,337, and in PCT application No. US08/088339, all of which are
expressly incorporated by reference herein.
[0134] Certain properties of the morpholino-based subunits include:
the ability to be linked in a oligomeric form by stable, uncharged
backbone linkages; the ability to support a nucleotide base (e.g.,
adenine, cytosine, guanine or uracil) such that the polymer formed
can hybridize with a complementary-base target nucleic acid,
including target RNA, with high Tm, even with oligomers as short as
10-14 bases; the ability of the oligomer to be actively transported
into mammalian cells; and the ability of the oligomer:RNA
heteroduplex to resist RNase degradation.
[0135] Examples of morpholino oligomers having
phosphorus-containing backbone linkages are illustrated in FIGS.
1A, 2A-B. Especially preferred is a phosphorodiamidate-linked
morpholino oligonucleotide such as shown in FIG. 2B, which is
modified, in accordance with one aspect of the present invention,
to contain positively charged groups at preferably about 10%-50% of
its backbone linkages. Morpholino oligonucleotides with uncharged
backbone linkages, including antisense oligonucleotides, are
detailed, for example, in (Summerton and Weller 1997) and in
co-owned U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,
5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are
expressly incorporated by reference herein.
[0136] Properties of the morpholino-based subunits include: 1) the
ability to be linked in a oligomeric form by stable, uncharged or
positively charged backbone linkages; 2) the ability to support a
nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil
and inosine) such that the polymer formed can hybridize with a
complementary-base target nucleic acid, including target RNA, Tm
values above about 45.degree. C. in relatively short
oligonucleotides (e.g., 10-15 bases); 3) the ability of the
oligonucleotide to be actively or passively transported into
mammalian cells; and 4) the ability of the antisense
oligonucleotide:RNA heteroduplex to resist RNAse and RNaseH
degradation, respectively.
[0137] Exemplary backbone structures for antisense oligonucleotides
of the claimed subject matter include the morpholino subunit types
shown in FIGS. 1B-E, each linked by an uncharged or positively
charged, phosphorus-containing subunit linkage. FIG. 1B shows a
phosphorus-containing linkage which forms the five atom
repeating-unit backbone, where the morpholino rings are linked by a
1-atom phosphoamide linkage. FIG. 1C shows a linkage which produces
a 6-atom repeating-unit backbone. In this structure, the atom Y
linking the 5' morpholino carbon to the phosphorus group may be
sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety
pendant from the phosphorus may be fluorine, an alkyl or
substituted alkyl, an alkoxy or substituted alkoxy, a thioalkoxy or
substituted thioalkoxy, or unsubstituted, monosubstituted, or
disubstituted nitrogen, including cyclic structures, such as
morpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferably
include 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and
are preferably oxygen.
[0138] The linkages shown in FIGS. 1D and 1E are designed for
7-atom unit-length backbones. In structure 1 D, the X moiety is as
in Structure 1C, and the Y moiety may be methylene, sulfur, or,
preferably, oxygen. In Structure 1 E, the X and Y moieties are as
in Structure 1C. Particularly preferred morpholino oligonucleotides
include those composed of morpholino subunit structures of the form
shown in FIG. 1C, where X.dbd.NH.sub.2, N(CH.sub.3).sub.2, or
1-piperazine or other charged group, Y.dbd.O, and Z.dbd.O.
[0139] As noted above, the substantially uncharged oligonucleotide
may be modified, in accordance with an aspect of the invention, to
include charged linkages, e.g. up to about 1 per every 2-5
uncharged linkages, such as about 4-5 per every 10 uncharged
linkages. Optimal improvement in antisense activity may be seen
when about 25% of the backbone linkages are cationic. In certain
embodiments, enhancement may be seen with a small number e.g.,
10-20% cationic linkages, or where the number of cationic linkages
are in the range 50-80%, such as about 60%.
[0140] Additional experiments conducted in support of the present
invention indicate that the enhancement seen with added cationic
backbone charges may, in some cases, be further enhanced by
distributing the bulk of the charges close of the "center-region"
backbone linkages of the antisense oligonucleotide, e.g., in a
20mer oligonucleotide with 8 cationic backbone linkages, having at
least 70% of these charged linkages localized in the 10 centermost
linkages.
[0141] In certain embodiments, the antisense compounds can be
prepared by stepwise solid-phase synthesis, employing methods
detailed in the references cited above, and below with respect to
the synthesis of oligonucleotides having a mixture or uncharged and
cationic backbone linkages. In some cases, it may be desirable to
add additional chemical moieties to the antisense compound, e.g. to
enhance pharmacokinetics or to facilitate capture or detection of
the compound. Such a moiety may be covalently attached, typically
to a terminus of the oligomer, according to standard synthetic
methods. For example, addition of a polyethyleneglycol moiety or
other hydrophilic polymer, e.g., one having 10-100 monomeric
subunits, may be useful in enhancing solubility. One or more
charged groups, e.g., anionic charged groups such as an organic
acid, may enhance cell uptake.
[0142] A reporter moiety, such as fluorescein or a radiolabeled
group, may be attached for purposes of detection. Alternatively,
the reporter label attached to the oligomer may be a ligand, such
as an antigen or biotin, capable of binding a labeled antibody or
streptavidin. In selecting a moiety for attachment or modification
of an antisense compound, it is generally of course desirable to
select chemical compounds of groups that are biocompatible and
likely to be tolerated by a subject without undesirable side
effects.
[0143] As noted above, the antisense compound can be constructed to
contain a selected number of cationic linkages interspersed with
uncharged linkages of the type described above. The intersubunit
linkages, both uncharged and cationic, preferably are
phosphorus-containing linkages, having the structure:
##STR00002##
where
[0144] W is S or O, and is preferably O,
[0145] X.dbd.NR.sup.1R.sup.2 or OR.sup.6,
[0146] Y.dbd.O or NR.sup.7,
[0147] and each said linkage in the oligomer is selected from:
[0148] (a) uncharged linkage (a), where each of R.sup.1, R.sup.2,
R.sup.6 and R.sup.7 is independently selected from hydrogen and
lower alkyl; [0149] (b1) cationic linkage (b1), where
X.dbd.NR.sup.1R.sup.2 and Y.dbd.O, and NR.sup.1R.sup.2 represents
an optionally substituted piperazino group, such that
R.sup.1R.sup.2=-CHRCHRN(R.sup.3)(R.sup.4)CHRCHR--, where
[0150] each R is independently H or CH.sub.3,
[0151] R.sup.4 is H, CH.sub.3, or an electron pair, and
[0152] R.sup.3 is selected from H, lower alkyl, e.g. CH.sub.3,
C(.dbd.NH)NH.sub.2, Z-L-NHC(.dbd.NH)NH.sub.2, and
[C(O)CHR'NH].sub.mH, where: Z is C(O) or a direct bond, L is an
optional linker up to 18 atoms in length, preferably up to 12
atoms, and more preferably up to 8 atoms in length, having bonds
selected from alkyl, alkoxy, and alkylamino, R' is a side chain of
a naturally occurring amino acid or a one- or two-carbon homolog
thereof, and m is 1 to 6, preferably 1 to 4; [0153] (b2) cationic
linkage (b2), where X.dbd.NR.sup.1R.sup.2 and Y.dbd.O,
R.sup.1.dbd.H or CH.sub.3, and R.sup.2=LNR.sup.3R.sup.4R.sup.5,
where L, R.sup.3, and R.sup.4 are as defined above, and R.sup.5 is
H, lower alkyl, or lower (alkoxy)alkyl; and [0154] (b3) cationic
linkage (b3), where Y.dbd.NR.sup.7 and X.dbd.OR.sup.6, and
R.sup.7=LNR.sup.3R.sup.4R.sup.5, where L, R.sup.3, R.sup.4 and
R.sup.5 are as defined above, and R.sup.6 is H or lower alkyl;
[0155] and at least one said linkage is selected from cationic
linkages (b1), (b2), and (b3).
[0156] In certain embodiments, the oligomer includes at least two
consecutive linkages of type (a) (i.e. uncharged linkages). In
further embodiments, at least 5% of the linkages in the oligomer
are cationic linkages (i.e. type (b1), (b2), or (b3)); for example,
10% to 60%, and preferably 20-50% linkages may be cationic
linkages.
[0157] In one embodiment, at least one linkage is of type (b1),
where, preferably, each R is H, R.sup.4 is H, CH.sub.3, or an
electron pair, and R.sup.3 is selected from H, lower alkyl, e.g.
CH.sub.3, C(.dbd.NH)NH.sub.2, and C(O)-L-NHC(.dbd.NH)NH.sub.2. The
latter two embodiments of R.sup.3 provide a guanidino moiety,
either attached directly to the piperazine ring, or pendant to a
linker group L, respectively. For ease of synthesis, the variable Z
in R.sup.3 is preferably C(O) (carbonyl), as shown.
[0158] The linker group L, as noted above, contains bonds in its
backbone selected from alkyl (e.g. --CH.sub.2--CH.sub.2--), alkoxy
(--C--O--), and alkylamino (e.g. --CH.sub.2--NH--), with the
proviso that the terminal atoms in L (e.g., those adjacent to
carbonyl or nitrogen) are carbon atoms. Although branched linkages
(e.g. --CH.sub.2--CHCH.sub.3--) are possible, the linker is
preferably unbranched. In one embodiment, the linker is a
hydrocarbon linker. Such a linker may have the structure
--(CH.sub.2).sub.n--, where n is 1-12, preferably 2-8, and more
preferably 2-6.
[0159] The morpholino subunits have the structure:
##STR00003##
[0160] (i) where Pi is a base-pairing moiety, and the linkages
depicted above connect the nitrogen atom of (i) to the 5' carbon of
an adjacent subunit. The base-pairing moieties Pi may be the same
or different, and are generally designed to provide a sequence
which binds to a target nucleic acid.
[0161] The use of embodiments of linkage types (b1), (b2) and (b3)
above to link morpholino subunits may be illustrated graphically as
follows:
[0162] where Pi is a base-pairing moiety, and the linkages depicted
above connect the nitrogen atom of (i) to the 5' carbon of an
adjacent subunit. The base-pairing moieties Pi may be the same or
different, and are generally designed to provide a sequence which
binds to a target nucleic acid.
[0163] The use of embodiments of linkage types (b1), (b2) and (b3)
above to link morpholino subunits may be illustrated graphically as
follows:
##STR00004##
[0164] Preferably, all cationic linkages in the oligomer are of the
same type; i.e. all of type (b1), all of type (b2), or all of type
(b3).
[0165] In further embodiments, the cationic linkages are selected
from linkages (b1') and (b1'') as shown below, where (b1') is
referred to herein as a "Pip" linkage and (b1'') is referred to
herein as a "GuX" linkage:
##STR00005##
[0166] In the structures above, W is S or O; each of R.sup.1 and
R.sup.2 is independently selected from hydrogen and lower alkyl,
and is preferably methyl; and A represents hydrogen or a
non-interfering substituent on one or more carbon atoms in (b1')
and (b1''). In certain embodiments, the ring carbons in the
piperazine ring are unsubstituted; however, they may include
non-interfering substituents, such as methyl or fluorine. In
certain embodiments, at most one or two carbon atoms is so
substituted.
[0167] In further embodiments, at least 10% of the linkages are of
type (b1') or (b1''); for example, 10%-60% and preferably 20% to
50%, of the linkages may be of type (b1') or (b1''). In certain
embodiments, the oligomer contains no linkages of the type (b1')
above. Alternatively, the oligomer contains no linkages of type
(b1) where each R is H, R.sup.3 is H or CH.sub.3, and R.sup.4 is H,
CH.sub.3, or an electron pair.
[0168] The morpholino subunits may also be linked by
non-phosphorus-based intersubunit linkages, as described further
below, where at least one linkage is modified with a pendant
cationic group as described above.
[0169] Other oligonucleotide analog linkages which are uncharged in
their unmodified state but which could also bear a pendant amine
substituent could be used. For example, a 5' nitrogen atom on a
morpholino ring could be employed in a sulfamide linkage or a urea
linkage (where phosphorus is replaced with carbon or sulfur,
respectively) and modified in a manner analogous to the 5'-nitrogen
atom in structure (b3) above.
