U.S. patent application number 17/117025 was filed with the patent office on 2021-06-24 for c/ebp alpha sarna compositions and methods of use.
The applicant listed for this patent is MiNA THERAPEUTICS LIMITED. Invention is credited to Robert Habib, Markus Hossbach, Hans E. Huber, Monika Krampert, Pal Saetrom, Endre Bakken Stovner, Hans-Peter Vornlocher, Andreas Wagner.
Application Number | 20210187007 17/117025 |
Document ID | / |
Family ID | 1000005436065 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210187007 |
Kind Code |
A1 |
Wagner; Andreas ; et
al. |
June 24, 2021 |
C/EBP ALPHA SARNA COMPOSITIONS AND METHODS OF USE
Abstract
The invention relates to saRNA targeting a C/EBP.alpha.
transcript and therapeutic compositions comprising said saRNA.
Methods of using the therapeutic compositions are also
provided.
Inventors: |
Wagner; Andreas; (Wien,
AT) ; Habib; Robert; (London, GB) ; Huber;
Hans E.; (Lansdale, PA) ; Saetrom; Pal;
(Trondheim, NO) ; Stovner; Endre Bakken;
(Trondheim, NO) ; Hossbach; Markus; (Kulmbach,
DE) ; Krampert; Monika; (Bamberg, DE) ;
Vornlocher; Hans-Peter; (Bayreuth, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MiNA THERAPEUTICS LIMITED |
LONDON |
|
GB |
|
|
Family ID: |
1000005436065 |
Appl. No.: |
17/117025 |
Filed: |
December 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15568139 |
Oct 20, 2018 |
10912790 |
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PCT/GB2016/051117 |
Apr 21, 2016 |
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17117025 |
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62150889 |
Apr 22, 2015 |
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62235778 |
Oct 1, 2015 |
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62308521 |
Mar 15, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/315 20130101; C12N 2310/14 20130101; A61K 31/7105
20130101; A61P 1/16 20180101; A61K 9/127 20130101; C12N 2310/113
20130101; A61P 35/00 20180101; C12N 2310/351 20130101; A61P 3/08
20180101 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; A61P 3/08 20060101 A61P003/08; A61P 1/16 20060101
A61P001/16; A61P 35/00 20060101 A61P035/00; A61K 9/127 20060101
A61K009/127; C12N 15/113 20060101 C12N015/113 |
Claims
1. A synthetic isolated saRNA which up-regulates expression of
C/EBP.alpha. gene, wherein the saRNA is at least 80% complement to
a region on SEQ ID No. 77, and wherein the saRNA has 14-30
nucleotides.
2. The saRNA of claim 1, wherein the saRNA is single stranded.
3. The saRNA of claim 2, wherein the saRNA comprises a 3'
overhang.
4. The saRNA of claim 2, wherein the saRNA is modified.
5. The saRNA of claim 4, wherein the saRNA comprises at least 2
modifications.
6. The saRNA of claim 4, wherein the modification comprises any of
2'-F, 2'-OMe, inverted deoxyribose, or phosphorothioate linkage
between nucleotides.
7. The saRNA of claim 2, wherein the saRNA comprises a sequence
selected from SEQ ID No. 35, 37, 39, 41, 43, 45, 47, 49, 93 (AW51)
and 109 (CEBPA51).
8. The saRNA of claim 1, wherein the saRNA is double-stranded and
comprises an antisense strand and a sense strand.
9. The saRNA of claim 5, wherein the antisense strand comprises a
sequence selected from SEQ ID No. 35, 37, 39, 41, 43, 45, 47, 49,
93 (AW51) and 109 (CEBPA51).
10. The saRNA of claim 6, wherein the sense strand comprises a
sequence selected from SEQ ID No. 34, 36, 38, 40, 42, 44, 48, 94
and 110.
11. The saRNA of claim 8, wherein the saRNA is modified.
12. The saRNA of claim 11, wherein the saRNA comprises at least 2
modifications.
13. The saRNA of claim 11, wherein the modification may comprise
any of 2'-F, 2'-OMe, inverted deoxyribose, or phosphorothioate
linkage between nucleotides.
14. The saRNA of claim 11, wherein the modification is on the sense
strand.
15. The saRNA of claim 11, wherein the modification is on both the
sense and antisense strand.
16. A method of up-regulating C/EBP.alpha. gene in a cell or
up-regulating the expression of a gene selected from,
alanine-glyoxylate aminotransferase (AGXT), cytochrome P450 3A4
(CYP3A4), ornithine transcarbamylase (OTC, or hepatocyte nuclear
factor 4-alpha (HNF4a) in a cell, comprising administering the
saRNA of claim 1 to the cell.
17. The method of claim 16, wherein the cell is a proliferating
cell.
18. The method of claim 17, wherein the cell is a cancer cell.
19. The method of claim 18, wherein the cell is a hepatocellular
carcinoma (HCC) cell.
20. The method of claim 16, wherein the cell is not
hyperproliferating cell.
21. The method of claim 20, wherein the cell is a primary human
hepatocyte cell.
22. A method of treating liver fibrosis, liver failure, or
nonalcoholic steatohepatitis (NASH) of a subject in need thereof
comprising administering the saRNA of claim 1 to the subject.
23. The method of claim 22, wherein the liver failure is acute
liver failure.
24. The method of claim 22, wherein the total bilirubin (TBIL)
level, circulating alanine aminotransferase (ALT) level, aspartate
aminotransferase (AST) level, alkaline phosphatase (ALP) level,
gamma-glutamyl-transpeptidase (GGT) level, liver hydroxyproline
level, prothrombin time, ammonia, or liver triglyceride (liver TG)
level of said subject is decreased.
25. The method of claim 22, wherein the serum albumin level, total
protein level of said subject is increased.
26. The method of claim 22, wherein the fibrous tissue or
peudolobule formation of said subject is reduced.
27. A method of treating type II diabetes or insulin resistance of
a subject in need thereof comprising administering the saRNA of
claim 1 to the subject.
28. The method of claim 27, wherein liver cholesterol level, serum
AST level, fasting glucose level, the ratio of triglycerides to
HDL-c, or liver to body ratio of said subject is decreased.
29. The method of claim 27, wherein the insulin level of said
subject is increased.
30. A method of encapsulating the saRNA of claim 1 in a liposome,
comprising: dissolving the saRNA in a first buffer to for a saRNA
solution, filtering the saRNA solution through a 0.2 .mu.m filter,
mixing the filtered saRNA solution with a lipid solution in an
injection module to form a liposome formulation, adding a second
buffer to the liposome formulation.
31. The method of claim 30, wherein the first buffer is
Na-Acetate/Sucrose.
32. The method of claim 30, wherein the pH for the saRNA solution
is around 4.0.
33. The method of claim 30, wherein the concentration of the saRNA
in the saRNA solution is around 2.38 mg/mL.
34. The method of claim 30, wherein the lipid solution comprises
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
cholesteryl-hemi succinate (CHEMS), and
4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MOCHOL).
35. The method of claim 34, wherein the molar ratio of
POPC:DOPE:CHEMS:MOCHOL is around 6:24:23:47.
36. The method of claim 30, wherein the second buffer has a pH of
around 9.
37. The method of claim 36, wherein the second buffer is
NaCl/Na2HPO4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application which claims
priority to U.S. Non-Prov. application Ser. No. 15/568,139 filed
Oct. 20, 2017, which is a National Phase entry of PCT Application
No. PCT/GB2016/051117 filed Apr. 21, 2016, which claims priority to
U.S. Prov. Application No. 62/150,889 filed Apr. 22, 2015, U.S.
Prov. Application No. 62/235,778 filed Oct. 1, 2015, and U.S. Prov.
Application No. 62/308,521 filed Mar. 15, 2016, the contents of
each of which are incorporated herein by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The sequence listing filed, entitled
20581015USCON_SEQLIST.txt, was created on Dec. 9, 2020 and is
80,920 bytes in size. The information in electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to polynucleotide, specifically saRNA,
compositions for the modulating C/EBP.alpha. and C/EBP.alpha.
pathways and to the methods of using the compositions in
therapeutic applications such as treating metabolic disorders,
hyperproliferative diseases, and regulating stem cell linage.
BACKGROUND OF THE INVENTION
[0004] CCAAT/enhancer-binding protein .alpha. (C/EBP.alpha., C/EBP
alpha, C/EBPA or CEBPA) is a leucine zipper protein that is
conserved across humans and rats. This nuclear transcription factor
is enriched in hepatocytes, myelomonocytes, adipocytes, as well as
other types of mammary epithelial cells [Lekstrom-Himes et al., J.
Bio. Chem, vol. 273, 28545-28548 (1998)]. It is composed of two
transactivation domains in the N-terminal part, and a leucine
zipper region mediating dimerization with other C/EBP family
members and a DNA-binding domain in the C-terminal part. The
binding sites for the family of C/EBP transcription factors are
present in the promoter regions of numerous genes that are involved
in the maintenance of normal hepatocyte function and response to
injury. C/EBP.alpha. has a pleiotropic effect on the transcription
of several liver-specific genes implicated in the immune and
inflammatory responses, development, cell proliferation,
anti-apoptosis, and several metabolic pathways [Darlington et al.,
Current Opinion of Genetic Development, vol. 5(5), 565-570 (1995)].
It is essential for maintaining the differentiated state of
hepatocytes. It activates albumin transcription and coordinates the
expression of genes encoding multiple ornithine cycle enzymes
involved in urea production, therefore playing an important role in
normal liver function.
[0005] In the adult liver, C/EBP.alpha. is defined as functioning
in terminally differentiated hepatocytes whilst rapidly
proliferating hepatoma cells express only a fraction of
C/EBP.alpha. [Umek et al., Science, vol. 251, 288-292 (1991)].
C/EBP.alpha. is known to up-regulate p21, a strong inhibitor of
cell proliferation through the up-regulation of retinoblastoma and
inhibition of Cdk2 and Cdk4 [Timchenko et al., Genes &
Development, vol. 10, 804-815 (1996); Wang et al., Molecular Cell,
vol. 8, 817-828 (2001)]. In hepatocellular carcinoma (HCC),
C/EBP.alpha. functions as a tumor suppressor with
anti-proliferative properties [Iakova et al., Seminars in Cancer
Biology, vol. 21(1), 28-34 (2011)].
[0006] Different approaches are carried out to study C/EBP.alpha.
mRNA or protein modulation. It is known that C/EBP.alpha. protein
is regulated by post-translational phosphorylation and sumoylation.
For example, FLT3 tyrosine kinase inhibitors and extra-cellular
signal-regulated kinases 1 and/or 2 (ERK1/2) block serine-21
phosphorylation of C/EBP.alpha., which increases the granulocytic
differentiation potential of the C/EBP.alpha. protein [Radomska et
al., Journal of Experimental Medicine, vol. 203(2), 371-381 (2006)
and Ross et al., Molecular and Cellular Biology, vol. 24(2),
675-686 (2004)]. In addition, C/EBP.alpha. translation can be
efficiently induced by 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid
(CDDO), which alters the ratio of the C/EBP.alpha. protein isoforms
in favor of the full-length p42 form over p30 form thereby inducing
granulocytic differentiation [Koschmieder et al., Blood, vol.
110(10), 3695-3705 (2007)]. The C/EBP.alpha. gene is an intronless
gene located on chromosome 19q13.1. Most eukaryotic cells use
RNA-complementarity as a mechanism for regulating gene expression.
One example is the RNA interference (RNAi) pathway which uses
double stranded short interfering RNAs to knockdown gene expression
via the RNA-induced silencing complex (RISC). It is now established
that short duplex RNA oligonucleotides also have the ability to
target the promoter regions of genes and mediate transcriptional
activation of these genes and they have been referred to as RNA
activation (RNAa), antigene RNA (agRNA) or short activating RNA
(saRNA) [Li et al., PNAS, vol. 103, 17337-17342 (2006)]. saRNA
induced activation of genes is conserved in other mammalian species
including mouse, rat, and non-human primates and is fast becoming a
popular method for studying the effects of endogenous up-regulation
of genes. Thus, there is a need for targeted modulation of
C/EBP.alpha. for therapeutic purposes with saRNA.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions, methods and
kits for the design, preparation, manufacture, formulation and/or
use of short activating RNA (saRNA) molecules that modulate
C/EBP.alpha. gene expression and/or function for therapeutic
purposes, including diagnosing and prognosis.
[0008] One aspect of the invention provides a pharmaceutical
composition comprising a saRNA that targets a C/EBP.alpha.
transcript and at least one pharmaceutically acceptable carrier.
Yet another aspect of the invention provides a method of regulating
stem cell differentiation and pluripotency comprising contact said
stem cell with a saRNA that targets a C/EBP.alpha. transcript.
[0009] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the invention, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead being placed upon illustrating the
principles of various embodiments of the invention.
[0011] FIG. 1 shows the primary effects of C/EBP.alpha. on the
liver.
[0012] FIG. 2 shows the secondary effects of C/EBP.alpha. on the
adipose tissue.
[0013] FIG. 3 is a schematic illustrating the relationships among
the nucleic acid moieties involved in the function of an saRNA of
the invention.
[0014] FIG. 4A-4D show upregulation of CEBPA in a panel of HCC
cells by CEBPA-saRNA.
[0015] FIG. 5A-5D show upregulation of albumin in a panel of HCC
cells by CEBPA-saRNA.
[0016] FIG. 6A shows CEBPA mRNA levels in DU145 cells transfected
with modified saRNA normalized to GAPDH. FIG. 6B shows GAPDH mRNA
levels in DU145 cells. FIG. 6C shows Aha1 mRNA levels as a
transfection control.
[0017] FIG. 7A-7B show CEBPA mRNA levels in DU145 cells transfected
with CEBPA-siRNA or Fluc normalized to GAPDH. FIG. 7C shows AhA-1
mRNA level in DU145 cells transfected with AhA-1-siRNA normalized
to GAPDH.
[0018] FIG. 8A-8C show CEBPA mRNA levels in DU145 cells transfected
with three saRNAs normalized to GAPDH.
[0019] FIG. 9A and FIG. 9B show expression levels. FIG. 9A shows
AhA1, albumin and CEBPA relative expression levels in hepatocytes
in non-proliferation media. FIG. 9B shows AhA1, albumin and CEBPA
relative expression levels in hepatocytes in proliferation
media.
[0020] FIG. 10A shows representative Western Blot showing
C/EBP-.alpha. protein levels in HepG2, Hep3B and PLCPRF5 cells
following transfection with CEBPA-51. FIG. 10B shows relative CEBPA
mRNA expression (*** p=0.0002; ** p=0.0012) in HepG2, Hep3B and
PLCPRF5 cells following transfection with CEBPA-51.
[0021] FIG. 11A-11C show WST-1 cell proliferation assay results of
AW51 in HEP3B, HEPG2, and PLCPRF5 cell lines. FIG. 11D-11F show
sulforhodamine B (SRB) cell number assay results of AW51 in HEP3B,
HEPG2 and PLCPRF5 cells.
[0022] FIG. 12A-12B show AW51 off-targets measured in HuH7 cells
(FIG. 12A) and Panc-1 cells (FIG. 12B).
[0023] FIG. 13A-13B show CEBPA mRNA and albumin mRNA levels in in
cells transfected with non-specific control (NC-500000), the
unmodified AW1-51 sequence, the AW1-51 with internal sequence
mutations (CEBPA-AW01-510500), the AW1-51 modified on SS
(CEBPA-AW01-510012), and the AW1-51 modified on AS
(CEBPA-AW01-510013), or modified on both strands
(CEBPA-AW01-510014).
[0024] FIG. 14A demonstrates CEBPA51 upregulates CEBPA in primary
human hepatocytes. FIG. 14B demonstrates CEBPA51 increases albumin
secretion in primary human hepatocytes. FIG. 14C shows Aha1 levels.
Aha1 siRNA was used as a positive control to determine transfection
efficiency in primary cells. All statistical significance follows a
non-parametric Mann Whitney U test at 95% confidence interval.
[0025] FIG. 15 shows albumin ELISA results from media of cultured
primary human hepatocytes transfected with CEBPA51.
[0026] FIG. 16A-16F show relative expression of (A)
Alanine-glyoxylate aminotransferase (AGXT); (B) Albumin; (C)
Cytochrome P450 3A4 (CYP3A4); (D) Ornithine transcarbamylase (OTC);
(E) Hepatocyte nuclear factor 4-alpha (HNF4A) and (F) CEBPA
transcript levels detected in primary human hepatocytes transfected
with CEBPA-51.
[0027] FIG. 17 shows CEBPA mRNA expression in cynomogus (CYNOM-K1)
fibroblasts 24 hours after second transfection of CEBPA-51.
[0028] FIG. 18A-18C show stability of CEBPA-51 in rat plasma, human
plasma and cynomolgus monkey plasma.
[0029] FIG. 19A-19C show stability of MTL-CEBPA in rat plasma,
human plasma and cynomolgus monkey plasma.
[0030] FIG. 20A shows mean concentration of CEBPA51 and
metabolites/impurities after IV administration of 1.5 mg/kg CEBPA51
in rat plasma.
[0031] FIG. 20B shows mean concentration of intact CEBPA51 in rat
plasma after IV administration of 1.5 mg/kg CEBPA51. 0.5 hours
after administration the concentration of intact CEBPA51 is below
detection limit.
[0032] FIG. 21A shows mean concentration of CEBPA51 after IV
administration of 2.175 mg/kg MTL-CEBPA in rat plasma. Comparison
of intact parent compound to metabolites of CEBPA51 shows a high
stability of MTL-CEBPA in plasma.
[0033] FIG. 21B shows mean concentration of intact CEBPA51 in rat
plasma after IV administration of 2.175 mg/kg MTL-CEBPA. 48 hours
after administration CEBPA51 is still found in plasma.
[0034] FIG. 22A-22K show body weight, ALT level, AST level, ALP
level, GGT level, bilirubin level, total protein level, albumin
level, prothrombin time, ammonia level, and hydroxyproline level
changes in CCL4-treated rats after administration of different
doses of MTL-CEBPA.
[0035] FIG. 23 is gross pathology of a healthy liver and livers
treated with CCL4 and control, CCL4 and 0.3 mg/kg MTL-CEBPA, and
CCL4 and 3.0 mg/kg MTL-CEBPA.
[0036] FIG. 24A-24C show histology staining including H&E
staining, Mason Trichrome staining, and Sirius red staining for
livers of naive rats, rats treated with CCL4 and control, and rats
treated with CCL4 and MTL-CEBPA. FIG. 23A is sham control. FIG. 23B
is CCL4-treated rats that received NOV340/siFluc treatment
(negative control). FIG. 23C is CCL4-treated rats that received
MTL-CEBPA treatment.
[0037] FIG. 25A-25H show effect of TAA injection on liver function
parameters such as ALT, AST, ALP, GGT, bilirubin, and other
parameters such as total protein, albumin and ammonia.
[0038] FIG. 26A-26N show serum and physical parameters of diabetes
rats treated with CEBPA-saRNA. FIG. 26A: triglyceride levels. FIG.
26B: total cholesterol levels. FIG. 26C: liver cholesterol levels.
FIG. 26D: HDL-c levels. FIG. 26E: LDL-c levels. FIG. 26F:
HDL-c/LDL-c ratios. FIG. 26G: AST levels. FIG. 26H: ALT levels.
FIG. 26I: TG/HDL-c ratios. FIG. 26J: fasting glucose levels. FIG.
26K: insulin levels. FIG. 26L: body weight changes. FIG. 26M: liver
weight changes. FIG. 26N: liver weight/body weight ratios.
[0039] FIG. 27A-27B show body weight and feed consumption changes
in an MCD-induced NASH study. FIG. 27C-27H showed ALT, AST, ALP,
bilirubin, albumin, and liver TG level changes in the study.
[0040] FIG. 28 shows CEBPA and Albumin mRNA expression in liver
tissue. Expression values are relative to pretreatment control
(DEN-induced HCC), **p<0.01 vs. NOV340/siFLUC.
[0041] FIG. 29A-29I show physical and Serum Parameters in
MTL-CEBPA-treated DEN-Rats. Values shown as mean.+-.SEM; p-values
shown for MTL-CEBPA: #p<0.1, *p<0.05 vs. NOV340/siFLUC;
$p<0.05 vs. Pretreatment control.
[0042] FIG. 30A shows co-immunoprecipitation results of Argonaute
proteins with Biotinylated strands of CEBPA51.
[0043] FIG. 30B shows CEBPA levels in wild type and Ago2 knock-out
mouse embryonic fibroblasts (MEF) cells both transfected with
CEBPA-saRNA.
[0044] FIG. 30C shows p21 levels in wild type and Ago2 knock-out
mouse embryonic fibroblasts (MEF) cells both transfected with
CEBPA-saRNA.
[0045] FIG. 31 is a general overview of MTL-CEBPA production
process.
[0046] FIG. 32A and FIG. 32B show TNF-.alpha. and IFN-.alpha.
secretion in huPBMCs after transfection with CEBPA-51 and control
oligos.
[0047] FIG. 33 is a dose escalation flowchart.
[0048] FIG. 34 shows encapsulation efficiency of CEBPA-51 into
liposomes versus API concentration in the injection buffer for two
different pH values of the API solution.
[0049] FIG. 35 shows encapsulation efficiency of CEBPA-51 into
liposomes versus API concentration in the injection buffer.
[0050] FIG. 36 shows ammonia levels after MTL-CEBPA treatment at
week 8 or week 11.
[0051] FIG. 37A and FIG. 37B show ascites scores after MTL-CEBPA
treatment at week 8 or week 11.
[0052] FIG. 38A and FIG. 38B show survival graphs after MTL-CEBPA
treatment at week 8 or week 11.
[0053] FIG. 39 shows a duplex of CEBPA-51 (sense and antisense
strands).
[0054] FIG. 40A is a general overview of CEBPA-51 synthesis.
[0055] FIG. 40B is a detailed flow chart of CEBPA-51 synthesis.
[0056] FIG. 41 is a flow chart of MTL-CEBPA production (Steps
1-9).
DETAILED DESCRIPTION
[0057] The present invention provides compositions, methods and
kits for modulating C/EBP.alpha. gene expression and/or function
for therapeutic purposes. These compositions, methods and kits
comprise nucleic acid constructs that target a C/EBP.alpha.
transcript.
[0058] C/EBP.alpha. protein is known as a critical regulator of
metabolic processes and cell proliferation. Modulating C/EBP.alpha.
gene has great potentials for therapeutic purposes. The present
invention addresses this need by providing nucleic acid constructs
targeting a C/EBP.alpha. transcript, wherein the nucleic acid
constructs may include single or double stranded DNA or RNA with or
without modifications.
[0059] C/EBP.alpha. gene as used herein is a double-stranded DNA
comprising a coding strand and a template strand. It may also be
referred to the target gene in the present application.
[0060] The terms "C/EBP.alpha. transcript", "C/EBP.alpha. target
transcript" or "target transcript" in the context may be
C/EBP.alpha. mRNA encoding C/EBP.alpha. protein. C/EBP.alpha. mRNA
is transcribed from the template strand of C/EBP.alpha. gene and
may exist in the mitochondria.
[0061] The antisense RNA of the C/EBP.alpha. gene transcribed from
the coding strand of the C/EBP.alpha. gene is called a target
antisense RNA transcript herein after. The target antisense RNA
transcript may be a long non-coding antisense RNA transcript.
[0062] The terms "small activating RNA", "short activating RNA", or
"saRNA" in the context of the present invention means a
single-stranded or double-stranded RNA that upregulates or has a
positive effect on the expression of a specific gene. The saRNA may
be single-stranded of 14 to 30 nucleotides. The saRNA may also be
double-stranded, each strand comprising 14 to 30 nucleotides. The
gene is called the target gene of the saRNA. A saRNA that
upregulates the expression of the C/EBP.alpha. gene is called an
"C/EBP.alpha.-saRNA" and the C/EBP.alpha. gene is the target gene
of the C/EBP.alpha.-saRNA.
[0063] In one embodiment, C/EBP.alpha.-saRNA targeting a
C/EBP.alpha. target antisense RNA transcript upregulates
C/EBP.alpha. gene expression and/or function.
[0064] The terms "target" or "targeting" in the context mean having
an effect on a C/EBP.alpha. gene. The effect may be direct or
indirect. Direct effect may be caused by complete or partial
hybridization with the C/EBP.alpha. target antisense RNA
transcript. Indirect effect may be upstream or downstream.
[0065] C/EBP.alpha.-saRNA may have a downstream effect on a
biological process or activity. In such embodiments,
C/EBP.alpha.-saRNA may have an effect (either upregulating or
downregulating) on a second, non-target transcript.
[0066] The term "gene expression" in the context may include the
transcription step of generating C/EBP.alpha. mRNA from
C/EBP.alpha. gene or the translation step generating C/EBP.alpha.
protein from C/EBP.alpha. mRNA. An increase of C/EBP.alpha. mRNA
and an increase of C/EBP.alpha. protein both indicate an increase
or a positive effect of C/EBP.alpha. gene expression.
[0067] By "upregulation" or "activation" of a gene is meant an
increase in the level of expression of a gene, or levels of the
polypeptide(s) encoded by a gene or the activity thereof, or levels
of the RNA transcript(s) transcribed from the template strand of a
gene above that observed in the absence of the saRNA of the present
invention. The saRNA of the present invention may have a direct or
indirect upregulating effect on the expression of the target
gene.
[0068] In one embodiment, the saRNA of the present invention may
show efficacy in proliferating cells. As used herein with respect
to cells, "proliferating" means cells which are growing and/or
reproducing rapidly.
I. Composition of the Invention
[0069] One aspect of the present invention provides pharmaceutical
compositions comprising a saRNA that upregulates CEBPA gene, and at
least one pharmaceutically acceptable carrier. Such a saRNA is
referred herein after as "C/EBP.alpha.-saRNA", or "saRNA of the
present invention", used interchangeably in this application.
saRNA Design
[0070] C/EBP.alpha.-saRNA upregulates C/EBP.alpha. gene. In one
embodiment, it is designed to be complementary to a target
antisense RNA transcript of C/EBP.alpha. gene, and it may exert its
effect on C/EBP.alpha. gene expression and/or function by
down-regulating the target antisense RNA transcript.
[0071] The term "complementary to" in the context means being able
to hybridize with the target antisense RNA transcript under
stringent conditions.
[0072] The term "sense" when used to describe a nucleic acid
sequence in the context of the present invention means that the
sequence has identity to a sequence on the coding strand of a gene.
The term "antisense" when used to describe a nucleic acid sequence
in the context of the present invention means that the sequence is
complementary to a sequence on the coding strand of a gene.
[0073] It is to be understood that thymidine of the DNA is replaced
by uridine in RNA and that this difference does not alter the
understanding of the terms "antisense" or "complementarity".
[0074] The target antisense RNA transcript may be transcribed from
a locus on the coding strand between up to 100, 80, 60, 40, 20 or
10 kb upstream of a location corresponding to the target gene's
transcription start site (TSS) and up to 100, 80, 60, 40, 20 or 10
kb downstream of a location corresponding to the target gene's
transcription stop site.
[0075] In one embodiment, the target antisense RNA transcript is
transcribed from a locus on the coding strand located within +/-1
kb of the target gene's transcription start site.
[0076] In another embodiment, the target antisense RNA transcript
is transcribed from a locus on the coding strand located within
+/-500, +/-250 or +/-100 of the target gene's transcription start
site.
[0077] In another embodiment, the target antisense RNA transcript
is transcribed from a locus on the coding strand located +/-2000
nucleotides of the target gene's transcription start site.
[0078] In another embodiment, the locus on the coding strand is no
more than 1000 nucleotides upstream or downstream from a location
corresponding to the target gene's transcription start site.
[0079] In another embodiment, the locus on the coding strand is no
more than 500 nucleotides upstream or downstream from a location
corresponding to the target gene's transcription start site.
[0080] The term "transcription start site" (TSS) as used herein
means a nucleotide on the template strand of a gene corresponding
to or marking the location of the start of transcription. The TSS
may be located within the promoter region on the template strand of
the gene.
[0081] The term "transcription stop site" as used herein means a
region, which can be one or more nucleotides, on the template
strand of a gene, which has at least one feature such as, but not
limited to, a region which encodes at least one stop codon of the
target transcript, a region encoding a sequence preceding the 3'UTR
of the target transcript, a region where the RNA polymerase
releases the gene, a region encoding a splice site or an area
before a splice site and a region on the template strand where
transcription of the target transcript terminates.
[0082] The phrase "is transcribed from a particular locus" in the
context of the target antisense RNA transcript of the invention
means the transcription of the target antisense RNA transcript
starts at the particular locus.
[0083] The target antisense RNA transcript is complementary to the
coding strand of the genomic sequence of the target gene, and any
reference herein to "genomic sequence" is shorthand for "coding
strand of the genomic sequence".
[0084] The "coding strand" of a gene has the same base sequence as
the mRNA produced, except T is replayed by U in the mRNA. The
"template strand" of a gene is therefore complementary and
antiparallel to the mRNA produced.
[0085] Thus, the target antisense RNA transcript may comprise a
sequence which is complementary to a genomic sequence located
between 100, 80, 60, 40, 20 or 10 kb upstream of the target gene's
transcription start site and 100, 80, 60, 40, 20 or 10 kb
downstream of the target gene's transcription stop site.
[0086] In one embodiment, the target antisense RNA transcript
comprises a sequence which is complementary to a genomic sequence
located between 1 kb upstream of the target gene's transcription
start site and 1 kb downstream of the target gene's transcription
stop site.
[0087] In another embodiment, the target antisense RNA transcript
comprises a sequence which is complementary to a genomic sequence
located between 500, 250 or 100 nucleotides upstream of the target
gene's transcription start site and ending 500, 250 or 100
nucleotides downstream of the target gene's transcription stop
site.
[0088] The target antisense RNA transcript may comprise a sequence
which is complementary to a genomic sequence which includes the
coding region of the CEBPA gene. The target antisense RNA
transcript may comprise a sequence which is complementary to a
genomic sequence that aligns with the target gene's promoter region
on the template strand. Genes may possess a plurality of promoter
regions, in which case the target antisense RNA transcript may
align with one, two or more of the promoter regions. An online
database of annotated gene loci may be used to identify the
promoter regions of genes. The terms `align` and `alignment` when
used in the context of a pair of nucleotide sequences mean the pair
of nucleotide sequences are complementary to each other or have
sequence identity with each other.
[0089] The region of alignment between the target antisense RNA
transcript and the promoter region of the target gene may be
partial and may be as short as a single nucleotide in length,
although it may be at least 15 or at least 20 nucleotides in
length, or at least 25 nucleotides in length, or at least 30, 35,
40, 45 or 50 nucleotides in length, or at least 55, 60, 65, 70 or
75 nucleotides in length, or at least 100 nucleotides in length.
Each of the following specific arrangements is intended to fall
within the scope of the term "alignment":
a) The target antisense RNA transcript and the target gene's
promoter region are identical in length and they align (i.e. they
align over their entire lengths). b) The target antisense RNA
transcript is shorter than the target gene's promoter region and
aligns over its entire length with the target gene's promoter
region (i.e. it aligns over its entire length to a sequence within
the target gene's promoter region). c) The target antisense RNA
transcript is longer than the target gene's promoter region and the
target gene's promoter region is aligned fully by it (i.e. the
target gene's promoter region is aligns over its entire length to a
sequence within the target antisense RNA transcript). d) The target
antisense RNA transcript and the target gene's promoter region are
of the same or different lengths and the region of alignment is
shorter than both the length of the target antisense RNA transcript
and the length of the target gene's promoter region.
[0090] The above definition of "align" and "alignment" applies
mutatis mutandis to the description of other overlapping, e.g.,
aligned sequences throughout the description. Clearly, if a target
antisense RNA transcript is described as aligning with a region of
the target gene other than the promoter region then the sequence of
the target antisense RNA transcript aligns with a sequence within
the noted region rather than within the promoter region of the
target gene.
[0091] In one embodiment, the target antisense RNA transcript is at
least 1 kb, or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 20, 25,
30, 35 or 40 kb long.
[0092] In one embodiment, the target antisense RNA transcript
comprises a sequence which is at least 75%, or at least 85%, or at
least 90%, or at least 95% complementary along its full length to a
sequence on the coding strand of the target gene.
[0093] The present invention provides saRNAs targeting the target
antisense RNA transcript and may effectively and specifically
down-regulate such target antisense RNA transcripts. This can be
achieved by saRNA having a high degree of complementarity to a
region within the target antisense RNA transcript. The saRNA will
have no more than 5, or no more than 4 or 3, or no more than 2, or
no more than 1, or no mismatches with the region within the target
antisense RNA transcript to be targeted.
