U.S. patent application number 11/908696 was filed with the patent office on 2011-12-22 for treatment of intestinal conditions.
This patent application is currently assigned to Sylentis S.A.U.. Invention is credited to Irene Gascon, Ana I. Jimenez, Maria Concepcion Jimenez, Jose P. Roman, Angela Sesto.
Application Number | 20110313016 11/908696 |
Document ID | / |
Family ID | 34508943 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110313016 |
Kind Code |
A1 |
Jimenez; Ana I. ; et
al. |
December 22, 2011 |
Treatment of Intestinal Conditions
Abstract
Methods and compositions for the treatment of intestinal
disorders, such as IBD and Crohn's disease, are disclosed.
Preferred compositions include siNA. Also disclosed is a method of
specifically targeting siNA to treat intestinal disorders by
intrarectal administration of siNA compounds.
Inventors: |
Jimenez; Ana I.; (Madrid,
ES) ; Gascon; Irene; (Madrid, ES) ; Jimenez;
Maria Concepcion; (Madrid, ES) ; Roman; Jose P.;
(Vitoria, ES) ; Sesto; Angela; (Madrid,
ES) |
Assignee: |
Sylentis S.A.U.
Midrid
ES
|
Family ID: |
34508943 |
Appl. No.: |
11/908696 |
Filed: |
March 14, 2006 |
PCT Filed: |
March 14, 2006 |
PCT NO: |
PCT/GB06/50051 |
371 Date: |
October 29, 2007 |
Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 3/10 20180101; A61P 25/00 20180101; A61P 35/00 20180101; A61P
1/12 20180101; A61K 31/00 20130101; A61P 1/10 20180101; A61P 33/04
20180101; Y02A 50/30 20180101; A61P 37/00 20180101; A61P 1/14
20180101; A61P 37/06 20180101; A61P 1/04 20180101; Y02A 50/486
20180101; A61P 35/04 20180101; A61P 29/00 20180101; A61K 31/7115
20130101; A61P 31/00 20180101; A61P 31/04 20180101; A61P 1/00
20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/44.A ;
536/24.5 |
International
Class: |
C07H 21/02 20060101
C07H021/02; A61P 1/00 20060101 A61P001/00; A61P 35/04 20060101
A61P035/04; A61P 1/12 20060101 A61P001/12; A61P 1/14 20060101
A61P001/14; A61P 37/00 20060101 A61P037/00; A61P 31/00 20060101
A61P031/00; A61K 31/7105 20060101 A61K031/7105; A61P 1/04 20060101
A61P001/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2005 |
GB |
0505081.0 |
Claims
1-47. (canceled)
48. A method of treating an intestinal disorder in an individual,
comprising administering to a patient a therapeutically effective
amount of a short interfering nucleic acid molecule (siNA), wherein
the siNA molecule targets Interleukin 12.
49. The method according to claim 48, wherein the siNA molecule is
administered intrarectally.
50. The method of claim 48, wherein the disorder is localized in an
organ selected from the group consisting of the small intestine,
the large intestine, and the rectum.
51. The method of claim 48, wherein the disorder is a disorder of
the small intestine.
52. The method of claim 48, wherein the disorder is selected from
the group consisting of hyperproliferative diseases, autoimmune and
inflammatory bowel diseases (IBD), infectious diseases of the
intestine, malabsorption syndromes, rectal diseases and
diarrhea.
53. The method of claim 48, wherein the disorder is selected from
the group consisting of colorectal cancer, Crohn's disease,
colitis, ulcerative colitis, irritable bowel syndrome,
pseudomembranous colitis, amebiasis, intestinal tuberculosis,
colonic polyps, diverticular disease, constipation, and intestinal
obstruction.
54. The method of claim 48, wherein the siNA molecule is selected
from the group consisting of dsRNA, siRNA, and shRNA.
55. The method of claim 48, wherein the siNA molecule is dsRNA.
56. The method of claim 48, wherein the siNA molecule modulates
miRNA levels.
57. The method of claim 48, wherein the siNA molecule comprises a
modified oligonucleotide.
58. The method of claim 48, wherein the siNA molecule is 40 base
pairs or fewer in length.
59. The method of claim 48, wherein the siNA molecule has 3'
overhangs.
60. The method of claim 59, wherein the 3' overhangs are
dinucleotides.
61. The method of claim 60, wherein the dinucleotide overhangs are
made of thymidine nucleotides.
62. The method of claim 48, wherein a plurality of siNA molecules
is used, and wherein the siNA molecules are targeted to the same
mRNA sequences or to different mRNA sequences.
63. The method of claim 48, wherein the siNA molecule comprises a
sequence selected from the group consisting of SEQ ID NO: 1 to SEQ
ID NO: 85.
64. The method of claim 48, wherein the siNA molecule is targeted
to the Interleukin-12 35 kDa subunit (IL12-p35).
65. The method of claim 64, wherein the siNA molecule comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1 to SEQ ID NO: 33.
66. The method of claim 48, wherein the siNA molecule is targeted
to the Interleukin-12 40 kDa subunit (IL12-p40).
67. The method of claim 66, wherein the siNA molecule comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 34 to SEQ ID NO: 85.
68. A method of treating an intestinal disorder in an individual,
comprising administering intrarectally to a patient a
therapeutically effective amount of a short interfering nucleic
acid molecule (siNA) that targets a gene associated with said
intestinal disorder.
69. The method of claim 68, wherein the disorder is selected from
the group consisting of hyperproliferative diseases, autoimmune and
inflammatory bowel diseases (IBD), infectious diseases of the
intestine, malabsorption syndromes, rectal diseases and
diarrhea.
70. The method of claim 68, wherein the disorder is selected from
the group consisting of colorectal cancer, Crohn's disease,
colitis, ulcerative colitis, irritable bowel syndrome,
pseudomembranous colitis, amebiasis, intestinal tuberculosis,
colonic polyps, diverticular disease, constipation, and intestinal
obstruction.
71. A pharmaceutical composition for the treatment of an intestinal
disorder comprising a short interfering nucleic acid molecule
(siNA) compound formulated for intrarectal administration.
72. The pharmaceutical composition of claim 71, wherein the siNA
molecule targets IL-12 mRNA expression.
73. The pharmaceutical composition of claim 72, wherein the siNA
molecule comprises a sequence selected from SEQ ID NO: 1 to SEQ ID
NO: 85.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for the treatment of intestinal pathologies by means of intrarectal
administration of the RNAi technology. The compositions of the
invention comprise short interfering nucleic acid molecules (siNA)
and related compounds including, but not limited to,
small-interfering RNAs (siRNA). In particular, the compositions of
the invention can be used for the treatment of intestinal
pathologies including: hyperproliferative diseases, in particular,
colorectal cancer; autoimmune and inflammatory bowel diseases
(IBD), in particular Crohn's disease; colitis, in particular
ulcerative colitis; irritable bowel syndrome; infectious diseases
of the intestine, such as pseudomembranous colitis, amebiasis or
intestinal tuberculosis; colonic polyps; diverticular disease;
constipation; intestinal obstruction; malabsorption syndromes;
rectal diseases and diarrhoea.
[0002] In certain embodiments, intestinal conditions caused by
increased levels of interleukin-12 (IL-12), a cytokine involved in
type 1 helper T (Th1) cells immune response are to be treated by
this approach: for example, autoimmune diseases and IBD.
