U.S. patent application number 14/139584 was filed with the patent office on 2014-11-27 for rtp801l sirna compounds and methods of use thereof.
This patent application is currently assigned to QUARK PHARMACEUTICALS, INC.. The applicant listed for this patent is Elena Feinstein, Hagar Kalinski, Igor Mett. Invention is credited to Elena Feinstein, Hagar Kalinski, Igor Mett.
Application Number | 20140350068 14/139584 |
Document ID | / |
Family ID | 48427524 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140350068 |
Kind Code |
A1 |
Feinstein; Elena ; et
al. |
November 27, 2014 |
RTP801L SIRNA COMPOUNDS AND METHODS OF USE THEREOF
Abstract
The invention provides chemically modified siRNA
oligonucleotides that target RTP801L, compositions comprising same
and to the use of such molecules to treat, inter alia, respiratory
diseases including acute and chronic pulmonary disorders, eye
diseases including glaucoma and ION, microvascular disorders,
angiogenesis- and apoptosis-related conditions, and hearing
impairments.
Inventors: |
Feinstein; Elena; (Rehovot,
IL) ; Mett; Igor; (Rehovot, IL) ; Kalinski;
Hagar; (Rishon-le-Zion, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Feinstein; Elena
Mett; Igor
Kalinski; Hagar |
Rehovot
Rehovot
Rishon-le-Zion |
|
IL
IL
IL |
|
|
Assignee: |
QUARK PHARMACEUTICALS, INC.
Fremont
CA
|
Family ID: |
48427524 |
Appl. No.: |
14/139584 |
Filed: |
December 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13651123 |
Oct 12, 2012 |
8614311 |
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14139584 |
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12735061 |
Jan 13, 2011 |
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PCT/IL2008/001606 |
Dec 11, 2008 |
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13651123 |
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61007480 |
Dec 12, 2007 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
C12N 2310/14 20130101;
C12N 2310/319 20130101; C12N 15/113 20130101; C07H 21/02 20130101;
C12N 2310/343 20130101; C12N 2310/321 20130101; C12N 2310/3521
20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1-28. (canceled)
29. A method for providing neuroprotection in a patient in need
thereof, comprising administering to the patient a therapeutically
effective dose of a RTP801L inhibitor, thereby providing
neuroprotection in the patient.
30. A method for treating a neurodegenerative disease in a patient
in need thereof, comprising administering to the patient a
therapeutically effective dose of a RTP801L inhibitor, thereby
treating the neurodegenerative disease in the patient.
31. The method of claim 29, wherein the neuroprotection comprises
neuroprotection of the retina.
32. The method of claim 30, wherein the neurodegenerative disease
comprises neurodegeneration of the retina and is associated with
neuroretinal apoptosis.
33. The method of claim 32, wherein the neurodegeneration is
associated with diseases of the optic nerve.
34. The method of claim 31, wherein the patient is suffering from
glaucoma.
35. The method of claim 31, wherein the patient is suffering from
optic neuritis.
36. The method of claim 31, wherein the patient is suffering from
ischemic optic neuropathy (ION).
37. The method of claim 36, wherein the ION is anterior ischemic
optic neuropathy (AION).
38. The method of claim 30, wherein the patient is suffering from
retinopathy.
39. The method of claim 38, wherein the retinopathy comprises
retinal vascular damage or occlusion, retinopathy associated with
systemic diseases, retinopathy associated with neurodegenerative
diseases in the CNS, hypertensive retinopathy, radiation
retinopathy, idiopathic retinal vasculitis, aneurysms,
neuroretinitis, Birdshot retinochoroidopathy, long-standing retinal
detachment acute glaucoma, wet age-related macular degeneration,
dry age-related macular degeneration, geographic atrophy or
retinitis pigmentosa.
40. The method of claim 39, wherein the retinal vascular damage or
occlusion comprises retinal artery occlusion, retinal vein
occlusion, branch retinal artery or vein occlusion, retinopathy of
prematurity, retinal embolization or Purtscher retinopathy.
41. The method of claim 38, wherein the retinopathy is associated
with a systemic disease selected from HIV retinopathy, retinopathy
associated with systemic vasculitis, retinopathy associated with
autoimmune diseases, or retinopathy associated with systemic
neurodegenerative diseases.
42. The method of claim 29, wherein the neuroprotection comprises
neuroprotection of the inner ear.
43. The method of claim 29, wherein the neuroprotection comprises
neuroprotection of the CNS.
44. A method for treating or preventing a disease associated with
ischemia or ischemia-reperfusion injury to a tissue in a patient in
need thereof, comprising administering to the patient a
therapeutically effective dose of a RTP801L inhibitor, thereby
treating or preventing the disease in the patient.
45. The method of claim 44, wherein the tissue comprises skin,
lung, heart or kidney.
46. The method of claim 29, wherein the RTP801L inhibitor comprises
an oligonucleotide.
47. The method of claim 46, wherein the oligonucleotide is selected
from an antisense oligonucleotide, a siRNA, an aptamer, or a
catalytic RNA.
48. A method for treating a disease associated with cell loss in a
patient in need thereof, comprising administering to the patient a
therapeutically effective dose of a RTP801L inhibitor, thereby
treating the disease in the patient.
Description
[0001] This application is a divisional of U.S. Ser. No.
13/651,123, filed Oct. 12, 2012, which is a continuation-in-part of
U.S. Ser. No. 12/735,061, filed Jan. 13, 2011, which is a .sctn.371
national stage of PCT International Application No.
PCT/IL2008/001606, filed Dec. 11, 2008, claiming the benefit of
U.S. Provisional Application No. 61/007,480, filed Dec. 12, 2007,
the content of each of which is hereby incorporated by reference
into the subject application.
[0002] This application incorporates-by-reference nucleotide and/or
amino acid sequences which are present in the file named
"131223.sub.--2094.sub.--76494-BZ-PCT-US_Sequence_Listing_LC.txt,"
which is 1.27 megabytes in size, was created on Dec. 19, 2013, in
the IBM-PCT machine format, having an operating system
compatibility with MS-Windows and is contained in the text file
submitted Oct. 12, 2012 as part of the above-identified
application.
FIELD OF THE INVENTION
[0003] The present invention relates to double stranded
oligonucleotide inhibitors of RTP801L (RTP801-like; REDD2,
DNA-damage-inducible transcript 4-like, DDIT4_L), pharmaceutical
compositions comprising same and methods of use thereof. The
compounds and compositions are thus useful in the treatment of
subjects suffering from diseases or conditions and or symptoms
associated with such diseases or conditions in which RTP801L
expression has adverse consequences. In particular embodiments, the
invention provides chemically modified siRNA oligonucleotides,
compositions comprising same and to the use of such molecules to
treat, inter alia, respiratory disorders of all types (including
acute and chronic pulmonary disorders), eye diseases including
glaucoma and ION, microvascular disorders, angiogenesis- and
apoptosis-related conditions, and hearing impairments.
BACKGROUND OF THE INVENTION
siRNAs and RNA Interference
[0004] RNA interference (RNAi) is a phenomenon involving
double-stranded (ds) RNA-dependent gene specific
posttranscriptional silencing. Originally, attempts to study this
phenomenon and to manipulate mammalian cells experimentally were
frustrated by an active, non-specific antiviral defense mechanism
which was activated in response to long dsRNA molecules (see Gil et
al. 2000, Apoptosis, 5:107-114). Later it was discovered that
synthetic duplexes of 21 nucleotide RNAs could mediate gene
specific RNAi in mammalian cells, without the stimulation of the
generic antiviral defense mechanisms (see Elbashir et al., 2001.
Nature, 411:494-498 and Caplen et al., 2001. PNAS, 98:9742-9747).
As a result, small interfering RNAs (siRNAs), which are short
double-stranded RNAs, have become powerful tools in attempting to
understand gene function. Thus RNA interference (RNAi) refers to
the process of sequence-specific post-transcriptional gene
silencing in mammals mediated by small interfering RNAs (siRNAs)
(Fire et al, 1998, Nature 391:806) or microRNAs (miRNAs) (Ambros,
2004. Nature 431:7006,350-355; and Bartel, 2004. Cell,
116(2):281-97). The corresponding process in plants is commonly
referred to as specific post transcriptional gene silencing or RNA
silencing and is also referred to as quelling in fungi.
[0005] An siRNA is a double-stranded RNA molecule which
down-regulates or silences (prevents) the expression of a gene/mRNA
of its endogenous or cellular counterpart. The mechanism of RNA
interference is detailed infra.
[0006] siRNA has been successfully used for inhibition in primates;
(for further details see Tolentino et al., 2004. Retina
24(1):132-138). Several studies have revealed that siRNA
therapeutic agents are effective in vivo in both mammals and in
humans. Bitko et al., have shown that specific siRNA molecules
directed against the respiratory syncytial virus (RSV) nucleocapsid
N gene are effective in treating mice when administered
intranasally (Bitko et al., 2005. Nat. Med. 11(1):50-55). Reviews
of the use of siRNA as a therapeutic agent recently published (see
for example Batik 2005. J. Mol. Med 83:764-773 and Dykxhoom et al.,
2006. Gene Therapy 13:541-552). In addition, clinical studies with
short siRNAs that target the VEGFR1 receptor for the treatment of
Age-Related Macular Degeneration (AMD) have been conducted in human
patients. (Kaiser, 2006. Am. J Ophthalmol. 142(4):660-8).
Chemically Modified siRNA
[0007] The selection and synthesis of siRNA corresponding to known
genes has been widely reported; (see for example Ui-Tei et al.,
2006. J Biomed Biotechnol.; 2006:65052; Chalk et al., 2004. BBRC.
319(1): 264-74; Sioud & Leirdal, 2004. Met. Mol Biol.;
252:457-69; Levenkova et al., 2004. Bioinform. 20(3):430-2; Ui-Tei
et al., 2004. NAR 32(3):936-48).
[0008] For examples of the use of, and production of, modified
siRNA see for example Braasch et al., 2003. Biochem.,
42(26):7967-75; Chiu et al., 2003, RNA, 9(9):1034-48; PCT
publications WO 2004/015107 (atugen AG) and WO 02/44321 (Tuschl et
al). U.S. Pat. Nos. 5,898,031 and 6,107,094 teach chemically
modified oligomers. US patent publication 2005/0080246 relates to
oligomeric compounds having an alternating motif. US patent
publication 2005/0042647 describes dsRNA compounds having
chemically modified internucleoside linkages.
[0009] The inclusion of a 5'-phosphate moiety was shown to enhance
activity of siRNAs in Drosophila embryos (Boutla, et al., 2001.
Curr. Biol. 11:1776-1780) and is required for siRNA function in
human HeLa cells (Schwarz et al., 2002, Mol. Cell, 10:537-548).
[0010] Amarzguoui et al., (2003, NAR, 31(2):589-595) showed that
siRNA activity depended on the positioning of the 2-O-methyl
modifications. Holen et al (2003, NAR, 31(9):2401-2407) report that
an siRNA having small numbers of 2'-O-methyl modified nucleosides
gave good activity compared to wild type but that the activity
decreased as the numbers of 2'-O-methyl modified nucleosides was
increased. Chiu and Rana (2003, RNA, 9:1034-1048) teach that
incorporation of 2'-O-methyl modified nucleosides in the sense or
antisense strand (fully modified strands) severely reduced siRNA
activity relative to unmodified siRNA. The placement of a
2'-O-methyl group at the 5'-terminus on the antisense strand was
reported to severely limit activity whereas placement at the
3'-terminus of the antisense and at both termini of the sense
strand was tolerated (Czauderna et al., 2003, NAR, 31(11),
2705-2716).
[0011] PCT Patent Publication Nos. PCT/IL2008/000248 and
PCT/IL2008/001197 assigned to the assignee of the present invention
disclose motifs useful in the preparation of chemically modified
siRNA compounds.
RTP801L
[0012] Gene RTP801 (REDD1, DDIT4), was first reported by the
assignee of the instant application. U.S. Pat. Nos. 6,455,674,
6,555,667, and 6,740,738, all assigned to the assignee of the
instant application, disclose and claim per se the RTP801
polynucleotide and polypeptide, and antibodies directed toward the
polypeptide. RTP801 represents a unique gene target for
hypoxia-inducible factor-1 (HIF-1) that may regulate
hypoxia-induced pathogenesis independent of growth factors such as
VEGF. Further discoveries relating to gene RTP801, as discovered by
the assignee of the instant application, were reported in Shoshani,
et al. 2002. Mol. Cell Biol., 22(7):2283-2293; this paper,
co-authored by the inventor of the present invention, details the
discovery of the RTP801 gene. Gene RTP801L, so named because of its
resemblance to RTP801, was also first reported by the assignee of
the instant application, and given Pubmed accession No.
NM.sub.--145244 subsequent to said report.
[0013] While RTP801 and RTP801L share sequence homology of about
65% at the amino acid level, indicating a possible similarity of
function, and while the assignee of the present invention has found
that both RTP801 and RTP801L interact with TSC2 and affect the mTOR
pathway, the inventors of the present invention have found that the
embryological expression pattern of the two polypeptides differs,
and that, contrary to RTP801, RTP801L is not induced by hypoxia in
all conditions which induce RTP801 expression; it is, however,
induced in MEFs as a result of H.sub.2O.sub.2 treatment (hypoxia
treatment), and the induction follows kinetics similar to those of
RTP801 expression induction under the same conditions.
Additionally, the inventors of the present invention have found
that RTP801 polypeptide is more abundantly expressed than RTP801L.
Thus, RTP801L may serve as a target in the treatment of conditions
for which RTP801 is a target, and may have the added benefit of a
similar, yet different, target.
[0014] The following patent applications and publications give
aspects of background information relating to RTP801L: Patent
application/publication Nos. EP1580263, WO2003029271, WO2001096391,
WO2003087768, WO2004048938, WO2005044981, WO2003025138,
WO2002068579, EP1104808 and CA2343602 all disclose a nucleic acid
or polypeptide which is homologous to RTP801L. Various groups have
studied the mechanism of action of RTP801L (Corradetti et al.,
2005. J Biol Chem. 280(11):9769-72; Pisani et al., 2005. BBRC
326(4):788-93; Cuaz-perolin et al., 2004 Arterioscler Thromb Vasc
Biol. 24(10):1830-5; Sofer et al., 2005 Mol Cell Biol.
25(14):5834-45).
[0015] Inhibitors of RTP801L are disclosed in PCT patent
publication WO 2007/141796, assigned to the assignee of the present
invention and incorporated herein by reference in its entirety. Use
of those inhibitors in treating numerous indications is disclosed
therein.
SUMMARY OF THE INVENTION
[0016] The present invention relates in part to chemically modified
RTP801L siRNA, and in particular to chemically modified RTP801L
siRNA oligonucleotides having sense and antisense sequences set
forth in Tables A-F. The chemically modified siRNA compounds
disclosed herein are useful in down regulating RTP801L expression.
The compounds according to the present invention exhibit properties
that render them useful as therapeutic agents for treatment of a
subject suffering from a disease or disorder associated with
RTP801L expression. Specifically the compounds exhibit high
activity, and/or serum stability and/or reduced off-target effects
and/or reduced adverse immune response as compared to an unmodified
siRNA compound. The present invention additionally provides novel
RTP801L siRNA oligonucleotide pairs shown in Tables B-F and set
forth in SEQ ID NOS:1852-6927. PCT patent publication WO
2007/141796, incorporated by reference herein, discloses the
oligonucleotide pairs shown in Table A set forth in SEQ ID NO:
2-1851.
[0017] The present invention provides pharmaceutical compositions
comprising one or more such oligonucleotides. The present invention
further relates to methods for treating or preventing the incidence
or severity of various diseases or conditions in a subject in need
thereof wherein the disease or condition and/or symptoms associated
therewith is selected from the group consisting of an ophthalmic
disease or condition, a respiratory disease, an ischemic disease, a
microvascular disease, an angiogenesis- and an apoptosis-related
condition, a hearing impairment or any other disease, condition or
combination of conditions as disclosed herein. Such methods involve
administering to a mammal in need of such treatment a
prophylactically or therapeutically effective amount of one or more
such chemically modified siRNA compound, which inhibits or reduces
expression or activity of RTP801L.
[0018] In one aspect the present invention provides a compound
having the following structure:
TABLE-US-00001 5' (N).sub.x-Z 3' (antisense strand) 3'
Z'-(N').sub.y-z'' 5' (sense strand)
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein Z
and Z' may be present or absent, but if present is independently
1-5 consecutive nucleotides covalently attached at the 3' terminus
of the strand in which it is present; wherein z'' may be present or
absent, but if present is a capping moiety covalently attached at
the 5' terminus of (N')y; each of x and y is independently an
integer between 18 and 40; wherein the sequence of (N)x is
substantially complementary to the sequence of (N')y; and the
sequence of (N')y is substantially identical to any one of the
sense sequences set forth in any one of Tables B-F.
[0019] In certain preferred embodiments the present invention
provides an siRNA compound comprising an antisense strand (N)x and
its substantially complementary sense strand (N')y, set forth in
any one of SEQ ID NOS:1852-6927. The siRNA compounds consist of
unmodified ribonucleotides or a combination of unmodified
ribonucleotides and ribonucleotides and or unconventional
moieties.
[0020] In some embodiments the chemically modified siRNA compounds
according to the present invention comprise the oligonucleotides
disclosed in Tables B-F.
[0021] In some embodiments, the present invention provides a
chemically modified siRNA compound having the following
structure:
TABLE-US-00002 5' (N)x-Z 3' antisense strand 3' Z'-(N')y-z'' 5'
sense strand
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide and a modified ribonucleotide; wherein
each of (N)x and (N')y is an oligomer in which each consecutive
ribonucleotide is joined to the next ribonucleotide by a covalent
bond; wherein each of x and y is independently an integer between
18 and 40; wherein in each of (N)x and (N')y the ribonucleotides
alternate between modified ribonucleotides and unmodified
ribonucleotides each modified ribonucleotide being modified so as
to have a 2'-O-methyl on its sugar and the ribonucleotide located
at the middle position of (N)x being unmodified and the
ribonucleotide located at the middle position of (N')y being
modified; wherein each of Z and Z' may be present or absent, but if
present is 1-5 deoxyribonucleotides covalently attached at the 3'
terminus of the oligomer to which it is attached; wherein the
sequence of (N)x is substantially complementary to the sequence of
(N')y; and wherein the sequence of (N')y is substantially identical
to any one of the sense sequences set forth in any one of Tables
B-F.
[0022] The present invention provides additional chemically
modified RTP801L siRNA compounds. In some embodiments, the present
invention provides a compound having the following structure:
TABLE-US-00003 5' (N)x-Z 3' (antisense strand) 3' Z'-(N')y-z'' 5'
(sense strand)
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein Z
and Z' may be present or absent, but if present is independently
1-5 consecutive nucleotides covalently attached at the 3' terminus
of the strand in which it is present; wherein z'' may be present or
absent, but if present is a capping moiety covalently attached at
the 5' terminus of (N')y; wherein each of x and y is independently
an integer between 18 and 40; wherein (N)x comprises modified and
unmodified ribonucleotides, each modified ribonucleotide having a
2'-O-methyl on its sugar, wherein N at the 3' terminus of (N)x is a
modified ribonucleotide, (N)x comprises at least five alternating
modified ribonucleotides beginning at the 3' end and at least nine
modified ribonucleotides in total and each remaining N is an
unmodified ribonucleotide; wherein in (N')y at least one
unconventional moiety is present, which unconventional moiety may
be a modified or unmodified deoxyribonucleotide, a mirror
nucleotide, or a nucleotide joined to an adjacent nucleotide by a
2'-5' internucleotide phosphate bond; wherein the sequence of (N)x
is substantially complementary to the sequence of (N')y; and the
sequence of (N')y is substantially identical to a sequence of
identical length of consecutive ribonucleotides in the mRNA set
forth in SEQ ID NO:1.
[0023] In various embodiments the compound of the invention
comprises an antisense sequence (N)x present in any one of Tables
A-F, set forth in SEQ ID NOS:2-6927. In certain embodiments (N)x
comprising the oligonucleotide set forth in SEQ ID NO:999 or SEQ ID
NO:1000. In other embodiments (N)x comprising the oligonucleotide
set forth in SEQ ID NO:6914 or SEQ ID NO:6915.
[0024] In some embodiments the covalent bond joining each
consecutive N or N' is a phosphodiester bond. In various
embodiments all the covalent bonds are phosphodiester bonds.
[0025] In various embodiments x=y and each of x and y is 19, 20,
21, 22 or 23. In some embodiments x=y=23. In other embodiments
x=y=19.
[0026] In one embodiment of the above structure, the compound
comprises at least one mirror nucleotide at one or both termini in
(N')y. In various embodiments the compound comprises two
consecutive mirror nucleotides, one at the 3' penultimate position
and one at the 3' terminus in (N')y. In one preferred embodiment
x=y=19 and (N')y comprises an L-deoxyribonucleotide at position
18.
[0027] In some embodiments the mirror nucleotide is selected from
an L-ribonucleotide and an L-deoxyribonucleotide. In various
embodiments the mirror nucleotide is an L-deoxyribonucleotide. In
some embodiments y=19 and (N')y, consists of unmodified
ribonucleotides at positions 1-17 and 19 and one L-DNA at the 3'
penultimate position (position 18). In other embodiments y=19 and
(N')y consists of unmodified ribonucleotides at position 1-16 and
19 and two consecutive L-DNA at the 3' penultimate position
(positions 17 and 18).
[0028] In another embodiment of the above structure, (N')y further
comprises one or more nucleotides containing an intra-sugar bridge
at one or both termini.
[0029] In another embodiment of the above structure, (N')y
comprises at least two consecutive nucleotide joined together to
the next nucleotide by a 2'-5' phosphodiester bond at one or both
termini. In certain preferred embodiments in (N')y the 3'
penultimate nucleotide is linked to the 3' terminal nucleotide with
a 2'-5' phosphodiester bridge.
[0030] In certain preferred embodiments the compound of the
invention is a blunt-ended (z'', Z and Z' are absent), double
stranded oligonucleotide structure, x=y and x=19 or 23, wherein
(N')y comprises unmodified ribonucleotides in which three
consecutive nucleotides at the 3' terminus are joined together by
two 2'-5' phosphodiester bonds; and an antisense strand (AS) of
alternating unmodified and 2'-O methyl sugar-modified
ribonucleotides.
[0031] In additional embodiments (N)x comprises modified
ribonucleotides in alternating positions wherein each N at the 5'
and 3' termini are modified in their sugar residues and the middle
ribonucleotide is not modified, e.g. ribonucleotide in position 10
in a 19-mer strand or position 12 in a 23-mer strand.
[0032] In some embodiments, neither (N)x nor (N')y are
phosphorylated at the 3' and 5' termini, In other embodiments
either or both (N)x and (N')y are phosphorylated at the 3'
termini.
[0033] In various embodiments the compound comprises an antisense
and sense oligonucleotide pair present in any one of Tables A-G set
forth in SEQ ID NOS:2-6927.
[0034] In certain embodiments for all the above-mentioned
structures, the compound is blunt ended, for example wherein both Z
and Z' are absent. In an alternative embodiment, the compound
comprises at least one 3' overhang, wherein at least one of Z or Z'
is present. Z and Z' can independently comprise one or more
covalently linked modified or non-modified nucleotides, for example
inverted dT or dA; dT, LNA, mirror nucleotide and the like. In some
embodiments each of Z and Z' are independently selected from dT and
dTdT.
[0035] In some embodiments the present invention provides an
expression vector comprising an antisense sequence present in any
one of Tables B-F.
[0036] In a second aspect the present invention provides a
pharmaceutical composition comprising one or more compounds of the
present invention, in an amount effective to inhibit RTP801L human
gene expression and a pharmaceutically acceptable carrier.
[0037] In another aspect, the present invention relates to a method
for the treatment of a subject in need of treatment for a disease
or disorder or symptoms or conditions associated with the disease
or disorder, associated with the expression of RTP801L comprising
administering to the subject an amount of an siRNA which reduces or
inhibits expression of RTP801L.
[0038] More specifically, the present invention provides methods,
compounds and compositions useful in therapy for treating a subject
suffering from acute renal failure (ARF), hearing loss, glaucoma,
acute respiratory distress syndrome (ARDS) and other acute lung and
respiratory injuries, injury (e.g. ischemia-reperfusion injury) in
organ transplant including lung, kidney, bone marrow, heart,
pancreas, cornea or liver transplantation, nephrotoxicity, spinal
cord injury, pressure sores, dry eye syndrome, oral mucositis and
chronic obstructive pulmonary disease (COPD). The methods of the
invention comprise administering to the subject one or more siRNA
compounds which inhibit expression of RTP801L in a therapeutically
effective dose so as to thereby treat the patient.
[0039] According to one embodiment the compound consists of an
antisense strand having an oligomer sequence set forth in SEQ ID
NO:1000 and a sense strand having an oligomer sequence set forth in
SEQ ID NO:75. According to one embodiment the compound consists of
an antisense strand having an oligomer sequence set forth in SEQ ID
NO:999 and a sense strand having an oligomer sequence set forth in
SEQ ID NO:74. According to another embodiment, the compound
consists of an antisense strand having an oligomer sequence set
forth in SEQ ID NO:6914 and a sense strand having an oligomer
sequence set forth in SEQ NO:6898 (DDIT4L.sub.--14 in Table F).
According to another embodiment, the compound consists of an
antisense strand having an oligomer sequence set forth in SEQ ID
NO:6915 and a sense strand having an oligomer sequence set forth in
SEQ ED NO:6899 (DDIT4L.sub.--15 in Table F).
[0040] In various embodiments the siRNA compound is selected from
any one of the compounds shown in Table G (FIG. 3).
[0041] In certain preferred embodiments the siRNA compounds
indicated above are modified so at to have antisense strand
comprising alternating 2'OMe and unmodified ribonucleotides, and a
sense strand comprising unmodified ribonucleotides and an L-DNA
moiety at position 18 or at positions 17 and 18. In some
embodiments the sense strand further includes a deoxyribonucleotide
at position 15.
[0042] In another aspect the present invention provides a
pharmaceutical composition comprising on or more RTP801L siRNA
inhibitors of the invention: and a pharmaceutically acceptable
excipient.
[0043] In another aspect, the present invention provides a method
of treating a patient suffering from a microvascular disorder,
macular degeneration or a respiratory disorder, comprising
administering to the patient a pharmaceutical composition
comprising one or more RTP801L inhibitor.
[0044] Another embodiment of the present invention concerns a
method for treating a patient suffering from COPD, comprising
administering to the patient a pharmaceutical composition
comprising a therapeutically effective amount of one or more siRNA
RTP801L inhibitor. In one embodiment the inhibitor is selected from
the group consisting of an siRNA molecule, an antisense molecule,
and a ribozyme or a combination thereof.
[0045] Another embodiment of the present invention concerns a
method for treating a patient suffering from Acute Lung Injury
(ALI), comprising administering to the patient a pharmaceutical
composition comprising a therapeutically effective amount of one or
more RTP801L inhibitor. In one embodiment the inhibitor selected
from the group consisting of an siRNA molecule, an antisense
molecule, and a ribozyme or a combination thereof.
[0046] Another embodiment of the present invention concerns a
method for treating a patient suffering from macular degeneration,
comprising administering to the patient a pharmaceutical
composition comprising a therapeutically effective amount of one or
more RTP801L inhibitor. In one embodiment the inhibitor is an siRNA
molecule, an antisense molecule, or a ribozyme or a combination
thereof.
[0047] Another embodiment of the present invention concerns a
method for treating a patient suffering from a microvascular
disorder, comprising administering to the patient a pharmaceutical
composition comprising a therapeutically effective amount of one or
more RTP801L inhibitor. In one embodiment the inhibitor is an siRNA
molecule, an antisense molecule, or a ribozyme or a combination
thereof.
[0048] An additional embodiment of the present invention provides
for the use of a therapeutically effective amount of an RTP801L
inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from a respiratory disorder. In one
embodiment the respiratory disorder is COPD and the inhibitor is
preferably one or more siRNA. In another embodiment the respiratory
disorder is ALI and the inhibitor is preferably one or more
siRNA.
[0049] An additional embodiment of the present invention provides
for the use of a therapeutically effective dose of one or more
RTP801L inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from macular degeneration. In one
embodiment the macular degeneration is AMD and the inhibitor is
preferably one or more siRNA.
[0050] An additional embodiment of the present invention provides
for the use of a therapeutically effective dose of one or more
RTP801L inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from glaucoma. In one embodiment
the inhibitor is preferably one or more siRNA. In various
embodiments the present invention is useful in therapy for treating
a patient in need of neuroprotection. In some embodiments the siRNA
compound is therapeutically effective in neuroprotection of the
optic nerve.
[0051] An additional embodiment of the present invention provides
for the use of a therapeutically effective dose of one or more
RTP801L inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from an eye disorder secondary to
diabetes. In one embodiment the inhibitor is preferably one or more
siRNA.
[0052] An additional embodiment of the present invention provides
for the use of a therapeutically effective amount of one or more
RTP801L inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from a microvascular disorder. In
one embodiment the microvascular disorder is diabetic retinopathy
and the inhibitor is preferably one or more siRNA. In another
embodiment the disorder is Acute Renal Failure and the inhibitor is
preferably one or more siRNA.
[0053] The present invention also relates generally to methods and
compositions for treating or preventing the incidence or severity
of hearing impairment (or balance impairment), particularly hearing
impairment associated with cell death of the inner ear hair cells.
The methods and compositions involve administering to a mammal in
need of such treatment a prophylactically or therapeutically
effective amount of one or more compounds which down-regulate
expression of the RTP801L gene, particularly novel small
interfering RNAs (siRNAs).
[0054] More specifically, the present invention provides methods
and compositions for treating a patient suffering from hearing
impairment, or other oto-pathologies associated with cell death of
inner ear hair cells. Such oto-pathologies may be the result of
acoustic trauma, mechanical trauma, age (presbycusis) or
ototoxin-induced hearing loss. The methods of the invention
comprising administering to the patient one or more compounds which
down-regulate expression of the RTP801L gene, particularly siRNAs
that inhibit RTP801L typically as a pharmaceutical composition, in
a therapeutically effective dose so as to thereby treat the
patient.
[0055] In one embodiment, the present invention provides for
improved compositions and methods for treatments requiring
administration of a pharmaceutical drug having an ototoxic,
hearing-impairing side-effect, in combination with a
therapeutically effective amount of one or more siRNA molecules
that inhibit RTP801L, to treat or prevent the ototoxicity induced
by the pharmaceutical drug. The compositions of the invention can
be administered at a suitable interval(s) either prior to,
subsequent to, or substantially concurrent with the administration
of the ototoxic, hearing-impairing drug that induces inner ear
apoptotic tissue damage.
[0056] Accordingly, it is an object of the invention to provide an
improved composition containing a therapeutically effective amount
of one or more siRNA molecules that inhibit RTP801L in combination
with an ototoxic, hearing-impairing pharmaceutical drug for
administration to a mammal. Said combination drugs may be
administered separately; the siRNA molecules that inhibit RTP801L
would then be administered locally while the ototoxic,
hearing-impairing pharmaceutical drug is administered systemically.
The siRNA molecules may be administered prior to, simultaneously
with or subsequent to the ototoxic drug. Such combination
compositions can further contain a pharmaceutically acceptable
carrier. The pharmaceutical composition will have lower ototoxicity
than the ototoxic pharmaceutical alone, and preferably, will have a
higher dosage of the ototoxic pharmaceutical than typically used.
Examples of such improved compositions include cisplatin or other
ototoxic neoplastic agent or an aminoglycoside antibiotic(s) in
combination with the therapeutically effective amount of one or
more siRNA molecules that inhibit RTP801L.
[0057] Still further, the invention relates to the use of the
compositions of the invention in cases where diuretics are needed.
The present invention provides a solution to the art that has long
sought a therapy and a medicament which can treat the ototoxic
effects currently associated with certain diuretics, and particular
with the more popular and commonly used loop-diuretics, without
sacrificing their diuretic effectiveness.
[0058] Still further, the invention relates to the use of the
compositions of the invention in cases where quinine or
quinine-like compounds are needed. The present invention provides a
solution to the art that has long sought a therapy and a medicament
which can treat the ototoxic effects currently associated with
certain quinines without sacrificing their effectiveness.
[0059] The present invention further relates to methods and
compositions for treating or preventing the incidence or severity
of pressure sores. The methods and compositions involve
administering to a mammal in need of such treatment a
prophylactically or therapeutically effective amount of one or more
compounds which down-regulate expression of the RTP801L gene,
particularly novel small interfering RNAs (siRNAs).
[0060] Further, the present invention relates to methods and
compositions for the treatment of any ischemic or
ischemia-reperfusion injuries or conditions, as described herein.
Said methods and compositions involve administering to a mammal in
need of such treatment a prophylactically or therapeutically
effective amount of one or more compounds which down-regulate
expression of the RTP801L gene, particularly novel small
interfering RNAs (siRNAs).
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIG. 1 provides the polynucleotide sequence of REDD2 full
length mRNA (SEQ ID NO:1);
[0062] FIG. 2 provides Table F, a list of certain preferred siRNA
RTP801L oligonucleotide pairs.
[0063] FIG. 3a-3c provide Table G, a list of certain preferred
chemically modified RTP801L siRNA. "r" preceeding A, C, G or U
refers to an unmodified ribonucleotide; "m" preceeding A, C, G or U
refers to a 2'OMe modified ribonucleotide; LAC refers to
L-deoxycytidine, which substituted some of the A, C, G or U in the
sense strands. FIG. 4 details the activity results of RTP801L
siRNAs on the endogenous RTP801L gene in wild type MEF cells
following H.sub.2O.sub.2 treatment;
[0064] FIG. 5 demonstrates dose dependent activity of RTP801L
siRNAs as measured in 801 wt MEF cells;
[0065] FIG. 6 shows activity results of RTP801L siRNAs on the
endogenous RTP801L gene in wt 293T cells;
[0066] FIG. 7 demonstrates dose dependent activity of RTP801L siRNA
as measured in 2931 cells,
[0067] FIGS. 8a-8b show structure, activity and stability results
for certain RTP801L siRNA compounds of the present invention.
[0068] FIG. 9 shows IC50 results for an siRNA compound of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The present invention, relates to novel oligonucleotide
compounds useful in inhibiting the RTP801L gene for the treatment
of eye diseases, respiratory disorders, microvascular disorders,
hearing disorders and ischemic conditions, inter alia. As will be
described herein, the preferred inhibitors to be used with the
present invention are chemically modified siRNA.
[0070] Compounds and compositions comprising same which inhibit
RTP801L are discussed herein at length, and any of said compounds
and/or compositions may be beneficially employed in the treatment
of a patient suffering from a disease or disorder associated with
RTP801L expression.
[0071] The siRNAs of the present invention possess structures and
modifications which may increase activity, increase stability, and
or minimize toxicity; the novel modifications of the siRNAs of the
present invention can be beneficially applied to double stranded
RNA useful in preventing or attenuating RTP801L expression.