[0170] Oligomers having any number of cationic linkages are
provided, including fully cationic-linked oligomers. Preferably,
however, the oligomers are partially charged, having, for example,
10%-80%. In preferred embodiments, about 10% to 60%, and preferably
20% to 50% of the linkages are cationic.
[0171] In one embodiment, the cationic linkages are interspersed
along the backbone. The partially charged oligomers preferably
contain at least two consecutive uncharged linkages; that is, the
oligomer preferably does not have a strictly alternating pattern
along its entire length.
[0172] Also considered are oligomers having blocks of cationic
linkages and blocks of uncharged linkages; for example, a central
block of uncharged linkages may be flanked by blocks of cationic
linkages, or vice versa. In one embodiment, the oligomer has
approximately equal-length 5', 3' and center regions, and the
percentage of cationic linkages in the center region is greater
than about 50%, preferably greater than about 70%.
[0173] Oligomers for use in antisense applications generally range
in length from about 10 to about 40 subunits, more preferably about
10 to 30 subunits, and typically 15-25 bases. For example, an
oligomer of the invention having 19-20 subunits, a useful length
for an antisense compound, may ideally have two to ten, e.g. four
to eight, cationic linkages, and the remainder uncharged linkages.
An oligomer having 14-15 subunits may ideally have two to seven,
e.g., 3, 4, or 5, cationic linkages and the remainder uncharged
linkages.
[0174] Each morpholino ring structure supports a base pairing
moiety, to form a sequence of base pairing moieties which is
typically designed to hybridize to a selected antisense target in a
cell or in a subject being treated. The base pairing moiety may be
a purine or pyrimidine found in native DNA or RNA (A, G, C, T, or
U) or an analog, such as hypoxanthine (the base component of the
nucleoside inosine) or 5-methyl cytosine.
RNA Intererence Agents
[0175] The CFLAR target regions described herein may also be
targeted by a variety of RNA interference-based methods. RNA
interference (RNAi) is an evolutionarily conserved gene-silencing
mechanism, originally discovered in studies of the nematode
Caenorhabditis elegans (Lee et al, Cell 75:843, 1993; Reinhart et
al., Nature 403:901, 2000). It may be triggered by introducing
dsRNA into cells expressing the appropriate molecular machinery,
which then degrades the corresponding endogenous mRNA. The
mechanism involves conversion of dsRNA into short RNAs that direct
ribonucleases to homologous mRNA targets (summarized, Ruvkun,
Science 2294:797, 2001).
[0176] In certain embodiments, the methods provided herein may
utilize double-stranded ribonucleic acid (dsRNA) molecules as
modulating agents, for reducing CFLAR expression, and thereby
reducing hypersensitivity or contact dermatitis. dsRNAs generally
comprise two single strands. One strand of the dsRNA comprises a
nucleotide sequence that is substantially identical to a portion of
the target gene or target region (the "sense" strand), and the
other strand (the "complementary" or "antisense" strand) comprises
a sequence that is substantially complementary to a portion of the
target region. The strands are sufficiently complementary to
hybridize to form a duplex structure. In certain embodiments, the
complementary RNA strand may be less than 30 nucleotides, less than
25 nucleotides in length, or even 19 to 24 nucleotides in length.
In certain aspects, the complementary nucleotide sequence may be
20-23 nucleotides in length, or 22 nucleotides in length.
[0177] In certain embodiments, at least one of the RNA strands
comprises a nucleotide overhang of 1 to 4 nucleotides in length. In
other embodiments, the dsRNA may further comprise at least one
chemically modified nucleotide. In certain aspects, a dsRNA
comprising a single-stranded overhang of 1 to 4 nucleotides may
comprise a molecule wherein the unpaired nucleotide of the
single-stranded overhang that is directly adjacent to the terminal
nucleotide pair contains a purine base. In other aspects, the last
complementary nucleotide pairs on both ends of a dsRNA are a G-C
pair, or, at least two of the last four terminal nucleotide pairs
are G-C pairs.
[0178] Certain embodiments of the present invention may comprise
microRNAs. Micro-RNAs represent a large group of small RNAs
produced naturally in organisms, some of which regulate the
expression of target genes. Micro-RNAs are formed from an
approximately 70 nucleotide single-stranded hairpin precursor
transcript by Dicer. (V. Ambros et al. Current Biology 13:807,
2003). Micro-RNAs are not translated into proteins, but instead
bind to specific messenger RNAs, thereby blocking translation. It
is thought that micro-RNAs base-pair imprecisely with their targets
to inhibit translation. Certain micro-RNAs may be transcribed as
hairpin RNA precursors, which are then processed to their mature
forms by Dicer enzyme.
[0179] In certain embodiments, the modulating agent, or RNAi
oligonucleotide, is single stranded. In other embodiments, the
modulating agent, or RNAi oligonucleotide, is double stranded.
Certain embodiments may also employ short-interfering RNAs (siRNA).
In certain embodiments, the first strand of the double-stranded
oligonucleotide contains two more nucleoside residues than the
second strand. In other embodiments, the first strand and the
second strand have the same number of nucleosides; however, the
first and second strands are offset such that the two terminal
nucleosides on the first and second strands are not paired with a
residue on the complimentary strand. In certain instances, the two
nucleosides that are not paired are thymidine resides.
[0180] In instances when the modulating agent comprises siRNA, the
agent should include a region of sufficient homology to the target
region, and be of sufficient length in terms of nucleotides, such
that the siRNA agent, or a fragment thereof, can mediate down
regulation of the target RNA. It will be understood that the term
"ribonucleotide" or "nucleotide" can, in the case of a modified RNA
or nucleotide surrogate, also refer to a modified nucleotide, or
surrogate replacement moiety at one or more positions. Thus, an
siRNA agent is or includes a region which is at least partially
complementary to the target RNA. It is not necessary that there be
perfect complementarity between the siRNA agent and the target, but
the correspondence must be sufficient to enable the siRNA agent, or
a cleavage product thereof, to direct sequence specific silencing,
such as by RNAi cleavage of the target RNA. Complementarity, or
degree of homology with the target strand, is most critical in the
antisense strand. While perfect complementarity, particularly in
the antisense strand, is often desired some embodiments include one
or more but preferably 10, 8, 6, 5, 4, 3, 2, or fewer mismatches
with respect to the target RNA. The mismatches are most tolerated
in the terminal regions, and if present are preferably in a
terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides
of the 5' and/or 3' terminus. The sense strand need only be
sufficiently complementary with the antisense strand to maintain
the over all double-strand character of the molecule.
[0181] In addition, an siRNA modulating agent may be modified or
include nucleoside surrogates. Single stranded regions of an siRNA
agent may be modified or include nucleoside surrogates, e.g., the
unpaired region or regions of a hairpin structure, e.g., a region
which links two complementary regions, can have modifications or
nucleoside surrogates. Modification to stabilize one or more 3'- or
5'-terminus of an siRNA agent, e.g., against exonucleases, or to
favor the antisense siRNA agent to enter into RISC are also useful.
Modifications can include C3 (or C6, C7, C12) amino linkers, thiol
linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9,
C12, abasic, triethylene glycol, hexaethylene glycol), special
biotin or fluorescein reagents that come as phosphoramidites and
that have another DMT-protected hydroxyl group, allowing multiple
couplings during RNA synthesis.
[0182] siRNA agents may include, for example, molecules that are
long enough to trigger the interferon response (which can be
cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and
enter a RISC(RNAi-induced silencing complex)), in addition to
molecules which are sufficiently short that they do not trigger the
interferon response
[0183] (which molecules can also be cleaved by Dicer and/or enter a
RISC), e.g., molecules which are of a size which allows entry into
a RISC, e.g., molecules which resemble Dicer-cleavage products.
Molecules that are short enough that they do not trigger an
interferon response are termed siRNA agents or shorter RNAi agents
herein. "siRNA agent or shorter RNAi agent" as used refers to an
siRNA agent that is sufficiently short that it does not induce a
deleterious interferon response in a human cell, e.g., it has a
duplexed region of less than 60 but preferably less than 50, 40, or
30 nucleotide pairs. An siRNA modulating agent, or a cleavage
product thereof, can down regulate a target gene, e.g., by inducing
RNAi with respect to a target RNA, preferably a CFLAR mRNA.
[0184] Each strand of an siRNA modulating agent can be equal to or
less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length.
The strand is preferably at least 19 nucleotides in length. For
example, each strand can be between 21 and 25 nucleotides in
length. Preferred siRNA agents have a duplex region of 17, 18, 19,
29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more
overhangs, preferably one or two 3' overhangs, of 2-3
nucleotides.
[0185] In addition to homology to target RNA and the ability to
down regulate a target gene, an siRNA modulating agent may have one
or more of the following properties: it may, despite modifications,
even to a very large number, or all of the nucleosides, have an
antisense strand that can present bases (or modified bases) in the
proper three dimensional framework so as to be able to form correct
base pairing and form a duplex structure with a homologous target
RNA which is sufficient to allow down regulation of the target,
e.g., by cleavage of the target RNA; it may, despite modifications,
even to a very large number, or all of the nucleosides, still have
"RNA-like" properties, i.e., it may possess the overall structural,
chemical and physical properties of an RNA molecule, even though
not exclusively, or even partly, of ribonucleotide-based content.
For example, an siRNA agent can contain, e.g., a sense and/or an
antisense strand in which all of the nucleotide sugars contain
e.g., 2' fluoro in place of 2' hydroxyl. This
deoxyribonucleotide-containing agent can still be expected to
exhibit RNA-like properties. While not wishing to be bound by
theory, the electronegative fluorine prefers an axial orientation
when attached to the C2' position of ribose. This spatial
preference of fluorine can, in turn, force the sugars to adopt a
C.sub.3'-endo pucker. This is the same puckering mode as observed
in RNA molecules and gives rise to the RNA-characteristic
A-family-type helix. Further, since fluorine is a good hydrogen
bond acceptor, it can participate in the same hydrogen bonding
interactions with water molecules that are known to stabilize RNA
structures. Generally, it is preferred that a modified moiety at
the 2' sugar position will be able to enter into H-bonding which is
more characteristic of the OH moiety of a ribonucleotide than the H
moiety of a deoxyribonucleotide.
[0186] A "single strand RNAi agent" as used herein, is an RNAi
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand RNAi
modulating agents are preferably antisense with regard to the
target molecule. A single strand RNAi agent should be sufficiently
long that it can enter the RISC and participate in RISC mediated
cleavage of a target mRNA. A single strand RNAi agent is at least
14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50
nucleotides in length. It is preferably less than 200, 100, or 60
nucleotides in length.
[0187] Hairpin RNAi modulating agents may have a duplex region
equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25
nucleotide pairs. The duplex region may preferably be equal to or
less than 200, 100, or 50, in length. Certain ranges for the duplex
region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides
pairs in length. The hairpin may have a single strand overhang or
terminal unpaired region, preferably the 3', and preferably of the
antisense side of the hairpin. In certain embodiments, overhangs
are 2-3 nucleotides in length.
[0188] Certain modulating agents utilized according to the methods
provided herein may comprise RNAi oligonucleotides such as chimeric
oligonucleotides, or "chimeras," which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. Consequently, comparable results can often
be obtained with shorter oligonucleotides when chimeric
oligonucleotides are used, compared to phosphorothioate
oligodeoxynucleotides. Chimeric oligonucleotides may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleotides and/or oligonucleotide mimetics
as described above. Such oligonucleotides have also been referred
to in the art as hybrids or gapmers. Representative United States
patents that teach the preparation of such hybrid structures
include, 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; 5,700,922; and
5,955,589, each of which is herein incorporated by reference. In
certain embodiments, the chimeric oligonucleotide is RNA-DNA,
DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or RNA-DNA-RNA-DNA, wherein the
oligonucleotide is between 5 and 60 nucleotides in length.