Compositions and methods comprising siRNA and related compounds
targeting IL12-p40 subunit and/or IL12-p35 subunit are provided for
the treatment of diseases associated with over-expression of IL-12,
in particular Crohn's disease.
BACKGROUND OF THE INVENTION
RNAi as a Tool to Modulate Gene Expression
[0003] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing mediated by double-stranded RNA
(dsRNA). After the discovery of the phenomenon in plants in the
early 1990s, Andy Fire and Craig Mello demonstrated that dsRNA
specifically and selectively inhibited gene expression in an
extremely efficient manner in Caenorhabditis elegans (Fire et al.,
1998). The sequence of the first strand (sense RNA) coincided with
that of the corresponding region of the target messenger RNA
(mRNA). The second strand (antisense RNA) was complementary to the
mRNA. The resulting dsRNA turned out to be several orders of
magnitude more efficient than the corresponding single-stranded RNA
molecules (in particular, antisense RNA).
[0004] The process of RNAi begins when the enzyme DICER encounters
dsRNA and chops it into pieces called small-interfering RNAs or
siRNA. This protein belongs to the RNase III nuclease family. A
complex of proteins gathers up these siRNAs and uses their code as
a guide to search out and destroy any RNAs in the cell with a
matching sequence, such as target mRNA (see Bosher & Labouesse,
2000; and Akashi et al., 2001).
[0005] In attempting to apply RNAi for gene knockdown, it was
recognized that mammalian cells have developed various protective
mechanisms against viral infections that could impede the use of
this approach. Indeed, the presence of extremely low levels of
viral dsRNA triggers an interferon response, resulting in a global
non-specific suppression of translation, which in turn triggers
apoptosis (Williams, 1997, Gil & Esteban, 2000).
[0006] In 2000, dsRNA was reported to specifically inhibit three
genes in the mouse oocyte and early embryo. Translational arrest,
and thus a PKR response, was not observed as the embryos continued
to develop (Wianny & Zernicka-Goetz, 2000). Research at
Ribopharma AG (Kulmbach, Germany) demonstrated the functionality of
RNAi in mammalian cells, using short (20-24 base pairs) dsRNAs to
switch off genes in human cells without initiating the acute-phase
response. Similar experiments carried out by other research groups
confirmed these results (Elbashir et al., 2001; Caplen et al.,
2001). Tested in a variety of normal and cancer human and mouse
cell lines, it was determined that short hairpin RNAs (shRNAs) can
silence genes as efficiently as their siRNA counterparts (Paddison
et al., 2002). Recently, another group of small RNAs (21-25 base
pairs) was shown to mediate downregulation of gene expression.
These RNAs, small temporally regulated RNAs (stRNAs), regulate
timing of gene expression during development in Caenorhabditis
elegans (for review see Banerjee & Slack, 2002 and Grosshans
& Slack, 2002).
[0007] Scientists have used RNAi in several systems, including
Caenorhabditis elegans, Drosophila, trypanosomes, and other
invertebrates. Several groups have recently presented the specific
suppression of protein biosynthesis in different mammalian cell
lines (specifically in HeLa cells) demonstrating that RNAi is a
broadly applicable method for gene silencing in vitro. Based on
these results, RNAi has rapidly become a well recognized tool for
validating (identifying and assigning) gene functions. RNAi
employing short dsRNA oligonucleotides will yield an understanding
of the function of genes being only partially sequenced.
[0008] Recently, Krutzfeldt and colleagues have shown that a class
of specially engineered compounds called `antagomirs` can
effectively silence the action of microRNAs (miRNAs), non-coding
pieces of RNA that regulate gene expression (Krutzfeldt et al.,
2005).
[0009] The preceding is a discussion of relevant art pertaining to
RNAi. The discussion is provided only for understanding of the
invention that follows, and is not an admission that any of the
work described is prior art to the claimed invention.
Interleukin-12 and Crohn's Disease.
[0010] Interleukin-12 (1L-12) is a heterodimeric 70 kDa
glycoprotein (IL12-p70) consisting of a 40 kDa subunit (designated
IL12-p40) and a 35 kDa subunit (designated IL12p35) linked by
disulfide bonds that are essential for the biological activity of
IL-12.
[0011] IL-12 is a key cytokine that regulates cell-mediated immune
responses and type 1 helper T (Th1) cells inflammatory reaction
(Gately et al., 1998; Trinchieri, 1998). The ability of IL-12 to
strongly promote the development of Th1 cells makes it an ideal
target for the treatment of Th1 cell-mediated diseases, such as
autoimmune diseases and inflammatory bowel disease (IBD).
[0012] One particular IBD is Crohn's disease, a pathology
characterized by an increased production of IL-12 by
antigen-presenting cells in intestinal tissue and
interferon-.gamma. and tumour necrosis factor .alpha. (TNF-.alpha.)
by intestinal lymphocytes and macrophages (Fais et al., 1994; Fuss
et al., 1996; Monteleone et al., 1997; Parronchi et al., 1997;
Plevy et al., 1997).
[0013] Crohn's disease causes inflammation in the small intestine.
The inflammation can cause pain and can make the intestines empty
frequently, resulting in diarrhoea. The most common symptoms of
Crohn's disease are abdominal pain and diarrhoea, although rectal
bleeding, weight loss and fever may also occur. Bleeding may be
serious and persistent, leading to anaemia. Children with Crohn's
disease may suffer delayed development and stunted growth.
[0014] Most people are first treated with drugs containing
mesalamine, a substance that helps control inflammation.
Sulfasalazine is the most commonly used of these drugs. Patients
who do not benefit from it or who cannot tolerate it may be put on
other mesalamine-containing drugs, generally known as 5-ASA agents,
such as Asacol, Dipentum or Pentasa. Possible side effects of
mesalamine preparations include nausea, vomiting, diarrhoea and
headache. Some patients take corticosteroids to control
inflammation. These drugs are the most effective for active Crohn's
disease, but they can cause serious side effects, including greater
susceptibility to infection. Drugs that suppress the immune system
are also used to treat Crohn's disease. Most commonly prescribed
are 6-mercaptopurine and a related drug, azathioprine.
Immunosuppressive agents work by blocking the immune reaction that
contributes to inflammation. These drugs may cause side effects
like nausea, vomiting, and diarrhoea and may lower a person's
resistance to infection. Surgery to remove part of the intestine
can help Crohn's disease but cannot cure it. Due to the side
effects and the lack of effectiveness of the current treatments for
Crohn's disease, researchers continue to look for more effective
treatments.
[0015] Inhibiting the action of IL-12 has been shown to suppress
development and clinical progression of disease in a multitude of
experimental models of autoimmunity and chronic inflammation
(Caspi, 1998). These models include experimental autoimmune
encephalomyelitis (EAE), experimental autoimmune uveitis (EAU),
collagen-induced arthritis (CIA), autoimmune nephritis,
insulin-dependent diabetes mellitus (IDDM) and different models for
IBD (Vandenbroeck et al., 2004). In these models, the role of
endogenous IL-12 has been addressed by using IL-12p40 knockout mice
or by administering anti-IL-12 antibodies.
[0016] In particular, targeting IL-12 with antibodies is an
effective treatment for the intestinal inflammation in animal
models of Crohn's disease (Mannon et al., 2004). Thus, mice with
trinitrobenzene sulfonate-induced colitis have a Th1-mediated gut
inflammation characterized by greatly increased production of
IL-12, interferon-.gamma. and tumour necrosis factor .alpha.