[0094] Referring to FIG. 3, as the target antisense RNA transcript
has sequence identity with a region of the template strand of the
target gene, the target antisense RNA transcript will be in part
identical to a region within the template strand of the target gene
allowing reference to be made either to the template strand of the
gene or to a target antisense RNA transcript. The location at which
the saRNA hybridizes or binds to the target antisense RNA
transcript (and hence the same location on the template strand) is
referred to as the "targeted sequence" or "target site".
[0095] The antisense strand of the saRNA (whether single- or
double-stranded) may be at least 80%, 90%, 95%, 98%, 99% or 100%
identical with the reverse complement of the targeted sequence.
Thus, the reverse complement of the antisense strand of the saRNA
has a high degree of sequence identity with the targeted sequence.
The targeted sequence may have the same length, i.e., the same
number of nucleotides, as the saRNA and/or the reverse complement
of the saRNA.
[0096] In some embodiments, the targeted sequence comprises at
least 14 and less than 30 nucleotides.
[0097] In some embodiments, the targeted sequence has 19, 20, 21,
22, or 23 nucleotides.
[0098] In some embodiments, the location of the targeted sequence
is situated within a promoter area of the template strand.
[0099] In some embodiments, the targeted sequence is located within
a TSS (transcription start site) core of the template stand. A "TSS
core" or "TSS core sequence" as used herein, refers to a region
between 2000 nucleotides upstream and 2000 nucleotides downstream
of the TSS (transcription start site). Therefore, the TSS core
comprises 4001 nucleotides and the TSS is located at position 2001
from the 5' end of the TSS core sequence. CEBPA TSS core sequence
is show in the table below:
TABLE-US-00001 CEBPA mRNA CEBPA protein CEBPA TSS core CEBPA TSS
core REF. No. REF. No. genomic location sequence ID No.
NM_001285829 NP_001272758 chr19:33302564 SEQ ID No. 77 NM_001287424
NP_001274353 minus strand NM_001287435 NP_001274364 NM_004364
NP_004355
[0100] In some embodiments, the targeted sequence is located
between 1000 nucleotides upstream and 1000 nucleotides downstream
of the TSS.
[0101] In some embodiments, the targeted sequence is located
between 500 nucleotides upstream and 500 nucleotides downstream of
the TSS.
[0102] In some embodiments, the targeted sequence is located
between 250 nucleotides upstream and 250 nucleotides downstream of
the TSS.
[0103] In some embodiments, the targeted sequence is located
between 100 nucleotides upstream and 100 nucleotides downstream of
the TSS.
[0104] In some embodiments, the targeted sequence is located
upstream of the TSS in the TSS core. The targeted sequence may be
less than 2000, less than 1000, less than 500, less than 250, or
less than 100 nucleotides upstream of the TSS.
[0105] In some embodiments, the targeted sequence is located
downstream of the TSS in the TSS core. The targeted sequence may be
less than 2000, less than 1000, less than 500, less than 250, or
less than 100 nucleotides downstream of the TSS.
[0106] In some embodiments, the targeted sequence is located +/-50
nucleotides surrounding the TSS of the TSS core. In some
embodiments, the targeted sequence substantially overlaps the TSS
of the TSS core. In some embodiments, the targeted sequence
overlaps begins or ends at the TSS of the TSS core. In some
embodiments, the targeted sequence overlaps the TSS of the TSS core
by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or
19 nucleotides in either the upstream or downstream direction.
[0107] The location of the targeted sequence on the template strand
is defined by the location of the 5' end of the targeted sequence.
The 5' end of the targeted sequence may be at any position of the
TSS core and the targeted sequence may start at any position
selected from position 1 to position 4001 of the TSS core. For
reference herein, when the 5' most end of the targeted sequence
from position 1 to position 2000 of the TSS core, the targeted
sequence is considered upstream of the TSS and when the 5' most end
of the targeted sequence is from position 2002 to 4001, the
targeted sequence is considered downstream of the TSS. When the 5'
most end of the targeted sequence is at nucleotide 2001, the
targeted sequence is considered to be a TSS centric sequence and is
neither upstream nor downstream of the TSS.
[0108] For further reference, for example, when the 5' end of the
targeted sequence is at position 1600 of the TSS core, i.e., it is
the 1600.sup.th nucleotide of the TSS core, the targeted sequence
starts at position 1600 of the TSS core and is considered to be
upstream of the TSS.
[0109] In one embodiment, the saRNA of the present invention may
have two strands that form a duplex, one strand being a guide
strand. The saRNA duplex is also called a double-stranded saRNA. A
double-stranded saRNA or saRNA duplex, as used herein, is a saRNA
that includes more than one, and preferably, two, strands in which
interstrand hybridization can form a region of duplex structure.
The two strands of a double-stranded saRNA are referred to as an
antisense strand or a guide strand, and a sense strand or a
passenger strand.
[0110] The antisense strand of a saRNA duplex, used interchangeably
with antisense strand saRNA or antisense saRNA, has a high degree
of complementarity to a region within the target antisense RNA
transcript. The antisense strand may have no more than 5, or no
more than 4 or 3, or no more than 2, or no more than 1, or no
mismatches with the region within the target antisense RNA
transcript or targeted sequence. Therefore, the antisense strand
has a high degree of complementary to the targeted sequence on the
template strand. The sense strand of the saRNA duplex, used
interchangeably with sense strand saRNA or sense saRNA, has a high
degree of sequence identity with the targeted sequence on the
template strand. In some embodiments, the targeted sequence is
located within the promoter area of the template strand. In some
embodiments, the targeted sequence is located within the TSS core
of the template stand.
[0111] The location of the antisense strand and/or sense strand of
the saRNA duplex, relative to the targeted sequence is defined by
making reference to the TSS core sequence. For example, when the
targeted sequence is downstream of the TSS, the antisense saRNA and
the sense saRNA start downstream of the TSS. In another example,
when the targeted sequence starts at position 200 of the TSS core,
the antisense saRNA and the sense saRNA start upstream of the
TSS.
[0112] The relationships among the saRNAs, a target gene, a coding
strand of the target gene, a template strand of the target gene, a
target antisense RNA transcript, a target transcript, a targeted
sequence/target site, and the TSS are shown in FIG. 3.
[0113] A "strand" in the context of the present invention means a
contiguous sequence of nucleotides, including non-naturally
occurring or modified nucleotides. Two or more strands may be, or
each form a part of, separate molecules, or they may be connected
covalently, e.g., by a linker such as a polyethyleneglycol linker.
At least one strand of a saRNA may comprise a region that is
complementary to a target antisense RNA. Such a strand is called an
antisense or guide strand of the saRNA duplex. A second strand of a
saRNA that comprises a region complementary to the antisense strand
of the saRNA is called a sense or passenger strand.
[0114] A saRNA duplex may also be formed from a single molecule
that is at least partly self-complementary forming a hairpin
structure, including a duplex region. In such case, the term
"strand" refers to one of the regions of the saRNA that is
complementary to another internal region of the saRNA. The guide
strand of the saRNA will have no more than 5, or no more than 4 or
3, or no more than 2, or no more than 1, or no mismatches with the
sequence within the target antisense RNA transcript.
[0115] In some embodiments, the passenger strand of a saRNA may
comprise at least one nucleotide that is not complementary to the
corresponding nucleotide on the guide strand, called a mismatch
with the guide strand. The mismatch with the guide strand may
encourage preferential loading of the guide strand (Wu et al., PLoS
ONE, vol. 6(12):e28580 (2011), the contents of which are
incorporated herein by reference in their entirety). In one
embodiment, the at least one mismatch with the guide strand may be
at 3' end of the passenger strand. In one embodiment, the 3' end of
the passenger strand may comprise 1-5 mismatches with the guide
strand. In one embodiment, the 3' end of the passenger strand may
comprise 2-3 mismatches with the guide strand. In one embodiment,
the 3' end of the passenger strand may comprise 6-10 mismatches
with the guide strand.
[0116] In one embodiment, an saRNA duplex may show efficacy in
proliferating cells
[0117] A saRNA duplex may have siRNA-like complementarity to a
region of a target antisense RNA transcript; that is, 100%
complementarity between nucleotides 2-6 from the 5' end of the
guide strand in the saRNA duplex and a region of the target
antisense RNA transcript. Other nucleotides of the saRNA may, in
addition, have at least 80%, 90%, 95%, 98%, 99% or 100%
complementarity to a region of the target antisense RNA transcript.
For example, nucleotides 7 (counted from the 5' end) until the 3'
end of the saRNA may have least 80%, 90%, 95%, 98%, 99% or 100%
complementarity to a region of the target antisense RNA
transcript.
[0118] The terms "small interfering RNA" or "siRNA" in the context
mean a double-stranded RNA typically 20-25 nucleotides long
involved in the RNA interference (RNAi) pathway and interfering
with or inhibiting the expression of a specific gene. The gene is
the target gene of the siRNA. For example, siRNA that interferes
the expression of APOA1 gene is called "APOA1-siRNA" and the APOA1
gene is the target gene. siRNA is usually about 21 nucleotides
long, with 3' overhangs (e.g., 2 nucleotides) at each end of the
two strands.
[0119] siRNA inhibits target gene expression by binding to and
promoting the cleavage of one or more RNA transcripts of the target
gene at specific sequences. Typically in RNAi the RNA transcripts
are mRNA, so cleavage of mRNA results in the down-regulation of
gene expression. In the present invention, not willing to be bound
with any theory, one of the possible mechanisms is that saRNA of
the present invention may modulate the target gene expression by
cleavage of the target antisense RNA transcript.
[0120] A double-stranded saRNA may include one or more
single-stranded nucleotide overhangs. The term "overhang" or "tail"
in the context of double-stranded saRNA and siRNA refers to at
least one unpaired nucleotide that protrudes from the duplex
structure of saRNA or siRNA. For example, when a 3'-end of one
strand of a saRNA extends beyond the 5'-end of the other strand, or
vice versa, there is a nucleotide overhang. A saRNA may comprise an
overhang of at least one nucleotide; alternatively the overhang may
comprise at least two nucleotides, at least three nucleotides, at
least four nucleotides, at least five nucleotides or more. A
nucleotide overhang may comprise of consist of a
nucleotide/nucleoside analog, including a
deoxynucleotide/nucleoside. The overhang(s) may be on the sense
strand, the antisense strand or any combination thereof.
Furthermore, the nucleotide(s) of an overhang can be present on the
5' end, 3' end or both ends of either an antisense or sense strand
of a saRNA. Where two oligonucleotides are designed to form, upon
hybridization, one or more single-stranded overhangs, such
overhangs shall not be regarded as mismatches with regard to the
determination of complementarity. For example, a saRNA comprising
one oligonucleotide 19 nucleotides in length and another
oligonucleotide 21 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 19 nucleotides that is
fully complementary to the shorter oligonucleotide, can yet be
referred to as "fully complementary" for the purposes described
herein.
[0121] In one embodiment, the antisense strand of a double-stranded
saRNA has a 1-10 nucleotide overhang at the 3' end and/or the 5'
end. In one embodiment, the antisense strand of a double-stranded
saRNA has 1-4 nucleotide overhang at its 3' end, or 1-2 nucleotide
overhang at its 3' end. In one embodiment, the sense strand of a
double-stranded saRNA has a 1-10 nucleotide overhang at the 3' end
and/or the 5' end. In one embodiment, the sense strand of a
double-stranded saRNA has 1-4 nucleotide overhang at its 3' end, or
1-2 nucleotide overhang at its 3' end. In one embodiment, both the
sense strand and the antisense strand of a double-stranded saRNA
have 3' overhangs. The 3' overhangs may comprise one or more
uracils, e.g., the sequences UU or UUU. In one embodiment, one or
more of the nucleotides in the overhang is replaced with a
nucleoside thiophosphate, wherein the internucleoside linkage is
thiophosphate. In one embodiment, the overhang comprises one or
more deoxyribonucleoside, e.g., the sequence dTdT or dTdTdT. In one
embodiments, the overhang comprises the sequence dT*dT, wherein *
is a thiophosphate internucleoside linkage.
[0122] The skilled person will appreciate that it is convenient to
define the saRNA of the present invention by reference to the
target antisense RNA transcript or the targeted sequence,
regardless of the mechanism by which the saRNA modulates the target
gene expression. However, the saRNA of the present invention may
alternatively be defined by reference to the target gene. The
target antisense RNA transcript is complementary to a genomic
region on the coding strand of the target gene, and the saRNA of
the present invention is in turn complementary to a region of the
target antisense RNA transcript, so the saRNA of the present
invention may be defined as having sequence identity to a region on
the coding strand of the target gene. All of the features discussed
herein with respect to the definition of the saRNA of the present
invention by reference to the target antisense RNA transcript apply
mutatis mutandis to the definition of the saRNA of the present
invention by reference to the target gene so any discussion of
complementarity to the target antisense RNA transcript should be
understood to include identity to the genomic sequence of the
target gene. Thus, the saRNA of the present invention may have a
high percent identity, e.g. at least 80%, 90%, 95%, 98% or 99%, or
100% identity, to a genomic sequence on the target gene. The
genomic sequence may be up to 2000, 1000, 500, 250, or 100
nucleotides upstream or downstream of the target gene's
transcription start site. It may align with the target gene's
promoter region. Thus, the saRNA may have sequence identity to a
sequence that aligns with the promoter region of the target
gene.
[0123] In one embodiment, the existence of the target antisense RNA
transcript does not need to be determined to design the saRNA of
the present invention. In another word, the design of the saRNA
does not require the identification of the target antisense RNA
transcript. For example, the nucleotide sequence of the TSS core,
i.e., the sequence in the region 2000 nucleotides upstream of the
target gene's transcription start site to 2000 nucleotides
downstream of the target gene's transcription start may be obtained
by the genomic sequence of the coding strand of the target gene, by
sequencing or by searching in a database. Targeted sequence within
the TSS core starting at any position from position 1 to position
4001 of the TSS core on the template strand can be selected and can
then be used to design saRNA sequences. As discussed above, the
saRNA has a high degree of sequence identity with the reverse
complement of the targeted sequence.
[0124] The saRNA sequence's off-target hit number in the whole
genome, 0 mismatch (0 mm) hit number, and 1 mismatch (1 mm) hit
number are then determined. The term "off-target hit number" refers
to the number of other sites in the whole genome that are identical
to the saRNA's targeted sequence on the template strand of the
target gene. The term "0 mm hit number" refers to the number of
known protein coding transcript other than the target transcript of
the saRNA, the complement of which the saRNA may hybridize with or
bind to with 0 mismatch. In another word, "0 mm hit number" counts
the number of known protein coding transcript, other than the
target transcript of the saRNA that comprises a region completely
identical with the saRNA sequence. The term "1 mm hit number"
refers to the number of known protein coding transcript other than
the target transcript of the saRNA, the complement of which the
saRNA may hybridize with or bind to with 1 mismatch. In another
word, "1 mm hit number" counts the number of known protein coding
transcript, other than the target transcript of the saRNA that
comprises a region identical with the saRNA sequence with only 1
mismatch. In one embodiment, only saRNA sequences that have no
off-target hit, no 0 mm hit and no 1 mm hit are selected. For those
saRNA sequences disclosed in the present application, each has no
off-target hit, no 0 mm hit and no 1 mm hit.
[0125] The method disclosed in US 2013/0164846 filed Jun. 23, 2011
(saRNA algorithm), the contents of which are incorporated herein by
reference in their entirety, may also be used to design saRNA. The
design of saRNA is also disclosed in U.S. Pat. Nos. 8,324,181 and
7,709,566 to Corey et al., US Pat. Pub. No. 2010/0210707 to Li et
al., and Voutila et al., Mol Ther Nucleic Acids, vol. 1, e35
(2012), the contents of each of which are incorporated herein by
reference in their entirety.
[0126] "Determination of existence" means either searching
databases of ESTs and/or antisense RNA transcripts around the locus
of the target gene to identify a suitable target antisense RNA
transcript, or using RT PCR or any other known technique to confirm
the physical presence of a target antisense RNA transcript in a
cell.
[0127] In some embodiments, the saRNA of the present invention may
be single or, double-stranded. Double-stranded molecules comprise a
first strand and a second strand. If double-stranded, each strand
of the duplex may be at least 14, or at least 18, e.g. 19, 20, 21
or 22 nucleotides in length. The duplex may be hybridized over a
length of at least 12, or at least 15, or at least 17, or at least
19 nucleotides. Each strand may be exactly 19 nucleotides in
length. Preferably, the length of the saRNA is less than 30
nucleotides since oligonucleotide duplex exceeding this length may
have an increased risk of inducing the interferon response. In one
embodiment, the length of the saRNA is 19 to 25 nucleotides. The
strands forming the saRNA duplex may be of equal or unequal
lengths.
[0128] In one embodiment, the saRNAs of the present invention
comprise a sequence of at least 14 nucleotides and less than 30
nucleotides which has at least 80%, 90%, 95%, 98%, 99% or 100%
complementarity to the targeted sequence. In one embodiment, the
sequence which has at least 80%, 90%, 95%, 98%, 99% or 100%
complementarity to the targeted sequence is at least 15, 16, 17, 18
or 19 nucleotides in length, or 18-22 or 19 to 21, or exactly
19.
[0129] The saRNA of the present invention may include a short 3' or
5' sequence which is not complementary to the target antisense RNA
transcript. In one embodiment, such a sequence is at 3' end of the
strand. The sequence may be 1-5 nucleotides in length, or 2 or 3.
The sequence may comprises uracil, so it may be a 3' stretch of 2
or 3 uracils. The sequence may comprise one or more
deoxyribonucleoside, such as dT. In one embodiment, one or more of
the nucleotides in the sequence is replaced with a nucleoside
thiophosphate, wherein the internucleoside linkage is
thiophosphate. As a non-limiting example, the sequence comprises
the sequence dT*dT, wherein * is a thiophosphate internucleoside
linkage. This non-complementary sequence may be referred to as
"tail". If a 3' tail is present, the strand may be longer, e.g., 19
nucleotides plus a 3' tail, which may be UU or UUU. Such a 3' tail
shall not be regarded as mismatches with regard to determine
complementarity between the saRNA and the target antisense RNA
transcript.
[0130] Thus, the saRNA of the present invention may consist of (i)
a sequence having at least 80% complementarity to a region of the
target antisense RNA transcript; and (ii) a 3' tail of 1-5
nucleotides, which may comprise or consist of uracil residues. The
saRNA will thus typically have complementarity to a region of the
target antisense RNA transcript over its whole length, except for
the 3' tail, if present. Any of the saRNA sequences disclosed in
the present application may optionally include such a 3' tail.
Thus, any of the saRNA sequences disclosed in the saRNA Tables and
Sequence Listing may optionally include such a 3' tail. The saRNA
of the present invention may further comprise Dicer or Drosha
substrate sequences.
[0131] The saRNA of the present invention may contain a flanking
sequence. The flanking sequence may be inserted in the 3' end or 5'
end of the saRNA of the present invention. In one embodiment, the
flanking sequence is the sequence of a miRNA, rendering the saRNA
to have miRNA configuration and may be processed with Drosha and
Dicer. In a non-limiting example, the saRNA of the present
invention has two strands and is cloned into a microRNA precursor,
e.g., miR-30 backbone flanking sequence.
[0132] The saRNA of the present invention may comprise a
restriction enzyme substrate or recognition sequence. The
restriction enzyme recognition sequence may be at the 3' end or 5'
end of the saRNA of the present invention. Non-limiting examples of
restriction enzymes include NotI and AscI.
[0133] In one embodiment, the saRNA of the present invention
consists of two strands stably base-paired together. In some
embodiments, the passenger strand may comprise at least one
nucleotide that is not complementary to the corresponding
nucleotide on the guide strand, called a mismatch with the guide
strand. In one embodiment, the at least one mismatch with the guide
strand may be at 3' end of the passenger strand. In one embodiment,
the 3' end of the passenger strand may comprise 1-5 mismatches with
the guide strand. In one embodiment, the 3' end of the passenger
strand may comprise 2-3 mismatches with the guide strand. In one
embodiment, the 3' end of the passenger strand may comprise 6-10
mismatches with the guide strand.
[0134] In some embodiments, the double-stranded saRNA may comprise
a number of unpaired nucleotides at the 3' end of each strand
forming 3' overhangs. The number of unpaired nucleotides forming
the 3' overhang of each strand may be in the range of 1 to 5
nucleotides, or 1 to 3 nucleotides, or 2 nucleotides. The 3'
overhang may be formed on the 3' tail mentioned above, so the 3'
tail may be the 3' overhang of a double-stranded saRNA.
[0135] Thus, the saRNA of the present invention may be
single-stranded and consists of (i) a sequence having at least 80%
complementarity to a region of the target antisense RNA transcript;
and (ii) a 3' tail of 1-5 nucleotides, which may comprise uracil
residues. The saRNA of the present invention may have
complementarity to a region of the target antisense RNA transcript
over its whole length, except for the 3' tail, if present. As
mentioned above, instead of "complementary to the target antisense
RNA transcript" the saRNA of the present invention may also be
defined as having "identity" to the coding strand of the target
gene. The saRNA of the present invention may be double-stranded and
consists of a first strand comprising (i) a first sequence having
at least 80% complementarity to a region of the target antisense
RNA transcript and (ii) a 3' overhang of 1-5 nucleotides; and a
second strand comprising (i) a second sequence that forms a duplex
with the first sequence and (ii) a 3' overhang of 1-5
nucleotides.
[0136] As described herein, the sequence for C/EBP.alpha. gene is
used to design C/EBP.alpha.-saRNA. The sequence of a target
antisense RNA transcript of CEBPA gene may be determined from the
sequence of C/EBP.alpha. gene for designing C/EBP.alpha.-saRNA.
However, the existence of such a target antisense RNA transcript
does not need to be determined. Sequences of suitable
C/EBP.alpha.-saRNA of the present invention are provided in Table
1. Thus, provided is C/EBP.alpha.-saRNA having a first strand
comprising a sequence selected from SEQ ID Nos: 2, 4, 6, 8, 10, and
12. Optionally, the C/EBP.alpha.-saRNA may comprise a 3' tail at
the 3' end of these sequences.
[0137] Single stranded C/EBP.alpha.-saRNA only consists of a first
strand, whereas double stranded C/EBP.alpha.-saRNA also has a
second strand. The single stranded CEBPA-saRNA comprises a sequence
selected from the anti-sense strands in Tables 1 and 1A. The
double-stranded C/EBP.alpha.-saRNA comprises a first strand,
wherein the first strand comprises a sequence selected from the
anti-sense strands in Tables 1 and 1A, and a second strand, wherein
the second strand comprises a sequence which is the corresponding
sense strand in Tables 1 and 1A. The anti-sense and/or sense
strands may comprise a 3' overhang.
TABLE-US-00002 TABLE 1 saRNA sequences Anti- Sense SEQ sense SEQ
strand ID strand ID ID (Passenger) NO (Guide) NO Human AW1
CGGUCAUUGU 1 UGACCAGUGA 2 C/EBP.alpha. CACUGGUCA CAAUGACCG AW2
AGCUGAAAGG 3 AGGAUGAAUC 4 AUUCAUCCU CUUCCAGCU NR1 ACAUAGUCCC 5
UUAAUCACUG 6 AGUGAUUAA GGACUAUGU NR2 GAAUAAGACU 7 AUUGGACAAA 8
UUGUCCAAU GUCUUAUUC PR1 GCGCGGAUUC 9 UUUGAAAGAG 10 UCUUUCAAA
AAUCCGCGC PR2 CCAGGAACUC 11 UUCAACGACGA 12 GUCGUUGAA GUUCCUGG
TABLE-US-00003 TABLE 1A additional saRNA sequences Anti- Sense
sense Strand SEQ strand SEQ (Passenger) ID NO (Guide) ID NO
GGUAUACAUC 34 AGCUCUGAGG 35 CUCAGAGCU AUGUAUACC CUAGCUUUCU 36
AGUCACACCA 37 GGUGUGACU GAAAGCUAG CGGGCUUGUC 38 UGAGAUCCCG 39
GGGAUCUCA ACAAGCCCG GCAUUGGAGC 40 AAACUCACCG 41 GGUGAGUUU CUCCAAUGC
GGCACAAGGU 42 UUUAGGAUAA 43 UAUCCUAAA CCUUGUGCC GCACAAGGUU 44
AUUUAGGAUA 45 AUCCUAAAU ACCUUGUGC CGGUCAUUGU 46 UGACCAGUGA 47
CACUGGUCA CAAUGACCG CCAGGAACUC 48 UUCAACGACG 49 GUCGUUGAA
AGUUCCUGG
[0138] Bifunction or dual-functional oligonucleotides are also
designed to up-regulate C/EBP.alpha. gene expression and
down-regulate C/EBP.beta. gene expression. One strand of the
dual-functional oligonucleotide activates C/EBP.alpha. gene
expression and the other inhibits C/EBP.beta. gene expression.
Preferred dual-functional oligonucleotide sequences are shown in
Table 2A. Each strand might further comprise a Dicer substrate
sequence as shown in Table 2B.
TABLE-US-00004 TABLE 2A Bifunction oligonucleotide sequences 19mer
1 19mer 2 (Target (Target C/EBP.beta. C/EBP.alpha.-AS ID
(NM_005194)) (NM_004364)) sa- AGAAGUUGGC AUGGAGUCG CEBPA_si-
CACUUCCAU GCCGACUUCU CEBPB-1 (SEQ ID (SEQ ID NO. 13) NO. 14) sa-
AAGAGGUCGG AGUUCCUGGC CEBPA_si- AGAGGAAGU CGACCUGUU CEBPB-2 (SEQ ID
(SEQ ID NO. 15) NO. 16) sa- UUGUACUCGU AGAACAGCAA CEBPA_si-
CGCUGUGCU CGAGUACCG CEBPB-3 (SEQ ID (SEQ ID NO. 17) NO. 18) sa-
UACUCGUCGC ACAAGAACAG CEBPA_si- UGUGCUUGU CAACGAGUA CEBPB-4 (SEQ ID
(SEQ ID NO. 19) NO. 20)
TABLE-US-00005 TABLE 2B Dice substrate sequences of bifunction
oligonucleotide sequences DicerSubstrate- DicerSubstrate- Strand1
Strand2 (RNAs in (RNAs in upper case; upper case; DNAs in DNAs in
underlined underlined ID lower case) lower case) sa- AGAAGUUGGCCAC
tcCCCCAUGGAGUC CEBPA_si- UUCCAUGGGGcga GGCCGACUUCUAC CEBPB-1 (SEQ
ID NO. 21) (SEQ ID NO. 22) sa- AAGAGGUCGGAGA acGACGAGUUCCUG
CEBPA_si- GGAAGUCGUCgt GCCGACCUGUUCC CEBPB-2 (SEQ ID NO. 23) (SEQ
ID NO. 24) sa- UUGUACUCGUCGC tqGACAAGAACAGC CEBPA_si- UGUGCUUGUCca
AACGAGUACCGGG CEBPB-3 (SEQ ID NO. 25) (SEQ ID NO. 26) sa-
UACUCGUCGCUGUG cgGUGGACAAGAAC CEBPA_si- CUUGUCCACcg AGCAACGAGUACC
CEBPB-4 (SEQ ID NO. 27) (SEQ ID NO. 28)
[0139] The saRNA of the present invention may be produced by any
suitable method, for example synthetically or by expression in
cells using standard molecular biology techniques which are
well-known to a person of ordinary skill in the art. For example,
the saRNA of the present invention may be chemically synthesized or
recombinantly produced using methods known in the art.
Chemical Modifications of saRNA
[0140] Herein, in saRNA, the terms "modification" or, as
appropriate, "modified" refer to structural and/or chemical
modifications with respect to A, G, U or C ribonucleotides.
Nucleotides in the saRNA of the present invention may comprise
non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. The saRNA of the present invention may include
any useful modification, such as to the sugar, the nucleobase, or
the internucleoside linkage (e.g. to a linking phosphate/to a
phosphodiester linkage/to the phosphodiester backbone). One or more
atoms of a pyrimidine nucleobase may be replaced or substituted
with optionally substituted amino, optionally substituted thiol,
optionally substituted alkyl (e.g., methyl or ethyl), or halo
(e.g., chloro or fluoro). In certain embodiments, modifications
(e.g., one or more modifications) are present in each of the sugar
and the internucleoside linkage. Modifications according to the
present invention may be modifications of ribonucleic acids (RNAs)
to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs),
glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked
nucleic acids (LNAs) or hybrids thereof.
[0141] In one embodiment, the saRNAs of the present invention may
comprise at least one modification described herein.
[0142] In another embodiment, the saRNA is an saRNA duplex and the
sense strand and antisense sequence may independently comprise at
least one modification. As a non-limiting example, the sense
sequence may comprises a modification and the antisense strand may
be unmodified. As another non-limiting example, the antisense
sequence may comprises a modification and the sense strand may be
unmodified. As yet another non-limiting example, the sense sequence
may comprises more than one modification and the antisense strand
may comprise one modification. As a non-limiting example, the
antisense sequence may comprises more than one modification and the
sense strand may comprise one modification.
[0143] The saRNA of the present invention can include a combination
of modifications to the sugar, the nucleobase, and/or the
internucleoside linkage. These combinations can include any one or
more modifications described herein or in International Application
Publication WO2013/052523 filed Oct. 3, 2012, in particular
Formulas (Ia)-(Ia-5), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2),
(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)),
the contents of which are incorporated herein by reference in their
entirety.
[0144] The saRNA of the present invention may or may not be
uniformly modified along the entire length of the molecule. For
example, one or more or all types of nucleotide (e.g., purine or
pyrimidine, or any one or more or all of A, G, U, C) may or may not
be uniformly modified in the saRNA of the invention. In some
embodiments, all nucleotides X in a saRNA of the invention are
modified, wherein X may be any one of nucleotides A, G, U, C, or
any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,
A+G+C, G+U+C or A+G+C.
[0145] Different sugar modifications, nucleotide modifications,
and/or internucleoside linkages (e.g., backbone structures) may
exist at various positions in a saRNA. One of ordinary skill in the
art will appreciate that the nucleotide analogs or other
modification(s) may be located at any position(s) of a saRNA such
that the function of saRNA is not substantially decreased. The
saRNA of the present invention may contain from about 1% to about
100% modified nucleotides (either in relation to overall nucleotide
content, or in relation to one or more types of nucleotide, i.e.
any one or more of A, G, U or C) or any intervening percentage
(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to
60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to
95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to
60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to
95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20%
to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20%
to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from
50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,
from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to
100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90%
to 95%, from 90% to 100%, and from 95% to 100%).
[0146] In some embodiments, the modification may be on the ribose
ring. The 2'--OH group on the ribose may be substituted to protect
saRNA against ribonucleases. For example, the 2'-OH group may be
substituted with 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F),
2'-O-methoxyethyl (2'-O-MOE), 2'-O-allyl (2'-O-allyl), etc.
[0147] In some embodiments, the modifications include bicyclic
derivatives of the nucleotides (LNA, ENA, CLNA, CENA, AENA etc.),
acyclic nucleotides (UNA, PNA, etc.) or nucleotides containing
pyranose ring (ANA, HNA) instead of ribose.
[0148] In some embodiments, the modification may be on the backbone
to increase nuclease resistance of the saRNA. Non-limiting examples
include the replacement of phosphate group (PO) with
phosphorothioate (PS) or boranophosphonate (PB) groups, the
replacement of the 3',5'-phosphodiester bond with 2',5'-bond or the
amide bond instead of the ester bond, etc.
[0149] In some embodiments, the modification may be on the
nucleobases. For example, uridine (U) may be replaced with
pseudouridine (.psi.), 2-thiouridine (s2U), dihydrouridine (D),
5-bromo-U, 5-iodo-U, etc. Purine may be replaced with
2,6-diaminopurine.
[0150] In some embodiments, the modification may be at the termini
of saRNA. Any termini modification may be used to increase nuclease
resistance, to facilitate asymmetric RISC assembly, to help saRNA
accumulation in cells, and to enable saRNA detection. For example,
fluorescence labels and biotin may be attached to a terminus of
saRNA. In another example, inverted deoxyribose may be employed at
a terminus of saRNA.
[0151] In some embodiments, the saRNA of the present invention may
be modified to be a spherical nucleic acid (SNA) or a circular
nucleic acid. The terminals of the saRNA of the present invention
may be linked by chemical reagents or enzymes, producing spherical
saRNA that has no free ends. Spherical saRNA is expected to be more
stable than its linear counterpart and to be resistant to digestion
with RNase R exonuclease. Spherical saRNA may further comprise
other structural and/or chemical modifications with respect to A,
G, U or C ribonucleotides.