[0189] In one aspect of the invention, modulating agents, such as
RNAi agents, relate to an oligonucleotide comprising at least one
ligand tethered to an altered or non-natural nucleobase. A large
number of compounds can function as the altered base. The structure
of the altered base is important to the extent that the altered
base should not substantially prevent binding of the
oligonucleotide to its target, e.g., mRNA. In certain embodiments,
the altered base is difluorotolyl, nitropyrrolyl, nitroimidazolyl,
nitroindolyl, napthalenyl, anthrancenyl, pyridinyl, quinolinyl,
pyrenyl, or the divalent radical of any one of the non-natural
nucleobases described herein. In certain embodiments, the
non-natural nucleobase is difluorotolyl, nitropyrrolyl, or
nitroimidazolyl. In certain embodiments, the non-natural nucleobase
is difluorotolyl. A wide variety of ligands are known in the art
and are amenable to the present invention. For example, the ligand
can be a steroid, bile acid, lipid, folic acid, pyridoxal, B12,
riboflavin, biotin, aromatic compound, polycyclic compound, crown
ether, intercalator, cleaver molecule, protein-binding agent, or
carbohydrate. In certain embodiments, the ligand is a steroid or
aromatic compound. In certain instances, the ligand is
cholesteryl.
[0190] In other embodiments, the RNAi agent is an oligonucleotide
tethered to a ligand for the purposes of improving cellular
targeting and uptake. For example, an RNAi agent may be tethered to
an antibody, or antigen binding fragment thereof. As an additional
example, an RNAi agent may be tethered to a specific ligand binding
molecule, such as a polypeptide or polypeptide fragment that
specifically binds a particular cell-surface receptor.
[0191] In other embodiments, the modulating agent comprises a
non-natural nucleobase. In certain embodiments, the non-natural
nucleobase is difluorotolyl, nitroimidazolyl, nitroindolyl, or
nitropyrrolyl. In certain embodiments, the modulating agents
provided herein relate to a double-stranded oligonucleotide
sequence, wherein only one of the two strands contains a
non-natural nucleobase. In certain embodiments, the modulating
agents as used herein relate to a double-stranded oligonucleotide
sequence, wherein both of the strands independently comprise at
least one non-natural nucleobase.
[0192] In certain instances, the ribose sugar moiety that naturally
occurs in nucleosides is replaced with a hexose sugar. In certain
aspects, the hexose sugar is an allose, altrose, glucose, mannose,
gulose, idose, galactose, talose, or a derivative thereof. In a
preferred embodiment, the hexose is a D-hexose. In certain
instances, the ribose sugar moiety that naturally occurs in
nucleosides is replaced with a polycyclic heteroalkyl ring or
cyclohexenyl group. In certain instances, the polycyclic
heteroalkyl group is a bicyclic ring containing one oxygen atom in
the ring. In certain instances, the polycyclic heteroalkyl group is
a bicyclo[2.2.1]heptane, a bicyclo[3.2.1]octane, or a
bicyclo[3.3.1]nonane. In certain embodiments, the backbone of the
oligonucleotide has been modified to improve the therapeutic or
diagnostic properties of the oligonucleotide compound. In certain
embodiments, at least one of the bases or at least one of the
sugars of the oligonucleotide has been modified to improve the
therapeutic or diagnostic properties of the oligonucleotide
compound. In instances when the oligonucleotide is double stranded,
the two strands are complementary, partially complementary, or
chimeric oligonucleotides.
[0193] Examples of modified RNAi agents envisioned for use in the
methods of the present invention include oligonucleotides
containing modified backbones or non-natural internucleoside
linkages. As defined here, oligonucleotides having modified
backbones or internucleoside linkages include those that retain a
phosphorus atom in the backbone and those that do not have a
phosphorus atom in the backbone. Modified oligonucleotides that do
not have a phosphorus atom in their intersugar backbone can also be
considered to be oligonucleotides. Specific oligonucleotide
chemical modifications are described below. It is not necessary for
all positions in a given compound to be uniformly modified, and in
fact more than one of the following modifications may be
incorporated in a single oligonucleotide compound or even in a
single nucleotide thereof.
[0194] Examples of modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalklyphosphotriesters, 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'. Various salts, mixed salts and free-acid forms are also
included.
[0195] Representative United States patents that teach the
preparation of the above phosphorus atom-containing linkages
include, but are not limited to, 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; 5,625,050;
and 5,697,248, each of which is herein incorporated by
reference.
[0196] Examples of modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleotides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
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 CH.sub.2
component parts.
[0197] Representative United States patents that teach the
preparation of the above oligonucleotides include, but are not
limited to, 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.
[0198] In other examples of oligonucleotide mimetics, both the
sugar and the internucleoside linkage, i.e., the backbone, of the
nucleoside units may be replaced with novel groups. The nucleobase
units are maintained for hybridization with an appropriate nucleic
acid target compound. One such oligonucleotide, an oligonucleotide
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 oligonucleotide is replaced
with an amide-containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds include, 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.
[0199] The present invention further encompasses oligonucleotides
employing ribozymes. Synthetic RNA molecules and derivatives
thereof that catalyze highly specific endoribonuclease activities
are known as ribozymes. (See, generally, U.S. Pat. No. 5,543,508 to
Haseloff et al., and U.S. Pat. No. 5,545,729 to Goodchild et al.)
The cleavage reactions are catalyzed by the RNA molecules
themselves. In naturally occurring RNA molecules, the sites of
self-catalyzed cleavage are located within highly conserved regions
of RNA secondary structure (Buzayan et al., Proc. Natl. Acad. Sci.
U.S.A., 1986, 83, 8859; Forster et al., Cell, 1987, 50, 9).
Naturally occurring autocatalytic RNA molecules have been modified
to generate ribozymes which can be targeted to a particular
cellular or pathogenic RNA molecule with a high degree of
specificity. Thus, ribozymes serve the same general purpose as
antisense oligonucleotides (i.e., modulation of expression of a
specific gene) and, like oligonucleotides, are nucleic acids
possessing significant portions of single-strandedness. That is,
ribozymes have substantial chemical and functional identity with
oligonucleotides and are thus considered to be equivalents for
purposes of the present invention.
[0200] In certain instances, the RNAi agents for use with the
methods provided herein may be modified by non-ligand group. A
number of non-ligand molecules have been conjugated to
oligonucleotides in order to enhance the activity, cellular
distribution, cellular targeting, or cellular uptake of the
oligonucleotide, and procedures for performing such conjugations
are available in the scientific literature. Such non-ligand
moieties have included lipid moieties, such as cholesterol
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553),
cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,
4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al.,
Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med.
Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,
Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk
et al., Biochimie, 1993, 75:49), 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; Shea et al., Nucl. Acids Res.,
1990, 18:3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,
1995, 1264:229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such oligonucleotide
conjugates have been listed above. Typical conjugation protocols
involve the synthesis of oligonucleotides bearing an aminolinker at
one or more positions of the sequence. The amino group is then
reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the oligonucleotide still bound to the solid
support or following cleavage of the oligonucleotide in solution
phase. Purification of the oligonucleotide conjugate by HPLC
typically affords the pure conjugate.
[0201] Additional examples of modulating agents, such as RNAi
oligonucleotides, may be found in U.S. Application Publication Nos.
2007/0275465, 2007/0054279, 2006/0287260, 2006/0035254,
2006/0008822, which are incorporated by reference.
Peptide Transporters
[0202] In certain embodiments, the antisense compounds described
herein may include an oligonucleotide moiety conjugated to a
cell-penetrating peptide that enhances uptake of the
oligonucleotide into a selected cell. Examples of such cells
include T-cells, such as activated T-cells and quiescent T-cells.
In certain embodiments, the antisense compounds of the invention
may include an oligonucleotide moiety conjugated to an
arginine-rich peptide transport moiety effective to enhance
transport of the compound into cells.
[0203] In certain embodiments, the transport moiety is attached to
a terminus of the oligomer, as illustrated, for example, in FIGS.
2A and 2B. In certain embodiments, the transport moiety is attached
to the 5'-terminus of the oligomer. In certain embodiments, the
transport moiety is attached to the 3-terminus of the oligomer.
[0204] In certain embodiments, the peptide transport moiety
comprises about 6 to 16 subunits selected independently from X'
subunits, Y' subunits, and Z' subunits,
[0205] where [0206] (a) each X' subunit independently represents
lysine, arginine or an arginine analog, said analog being a
cationic .alpha.-amino acid comprising a side chain of the
structure R.sup.1N.dbd.C(NH.sub.2)R.sup.2, where R.sup.1 is H or R;
R.sup.2 is R, NH.sub.2, NHR, or NR.sub.2, where R is lower alkyl or
lower alkenyl and may further include oxygen or nitrogen; R.sup.1
and R.sup.2 may together form a ring; and the side chain is linked
to said amino acid via R.sup.1 or R.sup.2; [0207] (b) each Y'
subunit independently represents a neutral amino acid
--C(O)--(CHR).sub.n--NH--, where n is 2 to 7 and each R is
independently H or methyl; and [0208] (c) each Z' subunit
independently represents an .alpha.-amino acid having a neutral
aralkyl side chain;
[0209] wherein the peptide comprises a sequence represented
independently by any one or more of (X'Y'X').sub.p, (X'Y').sub.m,
and (X'Z'Z').sub.p, where p is 2 to 5 and m is 2 to 8. Certain
embodiments include various combinations selected independently
from (X'Y'X').sub.p, (X'Y').sub.m, and/or (X'Z'Z').sub.p,
including, for example, peptides having the sequence
(X'Y'X')(X'Z'Z')(X'Y'X')(X'Z'Z') (SEQ ID NO:37).
[0210] In selected embodiments, for each X', the side chain moiety
is guanidyl, as in the amino acid subunit arginine (Arg). In
further embodiments, each Y' is --CO--(CH.sub.2).sub.n.CHR--NH--,
where n is 2 to 7 and R is H. For example, when n is 5 and R is H,
Y' is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx;
when n is 2 and R is H, Y' is a .beta.-alanine subunit, abbreviated
herein as B. Certain embodiments relate to carrier peptides having
a combination of different neutral amino acids, including, for
example, peptides comprising the sequence -RahxRRBRRAhxRRBRAhxB-
(SEQ ID NO:9), which contains both .beta.-alanine and
6-aminohexanoic acid.
[0211] Certain peptides of this type include those comprising
arginine dimers alternating with single Y' subunits, where Y' is
preferably Ahx. Examples include peptides having the formula
(RY'R).sub.p or the formula (RRY').sub.p, where Y' is preferably
Ahx.
[0212] In one embodiment, Y' is a 6-aminohexanoic acid subunit, R
is arginine and p is 4.
[0213] Certain embodiments include various linear combinations of
at least two of (RY'R).sub.p and (RRY').sub.p, including, for
example, illustrative peptides having the sequence
(RY'R)(RRY')(RY'R)(RRY') (SEQ ID NO:38), or (RRY')(RY'R)(RRY') (SEQ
ID NO:39). Other combinations are contemplated. In a further
embodiment, each Z' is phenylalanine, and m is 3 or 4.
[0214] In certain embodiments, the conjugated peptide is linked to
a terminus of the oligomer via a linker Ahx-B, where Ahx is a
6-aminohexanoic acid subunit and B is a .beta.-alanine subunit, as
shown, for example, in FIGS. 2A and 2B.
[0215] In certain embodiments, for each X', the side chain moiety
is independently selected from the group consisting of guanidyl
(HN.dbd.C(NH.sub.2)NH--), amidinyl (HN.dbd.C(NH.sub.2)C<),
2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl,
2-aminopyridinyl, and 2-aminopyrimidonyl, and it is preferably
selected from guanidyl and amidinyl. In one embodiment, the side
chain moiety is guanidyl, as in the amino acid subunit arginine
(Arg).
[0216] In certain embodiments, the Y' subunits may be either
contiguous, in that no X' subunits intervene between Y' subunits,
or interspersed singly between X' subunits. In certain embodiments,
the linking subunit may be between Y' subunits. In one embodiment,
the Y' subunits are at a terminus of the transporter; in other
embodiments, they are flanked by X' subunits. In further preferred
embodiments, each Y' is --CO(CH.sub.2).sub.n.CHR--NH--, where n is
2 to 7 and R is H. For example, when n is 5 and R is H, Y' is a
6-aminohexanoic acid subunit, abbreviated herein as Ahx.