(TNF-.alpha.). In mice, administration of a monoclonal antibody
against IL-12 can result in the resolution of established colitis
and, if given at the time of induction of colitis, can prevent
inflammation (Neurath et al., 1995).
[0017] Anti-interleukin-12 can also prevent and treat the
spontaneous colitis seen in models of Th1-mediated inflammation
such as mice that over-express the human CD3e gene and mice
deficient in interleukin-10 (Davidson et al., 1998; Simpson et al.,
1998).
[0018] Data from an early phase 2 study provide some evidence that
treatment with a monoclonal antibody against IL-12 p40 may induce
clinical response and remission in patients with active Crohn's
disease (Mannon et al., 2004). This treatment is associated with
decreases in Th1-mediated inflammatory cytokines at the site of
disease.
[0019] Previous evidence obtained from animal models, as well as
the clinical effects of anti-IL-12 in patients with Crohn's disease
(Mannon et al., 2004), highlight the importance of IL-12 as a
target for future treatments for Crohn's disease.
Modulation of IL-12 Levels by Means of siRNA.
[0020] siRNA targeting of IL-12 expression has already been used to
obtain modified dendritic cells (DC) that might be used in a
variety of therapeutic in vitro, ex vivo and in vivo methods to
modulate T cell activity, and thus have use in therapeutic
approaches for the treatment of immune disorders in a mammalian
subject (WO 03/104455; Hill et al., 2003). siRNA targeting of IL-12
expression in mature DC has revealed a critical role for IL-12 in
natural killer cell interferon .gamma. (IFN-.gamma.) secretion
promoted by mature DC (Borg et al., 2004). Further, IL-12 p35
inhibitors including siRNA have surprisingly demonstrated to block
differentiation of preadipocytes to adipocytes and triglyceride
accumulation in adipocytes (WO 03/104495).
[0021] siRNA targeting IL-12p40 has successfully been delivered by
means of liposome encapsulation to murine peritoneal cavity to
modulate the local and systemic inflammatory response after
endotoxin challenge (Flynn et al., 2004). However, to our
knowledge, there is no previous evidence of intrarectal
administration of siRNA for the downregulation of IL-12 nor of any
other gene involved in intestinal pathologies. We have developed
techniques for downregulation of IL-12 expression in vivo to treat
intestinal disorders; and we have also developed techniques for
targeting siRNAs to the intestine by intrarectal
administration.
SUMMARY OF THE INVENTION
[0022] The present invention provides methods and compositions for
the treatment of intestinal pathologies by means of intrarectal
administration of the RNAi technology. The compositions of the
invention comprise short interfering nucleic acid molecules (siNA)
and related compounds including, but not limited to, siRNA. In
particular, the compositions of the invention can be used in the
preparation of a medicament for the treatment of intestinal
pathologies including: hyperproliferative diseases, in particular,
colorectal cancer; autoimmune and inflammatory bowel diseases
(1BD), in particular Crohn's disease; colitis, in particular
ulcerative colitis; irritable bowel syndrome; infectious diseases
of the intestine, such as pseudomembranous colitis, amebiasis or
intestinal tuberculosis; colonic polyps; diverticular disease;
constipation; intestinal obstruction; malabsorption syndromes;
rectal diseases and diarrhoea. The present invention encompasses
compositions and methods of use of siNA including, but not limited
to, short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), antagomirs and short hairpin RNA (shRNA)
molecules capable of mediating RNA interference.
[0023] The methods of the invention comprise the administration to
a patient in the need thereof of an effective amount of one or more
siNA of the invention for the treatment of an intestinal condition.
In preferred embodiments, the methods of the invention comprise
intrarectal administration of the therapeutic siNA.
[0024] In one embodiment, the present invention relates to siNA or
similar chemically synthesized entities that are directed at
interfering with the mRNA expression of either the p35 or the p40
subunits of the cytokine IL-12, and that ultimately modulate the
amount of protein produced. Compositions and methods comprising
above-mentioned siRNA and related compounds are intended for the
treatment of diseases associated with over-expression of IL-12,
such as autoimmune diseases and inflammatory bowel diseases (IBD),
in particular, Crohn's disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. Oligonucleotide sequences for siRNA molecules
targeting IL-12 p35 and p40 subunits encompassed by the present
invention. The SEQ ID Nos given in the Figure refer to the sense
(5'->3') strand; typically sIRNA will be administered as dsRNA,
so will include both the sense strand and its complement.
[0026] FIG. 2. Effect of siRNA on IL-12 p35 subunit expression in
an in vitro system. siRNA treatment reduces the levels of IL-12 p35
gene transcript. RNA was prepared from SW480 cells treated with
siRNAs for different times. The samples were analyzed by RT-PCR
using specific primers. The values show the mean expression levels
of different transcripts normalized to 18S as housekeeping
gene.
[0027] FIG. 3. Effect of siRNA on IL-12 p40 subunit expression in
an in vitro system. A: siRNA treatment reduces the levels of IL-12
p40 gene transcript in human cells. RNA was prepared from SW480
cells treated with siRNA SEQ ID 67 and SEQ ID 79 at different
times, at dose treatment of 200 nM. The values show the mean
expression levels of different transcripts normalized to 18S as
housekeeping gene. The values represent the mean of the percentage
of the normalized mRNA levels upon siRNA interference over the
control gene expression and their medium standard deviations (SEM).
B: siRNA treatment reduces the levels of IL-12 p40 gene transcript
in murine cells. RNA was prepared from C2C12 cells treated with
siRNA SEQ ID 86 and SEQ ID 87 at different times, at dose treatment
of 100 nM. SEQ ID 86, which is homologous to human SEQ ID 67,
targets the mouse IL-12 p40 subunit. Further targeting the mouse
IL-12 p40 subunit, SEQ ID 87 is the siRNA with the best score in
mouse, and has no homologous siRNA duplex in human. siNA molecules
SEQ ID 86 and SEQ ID 87 are as described below, with 2 thymidine
nucleotide 3' overhangs. The values represent the mean of the
percentage of the normalized mRNA levels compared to 18S upon siRNA
interference over the control gene expression and their medium
standard deviations (SEM).
[0028] FIG. 4. siRNA treatment reduces the levels of GFP gene
transcript in small intestine. The collected tissue in OCT was
analyzed by microscopy and measured by photoshop program. Data show
single dose siRNA treatment (mice 2-3) and repeated dose treatment
(mice 4-5). The values show the expression levels of 25
representative images per mouse referred to control untreated
mouse. Standard deviation of the data is represented.
[0029] FIG. 5. siRNA treatment reduces the levels of GFP gene
transcript in small intestine. The tissue collected in RNA later
was analyzed by RT-PCR. Data show single dose siRNA treatment (mice
2-3) and repeated dose treatment (mice 4-5). Standard deviation is
represented.
[0030] FIG. 6. siRNA treatment reduces the levels of GFP gene
transcript in large intestine. The tissue collected in OCT was
analyzed by microscopy and measured by photoshop program. Data show
single dose siRNA treatment (mice 2-3) and repeated dose treatment
(mice 4-5). The values show the expression levels of 25
representative images per mouse referred to control untreated
mouse. Standard deviation of the data is represented.
[0031] FIG. 7: siRNA treatment reduces the levels of GFP gene
transcript in large intestine. The collected tissue in RNA later
was analyzed by RT-PCR. Data show single dose siRNA treatment (mice
2-3) and repeated dose treatment (mice 4-5). Standard deviation is
represented.
[0032] FIG. 8: Data of samples collected in OCT medium.