[0152] In some embodiments, the saRNA of the present invention may
comprise inverted dT modifications. The inverted modification may
be at 5' terminus or 3' terminus. In some embodiments, the 2'-OH of
a nucleotide is substituted with --OMe, referred to as 2'-OMe. In
some embodiments, the 2'-OH of a nucleotide is substituted with
--F, referred to as 2'-F. In some embodiments, there is
phosphorothioate linkage between nucleotides. In some embodiments,
the saRNA of the present invention may comprise abasic
modifications.
[0153] The saRNA of the present invention may comprise a
combination of modifications. The saRNA may comprise at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
modifications. For example, the saRNA may comprise alternating 2'-F
and 2'-OMe modifications. In some embodiments, the saRNA may be
modified across its whole length.
[0154] Any suitable modification to render the sense strand
inactive and/or to reduce off-targets, which does not interfere
with guide strand activity, may be used.
[0155] Table 3 includes non-limiting examples of modified
CEBPA-saRNA sequences and the unmodified CEBPA-saRNA sequences. In
Table 3, lower case letters refer to 2'-OMe modification. `(invdT)`
refers to inclusion of an inverted dT at 3' and/or 5' end. `f`
means the nucleotide preceding it has 2'-F modification. `s` means
there is a phosphorothioate linkage between the nucleotides. `dT`
refers to deoxy-thymine. `dG` refers to deoxy-guanosine. `dA`
refers to deoxy-adenosine.
TABLE-US-00006 TABLE 3-1 Modified saRNA sequences-sense sequences
Duplex- Sense- Sense SEQ ID ID Sequence ID Notes XD-03287 X09198
CGGUCAUUGUC 50 Unmodified ACUGGUCAUU XD-04353 X12716 cgGfuCfaUfu 52
GfuCfaCfuGf gUfcAfusu XD-04354 X12718 csgGfuCfaUf 54 uGfuCfaCfuG
fgUfcAf (invdT) XD-04355 X12720 (invdT)cgGf 56 uCfaUfuGfuC
faCfuGfgUfc Af(invdT) XD-04356 X12721 (invdT)CfdG 57 dGUfCfdAUfU
fdGUfCfdACf UfdGdGUfC fdA(invdT) XD-03302 X09316 GCGGUCAUUGU 73
Unmodified CACUGGUCUU XD-04358 X12723 gcGfgUfcAfu 59 UfgUfcAfcUf
gGfuCfuUfusu XD-04359 X12725 gscGfgUfcAf 61 uUfgUfcAfcU fgGfuCfuUf
(invdT) XD-04360 X12727 (invdT)gcGf 63 gUfcAfuUfgU fcAfcUfgGfu
CfuUf(invdT) XD-04361 X12728 (invdT)dGCf 64 dGdGUfCfdAU fUfdGUfCfdA
CfUfdGdG UfCfUfUf (invdT) XD-03317 X09346 UGAAAGGAUUC 74 Unmodified
AUCCUCCUUU XD-04363 X12730 ugAfaAfgGfa 66 UfuCfaUfcCf uCfcUfuUfusu
XD-04364 X12732 usgAfaAfgGfa 68 UfuCfaUfcCfu CfcUfuUf (invdT)
XD-04365 X12734 (invdT)ugAfa 70 AfgGfaUfuCfa UfcCfuCfcUfu Uf(invdT)
XD-04366 X12735 (invdT)UfdGd 71 AdAdAdGdGdAU fUfCfdAUfCfC
fUfCfCfUfU fUf(invdT)
TABLE-US-00007 TABLE 3-2 Modified saRNA sequences-antisense
sequences Duplex- Antisense- Antisense SEQ ID ID Sequence ID Notes
XD-03287 X09199 UGACCAGUG 51 Unmodified ACAAUGACC GUU XD-04353
X12717 UfGfaCfcA 53 fgUfgAfcA faUfgAfc Cfgusu XD-04354 X12719
UfsGfaCfc 55 AfgUfgAfc AfaUfgAfc Cfgsusu XD-04355 X12719 UfsGfaCfc
55 AfgUfgAfc AfaUfgAfc Cfgsusu XD-04356 X12722 UfgaCfCfa 58
gUfgaCfaa UfgaCfCfg usu XD-03302 X09317 GACCAGUGA 75 Unmodified
CAAUGACCG CUU XD-04358 X12724 AfAfgAfcC 60 faGfuGfaC faAfuGfaC
fcGfcusu XD-04359 X12726 AfAfgAfcC 62 faGfuGfaC faAfuGfaC fcGfscusu
XD-04360 X12726 AfAfgAfcC 62 faGfuGfaC faAfuGfaC fcGfscusu XD-04361
X12729 gaCfCfagU 65 fgaCfaaUf gaCfCfgCf UfUfusu XD-03317 X09347
AGGAGGAUG 76 Unmodified AAUCCUUUC AUU XD-04363 X12731 AfAfaGfgA 67
fgGfaUfgA faUfcCfuU fuCfausu XD-04364 X12733 AfAfaGfgA 69 fgGfaUfgA
faUfcCfuU fuCfasusu XD-04365 X12733 AfAfaGfgA 69 fgGfaUfgA
faUfcCfuU fuCfasusu XD-04366 X12736 aggaggaUf 72 gaaUfCfCf
UfUfUfCfa UfUfusu
saRNA Conjugates and Combinations
[0156] Conjugation may result in increased stability and/or half
life and may be particularly useful in targeting the saRNA of the
present invention to specific sites in the cell, tissue or
organism. The saRNA of the present invention can be designed to be
conjugated to other polynucleotides, dyes, intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), alkylating agents, phosphate, amino,
mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2, polyamino,
alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens
(e.g. biotin), transport/absorption facilitators (e.g., aspirin,
vitamin E, folic acid), synthetic ribonucleases, proteins, e.g.,
glycoproteins, or peptides, e.g., molecules having a specific
affinity for a co-ligand, or antibodies e.g., an antibody, that
binds to a specified cell type such as a cancer cell, endothelial
cell, or bone cell, hormones and hormone receptors, non-peptidic
species, such as lipids, lectins, carbohydrates, vitamins,
cofactors, or a drug. Suitable conjugates for nucleic acid
molecules are disclosed in International Publication WO 2013/090648
filed Dec. 14, 2012, the contents of which are incorporated herein
by reference in their entirety.
[0157] According to the present invention, C/EBP.alpha.-saRNA may
be administered with, or further encode one or more of RNAi agents,
small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), long
non-coding RNAs (lncRNAs), enhancer RNAs, enhancer-derived RNAs or
enhancer-driven RNAs (eRNAs), microRNAs (miRNAs), miRNA binding
sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that
induce triple helix formation, aptamers or vectors, and the like to
achieve different functions. The one or more RNAi agents, small
interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), long
non-coding RNAs (lncRNA), microRNAs (miRNAs), miRNA binding sites,
antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce
triple helix formation, aptamers or vectors may comprise at least
one modification or substitution. In some embodiments, the
modification is selected from a chemical substitution of the
nucleic acid at a sugar position, a chemical substitution at a
phosphate position and a chemical substitution at a base position.
In other embodiments, the chemical modification is selected from
incorporation of a modified nucleotide; 3' capping; conjugation to
a high molecular weight, non-immunogenic compound; conjugation to a
lipophilic compound; and incorporation of phosphorothioate into the
phosphate backbone. In a preferred embodiment, the high molecular
weight, non-immunogenic compound is polyalkylene glycol, and more
preferably is polyethylene glycol (PEG).
[0158] In one embodiment, C/EBP.alpha.-saRNA may be attached to a
transgene so it can be co-expressed from an RNA polymerase II
promoter. In a non-limiting example, C/EBP.alpha.-saRNA is attached
to green fluorescent protein gene (GFP).
[0159] In one embodiment, C/EBP.alpha.-saRNA may be attached to a
DNA or RNA aptamer, thereby producing C/EBP.alpha.-saRNA-aptamer
conjugate. Aptamers are oligonucleotides or peptides with high
selectivity, affinity and stability. They assume specific and
stable three-dimensional shapes, thereby providing highly specific,
tight binding to target molecules. An aptamer may be a nucleic acid
species that has been engineered through repeated rounds of in
vitro selection or equivalently, SELEX (systematic evolution of
ligands by exponential enrichment) to bind to various molecular
targets such as small molecules, proteins, nucleic acids, and even
cells, tissues and organisms. Nucleic acid aptamers have specific
binding affinity to molecules through interactions other than
classic Watson-Crick base pairing. Nucleic acid aptamers, like
peptides generated by phage display or monoclonal antibodies
(mAbs), are capable of specifically binding to selected targets
and, through binding, block their targets' ability to function. In
some cases, aptamers may also be peptide aptamers. For any specific
molecular target, nucleic acid aptamers can be identified from
combinatorial libraries of nucleic acids, e.g. by SELEX. Peptide
aptamers may be identified using a yeast two hybrid system. A
skilled person is therefore able to design suitable aptamers for
delivering the saRNAs or cells of the present invention to target
cells such as liver cells. DNA aptamers, RNA aptamers and peptide
aptamers are contemplated. Administration of saRNA of the present
invention to the liver using liver-specific aptamers is
particularly preferred.
[0160] As used herein, a typical nucleic acid aptamer is
approximately 10-15 kDa in size (20-45 nucleotides), binds its
target with at least nanomolar affinity, and discriminates against
closely related targets. Nucleic acid aptamers may be ribonucleic
acid, deoxyribonucleic acid, or mixed ribonucleic acid and
deoxyribonucleic acid. Aptamers may be single stranded ribonucleic
acid, deoxyribonucleic acid or mixed ribonucleic acid and
deoxyribonucleic acid. Aptamers may comprise at least one chemical
modification.
[0161] A suitable nucleotide length for an aptamer ranges from
about 15 to about 100 nucleotides (nt), and in various other
preferred embodiments, 15-30 nt, 20-25 nt, 30-100 nt, 30-60 nt,
25-70 nt, 25-60 nt, 40-60 nt, 25-40 nt, 30-40 nt, any of 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or
40 nt or 40-70 nt in length. However, the sequence can be designed
with sufficient flexibility such that it can accommodate
interactions of aptamers with two targets at the distances
described herein. Aptamers may be further modified to provide
protection from nuclease and other enzymatic activities. The
aptamer sequence can be modified by any suitable methods known in
the art.
[0162] The C/EBP.alpha.-saRNA-aptamer conjugate may be formed using
any known method for linking two moieties, such as direct chemical
bond formation, linkage via a linker such as streptavidin and so
on.
[0163] In one embodiment, C/EBP.alpha.-saRNA may be attached to an
antibody. Methods of generating antibodies against a target cell
surface receptor are well known. The saRNA molecules of the
invention may be attached to such antibodies with known methods,
for example using RNA carrier proteins. The resulting complex may
then be administered to a subject and taken up by the target cells
via receptor-mediated endocytosis.
[0164] In one embodiment, C/EBP.alpha.-saRNA may be conjugated with
lipid moieties such as a cholesterol moiety (Letsinger et al.,
Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid
(Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a
thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem.
Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,
Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J,
1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330;
Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995,
14:969-973), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra
et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an
octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937), the content
of each of which is herein incorporated by reference in its
entirety.
[0165] In one embodiment, the saRNA of the present invention is
conjugated with a ligand disclosed in US 20130184328 to Manoharan
et al., the contents of which are incorporated herein by reference
in their entirety. The conjugate has a formula of
Ligand-[linker].sub.optional-[tether].sub.optional-oligonucleotide
agent. The oligonucleotide agent may comprise a subunit having
formulae (I) disclosed by US 20130184328 to Manoharan et al., the
contents of which are incorporated herein by reference in their
entirety.
[0166] Representative U.S. patents that teach the preparation of
such nucleic acid/lipid conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;
5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;
5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;
5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941, the content of each of which is herein incorporated by
reference in its entirety.
[0167] In on embodiment, the saRNA is conjugated with a
carbohydrate ligand, such as any carbohydrate ligand disclosed in
U.S. Pat. Nos. 8,106,022 and 8,828,956 to Manoharan et al. (Alnylam
Pharmaceuticals), the contents of which are incorporated herein by
reference in their entirety. For example, the carbohydrate ligand
may be monosaccharide, disaccharide, trisaccharide,
tetrasaccharide, oligosaccharide, or polysaccharide. These
carbohydrate-conjugated RNA agents may target the parenchymal cells
of the liver. In one embodiment, the saRNA is conjugated with more
than one carbohydrate ligand, preferably two or three. In one
embodiment, the saRNA is conjugated with one or more galactose
moiety. In another embodiment, the saRNA is conjugated at least one
(e.g., two or three or more) lactose molecules (lactose is a
glucose coupled to a galactose). In another embodiment, the saRNA
is conjugated with at least one (e.g., two or three or more)
N-Acetyl-Galactosamine (GalNAc), N--Ac-Glucosamine (GluNAc), or
mannose (e.g., mannose-6-phosphate). In one embodiment, the saRNA
is conjugated with at least one mannose ligand, and the conjugated
saRNA targets macrophages.
[0168] The saRNA of the present invention may be provided in
combination with other active ingredients known to have an effect
in the particular method being considered. The other active
ingredients may be administered simultaneously, separately, or
sequentially with the saRNA of the present invention. In one
embodiment, C/EBP.alpha.-saRNA is administered with saRNA
modulating a different target gene. Non-limiting examples include
saRNA that modulates albumin, insulin or HNF4A genes. Modulating
any gene may be achieved using a single saRNA or a combination of
two or more different saRNAs. Non-limiting examples of saRNA that
can be administered with C/EBP.alpha.-saRNA of the present
invention include saRNA modulating albumin or HNF4A disclosed in
International Publication WO 2012/175958 filed Jun. 20, 2012, saRNA
modulating insulin disclosed in International Publications WO
2012/046084 and WO 2012/046085 both filed Oct. 10, 2011, saRNA
modulating human progesterone receptor, human major vault protein
(hMVP), E-cadherin gene, p53 gene, or PTEN gene disclosed in U.S.
Pat. No. 7,709,456 filed Nov. 13, 2006 and US Pat. Publication US
2010/0273863 filed Apr. 23, 2010, and saRNAs targeting p21 gene
disclosed in International Publication WO 2006/113246 filed Apr.
11, 2006, the contents of each of which are incorporated herein by
reference in their entirety.
[0169] In one embodiment, C/EBP.alpha.-saRNA is administered with a
small interfering RNA or siRNA that inhibits the expression of
C/EBP.beta. gene, i.e., C/EBP.beta.-siRNA. Preferred sequences of
suitable siRNAs of the invention are provided in Table 4.
TABLE-US-00008 TABLE 4 siRNA sequences ID C/EBP.beta.-si-1
C/EBP.beta.-si-2 Target Ctgagtaatc Gaaacttta gcttaaaga gcgagtcaga
Efficacy 0.7 0.52 Location 1892 239 Sense CUGAGUAAUCG GAAACUUUAGC
(passenger) CUUAAAGAUU GAGUCAGAUU (SEQ ID (SEQ ID NO. 29) NO. 31)
Antisense UCUUUAAGCGA UCUGACUCGCU (guide) UUACUCAGUU AAAGUUUCUU
(SEQ ID (SEQ ID NO. 30) NO. 32)
[0170] In one embodiment, C/EBP.alpha.-saRNA is administered with
one or more drugs that regulate metabolics, particularly liver
function. In a non-limiting example, C/EBP.alpha.-saRNA of the
present invention is administered with drugs that decrease low
density lipoprotein (LDL) cholesterol levels, such as statin,
simvastatin, atorvastatin, rosuvastatin, ezetimibe, niacin, PCSK9
inhibitors, CETP inhibitors, clofibrate, fenofibric, tocotrienols,
phytosterols, bile acid sequestrants, probucol, or a combination
thereof. C/EBP.alpha.-saRNA may also be administered with vanadium
biguanide complexes disclosed in U.S. Pat. No. 6,287,586 to Orvig
et al. In another example, C/EBP.alpha.-saRNA may be administered
with a composition disclosed in WO 201102838 to Rhodes, the
contents of which are incorporated by reference in their entirety,
to lower serum cholesterol. The composition comprises an antigen
binding protein that selectively binds to and inhibits a PCSK9
protein; and an RNA effector agent which inhibits the expression of
a PCSK9 gene in a cell. In yet another example, C/EBP.alpha.-saRNA
may be administered with an ABC1 polypeptide having ABC1 biological
activity, or a nucleic acid encoding an ABC1 polypeptide having
ABC1 activity to modulate cholesterol levels as described in
EP1854880 to Brooks-Wilson et al., the contents of which are
incorporated herein by reference in their entirety.
[0171] In another embodiment, C/EBP.alpha.-saRNA of the present
invention is administered with drugs that increase insulin
sensitivity or treat type II diabetes mellitus, such as metformin,
sulfonylurea, nonsulfonylurea secretagogues, .alpha. glucosidase
inhibitors, thiazolidinediones, pioglitazone, rosiglitazone,
glucagon-like peptide-1 analog, and dipeptidyl peptidase-4
inhibitors or a combination thereof. Other hepato-protective agents
that may be administered in combination with the saRNA of the
present invention are disclosed in Adams et al., Postgraduate
Medical Journal, vol. 82, 315-322 (2006), the contents of which are
incorporated herein by reference in their entirety.
Gankyrin and FXR Protein
[0172] The development of hepatocellular carcinoma (HCC) is a
multistep process which involves progressive changes of gene
expression leading to liver hyperproliferation and to liver cancer.
During carcinogenesis of liver cancer, tumor suppressor proteins
Rb, p53, hepatocyte nuclear factor 4a (HNF4a), and C/EBP-.alpha.
are neutralized. The elimination of these proteins is mediated by a
small subunit of 26S proteasome, gankyrin, which is activated by
cancer. Wang et al. discloses that gankyrin interacts with S193-ph
isoform of C/EBP.alpha. and targets it for ubiquitinproteasome
system (UPS)-mediated degradation. Gankyrin level is elevated
during the early stages of liver cancer development (Wang et al.,
J. Clin. Invest, vol. 120(7):2549-2562 (2010), the contents of
which are incorporated herein by reference in their entireties).
Inhibiting gankyrin, e.g., using siRNA of the gankyrin gene (also
known as PSMD10 gene) and/or gankyrin inhibitors, may prevent
and/or treat HCC.
[0173] Jiang et al. found that farnesoid X receptor (FXR), also
known as bile acid receptor (BAR) or NR1H4, inhibits expression of
gankyrin in quiescent livers by silencing the gankyrin promoter
through HDAC1-C/EBP.beta. complexes (Jiang et al., Hepatology, vol.
57(3):1098-1106 (2013), the contents of which are incorporated
herein by reference in their entireties). Deletion of FXR signaling
in mice leads to de-repression of the gankyrin promoter and to
spontaneous development of liver cancer at 12 months of age.
Diethylnitrosoamine (DEN)-mediated liver cancer in wild-type mice
also involves the reduction of FXR and activation of gankyrin.
Examination of liver cancer in old mice and liver cancer in human
patients revealed that FXR is reduced, while gankyrin is elevated
during spontaneous development of liver cancer. Jiang et al.
concluded that FXR prevents liver cancer by inhibiting the gankyrin
promoter via C/EBP.beta.-HDAC1 complexes leading to subsequent
protection of tumor suppressor proteins from degradation.
Stabilization and nuclear translocation of FXR inhibits gankyrin.
Activating FXR, e.g., using FXR agonists or activators, or
activator of NR1H4 gene, may prevent and/or treat HCC.
[0174] C/EBP.alpha.-saRNA of the present invention may be used in
combination with one or more of therapeutic agents that
down-regulate gankyrin or up-regulate FXR. The combination may have
synergistic effect on preventing and/or treating HCC. In some
embodiments, C/EBP.alpha.-saRNA of the present invention may be
used in combination with gankyrin-siRNA. Double-stranded
Gankyrin-siRNA may be produced using the method disclosed by
Higashitsuji et al. in the `Inhibition of endogenous gene
expression by RNAi` section (Higashitsuji et al., Cancer Cell, vol.
8:75-87 (2005), the contents of which are incorporated herein by
reference in their entireties). In some embodiments,
C/EBP.alpha.-saRNA of the present invention may be used in
combination with FXR agonists. Non-limiting examples of FXR
agonists or activators include taurocholic acid, obeticholic acid
(OCA), INT-767 (Intercept Pharmaceuticals), INT-777 (Intercept
Pharmaceuticals), and any FXR agonist or activator disclosed in US
Pat. App. No. 20140057886, U.S. Pat. Nos. 8,546,365, 7,932,244, US
Pat. App. No. 20140100209, U.S. Pat. Nos. 8,445,472, 8,114,862, US
Pat. App. No. 20140094443, U.S. Pat. Nos. 8,410,083, 8,796,249, US
Pat. App. No. 20140024631, U.S. Pat. Nos. 8,377,916, 8,258,267,
7,786,102, 7,138,390, 7,994,352, 7,858,608, 7,812,011, US Pat. App.
No. 20140148428, and US Pat. App. No. 20060252670 (the contents of
each of which are incorporated herein by reference in their
entirety).
Formulation, Delivery, Administration, and Dosing
[0175] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes, but is not limited to, any and all solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, and the like, as suited to the
particular dosage form desired. Various excipients for formulating
pharmaceutical compositions and techniques for preparing the
composition are known in the art (see Remington: The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro, Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference in its entirety). The use of a conventional excipient
medium may be contemplated within the scope of the present
disclosure, except insofar as any conventional excipient medium may
be incompatible with a substance or its derivatives, such as by
producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical composition.
[0176] In some embodiments, compositions are administered to
humans, human patients or subjects. For the purposes of the present
disclosure, the phrase "active ingredient" generally refers to
C/EBP.alpha.-saRNA to be delivered as described herein.
[0177] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to any other animal,
e.g., to non-human animals, e.g. non-human mammals. Modification of
pharmaceutical compositions suitable for administration to humans
in order to render the compositions suitable for administration to
various animals is well understood, and the ordinarily skilled
veterinary pharmacologist can design and/or perform such
modification with merely ordinary, if any, experimentation.
Subjects to which administration of the pharmaceutical compositions
is contemplated include, but are not limited to, humans and/or
other primates; mammals, including commercially relevant mammals
such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;
and/or birds, including commercially relevant birds such as
poultry, chickens, ducks, geese, and/or turkeys.
[0178] In one embodiment, the efficacy of the formulated saRNA
described herein may be determined in proliferating cells.
[0179] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, dividing, shaping and/or
packaging the product into a desired single- or multi-dose
unit.
[0180] A pharmaceutical composition in accordance with the
invention may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient which would be administered
to a subject and/or a convenient fraction of such a dosage such as,
for example, one-half or one-third of such a dosage.
[0181] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
invention will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
[0182] In some embodiments, the formulations described herein may
contain at least one saRNA. As a non-limiting example, the
formulations may contain 1, 2, 3, 4 or 5 saRNAs with different
sequences. In one embodiment, the formulation contains at least
three saRNAs with different sequences. In one embodiment, the
formulation contains at least five saRNAs with different
sequences.
[0183] The saRNA of the invention can be formulated using one or
more excipients to: (1) increase stability; (2) increase cell
transfection; (3) permit the sustained or delayed release (e.g.,
from a depot formulation of the saRNA); (4) alter the
biodistribution (e.g., target the saRNA to specific tissues or cell
types); (5) increase the translation of encoded protein in vivo;
and/or (6) alter the release profile of encoded protein in vivo. In
addition to traditional excipients such as any and all solvents,
dispersion media, diluents, or other liquid vehicles, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, excipients of the present
invention can include, without limitation, lipidoids, liposomes,
lipid nanoparticles, polymers, lipoplexes, core-shell
nanoparticles, peptides, proteins, cells transfected with saRNA
(e.g., for transplantation into a subject), hyaluronidase,
nanoparticle mimics and combinations thereof. Accordingly, the
formulations of the invention can include one or more excipients,
each in an amount that together increases the stability of the
saRNA and/or increases cell transfection by the saRNA. Further, the
saRNA of the present invention may be formulated using
self-assembled nucleic acid nanoparticles. Pharmaceutically
acceptable carriers, excipients, and delivery agents for nucleic
acids that may be used in the formulation with the saRNA of the
present invention are disclosed in International Publication WO
2013/090648 filed Dec. 14, 2012, the contents of which are
incorporated herein by reference in their entirety.
[0184] In one embodiment, the saRNA of the present invention
comprises two single RNA strands that are 21 nucleotides in length
each that are annealed to form a double stranded C/EBP.alpha.-saRNA
as the active ingredient. The composition further comprises a salt
buffer composed of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl and 5 mM
EDTA.
[0185] In another embodiment, the saRNA of the present invention
may be delivered with dendrimers. Dendrimers are highly branched
macromolecules. In a preferred embodiment, the saRNA of the present
invention is complexed with structurally flexible poly(amidoamine)
(PAMAM) dendrimers for targeted in vivo delivery. The complex is
called C/EBP.alpha.-saRNA-dendrimers. Dendrimers have a high degree
of molecular uniformity, narrow molecular weight distribution,
specific size and shape characteristics, and a
highly-functionalized terminal surface. The manufacturing process
is a series of repetitive steps starting with a central initiator
core. Each subsequent growth step represents a new generation of
polymers with a larger molecular diameter and molecular weight, and
more reactive surface sites than the preceding generation. PAMAM
dendrimers are efficient nucleotide delivery systems that bear
primary amine groups on their surface and also a tertiary amine
group inside of the structure. The primary amine group participates
in nucleotide binding and promotes their cellular uptake, while the
buried tertiary amino groups act as a proton sponge in endosomes
and enhance the release of nucleic acid into the cytoplasm. These
dendrimers protect the saRNA carried by them from ribonuclease
degradation and achieves substantial release of saRNA over an
extended period of time via endocytosis for efficient gene
targeting. The in vivo efficacy of these nanoparticles have
previously been evaluated where biodistribution studies show that
the dendrimers preferentially accumulate in peripheral blood
mononuclear cells and live with no discernible toxicity (see Zhou
et al., Molecular Ther. 2011 Vol. 19, 2228-2238, the contents of
which are incorporated herein by reference in their entirety).
PAMAM dendrimers may comprise a triethanolamine (TEA) core, a
diaminobutane (DAB) core, a cystamine core, a diaminohexane (HEX)
core, a diamonododecane (DODE) core, or an ethylenediamine (EDA)
core. Preferably, PAMAM dendrimers comprise a TEA core or a DAB
core.
Lipidoids
[0186] The synthesis of lipidoids has been extensively described
and formulations containing these compounds are particularly suited
for delivery of oligonucleotides or nucleic acids (see Mahon et
al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern
Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569;
Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart
et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which
are incorporated herein in their entireties).
[0187] While these lipidoids have been used to effectively deliver
double stranded small interfering RNA molecules in rodents and
non-human primates (see Akinc et al., Nat Biotechnol. 2008
26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008
105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et
al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al.,
Nat Biotechnol. 2011 29:1005-1010; all of which is incorporated
herein in their entirety), the present disclosure describes their
formulation and use in delivering saRNA. Complexes, micelles,
liposomes or particles can be prepared containing these lipidoids
and therefore, can result in an effective delivery of the saRNA
following the injection of a lipidoid formulation via localized
and/or systemic routes of administration. Lipidoid complexes of
saRNA can be administered by various means including, but not
limited to, intravenous, intramuscular, or subcutaneous routes.
[0188] In vivo delivery of nucleic acids may be affected by many
parameters, including, but not limited to, the formulation
composition, nature of particle PEGylation, degree of loading,
oligonucleotide to lipid ratio, and biophysical parameters such as,
but not limited to, particle size (Akinc et al., Mol Ther. 2009
17:872-879; the contents of which are herein incorporated by
reference in its entirety). As an example, small changes in the
anchor chain length of poly(ethylene glycol) (PEG) lipids may
result in significant effects on in vivo efficacy. Formulations
with the different lipidoids, including, but not limited to
penta[3-(1-laurylaminopropionyl)]-triethylenetetramine
hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al.,
Analytical Biochemistry, 401:61 (2010); the contents of which are
herein incorporated by reference in its entirety), C12-200
(including derivatives and variants), and MD1, can be tested for in
vivo activity.
[0189] The lipidoid referred to herein as "98N12-5" is disclosed by
Akinc et al., Mol Ther. 2009 17:872-879 and the contents of which
is incorporated by reference in its entirety. (See FIG. 2)
[0190] The lipidoid referred to herein as "C12-200" is disclosed by
Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG.
2) and Liu and Huang, Molecular Therapy. 2010 669-670 (see FIG. 2);
the contents of both of which are herein incorporated by reference
in their entirety. The lipidoid formulations can include particles
comprising either 3 or 4 or more components in addition to the
saRNA. As an example, formulations with certain lipidoids, include,
but are not limited to, 98N12-5 and may contain 42% lipidoid, 48%
cholesterol and 10% PEG (C14 alkyl chain length). As another
example, formulations with certain lipidoids, include, but are not
limited to, C12-200 and may contain 50% lipidoid, 10%
disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5%
PEG-DMG.
[0191] In one embodiment, a saRNA formulated with a lipidoid for
systemic intravenous administration can target the liver. For
example, a final optimized intravenous formulation using saRNA and
comprising a lipid molar composition of 42% 98N12-5, 48%
cholesterol, and 10% PEG-lipid with a final weight ratio of about
7.5 to 1 total lipid to saRNA and a C14 alkyl chain length on the
PEG lipid, with a mean particle size of roughly 50-60 nm, can
result in the distribution of the formulation to be greater than
90% to the liver. (see, Akinc et al., Mol Ther. 2009 17:872-879;
the contents of which are herein incorporated by reference in its
entirety). In another example, an intravenous formulation using a
C12-200 (see U.S. provisional application 61/175,770 and published
international application WO2010129709, the contents of each of
which is herein incorporated by reference in their entirety)
lipidoid may have a molar ratio of 50/10/38.5/1.5 of
C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a
weight ratio of 7 to 1 total lipid to nucleic acid and a mean
particle size of 80 nm may be effective to deliver saRNA (see, Love
et al., Proc Natl Acad Sci USA. 2010 107:1864-1869, the contents of
which are herein incorporated by reference in its entirety). In
another embodiment, an MD1 lipidoid-containing formulation may be
used to effectively deliver saRNA to hepatocytes in vivo. The
characteristics of optimized lipidoid formulations for
intramuscular or subcutaneous routes may vary significantly
depending on the target cell type and the ability of formulations
to diffuse through the extracellular matrix into the blood stream.
While a particle size of less than 150 nm may be desired for
effective hepatocyte delivery due to the size of the endothelial
fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879, the
contents of which are herein incorporated by reference in its
entirety), use of a lipidoid-formulated saRNA to deliver the
formulation to other cells types including, but not limited to,
endothelial cells, myeloid cells, and muscle cells may not be
similarly size-limited. Use of lipidoid formulations to deliver
siRNA in vivo to other non-hepatocyte cells such as myeloid cells
and endothelium has been reported (see Akinc et al., Nat
Biotechnol. 2008 26:561-569; Leuschner et al., Nat Biotechnol. 2011
29:1005-1010; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118;
8.sup.th International Judah Folkman Conference, Cambridge, Mass.
Oct. 8-9, 2010; the contents of each of which is herein
incorporated by reference in its entirety). Effective delivery to
myeloid cells, such as monocytes, lipidoid formulations may have a
similar component molar ratio. Different ratios of lipidoids and
other components including, but not limited to,
disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used
to optimize the formulation of saRNA for delivery to different cell
types including, but not limited to, hepatocytes, myeloid cells,
muscle cells, etc. For example, the component molar ratio may
include, but is not limited to, 50% C12-200, 10%
disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG
(see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; the
contents of which are herein incorporated by reference in its
entirety). The use of lipidoid formulations for the localized
delivery of nucleic acids to cells (such as, but not limited to,
adipose cells and muscle cells) via either subcutaneous or
intramuscular delivery, may not require all of the formulation
components desired for systemic delivery, and as such may comprise
only the lipidoid and saRNA.
Liposomes, Lipoplexes, and Lipid Nanoparticles
[0192] The saRNA of the invention can be formulated using one or
more liposomes, lipoplexes, or lipid nanoparticles. In one
embodiment, pharmaceutical compositions of saRNA include liposomes.