[0217] In certain embodiments of this group, each X' comprises a
guanidyl side chain moiety, as in an arginine subunit. Certain
peptides of this type include those comprising arginine dimers
alternating with single Y' subunits, where Y' is preferably Ahx.
Examples include peptides having the formula (RY'R).sub.4 or the
formula (RRY').sub.4, where Y' is preferably Ahx. In the latter
case, the nucleic acid analog is preferably linked to a terminal Y'
subunit, preferably at the C-terminus, as shown, for example, in
FIGS. 2A and 2B. The preferred linker is of the structure AhxB,
where Ahx is a 6-aminohexanoic acid subunit and B is a
.beta.-alanine subunit.
[0218] The transport moieties as described above have been shown to
greatly enhance cell entry of attached oligomers, relative to
uptake of the oligomer in the absence of the attached transport
moiety, and relative to uptake by an attached transport moiety
lacking the hydrophobic subunits Y'. Such enhanced uptake is
preferably evidenced by at least a two-fold increase, and
preferably a four-fold increase, in the uptake of the compound into
mammalian cells relative to uptake of the agent by an attached
transport moiety lacking the hydrophobic subunits Y'. Uptake is
preferably enhanced at least five-fold, ten-fold, twenty fold, and
more preferably forty fold, relative to the unconjugated
compound.
[0219] A further benefit of the transport moiety is its expected
ability to stabilize a duplex between an antisense compound and its
target nucleic acid sequence, presumably by virtue of electrostatic
interaction between the positively charged transport moiety and the
negatively charged nucleic acid. The number of charged subunits in
the transporter is less than 14, as noted above, and preferably
between 8 and 11, since too high a number of charged subunits may
lead to a reduction in sequence specificity.
[0220] The use of peptide transporters such as arginine-rich
peptide transporters (i.e., cell-penetrating peptides) are
particularly useful in practicing the present invention. Certain
peptide transporters have been shown to be highly effective at
delivery of antisense compounds into primary leukocytes (Marshall,
Oda et al. 2007). Furthermore, compared to other known peptide
transporters such as Penetratin, the peptide transporters described
herein, when conjugated to an antisense PMO, demonstrate an
enhanced ability to alter splicing of several gene transcripts
(Marshall, Oda et al. 2007). Especially preferred are the P007,
CP06062, and CP04057 transport peptides listed below in Table 3
(SEQ ID NOS: 4, 9, and 10 respectively).
[0221] Exemplary peptide transporters, including linkers (B or
AhxB) are given below in Table 3: Preferred sequences are those
designated P007 (SEQ ID NO: 4) and CP06062 (SEQ ID NO: 9).
TABLE-US-00003 TABLE 3 Exemplary Peptide Transporters for
Intracellular Delivery of PMO Sequence SEQ Peptide (N-terminal to
C-terminal) ID NO: rTAT RRRQRRKKRC 1 R.sub.9F.sub.2 RRRRRRRRRFFC 2
(RRAhx).sub.4B RRAhxRRAhxRRAhxRRAhxB 3 (RAhxR).sub.4AhxB;
RAhxRRAhxRRAhxRRAhxRAhxB 4 (P007) (AhxRR).sub.4AhxB
AhxRRAhxRRAhxRRAhxRRAhxB 5 (RAhx).sub.6B RAhxRAhxRAhxRAhxRAhxRAhxB
6 (RAhx).sub.8B RAhxRAhxRAhxRAhxRAhxRAhx 7 RAhxB (RAhxR).sub.3AhxB
RAhxRRAhxRRAhxRAhxB 8 (RAhxRRBR).sub.2AhxB; RAhxRRBRRAhxRRBRAhxB 9
(CPO6062) (RAhxR)5AhxB RAhxRRAhxRRAhxRRAhxRRAh 10 (CP04057)
xRAhxB
Contact Hypersensitivity and Methods of Use
[0222] Embodiments of the present invention include compositions
and methods of treating or reducing skin or mucous membrane
inflammation, including inflammation associated with contact
hypersensitivity or contact dermatitis. These inflammatory
conditions are typically associated with topical exposure to a
sensitizing agent, such as an antigen. By way of non-limiting
theory, activation-induced cell death (AICD) is a naturally
occurring process for regulating the resolution of T-cell
responses, and antisense targeting of cFLAR expression, alone or in
conjunction with a selected antigen, sensitizes certain T-cells to
undergo early AICD, resulting in tolerance to the sensitizing
agent. Hence, in certain embodiments, the T-cells targeted by the
antisense oligonucleotides described herein may be specific for or
activated by one or more selected sensitizing agents, including
foreign allergens and irritants, as compared to being specific for
or activated by alloantigens or self-antigens.
[0223] As noted above, CFLAR expression can be targeted a variety
of ways, such as by targeting the AUG start codon region or a
splice region of a CFLAR mRNA transcript. Hence, certain
embodiments include methods of inducing tolerance to a sensitizing
agent, comprising topically applying an effective amount of an
antisense composition containing an antisense oligonucleotide,
wherein the antisense oligonucleotide targets the start site of a
CFLAR mRNA or a splice site or branch point of a CFLAR mRNA. The
antisense agent is typically effective to reduce expression of a
functional human CFLAR in CFLAR-expressing lymphocytes, such as
CD4+ and CD8+ T-cells. Also included are methods of treating
contact hypersensitivity or contact dermatitis, comprising
contacting the skin or mucous membrane of a subject with an
effective amount of an antisense composition described herein.
[0224] Hypersensitivity relates to an undesirable reaction produced
by the immune system, often in response to contact with a
sensitizing agent such as an irritant or allergen. In a delayed
type hypersensitivity reaction, CD8+ cytotoxic T cells and CD4+
helper T cells recognize sensitizing agents such as antigen in a
complex with either type 1 or 2 major histocompatibility complex,
and activate an undesired immune response, typically near the site
of contact with the sensitizing agent. This process typically
results in localized inflammation at the site of exposure to the
agent, though certain reactions may produce a systemic reaction.
Accordingly, certain embodiments include the treatment of
hypersensitivity reactions, such as delayed type hypersensitivity
reactions, mainly associated with a sensitizing agent.
[0225] Certain embodiments include the treatment or reduction of
contact dermatitis, an inflammatory skin or mucous membrane
reaction that results from exposure to sensitizing agents such as
allergens (allergic contact dermatitis) or irritants (irritant
contact dermatitis). Photocontact dermatitis occurs when the
allergen or irritant is activated by sunlight Irritant dermatitis
relates generally to inflammation that is triggered by contact with
acids, alkaline materials such as soaps and detergents, solvents,
adhesives, or other chemicals. The skin reaction in irritant
dermatitis usually resembles a burn. Allergic contact dermatitis
relates generally to inflammation that is triggered by exposure to
a variety of different substances, typically a substance or
material to which a subject is extra sensitive or allergic. The
allergic reaction is often delayed, with the rash or other symptom
appearing about 24-48 hours after exposure. The skin reaction in
allergic dermatitis typically varies from mild irritation and
redness to open sores, depending on the type of irritant, the body
part affected, and the sensitivity of the individual.
[0226] Certain embodiments include the treatment of conditions
related to dermatitis more generally. Examples of such conditions
include psoriasis (i.e., a typically chronic, recurrent skin
disease in humans marked by discrete macules, papules or patches
covered with lamellated silvery scales resulting from an increased
turnover of epidermal cells), seborrheic dermatitis, atopic
dermatitis (eczema), thermal-induced dermatitis, drug-induced
dermatitis, dyshidrotic dermatitis (i.e., a type of eczema that
occurs on the palms of the hands, sides of the fingers, and soles
of the feet, and typically causes a burning or itching sensation
and a blistering rash), urticaria (i.e., a skin condition
characterized by welts that itch intensely, caused by an allergic
reaction, an infection, or a nervous condition; often called
"hives"), and bullous dermatitis.
[0227] The symptoms of contact dermatitis include itching
(pruritus) of the skin in exposed areas, redness or inflammation in
the exposed area, tenderness of the skin in the exposed area,
localized swelling of the skin, warmth of the exposed area, skin
lesion or rash at the site of exposure, including redness, rash,
papules (pimple-like), vesicles, and bullae (blisters). Also, the
lesions may involve oozing, draining, or crusting, or may become
scaly, raw, or thickened. The symptoms of contact dermatitis may
last from several days to several weeks. Chronic contact dermatitis
refers to inflammation that persists after removal of the offending
sensitizing agent.
[0228] Contact hypersensitivity or dermatitis may occur on any body
surface such as the skin or mucous membranes. Skin architecture is
well known. Briefly, epidermis, the skin outer layer, is covered by
the stratum corneum, a protective layer of dead epidermal skin
cells (e.g., keratinocytes) and extracellular connective tissue
proteins. The epidermis undergoes a continual process of being
sloughed off as it is replaced by new material pushed up from the
underlying epidermal granular cell, spinous cell, and basal cell
layers, where continuous cell division and protein synthesis
produce new skin cells and skin proteins (e.g., keratin, collagen).
The dermis lies underneath the epidermis, and is a site for the
elaboration by dermal fibroblasts of connective tissue proteins
(e.g., collagen, elastin, etc.) that assemble into extracellular
matrix and fibrous structures that confer flexibility, strength and
elasticity to the skin. Also present in the dermis are nerves,
blood vessels, smooth muscle cells, hair follicles and sebaceous
glands. Included are skin sites such as the head, face, ears (e.g.,
otitis externa), neck, arms, hands, underarms, chest, back, pelvis,
groin, buttocks, legs, and feet.
[0229] The mucous membranes (i.e., mucosae or mucosa) refer linings
of mostly endodermal origin, covered in epithelium, which are
involved in absorption and secretion. Mucous membranes line various
body cavities that are exposed to the external environment, and are
continuous with the skin at several places, including the nostrils,
the lips, the ears, the eyes, the genital area, and the anus.
Examples of mucosal membranes include the buccal mucosae (mucous
membrane of the inside of the cheek), esophageal mucosae, gastric
mucosae, intestinal mucosae, nasal mucosae, olfactory mucosae, oral
mucosae, bronchial mucosae, uterine mucosae (e.g., endometrium),
and penile mucosae. Also included are ophthalmologic tissues. For
instance, allergy to chemicals in ophthalmologic preparations may
provoke dermatitis around the eyes. Hence, certain embodiments
relate to treating hypersensitivity or contact dermatitis
associated with any one or more of these skin sites or mucosal
sites, and may include application of an antisense composition to
any one or more of these sites.
[0230] A sensitizing agent refers to any substance that causes a
hypersensitivity or other inflammatory reaction in skin or mucosal
membranes of a subject. In certain embodiments, the sensitizing
agent causes contact dermatitis in the subject. Included are
allergens and irritants, among others, such as haptens and
hapten-protein conjugates. Particular examples of sensitizing
agents include, without limitation, acids, alkalis (e.g., soaps,
detergents, drain cleaners, strong soap with lye residues),
solvents (e.g., alcohol, xylene, turpentine, esters, acetone,
ketones), heavy metals (e.g., nickel, gold, cobalt such as cobalt
chloride) rubber (e.g., mercaptobenzothiazole), latex, surfactants
(e.g., sodium lauryl sulfate), kerosene, chlorine, ethylene oxide,
cosmetics, antiseptics, insecticides, potassium dichromate (e.g.,
cements, household cleaners), paraphenylenediamine, certain dental
products, formaldehyde, and fragrances (e.g., Myroxolon pereirae).
Further examples of sensitizing agents include urushiol oil,
medications (e.g., antibiotics such as neomycin and bacitracin,
topical steroids, fungicides such as thiram), quaternium-15, and
thimerosal, a mercury compound used in local antiseptics and in
vaccines.
[0231] Also included are chromates, or compounds containing
chromium, which can be found in cement, leather, some matches,
paints and anti-rust compounds. Occupational exposure to chromium
is common in jobs in the automobile, welding, foundry, cement,
railroad and building repair industries.
[0232] In certain embodiments, contact dermatitis may result from
exposure to plants, including plants that contain a sensitizing
agent. Examples of plant-based sensitizing agents include a number
of alkaloids, glycosides, saponins, anthraquinones, irritant
calcium oxalate crystals, and urushiol oil. Examples of plants that
contain urushiol oil include poison oak, poison ivy, poison sumac,
and other Anacardiaceae plant family members or other plants that
elicit similar inflammatory responses.