[0033] FIG. 9: Data of samples collected in RNA later.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention relates to methods and compositions
for the treatment of intestinal pathologies by means of intrarectal
administration of the RNAi technology. The compositions of the
invention comprise short interfering nucleic acid molecules (siNA)
that modulate the expression of target genes associated with
conditions of the intestinal wall.
[0035] The methods of the invention comprise the administration to
a patient in need thereof of an effective amount of one or more
siNA of the invention.
Design of siRNA
[0036] A gene is "targeted" by siNA according to the invention
when, for example, the siNA selectively decrease or inhibit the
expression of the gene. Alternatively, siNA target a gene when the
siNA hybridize under stringent conditions to the gene transcript.
siNA can be tested either in vitro or in vivo for the ability to
target a gene.
[0037] In 1999, Tuschl et al. deciphered the silencing effect of
siRNAs showing that their efficiency is a function of the length of
the duplex, the length of the 3'-end overhangs, and the sequence in
these overhangs.
[0038] Selecting the right homologous region within the target gene
is of great relevance for accurate silencing. A short fragment of
the target gene sequence (e.g., 19-40 nucleotides in length) is
chosen as the sequence of the siNA of the invention. In one
embodiment, the siNA is siRNA. In such embodiments, the short
fragment of target gene sequence is a fragment of the target gene
mRNA. In preferred embodiments, the criteria for choosing a
sequence fragment from the target gene mRNA to be a candidate siRNA
molecule include: 1) a sequence from the target gene mRNA that is
at least 50-100 nucleotides from the 5' or 3' end of the native
mRNA molecule; 2) a sequence from the target gene mRNA that has a
G/C content of between 30% and 70%, most preferably around 50%; 3)
a sequence from the target gene mRNA that does not contain
repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG,
TTTT); 4) a sequence from the target gene mRNA that is accessible
in the mRNA; and 5) a sequence from the target gene mRNA that is
unique to the target gene. The sequence fragment from the target
gene mRNA may meet one or more of the above-mentioned identified
criteria. In preferred embodiments, the siRNA has a G/C content
below 60% and/or lacks repetitive sequences.
[0039] Practically, the gene of interest is introduced as a
nucleotide sequence in a prediction program that takes into account
all the variables described above for the design of optimal
oligonucleotides. This program scans any mRNA nucleotide sequence
for regions susceptible to be targeted by siRNA. The output of this
analysis is a score of possible siRNA oligonucleotides. The highest
scores are used to design double stranded RNA oligonucleotides
(typically 21 bp long, although other lengths are also possible)
that are typically made by chemical synthesis. We plan to test
several chemical modifications that are well known in the art.
These modifications are aimed at increasing stability or
availability of the dsRNA oligonucleotides.
[0040] Candidate oligonucleotides can further be filtered for
interspecies sequence conservation in order to facilitate the
transition from animal to human clinical studies.
[0041] In addition to siNA which is perfectly complementary to the
target region, degenerate siNA sequences may be used to target
homologous regions. WO2005/045037 describes the design of siNA
molecules to target such homologous sequences, for example by
incorporating non-canonical base pairs, for example mismatches
and/or wobble base pairs, that can provide additional target
sequences. In instances where mismatches are identified,
non-canonical base pairs (for example, mismatches and/or wobble
bases) can be used to generate siNA molecules that target more than
one gene sequence. In a non-limiting example, non-canonical base
pairs such as UU and CC base pairs are used to generate siNA
molecules that are capable of targeting sequences for differing
targets that share sequence homology. As such, one advantage of
using siNAs of the invention is that a single siNA can be designed
to include nucleic acid sequence that is complementary to the
nucleotide sequence that is conserved between homologous genes. In
this approach, a single siNA can be used to inhibit expression of
more than one gene instead of using more than one siNA molecule to
target different genes.
[0042] Sequence identity may be calculated by sequence comparison
and alignment algorithms known in the art (see Gribskov and
Devereux, Sequence Analysis Primer, Stockton Press, 1991, and
references cited therein) and calculating the percent difference
between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90%, 95%, or 99% sequence
identity between the siNA and the portion of the target gene is
preferred. Alternatively, the complementarity between the siNA and
native RNA molecule may be defined functionally by hybridisation as
well as functionally by its ability to decrease or inhibit the
expression of a target gene. The ability of a siNA to affect gene
expression can be determined empirically either in vivo or in
vitro.
[0043] Preferred siNA molecules of the invention are double
stranded. In one embodiment, double stranded siNA molecules
comprise blunt ends. In another embodiment, double stranded siNA
molecules comprise overhanging nucleotides (e.g., 1-5 nucleotide
overhangs, preferably 2 nucleotide overhangs). In a specific
embodiment, the overhanging nucleotides are 3' overhangs. In
another specific embodiment, the overhanging nucleotides are 5'
overhangs. Any type of nucleotide can be a part of the overhang. In
one embodiment, the overhanging nucleotide or nucleotides are
ribonucleic acids. In another embodiment, the overhanging
nucleotide or nucleotides are deoxyribonucleic acids. In a
preferred embodiment, the overhanging nucleotide or nucleotides are
thymidine nucleotides. In another embodiment, the overhanging
nucleotide or nucleotides are modified or non-classical
nucleotides. The overhanging nucleotide or nucleotides may have
non-classical internucleotide bonds (e.g., other than
phosphodiester bond).
Synthesis of siNA Duplexes
[0044] siNA can be synthesized by any method known in the art. RNAs
are preferably chemically synthesized using appropriately protected
ribonucleoside phosphoramidites and a conventional DNA/RNA
synthesizer. Additionally, siRNA can be obtained from commercial
RNA oligo synthesis suppliers, including, but not limited to,
Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo.,
USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland,
Mass., USA), and Cruachem (Glasgow, UK), Qiagen (Germany), Ambion
(USA) and Invitrogen (Scotland). Alternatively, siNA molecules of
the invention can be expressed in cells by transfecting the cells
with vectors containing the reverse complement siNA sequence under
the control of a promoter. Once expressed, the siNA can be isolated
from the cell using techniques well known in the art.
[0045] An annealing step is necessary when working with
single-stranded RNA molecules. To anneal the RNAs, 30 .mu.l of each
RNA oligo 50 .mu.M solution are to be combined in 100 mM potassium
acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate. The
solution is then incubated for 1 minute at 90.degree. C.,
centrifuged for 15 seconds, and incubated for 1 hour at 37.degree.
C.
[0046] In embodiments where the siRNA is a short hairpin RNA
(shRNA); the two strands of the siRNA molecule may be connected by
a linker region (e.g., a nucleotide linker or a non-nucleotide
linker).
Chemical Modification of siNA.
[0047] The siNAs of the invention may contain one or more modified
nucleotides and/or non-phosphodiester linkages. Chemical
modifications well known in the art are capable of increasing
stability, availability, and/or cell uptake of the siNA. The
skilled person will be aware of other types of chemical
modification which may be incorporated into RNA molecules (see
International Publications WO03/070744 and WO2005/045037 for an
overview of types of modifications).
[0048] In one embodiment, modifications can be used to provide
improved resistance to degradation or improved uptake. Examples of
such modifications include phosphorothioate internucleotide
linkages, 2'-O-methyl ribonucleotides (especially on the sense
strand of a double stranded siRNA), 2'-deoxy-fluoro
ribonucleotides, 2'-deoxy ribonucleotides, "universal base"
nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic
residue incorporation (see generally GB2406568).