Liposomes are artificially-prepared vesicles which may primarily be
composed of a lipid bilayer and may be used as a delivery vehicle
for the administration of nutrients and pharmaceutical
formulations. Liposomes can be of different sizes such as, but not
limited to, a multilamellar vesicle (MLV) which may be hundreds of
nanometers in diameter and may contain a series of concentric
bilayers separated by narrow aqueous compartments, a small
unicellular vesicle (SUV) which may be smaller than 50 nm in
diameter, and a large unilamellar vesicle (LUV) which may be
between 50 and 500 nm in diameter. Liposome design may include, but
is not limited to, opsonins or ligands in order to improve the
attachment of liposomes to unhealthy tissue or to activate events
such as, but not limited to, endocytosis. Liposomes may contain a
low or a high pH in order to improve the delivery of the
pharmaceutical formulations.
[0193] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical
formulation entrapped and the liposomal ingredients, the nature of
the medium in which the lipid vesicles are dispersed, the effective
concentration of the entrapped substance and its potential
toxicity, any additional processes involved during the application
and/or delivery of the vesicles, the optimization size,
polydispersity and the shelf-life of the vesicles for the intended
application, and the batch-to-batch reproducibility and possibility
of large-scale production of safe and efficient liposomal
products.
[0194] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA)
liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), and MC3 (US20100324120; the contents of which are
herein incorporated by reference in its entirety) and liposomes
which may deliver small molecule drugs such as, but not limited to,
DOXIL.RTM. from Janssen Biotech, Inc. (Horsham, Pa.).
[0195] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from the synthesis of stabilized plasmid-lipid particles
(SPLP) or stabilized nucleic acid lipid particle (SNALP) that have
been previously described and shown to be suitable for
oligonucleotide delivery in vitro and in vivo (see Wheeler et al.
Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999
6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et
al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature.
2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;
Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin
Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008
19:125-132; the contents of each of which are incorporated herein
in their entireties). The original manufacture method by Wheeler et
al. was a detergent dialysis method, which was later improved by
Jeffs et al. and is referred to as the spontaneous vesicle
formation method. The liposome formulations may be composed of 3 to
4 lipid components in addition to the saRNA. As an example a
liposome can contain, but is not limited to, 55% cholesterol, 20%
disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%
1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by
Jeffs et al. In another example, certain liposome formulations may
contain, but are not limited to, 48% cholesterol, 20% DSPC, 2%
PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be
1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA,
or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as
described by Heyes et al. In another example, the nucleic
acid-lipid particle may comprise a cationic lipid comprising from
about 50 mol % to about 85 mol % of the total lipid present in the
particle; a non-cationic lipid comprising from about 13 mol % to
about 49.5 mol % of the total lipid present in the particle; and a
conjugated lipid that inhibits aggregation of particles comprising
from about 0.5 mol % to about 2 mol % of the total lipid present in
the particle as described in WO2009127060 to Maclachlan et al, the
contents of which are incorporated herein by reference in their
entirety. In another example, the nucleic acid-lipid particle may
be any nucleic acid-lipid particle disclosed in US2006008910 to
Maclachlan et al., the contents of which are incorporated herein by
reference in their entirety. As a non-limiting example, the nucleic
acid-lipid particle may comprise a cationic lipid of Formula I, a
non-cationic lipid, and a conjugated lipid that inhibits
aggregation of particles.
[0196] In one embodiment, the saRNA may be formulated in a lipid
vesicle which may have crosslinks between functionalized lipid
bilayers.
[0197] In one embodiment, the liposome may contain a sugar-modified
lipid disclosed in U.S. Pat. No. 5,595,756 to Bally et al., the
contents of which are incorporated herein by reference in their
entirety. The lipid may be a ganglioside and cerebroside in an
amount of about 10 mol percent.
[0198] In one embodiment, the saRNA may be formulated in a liposome
comprising a cationic lipid. The liposome may have a molar ratio of
nitrogen atoms in the cationic lipid to the phosphates in the saRNA
(N:P ratio) of between 1:1 and 20:1 as described in International
Publication No. WO2013006825, the contents of which are herein
incorporated by reference in its entirety. In another embodiment,
the liposome may have a N:P ratio of greater than 20:1 or less than
1:1.
[0199] In one embodiment, the saRNA may be formulated in a
lipid-polycation complex. The formation of the lipid-polycation
complex may be accomplished by methods known in the art and/or as
described in U.S. Pub. No. 20120178702, the contents of which are
herein incorporated by reference in its entirety. As a non-limiting
example, the polycation may include a cationic peptide or a
polypeptide such as, but not limited to, polylysine, polyornithine
and/or polyarginine and the cationic peptides described in
International Pub. No. WO2012013326; herein incorporated by
reference in its entirety. In another embodiment, the saRNA may be
formulated in a lipid-polycation complex which may further include
a neutral lipid such as, but not limited to, cholesterol or
dioleoyl phosphatidylethanolamine (DOPE).
[0200] The liposome formulation may be influenced by, but not
limited to, the selection of the cationic lipid component, the
degree of cationic lipid saturation, the nature of the PEGylation,
ratio of all components and biophysical parameters such as size. In
one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176; the contents of which are herein incorporated by
reference in its entirety), the liposome formulation was composed
of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%
cholesterol, and 1.4% PEG-c-DMA.
[0201] In some embodiments, the ratio of PEG in the lipid
nanoparticle (LNP) formulations may be increased or decreased
and/or the carbon chain length of the PEG lipid may be modified
from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example,
LNP formulations may contain 1-5% of the lipid molar ratio of
PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol.
In another embodiment the PEG-c-DOMG may be replaced with a PEG
lipid such as, but not limited to, PEG-DSG
(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG
(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The
cationic lipid may be selected from any lipid known in the art such
as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and
DLin-KC2-DMA.
[0202] In one embodiment, the saRNA may be formulated in a lipid
nanoparticle such as the lipid nanoparticles described in
International Publication No. WO2012170930, the contents of which
are herein incorporated by reference in its entirety.
[0203] In one embodiment, the cationic lipid which may be used in
formulations of the present invention may be selected from, but not
limited to, a cationic lipid described in International Publication
Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965,
WO2011043913, WO2011022460, WO2012061259, WO2012054365,
WO2012044638, WO2010080724, WO201021865 and WO2008103276, U.S. Pat.
Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication
No. US20100036115 and US20120202871; the contents of each of which
is herein incorporated by reference in their entirety. In another
embodiment, the cationic lipid may be selected from, but not
limited to, formula A described in International Publication Nos.
WO2012040184, WO2011153120, WO2011149733, WO2011090965,
WO2011043913, WO2011022460, WO2012061259, WO2012054365 and
WO2012044638; the contents of each of which is herein incorporated
by reference in their entirety. In yet another embodiment, the
cationic lipid may be selected from, but not limited to, formula
CLI-CLXXIX of International Publication No. WO2008103276, formula
CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S.
Pat. No. 7,404,969 and formula I-VI of US Patent Publication No.
0520100036115; the contents of each of which is herein incorporated
by reference in their entirety. In yet another embodiment, the
cationic lipid may be a multivalent cationic lipid such as the
cationic lipid disclosed in U.S. Pat. No. 7,223,887 to Gaucheron et
al., the contents of which are incorporated herein by reference in
their entirety. The cationic lipid may have a positively-charged
head group including two quaternary amine groups and a hydrophobic
portion including four hydrocarbon chains as described in U.S. Pat.
No. 7,223,887 to Gaucheron et al., the contents of which are
incorporated herein by reference in their entirety. In yet another
embodiment, the cationic lipid may be biodegradable as the
biodegradable lipids disclosed in US20130195920 to Maier et al.,
the contents of which are incorporated herein by reference in their
entirety. The cationic lipid may have one or more biodegradable
groups located in a lipidic moiety of the cationic lipid as
described in formula I-IV in US 20130195920 to Maier et al., the
contents of which are incorporated herein by reference in their
entirety. As a non-limiting example, the cationic lipid may be
selected from (20Z,23Z)--N,N-dimethylnonacosa-20,23-dien-10-amine,
(17Z,20Z)--N,N-dimemylhexacosa-17,20-dien-9-amine,
(1Z,19Z)--N5N-dimethylpentacosa-16,19-dien-8-amine,
(13Z,16Z)--N,N-dimethyldocosa-13,16-dien-5-amine,
(12Z,15Z)--N,N-dimethylhenicosa-12,15-dien-4-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-6-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-7-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-10-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-5-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-4-amine,
(19Z,22Z)--N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21
Z)--N,N-dimethylheptacosa-18,21-dien-8-amine,
(17Z,20Z)--N,N-dimethylhexacosa-17,20-dien-7-amine,
(16Z,19Z)--N,N-dimethylpentacosa-16,19-dien-6-amine,
(22Z,25Z)--N,N-dimethylhentriaconta-22,25-dien-10-amine,
(21Z,24Z)--N,N-dimethyltriaconta-21,24-dien-9-amine,
(18Z)--N,N-dimetylheptacos-18-en-10-amine,
(17Z)--N,N-dimethylhexacos-17-en-9-amine,
(19Z,22Z)--N,N-dimethyloctacosa-19,22-dien-7-amine,
N,N-dimethylheptacosan-10-amine,
(20Z,23Z)--N-ethyl-N-methylnonacosa-20,23-dien-10-amine,
1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,
(20Z)--N,N-dimethylheptacos-20-en-10-amine, (15Z)--N,N-dimethyl
eptacos-15-en-10-amine, (14Z)--N,N-dimethylnonacos-14-en-10-amine,
(17Z)--N,N-dimethylnonacos-17-en-10-amine,
(24Z)--N,N-dimethyltritriacont-24-en-10-amine,
(20Z)--N,N-dimethylnonacos-20-en-10-amine,
(22Z)--N,N-dimethylhentriacont-22-en-10-amine,
(16Z)--N,N-dimethylpentacos-16-en-8-amine,
(12Z,15Z)--N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,
(13Z,16Z)--N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,
1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,
N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimeth-
yl-1-[(1S,2
S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,-
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,
N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,
N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,
1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,
1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,
R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-
n-2-amine,
S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-
loxy)propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrr-
olidine,
(2S)--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-
)-oct-5-en-1-yloxy]propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-
idine,
(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-
xy]propan-2-amine,
(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-
opan-2-amine,
N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-
-amine,
N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-
ine;
(2S)--N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-
ctyloxy)propan-2-amine,
(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-
pan-2-amine,
(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-
an-2-amine,
1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2--
amine,
1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-
opan-2-amine,
(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-
pan-2-amine,
(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-
e,
1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
(2R)--N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1--
yloxy]propan-2-amine,
(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-
en-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2
S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2--
amine,
N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propa-
n-2-amine and
(11E,20Z,23Z)--N,N-dimethylnonacosa-11,20,2-trien-10-amine or a
pharmaceutically acceptable salt or stereoisomer thereof.
[0204] In one embodiment, the lipid may be a cleavable lipid such
as those described in International Publication No. WO2012170889,
the contents of which is herein incorporated by reference in its
entirety.
[0205] In one embodiment, the nanoparticles described herein may
comprise at least one cationic polymer described herein and/or
known in the art.
[0206] In one embodiment, the cationic lipid may be synthesized by
methods known in the art and/or as described in International
Publication Nos. WO2012040184, WO2011153120, WO2011149733,
WO2011090965, WO2011043913, WO2011022460, WO2012061259,
WO2012054365, WO2012044638, WO2010080724 and WO201021865; the
contents of each of which is herein incorporated by reference in
their entirety.
[0207] In one embodiment, the LNP formulations of the saRNA may
contain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment,
the LNP formulations of the saRNA may contain PEG-c-DOMG at 1.5%
lipid molar ratio.
[0208] In one embodiment, the pharmaceutical compositions of the
saRNA may include at least one of the PEGylated lipids described in
International Publication No. 2012099755, the contents of which is
herein incorporated by reference in its entirety.
[0209] In one embodiment, the LNP formulation may contain PEG-DMG
2000
(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene
glycol)-2000). In one embodiment, the LNP formulation may contain
PEG-DMG 2000, a cationic lipid known in the art and at least one
other component. In another embodiment, the LNP formulation may
contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and
cholesterol. As a non-limiting example, the LNP formulation may
contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another
non-limiting example the LNP formulation may contain PEG-DMG 2000,
DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see
e.g., Geall et al., Nonviral delivery of self-amplifying RNA
vaccines, PNAS 2012; PMID: 22908294; herein incorporated by
reference in its entirety). As another non-limiting example, the
saRNA described herein may be formulated in a nanoparticle to be
delivered by a parenteral route as described in U.S. Pub. No.
20120207845; the contents of which is herein incorporated by
reference in its entirety. The cationic lipid may also be the
cationic lipids disclosed in US20130156845 to Manoharan et al. and
US 20130129785 to Manoharan et al., WO 2012047656 to Wasan et al.,
WO 2010144740 to Chen et al., WO 2013086322 to Ansell et al., or WO
2012016184 to Manoharan et al., the contents of each of which are
incorporated herein by reference in their entirety.
[0210] In one embodiment, the saRNA of the present invention may be
formulated with a plurality of cationic lipids, such as a first and
a second cationic lipid as described in US20130017223 to Hope et
al., the contents of which are incorporated herein by reference in
their entirety. The first cationic lipid can be selected on the
basis of a first property and the second cationic lipid can be
selected on the basis of a second property, where the properties
may be determined as outlined in US20130017223, the contents of
which are herein incorporated by reference in its entirety. In one
embodiment, the first and second properties are complementary.
[0211] In another embodiment, the saRNA may be formulated with a
lipid particle comprising one or more cationic lipids and one or
more second lipids, and one or more nucleic acids, wherein the
lipid particle comprises a solid core, as described in US Patent
Publication No. US20120276209 to Cullis et al., the contents of
which are incorporated herein by reference in their entirety.
[0212] In one embodiment, the saRNA of the present invention may be
complexed with a cationic amphiphile in an oil-in-water (o/w)
emulsion such as described in EP2298358 to Satishchandran et al.,
the contents of which are incorporated herein by reference in their
entirety. The cationic amphiphile may be a cationic lipid, modified
or unmodified spermine, bupivacaine, or benzalkonium chloride and
the oil may be a vegetable or an animal oil. As a non-limiting
example, at least 10% of the nucleic acid-cationic amphiphile
complex is in the oil phase of the oil-in-water emulsion (see e.g.,
the complex described in European Publication No. EP2298358 to
Satishchandran et al., the contents of which are herein
incorporated by reference in its entirety).
[0213] In one embodiment, the saRNA of the present invention may be
formulated with a composition comprising a mixture of cationic
compounds and neutral lipids. As a non-limiting example, the
cationic compounds may be formula (I) disclosed in WO 1999010390 to
Ansell et al., the contents of which are disclosed herein by
reference in their entirety, and the neutral lipid may be selected
from the group consisting of diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide and sphingomyelin.
[0214] In one embodiment, the LNP formulation may be formulated by
the methods described in International Publication Nos.
WO2011127255 or WO2008103276, each of which are herein incorporated
by reference in their entirety. As a non-limiting example, the
saRNA of the present invention may be encapsulated in any of the
lipid nanoparticle (LNP) formulations described in WO2011127255
and/or WO2008103276; the contents of each of which are herein
incorporated by reference in their entirety.
[0215] In one embodiment, the LNP formulations described herein may
comprise a polycationic composition. As a non-limiting example, the
polycationic composition may be selected from formula 1-60 of US
Patent Publication No. US20050222064; the contents of which is
herein incorporated by reference in its entirety. In another
embodiment, the LNP formulations comprising a polycationic
composition may be used for the delivery of the saRNA described
herein in vivo and/or in vitro.
[0216] In one embodiment, the LNP formulations described herein may
additionally comprise a permeability enhancer molecule.
Non-limiting permeability enhancer molecules are described in US
Patent Publication No. US20050222064; the contents of which is
herein incorporated by reference in its entirety.
[0217] In one embodiment, the pharmaceutical compositions may be
formulated in liposomes such as, but not limited to, DiLa2
liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES.RTM./NOV340
(Marina Biotech, Bothell, Wash.), neutral DOPC
(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,
siRNA delivery for ovarian cancer (Landen et al. Cancer Biology
& Therapy 2006 5(12)1708-1713); the contents of which is herein
incorporated by reference in its entirety) and hyaluronan-coated
liposomes (Quiet Therapeutics, Israel). In some embodiments, the
pharmaceutical compositions may be formulated with any amphoteric
liposome disclosed in WO 2008/043575 to Panzner and U.S. Pat. No.
8,580,297 to Essler et al., the contents of which are incorporated
herein by reference in their entirety. The amphoteric liposome may
comprise a mixture of lipids including a cationic amphiphile, an
anionic amphiphile and optional one or more neutral amphiphiles.
The amphoteric liposome may comprise amphoteric compounds based on
amphiphilic molecules, the head groups of which being substituted
with one or more amphoteric groups. In some embodiments, the
pharmaceutical compositions may be formulated with an amphoteric
lipid comprising one or more amphoteric groups having an
isoelectric point between 4 and 9, as disclosed in US 20140227345
to Essler et al., the contents of which are incorporated herein by
reference in their entirety.
[0218] The nanoparticle formulations may be a carbohydrate
nanoparticle comprising a carbohydrate carrier and a nucleic acid
molecule (e.g., saRNA). As a non-limiting example, the carbohydrate
carrier may include, but is not limited to, an anhydride-modified
phytoglycogen or glycogen-type material, phtoglycogen octenyl
succinate, phytoglycogen beta-dextrin, anhydride-modified
phytoglycogen beta-dextrin. (See e.g., International Publication
No. WO2012109121; the contents of which is herein incorporated by
reference in its entirety).
[0219] Lipid nanoparticle formulations may be improved by replacing
the cationic lipid with a biodegradable cationic lipid which is
known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable
cationic lipids, such as, but not limited to, DLinDMA,
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in
plasma and tissues over time and may be a potential source of
toxicity. The rapid metabolism of the rapidly eliminated lipids can
improve the tolerability and therapeutic index of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10
mg/kg dose in rat. Inclusion of an enzymatically degraded ester
linkage can improve the degradation and metabolism profile of the
cationic component, while still maintaining the activity of the
reLNP formulation. The ester linkage can be internally located
within the lipid chain or it may be terminally located at the
terminal end of the lipid chain. The internal ester linkage may
replace any carbon in the lipid chain.
[0220] In one embodiment, the saRNA may be formulated as a
lipoplex, such as, without limitation, the ATUPLEX.TM. system, the
DACC system, the DBTC system and other siRNA-lipoplex technology
from Silence Therapeutics (London, United Kingdom), STEMFECT.TM.
from STEMGENT.RTM. (Cambridge, Mass.), and polyethylenimine (PEI)
or protamine-based targeted and non-targeted delivery of nucleic
acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther
2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;
Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et
al. Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 2009
32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo
Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,
23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6;
104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; the
contents of each of which are incorporated herein by reference in
its entirety).
[0221] In one embodiment such formulations may also be constructed
or compositions altered such that they passively or actively are
directed to different cell types in vivo, including but not limited
to hepatocytes, immune cells, tumor cells, endothelial cells,
antigen presenting cells, and leukocytes (Akinc et al. Mol Ther.
2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717;
Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al.,
Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006
13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier
et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol.
Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv.
2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 2011 18:1127-1133; the contents of each of
which are incorporated herein by reference in its entirety). One
example of passive targeting of formulations to liver cells
includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid
nanoparticle formulations which have been shown to bind to
apolipoprotein E and promote binding and uptake of these
formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010
18:1357-1364; the contents of which is herein incorporated by
reference in its entirety). Formulations can also be selectively
targeted through expression of different ligands on their surface
as exemplified by, but not limited by, folate, transferrin,
N-acetylgalactosamine (GalNAc), and antibody targeted approaches
(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206;
Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et
al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther
Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules.
2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008
5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et
al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods
Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68;
Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et
al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol
Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005
23:709-717; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 2011 18:1127-1133; the contents of each of
which are incorporated herein by reference in its entirety).
[0222] In one embodiment, the saRNA is formulated as a solid lipid
nanoparticle. A solid lipid nanoparticle (SLN) may be spherical
with an average diameter between 10 to 1000 nm. SLN possess a solid
lipid core matrix that can solubilize lipophilic molecules and may
be stabilized with surfactants and/or emulsifiers. In a further
embodiment, the lipid nanoparticle may be a self-assembly
lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2
(8), pp 1696-1702; the contents of which are herein incorporated by
reference in its entirety).
[0223] In one embodiment, the saRNA of the present invention can be
formulated for controlled release and/or targeted delivery. As used
herein, "controlled release" refers to a pharmaceutical composition
or compound release profile that conforms to a particular pattern
of release to effect a therapeutic outcome. In one embodiment, the
saRNA may be encapsulated into a delivery agent described herein
and/or known in the art for controlled release and/or targeted
delivery. As used herein, the term "encapsulate" means to enclose,
surround or encase. As it relates to the formulation of the
compounds of the invention, encapsulation may be substantial,
complete or partial. The term "substantially encapsulated" means
that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98,
99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical
composition or compound of the invention may be enclosed,
surrounded or encased within the delivery agent. "Partially
encapsulated" means that less than 10, 10, 20, 30, 40 50 or less of
the pharmaceutical composition or compound of the invention may be
enclosed, surrounded or encased within the delivery agent.
Advantageously, encapsulation may be determined by measuring the
escape or the activity of the pharmaceutical composition or
compound of the invention using fluorescence and/or electron
micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70,
80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99%
of the pharmaceutical composition or compound of the invention are
encapsulated in the delivery agent.
[0224] In another embodiment, the saRNA may be encapsulated into a
lipid nanoparticle or a rapidly eliminated lipid nanoparticle and
the lipid nanoparticles or a rapidly eliminated lipid nanoparticle
may then be encapsulated into a polymer, hydrogel and/or surgical
sealant described herein and/or known in the art. As a non-limiting
example, the polymer, hydrogel or surgical sealant may be PLGA,
ethylene vinyl acetate (EVAc), poloxamer, GELSITE.RTM.
(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX.RTM. (Halozyme
Therapeutics, San Diego Calif.), surgical sealants such as
fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL.RTM.
(Baxter International, Inc Deerfield, Ill.), PEG-based sealants,
and COSEAL.RTM. (Baxter International, Inc Deerfield, Ill.).
[0225] In another embodiment, the lipid nanoparticle may be
encapsulated into any polymer known in the art which may form a gel
when injected into a subject. As another non-limiting example, the
lipid nanoparticle may be encapsulated into a polymer matrix which
may be biodegradable.
[0226] In one embodiment, the saRNA formulation for controlled
release and/or targeted delivery may also include at least one
controlled release coating. Controlled release coatings include,
but are not limited to, OPADRY.RTM., polyvinylpyrrolidone/vinyl
acetate copolymer, polyvinylpyrrolidone, hydroxypropyl
methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose,
EUDRAGIT RL.RTM., EUDRAGIT RS.RTM. and cellulose derivatives such
as ethylcellulose aqueous dispersions (AQUACOAT.RTM. and
SURELEASE.RTM.).
[0227] In one embodiment, the controlled release and/or targeted
delivery formulation may comprise at least one degradable polyester
which may contain polycationic side chains. Degradeable polyesters
include, but are not limited to, poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and
combinations thereof. In another embodiment, the degradable
polyesters may include a PEG conjugation to form a PEGylated
polymer.
[0228] In one embodiment, the saRNA of the present invention may be
formulated with a targeting lipid with a targeting moiety such as
the targeting moieties disclosed in US20130202652 to Manoharan et
al., the contents of which are incorporated herein by reference in
their entirety. As a non-limiting example, the targeting moiety of
formula I of US 20130202652 to Manoharan et al. may selected in
order to favor the lipid being localized with a desired organ,
tissue, cell, cell type or subtype, or organelle. Non-limiting
targeting moieties that are contemplated in the present invention
include transferrin, anisamide, an RGD peptide, prostate specific
membrane antigen (PSMA), fucose, an antibody, or an aptamer.
[0229] In one embodiment, the saRNA of the present invention may be
encapsulated in a therapeutic nanoparticle. Therapeutic
nanoparticles may be formulated by methods described herein and
known in the art such as, but not limited to, International Pub
Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723,
WO2012054923, US Pub. Nos. US20110262491, US20100104645,
US20100087337, US20100068285, US20110274759, US20100068286 and
US20120288541 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208
and 8,318,211; the contents of each of which are herein
incorporated by reference in their entirety. In another embodiment,
therapeutic polymer nanoparticles may be identified by the methods
described in US Pub No. US20120140790, the contents of which are
herein incorporated by reference in its entirety.
[0230] In one embodiment, the therapeutic nanoparticle may be
formulated for sustained release. As used herein, "sustained
release" refers to a pharmaceutical composition or compound that
conforms to a release rate over a specific period of time. The
period of time may include, but is not limited to, hours, days,
weeks, months and years. As a non-limiting example, the sustained
release nanoparticle may comprise a polymer and a therapeutic agent
such as, but not limited to, the saRNA of the present invention
(see International Pub No. 2010075072 and US Pub No. US20100216804,
US20110217377 and US20120201859, the contents of each of which are
herein incorporated by reference in their entirety).
[0231] In one embodiment, the therapeutic nanoparticles may be
formulated to be target specific. As a non-limiting example, the
therapeutic nanoparticles may include a corticosteroid (see
International Pub. No. WO2011084518; the contents of which are
herein incorporated by reference in its entirety). In one
embodiment, the therapeutic nanoparticles may be formulated to be
cancer specific. As a non-limiting example, the therapeutic
nanoparticles may be formulated in nanoparticles described in
International Pub No. WO2008121949, WO2010005726, WO2010005725,
WO2011084521 and US Pub No. US20100069426, US20120004293 and
US20100104655, the contents of each of which are herein
incorporated by reference in their entirety.
[0232] In one embodiment, the nanoparticles of the present
invention may comprise a polymeric matrix. As a non-limiting
example, the nanoparticle may comprise two or more polymers such
as, but not limited to, polyethylenes, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates,
polycaprolactones, polyamides, polyacetals, polyethers, polyesters,
poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polylysine, poly(ethylene imine), poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or
combinations thereof.
[0233] In one embodiment, the therapeutic nanoparticle comprises a
diblock copolymer. In one embodiment, the diblock copolymer may
include PEG in combination with a polymer such as, but not limited
to, polyethylenes, polycarbonates, polyanhydrides,
polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polylysine, poly(ethylene imine), poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or
combinations thereof.
[0234] As a non-limiting example the therapeutic nanoparticle
comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293
and U.S. Pat. No. 8,236,330, each of which is herein incorporated
by reference in their entirety). In another non-limiting example,
the therapeutic nanoparticle is a stealth nanoparticle comprising a
diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No.
8,246,968 and International Publication No. WO2012166923, the
contents of each of which is herein incorporated by reference in
its entirety).
[0235] In one embodiment, the therapeutic nanoparticle may comprise
a multiblock copolymer such as, but not limited to the multiblock
copolymers described in U.S. Pat. Nos. 8,263,665 and 8,287,910; the
contents of each of which is herein incorporated by reference in
its entirety.
[0236] In one embodiment, the block copolymers described herein may
be included in a polyion complex comprising a non-polymeric micelle
and the block copolymer. (See e.g., U.S. Pub. No. 20120076836; the
contents of which are herein incorporated by reference in its
entirety).
[0237] In one embodiment, the therapeutic nanoparticle may comprise
at least one acrylic polymer. Acrylic polymers include but are not
limited to, acrylic acid, methacrylic acid, acrylic acid and
methacrylic acid copolymers, methyl methacrylate copolymers,
ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl
methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),
polycyanoacrylates and combinations thereof.
[0238] In one embodiment, the therapeutic nanoparticles may
comprise at least one amine-containing polymer such as, but not
limited to polylysine, polyethylene imine, poly(amidoamine)
dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No.
8,287,849; the contents of which are herein incorporated by
reference in its entirety) and combinations thereof.
[0239] In one embodiment, the therapeutic nanoparticles may
comprise at least one degradable polyester which may contain
polycationic side chains. Degradable polyesters include, but are
not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),
poly(4-hydroxy-L-proline ester), and combinations thereof. In
another embodiment, the degradable polyesters may include a PEG
conjugation to form a PEGylated polymer.
[0240] In another embodiment, the therapeutic nanoparticle may
include a conjugation of at least one targeting ligand. The
targeting ligand may be any ligand known in the art such as, but
not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res.
2006 66:6732-6740; the contents of which are herein incorporated by
reference in its entirety).
[0241] In one embodiment, the therapeutic nanoparticle may be
formulated in an aqueous solution which may be used to target
cancer (see International Pub No. WO2011084513 and US Pub No.
US20110294717, the contents of each of which is herein incorporated
by reference in their entirety).
[0242] In one embodiment, the saRNA may be encapsulated in, linked
to and/or associated with synthetic nanocarriers. Synthetic
nanocarriers include, but are not limited to, those described in
International Pub. Nos. WO2010005740, WO2010030763, WO201213501,
WO2012149252, WO2012149255, WO2012149259, WO2012149265,
WO2012149268, WO2012149282, WO2012149301, WO2012149393,
WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US
Pub. Nos. US20110262491, US20100104645, US20100087337 and
US20120244222, the contents of each of which are herein
incorporated by reference in their entirety. The synthetic
nanocarriers may be formulated using methods known in the art
and/or described herein. As a non-limiting example, the synthetic
nanocarriers may be formulated by the methods described in
International Pub Nos. WO2010005740, WO2010030763 and WO201213501
and US Pub. Nos. US20110262491, US20100104645, US20100087337 and
US2012024422, the contents of each of which are herein incorporated
by reference in their entirety. In another embodiment, the
synthetic nanocarrier formulations may be lyophilized by methods
described in International Pub. No. WO2011072218 and U.S. Pat. No.
8,211,473; the contents of each of which are herein incorporated by
reference in their entirety.
[0243] In one embodiment, the synthetic nanocarriers may contain
reactive groups to release the saRNA described herein (see
International Pub. No. WO20120952552 and US Pub No. US20120171229,
the contents of each of which are herein incorporated by reference
in their entirety).
[0244] In one embodiment, the synthetic nanocarriers may be
formulated for targeted release. In one embodiment, the synthetic
nanocarrier may be formulated to release the saRNA at a specified
pH and/or after a desired time interval. As a non-limiting example,
the synthetic nanoparticle may be formulated to release the saRNA
after 24 hours and/or at a pH of 4.5 (see International Pub. Nos.
WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and
US20110027217, the contents of each of which is herein incorporated
by reference in their entireties).
[0245] In one embodiment, the synthetic nanocarriers may be
formulated for controlled and/or sustained release of the saRNA
described herein. As a non-limiting example, the synthetic
nanocarriers for sustained release may be formulated by methods
known in the art, described herein and/or as described in
International Pub No. WO2010138192 and US Pub No. 20100303850, the
contents each of which is herein incorporated by reference in their
entirety.
[0246] In one embodiment, the nanoparticle may be optimized for
oral administration. The nanoparticle may comprise at least one
cationic biopolymer such as, but not limited to, chitosan or a
derivative thereof. As a non-limiting example, the nanoparticle may
be formulated by the methods described in U.S. Pub. No.
20120282343; the contents of which are herein incorporated by
reference in its entirety.
[0247] In one embodiment, the saRNA of the present invention may be
formulated in a modular composition such as described in U.S. Pat.
No. 8,575,123 to Manoharan et al., the contents of which are herein
incorporated by reference in their entirety. As a non-limiting
example, the modular composition may comprise a nucleic acid, e.g.,
the saRNA of the present invention, at least one endosomolytic
component, and at least one targeting ligand. The modular
composition may have a formula such as any formula described in
U.S. Pat. No. 8,575,123 to Manoharan et al., the contents of which
are herein incorporated by reference in their entirety.
[0248] In one embodiment, the saRNA of the present invention may be
encapsulated in the lipid formulation to form a stable nucleic
acid-lipid particle (SNALP) such as described in U.S. Pat. No.
8,546,554 to de Fougerolles et al., the contents of which are
incorporated here by reference in their entirety. The lipid may be
cationic or non-cationic. In one non-limiting example, the lipid to
nucleic acid ratio (mass/mass ratio) (e.g., lipid to saRNA ratio)
will be in the range of from about 1:1 to about 50:1, from about
1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to
about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1,
or 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1. In another example, the
SNALP includes 40%
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A), 10%
dioleoylphosphatidylcholine (DSPC), 40% cholesterol, 10%
polyethyleneglycol (PEG)-C-DOMG (mole percent) with a particle size
of 63.0.+-.20 nm and a 0.027 nucleic acid/lipid ratio. In another
embodiment, the saRNA of the present invention may be formulated
with a nucleic acid-lipid particle comprising an endosomal membrane
destabilizer as disclosed in U.S. Pat. No. 7,189,705 to Lam et al.,
the contents of which are incorporated herein by reference in their
entirety. As a non-limiting example, the endosomal membrane
destabilizer may be a Ca.sup.2+ ion.