[0233] Also included is the treatment of contact dermatitis
associated with low humidity. For instance, low humidity from air
conditioning has been shown to cause irritant contact dermatitis,
mainly due to the lack of sufficient water vapor.
[0234] Certain embodiments included treatment of photocontact
dermatitis, or photoaggravated dermatitis. This type of dermatitis
may be triggered by an interaction between an otherwise benign or
less harmful substance (e.g., a sensitizing agent) and a source of
ultraviolet light, such as the sun or a tanning bed lamp.
Typically, the ultraviolet light is in the range of about 320-400
nm.
[0235] Certain embodiments include reducing the risk of secondary
conditions or complications associated with contact dermatitis. For
instance, the methods provided herein may reduce secondary
bacterial skin infections that often occur during or following
contact dermatitis.
[0236] Also included are combination therapies. For instance, the
antisense oligonucleotides described herein may be combined with
one or more standard treatment agents or modalities. Examples of
standard treatment agents include calamine lotion, steroids such as
corticosteroids (e.g., hydrocortisone cream), antihistamines, and
barrier creams such as creams that contain zinc oxide. Included are
compositions that comprise an antisense agent and at least one of
these standard treatment agents, as well as methods of combination
therapy using said treatment agents, whether by applying them
sequentially with or at the same time as the antisense agent.
[0237] In certain embodiments, antisense oligonucleotides may be
administered simultaneously, separately, or over a period of time
in association with one or several allergens. Administration
includes applying the composition to the affected area skin or
mucous membrane area or area at risk of exposure to the agent, and
rubbing the composition into the skin or mucous membrane.
Application may be once a day or less often, or two or more times a
day, e.g., every 8 hours. In certain embodiments, the
administration of the pharmaceutical composition and the allergen
will be localized. In another embodiment, the allergen is a
component of the pharmaceutical composition.
[0238] The time course of administration may be similar to current
allergen desensitization treatment regimens. For illustration only,
the treatment regimen may range from a single treatment to daily
treatments for one to 12 weeks or until the clinical criteria for
contact dermatitis has been resolved. In certain embodiments, the
composition may be applied to a skin area of the subject prior to
contact with the sensitizing agent. In certain embodiments, the
composition may be applied to a skin area of the subject at the
same time as the sensitizing agent. In certain embodiments, the
composition may be applied to a skin area of the subject after
contact with the sensitizing agent.
Topical Compositions
[0239] Also included are pharmaceutical compositions or
formulations that comprise the CFLAR-targeted antisense agents
described herein. In certain embodiments, the pharmaceutical
compositions are adapted for topical administration, and include
compositions suitable for application to skin or mucous
membranes.
[0240] The step of administering may be performed by any means
known to the art, for example, topically, transdermally,
sublingually, subcutaneously, transbuccally, intranasally, via
inhalation, and intraoccularly. In preferred embodiments
administering may be performed topically, where pharmaceutical
excipients or carriers for topical use are described herein and
known in the art. Certain other embodiments contemplate
administration of the formulations described herein as a bulk
deposition, which may be, for example time-released or
alternatively immediately available
[0241] As noted above, certain invention embodiments described
herein relate to topical formulations of the described antisense
oligonucleotides, which formulations comprise the oligonucleotides
in a pharmaceutically acceptable carrier, excipient or diluent and
in a therapeutic amount, as disclosed herein, when administered
topically to an animal, preferably a mammal, and most preferably a
human.
[0242] Topical administration of the oligonucleotides described
herein, or their pharmaceutically acceptable salts, in pure form or
in an appropriate pharmaceutical composition, can be carried out
via any of the accepted modes of topical administration of agents
for serving similar utilities. Topical application or
administration of a composition means, in preferred embodiments,
directly contacting the composition (e.g., a topical formulation)
with skin or mucosa of the subject undergoing treatment, which may
be at one or more localized or widely distributed skin or mucosal
sites and which may generally refer to contacting the topical
formulation with intact stratum corneum or epidermis but need not
be so limited; for instance, certain embodiments contemplate as a
topical application the administration of a topical formulation
described herein to injured, abraded, wrinkled or damaged skin
(including photodamaged skin), or skin of a subject undergoing
surgery, such that contact of the topical formulation may take
place not only with stratum corneum or epidermis but also with skin
granular cell, spinous cell, and/or basal cell layers, and/or with
dermal or underlying tissues, for example, as may accompany certain
types of skin tissue remodeling.
[0243] The topical formulations with an appropriate
pharmaceutically acceptable carrier, diluent or excipient for use
in a topical formulation preparation, and may be formulated into
preparations in solid, semi-solid, gel, cream, colloid, suspension
or liquid or other topically applied forms, such as powders,
granules, ointments, solutions, washes, gels, pastes, plasters,
paints, bioadhesives, microsphere suspensions, and aerosol sprays.
Pharmaceutical compositions of these and related embodiments are
formulated so as to allow the active ingredients contained therein
to be bioavailable upon topical administration of the composition
to skin of a subject, such as a mammal, including a human.
[0244] Depending on the particular embodiments, which may vary as
will be appreciated by the skilled artisan in part as a function of
the condition to be treated in a given subject, the topical
formulations described herein deliver a therapeutically effective
amount of, e.g., the antisense oligonucleotides or other active
compound(s) to skin cells such as epithelial cells, keratinocytes,
cells of the scalp (including in certain embodiments cells such as
follicular cells and/or melanocytes), dermal fibroblasts, and/or
mucosal tissue. Preferred formulations may exhibit ready
permeability into the skin or mucosa, as can be determined
according to any of a number of established methodologies known to
the art for testing the skin or mucosal permeability of a drug
composition (see, e.g., Wagner et al., 2002 J. Invest. Dermatol.
118:540, and references cited therein; Bronaugh et al., 1985 J.
Pharm. Sci. 74:64; Bosman et al., 1998 J. Pharm. Biomed. Anal.
17:493-499; Bosman et al., 1996 J. Pharm Biomed Anal. 1996
14:1015-23; Bonferoni et al., 1999 Pharm Dev Technol. 4:45-53;
Frantz, Instrumentation and methodology for in vitro skin diffusion
cells in methodology for skin absorption. In: Methods for Skin
Absorption (Kemppainen & Reifenrath, Eds), CRC Press, Florida,
1990, pp. 35-59; Tojo, Design and calibration of in vitro
permeation apparatus. In: Transdermal Controlled Systemic
Medications (Chien Y W, Ed), Marcel Dekker, New York, 1987,
127-158; Barry, Methods for studying percutaneous absorption. In:
Dermatological Formulations: Percutaneous absorption, Marcel
Dekker, New York, 1983, 234-295).
[0245] Compositions that will be administered to the skin of a
subject may in certain embodiments take the form of one or more
dosage units, where for example, a liquid-filled capsule or ampule
may contain a single dosage unit, and a container of a topical
formulation as described herein in aerosol form may hold a
plurality of dosage units. Methods of preparing such dosage forms
are known to those skilled in the art; for example, see The Science
and Practice of Pharmacy, 20th Edition (Philadelphia College of
Pharmacy and Science, 2000). The composition to be administered may
contain an effective amount of a CFLAR-targeted antisense
oligonucleotide or a pharmaceutically acceptable salt thereof, as
described herein.
[0246] As noted above, the present topical formulations may take
any of a wide variety of forms, and include, for example, creams,
lotions, solutions, sprays, gels, ointments, pastes or the like,
and/or may be prepared so as to contain liposomes, micelles, and/or
microspheres. See, e.g., U.S. Pat. No. 7,205,003. For instance,
creams, as is well known in the arts of pharmaceutical and
cosmeceutical formulation, are viscous liquids or semisolid
emulsions, either oil-in-water or water-in-oil. Cream bases are
water-washable, and contain an oil phase, an emulsifier, and an
aqueous phase. The oil phase, also called the "internal" phase, is
generally comprised of petrolatum and a fatty alcohol such as cetyl
or stearyl alcohol. The aqueous phase usually, although not
necessarily, exceeds the oil phase in volume, and generally
contains a humectant. The emulsifier in a cream formulation is
generally a nonionic, anionic, cationic or amphoteric
surfactant.
[0247] Lotions are preparations to be applied to the skin surface
without friction, and are typically liquid or semi-liquid
preparations in which solid particles, including the active agent,
are present in a water or alcohol base. Lotions are usually
suspensions of solids, and preferably comprise a liquid oily
emulsion of the oil-in-water type. Lotions are preferred
formulations herein for treating large body areas, because of the
ease of applying a more fluid composition. It is generally
preferred that the insoluble matter in a lotion be finely divided.
Lotions typically contain suspending agents to produce better
dispersions as well as compounds useful for localizing and holding
the active agent in contact with the skin or mucosa, e.g.,
methylcellulose, sodium carboxymethyl-cellulose, or the like.
[0248] Solutions refer to homogeneous mixtures prepared by
dissolving one or more chemical substances (solutes) in a liquid
such that the molecules of the dissolved substance are dispersed
among those of the solvent. The solution may contain other
pharmaceutically acceptable and/or cosmeceutically acceptable
chemicals to buffer, stabilize or preserve the solute. Common
examples of solvents used in preparing solutions are ethanol,
water, propylene glycol or any other pharmaceutically acceptable
and/or cosmeceutically acceptable vehicles.
[0249] Gels are semisolid, suspension-type systems. Single-phase
gels contain organic macromolecules distributed substantially
uniformly throughout the carrier liquid, which is typically
aqueous, but also, preferably, contain an alcohol, and, optionally,
an oil. Preferred "organic macromolecules," i.e., gelling agents,
may be chemically crosslinked polymers such as crosslinked acrylic
acid polymers, for instance, the "carbomer" family of polymers,
e.g., carboxypolyalkylenes, that may be obtained commercially under
the Carbopol.RTM. trademark. Also preferred in certain embodiments
may be hydrophilic polymers such as polyethylene oxides,
polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol;
cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, and methyl cellulose; gums such as
tragacanth and xanthan gum; sodium alginate; and gelatin. In order
to prepare a uniform gel, dispersing agents such as alcohol or
glycerin can be added, or the gelling agent can be dispersed by
trituration, mechanical mixing or stirring, or combinations
thereof.
[0250] Ointments, as also well known in the art, are semisolid
preparations that are typically based on petrolatum or other
petroleum derivatives. The specific ointment base to be used, as
will be appreciated by those skilled in the art, is one that will
provide for a number of desirable characteristics, e.g., emolliency
or the like. As with other carriers or vehicles, an ointment base
should be inert, stable, nonirritating, and nonsensitizing. As
explained in Remington: The Science and Practice of Pharmacy, 19th
Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404,
ointment bases may be grouped in four classes: oleaginous bases;
emulsifiable bases; emulsion bases; and water-soluble bases.
Oleaginous ointment bases include, for example, vegetable oils,
fats obtained from animals, and semisolid hydrocarbons obtained
from petroleum. Emulsifiable ointment bases, also known as
absorbent ointment bases, contain little or no water and include,
for example, hydroxystearin sulfate, anhydrous lanolin, and
hydrophilic petrolatum. Emulsion ointment bases are either
water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and
include, for example, cetyl alcohol, glyceryl monostearate,
lanolin, and stearic acid. Preferred water-soluble ointment bases
are prepared from polyethylene glycols of varying molecular weight
(see, e.g., Remington, Id.).
[0251] Pastes are semisolid dosage forms in which the active agent
is suspended in a suitable base. Depending on the nature of the
base, pastes are divided between fatty pastes or those made from
single-phase aqueous gels. The base in a fatty paste is generally
petrolatum or hydrophilic petrolatum or the like. The pastes made
from single-phase aqueous gels generally incorporate
carboxymethylcellulose or the like as a base.