[0049] In another embodiment, modifications can be used to enhance
the stability of the siRNA or to increase targeting efficiency.
Modifications include chemical cross linking between the two
complementary strands of an siRNA, chemical modification of a 3' or
5' terminus of a strand of an siRNA, sugar modifications,
nucleobase modifications and/or backbone modifications, 2'-fluoro
modified ribonucleotides and 2'-deoxy ribonucleotides (see
generally International Publication WO2004/029212).
[0050] In another embodiment, modifications can be used to increase
or decrease affinity for the complementary nucleotides in the
target mRNA and/or in the complementary siNA strand (see generally
International Publication WO2005/044976). For example, an
unmodified pyrimidine nucleotide can be substituted for a 2-thio,
5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an
unmodified purine can be substituted with a 7-deza, 7-alkyl, or
7-alkenyl purine.
[0051] In another embodiment, when the siNA is a double-stranded
siRNA, the 3'-terminal nucleotide overhanging nucleotides are
replaced by deoxyribonucleotides (see generally Elbashir et al.,
2001).
In Vitro Testing of siRNA Duplexes.
[0052] To check the specificity of the siRNA interference, cell
cultures expressing the target genes were employed.
[0053] In the case of IL-12 p35 and p40 subunits, the cells used
for the experiments were human SW480 cells and murine muscle cells
C2C12. After incubation of the cells with the corresponding siRNA
duplexes, the levels of p35 and p40 expression were analyzed. For
linking siRNA knockdown to specific phenotypes in cultured cells,
it is necessary to demonstrate the decrease of the targeted protein
or at least to demonstrate the reduction of the targeted mRNA.
[0054] mRNA levels of the target gene can be quantitated by real
time PCR(RT-PCR). Further, the protein levels can be determined in
a variety of ways well known in the art, such as Western blot
analysis with specific antibodies to the different target allow
direct monitoring of the reduction of targeted protein.
[0055] siRNA are introduced into cells by means of any transfection
technique well known in the art. A single transfection of siRNA
duplex can be performed, for instance, by using a cationic lipid,
such as Lipofectamine 2000 Reagent (Invitrogen), followed by an
assay of silencing efficiency 24, 48 and 72 hours after
transfection.
[0056] A typical transfection protocol can be performed as follows:
for one well of a E-well plate, we transfect using 100 nM for
murine C2C12 cells or 200 nM for human SW480 cells as final
concentration of siRNA. Following Lipofectamine 2000 Reagent
protocol, the day before transfection, we seed 2-4.times.10.sup.5
cells per well in 3 ml of an appropriate growth medium, containing
DMEM, 10% serum, antibiotics and glutamine, and incubate cells
under normal growth conditions (37.degree. C. and 5% CO.sub.2). On
the day of transfection, cells have to be at 30-50% confluence. We
dilute 12.5 .mu.l of 20 .mu.M siRNA duplex (corresponding to 100 nM
final concentration) or 25 .mu.l of 20 .mu.M siRNA duplex
(corresponding to 200 nM final concentration) in 250 .mu.l of DMEM
and mix. Also, 6 .mu.l of Lipofectamine 2000 is diluted in 250
.mu.l of DMEM and mixed. After a 5 minute incubation at room
temperature, the diluted oligomer (siRNA duplex) and the diluted
Lipofectamine are combined to allow complex formation during a 20
minutes incubation at room temperature. Afterwards, we add the
complexes drop-wise onto the cells with 2 ml of fresh growth medium
low in antibiotics and mix gently by rocking the plate back and
forth, to ensure uniform distribution of the transfection
complexes. We incubate the cells under their normal growth
conditions and the day after, the complexes are removed and fresh
and complete growth medium is added. To monitor gene silencing,
cells are collected at 24, 48 and 72 h post-transfection.
[0057] The efficiency of transfection may depend on the cell type,
but also on the passage number and the confluency of the cells. The
time and the manner of formation of siRNA-liposome complexes (e.g.
inversion versus vortexing) are also critical. Low transfection
efficiencies are the most frequent cause of unsuccessful silencing.
Good transfection is a non-trivial issue and needs to be carefully
examined for each new cell line to be used. Transfection efficiency
may be tested transfecting reporter genes, for example a CMV-driven
EGFP-expression plasmid (e.g. from Clontech) or a B-Gal expression
plasmid, and then assessed by phase contrast and/or fluorescence
microscopy the next day.
[0058] Depending on the abundance and the life time (or turnover)
of the targeted protein, a knock-down phenotype may become apparent
after 1 to 3 days, or even later. In cases where no phenotype is
observed, depletion of the protein may be observed by
immunofluorescence or Western blotting.
[0059] After transfections, total RNA fractions extracted from
cells are pre-treated with DNase I and used for reverse
transcription using a random primer. PCR-amplified with a specific
primer pair covering at least one exon-exon junction is used as
control for amplification of pre-mRNAs. RT-PCR of a non-targeted
mRNA is also needed as control. Effective depletion of the mRNA yet
undetectable reduction of target protein may indicate that a large
reservoir of stable protein may exist in the cell. Alternatively,
RT-PCR amplification can be used to test in a more precise way the
mRNA decrease or disappearance. RT-PCR quantitates the initial
amount of the template most specifically, sensitively and
reproducibly. RT-PCR monitors the fluorescence emitted during the
reaction as an indicator of amplicon production during each PCR
cycle, in a light cycler apparatus. This signal increases in direct
proportion to the amount of PCR product in a reaction. By recording
the amount of fluorescence emission at each cycle, it is possible
to monitor the PCR reaction during exponential phase where the
first significant increase in the amount of PCR product correlates
to the initial amount of target template.
[0060] To verify the interference pattern of the differentially
expressed IL-p35 and p40 genes in the cell cultures, quantitative
RT-PCR was performed. For quantitative RT-PCR, approximately 500 ng
of total RNA was used for reverse transcription, followed by PCR
amplification with specific primers for each gene in reaction. The
PCR conditions were an initial step of 30 s at 95.degree. C.,
followed by 40 cycles of 5 s at 95.degree. C., 10 s at 62.degree.
C. and 15 s at 72.degree. C. Quantification of 18S mRNA was used as
a housekeeping gene as a control for data normalization. Relative
gene expression comparisons work best when the gene expression of
the chosen endogenous/internal control is more abundant and remains
constant, in proportion to total RNA, among the samples. By using
an invariant endogenous control as an active reference,
quantitation of an mRNA target can be normalised for differences in
the amount of total RNA added to each reaction. The amplification
curves obtained with the light cycler were analyzed in combination
with the control kit DNA, which targets in vitro transcribed
beta-globin DNA template, according to the manufacturer protocol.
In order to assess the specificity of the amplified PCR product a
melting curve analysis was performed. The resulting melting curves
allow discrimination between primer-dimers and specific PCR
product.
Intrarectal Administration of siNA.
[0061] Intrarectal siNA delivery studies were carried out in GFP
C57BL/6-TG (ACTB-EGFP) mice. This transgenic mouse line was bought
from "The Jackson Laboratory". Transgenic mice have been used
because homozygous mice for this transgene die within the first two
weeks following birth. The transgenic mouse line with an "enhanced"
GFP (EGFP) cDNA under the control of a chicken beta-actin promoter
and cytomegalovirus enhancer makes all of the tissues, with the
exception of erythrocytes and hair, appear green under excitation
light. This strain was generated in C57BL/6 mice. The strain cDNA
encoding enhanced green fluorescent protein (EGFP) was adjoined to
the chicken beta actin promoter and cytomegalovirus enhancer. A
bovine globin polyadenylation signal was also included in the
construct. The EcoR1 sites included in the PCR primers were used to
introduce the amplified EGFP cDNA into a pCAGGS expression vector
containing the chicken beta-actin promoter and cytomegalovirus
enhancer, beta-actin intron and bovine globin poly-adenylation
signal. The entire insert with the promoter and coding sequence was
excised with Bam-HI and SalI and gel-purified.