[0249] In one embodiment, the saRNA of the present invention may be
formulated with formulated lipid particles (FLiPs) disclosed in
U.S. Pat. No. 8,148,344 to Akine et al., the contents of which are
herein incorporated by reference in their entirety. Akine et al.
teach that FLiPs may comprise at least one of a single or double
stranded oligonucleotide, where the oligonucleotide has been
conjugated to a lipophile and at least one of an emulsion or
liposome to which the conjugated oligonucleotide has been
aggregated, admixed or associated. These particles have
surprisingly been shown to effectively deliver oligonucleotides to
heart, lung and muscle disclosed in U.S. Pat. No. 8,148,344 to
Akine et al., the contents of which are herein incorporated by
reference in their entirety.
[0250] In one embodiment, the saRNA of the present invention may be
delivered to a cell using a composition comprising an expression
vector in a lipid formulation as described in U.S. Pat. No.
6,086,913 to Tam et al., the contents of which are incorporated
herein by reference in their entirety. The composition disclosed by
Tam is serum-stable and comprises an expression vector comprising
first and second inverted repeated sequences from an adeno
associated virus (AAV), a rep gene from AAV, and a nucleic acid
fragment. The expression vector in Tam is complexed with
lipids.
[0251] In one embodiment, the saRNA of the present invention may be
formulated with a lipid formulation disclosed in US 20120270921 to
de Fougerolles et al., the contents of which are incorporated
herein by reference in their entirety. In one non-limiting example,
the lipid formulation may include a cationic lipid having the
formula A described in US 20120270921, the contents of which are
herein incorporated by reference in its entirety. In another
non-limiting example, the compositions of exemplary nucleic
acid-lipid particles disclosed in Table A of US 20120270921, the
contents of which are incorporated herein by reference in their
entirety, may be used with the saRNA of the present invention.
[0252] In one embodiment, the saRNA of the present invention may be
fully encapsulated in a lipid particle disclosed in US 20120276207
to Maurer et al., the contents of which are incorporated herein by
reference in their entirety. The particles may comprise a lipid
composition comprising preformed lipid vesicles, a charged
therapeutic agent, and a destabilizing agent to form a mixture of
preformed vesicles and therapeutic agent in a destabilizing
solvent, wherein said destabilizing solvent is effective to
destabilize the membrane of the preformed lipid vesicles without
disrupting the vesicles.
[0253] In one embodiment, the saRNA of the present invention may be
formulated with a conjugated lipid. In a non-limiting example, the
conjugated lipid may have a formula such as described in US
20120264810 to Lin et al., the contents of which are incorporated
herein by reference in their entirety. The conjugate lipid may form
a lipid particle which further comprises a cationic lipid, a
neutral lipid, and a lipid capable of reducing aggregation.
[0254] In one embodiment, the saRNA of the present invention may be
formulated in a neutral liposomal formulation such as disclosed in
US 20120244207 to Fitzgerald et al., the contents of which are
incorporated herein by reference in their entirety. The phrase
"neutral liposomal formulation" refers to a liposomal formulation
with a near neutral or neutral surface charge at a physiological
pH. Physiological pH can be, e.g., about 7.0 to about 7.5, or,
e.g., about 7.5, or, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or,
e.g., 7.3, or, e.g., 7.4. An example of a neutral liposomal
formulation is an ionizable lipid nanoparticle (iLNP). A neutral
liposomal formulation can include an ionizable cationic lipid,
e.g., DLin-KC2-DMA.
[0255] In one embodiment, the saRNA of the present invention may be
formulated with a charged lipid or an amino lipid. As used herein,
the term "charged lipid" is meant to include those lipids having
one or two fatty acyl or fatty alkyl chains and a quaternary amino
head group. The quaternary amine carries a permanent positive
charge. The head group can optionally include an ionizable group,
such as a primary, secondary, or tertiary amine that may be
protonated at physiological pH. The presence of the quaternary
amine can alter the pKa of the ionizable group relative to the pKa
of the group in a structurally similar compound that lacks the
quaternary amine (e.g., the quaternary amine is replaced by a
tertiary amine) In some embodiments, a charged lipid is referred to
as an "amino lipid." In a non-limiting example, the amino lipid may
be amino lipids described in US20110256175 to Hope et al., the
contents of which are incorporated herein by reference in their
entirety. For example, the amino lipids may have the structure
disclosed as structure (II), DLin-K-C2-DMA, DLin-K2-DMA,
DLin-K6-DMA disclosed in US20110256175 to Hope et al., the contents
of which are incorporated herein by reference in their entirety. In
another example, the amino lipid may have the structure (I), (II),
(III), or (IV), or 4-(R)-DUn-K-DMA (VI), 4-(S)-DUn-K-DMA (V) as
described in WO2009132131 to Muthiah et al., the contents of which
are incorporated herein by reference in their entirety. In another
example, the charged lipid used in any of the formulations
described herein may be any charged lipid described in EP2509636 to
Manoharan et al., the contents of which are incorporated herein by
reference in their entirety.
[0256] In one embodiment, the saRNA of the present invention may be
formulated with an association complex containing lipids,
liposomes, or lipoplexes. In a non-limiting example, the
association complex comprises one or more compounds each having a
structure defined by formula (I), a PEG-lipid having a structure
defined by formula (XV), a steroid and a nucleic acid disclosed in
U.S. Pat. No. 8,034,376 to Manoharan et al., the contents of which
are incorporated herein by reference in their entirety. The saRNA
may be formulated with any association complex described in U.S.
Pat. No. 8,034,376, the contents of which are herein incorporated
by reference in its entirety.
[0257] In one embodiment, the saRNA of the present invention may be
formulated with reverse head group lipids. As a non-limiting
example, the saRNA may be formulated with a zwitterionic lipid
comprising a headgroup wherein the positive charge is located near
the acyl chain region and the negative charge is located at the
distal end of the head group, such as a lipid having structure (A)
or structure (I) described in WO2011056682 to Leung et al., the
contents of which are incorporated herein by reference in their
entirety.
[0258] In one embodiment, the saRNA of the present invention may be
formulated in a lipid bilayer carrier. As a non-limiting example,
the saRNA may be combined with a lipid-detergent mixture comprising
a lipid mixture of an aggregation-preventing agent in an amount of
about 5 mol % to about 20 mol %, a cationic lipid in an amount of
about 0.5 mol % to about 50 mol %, and a fusogenic lipid and a
detergent, to provide a nucleic acid-lipid-detergent mixture; and
then dialyzing said nucleic acid-lipid-detergent mixture against a
buffered salt solution to remove said detergent and to encapsulate
said nucleic acid in a lipid bilayer carrier and provide a lipid
bilayer-nucleic acid composition, wherein said buffered salt
solution has an ionic strength sufficient to encapsulate of from
about 40% to about 80% of said nucleic acid, described in
WO1999018933 to Cullis et al., the contents of which are
incorporated herein by reference in their entirety.
[0259] In one embodiment, the saRNA of the present invention may be
formulated in a nucleic acid-lipid particle capable of selectively
targeting the saRNA to a heart, liver, or tumor tissue site. For
example, the nucleic acid-lipid particle may comprise (a) a nucleic
acid; (b) 1.0 mole % to 45 mole % of a cationic lipid; (c) 0.0 mole
% to 90 mole % of another lipid; (d) 1.0 mole % to 10 mole % of a
bilayer stabilizing component; (e) 0.0 mole % to 60 mole %
cholesterol; and (f) 0.0 mole % to 10 mole % of cationic polymer
lipid as described in EP1328254 to Cullis et al., the contents of
which are incorporated herein by reference in their entirety.
Cullis teaches that varying the amount of each of said cationic
lipid, bilayer stabilizing component, another lipid, cholesterol,
and cationic polymer lipid can impart tissue selectivity for heart,
liver, or tumor tissue site, thereby identifying a nucleic
acid-lipid particle capable of selectively targeting a nucleic acid
to said heart, liver, or tumor tissue site.
Delivery
[0260] The present disclosure encompasses the delivery of saRNA for
any of therapeutic, pharmaceutical, diagnostic or imaging by any
appropriate route taking into consideration likely advances in the
sciences of drug delivery. Delivery may be naked or formulated.
[0261] The saRNA of the present invention may be delivered to a
cell naked. As used herein in, "naked" refers to delivering saRNA
free from agents which promote transfection. For example, the saRNA
delivered to the cell may contain no modifications. The naked saRNA
may be delivered to the cell using routes of administration known
in the art and described herein.
[0262] The saRNA of the present invention may be formulated, using
the methods described herein. The formulations may contain saRNA
which may be modified and/or unmodified. The formulations may
further include, but are not limited to, cell penetration agents, a
pharmaceutically acceptable carrier, a delivery agent, a
bioerodible or biocompatible polymer, a solvent, and a
sustained-release delivery depot. The formulated saRNA may be
delivered to the cell using routes of administration known in the
art and described herein.
[0263] The compositions may also be formulated for direct delivery
to an organ or tissue in any of several ways in the art including,
but not limited to, direct soaking or bathing, via a catheter, by
gels, powder, ointments, creams, gels, lotions, and/or drops, by
using substrates such as fabric or biodegradable materials coated
or impregnated with the compositions, and the like. The saRNA of
the present invention may also be cloned into a retroviral
replicating vector (RRV) and transduced to cells.
Administration
[0264] The saRNA of the present invention may be administered by
any route which results in a therapeutically effective outcome.
These include, but are not limited to enteral, gastroenteral,
epidural, oral, transdermal, epidural (peridural), intracerebral
(into the cerebrum), intracerebroventricular (into the cerebral
ventricles), epicutaneous (application onto the skin), intradermal,
(into the skin itself), subcutaneous (under the skin), nasal
administration (through the nose), intravenous (into a vein),
intraarterial (into an artery), intramuscular (into a muscle),
intracardiac (into the heart), intraosseous infusion (into the bone
marrow), intrathecal (into the spinal canal), intraperitoneal,
(infusion or injection into the peritoneum), intravesical infusion,
intravitreal, (through the eye), intracavernous injection, (into
the base of the penis), intravaginal administration, intrauterine,
extra-amniotic administration, transdermal (diffusion through the
intact skin for systemic distribution), transmucosal (diffusion
through a mucous membrane), insufflation (snorting), sublingual,
sublabial, enema, eye drops (onto the conjunctiva), or in ear
drops. In specific embodiments, compositions may be administered in
a way which allows them cross the blood-brain barrier, vascular
barrier, or other epithelial barrier. Routes of administration
disclosed in International Publication WO 2013/090648 filed Dec.
14, 2012, the contents of which are incorporated herein by
reference in their entirety, may be used to administer the saRNA of
the present invention.
Dosage Forms
[0265] A pharmaceutical composition described herein can be
formulated into a dosage form described herein, such as a topical,
intranasal, intratracheal, or injectable (e.g., intravenous,
intraocular, intravitreal, intramuscular, intracardiac,
intraperitoneal, subcutaneous). Liquid dosage forms, injectable
preparations, pulmonary forms, and solid dosage forms described in
International Publication WO 2013/090648 filed Dec. 14, 2012, the
contents of which are incorporated herein by reference in their
entirety may be used as dosage forms for the saRNA of the present
invention.
II. Methods of Use
[0266] One aspect of the present invention provides methods of
using C/EBP.alpha.-saRNA and pharmaceutical compositions comprising
said C/EBP.alpha.-saRNA and at least one pharmaceutically
acceptable carrier. C/EBP.alpha.-saRNA modulates C/EBP.alpha. gene
expression. In one embodiment, the expression of C/EBP.alpha. gene
is increased by at least 20, 30, 40%, more preferably at least 45,
50, 55, 60, 65, 70, 75%, even more preferably at least 80% in the
presence of the saRNA of the present invention compared to the
expression of C/EBP.alpha. gene in the absence of the saRNA of the
present invention. In a further preferable embodiment, the
expression of C/EBP.alpha. gene is increased by a factor of at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably by a factor of at
least 15, 20, 25, 30, 35, 40, 45, 50, even more preferably by a
factor of at least 60, 70, 80, 90, 100, in the presence of the
saRNA of the present invention compared to the expression of
C/EBP.alpha. gene in the absence of the saRNA of the present
invention.
[0267] In one embodiment, the increase in gene expression of the
saRNA descried herein is shown in proliferating cells.
Metabolics Regulation
[0268] Hepatocytes are generally perceived as being important for
maintenance of several vital functions. For example, they can
regulate carbohydrate and lipid metabolism and detoxification of
exogenous and endogenous compounds. C/EBP.alpha. is expressed in a
variety of tissues where it plays an important role in the
differentiation of many cell types including adipocytes, type II
alveolar cells and hepatocytes. In the mouse, C/EBP.alpha. is found
most abundantly in fat, liver and lung tissues. The function role
of C/EBP.alpha. includes, but not limited to, regulation of
alpha-1-antitrypsin, transthyretin and albumin. Furthermore,
expression of C/EBP.alpha. gene in the liver cell line (HepG2)
results in increased levels of cytochrome P450 (CYP), a superfamily
of monooxygenases that participates in the metabolism of endogenous
substrates and plays a key role in detoxification and metabolic
activation of key xenobiotics [Jover et al., FEBS Letters, vol.
431(2), 227-230 (1998), the contents of which are incorporated
herein by reference in their entirety].
[0269] Non-alcoholic fatty liver disease (NAFLD) is a major global
health concern and affects 1 in 3 people in the United States.
NAFLD is the build-up of extra fat (lipid) in liver cells that is
not caused by excessive alcohol use. It is called a fatty liver
(steatosis) if more than 5%-10% of the liver's weight is fat. NAFLD
may progress to steatoheptitis, cirrhosis, and liver cancer. It is
associated with metabolic disorders, such as metabolic syndrome,
insulin resistance, type II diabetes, hyperlipidemia, hypertension,
obesity, etc. Treatment methods include lowering low-density
lipoprotein (LDL) cholesterol levels, improving insulin
sensitivity, treating metabolic risk factors, weight loss and so
on. [Adams et al., Postgraduate Medical Journal, vol. 82, 315-322
(2006); Musso et al., Curr. Opin. Lipidol., vol. 22(6), 489-496
(2011), the contents of which are incorporated herein by reference
in their entirety]
[0270] C/EBP.alpha. protein plays an important role in regulating
liver function and metabolics. The primary effects of C/EBP.alpha.
on the liver are shown in FIG. 1, including decreasing fatty acid
uptake by lowering CD36 protein level, decreasing de novo
lipogenesis by lowering sterol regulatory element-binding proteins
(SREBP), carbohydrate-responsive element-binding protein (ChREBP)
and fatty acid synthase (FAS) protein levels, increasing
.beta.-oxidation by increasing peroxisome proliferator-activated
receptor alpha (PPAR.alpha.) and peroxisome proliferator-activated
receptor gamma coactivator 1-alpha & -beta (PGC-1.alpha. &
.beta.) protein levels, decreasing hepatic lipid overload by
lowering apolipoprotein C-III (APOC3) and low density lipoprotein
receptor (LDLR) protein levels, decreasing progression to fibrosis
by increasing PGC-1.beta. protein level, and decreasing insulin
resistance by increasing peroxisome proliferator-activated receptor
gamma (PPAR.gamma.) protein level. Furthermore, C/EBP.alpha. has
secondary effects on adipose tissues as shown in FIG. 2. White
adipose tissue (WAT) is not only a lipogenic and fat storage tissue
but also an important endocrine organ that regulates energy
homeostasis, lipid metabolism, appetite, fertility, and immune and
stress responses. Brown adipose tissue (BAT) contains numerous
smaller lipid droplets and a much higher number of iron-containing
mitochondria compared with WAT. It plays a significant role in
nutritional energetics, energy balance and body weight. There is
evidence that the atrophy of BAT is related to obesity. In
particular, studies have indicated that impaired thermogenesis in
BAT is important in the aetiology of obesity in rodents [Trayhurn
P., J. Biosci., vol. 18(2), 161-173 (1993)]. C/EBP.alpha. decreases
hepatic steatosis and insulin resistance and increases PGC-1.alpha.
protein level, which may in turn cause browning of WAT, turn WAT
into BAT, and then activate BAT, thereby reducing body fat and
weight. Therefore, C/EBP.alpha.-saRNA of the present invention may
be used to regulate liver function, reduce steatosis, reduce serum
lipids, treat NAFLD, treat insulin resistance, increase energy
expenditure, and treat obesity.
[0271] In one embodiment, provided is a method of regulating liver
metabolism genes in vitro and in vivo by treatment of
C/EBP.alpha.-saRNA of the present invention. Also provided is a
method of regulating liver genes involved in NAFLD in vitro and in
vivo by treatment of C/EBP.alpha.-saRNA of the present invention.
The genes include, but are not limited to sterol regulatory
element-binding factor 1 (SREBF-1 or SREBF), cluster of
differentiation 36 (CD36), acetyl-CoA carboxylase 2 (ACACB),
apolipoprotein C-III (APOC3), microsomal triglyceride transfer
protein (MTP), peroxisome proliferator-activated receptor gamma
coactivator 1 alpha (PPAR.gamma.-CoA1.alpha. or PPARGC1A), low
density lipoprotein receptor (LDLR), peroxisome
proliferator-activated receptor gamma coactivator 1 beta
(PPAR.gamma.-CoA1.beta. or PERC), peroxisome proliferator-activated
receptor gamma (PPAR.gamma.), acetyl-CoA carboxylase 1 (ACACA),
carbohydrate-responsive element-binding protein (ChREBP or MLX1PL),
peroxisome proliferator-activated receptor alpha (PPAR.alpha. or
PPARA), FASN (fatty acid synthase), diglyceride acyltransferase-2
(DGAT2), and mammalian target of rapamycin (mTOR). In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of SREBF-1
gene in liver cells by at least 20%, 30%, preferably at least 40%.
In one embodiment, C/EBP.alpha.-saRNA decreases the expression of
CD36 gene in liver cells by at least 20%, 30%, 40%, 50%, preferably
at least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA increases
the expression of ACACB gene in liver cells by at least 20%, 30%,
40%, 50%, preferably at least 75%, 90%, 100%, 125%, 150%. In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of APOC3
gene in liver cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of MTP gene in liver cells by at least 20%, 30%, 40%,
50%, preferably at least 75%, 90%. In one embodiment,
C/EBP.alpha.-saRNA increases the expression of
PPAR.gamma.-CoA1.alpha. gene in liver cells by at least 20%, 30%,
40%, 50%, preferably at least 75%, 90%, 100%, 125%, 150%, more
preferably at least 175%, 200%, 250%, 300%. In one embodiment,
C/EBP.alpha.-saRNA increases the expression of PPAR.gamma. gene in
liver cells by at least 20%, 30%, 40%, 50%, preferably at least
75%, 90%, 100%, 125%, 150%, more preferably at least 175%, 200%,
250%, 300%. In one embodiment, C/EBP.alpha.-saRNA increases the
expression of PPAR.alpha. gene in liver cells by at least 20%, 30%,
40%, 50%, preferably at least 75%, 90%, 100%, 125%, 150%, more
preferably at least 175%, 200%, 250%, 300%. In one embodiment,
C/EBP.alpha.-saRNA decreases the expression of MLXIPL gene in liver
cells by at least 20%, 30%, 40%, 50%, preferably at least 75%. In
one embodiment, C/EBP.alpha.-saRNA decreases the expression of FASN
gene in liver cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of DGAT2 gene in liver cells by at least 10%, 20%,
preferably at least 30%, 40%, 50%.
[0272] C/EBP.alpha.-saRNA also modulates the expression of liver
metabolism genes disclosed above in BAT cells. In another
embodiment, C/EBP.alpha.-saRNA decreases the expression of SREBP
gene in BAT cells by at least 20%, 30%, preferably at least 40%. In
one embodiment, C/EBP.alpha.-saRNA decreases the expression of CD36
gene in BAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of LDLR gene in BAT cells by at least 20%, 30%, 40%,
50%, preferably at least 75%, 90%. In one embodiment,
C/EBP.alpha.-saRNA increases the expression of PPARGC1A gene in BAT
cells by at least 20%, 30%, preferably at least 40%. In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of APOC
gene in BAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%, more preferably at least 95%, 99%. In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of ACACB
gene in BAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of PERC gene in BAT cells by at least 20%, 30%, 40%,
50%, preferably at least 75%. In one embodiment, C/EBP.alpha.-saRNA
increases the expression of ACACA gene in BAT cells by at least
20%, 30%, 40%, 50%, preferably at least 75%, 90%, 100%, 125%, 150%.
In one embodiment, C/EBP.alpha.-saRNA decreases the expression of
MLXP1 gene in BAT cells by at least 20%, 30%, 40%, preferably at
least 50%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of MTOR gene in BAT cells by at least 20%, 30%, 40%,
preferably at least 50%, 75%. In one embodiment, C/EBP.alpha.-saRNA
increases the expression of PPARA gene in BAT cells by at least
20%, 30%, 40%, 50%, preferably at least 75%, 90%, 100%, 125%, 150%,
more preferably at least 200%, 250%, 300%, 350%, 400%. In one
embodiment, C/EBP.alpha.-saRNA increases the expression of FASN
gene in BAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA increases the
expression of DGAT gene in BAT cells by at least 20%, 30%, 40%,
50%, preferably at least 75%, 90%, 100%, 125%, 150%, more
preferably at least 200%, 250%, 300%.
[0273] C/EBP.alpha.-saRNA also modulates the expression of liver
metabolism genes disclosed above in WAT cells. In another
embodiment, C/EBP.alpha.-saRNA decreases the expression of SREBP
gene in WAT cells by at least 20%, 30%, preferably at least 40%. In
one embodiment, C/EBP.alpha.-saRNA decreases the expression of CD36
gene in WAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%, 90%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of LDLR gene in WAT cells by at least 20%, 30%, 40%,
50%, preferably at least 75%, 90%. In one embodiment,
C/EBP.alpha.-saRNA increases the expression of PPARGC1A gene in WAT
cells by at least 20%, 30%, preferably at least 40%. In one
embodiment, C/EBP.alpha.-saRNA increases the expression of MTP gene
in WAT cells by at least 20%, 30%, 40%, 50%, preferably at least
75%, 90%, more preferably at least 95%, more preferably at least by
a factor of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, more preferably by at
least a factor of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0. In one embodiment,
In one embodiment, C/EBP.alpha.-saRNA increases the expression of
APOC gene in WAT cells by at least 20%, 30%, 40%, 50%, preferably
at least 75%, 90%, more preferably at least 95%, 99%. In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of ACACB
gene in WAT cells by at least 20%, 30%, 40%, 50%, preferably at
least 75%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of PERC gene in WAT cells by at least 20%, 30%, 40%,
50%, preferably at least 75%. In one embodiment, C/EBP.alpha.-saRNA
decreases the expression of ACACA gene in WAT cells by at least
20%, 30%, 40%, 50%, preferably at least 75%, 90%, 95%. In one
embodiment, C/EBP.alpha.-saRNA decreases the expression of MLX1PL
gene in WAT cells by at least 20%, 30%, 40%, preferably at least
50%. In one embodiment, C/EBP.alpha.-saRNA decreases the expression
of MTOR gene in WAT cells by at least 20%, 30%, 40%, preferably at
least 50%, 75%. In one embodiment, C/EBP.alpha.-saRNA decreases the
expression of FASN gene in WAT cells by at least 5%, 10%,
preferably at least 15%, 20%. In one embodiment, C/EBP.alpha.-saRNA
decreases the expression of DGAT gene in WAT cells by at least 10%,
20%, 30%, more preferably 40%, 50%.
[0274] In another embodiment, provided is a method of reducing
insulin resistance (IR) or increasing insulin sensitivity by
administering C/EBP.alpha.-saRNA of the present invention to a
patient in need thereof. Also provided is a method of treating type
II diabetes, hyperinsulinaemia and steatosis by administering
C/EBP.alpha.-saRNA of the present invention to a patient in need
thereof. If liver cells are resistance to insulin and cannot use
insulin effectively, hyperglycemia develops. Subsequently, beta
cells in pancreas increase their production of insulin leading to
hyperinsulinemia and type II diabetes. Many regulators affect
insulin resistance of liver cells. For example, sterol regulatory
element-binding proteins 1 (SREBP1 or SREBP) is the master
regulator of cholesterol and associated with increased insulin
resistance. The up-regulation of cholesteryl ester transfer protein
(CETP) is associated with increased insulin resistance. The
up-regulation of hepatic fatty acid translocase/cluster of
differentiation 36 (FAT/CD36) is associated with insulin
resistance, hyperinsulinaemia, increased steatosis in patients with
non-alcoholic steatohepatitis (NASH). Liver-specific overexpression
of lipoprotein lipase gene (LPL) causes liver-specific insulin
resistance. Liver X receptor gene (LXR) has a central role in
insulin-mediated activation of sterol regulatory element-binding
protein (SREBP)-1c-induced fatty acid synthesis in liver. Other
factors include diglyceride acyltransferase-2 (DGAT2) that
regulates triglyceride synthesis and fatty acid synthase (FASN)
that regulates fatty acid biosynthesis. In one embodiment,
C/EBP.alpha.-saRNA reduces the expression of FAT/CD36 gene in liver
cells by at least 25%, preferably at least 50%, more preferably at
least 75%, even more preferably 90% compared to liver cells with no
treatment. In another embodiment, C/EBP.alpha.-saRNA increases the
expression of LPL gene in liver cells by at least 20, 30, 40%,
preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95%,
more preferably at least 100, 150, 200, 250, 300, 350 and 400%
compared to liver cells with no treatment. In another embodiment,
C/EBP.alpha.-saRNA increases the expression of LXR gene in liver
cells by at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95%, more
preferably at least 100, 150, 200, 250, 300, 350 and 400%, even
more preferably at least 450, 500, 550, 600% compared to liver
cells with no treatment. In another embodiment, C/EBP.alpha.-saRNA
decreases SREBP1 gene expression. In another embodiment,
C/EBP.alpha.-saRNA decreases DGAT2 gene expression. In another
embodiment, C/EBP.alpha.-saRNA decreases CETP gene expression. In
yet another embodiment, C/EBP.alpha.-saRNA decreases FASN gene
expression.
[0275] A summary of NAFLD and IR genes that may be modulated with
C/EBP.alpha.-saRNA is shown in Table 5. Abbreviations in Table 5:
NAFLD: non-alcoholic fatty liver disease; IR: insulin resistance;
DNL: de novo lipogenesis; FA: fatty acid; TG: triglycerides; LPL:
lipoprotein lipase; HP: hepatic lipase; CHOL: cholesterol.
TABLE-US-00009 TABLE 5 NAFLD and IR genes that may be modulated
with C/EBP.alpha.-saRNA Gene Deregulation Deregulation name
Mechanism Function/encoded products-References in NAFLD in IR CD36
FAs uptake Scavenger receptor, free FAs transporter up up in liver
and adipose tissue; regulates adipose tissue apoptosis and
inflammation PPAR.gamma. DNL Activates genes involved in lipid
storage up down and metabolism; required for lipid homeostasis;
high expressed in adipose tissue and very low in the liver;
implicated in adipocyte differentiation and insulin sensitivity
PPAR.gamma.- DNL Transcriptional coactivator for SREBP-1 ; up up
CoA 1.beta. enhances lipogenesis and VLDL synthesis; (PERC) highly
expressed in brown fat and heart and induced in the liver during
fasting; master regulator of mitochondrial biogenesis and oxidative
metabolism, lipogenesis, and TG secretion SREBP-1c DNL
Transcription factor, induces genes up up involved in glucose
utilization and FA synthesis; major mediator of insulin action on
lipogenic genes; regulates adipogenesis ChREBP DNL Transcription
factors activated by glucose; up up (MLX1PL) induces glycolytic and
lipogenic genes; major determinant of adipose tissue fatty acid
synthesis and systemic insulin sensitivity FAS DNL Enzyme that
catalyzes the last step in FA up up biosynthesis ACACA DNL Enzyme
that catalyzes the synthesis of up up (ACC1) malonyl-CoA for the
synthesis of FAs in the cytosol ACACB .beta.-oxidation Enzyme that
catalyzes the synthesis of up up (ACC2) malonyl-CoA, which
functions as inhibitor of mitochondrial .beta.-oxidation
PPAR.alpha. .beta.-oxidation Activates the genes involved in the
down down oxidation of FAs, major regulator of lipid metabolism in
the liver; predominantly expressed in the liver; involved in the
regulation of glucose homeostasis, insulin sensitivity, fat
accumulation, and adipose tissue glucose use PPAR.gamma.-
.beta.-oxidation Transcriptional co-activator that regulates down
down CoA 1.alpha. mitochondrial biology and energy homeostasis;
crucial role in mitochondrial biogenesis; interacts with
PPAR.alpha. to increase the mitochondrial .beta.-oxidation of FAs
DGAT2 TG synthesis Enzyme that catalyzes the final reaction in up
up the synthesis of TG APOC3 TG Protein that inhibits LPL and HP;
involved up up concentration in the regulation of plasma TG
concentrations; pro-steatosic LDLR CHOL Low-density lipoprotein
receptor; critical down no change concentration role in regulating
blood CHOL levels; abundant in the liver, which is the organ
responsible for removing most excess CHOL from the body MTP
Lipoprotein Carrier of TG; central role in VLDL down no change
(MTTP1) assembly assembly; prevalently expressed in the liver mTOR
Adipose Possible regulator of adipose tissue mass; up up mass
central role in lipolysis, lipogenesis, and adipogenesis Effects of
Ezetimibe Effects of C/EBP.alpha. Gene name in the liver Liver WAT
BAT CD36 minor down down down down PPAR.gamma. up up no change no
change PPAR.gamma.-CoA up up down up 10 (PERC) SREBP-1c up down
down down ChREBP up down up up (MLX1PL) FAS down down minor up up
ACACA minor up no change down up (ACC1) ACACB up up down down
(ACC2) PPAR.alpha. up up down up PPAR.gamma.-CoA up up up up
1.alpha. DGAT2 minor down minor down down up APOC3 down down up
down LDLR minor down down up minor down MTP (MTTP1) up down up down
mTOR no change no change down down
[0276] In one embodiment of the present invention, provided is a
method of lowering serum cholesterol level in vitro by treatment of
C/EBP.alpha.-saRNA of the present invention. The serum cholesterol
level with C/EBP.alpha.-saRNA reduces at least 25%, preferably 50%,
more preferably 75% compared to serum cholesterol level with no
treatment. Also provided is a method of lowering LDL and
triglyceride levels in hepatocyte cells and increasing circulating
levels of LDL in vivo by administering C/EBP.alpha.-saRNA of the
present invention. The circulation LDL level may increase at least
by a factor of 2, preferably by a factor of 3, preferably by a
factor of 4, preferably by a factor of 5, preferably by a factor of
10, and preferably by a factor of 15 compared to circulating LDL
level in the absence of C/EBP.alpha.-saRNA. The liver triglyceride
level may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, or
70% compared to the liver triglyceride level in the absence of
C/EBP.alpha.-saRNA. The liver LDL level may be reduced by at least
10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to the liver LDL
level in the absence of C/EBP.alpha.-saRNA.
[0277] In one embodiment of the present invention, provided is a
method of treating NAFLD and reducing fatty liver size by
administering C/EBP.alpha.-saRNA of the present invention to a
patient in need thereof. The size of a fatty liver of a patient
treated with C/EBP.alpha.-saRNA is reduced by at least 10%, 20%,
30%, 40%, or 50% compared with a patient without treatment. Also
provided is a method of reducing body weight and treating obesity
by administering C/EBP.alpha.-saRNA of the present invention to a
patient in need thereof. The body weight of a patient treated with
C/EBP.alpha.-saRNA is lower than the body weight of a patient
without treatment of C/EBP.alpha.-saRNA by at least 10%, 20%, 30%,
40%, 50%, 60%, or 70%. C/EBP.alpha.-saRNA of the present invention
may be administered in a dose, 2 doses, 3 does or more. Also
provided is a method of decreasing hepatic uptake of free fatty
acids by treatment of C/EBP.alpha.-saRNA of the present invention.
Also provided is a method of reducing white adipose tissue (WAT)
inflammation by treatment of C/EBP.alpha.-saRNA of the present
invention. Also provided is a method of reducing de novo
lipogenesis by treatment of C/EBP.alpha.-saRNA of the present
invention. Also provided is a method of increasing beta-oxidation
in the liver by treatment of C/EBP.alpha.-saRNA of the present
invention. Also provided is a method of increasing brown adipose
tissue (BAT) in the liver by treatment of C/EBP.alpha.-saRNA of the
present invention. Also provided is a method of reducing hepatic
lipid uptake by treatment of C/EBP.alpha.-saRNA of the present
invention. Also provided is a method of decreasing lipogenesis in
WAT by treatment of C/EBP.alpha.-saRNA of the present invention.