[0252] Formulations may also be prepared with liposomes, micelles,
and microspheres. Liposomes are microscopic vesicles having one
(unilamellar) or a plurality (multilamellar) of lipid walls
comprising a lipid bilayer, and, in the present context, may
encapsulate and/or have adsorbed to their lipid membranous surfaces
one or more components of the topical formulations herein
described, such as the antisense oligonucleotides certain carriers
or excipients. Liposomal preparations herein include cationic
(positively charged), anionic (negatively charged), and neutral
preparations. Cationic liposomes are readily available. For
example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA)
liposomes are available under the tradename Lipofectin.RTM. (GIBCO
BRL, Grand Island, N.Y.). Similarly, anionic and neutral liposomes
are readily available as well, e.g., from Avanti Polar Lipids
(Birmingham, Ala.), or can be easily prepared using readily
available materials. Such materials include phosphatidyl choline,
cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl
choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphoshatidyl ethanolamine (DOPE), among others. These
materials can also be mixed with DOTMA in appropriate ratios.
Methods for making liposomes using these materials are well known
in the art.
[0253] Micelles are known in the art as comprised of surfactant
molecules arranged so that their polar headgroups form an outer
spherical shell, while the hydrophobic, hydrocarbon chains are
oriented towards the center of the sphere, forming a core. Micelles
form in an aqueous solution containing surfactant at a high enough
concentration so that micelles naturally result. Surfactants useful
for forming micelles include, but are not limited to, potassium
laurate, sodium octane sulfonate, sodium decane sulfonate, sodium
dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium
chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether,
polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.
Microspheres, similarly, may be incorporated into the presently
described topical formulations.
[0254] Like liposomes and micelles, microspheres essentially
encapsulate one or more components of the present formulations.
They are generally, but not necessarily, formed from lipids,
preferably charged lipids such as phospholipids. Preparation of
lipidic microspheres is well known in the art.
[0255] Various additives, as known to those skilled in the art, may
also be included in the topical formulations. For example,
solvents, including relatively small amounts of alcohol, may be
used to solubilize certain formulation components. It may be
desirable, for certain topical formulations or in cases of
particularly severe inflammatory conditions of the skin, to include
in the topical formulation an added skin permeation enhancer in the
formulation. Examples of suitable enhancers include, but are not
limited to, ethers such as diethylene glycol monoethyl ether
(available commercially as Transcutol.RTM.) and diethylene glycol
monomethyl ether; surfactants such as sodium laurate, sodium lauryl
sulfate, cetyltrimethylammonium bromide, benzalkonium chloride,
Poloxamer.RTM. (231, 182, 184), Tween.RTM. (20, 40, 60, 80), and
lecithin (U.S. Pat. No. 4,783,450); alcohols such as ethanol,
propanol, octanol, benzyl alcohol, and the like; polyethylene
glycol and esters thereof such as polyethylene glycol monolaurate
(PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and other
nitrogenous compounds such as urea, dimethylacetamide (DMA),
dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone,
ethanolamine, diethanolamine, and triethanolamine; terpenes;
alkanones; and organic acids, particularly citric acid and succinic
acid. Azone.RTM. and sulfoxides such as DMSO and C.sub.10MSO may
also be used, but are less preferred.
[0256] Certain skin permeation enhancers include lipophilic
co-enhancers typically referred to as "plasticizing" enhancers,
i.e., enhancers that have a molecular weight in the range of about
150 to 1000 daltons, an aqueous solubility of less than about 1 wt
%, preferably less than about 0.5 wt %, and most preferably less
than about 0.2 wt %. The Hildebrand solubility parameter of
plasticizing enhancers is in the range of about 2.5 to about 10,
preferably in the range of about 5 to about 10. Preferred
lipophilic enhancers are fatty esters, fatty alcohols, and fatty
ethers. Examples of specific and most preferred fatty acid esters
include methyl laurate, ethyl oleate, propylene glycol monolaurate,
propylene glycerol dilaurate, glycerol monolaurate, glycerol
monooleate, isopropyl n-decanoate, and octyldodecyl myristate.
Fatty alcohols include, for example, stearyl alcohol and oleyl
alcohol, while fatty ethers include compounds wherein a diol or
triol, preferably a C.sub.2-C.sub.4 alkane diol or triol, are
substituted with one or two fatty ether substituents. Additional
skin permeation enhancers will be known to those of ordinary skill
in the art of topical drug delivery, and/or are described in the
relevant literature. See, e.g., Percutaneous Penetration Enhancers,
eds. Smith et al. (CRC Press, 1995).
[0257] Various other additives may be included in the topical
formulations according to certain embodiments of the present
invention, in addition to those identified above. These include,
but are not limited to, antioxidants, astringents, perfumes,
preservatives, emollients, pigments, dyes, humectants, propellants,
and sunscreen agents, as well as other classes of materials whose
presence may be cosmetically, medicinally or otherwise desirable.
Typical examples of optional additives for inclusion in the
formulations of the invention are as follows: preservatives such as
sorbate; solvents such as isopropanol and propylene glycol;
astringents such as menthol and ethanol; emollients such as
polyalkylene methyl glucosides; humectants such as glycerine;
emulsifiers such as glycerol stearate, PEG-100 stearate,
polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol
and other polyhydroxyalcohols such as polyethylene glycol;
sunscreen agents such as octyl methoxyl cinnamate (available
commercially as Parsol MCX) and butyl methoxy benzoylmethane
(available under the tradename Parsol 1789); antioxidants such as
ascorbic acid (vitamin C), .alpha.-tocopherol (Vitamin E),
.beta.-tocopherol, .gamma.-tocopherol, .delta.-tocopherol,
.epsilon.-tocopherol, .lamda..sub.1-tocopherol,
.lamda..sub.2-tocopherol, .eta.-tocopherol, and retinol (vitamin
A); essential oils, ceramides, essential fatty acids, mineral oils,
vegetable oils (e.g., soy bean oil, palm oil, liquid fraction of
shea butter, sunflower oil), animal oils (e.g., perhydrosqualene),
synthetic oils, silicone oils or waxes (e.g., cyclomethicone and
dimethicone), fluorinated oils (generally perfluoropolyethers),
fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax,
carnauba wax, and paraffin wax); skin-feel modifiers; and
thickeners and structurants such as swelling clays and cross-linked
carboxypolyalkylenes that may be obtained commercially under the
Carbopol.RTM. trademark.
[0258] Other additives include beneficial agents such as those
materials that condition the skin (particularly, the upper layers
of the skin in the stratum corneum) and keep it soft by retarding
the decrease of its water content and/or protect the skin. Such
conditioners and moisturizing agents include, by way of example,
pyrrolidine carboxylic acid and amino acids; organic antimicrobial
agents such as 2,4,4'-trichloro-2-hydroxy diphenyl ether
(triclosan) and benzoic acid; anti-inflammatory agents such as
acetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeic
agents such as retinoic acid; vasodilators such as nicotinic acid;
inhibitors of melanogenesis such as kojic acid; and mixtures
thereof. Other advantageously included cosmeceutically active
agents may be present, for example, .alpha.-hydroxyacids,
.alpha.-ketoacids, polymeric hydroxyacids, moisturizers, collagen,
marine extracts, and antioxidants such as ascorbic acid (vitamin
C), .alpha.-tocopherol (Vitamin E) or other tocopherols such as
those described above, and retinol (vitamin A), and/or cosmetically
acceptable salts, esters, amides, or other derivatives thereof.
Additional cosmetic agents include those that are capable of
improving oxygen supply in skin tissue, as described, for example,
in WO 94/00098 and WO 94/00109. Sunscreens may also be
included.
[0259] Other embodiments may include a variety of non-carcinogenic,
nonirritating healing materials that facilitate treatment with the
formulations of the invention. Such healing materials may include
nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant
extracts, glandular or animal extracts, or safe therapeutic agents
that may be added to the formulation to facilitate dermal healing.
The amounts of these various additives are those conventionally
used in the cosmetics field, and range, for example, from about
0.01% to about 20% of the total weight of the topical
formulation.
[0260] The formulations of the invention may also include
conventional additives such as opacifiers, fragrance, colorant,
gelling agents, thickening agents, stabilizers, surfactants, and
the like. Other agents may also be added, such as antimicrobial
agents, to prevent spoilage upon storage, i.e., to inhibit growth
of microbes such as yeasts and molds. Suitable antimicrobial agents
are typically selected from methyl and propyl esters of
p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium
benzoate, sorbic acid, imidurea, and combinations thereof. The
formulations may also contain irritation-mitigating additives to
minimize or eliminate the possibility of skin irritation or skin
damage resulting from the skin tissue repair-promoting compound to
be administered, or from other components of the composition.
Suitable irritation-mitigating additives include, for example:
.alpha.-tocopherol; monoamine oxidase inhibitors, particularly
phenyl alcohols such as 2-phenyl-1-ethanol; glycerin; salicylates;
ascorbates; ionophores such as monensin; amphiphilic amines;
animonium chloride; N-acetylcysteine; capsaicin; and chloroquine.
The irritation-mitigating additive, if present, may be incorporated
into the topical formulation at a concentration effective to
mitigate irritation or skin damage, typically representing not more
than about 20 wt %, more typically not more than about 5 wt %, of
the formulation.
[0261] In certain embodiments, a topical composition may include
any normally used galenic formulation, such as an aqueous,
hydroalcoholic or oily solution, an oil-in-water or water-in-oil or
multiple emulsion, an aqueous or oily gel, a liquid, paste or solid
anhydrous product, an oil dispersion in a polymeric phase such as
nanospheres and nanocapsules and/or non-ionic type lipidic
vesicles. Such compositions may be more or less fluid and may be in
the form of a white or colored cream, a pomade, a milk, a lotion, a
serum, a paste or a foam. It may even be applied on the skin in the
form of an aerosol. It may also be in powder or other solid form,
for example in stick form. Such compositions may also be in the
form of patches, pencils, brushes or applicators used for local
application on spots on the face or hands. The compositions
provided herein may also contain additives normally used in the
cosmetic field, such as hydrophilic or lipophilic gels, hydrophilic
or lipophilic active constituents, preservation agents,
antioxidants, solvents, odorants, fillers, filters, pigments, odor
absorbers and coloring material. The quantities of these different
additives are as conventionally used in the fields considered.
Depending on the nature, these additives may be added in the fatty
phase, in the aqueous phase, in lipidic vesicles and/or in
nanoparticles.
[0262] In one embodiment of the invention, the pharmaceutical
composition is in the form of an emulsion containing an oil, an
emulsifier chosen from among fatty acid and polyethylene glycol
esters such as PEG-20 stearate, and fatty acid and glycerine esters
such as glycerine stearate, and a co-emulsifier. When the cosmetic
composition of the invention is an emulsion, the proportion of the
fatty phase can vary from 5 to 80% by weight, and preferably from 5
to 50% by weight with reference to the total weight of the
composition. The oils, emulsifiers and co-emulsifiers used in the
composition in emulsion form are chosen from among those
conventionally used in the field considered. The emulsifying agent
and the co-emulsifying agent are present in the composition in a
proportion varying from 0.3% to 30% by weight, and preferably from
0.5% to 20% by weight compared with the total weight of the
composition. Oils that can be used in association with oligomer
conjugates according to the invention include mineral oils
(Vaseline oil), vegetable origin oils (avocado oil, Soya oil),
animal origin oils (lanoline), synthetic oils (perhydrosqualene),
silicone oils (cyclomethicone) and fluorine oils
(perfluoropolyethers). Fatty alcohols (cetylic alcohol), fatty
acids, waxes (Carnauba wax, ozokerite) can also be used as fatty
materials. For example, emulsifiers and coemulsifiers that can be
used in association with oligomer conjugates according to the
invention include fatty acid and polyethylene glycol esters such as
PEG-20 stearate and fatty acid and glycerine esters such as
glyceryl stearate. Hydrophilic gelifiers that can be used in
association with oligomer conjugates according to the invention
include in particular carboxyvinylic polymers (carbomer), acrylic
copolymers such as acrylate/alkylacrylate copolymers,
polyacrylamides, polysaccharides, natural gums and clays.
Lipophilic gelifiers include modified clays like bentones, metallic
salts of fatty acids, hydrophobic silica and polyethylenes.