[0062] The siRNA duplex used for intrarectal injection in mice was
purchased from Dharmacon. Dharmacon Research Inc (Lafayette, Colo.)
have developed a new generation of modified siRNA for in vivo use
as a therapeutic, named siSTABLEv2. Dharmacon's siSTABLEv2 siRNA
have demonstrated an enhanced stability in serum with respect to
that of non-modified siRNA. Conventional siRNA are typically
degraded within minutes in serum-containing environments, making in
vivo use of siRNA problematic. The siSTABLEv2 modification
dramatically extends the siRNA stability in serum as described in
Dharmacon's web page (http://www.dharmacon.com/docs/siSTABLE
%20v2%20Flier.pdf).
[0063] The siRNA used to downregulate EGFP mRNA expression targeted
the following sequence in EGFP mRNA: 5'-GGC UAC GUC CAG GAG CGC
ACC-3' (SEQ ID No 88). The sense strand of the siRNA duplex was
5'-P GGC UAC GUC CAG CGC ACC-3' (SEQ ID No 89) and the antisense
strand was 5'-P U GCG CUC CUG GAC GUA GCC UU-3' (SEQ ID No 90).
This sequence is distributed by Dharmacon as pre-synthesized
control siRNA green fluorescent protein duplex.
[0064] For the intrarectal delivery experiments C57BL/6-TG
(ACTB-EGFP) mice (males, 8 weeks old) were used. The animals were
kept in cages with free access to food and water until one day
before the experimental protocol. For intrarectal therapeutic
silencing, mice were fasted for one day prior to the treatment. The
drugs are typically administered by injecting a small volume (120
.mu.L) in the rectum. Control mouse is treated with the vehicle
alone. In all cases animals were sacrificed two days after the
first injection by cervical dislocation. The protocol for the siRNA
application in mouse is as follows. For each experimental
administration, 60 .mu.l siRNA duplexes were premixed with 60 .mu.l
of NaCl (1.8% w/v) up to physiological levels. In all cases animals
were sacrificed two days after the first injection.
[0065] Experimental conditions used are indicated in the Table
below. Each condition was analyzed in duplicate. Mice 2 and 3 were
treated intrarectally with one dose of 250 .mu.g (19 nanomols) of
the siRNA vs GFP, while mice 4 and 5 were treated with two doses of
125 .mu.g of siRNA during two consecutive days.
TABLE-US-00001 TABLE Schematic distribution of experimental
conditions for intrarectal siRNA delivery. Mouse number Intrarectal
Therapeutic Treatment 1 Vehicle control dose 2 Single dose of 250
.mu.g of siRNA 3 Single dose of 250 .mu.g of siRNA 4 Two doses of
125 .mu.g of siRNA each 24 h 5 Two doses of 125 .mu.g of siRNA each
24 h Doses of siRNA are indicated in the table.
[0066] The sample tissues were collected and analyzed by two
methods: One in OCT medium and another in RNA later (Ambion). OCT
blocks were storaged at -80.degree. C. until data processing. OCT
blocks were cut in slices of 12 .mu.m by a cryostat (Leica CM 1850)
at -20.degree. C. The collected slices were analyzed on a
fluorescence microscope (Olympus BX51) coupled to a digital camera
(DP70), using a filter of 488 nm. The sensitive conditions
(ISO200), resolution image size (2040.times.1536) and time exposure
(1 second) were set up for all the samples in order to be compared
between them. Green fluorescence was measured as an index of GFP
expression by an Adobe Photoshop program (version 8.0). By this
method, 25 different data were collected for each analyzed tissue.
Tissues isolated in RNA later were stored at -20.degree. C. RNA
later was removed before RNA extraction. RNA was isolated with the
Trizol Reagent (Invitrogen) according to the manufacturer protocol.
DNAse treatment was done before measurement of GFP expression by
RT-PCR as described above.
Pharmaceutical Formulations and Routes of Administration.
[0067] The present invention may comprise the administration of one
or more species of siNA molecule simultaneously. These species may
be selected to target one or more target genes.
[0068] In one embodiment, a single type of siNA is administered in
the therapeutic methods of the invention. In another embodiment, a
siNA of the invention is administered in combination with another
siNA of the invention and/or with one or more other non-siNA
therapeutic agents useful in the treatment, prevention or
management of a disease condition of the intestine wall. The term
"in combination with" is not limited to the administration of
therapeutic agents at exactly the same time, but rather it is meant
that the siNAs of the invention and the other agent are
administered to a patient in a sequence and within a time interval
such that the benefit of the combination is greater than the
benefit if they were administered otherwise. For example, each
therapeutic agent may be administered at the same time or
sequentially in any order at different points in time; however, if
not administered at the same time, they should be administered
sufficiently close in time so as to provide the desired therapeutic
effect. Each therapeutic agent can be administered separately, in
any appropriate form and by any suitable route.
[0069] The siNAs of the invention may be formulated into
pharmaceutical compositions by any of the conventional techniques
known in the art (see for example, Alfonso, G. et al., 1995, in:
The Science and Practice of Pharmacy, Mack Publishing, Easton Pa.,
19th ed.). Formulations comprising one or more siNAs for use in the
methods of the invention may be in numerous forms, and may depend
on the various factors specific for each patient (e.g., the type
and severity of disorder, type of siNA administered, age, body
weight, response, and the past medical history of the patient), the
number and type of siNAs in the formulation, the form of the
composition (e.g., in liquid, semi-liquid or solid form), the
therapeutic regime (e.g. whether the therapeutic agent is
administered over time as a slow infusion, a single bolus, once
daily, several times a day or once every few days), and/or the
route of administration (e.g., topical, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral, or
sublingual means).
[0070] The siNA molecules of the invention and formulations or
compositions thereof may be administered directly or topically as
is generally known in the art. For example, a siNA molecule can
comprise a delivery vehicle, including liposomes, for
administration to a subject. Carriers and diluents and their salts
can be present in pharmaceutically acceptable formulations. Nucleic
acid molecules can be administered to cells by a variety of methods
known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as biodegradable polymers,
hydrogels, cyclodextrins poly (lactic-co-glycolic) acid (PLGA) and
PLCA microspheres, biodegradable nanocapsules, and bioadhesive
microspheres, or by proteinaceous vectors. In another embodiment,
the nucleic acid molecules of the invention can also be formulated
or complexed with polyethyleneimine and derivatives thereof, such
as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine
(PEI-PEG-GAL) or
polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine
(PEI-PEG-triGAL) derivatives.
[0071] A siNA molecule of the invention may be complexed with
membrane disruptive agents and/or a cationic lipid or helper lipid
molecule.
[0072] Delivery systems which may be used with the invention
include, for example, aqueous and non aqueous gels, creams,
multiple emulsions, microemulsions, liposomes, ointments, aqueous
and non aqueous solutions, lotions, aerosols, hydrocarbon bases and
powders, and can contain excipients such as solubilizers,
permeation enhancers (e.g., fatty acids, fatty acid esters, fatty
alcohols and amino acids), and hydrophilic polymers (e.g.,
polycarbophil and polyvinylpyrolidone). In one embodiment, the
pharmaceutically acceptable carrier is a liposome or a transdermal
enhancer.