Also provided is a method of decreasing lipid storage in liver by
treatment of C/EBP.alpha.-saRNA of the present invention. Also
provided is a method of reducing lipid overload in the liver by
treatment of C/EBP.alpha.-saRNA of the present invention.
[0278] In another embodiment, C/EBP.alpha.-saRNA of the present
invention is used to increase liver function. In one non-limiting
example, C/EBP.alpha.-saRNA increases albumin gene expression and
thereby increasing serum albumin and unconjugated bilirubin levels.
The expression of albumin gene may be increased by at least 20, 30,
40%, more preferably at least 45, 50, 55, 60, 65, 70, 75%, even
more preferably at least 80% in the presence of the saRNA of the
present invention compared to the expression of albumin gene in the
absence of the saRNA of the present invention. In a further
preferable embodiment, the expression of albumin gene is increased
by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably
by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, even more
preferably by a factor of at least 60, 70, 80, 90, 100, in the
presence of the saRNA of the present invention compared to the
expression of albumin gene in the absence of the saRNA of the
present invention. In another non-limiting example,
C/EBP.alpha.-saRNA decreases the amount of alanine transaminase
(ALT), aspartate aminotransferase (AST), gamma glutamyl
transpeptidase (GGT), alphafectoprotein (AFP) and hepatocyte growth
factor (HGF). The amount of ALT, AST, GGT, AFP, or HGF may be
decreased by at least 20, 30, 40%, more preferably at least 45, 50,
55, 60, 65, 70, 75%, even more preferably at least 80% in the
presence of the saRNA of the present invention compared to the
amount of any of ALT, AST, GGT, AFP, or HGF in the absence of the
saRNA of the present invention.
[0279] In another embodiment, C/EBP.alpha.-saRNA of the present
invention is administered to regulate the levels of other members
of the C/EBP family. C/EBP.alpha.-saRNA increases the expression of
C/EBP.beta., C/EBP.gamma., C/EBP.delta. and C/EBP.zeta. depending
on the dose of C/EBP.alpha.-saRNA. In yet another embodiment, the
ratio of C/EBP.alpha. or C/EBP.beta. protein isoforms in a cell is
regulated by contacting said cell with C/EBP.alpha.-saRNA of the
present invention. In one embodiment, the 42 KDa isoform of
C/EBP.alpha. is increased. In one embodiment, the 30 kDa isoform of
C/EBP.beta. is increased.
ecCEBPA
[0280] Extra coding CEBPA (ecCEBPA), a functional ncRNA transcribed
from the CEBPA locus, regulates CEBPA methylation by interacting
with DNA methyltransferase (DNMT1) thus preventing CEBPA gene
methylation. It has been found that ecCEBPA knockdown led to a
decrease of CEBPA mRNA expression and to a significant increase in
DNA methylation (Ruscio et al., Nature, vol. 503:371-376 (2013),
the contents of which are incorporated herein by reference in their
entirety). In another embodiment, C/EBP.alpha.-saRNA of the present
invention is used to upregulate ecCEBPA levels.
Surgical Care
[0281] Hepatectomy, surgical resection of the liver or hepatic
tissue might cause liver failure, reduced production of albumin and
coagulation factors. Proper surgical care after hepatectomy is
needed. In some embodiments, C/EBP.alpha.-saRNA of the present
invention is used for surgical care after hepatectomy to promote
liver regeneration and increase survival rate.
Hyperproliferation Disorders
[0282] In one embodiment of the invention, C/EBP.alpha.-saRNA of
the present invention is used to reduce cell proliferation of
hyperproliferative cells. Examples of hyperproliferative cells
include cancerous cells, e.g., carcinomas, sarcomas, lymphomas and
blastomas. Such cancerous cells may be benign or malignant.
Hyperproliferative cells may result from an autoimmune condition
such as rheumatoid arthritis, inflammatory bowel disease, or
psoriasis. Hyperproliferative cells may also result within patients
with an oversensitive immune system coming into contact with an
allergen. Such conditions involving an oversensitive immune system
include, but are not limited to, asthma, allergic rhinitis, eczema,
and allergic reactions, such as allergic anaphylaxis. In one
embodiment, tumor cell development and/or growth is inhibited. In a
preferred embodiment, solid tumor cell proliferation is inhibited.
In another preferred embodiment, metastasis of tumor cells is
prevented. In another preferred example, undifferentiated tumor
cell proliferation is inhibited.
[0283] Inhibition of cell proliferation or reducing proliferation
means that proliferation is reduced or stops altogether. Thus,
"reducing proliferation" is an embodiment of "inhibiting
proliferation". Proliferation of a cell is reduced by at least 20%,
30% or 40%, or preferably at least 45, 50, 55, 60, 65, 70 or 75%,
even more preferably at least 80, 90 or 95% in the presence of the
saRNA of the invention compared to the proliferation of said cell
prior to treatment with the saRNA of the invention, or compared to
the proliferation of an equivalent untreated cell. In embodiments
wherein cell proliferation is inhibited in hyperproliferative
cells, the "equivalent" cell is also a hyperproliferative cell. In
preferred embodiments, proliferation is reduced to a rate
comparable to the proliferative rate of the equivalent healthy
(non-hyperproliferative) cell. Alternatively viewed, a preferred
embodiment of "inhibiting cell proliferation" is the inhibition of
hyperproliferation or modulating cell proliferation to reach a
normal, healthy level of proliferation.
[0284] In one non-limiting example, C/EBP.alpha.-saRNA is used to
reduce the proliferation of leukemia and lymphoma cells.
Preferably, the cells include Jurkat cells (acute T cell lymphoma
cell line), K562 cells (erythroleukemia cell line), U373 cells
(glioblastoma cell line), and 32Dp210 cells (myeloid leukemia cell
line).
[0285] In another non-limiting example, C/EBP.alpha.-saRNA is used
to reduce the proliferation of ovarian cancer cells, liver cancer
cells, pancreatic cancer cells, breast cancer cells, prostate
cancer cells, rat liver cancer cells, and insulinoma cells.
Preferably, the cells include PEO1 and PEO4 (ovarian cancer cell
line), HepG2 (hepatocellular carcinoma cell line), Panc1 (human
pancreatic carcinoma cell line), MCF7 (human breast adenocarcinoma
cell line), DU145 (human metastatic prostate cancer cell line), rat
liver cancer cells, and MING (rat insulinoma cell line).
[0286] In another non-limiting example, C/EBP.alpha.-saRNA is used
in combination with a siRNA targeting C/EBP.beta. gene to reduce
tumor cell proliferation. Tumor cell may include hepatocellular
carcinoma cells such as HepG2 cells and breast cancer cells such as
MCF7 cells.
[0287] In one embodiment, the saRNA of the present invention is
used to treat hyperproliferative disorders. Tumors and cancers
represent a hyperproliferative disorder of particular interest, and
all types of tumors and cancers, e.g. solid tumors and
haematological cancers are included. Examples of cancer include,
but not limited to, cervical cancer, uterine cancer, ovarian
cancer, kidney cancer, gallbladder cancer, liver cancer, head and
neck cancer, squamous cell carcinoma, gastrointestinal cancer,
breast cancer, prostate cancer, testicular cancer, lung cancer,
non-small cell lung cancer, non-Hodgkin's lymphoma, multiple
myeloma, leukemia (such as acute lymphocytic leukemia, chronic
lymphocytic leukemia, acute myelogenous leukemia, and chronic
myelogenous leukemia), brain cancer (e.g. astrocytoma,
glioblastoma, medulloblastoma), neuroblastoma, sarcomas, colon
cancer, rectum cancer, stomach cancer, anal cancer, bladder cancer,
endometrial cancer, plasmacytoma, lymphomas, retinoblastoma, Wilm's
tumor, Ewing sarcoma, melanoma and other skin cancers. The liver
cancer may include, but not limited to, cholangiocarcinoma,
hepatoblastoma, haemangiosarcoma, or hepatocellular carcinoma
(HCC). HCC is of particular interest.
[0288] Primary liver cancer is the fifth most frequent cancer
worldwide and the third most common cause of cancer-related
mortality. HCC represents the vast majority of primary liver
cancers [El-Serag et al., Gastroenterology, vol. 132(7), 2557-2576
(2007), the contents of which are disclosed herein in their
entirety]. HCC is influenced by the interaction of several factors
involving cancer cell biology, immune system, and different
aetiologies (viral, toxic and generic). The majority of patients
with HCC develop malignant tumors from a background of liver
cirrhosis. Currently most patients are diagnosed at an advanced
stage and therefore the 5 year survival for the majority of HCC
patients remains dismal. Surgical resection, loco-regional ablation
and liver transplantation are currently the only therapeutic
options which have the potential to cure HCC. However, based on the
evaluation of individual liver function and tumor burden only about
5-15% of patients are eligible for surgical intervention. The
binding sites for the family of C/EBP transcription factors are
present in the promoter regions of numerous genes that are involved
in the maintenance of normal hepatocyte function and response to
injury (including albumin, interleukin 6 response, energy
homeostasis, ornithine cycle regulation and serum amyloid A
expression). The present invention utilizes C/EBP.alpha.-saRNA to
modulate the expression of C/EBP.alpha. gene and treat liver
cirrhosis and HCC.
[0289] The method of the present invention may reduce tumor volume
by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%. Preferably, the
development of one or more new tumors is inhibited, e.g. a subject
treated according to the invention develops fewer and/or smaller
tumors. Fewer tumors means that he develops a smaller number of
tumors than an equivalent subject over a set period of time. For
example, he develops at least 1, 2, 3, 4 or 5 fewer tumors than an
equivalent control (untreated) subject. Smaller tumor means that
the tumors are at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%
smaller in weight and/or volume than tumors of an equivalent
subject. The method of the present invention reduces tumor burden
by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%.
[0290] The set period of time may be any suitable period, e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 months or years.
[0291] In one non-limiting example, provided is a method of
treating an undifferentiated tumor, comprising contacting a cell,
tissue, organ or subject with C/EBP.alpha.-saRNA of the present
invention. Undifferentiated tumors generally have a poorer
prognosis compared to differentiated ones. As the degree of
differentiation in tumors has a bearing on prognosis, it is
hypothesized that the use of a differentiating biological agent
could be a beneficial anti-proliferative drug. C/EBP.alpha. is
known to restore myeloid differentiation and prevent
hyperproliferation of hematopoietic cells in acute myeloid
leukemia. Preferably, undifferentiated tumors that may be treated
with C/EBP.alpha.-saRNA include undifferentiated small cell lung
carcinomas, undifferentiated pancreatic adenocarcinomas,
undifferentiated human pancreatic carcinoma, undifferentiated human
metastatic prostate cancer, and undifferentiated human breast
cancer.
[0292] In one non-limiting example, C/EBP.alpha.-saRNA is complexed
into PAMAM dendrimer, referred to as C/EBP.alpha.-saRNA-dendrimer
for targeted in vivo delivery. The therapeutic effect of
intravenously injected C/EBP.alpha.-saRNA-dendrimers is
demonstrated in a clinically relevant rat liver tumor model as
shown in Example 1. After three doses through tail vein injection
at 48 hour intervals, the treated cirrhotic rats showed
significantly increased serum albumin levels within one week. The
liver tumor burden was significantly decreased in the
C/EBP.alpha.-saRNA dendrimer treated groups. This study
demonstrates, for the first time, that gene targeting by small
activating RNA molecules can be used by systemic intravenous
administration to simultaneously ameliorate liver function and
reduce tumor burden in cirrhotic rats with HCC.
[0293] In one embodiment, C/EBP.alpha.-saRNA is used to regulate
oncogenes and tumor suppressor genes. Preferably, the expression of
the oncogenes may be down-regulated. The expression of the
oncogenes reduces by at least 20, 30, 40%, more preferably at least
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% in the presence of
C/EBP.alpha.-saRNA of the invention compared to the expression in
the absence of C/EBP.alpha.-saRNA of the invention. In a further
preferable embodiment, the expression of the oncogenes is reduced
by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably
by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, even more
preferably by a factor of at least 60, 70, 80, 90, 100, in the
presence of C/EBP.alpha.-saRNA of the invention compared to the
expression in the absence of C/EBP.alpha.-saRNA of the invention.
Preferably, the expressions of tumor suppressor genes may be
inhibited. The expression of the tumor suppressor genes increase by
at least 20, 30, 40%, more preferably at least 45, 50, 55, 60, 65,
70, 75, 80, 85,90, 95%, even more preferably at least 100% in the
presence of C/EBP.alpha.-saRNA of the invention compared to the
expression in the absence of C/EBP.alpha.-saRNA of the invention.
In a further preferable embodiment, the expression of tumor
suppressor genes is increased by a factor of at least 2, 3, 4, 5,
6, 7, 8, 9, 10, more preferably by a factor of at least 15, 20, 25,
30, 35, 40, 45, 50, even more preferably by a factor of at least
60, 70, 80, 90, 100 in the presence of C/EBP.alpha.-saRNA of the
invention compared to the expression in the absence of
C/EBP.alpha.-saRNA of the invention. Non-limiting examples of
oncogenes and tumor suppressor genes include Bcl-2-associated X
protein (BAX), BH3 interacting domain death agonist (BID), caspase
8 (CASP8), disabled homolog 2-interacting protein (DAB21P), deleted
in liver cancer 1 (DLC1), Fas surface death receptor (FAS), fragile
histidine triad (FHIT), growth arrest and DNA-damage-inducible-beta
(GADD45B), hedgehog interacting protein (HHIP), insulin-like growth
factor 2 (IGF2), lymphoid enhancer-binding factor 1 (LEF1),
phosphatase and tensin homolog (PTEN), protein tyrosine kinase 2
(PTK2), retinoblastoma 1 (RB1), runt-related transcription factor 3
(RUNX3), SMAD family member 4 (SMAD4), suppressor of cytokine
signaling (3SOCS3), transforming growth factor, beta receptor II
(TGFBR2), tumor necrosis factor (ligand) superfamily, member 10
(TNF SF10), P53, disintegrin and metalloproteinase
domain-containing protein 17 (ADAM17), v-akt murine thymoma viral
oncogene homolog 1 (AKT1), angiopoietin 2 (ANGPT2), B-cell
CLL/lymphoma 2 (BCL2), BCL2-like 1 (BCL2L1), baculoviral IAP repeat
containing 2 (BIRC2), baculoviral IAP repeat containing 5 (BIRCS),
chemokine (C-C motif) ligand 5 (CCL5), cyclin D1 (CCND1), cyclin D2
(CCND2), cadherin 1 (CDH1), cadherin 13 (CDH13), cyclin-dependent
kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B
(CDKN1B), cyclin-dependent kinase inhibitor 2A (CDKN2A), CASP8 and
FADD-like apoptosis regulator (CFLAR), catenin (cadherin-associated
protein) beta 1 (CTNNB1), chemokine receptor 4 (CXCR4), E2F
transcription factor 1 (E2F1), epidermal growth factor (EGF),
epidermal growth factor receptor (EGFR), E1A binding protein p300
(EP300), Fas (TNFRSF6)-associated via death domain (FADD),
fms-related tyrosine kinase 1 (FLT1), frizzled family receptor 7
(FZD7), glutathione S-transferase pi 1 (GSTP1), hepatocyte growth
factor (HGF), Harvey rat sarcoma viral oncogene homolog (HRAS),
insulin-like growth factor binding protein 1 (IGFBP1), insulin-like
growth factor binding protein 3 (IGFBP3), insulin receptor
substrate 1 (IRS1), integrin beta 1 (ITGB1), kinase insert domain
receptor (KDR), myeloid cell leukemia sequence 1 (MCL1), met
proto-oncogene (MET), mutS homolog 2 (MSH2), mutS homolog 3 (MSH3),
metadherin (MTDH), v-myc avian myelocytomatosis viral oncogene
homolog (MYC), nuclear factor of kappa light polypeptide gene
enhancer in B-cells 1 (NFKB1), neuroblastoma RAS viral (v-ras)
oncogene homolog (NRAS), opioid binding protein/cell adhesion
molecule-like (OPCML), platelet-derived growth factor receptor,
alpha polypeptide (PDGFRA), peptidylprolyl cis/trans isomerase,
NIMA-interacting 1 (PIN1), prostaglandin-endoperoxide synthase 2
(PTGS2), PYD and CARD domain containing (PYCARD), ras-related C3
botulinum toxin substrate 1 (RAC1), Ras association (RalGDS/AF-6)
domain family member 1 (RASSF1), reelin (RELN), ras homolog family
member A (RHOA), secreted frizzled-related protein 2 (SFRP2), SMAD
family member 7 (SMAD7), suppressor of cytokine signaling 1
(SOCS1), signal transducer and activator of transcription 3
(STAT3), transcription factor 4 (TCF4), telomerase reverse
transcriptase (TERT), transforming growth factor alpha (TGFA),
transforming growth factor beta 1 (TGFB1), toll-like receptor 4
(TLR4), tumor necrosis factor receptor superfamily member 10b
(TNFRSF10B), vascular endothelial growth factor A (VEGFA), Wilms
tumor 1 (WT1), X-linked inhibitor of apoptosis (XIAP), and
Yes-associated protein 1 (YAP1).
[0294] In one embodiment, provided is a method of increasing white
blood cell count by administering C/EBP.alpha.-saRNA of the present
invention to a patient in need thereof. Also provided is a method
of treating leukopaenia for patients having sepsis or chronic
inflammation diseases (e.g., hepatitis and liver cirrhosis) and for
immunocompromised patients (e.g., patients undergoing chemotherapy)
by administering C/EBP.alpha.-saRNA of the present invention to
said patient. Also provided is a method of treating pre B cell and
B cell malignancies including leukaemia and lymphoma by
administering C/EBP.alpha.-saRNA of the present invention to a
patient in need thereof. Also provided is a method of mobilize
white blood cells, haematopoietic or mesenchymal stem cells by
administering C/EBP.alpha.-saRNA of the present invention to a
patient in need thereof. In one embodiment, the white blood cell
count in a patient treated with C/EBP.alpha.-saRNA is increased by
at least 50%, 75%, 100%, more preferably by at least a factor of
1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, more preferably by at least a
factor of 6, 7, 8, 9, 10 compared to no C/EBP.alpha.-saRNA
treatment.
[0295] In one embodiment, C/EBP.alpha.-saRNA is used to regulate
micro RNAs (miRNA or miR) in the treatment of hepatocellular
carcinoma. MicroRNAs are small non-coding RNAs that regulate gene
expression. They are implicated in important physiological
functions and they may be involved in every single step of
carcinogenesis. They typically have 21 nucleotides and regulate
gene expression at the post transcriptional level via blockage of
mRNA translation or induction of mRNA degradation by binding to the
3'-untranslated regions (3'-UTR) of said mRNA.
[0296] In tumors, regulation of miRNA expression affects tumor
development. In HCC, as in other cancers, miRNAs function either as
oncogenes or tumor suppressor genes influencing cell growth and
proliferation, cell metabolism and differentiation, apoptosis,
angiogenesis, metastasis and eventually prognosis. [Lin et al.,
Biochemical and Biophysical Research Communications, vol. 375,
315-320 (2008); Kutay et al., J. Cell. Biochem., vol. 99, 671-678
(2006); Meng et al., Gastroenterology, vol. 133(2), 647-658 (2007),
the contents of each of which are incorporated herein by reference
in their entirety] C/EBP.alpha.-saRNA of the present invention
modulates C/EBP.alpha. gene expression and/or function and also
regulates miRNA levels in HCC cells. Non-limiting examples of
miRNAs that may be regulated by C/EBP.alpha.-saRNA of the present
invention include hsa-let-7a-5p, hsa-miR-133b, hsa-miR-122-5p,
hsa-miR-335-5p, hsa-miR-196a-5p, hsa-miR-142-5p, hsa-miR-96-5p,
hsa-miR-184, hsa-miR-214-3p, hsa-miR-15a-5p, hsa-let-7b-5p,
hsa-miR-205-5p, hsa-miR-181a-5p, hsa-miR-140-5p, hsa-miR-146b-5p,
hsa-miR-34c-5p, hsa-miR-134, hsa-let-7g-5p, hsa-let-7c,
hsa-miR-218-5p, hsa-miR-206, hsa-miR-124-3p, hsa-miR-100-5p,
hsa-miR-10b-5p, hsa-miR-155-5p, hsa-miR-1, hsa-miR-150-5p,
hsa-let-7i-5p, hsa-miR-27b-3p, hsa-miR-127-5p, hsa-miR-191-5p,
hsa-let-7f-5p, hsa-miR-10a-5p, hsa-miR-15b-5p, hsa-miR-16-5p,
hsa-miR-34a-5p, hsa-miR-144-3p, hsa-miR-128, hsa-miR-215,
hsa-miR-193a-5p, hsa-miR-23b-3p, hsa-miR-203a, hsa-miR-30c-5p,
hsa-let-7e-5p, hsa-miR-146a-5p, hsa-let-7d-5p, hsa-miR-9-5p,
hsa-miR-181b-5p, hsa-miR-181c-5p, hsa-miR-20b-5p, hsa-miR-125a-5p,
hsa-miR-148b-3p, hsa-miR-92a-3p, hsa-miR-378a-3p, hsa-miR-130a-3p,
hsa-miR-20a-5p, hsa-miR-132-3p, hsa-miR-193b-3p, hsa-miR-183-5p,
hsa-miR-148a-3p, hsa-miR-138-5p, hsa-miR-3'73-3p, hsa-miR-29b-3p,
hsa-miR-135b-5p, hsa-miR-21-5p, hsa-miR-181d, hsa-miR-301a-3p,
hsa-miR-200c-3p, hsa-miR-7-5p, hsa-miR-29a-3p, hsa-miR-210,
hsa-miR-17-5p, hsa-miR-98-5p, hsa-miR-25-3p, hsa-miR-143-3p,
hsa-miR-19a-3p, hsa-miR-18a-5p, hsa-miR-125b-5p, hsa-miR-126-3p,
hsa-miR-27a-3p, hsa-miR-372, hsa-miR-149-5p, and hsa-miR-32-5p.
[0297] In one non-limiting example, the miRNAs are oncogenic miRNAs
and are downregulated by a factor of at least 0.01, 0.02, 0.05,
0.1, 0.2, 0.3, 0.5, 1, 1.5, 2, 2.5, and 3, in the presence of
C/EBP.alpha.-saRNA of the invention compared to in the absence of
C/EBP.alpha.-saRNA. In another non-limiting example, the miRNAs are
tumor suppressing miRNAs and are upregulated by a factor of at
least 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.5, 1, more preferably by a
factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably by a
factor of at least 15, 20, 25, 30, 35, 40, 45, 50, even more
preferably by a factor of at least 60, 70, 80, 90, 100, in the
presence of C/EBP.alpha.-saRNA of the invention compared to in the
absence of C/EBP.alpha.-saRNA.
Stem Cell Regulation
[0298] In some embodiments of the present invention,
C/EBP.alpha.-saRNA is used to regulate self-renewal pluripotency
factors and affect stem cell differentiation. Altering the
phenotype of cells in order to express a protein of interest or to
change a cell to a different cell phenotype has been used in
different clinical, therapeutic and research settings. Altering a
phenotype of a cell is currently accomplished by expressing protein
from DNA or viral vectors. Currently there are studies being done
to evaluate the use of human embryonic stem cells as a treatment
option for various diseases such as Parkinson's disease and
diabetes and injuries such as a spinal cord injury. Embryonic stem
cells have the ability to grow indefinitely while maintaining
Pluripotency to generate any differentiated cell type.
[0299] Many factors such as pluripotency factors, cell phenotype
altering factors, transdifferentiation factors, differentiation
factors and dedifferentiation factors, are utilized to alter cell
phenotype, which is useful in the field of personal regenerative
medicine, cell therapy and therapies for other diseases. For
example, the self-renewal and pluripotency properties of stem cells
are regulated by an array of genes, such as transcription factors
and chromatin remodeling enzymes, in a core regulatory circuitry
including OCT4, SOX2, NANOG, and KLF genes [Bourillot et al., BMC
Biology, 8:125 (2010), the contents of which are incorporated
herein by reference in their entirety]. This regulatory circuitry
for self-regulatory networks also affects downstream genes.
Oligonucleotides have been utilized to regulate the core regulatory
circuitry. Xu et al. disclosed that miRNA-145 targets the 3'-UTR of
OCT4, SOX2, and KLF4. Reducing miRNA-145 impairs differentiation
and elevates OCT4, SOX2, and KLF4. [Xu et al., Cell, vol. 137, 1-12
(2009), the contents of which are incorporated herein by reference
in their entirety]
[0300] In one embodiment, C/EBP.alpha.-saRNA of the present
invention is used to regulate self-renewal pluripotency genes.
Non-limiting examples of pluripotency genes include SOX2, OCT4,
cKit, KLF4, KLF2, KLF5, NANOG, CDX2, and SALL4. In one embodiment,
the expression of the pluripotency gene is reduced by at least 20%,
30% or 40%, or preferably at least 45, 50, 55, 60, 65, 70 or 75%,
even more preferably at least 80, 90 or 95%, in the presence of
C/EBP.alpha.-saRNA of the invention compared to in the absence of
C/EBP.alpha.-saRNA. In another embodiment, the expression of the
pluripotency gene is increased by at least 20, 30, 40%, more
preferably at least 45, 50, 55, 60, 65, 70, 75%, even more
preferably at least 80%, in the presence of C/EBP.alpha.-saRNA of
the invention compared to in the absence of C/EBP.alpha.-saRNA. In
a preferable embodiment, the expression of the pluripotency gene is
increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, more
preferably by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50,
even more preferably by a factor of at least 60, 70, 80, 90, 100,
in the presence of C/EBP.alpha.-saRNA of the invention compared to
the expression in the absence of C/EBP.alpha.-saRNA.
[0301] In one embodiment, C/EBP.alpha.-saRNA is used to regulate
epithelial-mesenchymal transition (EMT) of a cell. Some tumors
contain cancer stem cells or cancer stem-like cells that can
self-renew and maintain tumor-initiating capacity through
differentiation into a different lineage of cancer cells. It has
been demonstrated that EMT is associated with cancer stem-like
cells, tumor aggressiveness and metastasis, and tumor recurrence.
[Kong et al., Cancers, vol. 3(1), 716-729 (2011)] There are many
factors that regulate EMT, including miRNAs such as miR-200 and
miR-134, growth factors such as fibroblast growth factor (FGF),
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), as well as factors such as Notch-1 and Wnt signaling
pathway. In one non-limiting example, C/EBP.alpha.-saRNA regulates
EMT by modulating the expression of miR-134. In another
non-limiting example, C/EBP.alpha.-saRNA regulates EMT by
modulating the expression of RUNX3, CTNB1, HGF, SMAD7 or TGFB1
genes.
III. Kits and Devices
Kits
[0302] The invention provides a variety of kits for conveniently
and/or effectively carrying out methods of the present invention.
Typically kits will comprise sufficient amounts and/or numbers of
components to allow a user to perform multiple treatments of a
subject(s) and/or to perform multiple experiments.
[0303] In one embodiment, the kits comprising saRNA described
herein may be used with proliferating cells to show efficacy.
[0304] In one embodiment, the present invention provides kits for
regulate the expression of genes in vitro or in vivo, comprising
C/EBP.alpha.-saRNA of the present invention or a combination of
C/EBP.alpha.-saRNA, saRNA modulating other genes, siRNAs, or
miRNAs. The kit may further comprise packaging and instructions
and/or a delivery agent to form a formulation composition. The
delivery agent may comprise a saline, a buffered solution, a
lipidoid, a dendrimer or any delivery agent disclosed herein.
Non-limiting examples of genes include C/EBP.alpha., other members
of C/EBP family, albumin gene, alphafectoprotein gene, liver
specific factor genes, growth factors, nuclear factor genes, tumor
suppressing genes, pluripotency factor genes.
[0305] In one non-limiting example, the buffer solution may include
sodium chloride, calcium chloride, phosphate and/or EDTA. In
another non-limiting example, the buffer solution may include, but
is not limited to, saline, saline with 2 mM calcium, 5% sucrose, 5%
sucrose with 2 mM calcium, 5% Mannitol, 5% Mannitol with 2 mM
calcium, Ringer's lactate, sodium chloride, sodium chloride with 2
mM calcium and mannose (See U.S. Pub. No. 20120258046; herein
incorporated by reference in its entirety). In yet another
non-limiting example, the buffer solutions may be precipitated or
it may be lyophilized. The amount of each component may be varied
to enable consistent, reproducible higher concentration saline or
simple buffer formulations. The components may also be varied in
order to increase the stability of saRNA in the buffer solution
over a period of time and/or under a variety of conditions.
[0306] In another embodiment, the present invention provides kits
to regulate the proliferation of cells, comprising
C/EBP.alpha.-saRNA of the present invention, provided in an amount
effective to inhibit the proliferation of cells when introduced
into said cells; optionally siRNAs and miRNAs to further regulate
the proliferation of target cells; and packaging and instructions
and/or a delivery agent to form a formulation composition.
[0307] In another embodiment, the present invention provides kits
for reducing LDL levels in cells, comprising saRNA molecules of the
present invention; optionally LDL reducing drugs; and packaging and
instructions and/or a delivery agent to form a formulation
composition.
[0308] In another embodiment, the present invention provides kits
for regulating miRNA expression levels in cells, comprising
C/EBP.alpha.-saRNA of the present invention; optionally siRNAs,
eRNAs and lncRNAs; and packaging and instructions and/or a delivery
agent to form a formulation composition.
Devices
[0309] The present invention provides for devices which may
incorporate C/EBP.alpha.-saRNA of the present invention. These
devices contain in a stable formulation available to be immediately
delivered to a subject in need thereof, such as a human patient.
Non-limiting examples of such a subject include a subject with
hyperproliferative disorders such as cancer, tumor, or liver
cirrhosis; and metabolics disorders such as NAFLD, obesity, high
LDL cholesterol, or type II diabetes.
[0310] Non-limiting examples of the devices include a pump, a
catheter, a needle, a transdermal patch, a pressurized olfactory
delivery device, iontophoresis devices, multi-layered microfluidic
devices. The devices may be employed to deliver C/EBP.alpha.-saRNA
of the present invention according to single, multi- or
split-dosing regiments. The devices may be employed to deliver
C/EBP.alpha.-saRNA of the present invention across biological
tissue, intradermal, subcutaneously, or intramuscularly. More
examples of devices suitable for delivering oligonucleotides are
disclosed in International Publication WO 2013/090648 filed Dec.
14, 2012, the contents of which are incorporated herein by
reference in their entirety.
Definitions
[0311] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0312] About: As used herein, the term "about" means+/-10% of the
recited value.
[0313] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents, e.g., saRNA, are administered to a subject
at the same time or within an interval such that there may be an
overlap of an effect of each agent on the patient. In some
embodiments, they are administered within about 60, 30, 15, 10, 5,
or 1 minute of one another. In some embodiments, the
administrations of the agents are spaced sufficiently close
together such that a combinatorial (e.g., a synergistic) effect is
achieved.
[0314] Amino acid: As used herein, the terms "amino acid" and
"amino acids" refer to all naturally occurring L-alpha-amino acids.
The amino acids are identified by either the one-letter or
three-letter designations as follows: aspartic acid (Asp:D),
isoleucine threonine (Thr:T), leucine (Leu:L), serine (Ser:S),
tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F),
proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine
(Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C),
tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine
(Met:M), asparagines (Asn:N), where the amino acid is listed first
followed parenthetically by the three and one letter codes,
respectively.
[0315] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans at any stage of development. In some embodiments,
"animal" refers to non-human animals at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[0316] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0317] Associated with: As used herein, the terms "associated
with," "conjugated," "linked," "attached," and "tethered," when
used with respect to two or more moieties, means that the moieties
are physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. An
"association" need not be strictly through direct covalent chemical
bonding. It may also suggest ionic or hydrogen bonding or a
hybridization based connectivity sufficiently stable such that the
"associated" entities remain physically associated.
[0318] Bifunctional: As used herein, the term "bifunctional" refers
to any substance, molecule or moiety which is capable of or
maintains at least two functions. The functions may affect the same
outcome or a different outcome. The structure that produces the
function may be the same or different.
[0319] Biocompatible: As used herein, the term "biocompatible"
means compatible with living cells, tissues, organs or systems
posing little to no risk of injury, toxicity or rejection by the
immune system.
[0320] Biodegradable: As used herein, the term "biodegradable"
means capable of being broken down into innocuous products by the
action of living things.