[0072] According to one aspect the present invention provides
inhibitory oligonucleotide compounds comprising unmodified and
modified nucleotides and or unconventional moieties. The compound
comprises at least one modified nucleotide selected from the group
consisting of a sugar modification, a base modification and an
internucleotide linkage modification and may contain DNA, and
modified nucleotides such as LNA (locked nucleic acid), ENA
(ethylene-bridged nucleic acid, PNA (peptide nucleic acid),
arabinoside, PACE, mirror nucleotide, or nucleotides with a 6
carbon sugar.
[0073] In one embodiment the compound comprises a 2' modification
on the sugar moiety of at least one ribonucleotide ("2' sugar
modification"). In certain embodiments the compound comprises
2'O-alkyl or 2'-tluoro or 2'O-allyl or any other 2' modification,
optionally on alternate positions. Other stabilizing modifications
are also possible (e.g. terminal modifications). In some
embodiments the backbone of the oligonucleotides is modified and
comprises phosphate-D-ribose entities but may also contain
thiophosphate-D-ribose entities, triester, thioate. 2'-5' bridged
backbone (also may be referred to as 5'-2'), PACE or any other type
of modification.
[0074] Other modifications include terminal modifications on the 5'
and/or 3' part of the oligonucleotides. Such terminal modifications
may be lipids, peptides, sugars or other molecules.
[0075] Further, an additional embodiment of the present invention
concerns a method for treating a patient suffering from a
microvascular disorder, an eye disease, a respiratory disorder, a
hearing disorder or a spinal cord injury or other wound, comprising
administering to the patient a pharmaceutical composition
comprising a therapeutically effective dose of an RTP801L inhibitor
comprising an siRNA molecule, optionally an siRNA molecule detailed
in any one of Tables A-G, in a dosage and over a period of time so
as to thereby treat the patient.
[0076] An additional method for treating a patient suffering from a
microvascular disorder, an eye disease, a respiratory disorder, a
hearing disorder or a spinal cord injury or other wound is
provided, comprising administering to the patient a pharmaceutical
composition comprising a therapeutically effective dose of an RNA
molecule which targets the RTP801L gene rnRNA in a dosage and over
a period of time so as to thereby treat the patient. The RNA
molecule may be an siRNA molecule, such as an siRNA molecule
detailed in any one of Tables A-G, preferably siRNA Nos:72 or 73.
In one embodiment, the compound consists of an antisense strand
having an oligomer sequence set forth in SEQ ID NO:6914 and a sense
strand having an oligomer sequence set forth in SEQ ID NO:6898
(DDIT4L.sub.--14 in Table F). According to another embodiment, the
compound consists of an antisense strand having an oligomer
sequence set forth in SEQ ID NO:6915 and a sense strand having an
oligomer sequence set forth in SEQ ED NO:6899 (DDIT4L.sub.--15 in
Table F). In certain preferred embodiments the siRNA is modified so
at to have antisense strand comprising alternating 2'OMe and
unmodified ribonucleotides, and a sense strand comprising
unmodified ribonucleotides and an L-DNA moiety at position 18 or at
positions 17 and 18. In some embodiments the sense strand further
includes a deoxyribonucleotide at position 15.
[0077] The present invention further provides a method for treating
a patient suffering from a microvascular disorder, an eye disease,
an ischemic disease, a kidney disorder, a respiratory disorder, a
hearing disorder or a spinal cord injury or other wound or any of
the conditions disclosed herein, comprising administering to the
patient a pharmaceutical composition comprising a therapeutically
effective dose of an siRNA molecule which targets the RTP801L gene
mRNA, optionally an siRNA molecule detailed in any one of Tables
A-G, in a dosage and over a period of time so as to thereby treat
the patient. Further, the eye disease may be macular degeneration
such as age-related macular degeneration (AMD) or glaucoma; the
microvascular disorder may be diabetic retinopathy or acute renal
failure; the ischemic disease may be ischemia--reperfusion or organ
transplant related; the kidney disorder may be nephrotoxicity; the
respiratory disorder may be COPD or ALI; and the hearing disorder
may be noise--induced deafness, chemically induced deafness such as
cisplatin-induced deafness or age-related deafness.
[0078] The present invention additionally relates to the use of the
novel siRNAs disclosed herein in the treatment of hearing
impairment in which inhibition of RTP801L expression is beneficial.
In one embodiment, the present invention constitutes a method for
treating a mammal having or prone to a hearing (or balance)
impairment or treating a mammal prophylactically in conditions
where inhibition of RTP801L expression is beneficial. The method of
this embodiment of the present invention would prevent or reduce
the occurrence or severity of a hearing (or balance) impairment
that would result from inner ear cell injury, loss, or
degeneration, in particular caused by an ototoxic agent or by
aging. In this embodiment, the method of the invention includes
administering a therapeutically effective amount of one or more
compounds which down-regulate expression of the RTP801L gene,
particularly the novel siRNAs of the present invention.
[0079] In one embodiment, it is the object of the present invention
to provide a method for treating a mammal, to prevent, reduce, or
treat a hearing impairment, disorder or imbalance, preferably an
ototoxin-induced hearing condition, by administering to a mammal in
need of such treatment a composition of the invention. One
embodiment is a method for treating a hearing disorder or
impairment wherein the ototoxicity results from administration of a
therapeutically effective amount of an ototoxic pharmaceutical
drug. Typical ototoxic drugs are chemotherapeutic agents, e.g.
antineoplastic agents, and antibiotics. Other possible candidates
include loop-diuretics, quinines or a quinine-like compound, and
salicylate or salicylate-like compounds. These methods are
especially effective when the ototoxic compound is an antibiotic,
preferably an aminoglycoside antibiotic. Ototoxic aminoglycoside
antibiotics include but are not limited to neomycin, paromomycin,
ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,
viomycin, gentamycin, sisomycin, netilmycin, streptomycin,
dibekacin, fortimycin, and dihydrostreptomycin, or combinations
thereof. Particular antibiotics include neomycin B, kanarnycin A,
kanamycin B, gentamycin C1, gentamycin C1a, and gentamycin C2. The
methods of the invention are also effective when the ototoxic
compound is a neoplastic agent such as vincristine, vinblastine,
cisplatin and cisplatin-like compounds and taxol and taxol-like
compounds
[0080] In some embodiments aimed at treating or preventing a
hearing disorder, the composition of the invention is
co-administered with an ototoxin. For example, an improved method
is provided for treatment of infection of a mammal by
administration of an aminoglycoside antibiotic, the improvement
comprising administering a therapeutically effective amount of one
or more compounds which down-regulate expression of the RTP801L
gene particularly novel siRNAs, to the patient in need of such
treatment to reduce or prevent ototoxin-induced hearing impairment
associated with the antibiotic. The compounds which reduce or
prevent the ototoxin-induced hearing impairment, particularly the
novel siRNAs are preferably administered locally within the inner
ear. In yet another embodiment is provided an improved method for
treatment of cancer in a mammal by administration of a
chemotherapeutic compound, the improvement comprises administering
a therapeutically effective amount of a composition of the
invention to the patient in need of such treatment to reduce or
prevent ototoxin-induced hearing impairment associated with the
chemotherapeutic drug. In another embodiment the methods of
treatment are applied to hearing impairments resulting from the
administration of a chemotherapeutic agent to treat its ototoxic
side effect. Ototoxic chemotherapeutic agents amenable to the
methods of the invention include, but are not limited to an
antineoplastic agent, including cisplatin or cisplatin-like
compounds, taxol or taxol-like compounds, and other
chemotherapeutic agents believed to cause ototoxin-induced hearing
impairments, e.g., vincristine, an antineoplastic drug used to
treat hematological malignancies and sarcomas. Cisplatin-like
compounds include carboplatin (Paraplatin.RTM.), tetraplatin,
oxaliplatin, aroplatin and transplatin inter alia in another
embodiment the methods of the invention are applied to hearing
impairments resulting from the administration of quinine and its
synthetic substitutes, typically used in the treatment of malaria,
to treat its ototoxic side-effect. In another embodiment the
methods of the invention are applied to hearing impairments
resulting from administration of a diuretic. Diuretics,
particularly "loop" diuretics, i.e. those that act primarily in the
Loop of Henle, are candidate ototoxins. Illustrative examples, not
limiting to the invention method, include furosemide, ethacrylic
acid, and mercurials. Diuretics are typically used to prevent or
eliminate edema. Diuretics are also used in nonedematous states for
example hypertension, hypercalcemia, idiopathic hypercalciuria, and
nephrogenic diabetes insipidus.
[0081] In another embodiment, the methods of the invention are
applied to treating or preventing the incidence or severity of
pressure sores. The methods and compositions involve administering
to a mammal in need of such treatment a prophylactically or
therapeutically effective amount of one or more compounds which
down-regulate expression of the RTP801L gene, particularly novel
small interfering RNAs (siRNAs). The compounds which treat or
prevent the incidence or severity of pressure sores, particularly
the novel siRNAs are preferably administered locally within the
damaged area. The methods and compositions of the present invention
are effective in the treatment and prevention of pressure sores or
pressure ulcers developed when sustained pressure (usually from a
bed or wheelchair) cuts off circulation to vulnerable parts of the
body. The methods and compositions are effective in patients with
diminished or absent sensation or who are debilitated, emaciated,
paralyzed, or long bedridden. The compositions of the present
invention are effective also in improving the healing of pressure
sores using the compositions. The compositions may be used at any
particular stage in the healing process including the stage before
any healing has initiated or even before a specific sore is made
(prophylactic treatment).
[0082] Other kinds of wounds to be treated according to the
invention include i) general wounds such as, e.g., surgical,
traumatic, infectious, ischemic, thermal, chemical and bullous
wounds; ii) wounds specific for the oral cavity such as, e.g.,
post-extraction wounds, endodontic wounds especially in connection
with treatment of cysts and abscesses, ulcers and lesions of
bacterial, viral or autoimmunological origin, mechanical, chemical,
thermal, infectious and lichenoid wounds; herpes ulcers, stomatitis
aphthosa, acute necrotising ulcerative gingivitis and burning mouth
syndrome are specific examples; and iii) wounds on the skin such
as, e.g., neoplasm, burns (e.g. chemical, the bacterial, viral,
autoimmunological), bites and surgical incisions.
[0083] The methods and compositions of the present invention are
also effective in the treatment and prevention of any chronic
wounds including inter alia pressure sores, venous ulcers, and
diabetic ulcers. In all these chronic wound types, the underlying
precipitating event is a period of ischemia followed by a period of
reperfusion. These ischemia-reperfusion events are usually
repetitive, which means the deleterious effects of
ischemia-reperfusion are potentiated and eventually sufficient to
cause ulceration. For both pressure sores and diabetic foot ulcers,
the ischemic event is the result of prolonged pressure sufficient
to prevent tissue perfusion, and when the pressure is finally
relieved, the reperfusion injury occurs. The present compositions
are effective in inhibiting the damage caused by
ischemia-reperfusion in chronic wounds.
[0084] The present compositions are also effective in other
conditions associated with ischemia-reperfusion such as but not
limited to: organ transplantation, intestinal and colon
anastamoses, operations on large blood vessels, stitching detached
limbs, balloon angioplasty or any cardiac surgery, stroke or brain
trauma, limb transplantation, pulmonary hypertension, hypoxemia,
and noncardiogenic pulmonary edema, acute renal failure, acute
glaucoma, diabetic retinopathy, hypertensive retinopathy, and
retinal vascular occlusion, cochlear ischemia, microvascular
surgery and ischemic lesions in scleroderma.
[0085] The methods and compositions of the present invention are
also effective in the treatment of acoustic trauma or mechanical
trauma, preferably acoustic or mechanical trauma that leads to
inner ear hair cell loss. Acoustic trauma to be treated in the
present invention may be caused by a single exposure to an
extremely loud sound, or following long-term exposure to everyday
loud sounds above 85 decibels. Mechanical inner ear trauma to be
treated in the present invention is for example the inner ear
trauma following an operation of electronic device insertion in the
inner ear. The compositions of the present invention prevent or
minimize the damage to inner ear hair cells associated with the
operation. The compounds which reduce or prevent the
ototoxin-induced hearing impairment, particularly the novel siRNAs
are preferably administered locally within the inner ear.
[0086] Additionally, as detailed above, the compound of the present
invention can be used to treat any condition in which ischemia is
involved, optionally ischemia-reperfusion. Such condition include
ischemia or ischemia-reperfusion resulting from an angioplasty,
cardiac surgery or thrombolysis; organ transplant; as a result of
plastic surgery; during severe compartment syndrome; during
re-attachment of severed limbs; as a result of multiorgan failure
syndrome; in the brain as a result of stroke or brain trauma; in
connection with chronic wounds such as pressure sores, venous
ulcers and diabetic ulcers; during skeletal muscle ischemia or limb
transplantation; as a result of mesenteric ischemia or acute
ischemic bowel disease; respiratory failure as a result of lower
torso ischemia, leading to pulmonary hypertension, hypoxemia, and
noncardiogenic pulmonary edema; acute renal failure as observed
after renal transplantation, major surgery, trauma, and septic as
well as hemorrhagic shock; Sepsis; Retinal ischemia occurring as a
result of acute vascular occlusion, leading to loss of vision in a
number of ocular diseases such as acute glaucoma, diabetic
retinopathy, hypertensive retinopathy, and retinal vascular
occlusion; Cochlear ischemia; flap failure in microvascular surgery
for head and neck defects; Raynaud's phenomenon and the associated
digital ischemic lesions in scleroderma; spinal cord injury;
vascular surgery; Traumatic rhabdomyolysis (crush syndrome); and
myoglobinuria. Further, ischemia/reperfusion may be involved in the
following conditions: hypertension, hypertensive cerebral vascular
disease, rupture of aneurysm, a constriction or obstruction of a
blood vessel--as occurs in the case of a thrombus or embolus,
angioma, blood dyscrasias, any form of compromised cardiac function
including cardiac arrest or failure, systemic hypotension, cardiac
arrest, cardiogenic shock, septic shock, spinal cord trauma, head
trauma, seizure, bleeding from a tumor; and diseases such as
stroke, Parkinson's disease, epilepsy, depression, ALS, Alzheimer's
disease, Huntington's disease and any other disease-induced
dementia (such as HIV induced dementia for example). Additionally,
an ischemic episode may be caused by a mechanical injury to the
Central Nervous System, such as results from a blow to the head or
spine. Trauma can involve a tissue insult such as an abrasion,
incision, contusion, puncture, compression, etc., such as can arise
from traumatic contact of a foreign object with any locus of or
appurtenant to the head, neck, or vertebral column. Other forms of
traumatic injury can arise from constriction or compression of CNS
tissue by an inappropriate accumulation of fluid (for example, a
blockade or dysfunction of normal cerebrospinal fluid or vitreous
humor fluid production, turnover, or volume regulation, or a
subdural or intracarnial hematoma or edema). Similarly, traumatic
constriction or compression can arise from the presence of a mass
of abnormal tissue, such as a metastatic or primary tumor.
[0087] "Treating a disease" refers to administering a therapeutic
substance effective to ameliorate symptoms associated with a
disease, to lessen the severity or cure the disease, or to prevent
the disease from occurring. "Treatment" refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) a disease or
disorder.
[0088] A "therapeutically effective dose" refers to an amount of a
pharmaceutical compound or composition which is effective to
achieve an improvement in a patient or his physiological systems
including, but not limited to, improved survival rate, more rapid
recovery, or improvement or elimination of symptoms, and other
indicators as are selected as appropriate determining measures by
those skilled in the art.
[0089] The methods of treating the diseases disclosed herein and
included in the present invention may include administering an
RTP801L inhibitor in conjunction with an additional RTP801L
inhibitor, a substance which improves the pharmacological
properties of the active ingredient as detailed below, or an
additional compound known to be effective in the treatment of the
disease to be treated, such as macular degeneration, glaucoma,
COPD, ALI, ARF, DR, cisplatin-induced deafness, and age-related
deafness, inter Win. By "in conjunction with" is meant prior to,
simultaneously or subsequent to. Further detail on exemplary
conjoined therapies is given below.
[0090] In another embodiment, the present invention provides for
the use of a therapeutically effective dose of an RTP801L inhibitor
for the preparation of a medicament for promoting recovery in a
patient suffering from macular degeneration, glaucoma, COPD, ALI,
ARF, DR, cisplatin-induced deafness, age-related deafness or any
eye disease, microvascular or respiratory condition or hearing
disorder as detailed above, and the use of a therapeutically
effective dose of an RTP801L inhibitor for the preparation of a
medicament for treating said diseases and conditions. In this
embodiment, the RTP801L inhibitor may comprise a polynucleotide
which comprises consecutive nucleotides having a sequence which
comprises an antisense sequence to the sequence set forth in FIG. 1
(SEQ ID No: 1). Additionally, the RTP801L inhibitor may be an
expression vector comprising a polynucleotide having a sequence
which is an antisense sequence to the sequence set forth in FIG. 1
(SEQ ID No:1). Additionally, the RTP801L inhibitor may be an RNA
molecule which targets the RTP801L gene mRNA such as a ribozyme or
an siRNA, optionally an siRNA comprising consecutive nucleotides
having a sequence identical to any one of the sequences set forth
in any one of Tables A-G (SEQ ID NOs:3-6927) and preferably, siRNA
Nos:72 and 73 of Table A, or DDIT4L.sub.--14 or DDIT4L.sub.--15 in
Table F.
[0091] Thus, according to the information disclosed herein, the
RTP801L inhibitor to be used with any of the methods disclosed
herein, in any of the uses disclosed herein and in any of the
pharmaceutical compositions disclosed herein, may be selected from
the group consisting of an siRNA molecule, a vector comprising an
siRNA molecule, a vector which can express an siRNA molecule and
any molecule which is endogenously processed into an siRNA
molecule. As detailed herein, said siRNA molecule is preferably an
siRNA comprising consecutive nucleotides having a sequence
identical to any one of the sequences set forth in any one of
Tables A-G and preferably siRNA Nos:72 and 73 of Table A, or
DDIT4L.sub.--14 or DDIT4L.sub.--15 in Table F.
[0092] "Respiratory disorder" refers to conditions, diseases or
syndromes of the respiratory system including but not limited to
pulmonary disorders of all types including chronic obstructive
pulmonary disease (COPD), acute lung injury (ALI), emphysema,
chronic bronchitis, asthma and lung cancer, inter alia. Emphysema
and chronic bronchitis may occur as part of COPD or independently.
Conditions resulting from lung transplantation may also be viewed
as such.
[0093] "Ischemic diseases/conditions" relates to any disease in
which ischemia is involved, as well as ischemia-reperfusion injury
and ischemia in connection with organ transplantation.
[0094] "Microvascular disorder" refers to any condition that
affects microscopic capillaries and lymphatics, in particular
vasospastic diseases, vasculitic diseases and lymphatic occlusive
diseases. Examples of microvascular disorders include, inter alia:
eye disorders such as Amaurosis Fugax (embolic or secondary to
SLE), apla syndrome, Prot CS and ATIII deficiency, microvascular
pathologies caused by IV drug use, dysproteinemia, temporal
arteritis, anterior ischemic optic neuropathy, optic neuritis
(primary or secondary to autoimmune diseases), glaucoma, von Hippel
Lindau syndrome, corneal disease, corneal transplant rejection
cataracts, Eales' disease, frosted branch angiitis, encircling
buckling operation, uveitis including pars planitis, choroidal
melanoma, choroidal hemangioma, optic nerve aplasia; retinal
conditions such as retinal artery occlusion, retinal vein
occlusion, retinopathy of prematurity, HIV retinopathy, Purtscher
retinopathy, retinopathy of systemic vasculitis and autoimmune
diseases, diabetic retinopathy, hypertensive retinopathy, radiation
retinopathy, branch retinal artery or vein occlusion, idiopathic
retinal vasculitis, aneurysms, neuroretinitis, retinal
embolization, acute retinal necrosis, Birdshot retinochoroidopathy,
long-standing retinal detachment; systemic conditions such as
Diabetes mellitus, diabetic retinopathy (DR), diabetes-related
microvascular pathologies (as detailed herein), hyperviscosity
syndromes, aortic arch syndromes and ocular ischemic syndromes,
carotid-cavernous fistula, multiple sclerosis, systemic lupus
erythematosus, arteriolitis with SS-A autoantibody, acute
multifocal hemorrhagic vasculitis, vasculitis resulting from
infection, vasculitis resulting from Behcet's disease, sarcoidosis,
coagulopathies, neuropathies, nephropathies, microvascular diseases
of the kidney, acute renal failure and ischemic microvascular
conditions, inter alia.
[0095] Microvascular disorders may comprise a neovascular element.
The term "neovascular disorder" refers to those conditions where
the formation of blood vessels (neovascularization) is harmful to
the patient. Examples of ocular neovascularization include: retinal
diseases (diabetic retinopathy, diabetic Macular Edema, chronic
glaucoma, retinal detachment, and sickle cell retinopathy);
rubeosis iritis; proliferative vitreo-retinopathy; inflammatory
diseases; chronic uveitis; neoplasms (retinoblastoma, pseudoglioma
and melanoma); Fuchs' heterochromic iridocyclitis; neovascular
glaucoma; corneal neovascularization (inflammatory, transplantation
and developmental hypoplasia of the iris); neovascularization
following a combined vitrectomy and lensectomy; vascular diseases
(retinal ischemia, choroidal vascular insufficiency, choroidal
thrombosis and carotid artery ischemia); neovascularization of the
optic nerve; and neovascularization due to penetration of the eye
or contusive ocular injury. All these neovascular conditions may be
treated using the compounds and pharmaceutical compositions of the
present invention.
[0096] "Eye disease" refers to refers to conditions, diseases or
syndromes of the eye including but not limited to any conditions
involving choroidal neovascularization (CNV), wet and dry AMD,
ocular histoplasmosis syndrome, angiod streaks, ruptures in Bruch's
membrane, myopic degeneration, ocular tumors, retinal degenerative
diseases, glaucoma, ION, and retinal vein occlusion (RVO). Some
conditions disclosed herein, such as DR, which may be treated
according to the methods of the present invention have been
regarded as either a microvascular disorder and an eye disease, or
both, under the definitions presented herein.
[0097] Hearing impairments relevant to the invention may be due to
end-organ lesions involving inner ear hair cells, e.g., acoustic
trauma, viral endolymphatic labyrinthitis, Meniere's disease.
Hearing impairments include tinnitus, which is a perception of
sound in the absence of an acoustic stimulus, and may be
intermittent or continuous, wherein there is diagnosed a
sensorineural loss. Hearing loss may be due to bacterial or viral
infection, such as in herpes zoster oticus, purulent labyrinthitis
arising from acute otitis media, purulent meningitis, chronic
otitis media, sudden deafness including that of viral origin, e.g.,
viral endolymphatic labyrinthitis caused by viruses including
mumps, measles, influenza, chicken pox, mononucleosis and
adenoviruses. The hearing loss can be congenital, such as that
caused by rubella, anoxia during birth, bleeding into the inner ear
due to trauma during delivery, ototoxic drugs administered to the
mother, erythroblastosis fetalis, and hereditary conditions
including Waardenburg's syndrome and Hurler's syndrome. The hearing
loss can be noise-induced, generally due to a noise greater than 85
decibels (db) that damages the inner ear. Preferably, the hearing
loss is caused by aging (presbycusis) or an ototoxic drug that
affects the auditory portion of the inner ear, particularly inner
ear hair cells. Incorporated herein by reference are Chapters 196,
197, 198 and 199 of The Merck Manual of Diagnosis and Therapy, 14th
Edition, (1982), Merck Sharp & Dome Research Laboratories, N.J.
and corresponding chapters in the most recent 16th edition,
including Chapters 207 and 210) relating to description and
diagnosis of hearing and balance impairments.
[0098] Hearing disorders or impairments (or balance impairment) to
be treated or prevented in the context of the present invention are
preferably, without being bound by theory, trauma-induced deafness,
age-related deafness and ototoxin-induced inner ear hair cells
apoptotic damage. Those in need of treatment include those already
experiencing a hearing impairment, those prone to having the
impairment, and those in which the impairment is to be prevented.
Without being bound by theory, the hearing impairments may be due
to apoptotic inner ear hair cell damage or loss, wherein the damage
or loss is caused by infection, mechanical injury, loud sound,
aging, or, in particular, chemical-induced ototoxicity. Ototoxins
include therapeutic drugs including antineoplastic agents,
salicylates, quinines, and aminoglycoside antibiotics, contaminants
in foods or medicinals, and environmental or industrial pollutants.
Typically, treatment is performed to prevent or reduce ototoxicity,
especially resulting from or expected to result from administration
of therapeutic drugs. Preferably a therapeutically effective
composition is given immediately after the exposure to prevent or
reduce the ototoxic effect. More preferably, treatment is provided
prophylactically, either by administration of the composition prior
to or concomitantly with the ototoxic pharmaceutical or the
exposure to the ototoxin.
[0099] The hearing impairment may be induced by chemotherapy. In
more detail, hearing impairment may be caused by chemotherapeutic
agents such as etoposide, 5-FU (5-fluorouracil), cis-platinum,
doxorubicin, a vinca alkaloid, vincristine, vinblastine,
vinorelbine, taxol, cyclophosphamide, ifosfamide, chlorambucil,
busulfan, mechlorethamine, mitomycin, dacarbazine, carboplatinum,
thiotepa, daunorubicin, idarubicin, mitoxantrone, bleomycin,
esperamicin A1, dactinomycin, plicamycin, carmustine, lomustine,
tauromustine, streptozocin, melphalan, dactinomycin, procarbazine,
dexamethasone, prednisone, 2-chlorodeoxyadenosine, cytarabine,
docetaxel, fludarabine, gemcitabine, herceptin, hydroxyurea,
irinotecan, methotrexate, oxaliplatin, rituxin, semustine,
epirubicin, etoposide, tomudex and topotecan, or a chemical analog
of one of these chemotherapeutic agents. The chemotherapeutic
agents most likely to cause hearing impairment is cisplatinum
(cisplatin) and cisplatin-like compounds.
[0100] By "ototoxin" in the context of the present invention is
meant a substance that through its chemical action injures, impairs
or inhibits the activity of the sound receptors of the nervous
system related to hearing, which in turn impairs hearing (and/or
balance). In the context of the present invention, ototoxicity
includes a deleterious effect on the inner ear hair cells. Ototoxic
agents that cause hearing impairments include, but are not limited
to, neoplastic agents such as vincristine, vinblastine, cisplatin
and cisplatin-like compounds, taxol and taxol-like compounds,
dideoxy-compounds, e.g., dideoxyinosine; alcohol; metals;
industrial toxins involved in occupational or environmental
exposure; contaminants of food or medicinals; and over-doses of
vitamins or therapeutic drugs, e.g., antibiotics such as penicillin
or chloramphenicol, and megadoses of vitamins A, D, or B6,
salicylates, quinines and loop diuretics. By "exposure to an
ototoxic agent" is meant that the ototoxic agent is made available
to, or comes into contact with, a mammal. Exposure to an ototoxic
agent can occur by direct administration, e.g., by ingestion or
administration of a food, medicinal, or therapeutic agent, e.g., a
chemotherapeutic agent, by accidental contamination, or by
environmental exposure, e g., aerial or aqueous exposure.
[0101] An "inhibitor" is a compound which is capable of reducing
the expression of a gene or the activity of the product of such
gene to an extent sufficient to achieve a desired biological or
physiological effect. The term "inhibitor" as used herein refers to
one or more of an oligonucleotide inhibitor, including siRNA,
shRNA, aptamers, antisense molecules, miRNA and ribozymes, as well
as antibodies. The inhibitor may cause complete or partial
inhibition.
[0102] The term "inhibit" as used herein refers to reducing the
expression of a gene or the activity of the product of such gene to
an extent sufficient to achieve a desired biological or
physiological effect. Inhibition may be complete or partial.
[0103] As used herein, the terms "polynucleotide" and "nucleic
acid" may be used interchangeably and refer to nucleotide sequences
comprising deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).
The terms should also be understood to include, as equivalents,
analogs of either RNA or DNA made from nucleotide analogs.
Throughout this application mRNA sequences are set forth as
representing the corresponding genes.
[0104] "Oligonucleotide" or "oligomer" refers to a
deoxyribonucleotide or ribonucleotide sequence from about 2 to
about 50 nucleotides. Each DNA or RNA nucleotide may be
independently natural or synthetic, and or modified or unmodified.
Modifications include changes to the sugar moiety, the base moiety
and or the linkages between nucleotides in the oligonucleotide. The
compounds of the present invention encompass molecules comprising
deoxyribonucleotides, ribonucleotides, modified
deoxyribonucleotides, modified ribonucleotides and combinations
thereof. See below for in depth description of
oligonucleotides.
[0105] RTP801L mRNA sequence, refers to the mRNA sequence shown in
FIG. 1 (SEQ ID NO:1), or any homologous sequence thereof preferably
having at least 70% identity, more preferable 80% identity, even
more preferably 90% or 95% identity. This encompasses any sequences
derived from SEQ ID NO:1 which have undergone mutations,
alterations or modifications as described herein. Thus, in a
preferred embodiment RTP801L is encoded by a nucleic acid sequence
according to SEQ. ID. NO. 1. It is also within the present
invention that the nucleic acids according to the present invention
are only complementary and identical, respectively, to a part of
the nucleic acid coding for RTP801L as, preferably, the first
stretch and first strand is typically shorter than the nucleic acid
according to the present invention. It is also to be acknowledged
that based on the amino acid sequence of RTP801L any nucleic acid
sequence coding for such amino acid sequence can be perceived by
the one skilled in the art based on the genetic code. However, due
to the assumed mode of action of the nucleic acids according to the
present invention, it is most preferred that the nucleic acid
coding for RTP801L, preferably the mRNA thereof, is the one present
in the organism, tissue and/or cell, respectively, where the
expression of RTP801L is to be reduced.
[0106] "RTP801L polypeptide" refers to the polypeptide of the
RTP801L gene, and is understood to include, for the purposes of the
instant invention, the terms "RTP777", "DDIT4L" "REDD2", and
"SMHS1", derived from any organism, optionally man, splice variants
and fragments thereof retaining biological activity, and homologs
thereof, preferably having at least 70%, more preferably at least
80%, even more preferably at least 90% or 95% homology thereto. In
addition, this term is understood to encompass polypeptides
resulting from minor alterations in the RTP801L coding sequence,
such as, inter alia, point mutations, substitutions, deletions and
insertions which may cause a difference in a few amino acids
between the resultant polypeptide and the naturally occurring
RTP801L. Polypeptides encoded by nucleic acid sequences which bind
to the RTP801L coding sequence or genomic sequence under conditions
of highly stringent hybridization, which are well-known in the art
(for example Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in
1995 and 1998), are also encompassed by this term. Chemically
modified RTP801L or chemically modified fragments of RTP801L are
also included in the term, so long as the biological activity is
retained. RTP801L preferably has or comprises an amino acid
sequence according to SEQ. ID. NO. 2. It is acknowledged that there
might be differences in the amino acid sequence among various
tissues of an organism and among different organisms of one species
or among different species to which the nucleic acid according to
the present invention can be applied in various embodiments of the
present invention. However, based on the technical teaching
provided herein, the respective sequence can be taken into
consideration accordingly when designing any of the nucleic acids
according to the present invention.
[0107] Without being bound by theory, RTP801L may be a factor
acting in the fine-tuning of cell response to energy disbalance. As
such, it is a target suitable for treatment of any disease where
cells should be rescued from apoptosis due to stressful conditions
(e.g. diseases accompanied by death of normal cells) or where
cells, which are adapted to stressful conditions due to changes in
RTP801L expression (e.g. cancer cells), should be killed. In the
latter case, RTP801L may be viewed as a survival factor for cancer
cells and its inhibitors may treat cancer as a monotherapy or as
sensitising drugs in combination with chemotherapy or radiotherapy.
The assignee of the present invention has previously discovered
gene RTP801 (see above) and molecules effective in inhibiting gene
RTP801 (see co-assigned PCT publication No. WO06/023544A2 and PCT
Application No. PCT/US2007/01468, hereby incorporated by reference
in their entirety). Although RTP801L shares sequence and functional
homology with RTP801, the assignee of the present invention has
discovered that inhibition of RTP801 does not cause simultaneous
inhibition of RTP801L, and vice versa. Therefore, RTP801L is an
excellent target for inhibition in the conditions disclosed herein,
and its inhibition is gene-specific. Tandem therapies which inhibit
both RTP801 and RTP801L can have additional advantages and are
discussed herein blow.
[0108] The term "amino acid" refers to a molecule which consists of
any one of the 20 naturally occurring amino acids, amino acids
which have been chemically modified (see below), or synthetic amino
acids.
[0109] The term "polypeptide" refers to a molecule composed of two
or more amino acids residues. The term includes peptides,
polypeptides, proteins and peptidomimetics.
[0110] By "biological effect of RTP801L" or "RTP801L biological
activity" is meant, without being bound by theory, the effect of
RTP801L on apoptosis, such as apoptosis of alveolar cells in
respiratory disorders; apoptosis of inner ear hair cells in hearing
disorders, apoptosis of macular cells in macular degeneration,
apoptosis of cells related to ischemia in any diseases or
conditions, inter alia. The effect of RTP801L on apoptosis may be
direct or indirect, and includes, without being bound by theory,
any effect of RTP801L of induced by hypoxic or hyperoxic
conditions. The indirect effect includes, but is not limited to,
RTP801L binding to or having an effect on one of several molecules,
which are involved in a signal transduction cascade resulting in
apoptosis.
[0111] "Apoptosis" refers to a physiological type of cell death
which results from activation of some cellular mechanisms, i.e.
death that is controlled by the machinery of the cell. Apoptosis
may, for example, be the result of activation of the cell machinery
by an external trigger, e.g. a cytokine or anti-FAS antibody, which
leads to cell death or by an internal signal. The term "programmed
cell death" may also be used interchangeably with "apoptosis".
[0112] "Apoptosis-related disease" refers to a disease whose
etiology is related either wholly or partially to the process of
apoptosis. The disease may be caused either by a malfunction of the
apoptotic process (such as in cancer or an autoimmune disease) or
by overactivity of the apoptotic process (such as in certain
neurodegenerative diseases). Many diseases in which RTP801L is
involved are apoptosis-related diseases. For example, apoptosis is
a significant mechanism in dry AMD, whereby slow atrophy of
photoreceptor and pigment epithelium cells, primarily in the
central (macular) region of retina takes place. Neuroretinal
apoptosis is also a significant mechanism in diabetic
retinopathy.
[0113] An "RTP801L inhibitor" is a compound which is capable of
inhibiting the activity of the RTP801L gene or RTP801L gene
product, particularly the human RTP801L gene or gene product. Such
inhibitors include substances that affect the transcription or
translation of the gene as well as substances that affect the
activity of the gene product. An RTP801L inhibitor may also be an
inhibitor of the RTP801L promoter. Examples of such inhibitors may
include, inter alias polynucleotides such as antisense (AS)
fragments, siRNA, or vectors comprising them; catalytic RNAs such
as ribozymes. Specific siRNA RTP801L inhibitors are provided
herein.