[0073] A pharmaceutical formulation of the invention is in a form
suitable for administration, e.g., systemic or local
administration, into a cell or subject, including for example a
human. Suitable forms, in part, depend upon the use or the route of
entry, for example oral, transdermal, or by injection. Other
factors are known in the art, and include considerations such as
toxicity and forms that prevent the composition or formulation from
exerting its effect.
[0074] The present invention also includes compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art. For
example, preservatives, stabilizers, dyes and flavouring agents can
be provided. These include sodium benzoate, sorbic acid and esters
of p-hydroxybenzoic acid. In addition, antioxidants and suspending
agents can be used.
[0075] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors that those skilled in the medical arts will recognize.
[0076] The formulations of the invention can be administered in
dosage unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and/or vehicles.
Formulations can be in a form suitable for oral use, for example,
as tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsion, hard or soft capsules,
or syrups or elixirs. Compositions intended for oral use can be
prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
can contain one or more such sweetening agents, flavoring agents,
coloring agents or preservative agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets
contain the active ingredient in admixture with non-toxic
pharmaceutically acceptable excipients that are suitable for the
manufacture of tablets.
[0077] These excipients can be, for example, inert diluents; such
as calcium carbonate, sodium carbonate, lactose, calcium phosphate
or sodium phosphate; granulating and disintegrating agents, for
example, corn starch, or alginic acid; binding agents, for example
starch, gelatin or acacia; and lubricating agents, for example
magnesium stearate, stearic acid or talc. The tablets can be
uncoated or they can be coated by known techniques. In some cases
such coatings can be prepared by known techniques to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate can be employed.
[0078] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0079] Aqueous suspensions contain the active materials in a
mixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more colouring agents, one or more flavouring agents, and
one or more sweetening agents, such as sucrose or saccharin.
[0080] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0081] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0082] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and
flavouring agents.
[0083] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension.
[0084] This suspension can be formulated according to the known art
using those suitable dispersing or wetting agents and suspending
agents that have been mentioned above.
[0085] A sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parentally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that can
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0086] The nucleic acid molecules of the invention can also be
administered in the form of suppositories, e.g., for rectal
administration of the drug. These compositions can be prepared by
mixing the drug with a suitable non-irritating excipient that is
solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Such
materials include cocoa butter and polyethylene glycols.
[0087] Nucleic acid molecules of the invention can be administered
parenterally in a sterile medium. The drug, depending on the
vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anaesthetics, preservatives and buffering agents can be dissolved
in the vehicle.
[0088] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0089] The nucleic acid molecules of the present invention can also
be administered to a subject in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
[0090] Alternatively, certain siNA molecules of the invention can
be expressed within cells from eukaryotic promoters. Recombinant
vectors capable of expressing the siNA molecules can be delivered
and persist in target cells. Alternatively, vectors can be used
that provide for transient expression of nucleic acid molecules.
Such vectors can be repeatedly administered as necessary. Once
expressed, the siNA molecule interacts with the target mRNA and
generates an RNAi response. Delivery of siNA molecule expressing
vectors can be systemic, such as by intravenous or intramuscular
administration, by administration to target cells ex-planted from a
subject followed by reintroduction into the subject, or by any
other means that would allow for introduction into the desired
target cell.
[0091] Results.
Example 1
Design of siNA
[0092] GenBank Accession numbers corresponding to IL-12 p35
(Interleukin 12A, natural killer cell stimulatory factor 1,
cytotoxic lymphocyte maturation factor 1, p35) and p40 (Interleukin
12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte
maturation factor 2, p40) subunits are NM 000882 and NM 002187,
respectively.
[0093] Corresponding mRNA nucleotide sequences were introduced
within the proprietary prediction program described above, and siNA
molecules directed to target IL-12 p35 and p40 subunits were
obtained. The output of this analysis was a score of possible siNA
oligonucleotides, the highest scores being used to design double
stranded RNA oligonucleotides (typically 19 bp long) that were
typically made by chemical synthesis.
[0094] In preferred embodiments, siNA compositions of the invention
are any of SEQ ID NOS:1-81 of FIG. 1; typically administered as a
duplex of the sense strand and the antisense strand. The invention
also encompasses siNA that are 40 nucleotides or less and comprise
a nucleotide sequence of any of SEQ ID NOS:1-81. In a specific
embodiment, the siNA is 21-30 nucleotides long and comprises any
one of SEQ ID NOS:1-81 of FIG. 1. All siNA molecules used in the
experiments described below were designed to have a 2 thymidine
nucleotide 3' overhang.
Example 2
In Vitro Assays for IL-12 p35
[0095] To determine the inhibition of the IL-12 p35 target gene, a
panel of siRNA contained within FIG. 1 has been analysed in cell
cultures. SiRNA with the best characteristics were selected to be
tested and were applied to proper cell cultures, such as SW480. The
effect of siRNA over the target gene was analyzed by RT-PCR
according to the manufacturer's protocol. The gene target
transcript levels were normalized using 18S as housekeeping gene.
Some of the different siRNA that were tested and their different
efficacies in the interference of the target gene are included in
the FIG. 2. These results correspond to SEQ ID 8 and SEQ ID 17 of
FIG. 1 in SW480 cells expressing p35. The values represent the mean
of the percentage of the normalized mRNA levels upon siRNA
interference over the control gene expression and their medium
standard deviations (SEM). The level of the p35 transcript after
the siRNA treatment was highly reduced with the siRNAs
corresponding to SEQ ID 8 and SEQ ID 17, in SW480 compared to the
control cells. The decrease of the gene expression depends on the
efficiency in siRNA silencing. In fact, siRNA SEQ ID 8 treatment
decreased the p35 gene expression to 56% at 24 h compared to the
control.
Example 3
In Vitro Assays for IL-12 p40
[0096] To determine the inhibition of the IL-12 p40 target gene, a
panel of siRNA contained within FIG. 1 has been analyzed. The siRNA
with the best characteristics designed as described before, were
tested in human and murine cells. The p40 transcript level was
analyzed by RT-PCR and normalized using 18S as housekeeping gene.
These results correspond to SEQ ID 67, SEQ ID 79 in SW480 cells
expressing p40 (FIG. 3A); and SEQ ID 86 and SEQ ID 87 in C2C12
cells expressing p40 (FIG. 3B). siNA molecules SEQ ID 86 and SEQ ID
87 are as described in the figure, with 2 thymidine nucleotide 3'
overhangs.
[0097] The level of the p40 transcript was highly reduced after the
treatment with the siRNA corresponding to SEQ ID 67 in SW480, up to
65% compared to control cells. In C2C12 cells siRNA corresponding
to SEQ ID 86 decreased the gene expression to 61% at 48 h compared
to the control. It is important to note that SEQ ID 67 and SEQ ID
86 correspond to homologous regions of human and mouse IL-12 p40
gene, respectively.