[0263] Antisense oligonucleotides for use in the present
formulations, or their pharmaceutically acceptable salts, are
administered in an effective amount, which will vary depending upon
a variety of factors including the activity of the specific
oligonucleotide employed; the metabolic stability and length of
action of the oligonucleotide; the age, body weight, general
health, sex, skin type and diet of the subject; the mode and time
of administration; the rate of excretion; the drug combination; the
severity of the particular inflammatory condition for which
treatment is desired; and the subject undergoing therapy. In
certain embodiments, an effective or therapeutically effective
daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e.,
0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a
therapeutically effective dose is (for a 70 kg mammal) from about
0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more
preferably a therapeutically effective dose is (for a 70 kg mammal)
from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).
In certain embodiments, treatment is characterized in that the
antisense oligomer or conjugate thereof represent(s) 0.0001% to
10%, preferably 0.003% to 3% of the total weight of the topical
pharmaceutical composition.
[0264] The ranges of effective doses provided herein are not
intended to be limiting and represent preferred dose ranges.
However, the most preferred dosage will be tailored to the
individual subject, as is understood and determinable by one
skilled in the relevant arts. (see, e.g., Berkow et al., eds., The
Merck Manual, 16.sup.th edition, Merck and Co., Rahway, N.J., 1992;
Goodman et al., eds., Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 10.sup.th edition, Pergamon Press, Inc.,
Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3rd edition,
ADIS Press, Ltd., Williams and Wilkins, Baltimore, Md. (1987);
Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci
al., eds., Remington's Pharmaceutical Sciences, 18.sup.th edition,
Mack Publishing Co., Easton, Pa. (1990); Katzung, Basic and
Clinical Pharmacology, Appleton and Lange, Norwalk, Conn.
(1992)).
[0265] The total dose required for each treatment can be
administered by multiple doses or in a single dose over the course
of the day, if desired. Certain preferred embodiments contemplate a
single administration of the formulation per day. Generally, and in
distinct embodiments, treatment may be initiated with smaller
dosages, which are less than the optimum dose of the
oligonucleotide. Thereafter, the dosage may be increased by small
increments until the optimum effect under the circumstances is
reached.
[0266] The compositions described herein can be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art. Controlled release drug delivery
systems include osmotic pump systems and dissolutional systems
containing polymer-coated reservoirs or drug-polymer matrix
formulations. Examples of controlled release systems are given in
U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al,
Regional Anesthesia 22 (6): 543-551 (1997), all of which are
incorporated herein by reference.
[0267] The most suitable route will depend on the nature and
severity of the condition being treated. Those skilled in the art
are also familiar with determining topical administration methods
(sprays, creams, open application, occlusive dressing, soaks,
washes, etc.), dosage forms, suitable pharmaceutical excipients and
other matters relevant to the delivery of the oligonucleotides to a
subject in need thereof.
[0268] All of the U.S. patents, U.S. patent applications, foreign
patents, foreign patent applications, and non-patent applications
referred to in this specification and/or listed in the Application
Data Sheet are incorporated herein by reference in their
entirety.
[0269] The following Examples are presented by way of illustration
and not limitation.
EXAMPLES
[0270] The following examples illustrate the method of the
invention in reducing skin inflammation when topically applied in a
contact hypersensitivity model.
[0271] They are presented to further illustrate and explain the
present invention and should not be taken as limiting in any
regard.
Materials and Methods
[0272] Peptide-Conjugated PMO (PPMO) Synthesis.
[0273] A PMO targeting the AUG translation inititation sequence of
murine CFLAR (SEQ ID NO:28) and a scrambled control sequence (SEQ
ID NO:35) were synthesized at AVI BioPharma (Corvallis, Oreg.).
Purity of full length oligomers was >95% as determined by
reverse-phase high-pressure liquid chromatography (HPLC) and MALDI
TOF mass spectroscopy. Peptide-conjugated PMO (PPMO) were produced
by attaching the carboxy terminal cysteine of the peptide R9F2 (SEQ
ID NO:2) to the 5' end of the CFLAR and Scrambled control PMOs
through a cross-linker N-[.alpha.-maleimidobutyryloxy] succinimide
ester (BGBS). The lyophilized PPMOs (SEQ ID NOS:28 and 35) were
dissolved in sterile H.sub.2O prior to use in cell cultures,
dissolved in PBS prior to intraperitoneal (i.p.) injection in mice,
or dissolved in sterile H.sub.2O or 95% propylene glycol/5%
linoleic acid prior to topical administration on mice ear skin.
[0274] Mice.
[0275] BALB/c and D0.11 mice 6-12 weeks of age were obtained from
Simonsen or Charles River laboratories and housed in micro-isolator
cages 3-5 per cage during acclimatization and treatment periods.
Animals were exposed to a 12-hr light/dark cycle in a temperature-
and humidity-controlled environment and allowed access to
commercially available pre-autoclaved sterilized rodent diet and
autoclaved sterilized water. Temperature controls were set to
maintain temperatures at 18.degree. to 26.degree.C. Autoclaved
caging, water bottles and storage containers were used to avoid
exposure of animals to any contaminated materials.
[0276] Contact Hypersensitivity Model.
[0277] On day 0 the bellies of BALB/c mice were shaved using a
small animal clipper (Oster, 40) and mice were sensitized by
pipeting either 20 uL of 0.5% FITC diluted 4:1 acetone/dibutyl
phthalate or 2% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5)
diluted in 4:1 acetone/olive oil onto the shaved region. On day 5
animals were pre-treated with 20 uL of cFLAR PPMO (SEQ ID NO: 28)
or scrambled sequence PPMO (SEQ ID NO: 35) dissolved in 95%
propylene glycol and 5% linoleic acid, and applied topically to an
area of the animal to be tested, in the present method, to each
side of one ear. On day 6 mice were similarly pretreated by topical
administration of PMO. Approximately 15 minutes later, an eliciting
dosage of either 10 uL of 0.2% FITC diluted 4:1 acetone/dibutyl
phthalate or 1% oxazolone in 4:1 acetone/olive oil was
epicutaneously applied to each side of the treated ear. Twenty four
hours later ear thickness measurements were taken on both ears
using a Starrett Caliper (#1015 MH, Athol, Mass.). Each reading was
performed three times and the median value was used for all
analysis.
[0278] In a Second Study, the Memory Response was Tested by Giving
a Second Eliciting Dose.
[0279] On day 21, 15 days after the initial elicitation, animals
were treated with either 10 uL of 0.2% FITC in 4:1 acetone/dibutyl
phthalate or 1% oxazolone in 4:1 acetone/olive, epicutaneously
applied to each side of the opposite ear. On day 26, five days
after second eliciting dose, the thickness of each ear was measured
using caliper. Right and left ears were both measured as described
earlier. Animals were euthanized, ears were removed and placed into
10% buffered formalin solution for 24 hours for paraffin embedding,
sectioning, H&E staining and immunohistochemistry. The
differences between thickness of treated and untreated ears for
each mouse were determined. The mean and the standard error were
then calculated and Analysis of Variance followed by a Newman-Keuls
Post test was performed with significance set at p<0.05 using
GraphPad Prism (GraphPad, San Diego, Calif.).
Example 1
Inhibition of CFLAR Long and Short Isomer Protein Expression in
Activated T Cells with CFLAR PPMO
[0280] The effect of an AUG targeted CFLAR PPMO on protein
expression of CFLAR long (CFLAR.sub.L) and short (CFLAR.sub.S)
isoforms in activated T cells was determined in vitro. The
CFLAR.sub.L and CFLAR.sub.S proteins are isoforms of CFLAR which
share the same translation initiation sequence within the mature
mRNA and are therefore both complementary with the AUG targeted
CFLAR PPMO. Purified BALB/c splenic T cells were cultured with
plate bound anti-CD3 [5 .mu.g/ml] and treated with 5 .mu.M CFLAR
PPMO (SEQ ID NO: 28), scrambled control sequence PPMO (SEQ ID NO:
35), or no treatment and analyzed for CFLAR long (cFLAR.sub.L) and
short (CFLAR.sub.S) isomer protein expression after 24 hours.
[0281] Immunoblot analysis revealed diminished levels of both
CFLAR.sub.L and CFLAR.sub.S protein in cells treated with CFLAR
PPMO while scrambled control PPMO treatment and untreated cells
showed no effect (FIG. 3). In addition, cells treated with CFLAR
PPMO displayed a higher molecular weight band in the GAPDH
immunoblot not present in scrambled PPMO treated or untreated
cells, which may be indicative of stress-induced insoluble
aggregation of GAPDH in cells undergoing activation-induced cell
death due to CFLAR antisense blockade (FIG. 3). Although such
results have not previously been described for cultured T cells, it
has been shown in cultured nerve cells that formation of an
oxidative stress-induced insoluble aggregate of GAPDH due to
intermolecular disulfide bonding is visible as a high molecular
weight GAPDH band (Nakajima et al., 2007, J. Biol. Chem.
282:26562-74).
Example 2
Antigen-Specific Induction of Apoptosis in T Cells with CFLAR
PPMO
[0282] Evidence of antigen-specific activation induced cell death
(AICD) in ovalbumin-specific CD4+ T cells treated with cFLAR PPMO
was examined in vitro. Freshly isolated splenocytes from
ovalbumin-specific T cell receptor (TCR) transgenic (DO.11) mice
were treated with 2.5 .mu.M CFLAR PPMO (SEQ ID NO: 28), scrambled
control PPMO (SEQ ID NO: 35) or media control overnight then
co-cultured with BALB/c bone marrow derived lipopolysaccharide
(Ips) matured ovalbumin pulsed DCs or control Ips stimulated DCs
for 24 hours. Apoptotic indicators were then examined by flow
cytometry using propidium iodide with anti-TCR KJ26+ staining to
examine loss of cell membrane integrity in KJ26+ cells and using a
caspase-3 fluorescing substrate to examine caspase activation.
[0283] Antigen-specific activated T cells treated with CFLAR PPMO
displayed a marked affect on apoptotic indicators as evidenced by
decreased membrane integrity and caspase activation versus
activated T cells treated with scrambled control PPMO or
non-activated T cells treated with CFLAR PPMO (data not shown).
Example 3
Inhibition of Fitc-Induced Dermatitis with Topical Application of
CFLAR PPMO
[0284] Contact hypersensitivity responses induced in mice by
epicutaneous application of fluorescein isothiocyanate (FITC) were
examined to determine if topical application of CFLAR PPMO could
reduce skin inflammation. The delayed-type hypersensitivity (DTH)
response was first tested in FITC pre-sensitized mice followed by
topical administration of 3, 30, 300 and 3000 .mu.g/ear of CFLAR
PPMO (SEQ ID NO: 28), 300 .mu.g/ear scrambled control PPMO (SEQ ID
NO: 35), or 95% propylene glycol/5% linoleic acid vehicle alone to
both ears for two days followed immediately by an eliciting dosage
of FITC applied to each side of one ear (N=6-9 per treatment
group). Both ears were measured 24 hours later and the difference
between thickness of FITC treated and untreated ears for each mouse
was determined. The memory response was then tested 15 days later
with a second eliciting dose of FITC applied to each side of the
opposite ear followed by right and left ear thickness measurements
five days later. Treatment effectiveness was compared to ears from
FITC sensitized mice not receiving topical PPMO or vehicle alone.
Mice were euthanized for removal of ears for examination of
leukocyte infiltration and CFLAR protein via H&E staining and
immunohistochemistry, respectively.
[0285] Ear thickness measurements demonstrated that topical CFLAR
PPMO reduced FITC induced DTH in a dose dependent manner, with
topical 3, 30, 300 and 3000 .mu.g/ear dosages causing a 21%, 57%,
70% and 96% decrease in ear thickening, respectively (p<0.001
for 30, 300 and 3000 .mu.g/ear treatment groups), versus FITC
sensitization alone. Topical scrambled control PPMO and vehicle
alone treatments caused a non-significant 12% and 17% reduction,
respectively (FIG. 4A).
[0286] Memory response after the second FITC challenge given 15
days later showed less of a CFLAR PPMO dose response effect versus
the effect on the first FITC challenge, although the second FITC
challenge generated a smaller change in ear thickness versus the
first challenge (0.043 versus 0.08 mm) in controls. Mice receiving
the 3, 30, 300, and 3000 .mu.g/ear CFLAR PPMO treatments 15 days
earlier displayed a 23%, 47% (p<0.05), 35% (p>0.05), and 54%
(p<0.01) decrease in ear thickening, respectively, versus FITC
sensitization alone (FIG. 4B).