[0114] "Expression vector" refers to a vector that has the ability
to incorporate and express heterologous DNA fragments in a foreign
cell. Many prokaryotic and eukaryotic expression vectors are known
and/or commercially available. Selection of appropriate expression
vectors is within the knowledge of those having skill in the
art.
[0115] The present invention also relates to functional nucleic
acids comprising a double-stranded structure, their use for the
manufacture of a medicament, a pharmaceutical composition
comprising such functional nucleic acids and a method for the
treatment of a patient.
[0116] Hypoxia has been recognised as a key element in the
pathomechanism of quite a number of diseases such as stroke,
emphysema and infarct which are associated with sub-optimum oxygen
availability and tissue damaging responses to the hypoxia
conditions. In fast-growing tissues, including tumor, a sub-optimum
oxygen availability is compensated by undesired neo-angiogenesis.
Therefore, at least in case of cancer diseases, the growth of
vasculature is undesired.
[0117] In view of this, the inhibition of angiogenesis and vascular
growth, respectively, is subject to intense research. Already today
some compounds are available which inhibit undesired angiogenesis
and vascular growth. Some of the more prominent compounds are those
inhibiting VEGF and the VEGF receptor. In both cases, the effect of
VEGF is avoided by either blocking VEGF as such, for example by
using an antibody directed against VEGF such as pursued by
Genentech's AVASTINT.TM. (monoclonal AB specific for VEGF) (Ferrara
N.; Endocr Rev. 2004 25(4):581-611), or by blocking the
corresponding receptor, i. e. the VEGF receptor (Traxler P; Cancer
Res. 2004 64(14):4931-41; or Stadler W M et al., Clin Cancer Res.
2004; 10(10):3365-70).
[0118] As, however, angiogenesis and the growth of vasculature is a
very basic and vital process in any animal and human being, the
effect of this kind of compound has to be focused at the particular
site where angiogenesis and vascular growth is actually undesired
which renders appropriate targeting or delivery a critical issue in
connection with this kind of therapeutic approach.
[0119] It is thus an objective of the present invention to provide
further means for the treatment of diseases involving undesired
growth of vasculature and angiogenesis, respectively.
[0120] By "small interfering RNA" (siRNA) is meant an RNA molecule
which decreases or silences (prevents) the expression of a
gene/mRNA of its endogenous cellular counterpart. The term is
understood to encompass "RNA interference" (RNAi). RNA interference
(RNAi) refers to the process of sequence-specific post
transcriptional gene silencing in mammals mediated by small
interfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806). The
corresponding process in plants is commonly referred to as specific
post transcriptional gene silencing or RNA silencing and is also
referred to as quelling in fungi. The RNA interference response may
feature an endonuclease complex containing an siRNA, commonly
referred to as an RNA-induced silencing complex (RISC), which
mediates cleavage of single-stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage
of the target RNA may take place in the middle of the region
complementary to the antisense strand of the siRNA duplex (Elbashir
et al 2001, Genes Dev., 15. 188). For recent information on these
terms and proposed mechanisms, see Bernstein E., et al., 2001
November; 7(11):1509-21; and Nishikura K.: Cell. 2001.
107(4):415-8. Examples of siRNA molecules which are used in the
present invention are given in Tables A-G.
[0121] During recent years, RNAi has emerged as one of the most
efficient methods for inactivation of genes (Nature Reviews, 2002,
v.3, p. 737-47; Nature. 2002, v.418, p. 244-51). As a method, it is
based on the ability of dsRNA species to enter a specific protein
complex, where it is then targeted to the complementary cellular
RNA and specifically degrades it. In more detail, dsRNAs are
digested into short (17-29 bp) inhibitory RNAs (siRNAs) by type III
RNAses (DICER, Drosha, etc) (Nature, 2001, v.409, p. 363-6; Nature,
2003, 0.425, p.415-9). These fragments and complementary mRNA are
recognized by the specific RISC protein complex. The whole process
is culminated by endonuclease cleavage of target mRNA (Nature
Reviews, 2002, v.3, p. 737-47; Curr Opin Mol Ther. 2003
5(3):217-24).
[0122] For delivery of siRNAs, see, for example, Shen et al FEBS
letters 539: 111-114 (2003), Xia et al., Nat Biotech 20: 1006-1010
(2002), Reich et al., Molec. Vision 9: 210-216 (2003), Sorensen et
al. J. Mol. Biol. 327: 761-766 (2003), Lewis et al., Nat Genet 32:
107-108 (2002) and Simeoni et al., NAR 31, 11: 2717-2724 (2003).
siRNA has been successfully used for inhibition in primates; for
further details see Tolentino et al., 2004 Retina
24(1):132-138.
[0123] A number of PCT applications have recently been published
that relate to the RNAi phenomenon. These include: PCT publication
WO 00/44895; PCT publication WO 00/49035; PCT publication WO
00/63364; PCT publication WO 01/36641; PCT publication WO 01/36646;
PCT publication WO 99/32619; PCT publication WO 00/44914; PCT
publication WO 01/29058; and PCT publication WO 01/75164.
[0124] RNA interference (RNAi) is based on the ability of dsRNA
species to enter a cytoplasmic protein complex, where it is then
targeted to the complementary cellular RNA and specifically degrade
it. The RNA interference response features an endonuclease complex
containing an siRNA, commonly referred to as an RNA-induced
silencing complex (RISC), which mediates cleavage of
single-stranded RNA having a sequence complementary to the
antisense strand of the siRNA duplex. Cleavage of the target RNA
may take place in the middle of the region complementary to the
antisense strand of the siRNA duplex (Elbashir et al., Genes Dev.,
2001, 15(2):188-200). In more detail, longer dsRNAs are digested
into short (17-29 bp) dsRNA fragments (also referred to as short
inhibitory RNAs, "siRNAs") by type III RNAses (DICER, DROSHA, etc.;
Bernstein et al., Nature, 2001, 409(6818):363-6; Lee et al.,
Nature, 2003, 425(6956):415-9). The RISC protein complex recognizes
these fragments and complementary mRNA. The whole process is
culminated by endonuclease cleavage of target mRNA (McManus &
Sharp, Nature Rev Genet, 2002, 3(10):737-47; Paddison & Hannon,
Curr Opin Mol Ther. 2003, 5(3):217-24). (For additional information
on these terms and proposed mechanisms, see for example Bernstein
et al. RNA 2001, 7(11):1509-21; Nishikura, Cell 2001, 107(4):415-8
and PCT publication WO 01/36646).
[0125] "Nucleotide" is meant to encompass deoxyribonucleotides and
ribonucleotides, which may be natural or synthetic, and or modified
or unmodified. Modifications include changes and substitutions to
the sugar moiety, the base moiety and/or the internucleotide
linkages.
[0126] All analogs of, or modifications to, a
nucleotide/oligonucleotide may be employed with the present
invention, provided that said analog or modification does not
substantially adversely affect the function of the
nucleotide/oligonucleotide. Acceptable modifications include
modifications of the sugar moiety, modifications of the base
moiety, modifications in the internucleotide linkages and
combinations thereof.
[0127] The nucleotides can be selected from naturally occurring or
synthetic modified bases. Naturally occurring bases include
adenine, guanine, cytosine, thymine and uracil. Modified bases of
nucleotides include inosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo
uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo
uracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, adenine,
8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted
adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine,
8-thioalkyl guanines, 8-hydroxyl guanine and other substituted
guanines, other aza and deaza adenines, other aza and deaza
guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine. Abasic
nucleotides are encompassed by the present invention, as well as
molecules comprising alternating RNA and DNA nucleotides.
[0128] In addition, analogs of polynucleotides can be prepared
wherein the structure of one or more nucleotide is fundamentally
altered and better suited as therapeutic or experimental reagents.
An example of a nucleotide analog is a peptide nucleic acid (PNA)
wherein the deoxyribose (or ribose) phosphate backbone in DNA (or
RNA is replaced with a polyamide backbone which is similar to that
found in peptides. PNA analogs have been shown to be resistant to
enzymatic degradation and to have extended lives in vivo and in
vitro. Mirror nucleotides ("L-nucleotides") may also be
employed.
[0129] Possible modifications to the sugar residue are manifold and
include 2'-O alkyl, locked nucleic acid (LNA), glycol nucleic acid
(GNA), threose nucleic acid (TNA), arabinoside, altritol (ANA) and
other 6-membered sugars including morpholinos, and
cyclohexinyls.
[0130] Examples of siRNA compounds comprising LNA nucleotides are
disclosed in Elmen et al., (NAR 2005. 33(1):439-447).
[0131] The compounds of the present invention can be synthesized
using one or more inverted nucleotides, for example inverted
thymidine or inverted adenine (for example see Takei, et al., 2002.
JBC 277(26):23800-06.)
[0132] Certain structures include siRNA compounds having one or a
plurality of 2'-5' internucleotide linkages (bridges or
backbone).
[0133] In the context of the present invention, a "mirror"
nucleotide also referred to as a Spiegelmer, is a nucleotide with
reverse chirality to the naturally occurring or commonly employed
nucleotide, i.e., a mirror image of the naturally occurring or
commonly employed nucleotide. The mirror nucleotide can be a
ribonucleotide (L-RNA) or a deoxyribonucleotide (L-DNA) and may
further comprise at least one sugar, base and or backbone
modification. U.S. Pat. No. 6,602,858 discloses nucleic acid
catalysts comprising at least one L-nucleotide substitution.
[0134] Backbone modifications, such as ethyl (resulting in a
phospho-ethyl triester); propyl (resulting in a phospho-propyl
triester); and butyl (resulting in a phospho-butyl triester) are
also possible. Other backbone modifications include polymer
backbones, cyclic backbones, acyclic backbones,
thiophosphate-D-ribose backbones, amidates, phosphonoacetates.
[0135] Other possible backbone modifications include thioate
modifications or 2'-5' bridged backbone modifications.
[0136] Additional modifications which may be present in the
molecules of the present invention include nucleoside modifications
such as artificial nucleic acids, peptide nucleic acid (PNA),
morpholino and locked nucleic acid (LNA), glycol nucleic acid
(GNA), threose nucleic acid (TNA), arabinoside, and mirror
nucleoside (for example, beta-L-deoxynucleoside instead of
beta-D-deoxynucleoside Further, said molecules may additionally
contain modifications on the sugar, such as 2' alkyl, 2' fluoro,
2'O allyl 2'amine and 2'alkoxy. Many additional sugar modifications
are discussed herein.
[0137] Further, the inhibitory nucleic acid molecules of the
present invention may comprise one or more gaps and/or one or more
nicks and/or one ore more mismatches. Without wishing to be bound
by theory, gaps, nicks and mismatches have the advantage of
partially destabilizing the nucleic acid/siRNA, so that it may be
more easily processed by endogenous cellular machinery such as
DICER, DROSHA or RISC into its inhibitory components.
[0138] In the context of the present invention, a gap in a nucleic
acid refers to the absence of one or more internal nucleotides in
one strand, while a nick in a nucleic acid refers to the absence of
a internucleotide linkage between two adjacent nucleotides in one
strand. Any of the molecules of the present invention may contain
one or more gaps and/or one or more nicks.
[0139] Further provided by the present invention is an siRNA
encoded by any of the molecules disclosed herein, a vector encoding
any of the molecules disclosed herein, and a pharmaceutical
composition comprising any of the molecules disclosed herein or the
vectors encoding them; and a pharmaceutically acceptable
carrier.
Oligonucleotides
[0140] The siRNA compounds useful in the present invention include
unmodified and chemically and/or structurally modified
compounds.
[0141] The selection and synthesis of siRNA corresponding to known
genes has been widely reported; see for example Ui-Tei et al., J
Biomed Biotechnol. 2006; 65052; Chalk et al., BBRC. 2004,
319(1):264-74; Sioud & Leirdal, Met. Mol Biol. 2004,
252:457-69; Levenkova et al., Bioinform. 2004, 20(3):430-2; Ui-Tei
et al., NAR. 2004, 32(3):936-48. For examples of the use and
production of modified siRNA see for example Braasch et al.,
Biochem. 2003, 42(26):7967-75; Chiu et al., RNA. 2003,
9(9):1034-48; PCT Publication Nos. WO 2004/015107 and WO 02/44321
and U.S. Pat. Nos. 5,898,031 and 6,107,094.
[0142] Tables A-G comprise nucleic acid sequences of sense and
corresponding antisense oligonucleotides useful in preparing
corresponding siRNA compounds.
[0143] The present invention provides double-stranded
oligonucleotides (e.g. siRNAs), which down-regulate the expression
of RTP801L. A siRNA of the invention is a duplex
oligoribonucleotide in which the sense strand is derived from the
mRNA sequence of RTP801L, and the antisense strand is complementary
to the sense strand. In general, some deviation from the target
mRNA sequence is tolerated without compromising the siRNA activity
(see e.g. Czauderna et al., 2003, NAR 31(11), 2705-2716). An siRNA
of the invention inhibits RTP801L gene expression on a
post-transcriptional level with or without destroying the mRNA.
Without being bound by theory, siRNA may target the mRNA for
specific cleavage and degradation and/or may inhibit translation
from the targeted message.
[0144] In some embodiments the oligonucleotide according to the
present invention comprises modified siRNA, having one or more of
any of the modifications disclosed herein. In various embodiments
the siRNA comprises an RNA duplex comprising a first strand and a
second strand, whereby the first strand comprises a ribonucleotide
sequence at least partially complementary to about 18 to about 40
consecutive nucleotides of a target nucleic acid which is mRNA
transcribed from RTP801L, and the second strand comprises a
ribonucleotide sequence at least partially complementary to the
first strand and wherein said first strand or said second strand
comprises a plurality of groups of modified ribonucleotides,
optionally having a modification at the 2'-position of the sugar
moiety whereby within each strand each group of modified
ribonucleotides is flanked on one or both sides by a group of
flanking nucleotides, optionally ribonucleotides, whereby each
ribonucleotide forming the group of flanking ribonucleotides is
selected from an unmodified ribonucleotide or a ribonucleotide
having a modification different from the modification of the groups
of modified ribonucleotides.
[0145] The group of modified ribonucleotides and/or the group of
flanking nucleotides may comprise a number of ribonucleotides
selected from the group consisting of an integer from 1 to 12.
Accordingly, the group thus comprises one nucleotide, two
nucleotides, three nucleotides, four nucleotides, five nucleotides,
six nucleotides, seven nucleotides, eight nucleotides, nine
nucleotides, ten nucleotides, eleven nucleotides or twelve
nucleotides.
[0146] The groups of modified nucleotides and flanking nucleotides
may be organized in a pattern on only one of the strands. In some
embodiments the sense or the antisense strand comprises a pattern
of modified nucleotides. In some preferred embodiments the middle
ribonucleotide in the antisense strand is an unmodified nucleotide.
For example, in a 19-oligomer antisense strand, ribonucleotide
number 10 is unmodified; in a 21-oligomer antisense strand,
ribonucleotide number 11 is unmodified; and in a 23-oligomer
antisense strand, ribonucleotide number 12 is unmodified. The
modifications or pattern of modification, if any, of the siRNA must
be planned to allow for this. In an even numbered oligomer e.g. a
22 mer, the middle nucleotide may be number 11 or 12.
[0147] Possible modifications on the 2' moiety of the sugar residue
include amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro,
chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl,
OCF.sub.3, OCN, O--, S--, or N-alkyl; O--, S, or N-alkenyl;
SOCH.sub.3; SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2, N.sub.3;
heterozycloalkyl; heterozycloalkaryl; aminoalkylamino;
polyalkylamino or substituted silyl, as, among others, described in
European patents EP 0 586 520 B1 or EP 0 618 925 131. One or more
deoxyribonucleotides are also tolerated in the compounds of the
present invention. As used herein, in the description of any
strategy for the design of molecules, RNAi or any embodiment of
RNAi disclosed herein, the term "end modification" means a chemical
entity added to the terminal 5' or 3' nucleotide of the sense
and/or antisense strand. Examples for such end modifications
include, but are not limited to, 3' or 5' phosphate, inverted
abasic, abasic, amino, fluoro, chloro, bromo, CN, CF.sub.3,
methoxy, imidazolyl, carboxylate, phosphorothioate, C.sub.1 to
C.sub.22 and lower alkyl, lipids, sugars and polyaminoacids (i.e.
peptides), substituted lower alkyl, alkaryl or aralkyl, OCF.sub.3,
OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH.sub.3;
SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2, N.sub.3; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted
silyl, as, among others, described in European patents EP 0 586 520
131 or EP 0 618 925 B1.
[0148] In some embodiments the siRNA is blunt ended, i.e. Z and Z'
are absent, on one or both ends. More specifically, the siRNA may
be blunt ended on the end defined by the 5'-terminus of the first
strand and the 3'-terminus of the second strand, and/or the end
defined by the 3'-terminus of the first strand and the 5'-terminus
of the second strand.
[0149] In other embodiments at least one of the two strands may
have an overhang of at least one nucleotide at the 5'-terminus; the
overhang may consist of at least one deoxyribonucleotide. At least
one of the strands may also optionally have an overhang of at least
one nucleotide at the 3'-terminus. The overhang may consist of from
about 1 to about 5 nucleotides.
[0150] The length of RNA duplex is from about 18 to about 40
ribonucleotides, preferably 19, 21 or 23 ribonucleotides. Further,
the length of each strand may independently have a length selected
from the group consisting of about 15 to about 40 bases, preferably
18 to 23 bases and more preferably 19, 21 or 23
ribonucleotides.
[0151] In certain embodiments the complementarity between said
first strand and the target nucleic acid is perfect. In some
embodiments, the strands are substantially complementary, i.e.
having one, two or up to three mismatches between said first strand
and the target nucleic acid. Substantially complementary refers to
complementarity of greater than about 84%, to another sequence. For
example in a duplex region consisting of 19 base pairs one mismatch
results in 94.7% complementarity, two mismatches results in about
89.5% complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0152] In some embodiments the first strand and the second strand
are linked by a loop structure, which is comprised of a non-nucleic
acid polymer such as, inter alia, polyethylene glycol.
Alternatively, the loop structure is comprised of a nucleic acid,
including modified and non-modified ribonucleotides and modified
and non-modified deoxyribonucleotides.
[0153] Further, the 5'-terminus of the first strand of the siRNA
may be linked to the 3'-terminus of the second strand, or the
3'-terminus of the first strand are linked to the 5'-terminus of
the second strand, said linkage being via a nucleic acid linker
typically having a length between 2-100 nucleobases, preferably
about 2 to about 30 nucleobases.
[0154] In preferred embodiments of the compounds of the invention
having alternating ribonucleotides modified in at least one of the
antisense and the sense strands of the compound, for 19 mer and 23
mer oligomers the ribonucleotides at the 5' and 3' termini of the
antisense strand are modified in their sugar residues, and the
ribonucleotides at the 5' and 3' termini of the sense strand are
unmodified in their sugar residues. For 21 mer oligomers the
ribonucleotides at the 5' and 3' termini of the sense strand are
modified in their sugar residues, and the ribonucleotides at the 5'
and 3' termini of the antisense strand are unmodified in their
sugar residues, or may have an optional additional modification at
the 3' terminus. As mentioned above, it is preferred that the
middle nucleotide of the antisense strand is unmodified.
[0155] Additionally, the invention provides siRNA comprising a
double stranded nucleic acid molecule wherein 1, 2, or 3 of the
nucleotides in one strand or both strands are substituted thereby
providing at least one base pair mismatch. The substituted
nucleotides in each strand are preferably in the terminal region of
one strand or both strands.
[0156] According to one preferred embodiment of the invention, the
antisense and the sense strands of the oligonucleotide/siRNA are
phosphorylated only at the 3'-terminus and not at the 5'-terminus.
According to another preferred embodiment of the invention, the
antisense and the sense strands are non-phosphorylated. According
to yet another preferred embodiment of the invention, the 5' most
ribonucleotide in the sense strand is modified to abolish any
possibility of in vivo 5'-phosphorylation.
[0157] Any siRNA sequence disclosed herein can be prepared having
any of the modifications/structures disclosed herein. The
combination of sequence plus structure is novel and can be used in
the treatment of the conditions disclosed herein.
[0158] Other modifications have been disclosed. The inclusion of a
5'-phosphate moiety was shown to enhance activity of siRNAs in
Drosophila embryos (Boutla, et al., Curr. Biol. 2001, 11:1776-1780)
and is required for siRNA function in human HeLa cells (Schwarz et
al., Mol. Cell, 2002, 10:537-48). Amarzguioui et al., (NAR, 2003,
31(2):589-95) showed that siRNA activity depended on the
positioning of the 2'-O-methyl modifications. Holen et al (NAR.
2003, 31(9):2401-07) report that an siRNA having small numbers of
2'-O-methyl modified nucleosides gave good activity compared to
wild type but that the activity decreased as the numbers of
2'-O-methyl modified nucleosides was increased. Chiu and Rana (RNA,
2003, 9:1034-48) teach that incorporation of 2'-O-methyl modified
nucleosides in the sense or antisense strand (fully modified
strands) severely reduced siRNA activity relative to unmodified
siRNA. The placement of a 2'-O-methyl group at the 5'-terminus on
the antisense strand was reported to severely limit activity
whereas placement at the 3'-terminus of the antisense and at both
termini of the sense strand was tolerated (Czauderna et al., NAR.
2003, 31(11):2705-16). The molecules of the present invention offer
an advantage in that they are active and or stable, are non-toxic
and may be formulated as pharmaceutical compositions for treatment
of various diseases.
[0159] In addition, analogues of polynucleotides can be prepared
wherein the structure of one or more nucleotide is fundamentally
altered and better suited as therapeutic or experimental
reagents.
[0160] Possible modifications to the sugar residue are manifold and
include 2'-O alkyl, locked nucleic acid (LNA), glycol nucleic acid
(GNA), threose nucleic acid (TNA), arabinoside, altritol (ANA) and
other, 6-membered sugars including morpholinos, and
cyclohexinyls.
[0161] LNA compounds are disclosed in International Patent
Publication Nos. WO 00/47599, WO 99/14226, and WO 98/39352.
Examples of siRNA compounds comprising LNA nucleotides are
disclosed in Elmen et al., (NAR 2005. 33(1):439-447) and in PCT
Patent Publication No. WO 2004/083430.
[0162] The compounds of the present invention can be synthesized
using one or more inverted nucleotides, for example inverted
thymidine or inverted adenine (for example see Takei, et al., 2002.
JBC 277(26):23800-06.
[0163] Backbone modifications, such as ethyl (resulting in a
phospho-ethyl triester); propyl (resulting in a phospho-propyl
triester); and butyl (resulting in a phospho-butyl triester) are
also possible. Other backbone modifications include polymer
backbones, cyclic backbones, acyclic backbones,
thiophosphate-D-ribose backbones, amidates, phosphonoacetate
derivatives. Certain structures include siRNA compounds having one
or a plurality of 2'-5' internucleotide linkages (bridges or
backbone).
[0164] The term "unconventional moiety" as used herein refers to
abasic ribose moiety, an abasic deoxyribose moiety, a
deoxyribonucleotide, a modified deoxyribonucleotide, a mirror
nucleotide, a non-base pairing nucleotide analog and a nucleotide
joined to an adjacent nucleotide by a 2'-5' internucleotide
phosphate bond; bridged nucleic acids including LNA and ethylene
bridged nucleic acids.
[0165] The term "capping moiety" as used herein includes abasic
ribose moiety, abasic deoxyribose moiety, modifications abasic
ribose and abasic deoxyribose moieties including 2' O alkyl
modifications; inverted abasic ribose and abasic deoxyribose
moieties and modifications thereof; C6-imino-Pi; a mirror
nucleotide including L-DNA and L-RNA; 5'OMe nucleotide; and
nucleotide analogs including 4',5'-methylene nucleotide;
1-(.beta.-D-erythrofuranosyl)nucleotide; 4'-thio nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted abasic moiety; 1,4-butanediol phosphate; 5'-amino;
and bridging or non bridging methylphosphonate and 5'-mercapto
moieties.
[0166] Abasic deoxyribose moiety includes for example abasic
deoxyribose-3'-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;
1,4-anhydro-2-deoxy-D-ribitol-3-phosphate, Inverted abasic
deoxyribose moiety includes inverted deoxyriboabasic; 3%5' inverted
deoxyriboabasic 5'-phosphate.
[0167] Mirror nucleotide includes for example L-DNA
(L-deoxyriboadenosine-3'-phosphate (mirror dA);
L-deoxyribocytidine-3'-phosphate (mirror dC);
L-deoxyriboguanosine-3'-phosphate (mirror dG);
L-deoxyribothymidine-3'-phosphate (mirror image dT)) and L-RNA
(L-riboadenosine-3'-phosphate (mirror rA);
L-ribocytidine-3'-phosphate (mirror rC);
L-riboguanosine-3'-phosphate (mirror rG); L-ribouracil-3'-phosphate
(mirror dU).
[0168] Further, the inhibitory nucleic acid molecules of the
present invention may comprise one or more gaps and/or one or more
nicks and/or one ore more mismatches. Without wishing to be bound
by theory, gaps, nicks and mismatches have the advantage of
partially destabilizing the nucleic acid/siRNA, so that it may be
more easily processed by endogenous cellular machinery such as
DICER, DROSHA or RISC into its inhibitory components.
[0169] The molecules of the present invention may comprise siRNAs,
synthetic siRNAs, shRNAs and synthetic shRNAs, in addition to other
nucleic acid sequences or molecules which encode such molecules or
other inhibitory nucleotide molecules.
[0170] The compounds of the present invention may further comprise
an end modification. A biotin group may be attached to either the
most 5' or the most 3' nucleotide of the first and/or second strand
or to both ends. In a more preferred embodiment the biotin group is
coupled to a polypeptide or a protein. It is also within the scope
of the present invention that the polypeptide or protein is
attached through any of the other aforementioned modifications.
[0171] The various end modifications as disclosed herein are
preferably located at the ribose moiety of a nucleotide of the
nucleic acid according to the present invention. More particularly,
the end modification may be attached to or replace any of the
OH-groups of the ribose moiety, including but not limited to the
2'OH, 3'OH and 5'OH position, provided that the nucleotide thus
modified is a terminal nucleotide. Inverted abasic or abasic are
nucleotides, either deoxyribonucleotides or ribonucleotides which
do not have a nucleobase moiety. This kind of compound is, inter
alia, described in Sternberger, et al., (Antisense Nucleic Acid
Drug Dev, 2002.12, 131-43).
[0172] In the context of the present invention, a gap in a nucleic
acid refers to the absence of one or more internal nucleotides in
one strand, while a nick in a nucleic acid refers to the absence of
an internucleotide linkage between two adjacent nucleotides in one
strand. Any of the molecules of the present invention may contain
one or more gaps and/or one or more nicks. Further provided by the
present invention is an siRNA encoded by any of the molecules
disclosed herein, a vector encoding any of the molecules disclosed
herein, and a pharmaceutical composition comprising any of the
molecules disclosed herein or the vectors encoding them; and a
pharmaceutically acceptable carrier.
[0173] Particular molecules to be administered according to the
methods of the present invention are disclosed below under the
heading "structural motifs". For the sake of clarity, any of these
molecules can be administered according to any of the methods of
the present invention.
Structural Motifs
[0174] According to the present invention the siRNA compounds are
chemically and or structurally modified according to one of the
following modifications set forth in Structures (A)-(P) or as
tandem siRNA or RNAstar.
[0175] In one aspect the present invention provides a compound set
forth as Structure (A):
TABLE-US-00004 (A) 5' (N).sub.x-Z 3' (antisense strand) 3'
Z'-(N').sub.y 5' (sense strand)
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of (N).sub.x and (N').sub.y is an oligonucleotide in which
each consecutive N or N' is joined to the next N or N' by a
covalent bond; wherein each of x and y is an integer between 18 and
40; wherein each of Z and Z' may be present or absent, but if
present is 1-5 consecutive nucleotides covalently attached at the
3' terminus of the strand in which it is present; and and wherein
the sequence of (N).sub.x comprises an antisense sequence
substantially complementary to about 18 to about 40 consecutive
ribonucleotides in an mRNA transcribed from the RTP801L gene.
[0176] In certain embodiments the present invention provides a
compound having structure B (structures having alternating
2'-O-methyl modification in both strands):
TABLE-US-00005 (B) antisense strand 5' (N)x 3' sense strand 3'
(N')y 5'
wherein each of (N).sub.x and (N').sub.y is an oligomer in which
each consecutive N or N' is an unmodified ribonucleotide or a
modified ribonucleotide joined to the next N or N' by a covalent
bond; wherein each of x and y=19, 21 or 23 and (N).sub.x and
(N').sub.y, are fully complementary wherein alternating
ribonucleotides in each of (N).sub.x and (N').sub.y, are modified
to result in a 2'-O-methyl modification in the sugar residue of the
ribonucleotides; wherein the sequence of (N').sub.y is a sequence
complementary to (N)x; and wherein the sequence of (N).sub.x,
comprises an antisense sequence substantially complementary to
about 18 to about 40 consecutive ribonucleotides in an mRNA
transcribed from the RTP801L gene.
[0177] In some embodiments each of (N).sub.x, and (N').sub.y, is
independently phosphorylated or non-phosphorylated at the 3' and 5'
termini.
[0178] In certain embodiments of the invention, alternating
ribonucleotides are modified in both the antisense and the sense
strands of the compound.
[0179] In certain embodiments wherein each of x and y=19 or 23,
each N at the 5' and 3' termini of (N).sub.x, is modified; and
each N' at the 5' and 3' termini of (N').sub.y, is unmodified.
[0180] In certain embodiments wherein each of x and y=21, each N at
the 5' and 3' termini of (N).sub.x is unmodified; and
each N' at the 5' and 3' termini of (N').sub.y is modified.
[0181] In particular embodiments, when x and y=19, the siRNA is
modified such that a 2'-O-methyl (2'-OMe) group is present on the
first, third, fifth, seventh, ninth, eleventh, thirteenth,
fifteenth, seventeenth and nineteenth nucleotide of the antisense
strand (N).sub.x, and whereby the very same modification, i. e. a
2'-OMe group, is present at the second, fourth, sixth, eighth,
tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of
the sense strand (N').sub.y. In various embodiments these
particular siRNA compounds are blunt ended at both termini.
[0182] In some embodiments, the present invention provides a
compound having Structure (C):
TABLE-US-00006 (C) antisense strand 5' (N)x -Z 3' sense strand 3'
Z' -(N')y 5'
wherein each of N and N' is a nucleotide independently selected
from an unmodified ribonucleotide, a modified ribonucleotide, an
unmodified deoxyribonucleotide and a modified deoxyribonucleotide;
wherein each of (N)x and (N')y is an oligomer in which each
consecutive nucleotide is joined to the next nucleotide by a
covalent bond and each of x and y is an integer between 18 and 40;
wherein in (N)x the nucleotides are unmodified or (N)x comprises
alternating modified ribonucleotides and unmodified
ribonucleotides; each modified ribonucleotide being modified so as
to have a 2'-O-methyl on its sugar and the ribonucleotide located
at the middle position of (N)x being modified or unmodified
preferably unmodified; wherein (N')y comprises unmodified
ribonucleotides further comprising one modified nucleotide at a
terminal or penultimate position, wherein the modified nucleotide
is selected from the group consisting of a mirror nucleotide, a
bicyclic nucleotide, a 2'-sugar modified nucleotide, an altritol
nucleotide, or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage; wherein if more than one
nucleotide is modified in (N')y, the modified nucleotides may be
consecutive; wherein each of Z and Z' may be present or absent, but
if present is 1-5 deoxyribonucleotides covalently attached at the
3' terminus of any oligomer to which it is attached; wherein the
sequence of (N').sub.y comprises a sequence substantially
complementary to (N)x; and wherein the sequence of (N).sub.x
comprises an antisense sequence substantially complementary to
about 18 to about 40 consecutive ribonucleotides in an mRNA
transcribed from the RTP801L gene.
[0183] In particular embodiments, x=y=19 and in (N)x each modified
ribonucleotide is modified so as to have a 2'-O-methyl on its sugar
and the ribonucleotide located at the middle of (N)x is unmodified.
Accordingly, in a compound wherein x=19, (N)x comprises 2'-O-methyl
sugar modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13,
15, 17 and 19. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and
may further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 5. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 2, 4,
8, 11, 13, 15, 17 and 19 and may further comprise at least one
abasic or inverted abasic pseudo-nucleotide for example in position
6. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 2, 4, 6, 8, 11, 13, 17 and 19 and may
further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 15. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 2, 4,
6, 8, 11, 13, 15, 17 and 19 and may further comprise at least one
abasic or inverted abasic pseudo-nucleotide for example in position
14. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 1, 2, 3, 7, 9, 11, 13, 15, 17 and 19
and may further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 5. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 1, 2,
3, 5, 7, 9, 11, 13, 15, 17 and 19 and may further comprise at least
one abasic or inverted abasic pseudo-nucleotide for example in
position 6. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 1, 2, 3, 5, 7, 9, 11, 13, 17 and 19
and may further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 15. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 1, 2,
3, 5, 7, 9, 11, 13, 15, 17 and 19 and may further comprise at least
one abasic or inverted abasic pseudo-nucleotide for example in
position 14. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 2, 4, 6, 7, 9, 11, 13, 15, 17 and 19
and may further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 5. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 1, 2,
4, 6, 7, 9, 11, 13, 15, 17 and 19 and may further comprise at least
one abasic or inverted abasic pseudo-nucleotide for example in
position 5. In other embodiments, (N)x comprises 2'O Me modified
ribonucleotides at positions 2, 4, 6, 8, 11, 13, 14, 16, 17 and 19
and may further comprise at least one abasic or inverted abasic
pseudo-nucleotide for example in position 15. In other embodiments,
(N)x comprises 2'O Me modified ribonucleotides at positions 1, 2,
3, 5, 7, 9, 11, 13, 14, 16, 17 and 19 and may further comprise at
least one abasic or inverted abasic pseudo-nucleotide for example
in position 15. In other embodiments, (N)x comprises 2'O Me
modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17
and 19 and may further comprise at least one abasic or inverted
abasic pseudo-nucleotide for example in position 7. In other
embodiments, (N)x comprises 2'O Me modified ribonucleotides at
positions 2, 4, 6, 11, 13, 15, 17 and 19 and may further comprise
at least one abasic or inverted abasic pseudo-nucleotide for
example in position 8. In other embodiments, (N)x comprises 2'O Me
modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17
and 19 and may further comprise at least one abasic or inverted
abasic pseudo-nucleotide for example in position 9. In other
embodiments, (N)x comprises 2'O Me modified ribonucleotides at
positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further
comprise at least one abasic or inverted abasic pseudo-nucleotide
for example in position 10. In other embodiments, (N)x comprises
2'O Me modified ribonucleotides at positions 2, 4, 6, 8, 13, 15, 17
and 19 and may further comprise at least one abasic or inverted
abasic pseudo-nucleotide for example in position 11. In other
embodiments, (N)x comprises 2'O Me modified ribonucleotides at
positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further
comprise at least one abasic or inverted abasic pseudo-nucleotide
for example in position 12. In other embodiments, (N)x comprises
2'O Me modified ribonucleotides at positions 2, 4, 6, 8, 11, 15, 17
and 19 and may further comprise at least one abasic or inverted
abasic pseudo-nucleotide for example in position 13.