[0321] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
that has activity in a biological system and/or organism. For
instance, a substance that, when administered to an organism, has a
biological effect on that organism, is considered to be
biologically active. In particular embodiments, the saRNA of the
present invention may be considered biologically active if even a
portion of the saRNA is biologically active or mimics an activity
considered biologically relevant.
[0322] Cancer: As used herein, the term "cancer" in an individual
refers to the presence of cells possessing characteristics typical
of cancer-causing cells, such as uncontrolled proliferation,
immortality, metastatic potential, rapid growth and proliferation
rate, and certain characteristic morphological features. Often,
cancer cells will be in the form of a tumor, but such cells may
exist alone within an individual, or may circulate in the blood
stream as independent cells, such as leukemic cells.
[0323] Cell growth: As used herein, the term "cell growth" is
principally associated with growth in cell numbers, which occurs by
means of cell reproduction (i.e. proliferation) when the rate of
the latter is greater than the rate of cell death (e.g. by
apoptosis or necrosis), to produce an increase in the size of a
population of cells, although a small component of that growth may
in certain circumstances be due also to an increase in cell size or
cytoplasmic volume of individual cells. An agent that inhibits cell
growth can thus do so by either inhibiting proliferation or
stimulating cell death, or both, such that the equilibrium between
these two opposing processes is altered.
[0324] Cell type: As used herein, the term "cell type" refers to a
cell from a given source (e.g., a tissue, organ) or a cell in a
given state of differentiation, or a cell associated with a given
pathology or genetic makeup.
[0325] Chromosome: As used herein, the term "chromosome" refers to
an organized structure of DNA and protein found in cells.
[0326] Complementary: As used herein, the term "complementary" as
it relates to nucleic acids refers to hybridization or base pairing
between nucleotides or nucleic acids, such as, for example, between
the two strands of a double-stranded DNA molecule or between an
oligonucleotide probe and a target are complementary.
[0327] Condition: As used herein, the term "condition" refers to
the status of any cell, organ, organ system or organism. Conditions
may reflect a disease state or simply the physiologic presentation
or situation of an entity. Conditions may be characterized as
phenotypic conditions such as the macroscopic presentation of a
disease or genotypic conditions such as the underlying gene or
protein expression profiles associated with the condition.
Conditions may be benign or malignant.
[0328] Controlled Release: As used herein, the term "controlled
release" refers to a pharmaceutical composition or compound release
profile that conforms to a particular pattern of release to effect
a therapeutic outcome.
[0329] Cytostatic: As used herein, "cytostatic" refers to
inhibiting, reducing, suppressing the growth, division, or
multiplication of a cell (e.g., a mammalian cell (e.g., a human
cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a
combination thereof.
[0330] Cytotoxic: As used herein, "cytotoxic" refers to killing or
causing injurious, toxic, or deadly effect on a cell (e.g., a
mammalian cell (e.g., a human cell)), bacterium, virus, fungus,
protozoan, parasite, prion, or a combination thereof.
[0331] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound, substance, entity, moiety, cargo
or payload.
[0332] Delivery Agent: As used herein, "delivery agent" refers to
any substance which facilitates, at least in part, the in vivo
delivery of a saRNA of the present invention to targeted cells.
[0333] Destabilized: As used herein, the term "destable,"
"destabilize," or "destabilizing region" means a region or molecule
that is less stable than a starting, wild-type or native form of
the same region or molecule.
[0334] Detectable label: As used herein, "detectable label" refers
to one or more markers, signals, or moieties which are attached,
incorporated or associated with another entity that is readily
detected by methods known in the art including radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance and
the like. Detectable labels include radioisotopes, fluorophores,
chromophores, enzymes, dyes, metal ions, ligands such as biotin,
avidin, streptavidin and haptens, quantum dots, and the like.
Detectable labels may be located at any position in the peptides,
proteins or polynucleotides, e.g, saRNA, disclosed herein. They may
be within the amino acids, the peptides, proteins, or
polynucleotides located at the N- or C-termini or 5' or 3' termini
as the case may be.
[0335] Encapsulate: As used herein, the term "encapsulate" means to
enclose, surround or encase.
[0336] Engineered: As used herein, embodiments of the invention are
"engineered" when they are designed to have a feature or property,
whether structural or chemical, that varies from a starting point,
wild type or native molecule.
[0337] Equivalent subject: As used herein, "equivalent subject" may
be e.g. a subject of similar age, sex and health such as liver
health or cancer stage, or the same subject prior to treatment
according to the invention. The equivalent subject is "untreated"
in that he does not receive treatment with a saRNA according to the
invention. However, he may receive a conventional anti-cancer
treatment, provided that the subject who is treated with the saRNA
of the invention receives the same or equivalent conventional
anti-cancer treatment.
[0338] Exosome: As used herein, "exosome" is a vesicle secreted by
mammalian cells.
[0339] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an RNA into a polypeptide or protein; and (4)
post-translational modification of a polypeptide or protein.
[0340] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element.
[0341] Formulation: As used herein, a "formulation" includes at
least a saRNA of the present invention and a delivery agent.
[0342] Fragment: A "fragment," as used herein, refers to a portion.
For example, fragments of proteins may comprise polypeptides
obtained by digesting full-length protein isolated from cultured
cells.
[0343] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0344] Gene: As used herein, the term "gene" refers to a nucleic
acid sequence that comprises control and most often coding
sequences necessary for producing a polypeptide or precursor.
Genes, however, may not be translated and instead code for
regulatory or structural RNA molecules.
[0345] A gene may be derived in whole or in part from any source
known to the art, including a plant, a fungus, an animal, a
bacterial genome or episome, eukaryotic, nuclear or plasmid DNA,
cDNA, viral DNA, or chemically synthesized DNA. A gene may contain
one or more modifications in either the coding or the untranslated
regions that could affect the biological activity or the chemical
structure of the expression product, the rate of expression, or the
manner of expression control. Such modifications include, but are
not limited to, mutations, insertions, deletions, and substitutions
of one or more nucleotides. The gene may constitute an
uninterrupted coding sequence or it may include one or more
introns, bound by the appropriate splice junctions.
[0346] Gene expression: As used herein, the term "gene expression"
refers to the process by which a nucleic acid sequence undergoes
successful transcription and in most instances translation to
produce a protein or peptide. For clarity, when reference is made
to measurement of "gene expression", this should be understood to
mean that measurements may be of the nucleic acid product of
transcription, e.g., RNA or mRNA or of the amino acid product of
translation, e.g., polypeptides or peptides. Methods of measuring
the amount or levels of RNA, mRNA, polypeptides and peptides are
well known in the art.
[0347] Genome: The term "genome" is intended to include the entire
DNA complement of an organism, including the nuclear DNA component,
chromosomal or extrachromosomal DNA, as well as the cytoplasmic
domain (e.g., mitochondrial DNA).
[0348] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical
or similar. The term "homologous" necessarily refers to a
comparison between at least two sequences (polynucleotide or
polypeptide sequences). In accordance with the invention, two
polynucleotide sequences are considered to be homologous if the
polypeptides they encode are at least about 50%, 60%, 70%, 80%,
90%, 95%, or even 99% for at least one stretch of at least about 20
amino acids. In some embodiments, homologous polynucleotide
sequences are characterized by the ability to encode a stretch of
at least 4-5 uniquely specified amino acids. For polynucleotide
sequences less than 60 nucleotides in length, homology is
determined by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. In accordance with the invention,
two protein sequences are considered to be homologous if the
proteins are at least about 50%, 60%, 70%, 80%, or 90% identical
for at least one stretch of at least about 20 amino acids.
[0349] The term "hyperproliferative cell" may refer to any cell
that is proliferating at a rate that is abnormally high in
comparison to the proliferating rate of an equivalent healthy cell
(which may be referred to as a "control"). An "equivalent healthy"
cell is the normal, healthy counterpart of a cell. Thus, it is a
cell of the same type, e.g. from the same organ, which performs the
same functions(s) as the comparator cell. For example,
proliferation of a hyperproliferative hepatocyte should be assessed
by reference to a healthy hepatocyte, whereas proliferation of a
hyperproliferative prostate cell should be assessed by reference to
a healthy prostate cell.
[0350] By an "abnormally high" rate of proliferation, it is meant
that the rate of proliferation of the hyperproliferative cells is
increased by at least 20, 30, 40%, or at least 45, 50, 55, 60, 65,
70, 75%, or at least 80%, as compared to the proliferative rate of
equivalent, healthy (non-hyperproliferative) cells. The "abnormally
high" rate of proliferation may also refer to a rate that is
increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by
a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, or by a factor
of at least 60, 70, 80, 90, 100, compared to the proliferative rate
of equivalent, healthy cells.
[0351] The term "hyperproliferative cell" as used herein does not
refer to a cell which naturally proliferates at a higher rate as
compared to most cells, but is a healthy cell. Examples of cells
that are known to divide constantly throughout life are skin cells,
cells of the gastrointestinal tract, blood cells and bone marrow
cells. However, when such cells proliferate at a higher rate than
their healthy counterparts, then they are hyperproliferative.
[0352] Hyperproliferative disorder: As used herein, a
"hyperproliferative disorder" may be any disorder which involves
hyperproliferative cells as defined above. Examples of
hyperproliferative disorders include neoplastic disorders such as
cancer, psoriatic arthritis, rheumatoid arthritis, gastric
hyperproliferative disorders such as inflammatory bowel disease,
skin disorders including psoriasis, Reiter's syndrome, pityriasis
rubra pilaris, and hyperproliferative variants of the disorders of
keratinization.
[0353] The skilled person is fully aware of how to identify a
hyperproliferative cell. The presence of hyperproliferative cells
within an animal may be identifiable using scans such as X-rays, MM
or CT scans. The hyperproliferative cell may also be identified, or
the proliferation of cells may be assayed, through the culturing of
a sample in vitro using cell proliferation assays, such as MTT,
XTT, MTS or WST-1 assays. Cell proliferation in vitro can also be
determined using flow cytometry.
[0354] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g., between
oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two polynucleotide sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using methods such as those described
in Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleotide sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0355] Inhibit expression of a gene: As used herein, the phrase
"inhibit expression of a gene" means to cause a reduction in the
amount of an expression product of the gene. The expression product
can be an RNA transcribed from the gene (e.g., an mRNA) or a
polypeptide translated from an mRNA transcribed from the gene.
Typically a reduction in the level of an mRNA results in a
reduction in the level of a polypeptide translated therefrom. The
level of expression may be determined using standard techniques for
measuring mRNA or protein.
[0356] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[0357] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[0358] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been separated from at least some of
the components with which it was associated (whether in nature or
in an experimental setting). Isolated substances may have varying
levels of purity in reference to the substances from which they
have been associated. Isolated substances and/or entities may be
separated from at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or more of
the other components with which they were initially associated. In
some embodiments, isolated agents are more than about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, or more than about
99% pure. As used herein, a substance is "pure" if it is
substantially free of other components. Substantially isolated: By
"substantially isolated" is meant that the compound is
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in the compound of the present disclosure.
Substantial separation can include compositions containing at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
at least about 99% by weight of the compound of the present
disclosure, or salt thereof. Methods for isolating compounds and
their salts are routine in the art.
[0359] Label: The term "label" refers to a substance or a compound
which is incorporated into an object so that the substance,
compound or object may be detectable.
[0360] Linker: As used herein, a linker refers to a group of atoms,
e.g., 10-1,000 atoms, and can be comprised of the atoms or groups
such as, but not limited to, carbon, amino, alkylamino, oxygen,
sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be
attached to a modified nucleoside or nucleotide on the nucleobase
or sugar moiety at a first end, and to a payload, e.g., a
detectable or therapeutic agent, at a second end. The linker may be
of sufficient length as to not interfere with incorporation into a
nucleic acid sequence. The linker can be used for any useful
purpose, such as to form saRNA conjugates, as well as to administer
a payload, as described herein. Examples of chemical groups that
can be incorporated into the linker include, but are not limited
to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester,
alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can
be optionally substituted, as described herein. Examples of linkers
include, but are not limited to, unsaturated alkanes, polyethylene
glycols (e.g., ethylene or propylene glycol monomeric units, e.g.,
diethylene glycol, dipropylene glycol, triethylene glycol,
tripropylene glycol, tetraethylene glycol, or tetraethylene
glycol), and dextran polymers and derivatives thereof. Other
examples include, but are not limited to, cleavable moieties within
the linker, such as, for example, a disulfide bond (--S--S--) or an
azo bond (--N.dbd.N--), which can be cleaved using a reducing agent
or photolysis. Non-limiting examples of a selectively cleavable
bond include an amido bond can be cleaved for example by the use of
tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,
and/or photolysis, as well as an ester bond can be cleaved for
example by acidic or basic hydrolysis.
[0361] Metastasis: As used herein, the term "metastasis" means the
process by which cancer spreads from the place at which it first
arose as a primary tumor to distant locations in the body.
Metastasis also refers to cancers resulting from the spread of the
primary tumor. For example, someone with breast cancer may show
metastases in their lymph system, liver, bones or lungs.
[0362] Modified: As used herein "modified" refers to a changed
state or structure of a molecule of the invention. Molecules may be
modified in many ways including chemically, structurally, and
functionally. In one embodiment, the saRNA molecules of the present
invention are modified by the introduction of non-natural
nucleosides and/or nucleotides.
[0363] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[0364] Nucleic acid. The term "nucleic acid" as used herein, refers
to a molecule comprised of one or more nucleotides, i.e.,
ribonucleotides, deoxyribonucleotides, or both. The term includes
monomers and polymers of ribonucleotides and deoxyribonucleotides,
with the ribonucleotides and/or deoxyribonucleotides being bound
together, in the case of the polymers, via 5' to 3' linkages. The
ribonucleotide and deoxyribonucleotide polymers may be single or
double-stranded. However, linkages may include any of the linkages
known in the art including, for example, nucleic acids comprising
5' to 3' linkages. The nucleotides may be naturally occurring or
may be synthetically produced analogs that are capable of forming
base-pair relationships with naturally occurring base pairs.
Examples of non-naturally occurring bases that are capable of
forming base-pairing relationships include, but are not limited to,
aza and deaza pyrimidine analogs, aza and deaza purine analogs, and
other heterocyclic base analogs, wherein one or more of the carbon
and nitrogen atoms of the pyrimidine rings have been substituted by
heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the
like.
[0365] Patient: As used herein, "patient" refers to a subject who
may seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition.
[0366] Peptide: As used herein, "peptide" is less than or equal to
50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids long.
[0367] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0368] Pharmaceutically acceptable excipients: The phrase
"pharmaceutically acceptable excipient," as used herein, refers any
ingredient other than the compounds described herein (for example,
a vehicle capable of suspending or dissolving the active compound)
and having the properties of being substantially nontoxic and
non-inflammatory in a patient. Excipients may include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, and waters
of hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
and xylitol.
[0369] Pharmaceutically acceptable salts: The present disclosure
also includes pharmaceutically acceptable salts of the compounds
described herein. As used herein, "pharmaceutically acceptable
salts" refers to derivatives of the disclosed compounds wherein the
parent compound is modified by converting an existing acid or base
moiety to its salt form (e.g., by reacting the free base group with
a suitable organic acid). Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or organic acid
salts of basic residues such as amines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like.
Representative acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,
hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine, and the like. The pharmaceutically
acceptable salts of the present disclosure include the conventional
non-toxic salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. The pharmaceutically
acceptable salts of the present disclosure can be synthesized from
the parent compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are preferred. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 17.sup.th ed.,
Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical
Salts: Properties, Selection, and Use, P. H. Stahl and C. G.
Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of
Pharmaceutical Science, 66, 1-19 (1977), each of which is
incorporated herein by reference in its entirety.
[0370] Pharmaceutically acceptable solvate: The term
"pharmaceutically acceptable solvate," as used herein, means a
compound of the invention wherein molecules of a suitable solvent
are incorporated in the crystal lattice. A suitable solvent is
physiologically tolerable at the dosage administered. For example,
solvates may be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Examples of suitable solvents are
ethanol, water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the solvate is referred to as a "hydrate."
[0371] Pharmacologic effect: As used herein, a "pharmacologic
effect" is a measurable biologic phenomenon in an organism or
system which occurs after the organism or system has been contacted
with or exposed to an exogenous agent. Pharmacologic effects may
result in therapeutically effective outcomes such as the treatment,
improvement of one or more symptoms, diagnosis, prevention, and
delay of onset of disease, disorder, condition or infection.
Measurement of such biologic phenomena may be quantitative,
qualitative or relative to another biologic phenomenon.
Quantitative measurements may be statistically significant.
Qualitative measurements may be by degree or kind and may be at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
different. They may be observable as present or absent, better or
worse, greater or less. Exogenous agents, when referring to
pharmacologic effects are those agents which are, in whole or in
part, foreign to the organism or system. For example, modifications
to a wild type biomolecule, whether structural or chemical, would
produce an exogenous agent. Likewise, incorporation or combination
of a wild type molecule into or with a compound, molecule or
substance not found naturally in the organism or system would also
produce an exogenous agent. The saRNA of the present invention,
comprises exogenous agents. Examples of pharmacologic effects
include, but are not limited to, alteration in cell count such as
an increase or decrease in neutrophils, reticulocytes,
granulocytes, erythrocytes (red blood cells), megakaryocytes,
platelets, monocytes, connective tissue macrophages, epidermal
langerhans cells, osteoclasts, dendritic cells, microglial cells,
neutrophils, eosinophils, basophils, mast cells, helper T cells,
suppressor T cells, cytotoxic T cells, natural killer T cells, B
cells, natural killer cells, or reticulocytes. Pharmacologic
effects also include alterations in blood chemistry, pH,
hemoglobin, hematocrit, changes in levels of enzymes such as, but
not limited to, liver enzymes AST and ALT, changes in lipid
profiles, electrolytes, metabolic markers, hormones or other marker
or profile known to those of skill in the art.
[0372] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[0373] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of an infection, disease,
disorder and/or condition; partially or completely delaying onset
of one or more symptoms, features, or clinical manifestations of a
particular infection, disease, disorder, and/or condition;
partially or completely delaying onset of one or more symptoms,
features, or manifestations of a particular infection, disease,
disorder, and/or condition; partially or completely delaying
progression from an infection, a particular disease, disorder
and/or condition; and/or decreasing the risk of developing
pathology associated with the infection, the disease, disorder,
and/or condition.
[0374] Prodrug: The present disclosure also includes prodrugs of
the compounds described herein. As used herein, "prodrugs" refer to
any substance, molecule or entity which is in a form predicate for
that substance, molecule or entity to act as a therapeutic upon
chemical or physical alteration. Prodrugs may by covalently bonded
or sequestered in some way and which release or are converted into
the active drug moiety prior to, upon or after administered to a
mammalian subject. Prodrugs can be prepared by modifying functional
groups present in the compounds in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compounds. Prodrugs include compounds wherein
hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any
group that, when administered to a mammalian subject, cleaves to
form a free hydroxyl, amino, sulfhydryl, or carboxyl group
respectively. Preparation and use of prodrugs is discussed in T.
Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol.
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, ed. Edward B. Roche, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
incorporated by reference in their entirety.
[0375] Prognosing: As used herein, the term "prognosing" means a
statement or claim that a particular biologic event will, or is
very likely to, occur in the future.
[0376] Progression: As used herein, the term "progression" or
"cancer progression" means the advancement or worsening of or
toward a disease or condition.
[0377] Proliferate: As used herein, the term "proliferate" means to
grow, expand or increase or cause to grow, expand or increase
rapidly. "Proliferative" means having the ability to proliferate.
"Anti-proliferative" means having properties counter to or
inapposite to proliferative properties.
[0378] Protein: A "protein" means a polymer of amino acid residues
linked together by peptide bonds. The term, as used herein, refers
to proteins, polypeptides, and peptides of any size, structure, or
function. Typically, however, a protein will be at least 50 amino
acids long. In some instances the protein encoded is smaller than
about 50 amino acids. In this case, the polypeptide is termed a
peptide. If the protein is a short peptide, it will be at least
about 10 amino acid residues long. A protein may be naturally
occurring, recombinant, or synthetic, or any combination of these.
A protein may also comprise a fragment of a naturally occurring
protein or peptide. A protein may be a single molecule or may be a
multi-molecular complex. The term protein may also apply to amino
acid polymers in which one or more amino acid residues are an
artificial chemical analogue of a corresponding naturally occurring
amino acid.
[0379] Protein expression: The term "protein expression" refers to
the process by which a nucleic acid sequence undergoes translation
such that detectable levels of the amino acid sequence or protein
are expressed.
[0380] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection.
[0381] Regression: As used herein, the term "regression" or "degree
of regression" refers to the reversal, either phenotypically or
genotypically, of a cancer progression. Slowing or stopping cancer
progression may be considered regression.
[0382] Sample: As used herein, the term "sample" or "biological
sample" refers to a subset of its tissues, cells or component parts
(e.g. body fluids, including but not limited to blood, mucus,
lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,
amniotic fluid, amniotic cord blood, urine, vaginal fluid and
semen). A sample further may include a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, organs. A sample further refers to a medium, such as
a nutrient broth or gel, which may contain cellular components,
such as proteins or nucleic acid molecule.
[0383] Signal Sequences: As used herein, the phrase "signal
sequences" refers to a sequence which can direct the transport or
localization of a protein.
[0384] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administered in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[0385] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[0386] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0387] Stable: As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0388] Stabilized: As used herein, the term "stabilize",
"stabilized," "stabilized region" means to make or become
stable.
[0389] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition in accordance with
the invention may be administered, e.g., for experimental,
diagnostic, prophylactic, and/or therapeutic purposes. Typical
subjects include animals (e.g., mammals such as mice, rats,
rabbits, non-human primates, and humans) and/or plants.
[0390] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0391] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0392] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term means within 2 seconds.
[0393] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of a disease, disorder, and/or
condition.
[0394] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or may not exhibit symptoms of the disease, disorder, and/or
condition but harbors a propensity to develop a disease or its
symptoms. In some embodiments, an individual who is susceptible to
a disease, disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0395] Sustained release: As used herein, the term "sustained
release" refers to a pharmaceutical composition or compound release
profile that conforms to a release rate over a specific period of
time.
[0396] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or polypeptides or other molecules of the present
invention may be chemical or enzymatic.
[0397] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells may be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism may be an animal, preferably a mammal, more preferably a
human and most preferably a patient.
[0398] Therapeutic Agent: The term "therapeutic agent" refers to
any agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect.
[0399] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0400] Therapeutically effective outcome: As used herein, the term
"therapeutically effective outcome" means an outcome that is
sufficient in a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0401] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr period. It may be administered
as a single unit dose.
[0402] Transcription factor: As used herein, the term
"transcription factor" refers to a DNA-binding protein that
regulates transcription of DNA into RNA, for example, by activation
or repression of transcription. Some transcription factors effect
regulation of transcription alone, while others act in concert with
other proteins. Some transcription factor can both activate and
repress transcription under certain conditions. In general,
transcription factors bind a specific target sequence or sequences
highly similar to a specific consensus sequence in a regulatory
region of a target gene. Transcription factors may regulate
transcription of a target gene alone or in a complex with other
molecules.
[0403] Treating: As used herein, the term "treating" refers to
partially or completely alleviating, ameliorating, improving,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of a particular infection, disease, disorder, and/or
condition. For example, "treating" cancer may refer to inhibiting
survival, growth, and/or spread of a tumor. Treatment may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition and/or to a subject who exhibits only
early signs of a disease, disorder, and/or condition for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition.
[0404] The phrase "a method of treating" or its equivalent, when
applied to, for example, cancer refers to a procedure or course of
action that is designed to reduce, eliminate or prevent the number
of cancer cells in an individual, or to alleviate the symptoms of a
cancer. "A method of treating" cancer or another proliferative
disorder does not necessarily mean that the cancer cells or other
disorder will, in fact, be completely eliminated, that the number
of cells or disorder will, in fact, be reduced, or that the
symptoms of a cancer or other disorder will, in fact, be
alleviated. Often, a method of treating cancer will be performed
even with a low likelihood of success, but which, given the medical
history and estimated survival expectancy of an individual, is
nevertheless deemed an overall beneficial course of action.
[0405] Tumor growth: As used herein, the term "tumor growth" or
"tumor metastases growth", unless otherwise indicated, is used as
commonly used in oncology, where the term is principally associated
with an increased mass or volume of the tumor or tumor metastases,
primarily as a result of tumor cell growth.
[0406] Tumor Burden: As used herein, the term "tumor burden" refers
to the total Tumor Volume of all tumor nodules with a diameter in
excess of 3 mm carried by a subject.
[0407] Tumor Volume: As used herein, the term "tumor volume" refers
to the size of a tumor. The tumor volume in mm.sup.3 is calculated
by the formula: volume=(width).sup.2.times.length/2.
[0408] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in any way.
Unmodified may, but does not always, refer to the wild type or
native form of a biomolecule. Molecules may undergo a series of
modifications whereby each modified molecule may serve as the
"unmodified" starting molecule for a subsequent modification.
Equivalents and Scope
[0409] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0410] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0411] It is also noted that the term "comprising" is intended to
be open and permits the inclusion of additional elements or
steps.
[0412] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0413] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0414] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[0415] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
[0416] Materials and Procedures have been disclosed in PCT
Application No. PCT/M2014/003054.
Example 1. C/EBP.alpha.-saRNA In Vitro Studies
[0417] AW51 (aka CEBPA-AW1-510000) was transfected in a panel of
HCC cell lines such as Hep3B, HepG2, PLC/PRF/5, SNU475 cells. The
cells were reverse transfected with 50 nM AW51 at seeding, forward
transfected 24 hours later, and harvested at 72 hours. CEBPA mRNA
and albumin (ALB) mRNA levels were measured. Upregulation of CEBPA
and ALB mRNA were observed as shown in FIG. 4A-4D and FIG.
5A-5D.
TABLE-US-00010 AW51 GACCAGUGAC SEQ ID Antisense AAUGACCGCUU No. 93
sequence (X09317) AW51 GCGGUCAUUG SEQ ID Sense UCACUGGUCUU No. 94
sequence (X09316)
Example 2. Modified CEBPA-saRNA Upregulates CEBPA
[0418] Modified CEBPA-saRNAs in Table 3 were transfected in DU145
cells. The cells were reverse transfected with 2.5 nM and 10 nM
modified CEBPA-saRNA at seeding, forward transfected 24 hours
later, and harvested at 72 hours. CEBPA and GAPDH mRNA levels were
measured. Results in Table 6, FIG. 6A shows that CEPBA-saRNA could
tolerate heavy modifications.
TABLE-US-00011 TABLE 6-1 CEBPA mRNA levels in DU145 cells rel. mRNA
rel. mRNA CEBPA Duplex ID CEBPA 2.5 nM SD 10 nM SD XD-03287
2.47405519 0.35441301 4.807203057 1.77941471 XD-04353 2.240846151
0.56776333 2.948275905 0.55264149 XD-04354 2.847748677 0.7442053
3.130235184 0.54793146 XD-04355 2.946658233 0.66053501 2.892767048
0.37945239 XD-04356 1.864020365 0.49485233 2.109506219 0.55126016
XD-03302 3.271904091 0.84352676 9.550389237 1.59963498 XD-04358
1.35233741 0.28653345 1.384020564 0.13677222 XD-04359 1.399054988
0.26024787 1.486989819 0.07346068 XD-04360 1.211792463 0.06559519
1.721136011 0.54936887 XD-04361 1.221228236 0.05912314 1.802248329
0.58804132 XD-03317 3.170377201 0.54481336 7.878877604 2.12261544
XD-04363 1.041339997 0.06854357 1.025008603 0.10910861 XD-04364
0.810622945 0.15207354 0.917036666 0.07051729 XD-04365 0.892397193
0.11321896 0.960369198 0.11671288 XD-04366 1.235336205 0.24529118
1.031610064 0.10794732 scramble 1.50761893 0.49259555 0.977396047
0.25336725 F-Luc 1.679417472 0.53349959 1.177929847 0.22429314 MeF
1.519521651 0.23368363 1.418274737 0.26159698 Aha-1 1.400196465
0.28373253 1.44526965 0.20604682
TABLE-US-00012 TABLE 6-2 GAPDH mRNA levels in DU145 cells Duplex ID
gapdh 2.5 nM SD gapdh 10 nM SD XD-03287 0.30012213 0.04872709
0.15487859 0.021476125 XD-04353 0.38430764 0.05893013 0.21179771
0.023662437 XD-04354 0.278508 0.0151083 0.21589058 0.025144858
XD-04355 0.33891401 0.03684563 0.23096784 0.039633795 XD-04356
0.66440667 0.09665852 0.34165265 0.068757809 XD-03302 0.48401386
0.0938301 0.15074631 0.023836647 XD-04358 0.99270067 0.10679351
0.60215746 0.076308233 XD-04359 0.93338175 0.08896079 0.55308903
0.050195732 XD-04360 1.14035319 0.05727254 0.47614574 0.040400001
XD-04361 1.19595973 0.10526669 0.46229043 0.060483173 XD-03317
0.59089619 0.10261665 0.15368292 0.02885901 XD-04363 1.42967486
0.10493026 0.93230015 0.188124401 XD-04364 1.51612477 0.20993157
1.02248778 0.070857079 XD-04365 1.31858062 0.12189526 1.07045908
0.136617289 XD-04366 1.02824228 0.13557063 0.58889998 0.03921991
scramble 0.9596105 0.1991626 0.88815236 0.227402132 F-Luc
0.95681136 0.1749612 1.06219451 0.229513678 MeF 0.81949497
0.03521342 0.71848557 0.094709517 Aha-1 0.94853936 0.10607439
0.72142219 0.134524626
[0419] Following table includes the controls used in this example.
Aha1 siRNA was used as transfection control and was transfected at
concentrations of 2.5 nM and 10 nM.
TABLE-US-00013 TABLE 7-1 Controls-sense sequences Duplex- Sense-
Sense SEQ ID ID Sequence ID No. Notes XD-03291 X09206 ACUACUGAG 33
Scramble, UGACAGUA unmodified GAUU XD-03292 X09208 CuUACGcUG 78
Fluc, AGUACUUCG modified Asusu XD-00033 X00122 GGAuGAAGu 79 AHA1
GGAGAuuAG SiRNA, udTsdT transfection control XD-00376 X01162
GGAUfCfAU 80 MeF fCfUfCfAA design, GufCfUfUf neg. ACfdTsdT ctrl
TABLE-US-00014 TABLE 7-2 Controls-antisense sequences Anti- Anti-
SEQ Duplex- Sense- Sense ID ID ID Sequence No. Notes XD-03291
X09207 UCUACUGUC 81 Scramble. ACUCAGUAG unmodified UUU XD-03292
X09209 UcGAAGuAc 82 Fluc, UCAGcGUAA modified gsusu XD-00033 X00123
ACuAAUCUC 83 AHA1 CACUUcAUC siRNA, CdTsdT transfection control
XD-00376 X01163 GUfAAGACfU 84 MeF fUfGAGAUfG design, AufCfCfdTs
neg. dT ctrl
[0420] A CEBPA-saRNA is conjugated with GalNac clusters (referred
to as GalNac-CEBPA-saRNA) and is transfected in DU145 cells. The
cells are reverse transfected with 2.5 nM, 10 nM, or 50 nM
GalNac-CEBPA-saRNA at seeding, forward transfected 24 hours later,
and harvested at 72 hours. CEBPA and albumin mRNA levels are
measured.
[0421] AW51 (aka CEBPA-AW1-510000) is conjugated with GalNac
clusters and is transfected in DU145 cells. The cells are reverse
transfected with 2.5 nM, 10 nM, or 50 nM GalNac-CEBPA-saRNA at
seeding, forward transfected 24 hours later, and harvested at 72
hours. CEBPA and albumin mRNA levels are measured.
Example 3. In Vitro Dose Response and Potency Comparison of saRNA
and siRNA
[0422] EC50 in DU145 cells of three unmodified CEBPA-saRNA
(XD-03287, XD-03302, XD-03317) was compared with IC50 of siRNA to
AHA1 and CEBPA in DU145 cells.
[0423] For EC50 test of saRNA, DU145 cells (p15, 8000 cells/well)
were reversed transfected with CEBPA-saRNA at 0 hr (Lipofectamine
2000, 0.4 .mu.l/well), forward transfected at 24 hr, and harvested
at 72 hrs. XD-03287, XD-03302, XD-03317 were dosed in 2.times.
dilution series from 100 nM. CEBPA mRNA levels were normalized with
GAPDH.