[0287] Examination of leukocyte infiltration revealed that FITC
sensitization alone increased the number of leukocytes in the skin
by 37% (p<0.05 versus nonsensitized control ears), while topical
application of CFLAR PPMO prior to FITC sensitization reduced these
leukocyte numbers to just 5% above nonsensitized controls
(p<0.05 versus FITC sensitization alone) (FIG. 5A). The results
were generated from histological examination of skin under the
various treatment conditions, as shown in FIGS. 5B-5E. CFLAR
positive cell distribution within the skin for each treatment group
revealed that infiltration of CFLAR positive cells correlated with
ear swelling and topical CFLAR PPMO decreased CFLAR positive cells
in FITC sensitized ears (FIGS. 6A-6D).
Example 4
Inhibition of Oxazolone-Induced Dermatitis with Topical Application
of CFLAR PPMO
[0288] Contact hypersensitivity responses induced in mice by
epicutaneous application of oxazolone were examined to ensure that
topical application of CFLAR PPMO could reduce skin inflammation
after exposure to antigens other than FITC (see Example 3 above).
The delayed-type hypersensitivity (DTH) response was first tested
in oxazolone pre-sensitized mice followed by topical administration
of 300 .mu.g/ear of CFLAR PPMO (SEQ ID NO: 28), 300 .mu.g/ear
scrambled control PPMO (SEQ ID NO: 35), or 95% propylene glycol/5%
linoleic acid vehicle (PG/LA) alone to both ears for two days
followed immediately by an eliciting dosage of oxazolone applied to
each side of one ear (N=6-9 per treatment group). Both ears were
measured 24 hours later and the difference between thickness of
oxazolone treated and untreated ears for each mouse was determined.
The memory response was then tested 15 days later with a second
eliciting dose of oxazolone applied to each side of the opposite
ear followed by right and left ear thickness measurements five days
later. Treatment effectiveness was compared to ears from oxazolone
sensitized mice receiving vehicle alone.
[0289] Ear thickness measurements demonstrated that topical CFLAR
PPMO reduced oxazolone-induced DTH, with the topical 300 .mu.g/ear
dosage causing a 70% decrease in ear thickening (**p<0.001 vs.
oxazolone sensitized ears receiving vehicle alone). Topical
scrambled control PPMO was no different from vehicle alone (FIG.
7A).
[0290] Memory response after the second oxazolone challenge showed
that ear thickness was reduced 49% in mice that received topical
CFLAR PPMO 15 days earlier (**p<0.001 vs. oxazolone sensitized
ears receiving vehicle alone). Topical scrambled control PPMO was
again no different from vehicle alone (FIG. 7B).
[0291] In summary, the combined results presented in Examples 3 and
4 provide evidence that treatment of skin with topical CFLAR PPMO
produced a significant reduction in dermatitis and localized
infiltration of lymphocytes that was dose-dependent, target- and
antigen-specific, and capable of inducing long-lived tolerance.
TABLE-US-00004 SEQUENCE ID LISTING SEQ Name Sequences ID NO:
Peptide Transporters (NH.sub.2 to COOH) rTAT RRRQRRKKRC 1
R.sub.9F.sub.2 RRRRRRRRRFFC 2 (RRAhx).sub.4B RRAhxRRAhxRRAhxRRAhxB
3 P007 RAhxRRAhxRRAhxRRAhxRAhxB 4 (AhxRR).sub.4AhxB
AhxRRAhxRRAhxRRAhxRRAhxB 5 (RAhx).sub.6B RAhxRAhxRAhxRAhxRAhxRAhxB
6 (RAhx).sub.8B RAhxRAhxRAhxRAhxRAhxRAhxRAhxB 7 (RAhxR).sub.3AhxB
RAhxRRAhxRRAhxRAhxB 8 CPO6062 RAhxRRBRRAhxRRBRAhxB 9 (RAhxR)5AhxB
RAhxRRAhxRRAhxRRAhxRRAhxRAhxB 10 (CP04057) Target Sequences (5' to
3') Hu-AUG (.+-.30) CCTTGTGAGCTTCCCTAGTCTAAGAGTAGG 11
ATGTCTGCTGAAGTCATCCATCAGGTTGAA HU-AUG (.+-.12)
TCTAAGAGTAGGATGTCTGCTGAAG 12 Hu-Ex2SA
CAGAAAAATTCCCTTTTAACCACAG/AACT 13 CCCCCACTGGAAAGGATTCTG Hu-Ex3SA
CTAAATGAACTTGTCTGGTTTGCAG/ 14 AGTGCTGATGGCAGAGATTGGTGAG Hu-Ex4SA
TGTTTTTTGTTGGTGGTTCTCTTAG/AGTTTCTT 15 GGACCTTGTGGTTGAGT Hu-Ex2SD
ACCCTCACCTTGTTTCGGACTATAG/GTAATTC 16 ATCAACTCTTCCTGAGGC Hu-Ex3SD
CCGAGGCAAGATAAGCAAGGAGAAG/GTGAGT 17 TTTCTTCTTTTGGTTCATG Mu-Ex2SA
ATAAGAGGATTCTCTTTCACCACAG/AGTGTC 18 TCTATTGCAAGAACTCTGA Mu-Ex2SD
ACCCTCACCTGGTTTCTGATTATAG/GTAAGT 19 CATCCCCTGGGGGAGGGGA Mu-Ex3SA
CTGAAGACACTTTTATGGTTTACAG/GGTCCT 20 GCTGATGGAGATTGGTGAG Mu-Ex3SD
CAGAGGCAAGATAGCCAAGGACAAG/GTGA 21 GTTGTCTTTGCTCGGTGCCTG Mu-Ex4SA
CATTTCTTGTTCATGGCTTTCTTAG/AGTTTC 22 TTGGATCTGGTGATTGAAT Oligomer
Targeting and Control PMO and PPMO Sequences (5' to 3')
CFLAR-huAUG1 CTTCAGCAGACATCCTACTC 23 CFLAR-huAUG2
GACTAGGGAAGCTCACAAGG 24 CFLAR-huAUG3 TCAACCTGATGGATGACTTC 25
CFLAR-huAUG(-5) GATGACTTCAGCAGACATCCTAC 26 CFLAR-huAUG(-11)
CTTCAGCAGACATCCTACTCTTAG 27 CFLAR-muAUG CTGGGCCATGTTCAGAACC 28
CFLAR-huSA2 GGAGTTCTGTGGTTAAAAGG 29 CFLAR-huSD2
CTATAGTCCGAAACAAGGTGAGG 30 CFLAR-huSA3 CACCAATCTCTGCCATCAGCACT 31
CFLAR-huSA4 TCAACCACAAGGTCCAAGAAACT 32 CFLAR-huSD3
CTTCTCCTTGCTTATCTTGCCCT 33 R.sub.9F.sub.2-CFLARmuAUG;
RRRRRRRRRFFC-CTGGGCCATGTTCAGAACC 34 CFLAR PPMO Scrambled Control
TGCGCGTCATGTACGCCAA 35 R.sub.9F.sub.2-Scr.Control;
RRRRRRRRRFFC-TGCGCGTCATGTACGCCAA 36 Scrambled Control PPMO
Sequence CWU 1
1
39110PRTArtificial SequenceTranport peptide for cellular uptake
enhancement 1Arg Arg Arg Gln Arg Arg Lys Lys Arg Cys1 5 10
212PRTArtificial SequenceTranport peptide for cellular uptake
enhancement 2Arg Arg Arg Arg Arg Arg Arg Arg Arg Phe Phe Cys1 5 10
313PRTArtificial SequenceTranport peptide for cellular uptake
enhancement 3Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5
10 414PRTArtificial SequenceTranport peptide for cellular uptake
enhancement 4Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa
Xaa1 5 10 514PRTArtificial SequenceTranport peptide for cellular
uptake enhancement 5Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg
Xaa Xaa1 5 10 613PRTArtificial SequenceTranport peptide for
cellular uptake enhancement 6Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Xaa1 5 10 717PRTArtificial SequenceTranport peptide for
cellular uptake enhancement 7Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg Xaa Arg Xaa1 5 10 15 Xaa811PRTArtificial
SequenceTranport peptide for cellular uptake enhancement 8Arg Xaa
Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5 10 914PRTArtificial
SequenceTranport peptide for cellular uptake enhancement 9Arg Xaa
Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5 10
1017PRTArtificial SequenceTranport peptide for cellular uptake
enhancement 10Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg
Xaa Arg Xaa1 5 10 15 Xaa1160DNAHomo sapiens 11ccttgtgagc ttccctagtc
taagagtagg atgtctgctg aagtcatcca tcaggttgaa 601225DNAHomo sapiens
12tctaagagta ggatgtctgc tgaag 251350DNAHomo sapiens 13cagaaaaatt
cccttttaac cacagaactc ccccactgga aaggattctg 501450DNAHomo sapiens
14ctaaatgaac ttgtctggtt tgcagagtgc tgatggcaga gattggtgag
501550DNAHomo sapiens 15tgttttttgt tggtggttct cttagagttt cttggacctt
gtggttgagt 501650DNAHomo sapiens 16accctcacct tgtttcggac tataggtaat
tcatcaactc ttcctgaggc 501750DNAHomo sapiens 17ccgaggcaag ataagcaagg
agaaggtgag ttttcttctt ttggttcatg 501850DNAMus musculus 18ataagaggat
tctctttcac cacagagtgt ctctattgca agaactctga 501950DNAMus musculus
19accctcacct ggtttctgat tataggtaag tcatcccctg ggggagggga
502050DNAMus musculus 20ctgaagacac ttttatggtt tacagggtcc tgctgatgga
gattggtgag 502150DNAMus musculus 21cagaggcaag atagccaagg acaaggtgag
ttgtctttgc tcggtgcctg 502250DNAMus musculus 22catttcttgt tcatggcttt
cttagagttt cttggatctg gtgattgaat 502320DNAArtificial SequenceHuman
CFLAR antisense targeting sequence 23cttcagcaga catcctactc
202420DNAArtificial SequenceHuman CFLAR antisense targeting
sequence 24gactagggaa gctcacaagg 202520DNAArtificial SequenceHuman
CFLAR antisense targeting sequence 25tcaacctgat ggatgacttc
202623DNAArtificial SequenceHuman CFLAR antisense targeting
sequence 26gatgacttca gcagacatcc tac 232724DNAArtificial
SequenceHuman CFLAR antisense targeting sequence 27cttcagcaga
catcctactc ttag 242819DNAArtificial SequenceMurine CFLAR antisense
targeting sequence 28ctgggccatg ttcagaacc 192920DNAArtificial
SequenceHuman CFLAR antisense targeting sequence 29ggagttctgt
ggttaaaagg 203023DNAArtificial SequenceHuman CFLAR antisense
targeting sequence 30ctatagtccg aaacaaggtg agg 233123DNAArtificial
SequenceHuman CFLAR antisense targeting sequence 31caccaatctc
tgccatcagc act 233223DNAArtificial SequenceHuman CFLAR antisense
targeting sequence 32tcaaccacaa ggtccaagaa act 233322DNAArtificial
SequenceHuman CFLAR antisense targeting sequence 33cttctccttg
cttatcttgc ct 223419DNAArtificial SequenceCFLARmuAUG PPMO oligomer
34ctgggccatg ttcagaacc 193519DNAArtificial SequenceControl CFLAR
antisense targeting sequence 35tgcgcgtcat gtacgccaa
193619DNAArtificial SequenceScrambled control PPMO oligomer
36tgcgcgtcat gtacgccaa 193712PRTArtificial SequenceTranport peptide
for cellular uptake enhancement 37Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 10 3812PRTArtificial SequenceTranport peptide
for cellular uptake enhancement 38Arg Xaa Arg Arg Arg Xaa Arg Xaa
Arg Arg Arg Xaa1 5 10 399PRTArtificial SequenceTranport peptide for
cellular uptake enhancement 39Arg Arg Xaa Arg Xaa Arg Arg Arg Xaa1
5
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