[0184] In yet other embodiments (N)x comprises at least one
nucleotide mismatch relative to the RTP801L gene. In certain
preferred embodiments, (N)x comprises a single nucleotide mismatch
on position 5, 6, or 14. In one embodiment of Structure (C), at
least two nucleotides at either or both the 5' and 3' termini of
(N')y are joined by a 2'-5' phosphodiester bond. In certain
preferred embodiments x=y=19 or x=y=23; in (N)x the nucleotides
alternate between modified ribonucleotides and unmodified
ribonucleotides, each modified ribonucleotide being modified so as
to have a 2'-O-methyl on its sugar and the ribonucleotide located
at the middle of (N)x being unmodified; and three nucleotides at
the 3' terminus of (N')y are joined by two 2'-S' phosphodiester
bonds (set forth herein as Structure I). In other preferred
embodiments, x=y=19 or x=y=23; in (N)x the nucleotides alternate
between modified ribonucleotides and unmodified ribonucleotides,
each modified ribonucleotide being modified so as to have a
2'-O-methyl on its sugar and the ribonucleotide located at the
middle of (N)x being unmodified; and four consecutive nucleotides
at the 5' terminus of (N')y are joined by three 2'-5'
phosphodiester bonds. In a further embodiment, an additional
nucleotide located in the middle position of (N)y may be modified
with 2'-O-methyl on its sugar. In another preferred embodiment, in
(N)x the nucleotides alternate between 2'-O-methyl modified
ribonucleotides and unmodified ribonucleotides, and in (N')y four
consecutive nucleotides at the 5' terminus are joined by three
2'-5' phosphodiester bonds and the 5' terminal nucleotide or two or
three consecutive nucleotides at the 5' terminus comprise
3'-O-methyl modifications.
[0185] In certain preferred embodiments of Structure C, x=y=19 and
in (N')y, at least one position comprises an abasic or inverted
abasic pseudo-nucleotide, preferably five positions comprises an
abasic or inverted abasic pseudo-nucleotides. In various
embodiments, the following positions comprise an abasic or inverted
abasic: positions 1 and 16-19, positions 15-19, positions 1-2 and
17-19, positions 1-3 and 18-19, positions 1-4 and 19 and positions
1-5. (N')y may further comprise at least one LNA nucleotide.
[0186] In certain preferred embodiments of Structure C, x=y=19 and
in (N')y the nucleotide in at least one position comprises a mirror
nucleotide, a deoxyribonucleotide and a nucleotide joined to an
adjacent nucleotide by a 2'-5' internucleotide bond.
[0187] In certain preferred embodiments of Structure C, x=y=19 and
(N')y comprises a mirror nucleotide. In various embodiments the
mirror nucleotide is an L-DNA nucleotide. In certain embodiments
the L-DNA is L-deoxyribocytidine. In some embodiments (N')y
comprises L-DNA at position 18. In other embodiments (N')y
comprises L-DNA at positions 17 and 18. In certain embodiments
(N')y comprises L-DNA substitutions at positions 2 and at one or
both of positions 17 and 18. In certain embodiments (N')y further
comprises a 5' terminal cap nucleotide such as 5'-O-methyl DNA or
an abasic or inverted abasic pseudo-nucleotide as an overhang.
[0188] In yet other embodiments (N')y comprises at least one
nucleotide mismatch relative to the RTP801L gene. In certain
preferred embodiments, (N')y comprises a single nucleotide mismatch
on position 6, 14, or 15.
[0189] In yet other embodiments (N')y comprises a DNA at position
15 and L-DNA at one or both of positions 17 and 18. In that
structure, position 2 may further comprise an L-DNA or an abasic
pseudo-nucleotide.
[0190] Other embodiments of Structure C are envisaged wherein
x=y=21 or wherein x=y=23; in these embodiments the modifications
for (N')y discussed above instead of being on positions 15, 16, 17,
18 are on positions 17, 18, 19, 20 for 21 mer and on positions 19,
20, 21, 22 for 23 mer; similarly the modifications at one or both
of positions 17 and 18 are on one or both of positions 19 or 20 for
the 21 mer and one or both of positions 21 and 22 for the 23 mer.
All modifications in the 19 mer are similarly adjusted for the 21
and 23 mers.
[0191] According to various embodiments of Structure (C), in (N')y
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
ribonucleotides at the 3 terminus are linked by 2'-5'
internucleotide linkages In one preferred embodiment, four
consecutive nucleotides at the 3' terminus of (N')y are joined by
three 2'-5' phosphodiester bonds, wherein one or more of the 2'-5'
nucleotides which form the 2'-5' phosphodiester bonds further
comprises a 3'-O-methyl sugar modification. Preferably the 3'
terminal nucleotide of (N')y comprises a 2'-O-methyl sugar
modification. In certain preferred embodiments of Structure C,
x=y=19 and in (N')y two or more consecutive nucleotides at
positions 15, 16, 17, 18 and 19 comprise a nucleotide joined to an
adjacent nucleotide by a 2'-5' internucleotide bond. In various
embodiments the nucleotide forming the 2'-5' internucleotide bond
comprises a 3' deoxyribose nucleotide or a 3' methoxy nucleotide.
In some embodiments the nucleotides at positions 17 and 18 in (N')y
are joined by a 2'-5' internucleotide bond. In other embodiments
the nucleotides at positions 16, 17, 18, 16-17, 17-18, or 16-18 in
(N')y are joined by a 2'-5' internucleotide bond.
[0192] In certain embodiments (N')y comprises an L-DNA at position
2 and 2'-5' internucleotide bonds at positions 16, 17, 18, 16-17,
17-18, or 16-18. In certain embodiments (N')y comprises 2'-5'
internucleotide bonds at positions 16, 17, 18, 16-17, 17-18, or
16-18 and a 5' terminal cap nucleotide.
[0193] According to various embodiments of Structure (C), in (N')y
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
nucleotides at either terminus or 2-8 modified nucleotides at each
of the 5' and 3' termini are independently mirror nucleotides. In
some embodiments the mirror nucleotide is an L-ribonucleotide. In
other embodiments the mirror nucleotide is an
L-deoxyribonucleotide. The mirror nucleotide may further be
modified at the sugar or base moiety or in an internucleotide
linkage.
[0194] In one preferred embodiment of Structure (C), the 3'
terminal nucleotide or two or three consecutive nucleotides at the
3' terminus of (N')y are L-deoxyribonucleotides.
[0195] In other embodiments of Structure (C), in (N')y 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at
either terminus or 2-8 modified nucleotides at each of the 5' and
3' termini are independently 2' sugar modified nucleotides. In some
embodiments the 2' sugar modification comprises the presence of an
amino, a fluoro, an alkoxy or an alkyl moiety. In certain
embodiments the 2' sugar modification comprises a methoxy moiety
(2'-OMe). In one series of preferred embodiments, three, four or
five consecutive nucleotides at the 5' terminus of (N')y comprise
the 2'-OMe modification. In another preferred embodiment, three
consecutive nucleotides at the 3' terminus of (N')y comprise the
2'-O-methyl modification.
[0196] In some embodiments of Structure (C), in (N')y 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at
either or 2-8 modified nucleotides at each of the 5' and 3' termini
are independently bicyclic nucleotide. In various embodiments the
bicyclic nucleotide is a locked nucleic acid (LNA). A 2'-O,
4'-C-ethylene-bridged nucleic acid (ENA) is a species of LNA (see
below).
[0197] In various embodiments (N')y comprises modified nucleotides
at the 5' terminus or at both the 3' and 5' termini.
[0198] In some embodiments of Structure (C), at least two
nucleotides at either or both the 5' and 3' termini of (N')y are
joined by P-ethoxy backbone modifications. In certain preferred
embodiments x=y=19 or x=y=23; in (N)x the nucleotides alternate
between modified ribonucleotides and unmodified ribonucleotides,
each modified ribonucleotide being modified so as to have a
2'-O-methyl on its sugar and the ribonucleotide located at the
middle position of (N)x being unmodified; and four consecutive
nucleotides at the 3' terminus or at the 5' terminus of (N')y are
joined by three P-ethoxy backbone modifications. In another
preferred embodiment, three consecutive nucleotides at the 3'
terminus or at the 5' terminus of (N')y are joined by two P-ethoxy
backbone modifications.
[0199] In some embodiments of Structure (C), in (N')y 2, 3, 4, 5,
6, 7 or 8, consecutive ribonucleotides at each of the 5' and 3'
termini are independently mirror nucleotides, nucleotides joined by
2'-5' phosphodiester bond, 2' sugar modified nucleotides or
bicyclic nucleotide. In one embodiment, the modification at the 5'
and 3' termini of (N')y is identical. In one preferred embodiment,
four consecutive nucleotides at the 5' terminus of (N')y are joined
by three 2'-5' phosphodiester bonds and three consecutive
nucleotides at the 3' terminus of (N')y are joined by two 2'-5'
phosphodiester bonds. In another embodiment, the modification at
the 5' terminus of (N')y is different from the modification at the
3' terminus of (N')y. In one specific embodiment, the modified
nucleotides at the 5' terminus of (N')y are mirror nucleotides and
the modified nucleotides at the 3' terminus of (N')y are joined by
2'-5' phosphodiester bond. In another specific embodiment, three
consecutive nucleotides at the 5' terminus of (N')y are LNA
nucleotides and three consecutive nucleotides at the 3' terminus of
(N')y are joined by two 2'-5' phosphodiester bonds. In (N)x the
nucleotides alternate between modified ribonucleotides and
unmodified ribonucleotides, each modified ribonucleotide being
modified so as to have a 2'-O-methyl on its sugar and the
ribonucleotide located at the middle of (N)x being unmodified, or
the ribonucleotides in (N)x being unmodified
[0200] In another embodiment of Structure (C), the present
invention provides a compound wherein x=y=19 or x=y=23; in (N)x the
nucleotides alternate between modified ribonucleotides and
unmodified ribonucleotides, each modified ribonucleotide being
modified so as to have a 2'-O-methyl on its sugar and the
ribonucleotide located at the middle of (N)x being unmodified;
three nucleotides at the 3' terminus of (N')y are joined by two
2'-5' phosphodiester bonds and three nucleotides at the 5' terminus
of (N')y are LNA such as ENA.
[0201] In another embodiment of Structure (C), five consecutive
nucleotides at the 5' terminus of (N')y comprise the 2'-O-methyl
sugar modification and two consecutive nucleotides at the 3'
terminus of (N')y are L-DNA.
[0202] In yet another embodiment, the present invention provides a
compound wherein x=y=19 or x=y=23; (N)x consists of unmodified
ribonucleotides; three consecutive nucleotides at the 3' terminus
of (N')y are joined by two 2'-5' phosphodiester bonds and three
consecutive nucleotides at the 5' terminus of (N')y are LNA such as
ENA.
[0203] According to other embodiments of Structure (C), in (N')y
the 5' or 3' terminal nucleotide, or 2, 3, 4, 5 or 6 consecutive
nucleotides at either termini or 1-4 modified nucleotides at each
of the 5' and 3' termini are independently phosphonocarboxylate or
phosphinocarboxylate nucleotides (PACE nucleotides). In some
embodiments the PACE nucleotides are deoxyribonucleotides. In some
preferred embodiments in (N')y, 1 or 2 consecutive nucleotides at
each of the 5' and 3' termini are PACE nucleotides. Examples of
PACE nucleotides and analogs are disclosed in U.S. Pat. Nos.
6,693,187 and 7,067,641 both incorporated by reference.
[0204] In additional embodiments, the present invention provides a
compound having Structure (D):
TABLE-US-00007 (D) antisense strand 5' (N)x -Z 3' sense strand 3'
Z'-(N')y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide or a modified deoxyribonucleotide; wherein each
of (N)x and (N')y is an oligomer in which each consecutive
nucleotide is joined to the next nucleotide by a covalent bond and
each of x and y is an integer between 18 and 40; wherein (N)x
comprises unmodified ribonucleotides further comprising one
modified nucleotide at the 3' terminal or penultimate position,
wherein the modified nucleotide is selected from the group
consisting of a bicyclic nucleotide, a 2' sugar modified
nucleotide, a mirror nucleotide, an altritol nucleotide, or a
nucleotide joined to an adjacent nucleotide by an internucleotide
linkage selected from a 2'-5' phosphodiester bond, a P-alkoxy
linkage or a PACE linkage; wherein (N')y comprises unmodified
ribonucleotides further comprising one modified nucleotide at the
5' terminal or penultimate position, wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, an
altritol nucleotide, or a nucleotide joined to an adjacent
nucleotide by an internucleotide linkage selected from a 2'-5'
phosphodiester bond, a P-alkoxy linkage or a PACE linkage; wherein
in each of (N)x and (N')y modified and unmodified nucleotides are
not alternating; wherein each of Z and Z' may be present or absent,
but if present is 1-5 deoxyribonucleotides covalently attached at
the 3' terminus of any oligomer to which it is attached;
[0205] wherein the sequence of (N').sub.y, is a sequence
substantially complementary to (N)x; and wherein the sequence of
(N).sub.x comprises an antisense sequence having substantial
identity to about 18 to about 40 consecutive ribonucleotides in an
inRNA transcribed from the RTP801L gene.
[0206] In one embodiment of Structure (D), x=y=19 or x=y=23; (N)x
comprises unmodified ribonucleotides in which two consecutive
nucleotides linked by one 2'-5' internucleotide linkage at the 3'
terminus; and (N')y comprises unmodified ribonucleotides in which
two consecutive nucleotides linked by one 2'-5' internucleotide
linkage at the 5' terminus.
[0207] In some embodiments, x=y=19 or x=y=23; (N)x comprises
unmodified ribonucleotides in which three consecutive nucleotides
at the 3' terminus are joined together by two 2'-5' phosphodiester
bonds; and (N')y comprises unmodified ribonucleotides in which four
consecutive nucleotides at the 5' terminus are joined together by
three 2'-5' phosphodiester bonds (set forth herein as Structure
II).
[0208] According to various embodiments of Structure (D) 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
starting at the ultimate or penultimate position of the 3' terminus
of (N)x and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
consecutive ribonucleotides starting at the ultimate or penultimate
position of the 5' terminus of (N')y are linked by 2'-5'
internucleotide linkages.
[0209] According to one preferred embodiment of Structure (D), four
consecutive nucleotides at the 5' terminus of (N')y are joined by
three 2'-5' phosphodiester bonds and three consecutive nucleotides
at the 3' terminus of (N')x are joined by two 2'-5' phosphodiester
bonds. Three nucleotides at the 5' terminus of (N')y and two
nucleotides at the 3' terminus of (N')x may also comprise
3'-O-methyl modifications.
[0210] According to various embodiments of Structure (D), 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides
starting at the ultimate or penultimate position of the 3' terminus
of (N)x and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
consecutive ribonucleotides starting at the ultimate or penultimate
position of the 5' terminus of (N')y are independently mirror
nucleotides. In some embodiments the mirror is an
L-ribonucleotide.
[0211] In other embodiments the mirror nucleotide is
L-deoxyribonucleotide.
[0212] In other embodiments of Structure (D), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at the
ultimate or penultimate position of the 3' terminus of (N)x and 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
ribonucleotides starting at the ultimate or penultimate position of
the 5' terminus of (N')y are independently 2' sugar modified
nucleotides. In some embodiments the 2' sugar modification
comprises the presence of an amino, a fluoro, an alkoxy or an alkyl
moiety. In certain embodiments the 2' sugar modification comprises
a methoxy moiety (2'-OMe).
[0213] In one preferred embodiment of Structure (D), five
consecutive nucleotides at the 5' terminus of (N')y comprise the
2'-O-methyl modification and five consecutive nucleotides at the 3'
terminus of (N')x comprise the 2'-O-methyl modification. In another
preferred embodiment of Structure (D), ten consecutive nucleotides
at the 5' terminus of (N')y comprise the 2'-O-methyl modification
and five consecutive nucleotides at the 3' terminus of (N')x
comprise the 2'-O-methyl modification. In another preferred
embodiment of Structure (D), thirteen consecutive nucleotides at
the 5' terminus of (N')y comprise the 2'-O-methyl modification and
five consecutive nucleotides at the 3' terminus of (N')x comprise
the 2'-O-methyl modification.
[0214] In some embodiments of Structure (D), in (N')y 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
starting at the ultimate or penultimate position of the 3' terminus
of (N)x and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
consecutive ribonucleotides starting at the ultimate or penultimate
position of the 5' terminus of (N')y are independently a bicyclic
nucleotide. In various embodiments the bicyclic nucleotide is a
locked nucleic acid (LNA) such as a 2-O, 4'-C-ethylene-bridged
nucleic acid (ENA).
[0215] In various embodiments of Structure (D), (N')y comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide or
a nucleotide joined to an adjacent nucleotide by an internucleotide
linkage selected from a 2'-5' phosphodiester bond, a P-alkoxy
linkage or a PACE linkage;
[0216] In various embodiments of Structure (D), (N)x comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide or
a nucleotide joined to an adjacent nucleotide by an internucleotide
linkage selected from a 2'-5' phosphodiester bond, a P-alkoxy
linkage or a PACE linkage;
[0217] In embodiments wherein each of the 3' and 5' termini of the
same strand comprises a modified nucleotide, the modification at
the 5' and 3' termini is identical. In another embodiment, the
modification at the 5' terminus is different from the modification
at the 3' terminus of the same strand. In one specific embodiment,
the modified nucleotides at the 5' terminus are mirror nucleotides
and the modified nucleotides at the 3' terminus of the same strand
are joined by 2'-5' phosphodiester bond.
[0218] In one specific embodiment of Structure (D), five
consecutive nucleotides at the 5' terminus of (N')y comprise the
2'-O-methyl modification and two consecutive nucleotides at the 3'
terminus of (N')y are L-DNA. In addition, the compound may further
comprise five consecutive 2'-O-methyl modified nucleotides at the
3' terminus of (N')x.
[0219] In various embodiments of Structure (D), the modified
nucleotides in (N)x are different from the modified nucleotides in
(N')y. For example, the modified nucleotides in (N)x are 2' sugar
modified nucleotides and the modified nucleotides in (N')y are
nucleotides linked by 2'-5' internucleotide linkages. In another
example, the modified nucleotides in (N)x are mirror nucleotides
and the modified nucleotides in (N')y are nucleotides linked by
2'-5' internucleotide linkages. In another example, the modified
nucleotides in (N)x are nucleotides linked by 2'-5' internucleotide
linkages and the modified nucleotides in (N')y are mirror
nucleotides.
[0220] In additional embodiments, the present invention provides a
compound having Structure (E):
TABLE-US-00008 (E) antisense strand 5' (N)x -Z 3' sense strand 3'
Z'-(N')y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide or a modified deoxyribonucleotide; wherein each
of (N)x and (N')y is an oligomer in which each consecutive
nucleotide is joined to the next nucleotide by a covalent bond and
each of x and y is an integer between 18 and 40; wherein (N)x
comprises unmodified ribonucleotides further comprising one
modified nucleotide at the 5' terminal or penultimate position,
wherein the modified nucleotide is selected from the group
consisting of a bicyclic nucleotide, a 2' sugar modified
nucleotide, a mirror nucleotide, an altritol nucleotide, or a
nucleotide joined to an adjacent nucleotide by an internucleotide
linkage selected from a 2'-5' phosphodiester bond, a P-alkoxy
linkage or a PACE linkage; wherein (N')y comprises unmodified
ribonucleotides further comprising one modified nucleotide at the
3' terminal or penultimate position, wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, an
altritol nucleotide, or a nucleotide joined to an adjacent
nucleotide by an internucleotide linkage selected from a 2'-5'
phosphodiester bond, a P-alkoxy linkage or a PACE linkage; wherein
in each of (N)x and (N')y modified and unmodified nucleotides are
not alternating; wherein each of Z and Z' may be present or absent,
but if present is 1-5 deoxyribonucleotides covalently attached at
the 3' terminus of any oligomer to which it is attached; wherein
the sequence of (N').sub.y is a sequence substantially
complementary to (N)x; and wherein the sequence of (N).sub.x
comprises an antisense sequence having substantial identity to
about 18 to about 40 consecutive ribonucleotides in an mRNA
transcribed from the RTP801L gene.
[0221] In certain preferred embodiments the ultimate nucleotide at
the 5' terminus of (N)x is unmodified.
[0222] According to various embodiments of Structure (E) 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
starting at the ultimate or penultimate position of the 5' terminus
of (N)x, preferably starting at the 5' penultimate position, and 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
ribonucleotides starting at the ultimate or penultimate position of
the 3' terminus of (N')y are linked by 2'-5' internucleotide
linkages.
[0223] According to various embodiments of Structure (E), 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides
starting at the ultimate or penultimate position of the 5' terminus
of (N)x, preferably starting at the 5' penultimate position, and 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides
starting at the ultimate or penultimate position of the 3' terminus
of (N')y are independently mirror nucleotides. In some embodiments
the mirror is an L-ribonucleotide. In other embodiments the mirror
nucleotide is L-deoxyribonucleotide.
[0224] In other embodiments of Structure (E), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at the
ultimate or penultimate position of the 5' terminus of (N)x,
preferably starting at the 5' penultimate position, and 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
starting at the ultimate or penultimate position of the 3' terminus
of (N')y are independently 2' sugar modified nucleotides. In some
embodiments the 2' sugar modification comprises the presence of an
amino, a fluoro, an alkoxy or an alkyl moiety. In certain
embodiments the 2' sugar modification comprises a methoxy moiety
(2'-OMe).
[0225] In some embodiments of Structure (E), in (N')y 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
starting at the ultimate or penultimate position of the 5' terminus
of (N)x, preferably starting at the 5' penultimate position, and 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
ribonucleotides starting at the ultimate or penultimate position of
the 3' terminus of (N')y are independently a bicyclic nucleotide.
In various embodiments the bicyclic nucleotide is a locked nucleic
acid (LNA) such as a 2'-O, 4'-C-ethylene-bridged nucleic acid
(ENA).
[0226] In various embodiments of Structure (E), (N')y comprises
modified nucleotides selected from a bicyclic nucleotide, a 2'
sugar modified nucleotide, a mirror nucleotide, an altritol
nucleotide, a nucleotide joined to an adjacent nucleotide by a
P-alkoxy backbone modification or a nucleotide joined to an
adjacent nucleotide by an internucleotide linkage selected from a
2'-5' phosphodiester bond, a P-alkoxy linkage or a PACE linkage at
the 3' terminus or at each of the 3' and 5' termini.
[0227] In various embodiments of Structure (E), (N)x comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide,
or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage at the 5' terminus or at each
of the 3' and 5' termini.
[0228] In one embodiment where both 3' and 5' termini of the same
strand comprise a modified nucleotide, the modification at the 5'
and 3' termini is identical. In another embodiment, the
modification at the 5' terminus is different from the modification
at the 3' terminus of the same strand. In one specific embodiment,
the modified nucleotides at the 5' terminus are mirror nucleotides
and the modified nucleotides at the 3' terminus of the same strand
are joined by 2'-5' phosphodiester bond.
[0229] In various embodiments of Structure (E), the modified
nucleotides in (N)x are different from the modified nucleotides in
(N')y. For example, the modified nucleotides in (N)x are 2' sugar
modified nucleotides and the modified nucleotides in (N')y are
nucleotides linked by 2'-5' internucleotide linkages. In another
example, the modified nucleotides in (N)x are mirror nucleotides
and the modified nucleotides in (N')y are nucleotides linked by
2'-5' internucleotide linkages. In another example, the modified
nucleotides in (N)x are nucleotides linked by 2'-5' internucleotide
linkages and the modified nucleotides in (N')y are mirror
nucleotides.
[0230] In additional embodiments, the present invention provides a
compound having Structure (F):
TABLE-US-00009 (F) antisense strand 5' (N)x -Z 3' sense strand 3'
Z'-(N')y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide or a modified deoxyribonucleotide; wherein each
of (N)x and (N')y is an oligomer in which each consecutive
nucleotide is joined to the next nucleotide by a covalent bond and
each of x and y is an integer between 18 and 40; wherein each of
(N)x and (N')y comprise unmodified ribonucleotides in which each of
(N)x and (N')y independently comprise one modified nucleotide at
the 3' terminal or penultimate position wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, a
nucleotide joined to an adjacent nucleotide by a P-alkoxy backbone
modification or a nucleotide joined to an adjacent nucleotide by a
2'-5' phosphodiester bond; wherein in each of (N)x and (N')y
modified and unmodified nucleotides are not alternating; wherein
each of Z and Z' may be present or absent, but if present is 1-5
deoxyribonucleotides covalently attached at the 3' terminus of any
oligomer to which it is attached; wherein the sequence of
(N').sub.y is a sequence substantially complementary to (N)x; and
wherein the sequence of (N).sub.x comprises an antisense sequence
having substantial identity to about 18 to about 40 consecutive
ribonucleotides in an mRNA transcribed from the RTP801L gene.
[0231] In some embodiments of Structure (F), x=y=19 or x=y=23;
(N')y comprises unmodified ribonucleotides in which two consecutive
nucleotides at the 3' terminus comprises two consecutive mirror
deoxyribonucleotides; and (N)x comprises unmodified ribonucleotides
in which one nucleotide at the 3' terminus comprises a mirror
deoxyribonucleotide (set forth as Structure III).
[0232] According to various embodiments of Structure (F) 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
independently beginning at the ultimate or penultimate position of
the 3' termini of (N)x and (N')y are linked by 2'-5'
internucleotide linkages.
[0233] According to one preferred embodiment of Structure (F),
three consecutive nucleotides at the 3' terminus of (N')y are
joined by two 2'-5' phosphodiester bonds and three consecutive
nucleotides at the 3' terminus of (N')x are joined by two 2'-5'
phosphodiester bonds.
[0234] According to various embodiments of Structure (F), 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides
independently beginning at the ultimate or penultimate position of
the 3' termini of (N)x and (N')y are independently mirror
nucleotides. In some embodiments the mirror nucleotide is an
L-ribonucleotide. In other embodiments the mirror nucleotide is an
L-deoxyribonucleotide.
[0235] In other embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 3' termini
of (N)x and (N')y are independently 2' sugar modified nucleotides.
In some embodiments the 2' sugar modification comprises the
presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In
certain embodiments the 2' sugar modification comprises a methoxy
moiety (2'-OMe).
[0236] In some embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 3' termini
(N)x and (N')y are independently a bicyclic nucleotide. In various
embodiments the bicyclic nucleotide is a locked nucleic acid (LNA)
such as a 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA).
[0237] In various embodiments of Structure (F), (N')y comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide,
or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage at the 3' terminus or at both
the 3' and 5' termini.
[0238] In various embodiments of Structure (F), (N)x comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide,
or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage at the 3' terminus or at each
of the 3' and 5' termini.
[0239] In one embodiment where each of 3' and 5' termini e same
strand comprise a modified nucleotide, the modification at the 5'
and 3' termini is identical. In another embodiment, the
modification at the 5' terminus is different from the modification
at the 3' terminus of the same strand. In one specific embodiment,
the modified nucleotides at the 5' terminus are mirror nucleotides
and the modified nucleotides at the 3' terminus of the same strand
are joined by 2'-5' phosphodiester bond.
[0240] In various embodiments of Structure (F), the modified
nucleotides in (N)x are different from the modified nucleotides in
(N')y. For example, the modified nucleotides in (N)x are 2' sugar
modified nucleotides and the modified nucleotides in (N')y are
nucleotides linked by 2'-5' internucleotide linkages. In another
example, the modified nucleotides in (N)x are mirror nucleotides
and the modified nucleotides in (N')y are nucleotides linked by
2'-5' internucleotide linkages. In another example, the modified
nucleotides in (N)x are nucleotides linked by 2'-5' internucleotide
linkages and the modified nucleotides in (N')y are mirror
nucleotides.
[0241] In additional embodiments, the present invention provides a
compound having Structure (G):
TABLE-US-00010 (G) antisense strand 5' (N)x -Z 3' sense strand 3'
Z'-(N')y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide or a modified deoxyribonucleotide; wherein each
of (N)x and (N')y is an oligomer in which each consecutive
nucleotide is joined to the next nucleotide by a covalent bond and
each of x and y is an integer between 18 and 40; wherein each of
(N)x and (N')y comprise unmodified ribonucleotides in which each of
(N)x and (N')y independently comprise one modified nucleotide at
the 5' terminal or penultimate position wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, a
nucleotide joined to an adjacent nucleotide by a P-alkoxy backbone
modification or a nucleotide joined to an adjacent nucleotide by a
2'-5' phosphodiester bond; wherein for (N)x the modified nucleotide
is preferably at penultimate position of the 5' terminal; wherein
in each of (N)x and (N')y modified and unmodified nucleotides are
not alternating; wherein each of Z and Z' may be present or absent,
but if present is 1-5 deoxyribonucleotides covalently attached at
the 3' terminus of any oligomer to which it is attached; wherein
the sequence of (N').sub.y is a sequence substantially
complementary to (N)x; and wherein the sequence of (N).sub.x
comprises an antisense sequence having substantial identity to
about 18 to about 40 consecutive ribonucleotides in an mRNA
transcribed from the RTP801L gene.
[0242] In some embodiments of Structure (G), x=y=19 or x=y=23.
[0243] According to various embodiments of Structure (G) 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides
independently beginning at the ultimate or penultimate position of
the 5' termini of (N)x and (N')y are linked by 2'-5'
internucleotide linkages. For (N)x the modified nucleotides
preferably starting at the penultimate position of the 5'
terminal.
[0244] According to various embodiments of Structure (G), 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides
independently beginning at the ultimate or penultimate position of
the 5' termini of (N)x and (N')y are independently mirror
nucleotides. In some embodiments the mirror nucleotide is an
L-ribonucleotide. In other embodiments the mirror nucleotide is an
L-deoxyribonucleotide. For (N)x the modified nucleotides preferably
starting at the penultimate position of the 5' terminal.
[0245] In other embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 5' termini
of (N)x and (N')y are independently 2' sugar modified nucleotides.
In some embodiments the 2' sugar modification comprises the
presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In
certain embodiments the 2' sugar modification comprises a methoxy
moiety (2'-OMe). In some preferred embodiments the consecutive
modified nucleotides preferably begin at the penultimate position
of the 5' terminus of (N)x.
[0246] In one preferred embodiment of Structure (G), five
consecutive ribonucleotides at the 5' terminus of (N')y comprise a
2'-O-methyl modification and one ribonucleotide at the 5'
penultimate position of (N')x comprises a 2'-O-methyl modification.
In another preferred embodiment of Structure (G), five consecutive
ribonucleotides at the 5' terminus of (N')y comprise a 2'-O-methyl
modification and two consecutive ribonucleotides at the 5' terminal
position of (N')x comprise a 2'-O-methyl modification.
[0247] In some embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 5' termini
of (N)x and (N')y are bicyclic nucleotides. In various embodiments
the bicyclic nucleotide is a locked nucleic acid (LNA) such as a
2'-O, 4'-C-ethylene-bridged nucleic acid (ENA). In some preferred
embodiments the consecutive modified nucleotides preferably begin
at the penultimate position of the 5' terminus of (N)x.
[0248] In various embodiments of Structure (G), (N')y comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide,
or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage at the 5' terminus or at each
of the 3' and 5' termini.
[0249] In various embodiments of Structure (G), (N)x comprises a
modified nucleotide selected from a bicyclic nucleotide, a 2' sugar
modified nucleotide, a mirror nucleotide, an altritol nucleotide,
or a nucleotide joined to an adjacent nucleotide by an
internucleotide linkage selected from a 2'-5' phosphodiester bond,
a P-alkoxy linkage or a PACE linkage at the 5' terminus or at each
of the 3' and 5' termini.
[0250] In one embodiment where each of 3' and 5' termini of the
same strand comprise a modified nucleotide, the modification at the
5' and 3' termini is identical. In another embodiment, the
modification at the 5' terminus is different from the modification
at the 3' terminus of the same strand. In one specific embodiment,
the modified nucleotides at the 5' terminus are mirror nucleotides
and the modified nucleotides at the 3' terminus of the same strand
are joined by 2'-5' phosphodiester bond. In various embodiments of
Structure (G), the modified nucleotides in (N)x are different from
the modified nucleotides in (N')y. For example, the modified
nucleotides in (N)x are 2' sugar modified nucleotides and the
modified nucleotides in (N')y are nucleotides linked by 2'-5'
internucleotide linkages. In another example, the modified
nucleotides in (N)x are mirror nucleotides and the modified
nucleotides in (N')y are nucleotides linked by 2'-5'
internucleotide linkages. In another example, the modified
nucleotides in (N)x are nucleotides linked by 2'-5' internucleotide
linkages and the modified nucleotides in (N')y are mirror
nucleotides.
[0251] In additional embodiments, the present invention provides a
compound having Structure (H):
TABLE-US-00011 (H) antisense strand 5' (N)x -Z 3' sense strand 3'
Z'-(N')y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide or a modified deoxyribonucleotide; wherein each
of (N)x and (N')y is an oligomer in which each consecutive
nucleotide is joined to the next nucleotide by a covalent bond and
each of x and y is an integer between 18 and 40; wherein (N)x
comprises unmodified ribonucleotides further comprising one
modified nucleotide at the 3' terminal or penultimate position or
the 5' terminal or penultimate position, wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, an
altritol nucleotide, or a nucleotide joined to art adjacent
nucleotide by an internucleotide linkage selected from a 2'-5'
phosphodiester bond, a P-alkoxy linkage or a PACE linkage; wherein
(N')y comprises unmodified ribonucleotides further comprising one
modified nucleotide at an internal position, wherein the modified
nucleotide is selected from the group consisting of a bicyclic
nucleotide, a 2' sugar modified nucleotide, a mirror nucleotide, an
altritol nucleotide, or a nucleotide joined to an adjacent
nucleotide by an internucleotide linkage selected from a 2'-5'
phosphodiester bond, a P-alkoxy linkage or a PACE linkage; wherein
in each of (N)x and (N')y modified and unmodified nucleotides are
not alternating; wherein each of Z and Z' may be present or absent,
but if present is 1-5 deoxyribonucleotides covalently attached at
the 3' terminus of any oligomer to which it is attached; wherein
the sequence of (N').sub.y is a sequence substantially
complementary to (N)x; and wherein the sequence of (N).sub.x
comprises an antisense sequence having substantial identity to
about 18 to about 40 consecutive ribonucleotides in an mRNA
transcribed from the RTP801L gene.