[0098] A summary of the experiments of FIGS. 2 and 3 is displayed
in the following Table:
TABLE-US-00002 Gene Expression (%) SEM P35 (SW480) Control 100 0
SEQ ID 8, 24 h 56 13.9772455 SEQ ID 8, 48 h 73 13.6336806 SEQ ID
17, 24 h 67 17.8860754 SEQ ID 17, 48 h 73 20.7305043 p40 (SW480)
Control 100 0 SEQ ID 67, 24 h 65 8.58321816 SEQ ID 67, 48 h 86
20.4353968 SEQ ID 79, 24 h 69 17.8276602 SEQ ID 79, 48 h 84
13.1055338 p40 (C2C12) Control 100 0 SEQ ID 86, 24 h 121 13.8703694
SEQ ID 86, 4 8h 61 23.4419624 SEQ ID 87, 24 h 108 35.8061452 SEQ ID
87, 48 h 85 17.1974522
Example 4
In Vivo Assays. Analysis of the Small Intestine
[0099] The siRNA application is made in order to determine the
proper siRNA delivery in the intestine. To determine the siRNA
effect, small intestine samples were collected in OCT medium and
analyzed as previously described. Since the goal is to determine
the downregulation of GFP gene transcript, levels of fluorescence
were measured following siRNA application. No secondary effects
were observed in the animals during the experimental protocols.
[0100] The first group of work (animals 2 and 3) was treated with a
single dose of 250 .mu.g of siRNA and sacrificed 48 h later. The
results indicate a significant decrease of fluorescence when
compared with the control mouse. Moreover, when the siRNA (250
.mu.g) is administered in two doses of 125 .mu.g and analyzed 48 h
after the first injection, the decrease of GFP expression was
similar to that after a single application. The results are shown
in FIG. 4. For each experimental condition an average of the data
is represented.
[0101] At the same time, small intestine samples were collected in
RNA later to confirm previous data. mRNA levels were measured by
RT-PCR. These results, shown in FIG. 5, confirm the previous ones
obtained with fluorescence analysis.
[0102] As shown in FIG. 5, the administered dose of 250 .mu.g of
siRNA in one or two applications was enough and sufficient to
downregulate the level of GFP mRNA in small intestine, confirming
the delivery of the siRNA in small intestine by intrarectal
administration. The level of downregulation compared to the control
is higher when the analysis is done by RT-PCR, this being due to
the higher sensitivity of the technique.
Example 5
In Vivo Assays. Analysis of Large Intestine
[0103] Large intestine was further analyzed in the same way as
small intestine. To determine the siRNA effect in large intestine,
samples collected in OCT medium were analyzed to determine the
downregulation of GFP by measurement of fluorescence following
siRNA application. The results indicate a significant decrease of
fluorescence when compared to control mouse (FIG. 6). Moreover,
when the dose is administered in two applications of 125 .mu.g and
analyzed 48 h after the first injection, the decrease was very
similar to that obtained after a single siRNA administration,
demonstrating the effectiveness of the treatment.
[0104] As in small intestine, large intestine samples were
collected in RNA later and data of mRNA levels represented in FIG.
7. The data obtained by RT-PCR confirm the previous ones obtained
with fluorescence analysis. These results open a new route to
therapeutic siRNA administration to treatment of bowel
diseases.
[0105] Data of samples collected in OCT medium and in RNA later are
summarized in FIGS. 8 and 9 respectively.
[0106] We also investigated whether there was any downregulation of
GFP expression in other selected tissues of the mice; no
downregulation was observed in bladder, kidney, lung, ovary, and
liver tissues, suggesting that intrarectal administration of siRNA
can be used to specifically target intestinal tissue.
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Sequence CWU 1
1
90119RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1uguucccaug ccuucacca 19219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2aaccugcuga gggccguca 19319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3caugcuccag aaggccaga 19419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4ggccagacaa acucuagaa 19519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5aaccagcaca guggaggcc 19619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6accagcacag uggaggccu 19719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7ccaagaauga gaguugccu 19819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gaaugagagu ugccuaaau 19919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9ugagaguugc cuaaauucc 191019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10auuccagaga gaccucuuu 191119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11uuccagagag accucuuuc 191219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12cuaaugggag uugccuggc 191319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13gacuugaaga uguaccagg 191419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14gauguaccag guggaguuc 191519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15gaccaugaau gcaaagcuu 191619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16ugcaaagcuu cugauggau 191719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17agcuucugau ggauccuaa 191819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18gcuucugaug gauccuaag 191919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19gaggcagauc uuucuagau 192019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20aacaugcugg caguuauug 192119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21acaugcuggc aguuauuga 192219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22caugcuggca guuauugau 192319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23uuucaacagu gagacugug 192419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24cagugagacu gugccacaa 192519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25aaauccuccc uugaagaac 192619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26aauccucccu ugaagaacc 192719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27auccucccuu gaagaaccg 192819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28uccucccuug aagaaccgg 192919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29aaucaagcuc ugcauacuu 193019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30aucaagcucu gcauacuuc 193119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31ucaagcucug cauacuucu 193219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32gcucugcaua cuucuucau 193319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33uucgggcagu gacuauuga 193419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34uuggauuggu auccggaug 193519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35augguggucc ucaccugug 193619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36uggugguccu caccuguga 193719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 37gaagauggua ucaccugga 193819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38gaugguauca ccuggaccu 193919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 39aacccugacc auccaaguc 194019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 40acccugacca uccaaguca 194119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 41cccugaccau ccaagucaa 194219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 42gucaaagagu uuggagaug 194319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 43agaguuugga gaugcuggc 194419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 44gaguuuggag augcuggcc 194519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 45aggaggcgag guucuaagc 194619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 46ggaggcgagg uucuaagcc 194719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47gccauucgcu ccugcugcu 194819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 48aaaggaagau ggaauuugg 194919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 49aaggaagaug gaauuuggu 195019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50aggaagaugg aauuugguc 195119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 51ggaagaugga auuuggucc 195219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52gauggaauuu gguccacug 195319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 53aggaccagaa agaacccaa 195419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 54ggaccagaaa gaacccaaa 195519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55uaagaccuuu cuaagaugc 195619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56gaccuuucua agaugcgag 195719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 57gaugcgaggc caagaauua 195819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 58gaauuauucu ggacguuuc 195919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59uuauucugga cguuucacc 196019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 60aagcagcaga ggcucuucu 196119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 61agcagcagag gcucuucug 196219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62gcagcagagg cucuucuga 196319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 63caaggaguau gaguacuca 196419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 64ggaguaugag uacucagug 196519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 65aacuacacca gcagcuucu 196619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 66acuacaccag cagcuucuu 196719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67cuacaccagc agcuucuuc 196819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 68accugaccca cccaagaac 196919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 69ccugacccac ccaagaacu 197019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 70gaacuugcag cugaagcca 197119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 71cuugcagcug aagccauua 197219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 72gccauuaaag aauucucgg 197319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73agaauucucg gcaggugga 197419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 74gaauucucgg cagguggag 197519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 75uucucggcag guggagguc 197619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 76agaaagauag agucuucac 197719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77gaaagauaga gucuucacg 197819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78agauagaguc uucacggac 197919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79gauagagucu ucacggaca 198019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80gaccucagcc acggucauc 198119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81aaaugccagc auuagcgug 198219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 82aaugccagca uuagcgugc 198319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 83augccagcau uagcgugcg 198419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84ugccagcauu agcgugcgg 198519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85ugggcaucug ugcccugca 198619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 86cuacagcacc agcuucuuc 198719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 87gcacggcagc agaauaaau 198821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 88ggcuacgucc aggagcgcac c 218921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 89ggcuacgucc aggagcgcac c 219021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 90ugcgcuccug gacguagccu u 21
* * * * *
References