[0252] In one embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 3'
terminus or the 5' terminus or both termini of (N)x are
independently 2' sugar modified nucleotides, bicyclic nucleotides,
mirror nucleotides, altritol nucleotides or nucleotides joined to
an adjacent nucleotide by a 2'-5' phosphodiester bond and 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive internal
ribonucleotides in (N')y are independently 2' sugar modified
nucleotides, bicyclic nucleotides, mirror nucleotides, altritol
nucleotides or nucleotides joined to an adjacent nucleotide by a
2'-5' phosphodiester bond. In some embodiments the 2' sugar
modification comprises the presence of an amino, a fluoro, an
alkoxy or an alkyl moiety. In certain embodiments the 2' sugar
modification comprises a methoxy moiety (2'-OMe).
[0253] In another embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independently
beginning at the ultimate or penultimate position of the 3'
terminus or the 5' terminus or 2-8 consecutive nucleotides at each
of 5' and 3' termini of (N')y are independently 2' sugar modified
nucleotides, bicyclic nucleotides, mirror nucleotides, altritol
nucleotides or nucleotides joined to an adjacent nucleotide by a
2'-5' phosphodiester bond, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13 or 14 consecutive internal ribonucleotides in (N)x are
independently 2' sugar modified nucleotides, bicyclic nucleotides,
mirror nucleotides, altritol nucleotides or nucleotides joined to
an adjacent nucleotide by a 2'-5' phosphodiester bond.
[0254] In one embodiment wherein each of 3' and 5' termini of the
same strand comprises a modified nucleotide, the modification at
the 5' and 3' termini is identical. In another embodiment, the
modification at the 5' terminus is different from the modification
at the 3' terminus of the same strand. In one specific embodiment,
the modified nucleotides at the 5' terminus are mirror nucleotides
and the modified nucleotides at the 3' terminus of the same strand
are joined by 2'-5' phosphodiester bond.
[0255] In various embodiments of Structure (H), the modified
nucleotides in (N)x are different from the modified nucleotides in
(N')y. For example, the modified nucleotides in (N)x are 2' sugar
modified nucleotides and the modified nucleotides in (N')y are
nucleotides linked by 2'-5' internucleotide linkages. In another
example, the modified nucleotides in (N)x are mirror nucleotides
and the modified nucleotides in (N')y are nucleotides linked by
2'-5' internucleotide linkages. In another example, the modified
nucleotides in (N)x are nucleotides linked by 2'-5' internucleotide
linkages and the modified nucleotides in (N')y are mirror
nucleotides.
[0256] In one preferred embodiment of Structure (H), x=y=19; three
consecutive ribonucleotides at the 9-11 nucleotide positions 9-11
of (N')y comprise 2'-O-methyl modification and five consecutive
ribonucleotides at the 3' terminal position of (N')x comprise
2'-O-methyl modification.
[0257] For all the above Structures (A)-(H), in various embodiments
x=y and each of x and y is 19, 20, 21, 22 or 23. In certain
embodiments, x=y=19. In yet other embodiments x=y=23. In additional
embodiments the compound comprises modified ribonucleotides in
alternating positions wherein each N at the 5' and 3' termini of
(N)x are modified in their sugar residues and the middle
ribonucleotide is not modified, e.g. ribonucleotide in position 10
in a 19-mer strand, position 11 in a 21 mer and position 12 in a
23-mer strand.
[0258] In some embodiments where x=y=21 or x=y=23 the position of
modifications in the 19 mer are adjusted for the 21 and 23 mers
with the proviso that the middle nucleotide of the antisense strand
is preferably not modified.
[0259] In some embodiments, neither (N)x nor (N')y are
phosphorylated at the 3' and 5' termini. In other embodiments
either or both (N)x and (N')y are phosphorylated at the 3' termini.
In yet another embodiment, either or both (N)x and (N')y are
phosphorylated at the 3' termini using non-cleavable phosphate
groups. In yet another embodiment, either or both (N)x and (N')y
are phosphorylated at the terminal 2' termini position using
cleavable or non-cleavable phosphate groups. These particular siRNA
compounds are also blunt ended and are non-phosphorylated at the
termini; however, comparative experiments have shown that siRNA
compounds phosphorylated at one or both of the 3'-termini have
similar activity in viva compared to the non-phosphorylated
compounds.
[0260] In certain embodiments for all the above-mentioned
Structures, the compound is blunt ended, for example wherein both Z
and Z' are absent. In an alternative embodiment, the compound
comprises at least one 3' overhang, wherein at least one of Z or Z'
is present. Z and Z' independently comprises one or more covalently
linked modified or non-modified nucleotides, for example inverted
dT or dA; dT, LNA, mirror nucleotide and the like. In some
embodiments each of Z and Z' are independently selected from dT and
dTdT. siRNA in which Z and/or Z' is present have similar activity
and stability as siRNA in which Z and Z' are absent.
[0261] In certain embodiments for all the above-mentioned
Structures, the compound comprises one or more phosphonocarboxylate
and/or phosphinocarboxylate nucleotides (PACE nucleotides). In some
embodiments the PACE nucleotides are deoxyribonucleotides and the
phosphinocarboxylate nucleotides are phosphinoacetate nucleotides.
Examples of PACE nucleotides and analogs are disclosed in U.S. Pat.
Nos. 6,693,187 and 7,067,641, both incorporated herein by
reference.
[0262] In certain embodiments for all the above-mentioned
Structures, the compound comprises one or more locked nucleic acids
(LNA) also defined as bridged nucleic acids or bicyclic
nucleotides. Preferred locked nucleic acids are 2-O, 4'-C-ethylene
nucleosides (ENA) or 2'-O, 4'-C-methylene nucleosides. Other
examples of LNA and ENA nucleotides are disclosed in WO 98/39352,
WO 00/47599 and WO 99/14226, all incorporated herein by
reference.
[0263] In certain embodiments for all the above-mentioned
Structures, the compound comprises one or more altritol monomers
(nucleotides), also defined as 1,5
anhydro-2-deoxy-D-altrito-hexitol (see for example, Allart, et al.,
1998. Nucleosides & Nucleotides 17:1523-1526; Herdewijn et al.,
1999. Nucleosides & Nucleotides 18:1371-1376; Fisher et al.,
2007, NAR 35(4):1064-1074; all incorporated herein by
reference).
[0264] The present invention explicitly excludes compounds in which
each of N and/or N' is a deoxyribonucleotide (D-A, D-C, D-G, D-T).
In certain embodiments (N)x and (N')y may comprise independently 1,
2, 3, 4, 5, 6, 7, 8, 9 or more deoxyribonucleotides. In certain
embodiments the present invention provides a compound wherein each
of N is an unmodified ribonucleotide and the 3' terminal nucleotide
or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive
nucleotides at the 3' terminus of (N')y are deoxyribonucleotides.
In yet other embodiments each of N is an unmodified ribonucleotide
and the 5' terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13 or 14 consecutive nucleotides at the 5' terminus of (N')y
are deoxyribonucleotides. In further embodiments the 5' terminal
nucleotide or 2, 3, 4, 5, 6, 7, 8, or 9 consecutive nucleotides at
the 5' terminus and 1, 2, 3, 4, 5, or 6 consecutive nucleotides at
the 3' termini of (N)x are deoxyribonucleotides and each of N' is
an unmodified ribonucleotide. In yet further embodiments (N)x
comprises unmodified ribonucleotides and 1 or 2, 3 or 4 consecutive
deoxyribonucleotides independently at each of the 5' and 3' termini
and 1 or 2, 3, 4, 5 or 6 consecutive deoxyribonucleotides in
internal positions; and each of N' is an unmodified ribonucleotide.
In certain embodiments the 3' terminal nucleotide or 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12 13 or 14 consecutive nucleotides at the 3'
terminus of (N')y and the terminal 5' nucleotide or 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12 13 or 14 consecutive nucleotides at the 5'
terminus of (N)x are deoxyribonucleotides. The present invention
excludes compounds in which each of N and/or N' is a
deoxyribonucleotide. In some embodiments the 5' terminal nucleotide
of N or 2 or 3 consecutive of N and 1,2, or 3 of N' is a
deoxyribonucleotide. Certain examples of active DNA/RNA siRNA
chimeras are disclosed in US patent publication 2005/0004064, and
Ui-Tei, 2008 (NAR 36(7):2136-2151) incorporated herein by reference
in their entirety.
[0265] Unless otherwise indicated, in preferred embodiments of the
structures discussed herein the covalent bond between each
consecutive N or N' is a phosphodiester bond.
[0266] An additional novel molecule provided by the present
invention is an oligonucleotide comprising consecutive nucleotides
wherein a first segment of such nucleotides encode a first
inhibitory RNA molecule, a second segment of such nucleotides
encode a second inhibitory RNA molecule, and a third segment of
such nucleotides encode a third inhibitory RNA molecule. Each of
the first, the second and the third segment may comprise one strand
of a double stranded RNA and the first, second and third segments
may be joined together by a linker. Further, the oligonucleotide
may comprise three double stranded segments joined together by one
or more linker.
[0267] Thus, one molecule provided by the present invention is an
oligonucleotide comprising consecutive nucleotides which encode
three inhibitory RNA molecules; said oligonucleotide may possess a
triple stranded structure, such that three double stranded arms are
linked together by one or more linker, such as any of the linkers
presented hereinabove. This molecule forms a "star"-like structure,
and may also be referred to herein as RNAstar. Such structures are
disclosed in PCT patent publication WO 2007/091269, assigned to the
assignee of the present invention and incorporated herein in its
entirety by reference.
[0268] A covalent bond refers to an internucleotide linkage linking
one nucleotide monomer to an adjacent nucleotide monomer. A
covalent bond includes for example, a phosphodiester bond, a
phosphorothioate bond, a P-alkoxy bond, a P-carboxy bond and the
like. The normal internucleoside linkage of RNA and DNA is a 3' to
5' phosphodiester linkage. In certain preferred embodiments a
covalent bond is a phosphodiester bond. Covalent bond encompasses
non-phosphorous-containing internucleoside linkages, such as those
disclosed in WO 2004/041924 inter alia. Unless otherwise indicated,
in preferred embodiments of the structures discussed herein the
covalent bond between each consecutive N or N' is a phosphodiester
bond.
[0269] For all of the structures above, in some embodiments the
oligonucleotide sequence of (N)x is fully complementary to the
oligonucleotide sequence of (N')y. In other embodiments (N)x and
(N')y are substantially complementary. In certain embodiments (N)x
is fully complementary to a target sequence. In other embodiments
(N)x is substantially complementary to a target sequence.
[0270] In some embodiments, neither (N)x nor (N')y are
phosphorylated at the 3' and 5' termini. In other embodiments
either or both (N)x and (N')y are phosphorylated at the 3' termini
(3' Pi). In yet another embodiment, either or both (N)x and (N')y
are phosphorylated at the 3' termini with non-cleavable phosphate
groups. In yet another embodiment, either or both (N)x and (N')y
are phosphorylated at the terminal 2' termini position using
cleavable or non-cleavable phosphate groups. Further, the
inhibitory nucleic acid molecules of the present invention may
comprise one or more gaps and/or one or more nicks and/or one or
more mismatches. Without wishing to be bound by theory, gaps, nicks
and mismatches have the advantage of partially destabilizing the
nucleic acid/siRNA, so that it may be more easily processed by
endogenous cellular machinery such as DICER, DROSHA or RISC into
its inhibitory components.
[0271] In the context of the present invention, a gap in a nucleic
acid refers to the absence of one or more internal nucleotides in
one strand, while a nick in a nucleic acid refers to the absence of
an internucleotide linkage between two adjacent nucleotides in one
strand. Any of the molecules of the present invention may contain
one or more gaps and/or one or more nicks.
[0272] In one aspect the present invention provides a compound
having Structure (I) set forth below:
TABLE-US-00012 (I) (antisense strand) 5' (N)x - Z 3' (sense strand)
3' Z'-(N')y- z'' 5'
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein Z
and Z' may be present or absent, but if present is independently
1-5 consecutive nucleotides covalently attached at the 3' terminus
of the strand in which it is present; wherein z'' may be present or
absent, but if present is a capping moiety covalently attached at
the 5' terminus of (N')y; wherein x=18 to 27; wherein y=18 to 27;
wherein (N)x comprises modified and unmodified ribonucleotides,
each modified ribonucleotide having a 2'-O-methyl on its sugar,
wherein N at the 3' terminus of (N)x is a modified ribonucleotide.
(N)x comprises at least five alternating modified ribonucleotides
beginning at the 3' end and at least nine modified ribonucleotides
in total and each remaining N is an unmodified ribonucleotide;
wherein in (N')y at least one unconventional moiety is present,
which unconventional moiety may be an abasic ribose moiety, an
abasic deoxyribose moiety, a modified or unmodified
deoxyribonucleotide, a mirror nucleotide, and a nucleotide joined
to an adjacent nucleotide by a 2'-5' internucleotide phosphate
bond; and wherein the sequence of (N)x is substantially
complementary to the sequence of (N')y; and the sequence of (N')y
is substantially identical to the sequence of an mRNA encoded by
the RTP801L gene.
[0273] In some embodiments x=y=19. In other embodiments x=y=23. In
some embodiments the at least one unconventional moiety is present
at positions 15, 16, 17, or 18 in (N')y. In some embodiments the
unconventional moiety is selected from a mirror nucleotide, an
abasic ribose moiety and an abasic deoxyribose moiety. In some
preferred embodiments the unconventional moiety is a mirror
nucleotide, preferably an L-DNA moiety. In some embodiments an
L-DNA moiety is present at position 17, position 18 or positions 17
and 18.
[0274] In other embodiments the unconventional moiety is an abasic
moiety. In various embodiments (N')y comprises at least five abasic
ribose moieties or abasic deoxyribose moieties.
[0275] In yet other embodiments (N')y comprises at least five
abasic ribose moieties or abasic deoxyribose moieties and at least
one of N' is an LNA.
[0276] In some embodiments of Structure (DC) (N)x comprises nine
alternating modified ribonucleotides. In other embodiments of
Structure (I) (N)x comprises nine alternating modified
ribonucleotides further comprising a 2'O modified nucleotide at
position 2. In some embodiments (N)x comprises 2'O Me modified
ribonucleotides at the odd numbered positions 1, 3, 5, 7, 9, 11,
13, 15, 17, 19. In other embodiments (N)x further comprises a 2'O
Me modified ribonucleotide at one or both of positions 2 and 18. In
yet other embodiments (N)x comprises 2'O Me modified
ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.
[0277] In various embodiments z'' is present and is selected from
an abasic ribose moiety, a deoxyribose moiety; an inverted abasic
ribose moiety, a deoxyribose moiety; C6-amino-Pi; a mirror
nucleotide.
[0278] In another aspect the present invention provides a compound
having Structure (J) set forth below:
TABLE-US-00013 (J) (antisense strand) 5' (N)x - Z 3' (sense strand)
3' Z'-(N')y-z'' 5'
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein Z
and Z' may be present or absent, but if present is independently
1-5 consecutive nucleotides covalently attached at the 3' terminus
of the strand in which it is present; wherein z'' may be present or
absent but if present is a capping moiety covalently attached at
the 5' terminus of (N')y; wherein x=18 to 27; wherein y=18 to 27;
wherein (N)x comprises modified or unmocr ribonucleotides, and
optionally at least one unconventional moiety; wherein in (N')y at
least one unconventional moiety is present, which unconventional
moiety may be an abasic ribose moiety, an abasic deoxyribose
moiety, a modified or unmodified deoxyribonucleotide, a mirror
nucleotide, a non-base pairing nucleotide analog or a nucleotide
joined to an adjacent nucleotide by a 2'-5' internucleotide
phosphate bond; and wherein the sequence of (N)x is substantially
complementary to the sequence of (N')y; and the sequence of (N')y
is substantially identical to the sequence of an mRNA encoded by
the RTP801L gene.
[0279] In some embodiments x=y=19. In other embodiments x=y=23. In
some preferred embodiments (N)x comprises modified and unmodified
ribonucleotides, and at least one unconventional moiety.
[0280] In some embodiments in (N)x the N at the 3' terminus is a
modified ribonucleotide and (N)x comprises at least 8 modified
ribonucleotides. In other embodiments at least 5 of the at least 8
modified ribonucleotides are alternating beginning at the 3' end.
In some embodiments (N)x comprises an abasic moiety in one of
positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or
[0281] In some embodiments the at least one unconventional moiety
in (N')y is present at positions 15, 16, 17, or 18. In some
embodiments the unconventional moiety is selected from a mirror
nucleotide, an abasic ribose moiety and an abasic deoxyribose
moiety. In some preferred embodiments the unconventional moiety is
a mirror nucleotide, preferably an L-DNA moiety. In some
embodiments an L-DNA moiety is present at position 17, position 18
or positions 17 and 18. In other embodiments the at least one
unconventional moiety in (N')y is an abasic ribose moiety or an
abasic deoxyribose moiety.
[0282] In various embodiments of Structure (X) z'' is present and
is selected from an abasic ribose moiety, a deoxyribose moiety; an
inverted abasic ribose moiety, a deoxyribose moiety; C6-amino-Pi; a
mirror nucleotide.
[0283] In yet another aspect the present invention provides a
compound having Structure (K) set forth below:
TABLE-US-00014 (K) (antisense strand) 5' (N).sub.x -Z 3' (sense
strand) 3' Z'-(N').sub.y-z'' 5'
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein Z
and Z' may be present or absent, but if present is independently
1-5 consecutive nucleotides covalently attached at the 3' terminus
of the strand in which it is present; wherein z'' may be present or
absent but if present is a capping moiety covalently attached at
the 5' terminus of (N')y; wherein x=18 to 27; wherein y=18 to 27;
wherein (N)x comprises a combination of modified or unmodified
ribonucleotides and unconventional moieties, any modified
ribonucleotide having a 2'-O-methyl on its sugar; wherein (N')y
comprises modified or unmodified ribonucleotides and optionally an
unconventional moiety, any modified ribonucleotide having a 2'OMe
on its sugar; wherein the sequence of (N)x is substantially
complementary to the sequence of (N')y; and the sequence of (N')y
is substantially identical to the sequence of an mRNA encoded by
the RTP801L gene; and wherein there are less than 15 consecutive
nucleotides complementary to the mRNA.
[0284] In some embodiments x=y=19. In other embodiments x=y=23. In
some preferred embodiments the at least one preferred one
unconventional moiety is present in (N)x and is an abasic ribose
moiety or an abasic deoxyribose moiety. In other embodiments the at
least one unconventional moiety is present in (N)x and is a
non-base pairing nucleotide analog. In various embodiments (N')y
comprises unmodified ribonucleotides. In some embodiments (N)x
comprises at least five abasic ribose moieties or abasic
deoxyribose moieties or a combination thereof. In certain
embodiments (N)x and/or (N')y comprise modified ribonucleotides
which do not base pair with corresponding modified or unmodified
ribonucleotides in (N')y and/or (N)x.
[0285] In various embodiments the present invention provides an
siRNA set forth in Structure (L):
TABLE-US-00015 (L) (antisense strand) 5' (N).sub.x - Z 3' (sense
strand) 3' Z'-(N').sub.y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of (N).sub.x and (N').sub.y is an oligonucleotide in which
each consecutive N or N' is joined to the next N or N' by a
covalent bond; wherein Z and Z' are absent; wherein x=y=19; wherein
in (N')y the nucleotide in at least one of positions 15, 16, 17, 18
and 19 comprises a nucleotide selected from an abasic
pseudo-nucleotide, a mirror nucleotide, a deoxyribonucleotide and a
nucleotide joined to an adjacent nucleotide by a 2'-5'
internucleotide bond; wherein (N)x comprises alternating modified
ribonucleotides and unmodified ribonucleotides each modified
ribonucleotide being modified so as to have a 2'-O-methyl on its
sugar and the ribonucleotide located at the middle position of (N)x
being modified or unmodified, preferably unmodified; and wherein
the sequence of (N)x is substantially complementary to the sequence
of (N')y; and the sequence of (N')y is substantially identical to
the mRNA of the RTP801L gene.
[0286] In some embodiments of Structure (L), in (N')y the
nucleotide in one or both of positions 17 and 18 comprises a
modified nucleotide selected from an abasic pseudo-nucleotide, a
mirror nucleotide and a nucleotide joined to an adjacent nucleotide
by a 2'-5' internucleotide bond. In some embodiments the mirror
nucleotide is selected from L-DNA and L-RNA. In various embodiments
the mirror nucleotide is L-DNA.
[0287] In various embodiments (N')y comprises a modified nucleotide
at position 15 wherein the modified nucleotide is selected from a
mirror nucleotide and a deoxyribonucleotide.
[0288] In certain embodiments (N')y further comprises a modified
nucleotide or pseudo nucleotide at position 2 wherein the pseudo
nucleotide may be an abasic pseudo-nucleotide analog and the
modified nucleotide is optionally a mirror nucleotide.
[0289] In various embodiments the antisense strand (N)x comprises
2'O-Me modified ribonucleotides at the odd numbered positions (5'
to 3'; positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some
embodiments (N)x further comprises 2'O-Me modified ribonucleotides
at one or both positions 2 and 18. In other embodiments (N)x
comprises 2'O Me modified ribonucleotides at positions 2, 4, 6, 8,
11, 13, 15, 17, 19.
[0290] Other embodiments of Structures (L), (I) and (J) are
envisaged wherein x=y=21 or wherein x=y=23; in these embodiments
the modifications for (N')y discussed above instead of being in
positions 17 and 18 are in positions 19 and 20 for 21-mer
oligonucleotide and 21 and 22 for 23 mer oligonucleotide; similarly
the modifications in positions 15, 16, 17, 18 or 19 are in
positions 17, 18, 19, 20 or 21 for the 21-mer oligonucleotide and
positions 19, 20, 21, 22, or 23 for the 23-mer oligonucleotide. The
2'O Me modifications on the antisense strand are similarly
adjusted. In some embodiments (N)x comprises 2'O Me modified
ribonucleotides at the odd numbered positions (5' to 3'; positions
1, 3, 5, 7, 9, 12, 14, 16, 18, 20 for the 21 mer oligonucleotide
[nucleotide at position 11 unmodified] and 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23 for the 23 mer oligonucleotide [nucleotide at
position 12 unmodified]. In other embodiments (N)x comprises 2'O Me
modified ribonucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16,
18, 20 [nucleotide at position 11 unmodified for the 21 mer
oligonucleotide and at positions 2, 4, 6, 8, 10, 13, 15, 17, 19,
21, 23 for the 23 mer oligonucleotide [nucleotide at position 12
unmodified].
[0291] In some embodiments (N')y further comprises a 5' terminal
cap nucleotide. In various embodiments the terminal cap moiety is
selected from an abasic pseudo-nucleotide analog, an inverted
abasic pseudo-nucleotide analog, an L-DNA nucleotide, and a
C6-imine phosphate (C6 amino linker with phosphate at
terminus).
[0292] In other embodiments the present invention provides a
compound having Structure (M) set forth below:
TABLE-US-00016 (antisense strand) 5' (N).sub.x - Z 3' (sense
strand) 3' Z'-(N').sub.y 5'
wherein each of N and N' is selected from a pseudo-nucleotide and a
nucleotide; wherein each nucleotide is selected from an unmodified
ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of (N).sub.x and (N').sub.y is an oligonucleotide in which
each consecutive N or N' is joined to the next N or N' by a
covalent bond; wherein Z and Z' are absent; wherein x=18 to 27;
wherein y=18 to 27; wherein the sequence of (N)x is substantially
complementary to the sequence of (N')y; and the sequence of (N')y
is substantially identical to an mRNA of the RTP801L gene; wherein
at least one of N is selected from an abasic pseudo nucleotide, a
non-pairing nucleotide analog and a nucleotide mismatch to the mRNA
of the RTP801L gene in a position of (N)x such that (N)x comprises
less than 15 consecutive nucleotides complementary to the mRNA of
the RTP801L gene.
[0293] In other embodiments the present invention provides a double
stranded compound having Structure (N) set forth below:
TABLE-US-00017 (N) (antisense strand) 5' (N).sub.x - Z 3' (sense
strand) 3' Z'-(N').sub.y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of (N).sub.x and (N').sub.y is an oligonucleotide in which
each consecutive N or N' is joined to the next N or N' by a
covalent bond; wherein Z and Z' are absent; wherein each of x and y
is an integer between 18 and 40; wherein the sequence of (N)x is
substantially complementary to the sequence of (N')y; and the
sequence of (N')y is substantially identical to an mRNA of the
RTP801L gene; wherein (N)x, (N')y or (N)x and (N')y comprise non
base-pairing modified nucleotides such that (N)x and (N')y form
less than 15 base pairs in the double stranded compound.
[0294] In other embodiments the present invention provides a
compound having Structure (O) set forth below:
TABLE-US-00018 (O) (antisense strand) 5' (N).sub.x - Z 3' (sense
strand) 3' Z'-(N').sub.y 5'
wherein each of N is a nucleotide selected from an unmodified
ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of N' is a nucleotide analog selected from a six membered
sugar nucleotide, seven membered sugar nucleotide, morpholino
moiety, peptide nucleic acid and combinations thereof; wherein each
of (N).sub.x and (N').sub.y is an oligonucleotide in which each
consecutive N or N' is joined to the next N or N' by a covalent
bond; wherein Z and Z' are absent; wherein each of x and y is an
integer between 18 and 40; wherein the sequence of (N)x is
substantially complementary to the sequence of (N')y; and the
sequence of (N')y is substantially identical to an mRNA of the
RTP801L gene.
[0295] In other embodiments the present invention provides a
compound having Structure (P) set forth below:
TABLE-US-00019 (P) (antisense strand) 5' (N).sub.x - Z 3' (sense
strand) 3' Z'-(N').sub.y 5'
wherein each of N and N' is a nucleotide selected from an
unmodified ribonucleotide, a modified ribonucleotide, an unmodified
deoxyribonucleotide and a modified deoxyribonucleotide; wherein
each of (N).sub.x and (N').sub.y is an oligonucleotide in which
each consecutive N or N' is joined to the next N or N' by a
covalent bond; wherein Z and Z' are absent; wherein each of x and y
is an integer between 18 and 40; wherein one of N or N' in an
internal position of (N)x or (N')y or one or more of N or N' at a
terminal position of (N)x or (N')y comprises an abasic moiety or a
2' modified nucleotide; wherein the sequence of (N)x is
substantially complementary to the sequence of (N')y; and the
sequence of (N')y is substantially identical to an mRNA of the
RTP801L gene.
[0296] In various embodiments (N')y comprises a modified nucleotide
at position 15 wherein the modified nucleotide is selected from a
mirror nucleotide and a deoxyribonucleotide.
[0297] In certain embodiments (N')y further comprises a modified
nucleotide at position 2 wherein the modified nucleotide is
selected from a mirror nucleotide and an abasic pseudo-nucleotide
analog.
[0298] In various embodiments the antisense strand (N)x comprises
2'O-Me modified ribonucleotides at the odd numbered positions (5'
to 3'; positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some
embodiments (N).sub.x further comprises 2'O-Me modified
ribonucleotides at one or both positions 2 and 18. In other
embodiments (N)x comprises 2'O Me modified ribonucleotides at
positions 2, 4, 6, 8, 11, 13, 15, 17, 19.
[0299] The Structural motifs described above are useful with any
oligonucleotide pair (sense and antisense strands) to a mammalian
RTP801L gene, and preferably to the human RTP801L gene.
[0300] Any siRNA sequence disclosed herein can be prepared having
any of the modifications/structures disclosed herein.
[0301] In another aspect the present invention provides a
pharmaceutical composition comprising a modified or unmodified
compound of the present invention, in an amount effective to
inhibit human RTP801L gene expression wherein the compound
comprises an antisense sequence, (N).sub.x; and a pharmaceutically
acceptable carrier.
[0302] hi yet another aspect the present invention provides a
pharmaceutical composition comprising one or more modified
compounds of the present invention, in an amount effective to
inhibit human RTP801L gene expression wherein the compound
comprises an antisense sequence, (N).sub.x; and a pharmaceutically
acceptable carrier.
[0303] In another aspect, the present invention relates to a method
for the treatment of a subject in need of treatment for a disease
or disorder or symptoms associated with the disease or disorder,
associated with the expression of the RTP801L gene comprising
administering to the subject an amount of an siRNA, according to
the present invention, in a therapeutically effective dose so as to
thereby treat the subject.
[0304] The methods of the invention comprise administering to the
subject one or more siRNA compounds which inhibit expression of the
RTP801L gene. The novel structures disclosed herein, when
integrated into antisense and corresponding sense nucleic acid
sequences, provide siRNA compounds useful in reducing expression of
the RTP801L gene.
Pharmaceutical Compositions
[0305] The present invention provides a pharmaceutical composition
comprising one or more of the compounds of the invention; and a
pharmaceutically acceptable carrier. Such compositions may comprise
a mixture of two or more different oligonucleotides/siRNAs.
[0306] The invention further provides a pharmaceutical composition
comprising at least one compound of the invention covalently or
non-covalently bound to one or more compounds of the invention in
an amount effective to inhibit RTP801L; and a pharmaceutically
acceptable carrier. Endogenous cellular complexes to produce one or
more oligoribonucleotides of the invention may process the compound
intracellularly.
[0307] The present invention also provides for a process of
preparing a pharmaceutical composition, which comprises:
providing one or more siRNA compounds of the invention; and
admixing said compound with a pharmaceutically acceptable
carrier.
[0308] Substantially complementary refers to complementarity of
greater than about 84%, to another sequence. For example in a
duplex region consisting of 19 base pairs one mismatch results in
94.7% complementarity, two mismatches results in about 89.5%
complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0309] Additionally, the invention provides a method of inhibiting
the expression of the genes of the present invention by at least
50% as compared to a control comprising contacting an mRNA
transcript of the gene of the present invention with one or more of
the compounds of the invention.
[0310] In one embodiment the oligoribonucleotide is inhibiting the
RTP801L gene, whereby the inhibition is selected from the group
comprising inhibition of gene function, inhibition of polypeptide
and inhibition of mRNA expression.
[0311] In one embodiment the compound is inhibiting expression of a
polypeptide, whereby the inhibition is selected from the group
comprising inhibition of function (which may be examined by an
enzymatic assay or a binding assay with a known interactor of the
native gene/polypeptide, inter alia), inhibition of protein (which
may be examined by Western blotting, ELISA or immuno-precipitation,
inter alia) and inhibition of mRNA expression (which may be
examined by Northern blotting, quantitative RT-PCR, in-situ
hybridisation or microarray hybridisation, inter alia).
[0312] In additional embodiments the invention provides a method of
treating a subject suffering from a disease accompanied by an
elevated level of the RTP801L gene/polypeptide, the method
comprising administering to the subject a compound of the invention
in a therapeutically effective dose thereby treating the
subject.
[0313] More particularly, the invention provides a chemically
modified double stranded oligoribonucleotide wherein one strand
comprises consecutive nucleotides having, from 5' to 3', the
sequence set forth in any one of Tables A-G or a homolog thereof
wherein in up to two of the ribonucleotides in each terminal region
is altered.
[0314] Additionally, further nucleic acids according to the present
invention comprise at least 14 contiguous nucleotides of any one of
the oligomers set forth in any one of Tables A-G and more
preferably 14 contiguous nucleotide base pairs at any end of the
double-stranded structure comprised of the first strand and second
strand as described above.
Delivery
[0315] The siRNA molecules of the present invention may be
delivered to the target tissue by direct application of the naked
molecules prepared with a carrier or a diluent.
[0316] The term "naked siRNA" refers to siRNA molecules that are
free from any delivery vehicle that acts to assist, promote or
facilitate entry into the cell, including viral sequences, viral
particles, liposome formulations, lipofectin or precipitating
agents and the like. For example, siRNA in PBS is "naked
siRNA".
[0317] However, in some embodiments the siRNA molecules of the
invention are delivered in liposome formulations and lipofectin
formulations and the like and can be prepared by methods well known
to those skilled in the art. Such methods are described, for
example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859,
which are herein incorporated by reference.
[0318] Delivery systems aimed specifically at the enhanced and
improved delivery of siRNA into mammalian cells have been
developed, (see, for example, Shen et al FESS Let. 2003,
539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et
al., Mol. Vision 2003, 9: 210-216; Sorensen et al., J. Mol. Biol.
2003. 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and
Simeoni et al., NAR 2003, 31, 11:2717-2724).
[0319] The pharmaceutically acceptable carriers, solvents,
diluents, excipients, adjuvants and vehicles as well as implant
carriers generally refer to inert, non-toxic solid or liquid
fillers, diluents or encapsulating material not reacting with the
active ingredients of the invention and they include liposomes and
microspheres. Examples of delivery systems useful in the present
invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;
4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;
4,439,196; and 4,475,196. Many other such implants, delivery
systems, and modules are well known to those skilled in the art. In
one specific embodiment of this invention topical and transdermal
formulations may be selected. The siRNAs or pharmaceutical
compositions of the present invention are administered and dosed in
accordance with good medical practice, taking into account the
clinical condition of the individual patient, the disease to be
treated, the site and method of administration, scheduling of
administration, patient age, sex, body weight and other factors
known to medical practitioners.
[0320] The "therapeutically effective dose" for purposes herein is
thus determined by such considerations as are known in the art. The
dose must be effective to achieve improvement including but not
limited to improved survival rate or more rapid recovery, or
improvement or elimination of symptoms and other indicators as are
selected as appropriate measures by those skilled in the art.
[0321] In general, the active dose of compound for humans is in the
range of from 1 ng/kg to about 20-100 mg/kg body weight per day,
preferably about 0.01 mg to about 2-10 mg/kg body weight per day,
in a regimen of one dose per day or twice or three or more times
per day for a period of 1-4 weeks or longer.
[0322] The compounds of the present invention can be administered
by any of the conventional routes of administration. It should be
noted that the compound can be administered as the compound or as
pharmaceutically acceptable salt and can be administered alone or
as an active ingredient in combination with pharmaceutically
acceptable carriers, solvents, diluents, excipients, adjuvants and
vehicles. The compounds can be administered orally, subcutaneously
or parenterally including intravenous, intraarterial,
intramuscular, intraperitoneally, and intranasal, inhalation,
transtympanic administration as well as intrathecal and infusion
techniques. Implants of the compounds are also useful. Liquid forms
may be prepared for injection, the term including subcutaneous,
transdermal, intravenous, intramuscular, intrathecal, and other
parental routes of administration. The liquid compositions include
aqueous solutions, with and without organic co-solvents, aqueous or
oil suspensions, emulsions with edible oils, as well as similar
pharmaceutical vehicles. In a particular embodiment, the
administration comprises intravenous administration. In another
embodiment the administration comprises topical or local
administration.
[0323] In addition, in certain embodiments the compositions for use
in the novel treatments of the present invention may be formed as
aerosols, for example for intranasal administration.
[0324] In certain embodiments, oral compositions (such as tablets,
suspensions, solutions) may be effective for local delivery to the
oral cavity such as oral composition suitable for mouthwash for the
treatment of oral mucositis.
[0325] The compounds of the present invention can be administered
topically to the surface of the eye. It should be noted that the
compound is preferably administered as the compound or as
pharmaceutically acceptable salt active ingredient in combination
with pharmaceutically acceptable carriers, solvents, diluents,
excipients, adjuvants and or vehicles. According to the present
invention the preferred method of delivery is topical
administration for topical delivery to the eye.
[0326] Liquid forms are prepared for drops or spray. The liquid
compositions include aqueous solutions, with and without organic
co-solvents, aqueous or oil suspensions, emulsions with oils, as
well as similar pharmaceutical vehicles. In some embodiments
administration comprises topical or local administration.
[0327] These compounds are administered to humans and other animals
for therapy by any suitable route of administration to the eye, as
by, for example, a spray or drops, and topically, as by ointments,
suspensions or drops.
[0328] In preferred embodiments the subject being treated is a
warm-blooded animal and, in particular, mammals including
human.
Methods of Treatment
[0329] In one aspect, the present invention relates to a method for
the treatment of a subject in need of treatment for a disease or
disorder associated with expression of the RTP801L gene, comprising
administering to the subject an amount of at least one chemically
modified siRNA which inhibits expression of RTP801L. In certain
preferred embodiments more than one siRNA compound is
administered.
[0330] In preferred embodiments the subject being treated is a
warm-blooded animal and, in particular, mammals including
human.
[0331] The methods of the invention comprise administering to the
subject one or more RTP801L siRNA compounds which down-regulate
expression of the RTP801L gene; and in particular at least one
siRNA in a therapeutically effective dose to thereby treat the
subject.
[0332] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down, attenuate the related disorder as listed
above. Those in need of treatment include those already
experiencing the disease or condition, those prone to having the
disease or condition, and those in which the disease or condition
is to be prevented. The compounds of the invention may be
administered before, during or subsequent to the onset of the
disease or condition or symptoms associated therewith. In cases
where treatment is for the purpose of prevention, then the present
invention relates to a method for delaying the onset of or averting
the development of the disease or disorder.
[0333] In general, the method includes administering
oligoribonucleotides, such as small interfering RNAs (i. e.,
siRNAs) that are targeted to a particular mRNA and hybridize to it,
or nucleic acid material that can produce siRNAs in a cell, in an
amount sufficient to down-regulate expression of a target gene by
an RNA interference mechanism. In particular, the subject method
can be used to inhibit expression of the RTP801L gene for treatment
of respiratory disorders, microvascular disorders, eye disorders
and hearing impairments.
[0334] Thus, in one embodiment the present invention provides for a
method of treating a patient suffering from a microvascular
disorder, an eye disease a respiratory disorder, a hearing disorder
or a spinal cord injury or other wound, comprising administering to
the patient a pharmaceutical composition comprising an RTP801L
inhibitor in a therapeutically effective amount so as to thereby
treat the patient. The invention further provides a method of
treating a patient suffering from a microvascular disorder, an eye
disease, a respiratory disorder, a hearing disorder or a spinal
cord injury or other wound, or an ischemic disease, comprising
administering to the patient a pharmaceutical composition
comprising an RTP801L inhibitor, in a dosage and over a period of
time sufficient to promote recovery. The eye disease may be macular
degeneration such as age-related macular degeneration (AMD), or
glaucoma, inter cilia. The microvascular disorder may be diabetic
retinopathy or acute renal failure, inter cilia. The respiratory
disorder may be chronic obstructive pulmonary disease (COPD), acute
lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), Lung
transplantation, emphysema, chronic bronchitis, asthma and lung
cancer, inter alia. The hearing disorder may be trauma-induced
deafness, age-related deafness or cisplatin-induced deafness, inter
alia. Thus, a list of conditions to be treated includes ARF,
hearing loss, Acute Respiratory Distress Syndrome, Glaucoma. AMD,
COPD, nephrotoxicity, lung transplantation, and
Ischemia/reperfusion injury. Oligonucleotide sequences of RTP801L
siRNA inhibitors are set forth below and in any one of Tables A-G
(SEQ ID NOs:2-6927).
[0335] The present invention further relates to the use of any of
the compounds disclosed herein, particularly to novel small
interfering RNAs (siRNAs), in the treatment of diseases and
disorders associated with RTP801L expression.
[0336] A further end modification is a biotin group. Such biotin
group may preferably be attached to either the most 5' or the most
3' nucleotide of the first and/or second strand or to both ends. In
a more preferred embodiment the biotin group is coupled to a
polypeptide or a protein. It is also within the scope of the
present invention that the polypeptide or protein is attached
through any of the other aforementioned end modifications.
[0337] The various end modifications as disclosed herein are
preferably located at the ribose moiety of a nucleotide of the
nucleic acid according to the present invention. More particularly,
the end modification may be attached to or replace any of the
OH-groups of the ribose moiety, including but not limited to the
2'OH, 3'OH and 5'OH position, provided that the nucleotide thus
modified is a terminal nucleotide. Inverted abasic or abasic are
nucleotides, either deoxyribonucleotides or ribonucleotides which
do not have a nucleobase moiety. This kind of compound is, inter
alia, described in Sternberger, M., et al., (2002. Aniisense
Nucleic Acid Drug Dev, 12, 131-43).
[0338] A further form of nucleotides used may be siNA which is,
among others, described in international patent application WO
03/070918.
[0339] It is to be understood that, in the context of the present
invention, any of the siRNA molecules disclosed herein, or any long
double-stranded RNA molecules (typically 25-500 nucleotides in
length) which are processed by endogenous cellular complexes (such
as DICER--see above) to form the siRNA molecules disclosed herein,
or molecules which comprise the siRNA molecules disclosed herein,
can be incorporated into the molecules of the present invention to
form additional novel molecules, and can employed in the treatment
of the diseases or disorders described herein.
[0340] In particular, it is envisaged that a long oligonucleotide
(typically about 80-500 nucleotides in length) comprising one or
more stem and loop structures, where stem regions comprise the
oligonucleotides of the invention, may be delivered in a carrier,
preferably a pharmaceutically acceptable carrier, and may be
processed intracellularly by endogenous cellular complexes (e.g. by
DROSHA and DICER as described above) to produce one or more smaller
double stranded oligonucleotides (siRNAs) which are
oligonucleotides of the invention. This oligonucleotide can be
termed a tandem shRNA construct. It is envisaged that this long
oligonucleotide is a single stranded oligonucleotide comprising one
or more stem and loop structures, wherein each stem region
comprises a sense and corresponding antisense siRNA sequence. Any
molecules, such as, for example, antisense DNA molecules which
comprise the inhibitory sequences disclosed herein (with the
appropriate nucleic acid modifications) are particularly desirable
and may be used in the same capacity as their corresponding
RNAs/siRNAs for all uses and methods disclosed herein.
[0341] In addition, analogs of polynucleotides can be prepared
wherein the structure of the nucleotide is fundamentally altered
and that are better suited as therapeutic or experimental reagents.
An example of a nucleotide analog is a peptide nucleic acid (PNA)
wherein the deoxyribose (or ribose) phosphate backbone in DNA (or
RNA is replaced with a polyamide backbone which is similar to that
found in peptides. PNA analogs have been shown to be resistant to
degradation by enzymes and to extend lives in vivo and in vitro.
Further. PNAs have been shown to bind stronger to a complementary
DNA sequence than a DNA molecule. This observation is attributed to
the lack of charge repulsion between the PNA strand and the DNA
strand. Other modifications that can be made to oligonucleotides
include polymer backbones, cyclic backbones, or acyclic
backbones.
[0342] In a particularly preferred embodiment the compounds of the
present invention possess a sequence present in Table F (SEQ ID
NOS: 6896-6927). In other preferred embodiments the RTP801L
compound is selected from any one of the compounds set forth in
Table G.
[0343] In one preferred embodiment the siRNA used in the methods of
the present invention is one of a pair of oligonucleotides set
forth in Table F. In another preferred embodiment the siRNA
compound is selected from a compound set forth in Table G.
[0344] Thus, in a particularly preferred embodiment, the present
invention comprises a compound having an oligonucleotide sequence
and chemical modifications as shown in FIGS. 8a-8b. In some
preferred embodiments the compound is ID 128339 x=y=19; wherein
(N)x comprises alternating unmodified and 2'OMe sugar modified
ribonucleotides and wherein (N')y comprises unmodified
ribonucleotides and an L-deoxyribonucleotide at position 18 and an
optional deoxyribonucleotide at position 15. siRNA compound 128339
exhibits good activity (knockdown of about 75% at 20 nM) in human
cells and stability in human serum.
[0345] And a compound having the structure
##STR00001##
wherein in (N)x the ribonucleotides alternate between modified
ribonucleotides and unmodified ribonucleotides each modified
ribonucleotide being modified so as to have a 2'-O-methyl on its
sugar and the ribonucleotide located at the middle position being
unmodified; wherein in (N')y the nucleotide at position 18 or 17
and 18 is a mirror nucleotide and the nucleotide at position 15 is
optionally an unmodified ribonucleotide or a
deoxyribonucleotide;
[0346] and wherein the antisense and the sense strands are
unphosphorylated or phosphorylated at the 3' termini.
[0347] And a compound having the structure
##STR00002##
wherein in (N)x the ribonucleotides alternate between modified
ribonucleotides and unmodified ribonucleotides each modified
ribonucleotide being modified so as to have a 2'-O-methyl on its
sugar and the ribonucleotide located at the middle position being
unmodified; wherein in (N')y the nucleotide at position 18 or 17
and 18 is a mirror nucleotide and the nucleotide at position 15 is
optionally an unmodified ribonucleotide or a deoxyribonucleotide;
and wherein the antisense and the sense strands are
unphosphorylated or phosphorylated at the 3' termini.
[0348] And a compound having the structure
##STR00003##
wherein in (N)x the ribonucleotides alternate between modified
ribonucleotides and unmodified ribonucleotides each modified
ribonucleotide being modified so as to have a 2'-O-methyl on its
sugar and the ribonucleotide located at the middle position being
unmodified; wherein in (N')y the nucleotide at position 18 or 17
and 18 is a mirror nucleotide and the nucleotide at position 15 is
optionally an unmodified ribonucleotide or a deoxyribonucleotide;
and wherein the antisense and the sense strands are
unphosphorylated or phosphorylated at the 3' termini.
[0349] And a compound having the structure
##STR00004##
wherein in (N)x the ribonucleotides alternate between modified
ribonucleotides and unmodified ribonucleotides each modified
ribonucleotide being modified so as to have a 2'-O-methyl on its
sugar and the ribonucleotide located at the middle position being
unmodified; wherein in (N')y the nucleotide at position 18 or 17
and 18 is a mirror nucleotide and the nucleotide at position 15 is
optionally an unmodified ribonucleotide or a deoxyribonucleotide;
and wherein the antisense and the sense strands are
unphosphorylated or phosphorylated at the 3' termini.
[0350] Further, the present invention provides for a pharmaceutical
composition comprising any one of the above compounds and a
pharmaceutically acceptable excipient.
[0351] The siRNA molecules having antisense strand SEQ ID NO:999
and sense strand SEQ ID NO:74 or antisense strand SEQ ED NO:1000
and sense strand SEQ ID NO:75 or antisense strand SEQ ID NO:6914
and sense strand SEQ ID NO:6898 or antisense strand SEQ ID NO:6915
and sense strand SEQ ID NO:6899 comprise any of the additional
modifications disclosed herein, and are used in the treatment of
any of the indications disclosed herein, such as the following
diseases or conditions: hearing loss, acute renal failure (ARF),
glaucoma, acute respiratory distress syndrome (ARDS) and other
acute lung and respiratory injuries, ischemia-reperfusion injury
following lung transplantation, organ transplantation including
lung, liver, heart, bone marrow, pancreas, cornea and kidney
transplantation, spinal cord injury, pressure sores, age-related
macular degeneration (AMD), dry eye syndrome, oral mucositis and
chronic obstructive pulmonary disease (COPD). Other indications
include cancer of all types, chemical-induced nephrotoxicity and
chemical-induced neurotoxicity, for example toxicity induced by
cisplatin and cisplatin-like compounds, by aminoglycosides, by loop
diuretics, and by hydroquinone and their analogs.
[0352] These compounds and pharmaceuticals are used to treat a
patient suffering from any one of the diseases or conditions
disclosed herein; further, any of the siRNAs in any one of Tables
A-G are used in the same manner.
[0353] Additionally, the invention provides a method of
down-regulating the expression of the RTP801L gene by at least 50%
as compared to a control comprising contacting an mRNA transcript
of the RTP801L gene with one or more of the compounds of the
invention.
[0354] In one embodiment the oligoribonucleotide is down-regulating
the RTP801L gene, whereby the down-regulation is selected from the
group comprising down-regulation of gene function, down-regulation
of polypeptide and down-regulation of mRNA expression.
[0355] In one embodiment the compound is down-regulating the
RTP801L polypeptide, whereby the down-regulation is selected from
the group comprising down-regulation of function (which may be
examined by an enzymatic assay or a binding assay with a known
interactor of the native gene/polypeptide, inter alia),
down-regulation of protein (which may be examined by Western
blotting, ELISA or immuno-precipitation, inter alia) and
down-regulation of mRNA expression (which may be examined by
Northern blotting, quantitative RT-PCR, in-situ hybridization or
microarray hybridisation, inter alia).
[0356] In additional embodiments the invention provides a method of
treating a patient suffering from a disease accompanied by an
elevated level of RTP801L, the method comprising administering to
the patient a compound of the invention in a therapeutically
effective dose thereby treating the patient.
[0357] More particularly, the invention provides an
oligoribonucleotide wherein one strand comprises consecutive
nucleotides having, from 5' to 3', the sequence set forth in any
one of Tables A-G, or a homolog thereof wherein in up to two of the
ribonucleotides in each terminal region is altered.
[0358] The terminal region of the oligoribonucleotide refers to
bases 1-4 and/or 16-19 in the 19-mer sequence and to bases 1-4
and/or 18-21 in the 21-mer sequence, and to bases 1-4 and/or 20-23
in the 23mer sequence.
[0359] When the nucleic acid according to the present invention is
manufactured or expressed, preferably expressed in vivo, more
preferably in a patient who is in need of the nucleic acid
according to the present invention, such manufacture or expression
preferably uses an expression vector, preferably a mammalian
expression vector. Expression vectors are known in the art and
preferably comprise plasmids, cosmids, viral expression systems.
Preferred viral expression systems include, but are not limited to,
adenovirus, retrovirus and lentivirus.
[0360] Methods are known in the art to introduce the vectors into
cells or tissues. Such methods can be found generally described in
Sambrook et al., Molecular cloning: A Laboratory Manual, Cold
Springs Harbour Laboratory, New York (1983, 1992), or in Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md., 1998.
[0361] Suitable methods comprise, among others, transfection,
lipofection, electroporation and infection with recombinant viral
vectors. In connection with the present invention, an additional
feature of the vector is in one embodiment an expression limiting
feature such as a promoter and regulatory element, respectively,
that are specific for the desired cell type thus allowing the
expression of the nucleic acid sequence according to the present
invention only once the background is provided which allows the
desired expression.
[0362] In a further aspect the present invention is related to a
pharmaceutical composition comprising a nucleic acid according to
the present invention and/or a vector according to the present
invention and, optionally, a pharmaceutically acceptable carrier,
diluent or adjuvants or other vehicle(s). Preferably, such carrier,
diluents, adjuvants and vehicles are inert, and non-toxic. The
pharmaceutical composition is in its various embodiments adapted
for administration in various ways. Such administration comprises
systemic and local administration as well as oral, subcutaneous,
parenteral, intravenous, intraarterial, intramuscular,
intraperitonial, intranasal, and intrategral.
[0363] In particular embodiments the RTP801L siRNA is formulated
eye drops for administration to the surface of the eye. In other
embodiments the RTP801L siRNA compound is administered to the lung
by inhalation. In yet other embodiments the RTP801L siRNA compound
is formulated for delivery to the inner ear by transtympanic
injection or via ear drops.
[0364] It will be acknowledged by the one skilled in the art that
the amount of the pharmaceutical composition and the respective
siRNA depends on the clinical condition of the individual patient,
the site and method of administration, scheduling of
administration, patient age, sex, bodyweight and other factors
known to medical practitioners. The pharmaceutically effective
amount for purposes of prevention and/or treatment is thus
determined by such considerations as are known in the medical arts.
Preferably, the amount is effective to achieve improvement
including but limited to improve the diseased condition or to
provide for a more rapid recovery, improvement or elimination of
symptoms and other indicators as are selected as appropriate
measures by those skilled in the medical arts.
[0365] In a preferred embodiment, the pharmaceutical composition
according to the present invention may comprise other
pharmaceutically active compounds. Preferably, such other
pharmaceutically active compounds are selected from the group
comprising compounds which allow for uptake intracellular cell
delivery, compounds which allow for endosomal release, compounds
which allow for, longer circulation time and compounds which allow
for targeting of endothelial cells or pathogenic cells. Preferred
compounds for endosomal release are chloroquine, and inhibitors of
ATP dependent H.sup.+ pumps.
[0366] The pharmaceutical composition is preferably formulated so
as to provide for a single dosage administration or a multi-dosage
administration.
[0367] The pharmaceutical composition according to the present
invention can also be used in a method for preventing and/or
treating a disease as disclosed herein, whereby the method
comprises the administration of a nucleic acid according to the
present invention, a vector according to the present invention or a
pharmaceutical composition or medicament according to the present
invention for any of the diseases described herein.
[0368] In a further aspect, the present invention is related to a
method for designing or screening a nucleic acid which is suitable
to down-regulate RTP801L, more particularly to down-regulate
RTP801L function. This method comprises the use of a nucleic acid
sequence as disclosed herein and the assessment of such nucleic
acid in a suitable assay. Such assay is known in the art and, for
example, described in the example part of this application. In a
further step, a double-stranded nucleic acid is designed,
preferably according to the design principles as laid down herein,
which is suitable to down-regulate RTP801L, preferably in
connection with a post transcriptional gene silencing mechanism
such as RNA interference. Also the thus obtained, i. e. designed or
screened, nucleic acid is assessed in the respective assay and the
result, i. e. the effect of both the nucleic acid according to the
present invention as well as the newly designed or screened nucleic
acid in such assay compared. Preferably, the designed or screened
nucleic acid is more suitable in case it is either more stable or
more effective, preferably both. It will be acknowledged that the
method will be particularly effective if any of the nucleic acids
according to the present invention is used as a starting point. It
is thus within the present invention that new nucleic acid
molecules will be designed based on the principles disclosed
herein, whereby the target sequence on the RTP801L mRNA will be
slightly shifted relative to the target sequence on the RTP801L
mRNA for the corresponding nucleic acid according to the present
invention. Preferably the new nucleic acid will be shifted by at
least one or more nucleotides relative to the stretch on the target
mRNA in either the 5' or the 3' direction of the mRNA coding for
RTP801L. It is however with in the present invention that the shift
occurs in both directions simultaneously which means that the new
nucleic acid incorporates the nucleic acid according to the present
invention used as a starting point. It is also within the present
invention that the elongation of the nucleic acid according to the
present invention and used as a starting point is biased to either
the 3' end or the 5' end. In case of such as bias either the 3' end
or the 5' end of the new nucleic acid is longer, i.e more extended
than the other end. When the new nucleic acid molecule is generated
by extending either the 3' end of the 5' end of the antisense
strand and/or the sense strand, the following sequence of steps is
typically applied. If the shift is to the 5' end of the mRNA of
RTP801L, the 3' end of the antisense strand has to be extended by
the number of the nucleotides by which the 5' end of the mRNA of
RTP801L is shifted. The nucleotide(s) thus to be added to the 3'
end of the antisense strand of the new nucleic acid is/are
complementary to those nucleotides following at the 5' end of the
target sequence on the RTP801L mRNA used for the nucleic acid
molecule according to the present invention used as a starting
point. The same has to be done to the sense strand. However the
nucleotides to be added to the sense strand have to correspond,
i.e. be complementary to the nucleotides newly added to the 3' end
of the antisense strand which means that they have to be added to
the 5' end of the sense strand. The latter step on the sense
strand, however has to be done only to the extent that apart from
the antisense strand also the sense strand shall be shifted, which
is the case in preferred embodiments of the present invention.
Although this shifting can be done to an extent defined by the ones
skilled in the art, more preferably the shift shall be done such
that also the new nucleic acid still contains a stretch of at least
14 nucleotides, preferably 14 contiguous nucleotides as exhibited
by any of the nucleic acid molecules disclosed herein.
[0369] The synthesis of any of the nucleic acids described herein
is within the skills of the one of the art. Such synthesis is,
among others, described in Beaucage S. L. and Iyer R. P.,
Tetrahedron 1992; 48: 2223-2311, Beaucage S. L. and Iyer R. P.,
Tetrahedron 1993; 49: 6123-6194 and Caruthers M. H. et. al.,
Methods Enzymol. 1987; 154: 287-313, the synthesis of thioates is,
among others, described in Eckstein F., Annu. Rev. Biochem. 1985;
54: 367-402, the synthesis of RNA molecules is described in Sproat
B., in Humana Press 2005 Edited by Herdewijn P.; Kap. 2: 17-31 and
respective downstream processes are, among others, described in
Pingoud A. et. al., in IRL Press 1989 Edited by Oliver R. W. A.;
Kap. 7: 183-208 and Sproat B., in Humana Press 2005 Edited by
Herdewijn P.; Kap. 2: 17-31 (supra).
[0370] siRNA for RTP801L can be made using methods known in the art
as described above, based on the known sequence of RTP801L (SEQ II)
NO:1), and can be made stable by various modifications as described
above. For further information, see Example 5.
Combination Therapy
[0371] The present invention provides for combination therapy for
all the conditions disclosed herein and in particular conditions
involving choroidal neovascularization. In said combination
therapy, both the RTP801L and VEGFR genes are inhibited in order to
ameliorate the symptoms of the disease being treated. These genes
are inhibited with a combination of siRNAs or antibodies (including
aptamer antibodies) or both. The present invention therefore also
provides for a novel pharmaceutical composition comprising an
RTP801L inhibitor and a VEGF or VEGFR-1 inhibitor, the RTP801L
inhibitor preferable being an siRNA, more preferably an siRNA
molecule detailed in any one of Tables A-G, optionally-selected
from the group consisting of the siRNAs of Table F, and the
VEGF/VEGFR-1 inhibitor optionally being an antibody or aptamer. The
combined use of said compounds (i.e., RTP801L siRNA and VEGF
antibody or any other combined example disclosed herein) in the
preparation of a medicament is also part of the present
invention.
[0372] Thus, RTP801L siRNA such as an siRNA molecule detailed
herein and in any one of Tables A-G and optionally siRNA Nos:
DDIT4L.sub.--14 or DDIT4L.sub.--15 of Table F, are administered in
conjunction with agents which target VEGF or VEGF receptor 1
(VEGFR1). Such agents currently exist on the market or in various
stages of approval and work through different mechanisms.
Antibodies and antibody fragments such as ranibizumab (Lucentis,
Genentech) attach to released VEGF to inhibit binding of VEGF to
active receptors. An aptamer which can act like a ligand/antibody
(Macugen, Eyetech/Pfizer, approved recently by the FDA for wet AMD)
is also a possibility. Macugen bonds with extracellular VEGF to
block its activity. These drugs are administered locally by
intravitreal injection. Anti-VEGF siRNA based compounds (such as
Acuity's Cand5 inhibitor of VEGF or SIRNA's 027 inhibitor of
VEGFR-1) are also available. Additionally, the small molecule
aminosterol Squalamine (Genaera) which is administered systemically
reportedly interferes in multiple facets of the angiogenic process,
including inhibiting VEGF and other growth factor signaling in
endothelial cells.
[0373] The conjoined administration of an RTP801L siRNA, and any of
the above VEGF/VEGFR-1 inhibitory agents can have an additive or
even synergistic effect whereby said combined treatment is more
effective than treatment by any of these individual compositions,
irrespective of dosage in the single therapy option. RTP801L siRNA
has a different mechanism of action and is potentially additive or
even synergistic with VEGF-VEGFR inhibitors.
[0374] Additional disorders which can be treated by the molecules
and compositions of the present invention include all types of
choroidal neovascularization (CNV), which occurs not only in wet
AMD but also in other ocular pathologies such as ocular
histoplasmosis syndrome, angiod streaks, ruptures in Bruch's
membrane, myopic degeneration, ocular tumors and some retinal
degenerative diseases.
[0375] An additional aspect of the present invention provides for
methods of treating an apoptosis related disease. Methods for
therapy of diseases or disorders associated with uncontrolled,
pathological cell growth, e.g. cancer, psoriasis, autoimmune
diseases, inter alia, and methods for therapy of diseases
associated with ischemia and lack of proper blood flow, e.g.
myocardial infarction (MI) and stroke, are provided. "Cancer" or
"Tumor" refers to an uncontrolled growing mass of abnormal cells.
These terms include both primary tumors, which may be benign or
malignant, as well as secondary tumors, or metastases which have
spread to other sites in the body. Examples of cancer-type diseases
include, inter alia: carcinoma (e.g.: breast, colon and lung),
leukemia such as B cell leukemia, lymphoma such as B-cell lymphoma,
blastoma such as neuroblastoma and melanoma and sarcoma. It will be
acknowledged that the pharmaceutical composition according to the
present invention can be used for any disease which involves
undesired development or growth of vasculature including
angiogenesis, as well as any of the diseases and conditions
described herein. Preferably, these kind of diseases are tumor
diseases. Among tumor diseases, the following tumors are most
preferred: endometrial cancer, colorectal carcinomas, gliomas,
endometrial cancers, adenocarcinomas, endometrial hyperplasias,
Cowden's syndrome, hereditary non-polyposis colorectal carcinoma,
Li-Fraumene's syndrome, breast-ovarian cancer, prostate cancer
(Ali, I. U., Journal of the National Cancer Institute, Vol. 92, no.
11, Jun. 7, 2000, page 861-863), Bannayan-Zonana syndrome, LDD
(Lhermitte-Duklos' syndrome) (Macleod, K., supra)
hamartoma-macrocephaly diseases including Cow disease (CD) and
Bannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneous lesions (e.
g. trichilemmonmas), macrocephaly, mental retardation,
gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocystic
disease of the breast, cerebellar dysplastic gangliocytoma and
breast and thyroid malignancies (Vazquez, F., Sellers, W. R.,
supra).
[0376] The invention also provides a composition comprising one or
more of the compounds of the invention in a carrier, preferably a
pharmaceutically acceptable carrier. This composition may comprise
a mixture of two or more siRNAs for different genes or different
siRNAs for the same gene. A composition comprising siRNA for the
RTP801L gene and siRNA for the VEGF gene and/or the VEGF-R1 gene is
envisaged.
[0377] This invention also comprises a tandem double-stranded
structure which comprises two or more siRNA sequences, which is
processed intracellularly to form two or more different siRNAs, one
inhibiting RTP801L and a second inhibiting VEGF/VEGFR-1 In a
related aspect, this invention also comprises a tandem
double-stranded structure which comprises two or more siRNA
sequences, which is degraded intracellularly to form two or more
different siRNAs, both inhibiting RTP801L.
[0378] In particular, it is envisaged that a long oligonucleotide
(typically about 80-500 nucleotides in length) comprising one or
more stem and loop structures, where stem regions comprise the
sequences of the oligonucleotides of the invention, are delivered
in a carrier, preferably a pharmaceutically acceptable carrier, and
may be processed intracellularly by endogenous cellular complexes
(e.g. by DROSHA and DICER as described above) to produce one or
more smaller double stranded oligonucleotides (siRNAs) which are
oligonucleotides of the invention. This oligonucleotide can be
termed a tandem shRNA construct. It is envisaged that this long
oligonucleotide is a single stranded oligonucleotide comprising one
or more stem and loop structures, wherein each stem region
comprises a sense and corresponding antisense siRNA sequence of an
801 gene. In particular, it is envisaged that this oligonucleotide
comprises sense and antisense siRNA sequences as depicted in any
one of Tables A-G. Alternatively, the tandem shRNA construct may
comprise sense and complementary antisense siRNA sequence
corresponding to an 801L gene and additionally sense and
complementary antisense siRNA sequence corresponding to a different
gene such as 801, VEGF or VEGF-R1.
[0379] As mentioned herein, siRNA against RTP801L are the main
active component in a pharmaceutical composition, or are one active
component of a pharmaceutical composition containing two or more
siRNAs (or molecules which encode or endogenously produce two or
more siRNAs, be it a mixture of molecules or one or more tandem
molecule which encodes two or more siRNAs), said pharmaceutical
composition further being comprised of one or more additional siRNA
molecule which targets one or more additional gene. Simultaneous
inhibition of RTP801L and said additional gene(s) has an additive
or synergistic effect for treatment of the diseases disclosed
herein, according to the following:
[0380] Acute Renal Failure (ARF) and other microvascular disorders:
the pharmaceutical composition for treatment of ARF comprises of
the following compound combinations: 1) RTP801L siRNA and p53 siRNA
dimers; 2) RTP801L and Fas siRNA dimers; 3) RTP801L and Bax siRNA
dimers; 4) p53 and Fas siRNA dimers; 5) RTP801L and Bax siRNA
dimers; 6) RTP801L and Noxa siRNA dimers; 7) RTP801L and Puma siRNA
dimers; 8) RTP801L (REDD1) and RTP801LL (REDD2) siRNA dimers; 9)
RTP801LsiRNA, Fas siRNA and any of RTP801LL siRNA p53 siRNA, Bax
siRNA, Noxa siRNA or Puma siRNA to form trimers or polymers (i.e.,
tandem molecules which encode three siRNAs).
[0381] Macular degeneration (MD), diabetic retinopathy (DR), spinal
cord injury: pharmaceutical compositions for treatment of MD, DR
and spinal cord injury comprises of the following compound
combinations: 1) RTP801L siRNA combined with either of VEGF siRNA,
VEGF-R1 siRNA, VEGF R2 siRNA, PKCbeta siRNA, MCP1 siRNA, eNOS
siRNA, KLF2 siRNA, RTP801 siRNA (either physically mixed or in a
tandem molecule); 2) RTP801L siRNA in combination with two or more
siRNAs of the above list (physically mixed or in a tandem molecule
encoding three siRNAs, or a combination thereof).
[0382] COPD and respiratory disorders: the pharmaceutical
composition for treatment of respiratory disorders comprises of the
following compound combinations: RTP801L siRNA combined with siRNA
against one or more of the following genes: elastases, matrix
metalloproteases, phospholipases, caspases, sphingomyelinase,
RTP801 and ceramide synthase.
[0383] Further, a combination (tandem) siRNA directed against both
RTP801 and RTP801L can be used to treat any of the conditions
disclosed herein. For Example, the siRNA directed against RTP801
termed REDD14 (sense sequence: 5' GUGCCAACCUGAUGCAGCU 3' and
antisense sequence 5' AGCUGCAUCAGGUUGGCAC 3') can be joined in
tandem with any of the RTP801L siRNAs disclosed herein, such as an
siRNA of Table F, or any other siRNA present in any one of Tables
A-G.
[0384] Additionally, RTP801L siRNA or any nucleic acid molecule
comprising or encoding RTP801L siRNA can be linked (covalently or
non-covalently) to antibodies, in order to achieve enhanced
targeting for treatment of the diseases disclosed herein, according
to the following:
[0385] ARF: anti-Fas antibody (preferably neutralizing
antibodies).
[0386] Macular degeneration, diabetic retinopathy, spinal cord
injury: anti-Fas antibody, anti-MCP1 antibody, anti-VEGFR1 and
anti-VEGFR2 antibody. The antibodies should be preferably be
neutralizing antibodies.
[0387] Any molecules, such as, for example, antisense DNA molecules
which comprise the siRNA sequences disclosed herein (with the
appropriate nucleic acid modifications) are particularly desirable
and are used in the same capacity as their corresponding siRNAs for
all uses and methods disclosed herein.
[0388] The invention also comprises a method of treating a patient
suffering from a disorder such as the disorders described herein
comprising administering to the patient the above composition or
compound in a therapeutically effective dose so as to thereby treat
the patient.
Macular Degeneration
[0389] The most common cause of decreased best-corrected vision in
individuals over 65 years of age in the US is the retinal disorder
known as age-related macular degeneration (AMD). As AMD progresses,
the disease is characterized by loss of sharp, central vision. The
area of the eye affected by AMD is the Macula, a small area in the
center of the retina, composed primarily of photoreceptor cells.
So-called "dry" AMD, accounting for about 85%-90% of AMD patients,
involves alterations in eye pigment distribution, loss of
photoreceptors and diminished retinal function due to overall
atrophy of cells. So-called "wet" AMD involves proliferation of
abnormal choroidal vessels leading to clots or scars in the
sub-retinal space. Thus, the onset of wet AMD occurs because of the
formation of an abnormal choroidal neovascular network (choroidal
neovascularization, CNV) beneath the neural retina. The newly
formed blood vessels are excessively leaky. This leads to
accumulation of subretinal fluid and blood leading to loss of
visual acuity. Eventually, there is total loss of functional retina
in the involved region, as a large disciform scar involving
choroids and retina forms. While dry AMD patients may retain vision
of decreased quality, wet AMD often results in blindness. (Hamdi
& Kenney, May 2003. Frontiers in Bioscience, e305-314).
[0390] Acuity Pharmaceuticals and Sirna Therapeutics, have both
recently filed an IND for siRNA molecules inhibiting VEGF and
VEGF-R1 (Flt-1), respectively, for treatment of AMD. These
molecules are termed Cand5 and Sirna-027 respectively.
Glaucoma
[0391] Glaucoma is one of the leading causes of blindness in the
world. It affects approximately 66.8 million people worldwide. At
least 12,000 Americans are blinded by this disease each year (Kahn
and Milton, 1980. Am J Epidemiol. 111(6):769-76). Glaucoma is
characterized by the degeneration of axons in the optic nerve head,
primarily due to elevated intraocular pressure (IOP). One of the
most common forms of glaucoma, known as primary open-angle glaucoma
(POAG), results from the increased resistance of aqueous humor
outflow in the trabecular meshwork (TM), causing IOP elevation and
eventual optic nerve damage. Mucke (IDrugs 2007, 10(1):37-41)
reviews current therapeutics, including siRNA to various targets
for the treatment of ocular diseases, for example, age-related
macular degeneration (AMD) and glaucoma. Administration of
neuroprotective agents has also been shown to be a viable treatment
for glaucoma. The present invention provides RTP801L siRNA useful
as a neuroprotective agent in the treatment of ION, AEON, and
glaucoma.
Ischemic Optic Neuropathy (ION)
[0392] A severely blinding disease resulting from loss of the
arterial blood supply to the optic nerve (usually in one eye), as a
result of occlusive disorders of the nutrient arteries. Optic
neuropathy can be anterior (AION), which causes a pale edema of the
optic disc, or posterior, in which the optic disc is not swollen
and the abnormality occurs between the eyeball and the optic
chiasm. Ischemic anterior optic neuropathy usually causes a loss of
vision that may be sudden or occur over several days. Ischemic
posterior optic neuropathy is uncommon, and the diagnosis depends
largely upon exclusion of other causes, chiefly stroke and brain
tumor.
Dry-Eye Syndrome
[0393] Dry eye syndrome is a common problem usually resulting from
a decrease in the production of tear film that lubricates the eyes.
Most patients with dry eye experience discomfort, and no vision
loss; although in severe cases, the cornea may become damaged or
infected. Wetting drops (artificial tears) may be used for
treatment while lubricating ointments may help more severe
cases.
Microvascular Disorders
[0394] Microvascular disorders are composed of a broad group of
conditions that primarily affect the microscopic capillaries and
lymphatics and are therefore outside the scope of direct surgical
intervention. Microvascular disease can be broadly grouped into the
vasospastic, the vasculitis and lymphatic occlusive. Additionally,
many of the known vascular conditions have a microvascular element
to them.
Microvascular Pathologies Associated with Diabetes
[0395] Diabetes is the leading cause of blindness, the major cause
of amputations and impotence, and one of the most frequently
occurring chronic childhood diseases. Diabetes is also the leading
cause of end-stage renal disease in the United States, with a
prevalence rate of 31% compared with other renal diseases. Diabetes
is also the most frequent indication for kidney transplantation,
accounting for 22% of all transplantation operations.
[0396] In general, diabetic complications can be classified broadly
as microvascular or macrovascular disease. Microvascular
complications include neuropathy (nerve damage), nephropathy
(kidney disease) and vision disorders (eg retinopathy, glaucoma,
cataract and corneal disease). In the retina, glomerulus, and vasa
nervorum, similar pathophysiologic features characterize
diabetes-specific microvascular disease. All the above listed
conditions and pathologies are also be referred to herein as
conditions "secondary to diabetes",
Emphysema and COPD
[0397] Among the mechanisms that underlie lung destruction in
emphysema, excessive formation of reactive oxygen species (ROS)
should be first of all mentioned. It is well established that
prooxidant/antioxidant imbalance exists in the blood and in the
lung tissue of smokers (Hulea S A, et al: 1995. J Environ Pathol
Toxicol Oncol. 14(3-4):173-80.; Rahman I, MacNee W. 1999. Am J
Physiol. 277(6 Pt 1):L1067-88,; MacNee W. 2000 Chest. 117(5 Suppl
1):3035-17S; Marwick J A, et al., 2002. Ann N Y Acad Sci.
973:278-83; Aoshiba K, et al., 2003. Inhal Toxicol. (10):1029-38;
Dekhuijzen P N. 2004. Eur Respir J. 23(4):629-36; Tuder R M, et
al., 2003. Am J Respir Cell Mol Biol, 29:88-97). After one hour
exposure of mice to CS, there is a dramatic increase of
8-hydroxy-2'-deoxyguanosine (8-OHdG) in the alveolar epithelial
cells, particularly of type II (see Inhal Toxicol. 2003
15(10):1029-38, above).
[0398] Overproduced reactive oxygen species are known for their
cytotoxic activity, which stems from a direct DNA damaging effect
and from the activation of apoptotic signal transduction pathways
(Takahashi et al., 2004. Brain Res Bull. 62(6):497-504; Taniyama Y,
Griendling K K. 2003. Hypertension. 42(6):1075-81; Higuchi Y. 2003.
Biochem Pharmacol. 66(8):1527-35; Punj V, Chakrabarty A M. 2003.
Cell Microbial. (41:225-31; Ueda et al, 2002 Antioxid Redox Signal.
4(0.3):405-14),
[0399] Both reactive oxygen species (ROS) from inhaled cigarette
smoke and those endogenously formed by inflammatory cells
contribute to an increased intrapulmonary oxidant burden.
[0400] One additional pathogenic factor with regards to COPD
pathogenesis is the observed decreased expression of VEGF and
VEGFRII in lungs of emphysematous patients (Kasahara, et al., 2001,
Am J Respir Crit Care Med. 163:737-744). Moreover, inhibition of
VEGF signaling using chemical VEGFR inhibitor leads to alveolar
septal endothelial and then to epithelial cell apoptosis, probably
due to disruption of intimate structural/functional connection of
both types of cells within alveoli (Kasahara, et al., 2000. J.
Clin. Invest 106:1311-1319; Voelkel N F, Cool C D. 2003. Eur Respir
J Suppt 46:28s-32s).
Diabetic Neuropathy
[0401] Diabetic neuropathies are neuropathic disorders (peripheral
nerve damage) associated with diabetes mellitus. These conditions
usually result from diabetic microvascular injury involving small
blood vessels that supply nerves (vasa nervorum). Relatively common
conditions which are associated with diabetic neuropathy include
third nerve palsy; mononeuropathy; mononeuropathy multiplex;
diabetic amyotrophy; a painful polyneuropathy; autonomic
neuropathy; and thoracoabdominal neuropathy and the most common
form, peripheral neuropathy, which mainly affects the feet and
legs. There are four factors involved in the development of
diabetic neuropathy: microvascular disease, advanced glycated end
products, protein kinase C, and the polyol pathway. The compounds
of the present invention are useful in treating microvascular
disease in diabetic neuropathy.
[0402] Neuropathy is a common complication of diabetes occurring
over time in more than half of patients with type 2 diabetes. Nerve
conduction studies demonstrate that neuropathy is already present
in 10-18% of patients at the time of diabetes diagnosis, suggesting
that peripheral nerve injury occurs at early stages of disease and
with milder glycemic dysregulation. The concept that neuropathy is
an early clinical sign of diabetes was proposed >40 years ago,
and most studies report an association between IGT and neuropathy.
Most patients with IGT and associated neuropathy have a symmetric,
distal sensory polyneuropathy with prominent neuropathic pain. IGT
neuropathy (Singleton, J R et al. 2003. 1: Diabetes 52(12):2867-73)
is phenotypically similar to early diabetic neuropathy, which also
causes sensory symptoms, including pain, and autonomic
dysfunction.
[0403] Autonomic dysfunction, particularly erectile dysfunction and
altered cardiac vagal response, are common early features of
neuropathic injury in diabetes. Work with IGT patients also
suggests prevalent vagal dysautonoinia; separate studies have found
abnormal heart rate recovery following exercise, blunted R-R
interval variability to deep breathing, and reduced expiration to
inspiration ratio (all measures of vagal dysautonomia) in a greater
fraction of IGT patients than age-matched normoglycemic control
subjects.
[0404] For further information, see American Journal of Surgery,
Volume 187.cndot.Number 5 Suppl 1.cndot.May 1, 2004, Elsevier.
Coronary Microvascular Dysfunction in Diabetes
[0405] The correlation between histopathology and microcirculatory
dysfunction in diabetes is well known from old experimental studies
and from autopsy, where thickening of the basal membrane,
perivascular fibrosis, vascular rarefication, and capillary
hemorrhage are frequently found. The following papers relate to
microvascular dysfunction (Am J Physiol 2003:285; Hypert Res 2002;
25:893; Sambuceti et al., Circulation 2001.104:1129; Stone 2002;
Feldmann Circulation 2003; Herrmann, Circulation 2001).
Diabetic Nephropathy (Renal Dysfunction in Patients with
Diabetes)
[0406] Diabetic nephropathy encompasses microalbuminuria (a
microvascular disease effect), proteinuria and ESRD. Diabetes is
the most common cause of kidney failure, accounting for more than
40 percent of new cases. Even when drugs and diet are able to
control diabetes, the disease can lead to nephropathy and kidney
failure. Most people with diabetes do not develop nephropathy that
is severe enough to cause kidney failure. About 16 million people
in the United States have diabetes, and about 100,000 people have
kidney failure as a result of diabetes.
Diabetic Retinopathy
[0407] In the diabetic state, hyperglycemia leads to decreased
retinal blood flow, retinal hyperpermeability, delays in
photoreceptor nerve conduction, and retinal neuronal cell death. In
short duration diabetes, neuronal cell death has been identified
within the inner nuclear layer of the retina. Specifically,
apoptosis has been localized to glial cells such as Mueller cells
and astrocytes and has been shown to occur within 1 month of
diabetes in the STZ-induced diabetic rat model. The cause of these
events is multi-factorial including activation of the
diacylglycerol/PKC pathway, oxidative stress, and nonenzymatic
glycosylation. The combination of these events renders the retina
hypoxic and ultimately leads to the development of diabetic
retinopathy. One possible connection between retinal ischemia and
the early changes in the diabetic retina is the hypoxia-induced
production of growth factors such as VEGF. The master regulator of
the hypoxic response has been identified as hypoxia inducible
factor-1 (HIF-1), which controls genes that regulate cellular
proliferation and angiogenesis. Prior studies have demonstrated
that inhibition of HIF-1 ubiquitination leads to binding with
hypoxia responsive elements (HRE) and production of VEGF mRNA.
[0408] Diabetic Retinopathy is defined as the progressive
dysfunction of the retinal vasculature caused by chronic
hyperglycemia. Key features of diabetic retinopathy include
microaneurysms, retinal hemorrhages, retinal lipid exudates,
cotton-wool spots, capillary nonperfusion, macular edema and
neovascularization. Associated features include vitreous
hemorrhage, retinal detachment, neovascular glaucoma, premature
cataract and cranial nerve palsies.
[0409] A microvascular disease that primarily affects the
capillaries, diabetes mellitus affects the eye by destroying the
vasculature in the conjunctiva, retina and central nervous
system.
Neuropathy
[0410] Neuropathy affects all peripheral nerves: pain Fibers, motor
neurons, autonomic nerves. It therefore necessarily can affect all
organs and systems since all are innervated. There are several
distinct syndromes based on the organ systems and members affected,
but these are by no means exclusive. A patient can have
sensorimotor and autonomic neuropathy or any other combination.
Despite advances in the understanding of the metabolic causes of
neuropathy, treatments aimed at interrupting these pathological
processes have been limited by side effects and lack of efficacy.
Thus, treatments are symptomatic and do not address the underlying
problems. Agents for pain caused by sensorimotor neuropathy include
tricyclic antidepressants (TCAs), serotonin reuptake inhibitors
(SSRIs) and antiepileptic drugs (AEDs). None of these agents
reverse the pathological processes leading to diabetic neuropathy
and none alter the relentless course of the illness. Thus, it would
be useful to have a pharmaceutical composition that could better
treat these conditions and/or alleviate the symptoms.
Retinal Microvasculopathy (AIDS retinopathy)
[0411] Retinal microvasculopathy is seen in 100% of AIDS patients.
It is characterized by intraretinal hemorrhages, microaneurysms,
Roth spots, cotton-wool spots (microinfarctions of the nerve fiber
layer) and perivascular sheathing. The etiology of the retinopathy
is unknown though it has been thought to be due to circulating
immune complexes, local release of cytotoxic substances, abnormal
hemorheology, and HIV infection of endothelial cells. AIDS
retinopathy is now so common that cotton wool spots in a patient
without diabetes or hypertension but at risk for HIV should prompt
the physician to consider viral testing. There is no specific
treatment for AIDS retinopathy but its continued presence may
prompt a physician to reexamine the efficacy of the HIV therapy and
patient compliance.
Bone Marrow Transplantation (BMT) Retinopathy
[0412] Bone marrow transplantation retinopathy was first reported
in 1983. It typically occurs within six months, but it can occur as
late as 62 months after BMT. Risk factors such as diabetes and
hypertension may facilitate the development of BMT retinopathy by
heightening the ischemic microvasculopathy. There is no known age,
gender or race predilection for development of BMT retinopathy.
Patients present with decreased visual acuity and/or visual field
deficit. Posterior segment findings are typically bilateral and
symmetric. Clinical manifestations include multiple cotton wool
spots, telangiectasia, microaneurysms, macular edema, hard exudates
and retinal hemorrhages. Fluorescein angiography demonstrates
capillary nonperfusion and dropout, intraretinal microvascular
abnormalities, microaneurysms and macular edema. Although the
precise etiology of BMT retinopathy has not been elucidated, it
appears to be affected by several factors: cyclosporine toxicity,
total body irradiation (TBI), and chemotherapeutic agents.
Cyclosporine is a powerful immunomodulatory agent that suppresses
graft-versus-host immune response. It may lead to endothelial cell
injury and neurologic side effects, and as a result, it has been
suggested as the cause of BMT retinopathy. However, BMT retinopathy
can develop in the absence of cyclosporine use, and cyclosporine
has not been shown to cause BMT retinopathy in autologous or
syngeneic bone marrow recipients. Cyclosporine does not, therefore,
appear to be the sole cause of BMT retinopathy. Total body
irradiation (TBI) has also been implicated as the cause of BMT
retinopathy.
[0413] Radiation injures the retinal microvasculature and leads to
ischemic vasculopathy. Variables such as the total dose of
radiation and the time interval between radiation and bone marrow
ablation appear to be important. However, BMT retinopathy can occur
in patients who did not receive TBI, and BMT retinopathy is not
observed in solid organ transplant recipients who received similar
doses of radiation. Thus, TBI is not the sole cause, but it is
another contributing factor in development of BMT retinopathy.
Chemotherapeutic agents have been suggested as a potential
contributing factor in BMT retinopathy. Medications such as
cisplatin, carmustine, and cyclophosphamide can cause ocular side
effects including papilledema, optic neuritis, visual field deficit
and cortical blindness. It has been suggested that these
chemotherapeutic drugs may predispose patients to radiation-induced
retinal damages and enhance the deleterious effect of radiation. In
general, patients with BMT retinopathy have a good prognosis. The
retinopathy usually resolves within two to four months after
stopping or lowering the dosage of cyclosporine. In one report, 69
percent of patients experienced complete resolution of the retinal
findings, and 46 percent of patients fully recovered their baseline
visual acuity. Because of the favorable prognosis and relatively
non-progressive nature of BMT retinopathy, aggressive intervention
is usually not necessary.
Microvascular Diseases of the Kidney
[0414] The kidney is involved in a number of discreet
clinicopathologic conditions that affect systemic and renal
microvasculature. Certain of these conditions are characterized by
primary injury to endothelial cells, such as: Hemolytic-uremic
syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) and
Radiation nephritis--The long-term consequences of renal
irradiation in excess of 2500 rad.
[0415] In other kidney diseases, the microvasculature of the kidney
is involved in autoimmune disorders, such as systemic sclerosis
(scleroderma). Kidney involvement in systemic sclerosis manifests
as a slowly progressing chronic renal disease or as scleroderma
renal crisis (SRC), which is characterized by malignant
hypertension and acute azotemia. It is postulated that SRC is
caused by a Raynaud-like phenomenon in the kidney. Severe vasospasm
leads to cortical ischemia and enhanced production of renin and
angiotensin II, which in turn perpetuate renal vasoconstriction.
Hormonal changes (pregnancy), physical and emotional stress, or
cold temperature may trigger the Raynaud-like arterial vasospasm.
The role of the renin-angiotensin system in perpetuating renal
ischemia is underscored by the significant benefit of ACE
inhibitors in treating SRC. In patients with SRC who progress to
severe renal insufficiency despite antihypertensive treatment,
dialysis becomes a necessity. Both peritoneal dialysis and
hemodialysis have been employed. The End-Stage Renal Disease (ESRD)
Network report on 311 patients with systemic sclerosis-induced ESRD
dialyzed between 1983 and 1985 revealed a 33% survival rate at 3
years.
[0416] The renal microcirculation can also be affected in sickle
cell disease, to which the kidney is particularly susceptible
because of the low oxygen tension attained in the deep vessels of
the renal medulla as a result of countercurrent transfer of oxygen
along the vasa recta. The smaller renal arteries and arterioles can
also be the site of thromboembolic injury from
cholesterol-containing material dislodged from the walls of the
large vessels.
[0417] Taken as a group, diseases that cause transient or permanent
occlusion of renal microvasculature uniformly result in disruption
of glomerular perfusion, and hence of the glomerular filtration
rate, thereby constituting a serious threat to systemic
homeostasis.
[0418] An additional embodiment of the present invention provides
for the use of a therapeutically effective dose of an RTP801L
inhibitor for the preparation of a medicament for promoting
recovery in a patient suffering from any of the diseases or
conditions described herein eg spinal cord disease or injury. In
one embodiment the inhibitor is preferably an siRNA. In another
embodiment the inhibitor is preferably Structure A depicted
herein.
[0419] The invention has been described in an illustrative manner,
and it is to be understood that the terminology which has been used
is intended to be in the nature of words of description rather than
of limitation.
[0420] Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the appended
claims, the invention can be practiced otherwise than as
specifically described.
[0421] Throughout this application, various publications, including
United States patents, are referenced by author and year and
patents by number. The disclosures of these publications and
patents and patent applications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art to which this invention
pertains.
[0422] Citation of any document herein is not intended as an
admission that such document is pertinent prior art, or considered
material to the patentability of any claim of the present
application. Any statement as to content or a date of any document
is based on the information available to applicant at the time of
filing and does not constitute an admission as to the correctness
of such a statement.
[0423] The present invention is illustrated in detail below with
reference to examples, but is not to be construed as being limited
thereto.
EXAMPLES
[0424] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the claimed invention in any
way.
[0425] Standard molecular biology protocols known in the art not
specifically described herein are generally followed essentially as
in Sambrook et al., Molecular cloning: A laboratory manual. Cold
Springs Harbor Laboratory, New-York (1989, 1992), and in Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1988).
[0426] Standard organic synthesis protocols known in the art not
specifically described herein are generally followed essentially as
in Organic Syntheses: Vol. 1-79, editors vary, J. Wiley, New York,
(1941-2003); Gewert et al., Organic synthesis workbook, Wiley-VCH,
Weinheim (2000); Smith & March, Advanced Organic Chemistry,
Wiley-Interscience; 5th ed. (2001).
[0427] Standard medicinal chemistry methods known in the art not
specifically described herein are generally followed essentially as
in the series "Comprehensive Medicinal Chemistry", by various
authors and editors, published by Pergamon Press.
[0428] The features of the present invention disclosed in the
specification, the claims and/or the drawings may both separately
and in any combination thereof be material for realizing the
invention in various forms thereof.
General Materials and Methods
Cell Culture
[0429] The first human cell line, namely HeLa cells (American Type
Culture Collection) were cultured as follows: Hela cells (American
Type Culture Collection) were cultured as described in Czauderna F
et al. (Czauderna, F., et al., 2003. Nucleic Acids Res, 31,
670-82).
[0430] The second human cell line was a human keratinozyte cell
line which was cultivated as follows: Human keratinocytes were
cultured at 37.degree. C. in Dulbecco's modified Eagle medium
(DMEM) containing 10% FCS. The mouse cell line was B16V (American
Type Culture Collection) cultured at 37.degree. C. in Dulbecco's
modified Eagle medium (DMEM) containing 10% FCS. Culture conditions
were as described in Methods Find Exp Clin Pharmacol. 1997
19(4):231-9.
[0431] In each case, the cells were subject to the experiments as
described herein at a density of about 50,000 cells per well and
the double-stranded nucleic acid according to the present invention
was added at 20 nM, whereby the double-stranded nucleic acid was
complexed using 1 .mu.g/ml of a proprietary lipid.
Induction of Hypoxia-Like Condition
[0432] The cells were treated with CoCl.sub.2 for inducing a
hypoxia-like condition as follows: siRNA transfections were carried
out in 10-cm plates (30-50% confluency) as described by (Czauderna
et al., 2003; Kretschmer et al., 2003). Briefly, siRNA were
transfected by adding a preformed 10.times. concentrated complex of
GB and lipid in serum-free medium to cells in complete medium. The
total transfection volume was 10 ml. The final lipid concentration
was 1.0 .mu.g/ml; the final siRNA concentration was 20 nM unless
otherwise stated. Induction of the hypoxic responses was carried
out by adding CoCl.sub.2 (100 .mu.M) directly to the tissue culture
medium 24 h before lysis.
Preparation of Cell Extracts and Immuno Blotting
[0433] The preparation of cell extracts and immuno blot analysis
were carried out essentially as described by Klippel et al.
(Klippel, A., et al., 1998. Mol Cell Biol, 18, 5699-711; Klippel,
A., et al., 1996. Mol Cell Biol, 16, 4117-27). Polyclonal
antibodies against full length RTP801L were generated by immunising
rabbits with recombinant RTP801L protein producing bacteria from
pET19-b expression vector (Merck Biosciences GmbH, Germany). The
murine monoclonal anti-p110a and anti-p85 antibodies have been
described by Klippel et al. (supra).
In Vitro Testing of siRNA Compounds
[0434] About 1.5-2.times.10.sup.5 tested cells (HeLa cells and/or
293T cells for siRNA targeting human genes and NRK52 (normal rat
kidney proximal tubule cells) cells and/or NMuMG cells (mouse
mammary epithelial cell line) for siRNA targeting the rat/mouse
gene) were seeded per well in 6 wells plate (70-80% confluent). See
also Example 14 hereinbelow.
[0435] About 24 hours later, cells were transfected with siRNA
compounds using the Lipofectamine.TM. 2000 reagent (Invitrogen) at
final concentrations of 5 nM or 20 nM. The cells were incubated at
37.degree. C. in a CO.sub.2 incubator for 72 h.
[0436] As positive control for transfection PTEN-Cy3 labeled siRNA
compounds were used. Various chemically modified blunt ended siRNA
compounds having alternating modified and unmodified
ribonucleotides (modified at the 2' position of the sugar residue
in both the antisense and the sense strands, wherein the moiety at
the 2' position of the sugar is methoxy) and wherein the
ribonucleotides at the 5' and 3' termini of the antisense strand
are modified in their sugar residues, and the ribonucleotides at
the 5' and 3' termini of the sense strand are unmodified in their
sugar residues were tested. Another siRNA compound comprised a
blunt ended structure having an antisense with an alternating
pattern of methoxy moieties and a sense strand with three
ribonucleotides linked by two 2'5' bridges at the 3' terminus; and
another siRNA compound comprising antisense and sense strands
having three ribonucleotides linked by 2'5' bridges at the 3'
terminus was used. Some of the tested compounds comprised a blunt
ended structure having an antisense with an alternating pattern of
methoxy moieties and a sense strand with one or two
L-deoxynucleotides at the 3' terminal or 3' penultimate
positions.
[0437] GFP siRNA compounds were used as negative control for siRNA
activity.
[0438] At 72 h after transfection cells were harvested and RNA was
extracted from cells. Transfection efficiency was tested by
fluorescent microscopy.
[0439] The percent of inhibition of gene expression using specific
preferred siRNA structures was determined using qPCR analysis of a
target gene in cells expressing the endogenous gene.
[0440] In general, the siRNAs having specific sequences that were
selected for in vitro testing were specific for human and a second
species such as non-human primate, rat or rabbit genes. Similar
results are obtained using siRNAs having these RNA sequences and
modified as described herein.
Serum Stability Experiments
[0441] Chemically modified siRNA compounds according to the present
invention awere tested for duplex stability in human serum, as
follows:
[0442] siRNA molecules at final concentration of 7 uM were
incubated at 37.degree. C. in 100% human serum (Sigma Cat#114522).
(siRNA stock 100 uM diluted in human serum 1:14.29).
[0443] 5 ul were added to 15 ul 1.5.times.TBE-loading buffer at
different time points (0, 30 min, 1 h, 3 h, 6 h, 8 h, 10 h, 16 h
and 24 h)
[0444] Samples were immediately frozen in liquid nitrogen and were
kept at -20.degree. C.
[0445] Each sample was loaded onto a non-denaturing 20% acrylamide
gel, prepared according to methods known in the art.
[0446] The oligos were visualized with Ethidium bromide under UV
light.
Example 1
Chemically Modified RTP801L siRNA
[0447] Table 1 hereinbelow provides a code of the modified
nucleotides/unconventional moieties utilized in preparing the siRNA
oligonucleotides of the present invention
TABLE-US-00020 TABLE 1 Code modification Nuc 5medG
5-methyl-deoxyriboguanosine-3'-phosphate c6Np Amino modifier C6
(Glen Research 10-1906-xx) dA deoxyriboadenosine-3'-phosphate dB
abasic deoxyribose-3'-phosphate dC deoxyribocytidine-3'-phosphate
dG deoxyriboguanosine-3'-phosphate dT thymidine-3'-phosphate dT$
thymidine (no phosphate) enaA$ ethylene-bridged nucleic acid
adenosine (no phosphate) enaC ethylene-bridged nucleic acid
cytidine 3' phosphate enaG ethylene-bridged nucleic acid guanosine
3' phosphate enaT ethylene-bridged nucleic acid thymidine 3'
phosphate iB inverted deoxyabasic LdA
L-deoxyriboadenosine-3'-phosphate (mirror image dA) LdA$
L-deoxyriboadenosine (no phosphate) (mirror image dA) LdC
L-deoxyribocytidine-3'-phosphate (mirror image dC) LdC$
L-deoxyribocytidine (no phosphate) (mirror image dC) LdG
L-deoxyriboguanosine-3'-phosphate (mirror image dG) LdT
L-deoxyribothymidine-3'-phosphate (mirror image dT) LdT$
L-deoxyribothymidine (no phosphate) (mirror image dT) mA
2'-O-methyladenosine-3'-phosphate mA$ 2'-O-methyladenosine (no
phosphate) mC 2'-O-methylcytidine-3'-phosphate mC$
2'-O-methylcytidine (no 3'-phosphate) mG
2'-O-methylguanosine-3'-phosphate mG$ 2'-O-methylguanosine (no
phosphate) mU 2'-O-methyluridine-3'-phosphate mU$
2'-O-methyluridine (no phosphate) rA riboadenosine-3'-phosphate rA$
riboadenosine (no phosphate) rC ribocytidine-3'-phosphate rC$
ribocytidine (no phosphate) rC2p ribocytidine-2'-phosphate rG
riboguanosine-3'-phosphate rG2p riboguanosine-2'-phosphate rU
ribouridine-3'-phosphate rU$ ribouridine (no phosphate) rU2p
ribouridine-2'-phosphate
[0448] Table F (FIG. 2) provides certain preferred sense and
corresponding antisense oligonucleotides useful in the preparation
of siRNA compounds. Table G, (FIG. 3) provides chemically modified
siRNA compounds, whereby the sense and antisense sequences are
provided in code according to Table 1 above. In vitro activity of
the compounds is also presented in Table G. The legend for Table G
is as follows:
[0449] *1: Activity in 293 human cells at 20 nM. % residual
transcript relative to untreated control;
[0450] *2: Activity in 293 human cells at 5 nM-% residual
transcript relative to untreated control;
[0451] *3: Activity in rat C6 cells at 20 nM-% residual transcript
relative to untreated control
[0452] *4: Activity in rat C6 cells at 5 nM-% residual transcript
relative to untreated control;
[0453] *5. Stability in Human serum (hours) "-" refers to less than
3 hours duplex stability in 100% human serum.
[0454] FIGS. 8a-8b show the sequence, structure, activity and serum
stability of siRNA compounds according to the present invention.
The oligonucleotides are presented 5'-3', with sense strand above
the antisense strand.
Example 2
Experimental Models, Methods and Results Relating to Ocular
Disease
[0455] The compounds of the present invention are tested in the
following animal model of Choroidal neovascularization (CNV). This
hallmark of wet AMD is induced in model animals by laser
treatment.
A) Mouse Model
[0456] Choroidal neovascularization (CNV) induction: Choroid
neovascularization (CNV), a hallmark of wet AMD, is triggered by
laser photocoagulation (532 nm, 200 mW, 100 ms, 75 .mu.m) (OcuLight
GL, Iridex, Mountain View, Calif.) performed on both eyes of each
mouse on day 0 by a single individual masked to drug group
assignment. Laser spots are applied in a standardized fashion
around the optic nerve, using a slit lamp delivery system and a
cover slip as a contact lens.
Treatment Groups
[0457] CNV is induced in the following groups of mice (males 6-8
weeks of age): Both eyes of each mouse are laser-treated.
12 WT mice; 12 RTP801L Knock-Out mice; 12 WT mice injected with
0.25 .mu.g of synthetic stabilized active anti-RTP801L siRNA in one
eye and inactive anti-RTP801L siRNA (REDD8-negative control) in the
fellow eye, at days 0 and 7; 12 WT mice injected with 0.25 .mu.g of
synthetic stabilized active anti-RTP801L siRNA in one eye and
inactive anti-GFP siRNA (negative control) in the fellow eye at
days 0 and 7; 12 WT mice injected with either 0.1 .mu.g of
synthetic stabilized active anti-RTP801L siRNA in one eye and PBS
(negative control) in the fellow eye at days 0 and 7; 12 WT mice
injected with either 0.05 .mu.g of synthetic stabilized active
anti-RTP801L siRNA in one eye and PBS (negative control) in the
fellow eye at days 0 and 7.
[0458] Evaluation: The experiment is terminated at day 14. For
evaluation, the eyes are enucleated and fixed with 4%
paraformaldehyde for 30 min at 4.degree. C. The neurosensory retina
is detached and severed from the optic nerve. The remaining
RPE-choroid-sclera complex is flat mounted in Immu-Mount
(Vectashield Mounting Medium, Vector) and coverslipped. Flat mounts
are examined with a scanning laser confocal microscope (TCS SP,
Leica, Germany). Vessels are visualized by exciting with blue argon
laser. Horizontal optical sections (1 .mu.m step) are obtained from
the surface of the RPE-choroid-sclera complex. The deepest focal
plane in which the surrounding choroidal vascular network
connecting to the lesion can be identified is judged to be the
floor of the lesion. Any vessel in the laser treated area and
superficial to this reference plane is judged as CNV. Images of
each section are digitally stored. The area of CNV-related
fluorescence is measured by computerized image analysis using the
Leica TCS SP software. The summation of whole fluorescent area in
each horizontal section is used as an index for the volume of
CNV.
[0459] Separate WT mice are used for evaluating RTP801L mRNA
expression in CNV (as well as the expression of other genes
relevant to AMD) (untreated and treated with siRNA) using real-time
PCR on RNA extracted from RPE/choroids, or from neural retina.
[0460] Expression profiling conducted in the mouse model of CNV
revealed that the RTP801L transcript level is gradually increased
in mouse Retina following CNV induction, thus indicating that
RTP801L is a good target for inhibition in the treatment of AMD and
other conditions which involve choroidal neovascularization.
B) Non-Human Primate Model
[0461] CNV induction: Choroidal neovascularization (CNV) is induced
by perimacular laser treatment of both eyes prior to dose
administration. Nine lesions are placed in the macula with a laser
[OcuLight GL (532 nm) Laser Photo-coagulator with an IRIS
Medical.RTM. Portable Slit Lamp Adaptor], and laser spots in the
right eye mirror the placement in the left eye. The approximate
laser parameters are as follows: spot size: 50-100 .mu.m diameter;
laser power: 300-700 milliwatts; exposure time: 0.1 seconds.
[0462] Treatment: Immediately following laser treatment, both eyes
of all animals are subjected to a single intravitreal injection.
Left eye is typicality dosed with 350 ug of synthetic stabilized
siRNA against RTP801L in the final volume of 50 ul, whereas the
contralateral eye receives 50 ul of PBS (vehicle).
Evaluation
[0463] 1. All the animals are subjected to daily examination of
food consumption and body weight measurements. [0464] 2. two
monkeys are euthanized at day 6 following CNV induction. Their eyes
are enucleated and the posterior pole is flattened. Then the fovea
region is excised and separated into choroids and neuroretina which
are separately (for every animal) frozen in liquid nitrogen to be
subsequently used for RNA extraction and real time PCR evaluation
of RTP801L expression. [0465] 3. Fluorescein angiograms are
performed pre-study, and at the end of weeks 1, 2, and 3 following
CNV induction. Photographs are taken, using a fundus camera
(TRC-50EX Retina Camera). Images are captured using the TOPCON
IMAGEnet.TM. system. Fluorescein dye (10% fluorescein sodium,
approximately 0.1 mL/kg) is injected via vascular access ports.
Photographs are taken at several timepoints following dye
injection, to include the arterial phase, early arteriovenous phase
and several late arteriovenous phases in order to evaluate
neovascularization snd to monitor leakage of fluorescein associated
with CNV lesions. Interpretation and analysis of the fluorescein
angiograms is independently conducted by two ophthalmologists.
[0466] Neovascularization (NV) is assessed in early angiograms and
every spot is graded according to the following scheme: [0467]
0--no signs of NV [0468] 0.5--suspicious spot [0469] 1--"hot" spot
[0470] 2--NV in the laser burn [0471] 3--evident NV
[0472] Leakage is assessed according to the following scheme:
[0473] 0--no leakage [0474] 0.5--suspicious spot [0475] 1--evident
small spot leakage [0476] 2--leakage growing with time [0477]
3--leakage greater than previous borders (evidently)
[0478] In addition, the size of every spot is compared between the
early and the late angiograms using morphometric measurements, and
the increase in the spot's size resulting from the leakage is
calculated.
[0479] Electroretinograms (ERGs) are recorded using an Epic 2000
electroretinograph according to Sierra's SOPs and the
study-specific SOP, including the use of the Ganzfield apparatus,
at prestudy and in the end of week 3. The tabulated ERG data are
evaluated by a veterinary ophthalmologist.
C) Efficacy of Combination Therapy of RTP801L siRNA VEGF
Antibody
[0480] The efficacy of combination therapy of RTP801L siRNA and
anti-VEGF antibody or aptamer (such as macugen) in the treatment of
diseases in which CNV occurs is tested in the above mouse CNV
model.
[0481] A) CNV volume studies: The volume of choroidal
neovascularization (CNV) 3 weeks after laser injury is computed by
confocal fluorescence microscopy as previously described (Sakurai
et al. IOVS 2003; 44: 3578-85 & Sakurai et al. IOVS 2003; 44:
2743-2749).
B) CNV Leakage Studies
Experiment 1
[0482] This experiment was designed in order to identify a
potential additive or synergistic therapeutic effect of inhibition
of VEGF and RTP801L in the model of laser-induced choroid
neovascularization in mice
[0483] Materials: Chemically modified RTP801L siRNA; negative
control siRNA (GFP or scrambled); Anti-V EGF antibodies or
Macugen.TM. and negative control.
[0484] CNV is induced on day zero as described above; the test
material is injected to the subjects on day zero and day 7.
[0485] The results are evaluated by Fluorescein angiography on
weeks 1, 2, 3, and by CNV volume measurement on week 3.
Experimental Groups:
[0486] VEGF Ab or macugen 0.5 ng/eye VEGF Ab or macugen 1 ng/eye
VEGF Ab or macugen 2 ng/eye VEGF Ab 4 or macugen ng/eye RTP801L
siRNA 0.05 ug/eye RTP801L siRNA 0.1 ug/eye RTP801L siRNA 0.25
ug/eye RTP801L siRNA 0.05 ug/eye+VEGF Ab or macugen 1 ng/eye
RTP801L siRNA 0.1 ug/eye+VEGF Ab or macugen 1 ng/eye RTP801L siRNA
0.25 ug/eye+VEGF Ab or macugen 1 ng/eye
Control Groups:
PBS
[0487] Non-specific IgG 2 ng/eye negative control 0.1 ug/eye
negative control 0.1 ug/eye+VEGF Ab or macugen 1 ng/eye
[0488] The results show an additive or synergistic therapeutic
effect of inhibition of VEGF and RTP801L
Experiment 2
[0489] This experiment was designed in order to study the effect of
RTP801L siRNA on gene expression in RPE and neural retina.
Experimental Design
Groups:
PBS
[0490] RTP801L siRNA 0.25 mg
[0491] CNV is induced by laser treatment as described above on day
zero; the test material is also injected on day zero, and the
effect evaluated by qPCR analysis of gene expression in RPE and
neural retina on days zero and 5.
[0492] Additional AMD models which are used to test the methods of
the present invention:
[0493] Ccl-2 or Ccr-2 deficient animals--deficiency in either of
these proteins causes the development of some of the main features
of AMD. Animals deficient in these proteins can be used to test the
methods of the present invention.
[0494] For further information on AMD animal models, see: Chader,
Vision research 42 (2002) 393-399; Ambati et al., Nature Medicine
9(11) (2003) 1390-1397; Tolentino et at, Retina 24 (2004)
132-138.
Example 3
Models and Results Relating to COPD and Emphysema
[0495] The compounds of the present invention are tested in the
following models and are shown to prevent emphysema:
[0496] * Cigarette smoke-induced emphysema model: chronic exposure
to cigarette smoke causes emphysema in several animals such as,
inter alia, mouse, guinea pig.
[0497] * Lung protease vity as a trigger of emphysema.
[0498] * VEGFR inhibition model of emphysema.
[0499] * Bronchial instillation with human neutrophil/pancreatic
elastase in rodents.
[0500] * MMP (matrix metalloprotease)-induced enphysema.
[0501] *Inflammation-induced emphysema.
[0502] Additionally, emphysema models are generated through genetic
means (e.g., mice carrying the TSK mutation), and emphysematous
animals may be generated by known modifiers of susceptibility to
emphysema such as, inter alia, lung injury, alveolar hypoplasia,
hyperoxia, glucocorticoid treatment and nutrition.
[0503] Evaluation of the influence of lack of RTP801L on disease
progression in mouse models of emphysema by inhibiting endogenous
RTP801L employing intralung delivery RTP801L-inactivating siRNA
[0504] CS-induced inflammation is induced by 7 day smoking in 2
groups of C57BL6 mice, 10 mice per group. Group 1: CS+delivery of
control siRNA; Group 2: CS+RTP801L siRNA, Control groups of mice
are instilled with either type of siRNA but kept in room air
conditions. The lungs are subsequently agarose-inflated, fixed and
imbedded in paraffin, and development oxidative stress in the KO
mice is assessed by: [0505] a) immunohistochemical localization and
quantitation of 8-oxo-dG in the lung sections; [0506] b)
immunohistochemical localization and quantitation of active caspase
3 in the lung sections using specific antibodies, or quantitative
evaluation of the number of TUNEL-positive cells; [0507] c)
measurement of ceramide concentration in the lung extracts; [0508]
d) measurement of caspase activity in the lung extracts.
Methods
Exposure to Cigarette Smoking (CS)
[0509] Exposure is carried out (7 h/day, 7 days/week) by burning
2R4F reference cigarettes (2.45 mg nicotine per cigarette;
purchased from the Tobacco Research Institute, University of
Kentucky, Lexington, Ky., USA) using a smoking machine (Model
TE-10, Teague Enterprises, Davis, Calif., USA). Each smoldering
cigarette is puffed for 2 s, once every minute for a total of eight
puffs, at a flow rate of 1.05 L/min, to provide a standard puff of
35 cm3. The smoke machine is adjusted to produce a mixture of
sidestream smoke (89%) and mainstream smoke (11%) by burning five
cigarettes at one time. Chamber atmosphere is monitored for total
suspended particulates and carbon monoxide, with concentrations of
90 mg/m3 and 350 ppm, respectively.
Morphologic and Morphometric Analyses
[0510] After exposing the mice to CS or instillation of chemically
modified RTP801L the mice are anesthetized with halothane and the
lungs are inflated with 0.5% low-melting agarose at a constant
pressure of 25 cm as previously described. The inflated lungs are
fixed in 10% buffered formalin and embedded in paraffin. Sections
(5 .mu.m) are stained with hematoxylin and eosin. Mean alveolar
diameter, alveolar length, and mean linear intercepts are
determined by computer-assisted morphometry with the Image Pro Plus
software (Media Cybernetics, Silver Spring, Md., USA). The lung
sections in each group are coded and representative images (15 per
lung section) are acquired by an investigator masked to the
identity of the slides, with a Nikon E800 microscope, 20.times.
lens. The results show that siRNA to 801L prevents emphysema caused
by smoking as measured by the four parameters described above.
Bronchoalveolar Lavage (BAL) and Phenotyping
[0511] Following exposure to CS or instillation of chemically
modified RTP801L, the mice are anesthetized with sodium
pentobarbital. The BAL fluid collected from the lungs of the mice
is centrifuged (500 g at 4.degree. C.), and the cell pellet is
resuspended in phosphate-buffered saline. The total number of cells
in the lavage fluid is determined, and 2.times.104 cells are
cytocentrifuged (Shandon Southern Products, Pittsburgh, Pa., USA)
onto glass slides and stained with Wright-Giemsa stain.
Differential cell counts are performed on 300 cells, according to
standard cytologic techniques.
Identification of Alveolar Apoptotic Cell Populations in the
Lungs.
[0512] To identify the different alveolar cell types undergoing
apoptosis in the lungs, an immunohistochemical staining of active
caspase 3 is performed in the lung sections from the room air (RA)
as well as CS exposed mice. To identify the apoptotic type II
epithelial cells in the lungs, after active caspase 3 labeling, the
lung sections are incubated first with anti-mouse surfactant
protein C (SpC) antibody and then with an anti-rabbit Texas red
antibody. Apoptotic endothelial cells are identified by incubating
the sections first with the anti-mouse CD 31 antibody and then with
the biotinylated rabbit anti-mouse secondary antibody. The lung
sections are rinsed in PBS and then incubated with the
streptavidin-Texas red conjugated complex. The apoptotic
macrophages in the lungs are identified by incubating the sections
first with the rat anti-mouse Mac-3 antibody and then with the
anti-rat Texas red antibody. Finally, DAPI is applied to all lung
sections, incubated for 5 minutes, washed and mounted with
Vectashield HardSet mounting medium. DAPI and fluorescein are
visualized at 330-380 nm and 465-495 nm, respectively. Images of
the lung sections are acquired with the Nikon E800 microscope,
40.times. lens.
Immunohistochemical Localization of Active Caspase-3
[0513] Immunohistochemical staining of active caspase-3 assay is
performed using anti-active caspase-3 antibody and the active
caspase-3-positive cells are counted with a macro, using Image Pro
Plus program. The counts are normalized by the sum of the alveolar
profiles herein named as alveolar length and expressed in .mu.m.
Alveolar length correlates inversely with mean linear intercept,
i.e., as the alveolar septa are destroyed, mean linear intercepts
increases as total alveolar length, i.e., total alveolar septal
length decreases.
Caspase 3 Activity Assay
[0514] The caspase-3/7 activity is measured in lung tissue extracts
using a fluorometric assay according to the manufacturer's
instructions. Snap-frozen lung tissue (n=3 per group) was
homogenized with the assay buffer, followed by sonication and
centrifugation at 800.times.g. After removal of nuclei and cellular
debris, the supernatant (300 .mu.g protein) is then incubated with
the pro-fluorescent substrate at room temperature for 1 h and the
fluorescence intensity was measured utilizing a Typhoon
phosphoimager (Amersham Biosciences, Inc., Piscataway, N.J., USA).
The results are expressed as the rate of specific caspase-3
substrate cleavage, expressed in units of caspase 3 enzymatic
activity, normalized by total protein concentration. Active
recombinant caspase 3 was utilized as the assay standard (0-4 U).
Tissue lysates without substrate, assay buffer alone, and lysates
with caspase 3 inhibitor were utilized as negative controls,
Immunohistochemical Localization of 8-oxo-dG
[0515] For the immunohistochemical localization and quantification
of 8-oxo-dG, lung sections from the mice exposed to CS or instilled
with chemically modified RTP801L are incubated with anti-8-oxo-dG
antibody and stained using InnoGenex.TM. Iso-IHC DAB kit using
mouse antibodies. The 8-oxo-dG-positive cells are counted with a
macro (using Image Pro Plus), and the counts were normalized by
alveolar length as described.
Instillation of siRNA into Mouse Lungs
[0516] Chemically modified RTP801L (50 ug) is delivered in 80 ul
sterile perfluorocarbon. The oxygen carrying properties of
perfluorocarbon make it well-tolerated at these volumes, while its
physical-chemical properties allow for extremely efficient distal
long delivery when instilled intratracheally. Mice are anesthetized
by brief inhalational halothane exposure, the tongue is gently
pulled forward by forceps and the trachea instilled with
perfluorocarbon solution applied at the base of the tongue via a
blunt angiocatheter.
[0517] Mice are anesthetized with an intra-peritoneal injection of
Ketamine/Xylazine (115/22 mg/kg). 50 .mu.g of siRNA is instilled
intranasally in 50 .mu.l volume of 0.9% NaCl by delivering five
consecutive 10 .mu.l portions. At the end of the intranasal
instillation, the mouse's head is held straight up for 1 minute to
ensure that all the instilled solution drains inside.
[0518] For further information, see: Rangasamy T, et al., 2004.
J.C.I. 114(9):1248-59; Kasahara, Y et al., Am J Respir Crit Care
Med Vol 163. pp 737-744, 2001; Kasahara, Y et al., 2000. J. Clin.
Invest. 106:1311-1319; and Tuder, R M et al., Pulmonary
Pharmacology & Therpaeutics 2002.
Example 4
Models and Results Relating to Microvascular Disorders
[0519] The compounds of the present invention are tested in animal
models of a range of microvascular disorders as described
below.
1. Diabetic Retinopathy
[0520] RTP801L promotes neuronal cell apoptosis and generation of
reactive oxygen species in vitro. Experiment 1: Diabetes is induced
in 8 wk old RTP801L KO and C57/129sv wildtype (WT) littermate mice
by intraperitoneal injection of STZ. After 4 weeks, ERG (single
white flash, (0.4.times.10 4 ftc, 5 ms) is obtained from the left
eye after 1 hour of dark adaptation. RVP is assessed from both eyes
using the Evans-blue albumin permeation technique.
[0521] Experiment 2: Diabetes is induced in RTP801L knockout and in
control wild type mice with the matched genetic background. In
addition, it is induced in C57B16 mice, which are subsequently used
for intravitreal injection of anti-RTP801L and control siRNAs. For
diabetes induction, the mice are injected with streptozotocin (STZ
90 mg/kg/d for 2 days after overnight fast). Animal physiology is
monitored throughout the study for changes in blood glucose, body
weight, and hematocrit. Vehicle-injected mice serve as controls.
The appropriate animals are treated by intravitreal injections of 1
ug of RTP801L siRNA or 1 ug of GFP control siRNA. siRNA is injected
twice in the course of the study--on day 0, when the first STZ
injection is performed, and on day 14 after the STZ injection.
[0522] Retinal vascular leakage is measured using the Evans-blue
(EB) dye technique on the animals after 4 weeks duration of
diabetes. Mice have a catheter implanted into the right jugular
vein 24 hours prior to Evans Blue (EB) measurements. Retinal
permeability measurements in both eyes of each animal follows a
standard Evans-blue protocol.
2. Retinopathy of Prematurity
[0523] Retinopathy of prematurity is induced by exposing the test
animals to hypoxic and hyperoxic conditions, and subsequently
testing the effects on the retina.
3. Myocardial Infarction
[0524] Myocardial infarction is induced by Left Anterior Descending
artery ligation in mice, both short term and long term.
4. Microvascular Ischemic Conditions
[0525] Animal models for assessing ischemic conditions include:
[0526] 1. Closed Head Injury (CHI)--Experimental TBI produces a
series of events contributing to neurological and neurometabolic
cascades, which are related to the degree and extent of behavioral
deficits. CHI is induced under anesthesia, while a weight is
allowed to free-fall from a prefixed height (Chen et al, J.
Neurotrauma 13, 557, 1996) over the exposed skull covering the left
hemisphere in the midcoronal plane. [0527] 2. Transient middle
cerebral artery occlusion (MCAO)--a 90 to 120 minutes transient
focal ischemia is performed in adult, male Sprague Dawley rats,
300-370 gr. The method employed is the intraluminal suture MCAO
(Longa et al., Stroke, 30, 84, 1989, and Dogan et al., J.
Neurochem. 72, 765, 1999). Briefly, under halothane anesthesia, a
3-0-nylon suture material coated with Poly-L-Lysine is inserted
into the right internal carotid artery (ICA) through a hole in the
external carotid artery. The nylon thread is pushed into the ICA to
the right MCA origin (20-23 mm). 90-120 minutes later the thread is
pulled off, the animal is closed and allowed to recover. [0528] 3.
Permanent middle cerebral artery occlusion (MCAO)--occlusion is
permanent, unilateral-induced by electrocoagulation of MCA. Both
methods lead to focal brain ischemia of the ipsilateral side of the
brain cortex leaving the contralateral side intact (control). The
left MCA is exposed via a temporal craniectomy, as described for
rats by Tamura A., et al., J Cereb Blood Flow Metab. 1981; 1:53-60.
The MCA and its lenticulostriatal branch are occluded proximally to
the medial border of the olfactory tract with microbipolar
coagulation. The wound is sutured, and animals returned to their
home cage in a room warmed at 26.degree. C. to 28.degree. C. The
temperature of the animals is maintained all the time with an
automatic thermostat.
5. Acute Renal Failure (ARF)
[0529] Testing active siRNA for treating ARF is done using
sepsis-induced ARF or ischemia-reperfusion-induced ARF
1. Sepsis Induced ARF
[0530] Two predictive animal models of sepsis-induced ARF are
described by Miyaji T, et al., Kidney Int. 64(5):1620-31. These two
models are lipopolysaccharide administration and cecal ligation
puncture in mice, preferably in aged mice.
2. Ischemia-Reperfusion-Induced ARF
[0531] This predictive animal model is described by Kelly K J, et
al., 2003. J Am Soc Nephrol.; 14(1):128-38.
[0532] Ischemia-reperfusion injury is induced in rats following 45
minutes bilateral kidney arterial clamp and subsequent release of
the clamp to allow 24 hours of reperfusion. RTP801L siRNA or GFP
siRNA (negative control) are injected into the jugular vein 2 hrs
prior to and 30 minutes following the clamp. Additional siRNA is
given via the tail vein at 4 and 8 hrs after the clamp. ARF
progression is monitored by measurement of serum creatinine levels
before and 24 hrs post surgery. At the end of the experiment, the
rats are perfused via an indwelling femoral line with warm PBS
followed by 4% paraformaldehyde. The left kidneys are removed and
stored in 4% paraformaldehyde for subsequent histological analysis.
Acute renal failure is frequently defined as an acute increase of
the serum creatinine level from baseline. An increase of at least
0.5 mg per dL or 44.2 .mu.mol per L of serum creatinine is
considered as an indication for acute renal failure. Serum
creatinine is measured at time zero before the surgery and at 24
hours post ARF surgery. siRNA to 801L prevents production of ARF in
this model.
[0533] To study the distribution of siRNA in the rat kidney,
Cy3-labeled 19-mer blunt-ended siRNA molecules (2 mg/kg) having
alternating O-methyl modification in the sugar residues were
administered iv for 3-5 min, after which in vivo imaging was
conducted using two-photon confocal microscopy. The confocal
microscopy analysis revealed that the majority of siRNA in the
kidneys is concentrated in the endosomal compartment of proximal
tubular cells. Both endosomal and cytoplasmic siRNA fluorescence
were relatively stable during the first 2 hrs post delivery and
disappeared at 24 hrs.
[0534] The expression of RTP801L during ischemia-reperfusion
induced ARF was examined in rat kidneys. In both kidney regions,
cortex and medulla, RTP801L transcript level is decreased in the
ARF-10 hr group relative to the control group transcript level.
RTP801L transcript level is also elevated (up-regulated) in the
kidney medulla, 3 and 6 hrs following the ARF operation (bilateral
renal artery clamp).
Example 5
Selection and Preparation of siRNAs
[0535] Using proprietary algorithms and the known sequence of the
mRNA of gene RTP801L (SEQ ID NO:1), the sequences of many potential
siRNAs were generated. siRNA molecules according to the above
specifications were prepared essentially as described herein.
[0536] The siRNAs of the present invention can be synthesized by
any of the methods which are well-known in the art for synthesis of
ribonucleic (or deoxyribonucleic) oligonucleotides. For example, a
commercially available machine (available, inter alia, from Applied
Biosystems) can be used; the oligonucleotides are prepared
according to the sequences disclosed herein. Overlapping pairs of
chemically synthesized fragments can be ligated using methods well
known in the art (e.g., see U.S. Pat. No. 6,121,426). The strands
are synthesized separately and then are annealed to each other in
the tube. Then, the double-stranded siRNAs are separated from the
single-stranded oligonucleotides that were not annealed (e.g.
because of the excess of one of them) by 1-IPLC. In relation to the
siRNAs or siRNA fragments of the present invention, two or more
such sequences can be synthesized and linked together for use in
the present invention.
[0537] The siRNA molecules of the invention are synthesized by
procedures known in the art e.g. the procedures as described in
Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al.,
1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995, Nucleic
Acids Res. 23, 2677-2684; and Wincott et al., 1997, Methods Mol.
Bio., 74, 59, and may make use of common nucleic acid protecting
and coupling groups, such as dimethoxytrityl at the 5'-end, and
phosphoramidites at the 3'-end. The modified (e.g. 2'-O-methylated)
nucleotides and unmodified nucleotides are incorporated as
desired.
[0538] Alternatively, the nucleic acid molecules of the present
invention can be synthesized separately and joined together
post-synthetically, for example, by ligation (Moore et al., 1992,
Science 256, 9923; Draper et al., International PCT publication No.
WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19,
4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951;
Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by
hybridization following synthesis and/or deprotection.
[0539] The siRNA molecules of the invention can also be synthesized
via a tandem synthesis methodology, as described in US patent
application publication No. US2004/0019001 (McSwiggen) wherein both
siRNA strands are synthesized as a single contiguous
oligonucleotide fragment or strand separated by a cleavable linker
which is subsequently cleaved to provide separate siRNA fragments
or strands that hybridize and permit purification of the siRNA
duplex. The linker can be a polynucleotide linker or a
non-nucleotide linker. Note that in the attached Table A, the sense
and antisense strands of siRNAs have SEQ ID NOS: 2-1851.
[0540] Similarly, the sense and antisense strands of the siRNAs in
attached tables have SEQ ID NOS:1852-6927.
[0541] Further note that the coding region of gene RTP801L, as
presented in FIG. 1, is between nucleotides 204-785. Therefore, any
siRNA within this region targets the coding region of RTP801L, and
any siRNA outside this region targets the non-coding region of
RTP801L i.e. the 5'UTR or the 3' UTR. The exact region targeted by
each siRNA is given in the above Tables.
[0542] Additionally, sequences presented in the Tables are depicted
in the 5' to 3'direction.
Example 6
Pharmacology and Drug Delivery
[0543] The compounds or pharmaceutical compositions of the present
invention are administered and dosed in accordance with good
medical practice, taking into account the clinical condition of the
individual patient, the disease to be treated, the site and method
of administration, scheduling of administration, patient age, sex,
body weight and other factors known to medical practitioners.
[0544] The pharmaceutically "effective amount" for purposes herein
is thus determined by such considerations as are known in the art.
The amount must be effective to achieve improvement including but
not limited to improved survival rate or more rapid recovery, or
improvement or elimination of symptoms and other indicators as are
selected as appropriate measures by those skilled in the art.
[0545] The treatment generally has a length proportional to the
length of the disease process and drug effectiveness and the
patient species being treated. It is noted that humans are treated
generally longer than the mice or other experimental animals
exemplified herein.
[0546] The compounds of the present invention are administered by
any of the conventional routes of administration. It should be
noted that the compound can be administered as the compound or as
pharmaceutically acceptable salt and can be administered alone or
as an active ingredient in combination with pharmaceutically
acceptable carriers, solvents, diluents, excipients, adjuvants and
vehicles. The compounds can be administered orally, subcutaneously
or parenterally including intravenous, intraarterial,
intramuscular, intraperitoneally, and intranasal administration as
well as intrathecal and infusion techniques. Implants of the
compounds are also useful. Liquid forms are prepared for injection,
the term including subcutaneous, transdermal, intravenous,
intramuscular, intrathecal, and other parental routes of
administration. The liquid compositions include aqueous solutions,
with and without organic cosolvents, aqueous or oil suspensions,
emulsions with edible oils, as well as similar pharmaceutical
vehicles. In addition, under certain circumstances the compositions
for use in the novel treatments of the present invention are formed
as aerosols, for intranasal and like administration. The patient
being treated is a warm-blooded animal and, in particular, mammals
including man. The pharmaceutically acceptable carriers, solvents,
diluents, excipients, adjuvants and vehicles as well as implant
carriers generally refer to inert, non-toxic solid or liquid
fillers, diluents or encapsulating material not reacting with the
active ingredients of the invention.
[0547] When administering the compound of the present invention
parenterally, it is generally formulated in a unit dosage
injectable form (solution, suspension, emulsion). The
pharmaceutical formulations suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
The carrier can be a solvent or dispersing medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils.
[0548] Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, can also be used as solvent
systems for compound compositions. Additionally, various additives
which enhance the stability, sterility, and isotonicity of the
compositions, including antimicrobial preservatives, antioxidants,
chelating agents, and buffers, can be added. Prevention of the
action of microorganisms can be ensured by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, and the like. In many cases, it is desirable
to include isotonic agents, for example, sugars, sodium chloride,
and the like. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption,
for example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
have to be compatible with the compounds.
[0549] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various of the other ingredients, as desired.
[0550] A pharmacological formulation of the present invention can
be administered to the patient in an injectable formulation
containing any compatible carrier, such as various vehicle,
adjuvants, additives, and diluents; or the compounds utilized in
the present invention can be administered parenterally to the
patient in the form of slow-release subcutaneous implants or
targeted delivery systems such as monoclonal antibodies, vectored
delivery, iontophoretic, polymer matrices, liposomes, and
microspheres. Examples of delivery systems useful in the present
invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;
4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;
4,439,196; and 4,475,196. Many other such implants, delivery
systems, and modules are well known to those skilled in the
art.
[0551] A pharmacological formulation of the compound utilized in
the present invention can be administered orally to the patient.
Conventional methods such as administering the compound in tablets,
suspensions, solutions, emulsions, capsules, powders, syrups and
the like are usable. Known techniques which deliver it orally or
intravenously and retain the biological activity are preferred. In
one embodiment, the compound of the present invention can be
administered initially by intravenous injection to bring blood
levels to a suitable level. The patient's levels are then
maintained by an oral dosage form, although other forms of
administration, dependent upon the patient's condition and as
indicated above, can be used.
[0552] In general, the active dose of compound for humans is in the
range of from 1 ng/kg to about 20-100 mg/kg body weight per day,
preferably about 0.01 mg to about 2-10 mg/kg body weight per day,
in a regimen of one dose per day or twice or three or more times
per day for a period of 1-2 weeks or longer, preferably for 24- to
48 hrs or by continuous infusion during a period of 1-2 weeks or
longer.
Administration of Compounds of the Present Invention to the Eye
[0553] The compounds of the present invention can be administered
to the eye topically or in the form of an injection, such as an
intravitreal injection, a sub-retinal injection or a bilateral
injection. Preferred methods of delivery to the eye is using siRNA
dormulated as eye drops.
[0554] Further information on administration of the compounds of
the present invention can be found in Tolentino et al., Retina 24
(2004) 132-138; Reich et at, Molecular vision 9 (2003) 210-216,
Pulmonary Administration of Compounds of the Present Invention
[0555] The therapeutic compositions of the present invention are
preferably administered into the lung by inhalation of an aerosol
containing these compositions/compounds, or by intranasal or
intratracheal instillation of said compositions. Formulating the
compositions in liposomes may benefit absorption. Additionally, the
compositions may include a PFC liquid such as perflubron, and the
compositions formulated as a complex of the compounds of the
invention with polyethylemeimine (PEI).
[0556] For further information on pulmonary delivery of
pharmaceutical compositions see Weiss et al., Human gene therapy
10:2287-2293 (1999); Densmore et al., Molecular therapy 1:180-188
(1999); Gautam et al., Molecular therapy 3:551-556 (2001); and
Shahiwala Misra, AAPS PharmSciTech 5 (2004). Additionally,
respiratory formulations for siRNA are described in U.S. patent
application No. 2004/0063654 of Davis et el.
[0557] Further, the compounds of the present invention are
administered topically where appropriate (such as in the case of
diabetic foot ulcers for example), optionally in a lipid/liposome
formulation, or for use in iontophoresis.
[0558] A preferred administration mode is topical delivery of the
RTP801L inhibitors onto the round window membrane of the cochlea as
disclosed for example in Tanaka et al. (Hear Res. 2003;
177(1-2):21-31). Preferred delivery to the inner ear comprising
administering the siRNA as an ear drop formulation.
[0559] In the treatment of pressure sores or other wounds, the
administration of the pharmaceutical composition is preferably by
topical application to the damages area, but the compositions may
also be administered systemically.
[0560] Additional formulations for improved delivery of the
compounds of the present invention can include non-formulated
compounds, compounds covalently bound to cholesterol, and compounds
bound to targeting antibodies (Song et al., 2005. Nat Biotechnol.
23(6):709-17).
Example 7
Model Systems for Pressure Sores or Pressure Ulcers
[0561] Pressure sores or pressure ulcers including diabetic ulcers,
are areas of damaged skin and tissue that develop when sustained
pressure (usually from a bed or wheelchair) cuts off circulation to
vulnerable parts of the body, especially the skin on the buttocks,
hips and heels. The lack of adequate blood flow leads to ischemic
necrosis and ulceration of the affected tissue. Pressure sores
occur most often in patients with diminished or absent sensation or
who are debilitated, emaciated, paralyzed, or long bedridden.
Tissues over the sacrum, ischia, greater trochanters, external
malleoli, and heels are especially susceptible; other sites may be
involved depending on the patient's situation.
[0562] Testing the active inhibitors of the invention (such as
siRNA) for treating pressure sore, ulcers and similar wounds is
done in the mouse model described in Reid R R, et al., J Surgical
Research. 116: 172-180, 2004.
[0563] Additionally, a rabbit model is described by Mustoe et al,
JCI, 1991; Ahn & Mustoe, Ann Pl Surg, 1991 and is used for
testing the siRNAs of the invention.
Example 8
Model Systems for Spinal Cord Injury
[0564] Spinal cord injury, or myelopathy, is a disturbance of the
spinal cord that results in loss of sensation and/or mobility. The
two common types of spinal cord injury are due to trauma and
disease. Traumatic injury can be due to automobile accidents,
falls, gunshot, diving accidents inter alia, and diseases which can
affect the spinal cord include polio, spina bifida, tumors and
Friedreich's ataxia.
[0565] Testing the active inhibitors of the invention (such as
siRNA) for treating spinal cord injury is done in the rat spinal
cord contusion model as described by Young, W. in Prog Brain Res.
2002; 137:231-55. Other predictive animal models of spinal cord
injury are described in the following references: Gruner, J A 1992.
J Neurotrauma 9(2): 123; Hasegawa, K. and M. Grumet 2003. J
Neurosurg 98(5): 1065-71; and Huang, P P and W. Young (1994). J
Neurotrauma 11(5): 547.
Example 9
Model Systems for Glaucoma
[0566] Testing the active inhibitors of the invention (such as
siRNA) for treating or preventing Glaucoma is done in the animal
model for example as described by Pease et al., J. Glaucoma, 2006,
15(6):512-9 (Manometric calibration and comparison of TonoLab and
TonoPen tonometers in rats with experimental glaucoma and in normal
mice).
Rat Optic Nerve Crush (ONC) Model: Intravitreal siRNA Delivery and
Eye Drop Delivery
[0567] For optic nerve transsection the orbital optic nerve (ON) of
anesthetized rats is exposed through a supraorbital approach, the
meninges severed and all axons in the ON transected by crushing
with forceps for 10 seconds, 2 mm from the lamina cribrosa.
[0568] The siRNA compounds are delivered alone or in combination in
5 uL volume (10 ug/uL) as eye drops. Immediately after optic nerve
crush (ONC), 20 ug/10 ul test siRNA or 10 ul PBS is administered to
one or both eyes of adult Wistar rats and the levels of siRNA taken
up into the dissected and snap frozen whole retinae at 5 h and 1 d,
and later at 2 d, 4 d, 7 d, 14 d and 21 d post injection is
determined. Similar experiments are performed in order to test
activity and efficacy of siRNA administered via eye drops.
Example 10
Model Systems for Ischemia and Reperfusion Injury Following Lung
Transplantation in Rats
[0569] Testing the active inhibitors of the invention (such as
siRNA) for treating or preventing Ischemia and reperfusion injury
following lung transplantation is done in the animal model for
example as described by Mizobuchi et al., J. Heart Lung Transplant
2004:23:889-93.
Example 11
Model Systems for Acute Lung Injury (ALI)
[0570] Intratracheal (i.t) administration of LPS
(Lipopolysaccharide), a bacterial cell wall component, is an
accepted experimental model of acute lung injury (ALI), as LPS
stimulates profound lung recruitment of inflammatory cells and the
subsequent development of systemic inflammation.
[0571] (See, for example, Fang W F, et al., Am J Physiol Lung Cell
Mol Physiol. 2007 293(2):L336-44; Hagiwara S. Iwasaka H, Noguchi T.
J Anesth. 2007; 21(2):164-70).
[0572] Time-dependent changes of RTP801Lgene expression in mice
lungs during the first 24 hours (time points 0.5; 1; 2; 4; 8 &
24 hours), after Intratracheal (i.t) administration of LPS was
assessed. The assessment of gene expression was done using
qPCR.
[0573] The results indicate that the level of the RTP801L
transcript is gradually decreased following LPS instillation.
Example 12
Model Systems for Acute Respiratory Distress Syndrome
[0574] Testing the active inhibitors of the invention (such as
siRNA) for treating Acute respiratory distress syndrome is
performed, inter alia, in the animal model as described by Chen et
al. in J Biomed Sci. 2003; 10(6 Pt 1):588-92.
Example 13
Model Systems for Hearing Loss Conditions
(i) Animal Model of Carboplatin-Induced or Cisplatin-Induced Hair
Cell Death in the Cochlea of Chinchilla:
[0575] Chinchillas are pre-treated by direct administration of
specific siRNAs to RTP801L in saline to the left ear of each
animal. Saline is given to the right ear of each animal as placebo.
Two days following the administration of the specific siRNA, the
animals are treated with carboplatin (75 mg/kg ip) or cisplatin
(intraperitoneal infusion of 13 mg/kg over 30 minutes). After
sacrifice of the chinchillas (two weeks post carboplatin treatment)
the percentage of dead cells of inner hair cells (IHC) and outer
hair cells (OHC) is calculated in the left ear (siRNA treated) and
in the right ear (saline treated). The percentage of dead cells is
lower in the siRNA treated ear than in the control
(ii) Animal Model of Acoustic-Induced Hair Cell Death in the
Cochlea of Chinchilla:
[0576] The activity of specific siRNA to RTP801L (e.g. chemically
modified siRNA Nos: 72 or 73 in Table A) in an acoustic trauma
model is studied in chinchilla. The animals are exposed to an
octave band of noise centered at 4 kHz for 2.5 h at 105 dB. The
left ear of the noise-exposed chinchillas is pre-treated (48 h
before the acoustic trauma) with 30 .mu.g of either siRNA in
.about.10 .mu.L of saline; the right ear is pre-treated with
vehicle (saline). The compound action potential (CAP) is a
convenient and reliable electrophysiological method for measuring
the neural activity transmitted from the cochlea. The CAP is
recorded by placing an electrode near the base of the cochlea in
order to detect the local field potential that is generated when a
sound stimulus, such as click or tone burst, is abruptly turned on.
The functional status of each ear is assessed 2.5 weeks after the
acoustic trauma. Specifically, the mean threshold of the compound
action potential recorded from the round window is determined 2.5
weeks after the acoustic trauma in order to determine if the
thresholds in the siRNA-treated ear are lower (better) than the
untreated (saline) ear. In addition, the amount of inner and outer
hair cell loss is determined in the siRNA-treated and the control
ear. It is found that the thresholds in the siRNA-treated ear are
lower than the untreated (saline) ear Also, the amount of hair cell
loss is lower in the siRNA-treated ear than in the control ear.
Example 14
RTP801L siRNA Structures and Activity
[0577] In addition to Example 1 the siRNA compounds of the present
invention are tested in the following model. The following negative
controls were used:
a) Cy3-labeled synthetic stabilized siRNA against human, mouse and
rat PTEN gene (PTEN-Cy3). Stock solution 20 mg/ml in double
distilled. b) Synthetic stabilized siRNA against GFP (GFP siRNA).
Stock solution 20 mg/nil in double distilled.
[0578] The cells used in the experiment were 801 wt and Ko mouse
embryonic fibroblasts (MEF) cells and 293T embryonic kidney
cells.
[0579] The transfection reagent used was Lipofectamine.TM. 2000
(Invitrogene, Cat#11668-019).
[0580] Methods: 3.times.10.sup.5 and 1.times.10.sup.5 801 wt MEF
and 293T cells were seeded per well of the 6 wells plate,
respectively. 24 h subsequently, cells were transfected with:
RTP801L siRNA molecules at final concentrations per well of 0.5-40
nM GFP siRNA molecules at final concentrations per well of 0.5-40
nM PTEN-Cy3 siRNA at final concentrations per well of 20-40 nM
[0581] Transfection mixture per each well contained 3 ul
lipofectamine 2000 reagent (in 250 ul serum free medium).
[0582] RNA was extracted from cells 72 h following transfection. In
the last 8 h of incubation, 500 uM H.sub.2O.sub.2 was added to wt
MEF cells.
[0583] RNA was prepared from the cells and processed, and qPCR was
performed for the evaluation of RTP801L mRNA levels, using mouse or
human RTP801L-specific oligonucleotides and Cyclophylin as a
reference gene.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140350068A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140350068A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References