[0424] For IC50 test of siRNA, DU145 cells (p15, 8000 cells/well)
received a single transfection at 0 hr with harvest at 24 hrs. Life
Technologies CEBPA-siRNA and Axo unmod AHA1 were dosed in 5.times.
dilution series from 50 nM.
TABLE-US-00015 TABLE 8 Sequences of the saRNA, siRNA and controls:
Duplex- Sense- Sense Antisense- Antisense ID ID Sequence ID
Sequence Notes XD- X09198 CGGUCAU X09199 UGACCAGU CEBPA- 3287
UGUCACU GACAAUG saRNA GGUCAUU ACCGUU (SEQ ID (SEQ ID NO. 50) NO.
51) XD- X09316 GCGGUCA X09317 GACCAGUG CEBPA- 3302 UUGUCAC ACAAUGA
saRNA UGGUCUU CCGCUU (SEQ ID (SEQ ID NO. 85) NO. 86) XD- X09346
UGAAAGG X09347 AGGAGGAU CEBPA- 3317 AUUCAUC GAAUCCU saRNA CUCCUUU
UUCAUU (SEQ ID (SEQ ID NO. 87) NO. 88) XD- X02807 GGAUGAA X028I2
ACUAAUCU Aha-1 1030 GUGGAGA CCACUUC siRNA, UUAGUdT AUCCdTdT unmodi-
sdT (SEQ ID fied (SEQ ID NO. 90) NO. 89) XD- X00539 CuuAcGcu X00540
UCGAAGuA Fluc, 194 GAGuAcuu CUcAGCGu negative cGAdTsdT AAGdTsdT
control (SEQ ID (SEQ ID NO. 91) NO. 92) s2890 siRNA targeting CEBPA
from life techno- logies
TABLE-US-00016 TABLE 9 Concentrations of saRNA and siRNA in nM:
siRNA 50 10 2 0.4 0.08 0.016 0.0032 0.00064 0.000128 0.0000256
saRNA 100 50 25 12.5 6.25 3.125 1.5625 0.78125 0.390625
0.1953125
[0425] The IC50 values of siRNA were shown in Table 10 and FIG.
7A-7C. The EC50 values of saRNA were shown in Table 11 and FIG.
8A-8C. Slope ratio for saRNA/siRNA of around 5, suggesting a
different mechanism. For the calculation of the slope averages and
ratios, the slopes were normalized for a Y-axis range of 100%. This
results in average slopes of about 0.5 for siRNAs (CEBPA-siRNA and
Aha-1-siRNA) and about 2.7 for saRNAs (XD-03287, XD-03302 and
XD-03317). EC50 and IC50 are the inflection points (IP), a measure
of half-maximal activity. Therefore, CEBPA-saRNAs are highly potent
having IC50s in low nM range.
TABLE-US-00017 TABLE 10 IC50 values of siRNA and control: s2890
XD-01030 siRNA Duplex ID (CEBPA siRNA) XD-00194 (FLuc) (Aha-1
siRNA) IC50 (nM) 0.049 NA 0.0079 IP (nM) 0.0018 NA 0.0030 slope
-0.43 NA -0.59
TABLE-US-00018 TABLE 11 EC50 values of saRNA saRNA Duplex ID
XD-03287 XD-03302 XD-03317 EC50 (nM) 2.50 5.83 4.66 IP (nM) 5.42
6.24 5.58 slope 1.91 1.82 2.19
Example 4. In Vitro Studies with CEBPA-saRNA in Human
Hepatocytes
[0426] Primary human hepatocytes (LifeTechnologies, HMCPTS) were
placed in non-proliferation media. On the day of seeding, the cells
were subjected to a reverse transfection step where the saRNA
transfection complex was added to the cells before they adhered as
a monolayer. After 24 hours, the medium was changed and a forward
transfection was performed. The next day, the medium was changed
and the cells were incubated for a further 24 hours prior to
harvesting of cells for analysis. The hepatocytes were transfected
with AW51. CEBPA and albumin mRNA levels were measured at 48 hr and
72 hr. Aha-1-siRNA and Fluc were used as controls. Aha1, albumin,
CEBPA relative expressions were shown in Table 12 and FIG. 9A.
[0427] Materials
[0428] Primary hepatocyte thawing medium: Cryopreserved Hepatocyte
Recovery Medium (CHRM), 50 mL (Life Technologies Cat. No.
CM7000)
[0429] Primary hepatocyte plating medium: Fetal bovine serum, heat
inactivated--50 mL (Life Technologies Cat. No. 16140-071)
[0430] Insulin-Transferrin-Selenium (100.times.)--5 mL (Life
Technologies Cat. No. 41400-045)
[0431] HEPES (1 M)--5 mL (Life Technologies Cat. No. 15630-056)
[0432] L-Glutamine-Penicillin-Streptomycin solution--5 mL (Sigma
Cat. No. G1146)
[0433] Dexamethasone--to 40 ng/mL final concentration (Sigma Cat.
No. D8893)
[0434] William's E Medium, no phenol red--to 500 mL total (Life
Technologies Cat. No. A12176-01)
[0435] Primary hepatocyte maintenance medium: Primary Hepatocyte
Maintenance Supplement (Life Technologies Cat. No. CM4000)
[0436] William's E Medium, no phenol red--to 500 mL total (Life
Technologies Cat. No. A12176-01)
[0437] Primary hepatocyte culture plates:Collagen I, Coated Plate,
24 well (Life Technologies Cat. No. A11428-02)
[0438] Transfection reagents:HiPerFect Transfection Reagent (Qiagen
Cat. No. 301704)
[0439] Opti-MEM I Reduced Serum Medium, no phenol red (Life
Technologies Cat. No. 11058-021)
Protocol
[0440] saRNA Annealing:
[0441] Each lyophilised saRNA strand was resuspended to 1 mM in
RNase-free 10 mM Tris-HCl, 20 mM NaCl2, 1 mM EDTA. They were mixed
well to complete resuspension. Equal volumes of sense and antisense
strands were mixed together by gentle vortexing. The tube with
combined strands was placed in a beaker of water heated to
95.degree. C. The beaker was covered and allowed to cool to room
temperature. Subsequent dilutions were performed using RNAse-free
water. Generally for 24 well format, stock solution was diluted to
10 .mu.M. Aliquot annealed saRNA was aliquoted and stored at
-20.degree. C.
[0442] Thawing and Plating of Primary Hepatocytes:
[0443] CHRM and plating medium were warmed to 37.degree. C. in a
water bath. Cryopreserved hepatocytes were thawed in a 37.degree.
C. water bath until no ice crystals remain. The vial was
disinfected with 70% ethanol. In a sterile tissue culture hood,
thawed hepatocytes were transferred directly into CHRM. Hepatocytes
were centrifuged at 100.times.g (900 rpm in a Thermo F-G1
fixed-angle rotor) for 10 minutes at room temperature. Supernatant
was carefully poured off into a waste bottle. Pellet was
resuspended in 1 mL of Plating Medium per 1.times.106 cryopreserved
cells. Cells were counted using a NucleoCounter NC-200 aggregated
cells assay to determine cell viability. 2.0.times.105 viable cells
were plated in 500 .mu.L Plating Medium per well in a 24 well
plate.
[0444] Reverse Transfection (Immediately after Seeding):
[0445] For each well to be transfected, 12 .mu.L of 10 .mu.M saRNA
was diluted in 85 .mu.L Opti-MEM. For each well to be transfected,
3 .mu.L HiPerFect was added and mixed well by vortexing. The
transfection was incubated for 15 minutes at room temperature. 100
.mu.L transfection complexes was added to each well for a final
saRNA concentration of 200 nM. The plate was incubated at
37.degree. C. with 5% CO2 in a humidified incubator. After 5 hours,
the medium was changed to 500 .mu.L pre-warmed Maintenance
Medium.
[0446] Forward Transfection (24 Hours after Seeding):
[0447] For each well to be transfected, 12 .mu.L of 10 .mu.M saRNA
was diluted in 85 .mu.L Opti-MEM. For each well to be transfected,
3 .mu.L HiPerFect was added and mixed well by vortexing. The
transfection was incubated for 15 minutes at room temperature.
During incubation, medium was changed to 500 .mu.L fresh pre-warmed
Maintenance Medium per well. 100 .mu.L transfection complexes was
added to each well for a final saRNA concentration of 200 nM. The
plate was returned to incubator. After 24 hours, medium was changed
to 500 .mu.L of fresh pre-warmed Maintenance Medium. Peak gene
activation occurred 72 hours after cell seeding. Cells and/or
supernatant were collected for downstream analysis at this
time.
TABLE-US-00019 TABLE 12-1 Relative expression of Aha1, albumin and
CEBPA genes at 48 hr after aha1-siRNA, Fluc and AW51 transfection
Aha1 exprission Albumin expression CEBPA expression Untreated 1 1 1
Aha1 0.1 0.9 1.4 Flu 1.5 1.0 1.6 AW51 1.5 1.1 1.5
TABLE-US-00020 TABLE 12-2 Relative expression of Aha1, albumin and
CEBPA genes at 72 hr after aha1-siRNA, Fluc and AW51 transfection
Aha1 exprission Albumin expression CEBPA expression Untreated 1 1 1
Aha1 0.1 0.6 1.0 Flu 0.8 0.7 1.4 AW51 0.9 0.65 1.6
saRNA Transfection Protocol in Proliferating Primary Human
Hepatocytes
[0448] Primary human hepatocytes (LifeTechnologies, HMCPTS) were
placed in proliferation media. On the day of seeding, the cells are
subjected to a reverse transfection step where the saRNA
transfection complex was added to the cells before they adhere as a
monolayer. After 24 hours, the medium was changed and a forward
transfection is performed. The next day, the medium was changed and
the cells were incubated for a further 24 hours prior to harvesting
of cells for analysis. The hepatocytes were transfected with AW51.
CEBPA and albumin mRNA levels were measured at 48 hr and 72 hr.
Aha-1-siRNA and Fluc were used as controls. Aha1, albumin, CEBPA
relative expressions were shown in Table 13 and FIG. 9B.
Materials
[0449] Primary hepatocyte thawing medium:
[0450] Cryopreserved Hepatocyte Recovery Medium (CHRM), 50 mL (Life
Technologies Cat. No. CM7000)
[0451] Primary hepatocyte plating medium:
[0452] Fetal bovine serum, heat inactivated--50 mL (Life
Technologies Cat. No. 16140-071)
[0453] Insulin-Transferrin-Selenium (100.times.)--5 mL (Life
Technologies Cat. No. 41400-045)
[0454] HEPES (1 M) -5 mL (Life Technologies Cat. No. 15630-056)
[0455] L-Glutamine-Penicillin-Streptomycin solution--5 mL (Sigma
Cat. No. G1146)
[0456] Dexamethasone--to 40 ng/mL final concentration (Sigma Cat.
No. D8893)
[0457] William's E Medium, no phenol red--to 500 mL total (Life
Technologies Cat. No. A12176-01)
[0458] Primary hepatocyte maintenance medium:
[0459] Primary Hepatocyte Maintenance Supplement (Life Technologies
Cat. No. CM4000)
[0460] Hepatocyte Growth Factor human--to 40 ng/mL final
concentration (Sigma Cat. No. H5791)
[0461] Epidermal Growth Factor human--to 20 ng/mL final
concentration (Sigma Cat. No. E9644)
[0462] Nicotinamide--to 2.5 .mu.g/mL final concentration (Sigma
Cat. No. N0636)
[0463] William's E Medium, no phenol red--to 500 mL total (Life
Technologies Cat. No. A12176-01)
[0464] Primary hepatocyte culture plates:
[0465] Collagen I, Coated Plate, 24 well (Life Technologies Cat.
No. A11428-02)
[0466] Transfection reagents:
[0467] HiPerFect Transfection Reagent (Qiagen Cat. No. 301704)
[0468] Opti-MEM I Reduced Serum Medium, no phenol red (Life
Technologies Cat. No. 11058-021)
Protocol
[0469] saRNA Annealing:
[0470] Each lyophilised saRNA strand was resuspended to 1 mM in
RNase-free 10 mM Tris-HCl, 20 mM NaCl2, 1 mM EDTA. They were mixed
well to complete resuspension. Equal volumes of sense and antisense
strands were mixed together by gentle vortexing. The tube was
placed with combined strands in a beaker of water heated to
95.degree. C. The beaker was covered and allowed to cool to room
temperature. Subsequent dilutions were performed using RNAse-free
water. Generally for 24 well format, stock solution was diluted to
10 .mu.M. Annealed saRNA were aliquoted and store at -20.degree.
C.
[0471] Thawing and Plating of Primary Hepatocytes:
[0472] CHRM and Plating medium were warmed to 37.degree. C. in a
water bath. Cryopreserved hepatocytes were thawed in a 37.degree.
C. water bath until no ice crystals remain. The vial was
disinfected with 70% ethanol. In a sterile tissue culture hood,
thawed hepatocytes were transferred directly into CHRM. Hepatocytes
were centrifuged at 100.times.g (900 rpm in a Thermo F-G1
fixed-angle rotor) for 10 minutes at room temperature. Supernatant
was carefully poured off into a waste bottle. Resuspend pellet in 1
mL of Plating Medium per 1.times.106 cryopreserved cells. Cells
were counted using a NucleoCounter NC-200 aggregated cells assay to
determine cell viability. 1.0.times.105 viable cells were plated in
500 .mu.L Plating Medium per well in a 24 well plate.
[0473] Reverse Transfection (Immediately after Seeding):
[0474] For each well to be transfected, 3 .mu.L of 10 .mu.M saRNA
was diluted in 94 .mu.L Opti-MEM. For each well to be transfected,
3 .mu.L HiPerFect was added and mixed well by vortexing. The
transfection was incubated for 15 minutes at room temperature. 100
.mu.L transfection complexes were added to each well for a final
saRNA concentration of 50 nM.
[0475] The plate was incubated at 37.degree. C. with 5% CO2 in a
humidified incubator. After 5 hours, medium was changed to 500
.mu.L pre-warmed Maintenance Medium.
[0476] Forward Transfection (24 Hours after Seeding):
[0477] For each well to be transfected, 3 .mu.L of 10 .mu.M saRNA
was diluted in 94 .mu.L Opti-MEM. For each well to be transfected,
3 .mu.L HiPerFect was added and mixed well by vortexing.
[0478] The transfection was incubated for 15 minutes at room
temperature. During incubation, medium was changed to 500 .mu.L
fresh pre-warmed Maintenance Medium per well. 100 .mu.L
transfection complexes was added to each well for a final saRNA
concentration of 50 nM. Plate was returned to incubator. After 24
hours, medium was changed to 500 .mu.L of fresh pre-warmed
Maintenance Medium. Peak gene activation occurred 72 hours after
cell seeding. Cells and/or supernatant were collected for
downstream analysis at this time.
TABLE-US-00021 TABLE 13-1 Relative expression of Aha1, albumin and
CEBPA genes at 48 hr after aha1-siRNA, Fluc and AW51 transfection
Aha1 exprission Albumin expression CEBPA expression Untreated 1 1 1
Aha1 0.1 0.8 1.1 Flu 1.6 0.7 2.0 AW51 2.2 0.9 3.2
TABLE-US-00022 TABLE 13-2 Relative expression of Aha1, albumin and
CEBPA genes at 72 hr after aha1-siRNA, Fluc and AW51 transfection
Aha1 exprission Albumin expression CEBPA expression Untreated 1 1 1
Aha1 0.1 0.8 0.6 Flu 1.4 1.0 1.0 AW51 1.3 5.0 2.9
[0479] Tables 12-13 and FIG. 9A/9B show CEBPA-saRNA upregulates
CEBPA and albumin in hepatocytes when they are exposed to
proliferation media. Therefore, CEBPA-saRNA shows efficacy in
proliferating cells. siRNA shows efficacy in both proliferating
cells and non-proliferating cells.
Example 5. In Vitro Studies with CEBPA-saRNA
Biological Effects of CEBPA-51 in Hepatic Cell Lines:
[0480] Aim of Study: The aim of this study was to measure the
endogenous CEBPA transcript levels in HCC cell lines representative
of highly differentiated HCC (HepG2, Hep3B) or of poorly
differentiated HCC (PLCPRF5), and to determine the relative
increase in CEBPA mRNA and C/EBP-.alpha. protein expression
following transfection with CEBPA-51. In addition, the effect of
C/EBP-.alpha. upregulation on cell proliferation was assessed.
[0481] A panel of hepatic cell lines including HEP3B, HEPG, and
PLCPRF5 were transfected with CEBPA-51. C/EBP-.alpha. protein was
detected by Western Blot in cell lysates from 72 hours after
transfection (quantification: RC-DC Bradford assay, reference
protein: tubulin). The endogenous transcript levels of CEBPA were
significantly higher in Hep3B and HepG2 cells compared to PLCPRF5
cells. Treatment with CEBPA-51 led to a significant increase in
CEBPA mRNA transcript levels and increased C/EBP-.alpha. protein
levels in all 3 tested HCC cell lines as compared to untransfected
control and treatment with non-specific RNA duplex (siFLUC) (FIGS.
10A and 10B)
[0482] Cell proliferations were measured with WST-1 proliferation
assay and SRB colorimetric assay. Results were shown in FIG.
11A-11F. CEBPA-51 reduced cell proliferation compared with controls
in HEP3B and HEPG2 cell lines, but not in PLCPRF5 cells. Therefore,
the capacity of CEBPA-51 to inhibit cell proliferation was
confirmed in HepG2 and Hep3B cells. In contrast, PLCPRF5 cells were
not affected by CEBPA-51 treatment.
Off-Target Analysis of AW51:
[0483] Specificity of AW51 was confirmed from predicted off-target
sites. Bioinformatics off-target analysis was conducted. AW51 has
at least 2 mismatches of antisense strand with any other human
transcript. Only 4 off-targets were predicted with 2 mismatches to
antisense strand.-targets were measured in vitro in HuH7 cells and
Panc-1 cells with 24 hour incubation. mRNA levels were normalized
to gapdh and results were shown in FIG. 12A (HuH7 cells)-12B
(Panc-1 cells). Expression pattern of potential off-targets
following AW51 transfection showed that none of the genes is
significantly affected by AW51.
Example 6. Mechanism Studies of CEBPA-saRNA
Strand Selection/Identification and Cleavage Analysis
[0484] Inverted abasic modifications at 5' terminus have been shown
to prevent loading into the guide position in Ago2 complex.
Antisense strand (AS) and sense strand (SS) of C/EBPA-saRNA were
blocked with an inverted abasic modification at 5' end (b) and
C/EBPA mRNA expression was measured and the impact of blocking AS
and/or SS strands on C/EBPA mRNA expression was determined.
[0485] RNAi involves cleavage of target mRNAs. A non-cleaving
sequence, mutations of central 3 base pairs, was tested
(CEBPA-AW01-510500) to determine whether CEBPA-saRNA cleaves the
target EST (AW665812). Mutation of the central 3 base pairs creates
a non-cleavable saRNA, regardless of which strand serves as the
guide.
[0486] All saRNA were synthesized and annealed in water. RP-HPLC
has 90% purity. Sequences of the oligonucleotide samples were shown
in the following table.
TABLE-US-00023 SEQ Sequence ID Oligo ID (SS on top) No. Notes
NC-500000 5'-ACUACUGAGUG 95 Non-targeting ACAGUAGAUU-3' `scramble`
3'-UUUGAUGACUC 96 (negative ACUGUCAUCU-5' control) CEBPA-AW01-
5'-GCGGUCAUUGUC 97 Unmodified 510000 ACUGGUCUU-3' `AW1-51` (AW1-51,
3'-UUCGCCAGUAA 98 (positive AW-51 or CAGUGACCAG-5' control) AW51)
CEBPA-AW01- 5'-bGCGGUCAUUG 99 5' Inverted 510012 UCACUGGUCUU-3'
abasic 3'-UUCGCCAGUAAC 98 modification AGUGACCAG-5' on SS only
CEBPA-AW01- 5-GCGGUCAUUGUC 97 5' Inverted 510013 ACUGGUCUU-3'
abasic 3'-UUCGCCAGUAA 100 modification CAGUGACCAGb-5' on AS only
CEBPA-AW01- 5-bGCGGUCAUUGU 99 Inverted 510014 CACUGGUCUU-3' abasic
3'-UUCGCCAGUAA 100 modification CAGUGACCAGb-5' on both SS and AS
(negative control) CEBPA-AW01- 5-GCGGUCAUACA 101 Mutated 510500
CACUGGUCUU-3' central 3'-UUCGCCAGUA 102 three UGUGUGACCAG-5' base
pairs
Critical Reagents
[0487] Transfection. Cells were transfected at 100,000 cells per
well in a 24-well dish at a final oligonucleotide concentration of
10 nM with 1 .mu.L Lipofectamine 2000 (Life Technologies, Carlsbad,
Calif.). The same conditions were used for forward and reverse
transfections.
[0488] RNA Isolation. Total RNA was isolated with the RNeasy Mini
Kit according to the manufacturer's protocol (Qiagen, Venlo,
Netherlands).
[0489] Complementary DNA (cDNA) Synthesis. cDNA was synthesized
using the Quantitect Reverse Transcription kit according to the
manufacturer's protocol with 500 ng RNA in a 20 .mu.L reaction
(Qiagen).
[0490] Quantitative PCR. Quantitative PCR was performed with
QuantiFast SYBR Green PCR master mix (Qiagen) on an Applied
Biosystems 7900HT real-time PCR system (Life Technologies)
according to the manufacturer's protocol. Reactions were run in
triplicate wells with 12.5 ng cDNA in each reaction.
Cell Lines
[0491] HepG2 hepatocellular carcinoma cells were maintained in RPMI
medium supplemented with 10% Fetal BovineSerum and 1.times.
L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich, St.
Louis, Mo.) in an incubator maintained at 37.degree. C. with 5%
CO2.
Experimental Design
[0492] The experiment was performed in triplicate wells. HepG2
cells were seeded in 24-well dishes at 100,000 cells per well and
were reverse transfected with 10 nM (f.c.) of each test item using
Lipofectamine 2000. After an incubation period of 24 hours an
additional forward transfection step was conducted with 10 nM
(f.c.) of each test item using Lipofectamine 2000. Preliminary
experiments determined that maximal saRNA activity is observed
after a second transfection. Forty-eight hours after the second
transfection, cells were lysed and collected to determine the CEBPA
and albumin mRNA levels by quantitative reverse transcription-PCR
(qRT-PCR).
Data Evaluation
[0493] Real-time PCR results were analysed using the
.DELTA..DELTA.Ct method. Ct values were determined using SDS
software (Life Technologies) and relative quantities were
calculated by normalization to untransfected cells. Additionally,
the housekeeping gene GAPDH served as an internal control. The
transfection experiment was conducted with triplicates;
determination of qPCR was performed in triplicates. Statistical
significance was determined using a t-test with Welch's
correction
Results and Discussion
[0494] Compared to untransfected cells, a 2-2.5-fold CEBPA mRNA
upregulation was observed in cells transfected with the unmodified
AW1-51 sequence, the AW1-51 modified on SS (CEBPA-AW01-510012), and
AW1-51 with internal sequence mutations (CEBPA-AW01-510500) (FIG.
13A), all statistically significant at p<0.01. No upregulation
of CEBPA mRNA was observed after transfection with non-specific
control (NC-500000), the AW1-51 modified on AS (CEBPA-AW01-510013),
or modified on both strands (CEBPA-AW01-510014). Consistent with
this pattern of activation was the upregulation of albumin
expression, a downstream target for CEBPA transcriptional
activation, also statistically significant at p<0.05 (FIG.
13B).
[0495] Since 5' inverted abasic modification is known to block Ago2
from loading the strand, the oligo with this modification on both
strands, CEBPA-AW01-510014, is expected to be inactive. This could
be confirmed in the experiment. The observation of CEBPA activation
with the inverted abasic modification on the SS but not on the AS
therefore indicates that the AS is the guide strand, which is
loaded into Ago2 for triggering CEBPA mRNA expression.
[0496] Cleavage of the target by Ago2 is inhibited by central
mismatches in sequence between the guide strand and target sequence
(genomic DNA or antisense RNA transcripts arising from the gene).
The CEBPA-saRNA sequence containing central mutations
(CEBPA-AW01-510500) showed no difference in CEBPA activation
compared to non-mutated oligo, indicating that cleavage of the
CEBPA sequence is not necessary for saRNA activity.
Conclusion
[0497] It was demonstrated that the antisense strand of AW1-51 is
the guide strand that is responsible for saRNA activity. Further,
it was shown that the 5' inverted abasic modification on the sense
strand did not have any influence on CEBPA gene activation. In
addition, the target antisense RNA cleavage by Ago2 was not
necessary for triggering the saRNA activity.
Example 7. CEBPA-51 saRNA Activity in Primary Human Hepatocytes
[0498] As shown previously, CEBPA-saRNA upregulates CEBPA mRNA and
albumin mRNA in proliferating but not quiescent hepatocytes (FIG.
9A and FIG. 9B). In this study, the effect of CEBPA-saRNA on
proliferating hepatocytes was evaluated again (FIG. 14) and the
effect on albumin secretion (FIG. 15) and downstream markers (FIG.
16A-FIG. 16F) were also studied.
[0499] The effect of CEBPA-51 in normal human primary human
hepatocytes was evaluated. Since primary hepatocytes in culture do
not proliferate, this study demonstrates that CEBPA-51 upregulates
CEBPA transcript and albumin in primary hepatocytes that are
induced to proliferate in the presence of growth factors and
cytokines. Furthermore, this study shows CEBPA-51 causes regulation
of factors crucial for efficient liver function; these include
liver alanine glyoxylate aminotransferase, ornithine
transcarbamylases, albumin, CYP3A4 and HNF4A.
[0500] The objective of this study was to establish the efficacy of
CEBPA-51 in normal human primary hepatocytes on CEBPA and albumin
expression. Additionally, factors that are important for liver
function were also screened to assess if CEBPA-51 conferred a
favorable effect. These factors included: [0501] Alanine-glyoxylate
aminotransferase (AGXT). AGXT expression is liver specific and is
required for the metabolic function of hepatocytes. [0502] Albumin.
Serum albumin is the main protein of human blood plasma and is
exclusively synthesized by the liver. Its main function is to
regulate the colloidal osmotic pressure of blood as well as acting
as a carrier molecule for lipidsoluble hormones, bile salts,
unconjugated bilirubin, apoprotein, calcium and certain drugs
(warfarin, clofibrate etc). [0503] Cytochrome P450 3A4 (CYP3A4).
CYP3A4 is a member of the cytochrome P450 family of oxidizing
enzymes involved in drug metabolism. CYP3A4 is predominantly found
in the liver. There are several other members of this family of
Cytochrome P450, however CYP3A4 is the most common and versatile
member. [0504] Ornithine transcarbamylase (OTC). OTC is an enzyme
that catalyses the reaction between carbamoyl phosphate and
ornithine to form citrulline and phosphate in the mitochondria as
part of the urea cycle. [0505] Hepatocyte nuclear factor 4-alpha
(HNF4A). HNF4A is a liver specific transcription factor recognized
as being a master regulator of liver-specific gene expression for
genes involved in lipid transport and drug and glucose
metabolism.
[0506] To confirm target engagement of CEBPA-51, CEBPA and albumin
transcript levels were also confirmed along with the liver function
probes. Additionally an ELISA with albumin specific antibodies was
carried out to measure albumin secretion in the cell culture medium
following transfection with CEBPA-51.
Materials and Methods
[0507] The test item for this experiment was CEBPA-51, which is the
API of MTL-CEBPA. In addition, a non-targeting duplex, siFLUC, was
also used as a negative control and Aha-1 siRNA as a transfection
efficiency control. These RNA oligonucleotides (see table below)
were commercially synthesized (ST Pharm, Seoul, South Korea,
certificate of analysis in appendix), annealed, and stored in 10
.mu.M aliquots at -20.degree. C. in RNase-free H.sub.2O.
TABLE-US-00024 Oligo Sequence SEQ ID (SS on top) ID No. Notes
siFLUC 5'-mCmUmUAmCGm 103 Non- CmUGAGmUAmCmUm targeting
UmCGAdTpsdT-3' control 3'-dTpsdTGAAmU 104 GCGAmCUCAmUGAA GCU-5'
Aha1- 5'-GGAmUGAAGmU 105 Transfection siRNA GGAGAmUmUAGmUd
efficiency TpsdT-3' control 3'-dTpsdTCCUAm 106 CUUCAmCCUCUAAm
UCA-5' CEBPA-51 5'-bmGmCGmGUCA 107 API of UUmGUCAmCUGGUC MTL-
mUmU-3' CEBPA 3'-mUmUCGCCAGU 108 AACAGUGACCAG-5' b: 5' inverted
abasic sugar cap m: 2'-O-methyl modified base d:
deoxyribonucleotide ps: phosphorothioate
Critical Reagents
[0508] Primary hepatocyte thawing medium. Cryopreserved Hepatocyte
Recovery Medium (CHRM) was used for thawing each vial (Life
Technologies, CM7000).
[0509] Primary hepatocyte plating medium. Fetal bovine serum, heat
inactivated-50 ml (Life Technologies, 16140-071);
Insulin-Transferrin-Selenium (100.times.)-5 ml (Life Technologies,
41400-045); HEPES (1M)-5 ml (Life Technologies, 15630-056);
L-Glutamine-Penicillin-Streptomycin solution-5 ml (Sigma, G1146);
Dexamethasone-40 ng/ml final concentration (Sigma, D8893); Phenol
red free William's E Medium (Life Technologies, A12176-01).
[0510] Primary Hepatocyte Maintenance medium. Primary Hepatocyte
Maintenance Supplement (Life Technologies, CM4000); Human
Hepatocyte Growth Factor-40 ng/ml final concentration (Sigma,
H5791); Epidermal Growth Factor-20 ng/ml final concentration
(Sigma, E9644), Nicotinamide-2.5 ug/ml final concentration (Sigma
N0636); Phenol red free William's E Medium-500 ml (Life
Technologies, A12176-01).
[0511] Transfection. Cells were transfected at 100,000 cells per
well in a 24-well collagen coated dish at a final oligonucleotide
concentration of 50 nM with 3 .mu.L of HiPerFect transfection
reagent (Qiagen, 301704). The cells were incubated in plating media
for 5 hours to allow monolayer formation before replacing with
maintenance media. For the second (forward) transfection, the same
conditions were used as for reverse transfection. Maintenance media
was used for the remaining duration of the experiment.
[0512] RNA Isolation. Total RNA was isolated with the RNeasy Mini
Kit according to the manufacturer's protocol (Qiagen, Venlo,
Netherlands).
[0513] Complementary DNA (cDNA) Synthesis. cDNA was synthesized
using QuantiTect Reverse Transcription kit (Qiagen) according to
the manufacturer's protocol with 500 ng RNA in a 20 .mu.L
reaction.
[0514] Quantitative PCR. Quantitative PCR was performed with
Quantitect SYBR Master Mix (Qiagen) on an Applied Biosystems 7900HT
real-time PCR system (Life Technologies) according to the
manufacturer's protocol. Reactions were run in triplicate
wells.
[0515] Albumin Enzyme linked immunosorbent assay (ELISA). Culture
media from primary cells incubated within each experimental groups
were measured for albumin content using a human albumin ELISA
quantitation set (Bethyl Laboratories Inc, USA) following the
manufacturer's instructions. Human specific antibody against
albumin was immobilized onto each well of a Costar-3596-96 well
plate-(flat bottom, TC treated, nonpyrogenic, polystyrene, sterile
plates (Corning, USA). Reagents prepared in-house included:
ELISA plate coating buffer. 0.05M Carbonate-Bicarbonate, (Sigma,
C-3041) pH 9.6. ELISA wash buffer. 50 mM Tris; 0.14M NaCl; 0.05%
Tween 20 (Sigma, P1379) pH 8.0). ELISA blocking buffer. 50 mM Tris;
0.14M NaCl; 1% BSA (Sigma, A-4503), pH8.0). Sample/Conjugate
buffer. 50 nM Tris; 0.14M NaCl; 1% BSA (Sigma, A-4503); 0.05% Tween
20 (Sigma, P1379). Enzyme substrate buffer. 3,3',5,5'
Tetramethybenzidine (Sigma, T0440). ELISA Stop solution. (Sigma,
S5814).
Cell Lines
[0516] Human normal primary hepatocytes were purchased from Life
technologies (HMCPTS). All repeats were derived from the same batch
(HU8200-A).
Experimental Design
Relative Gene Expression
[0517] For relative quantitation of target transcript, the
experiment was performed in triplicate. Human primary hepatocytes
were seeded in 24 collagen-coated well-dishes (Life Technologies,
A11428-02) at a density of 100,000 cells per well in primary
hepatocyte plating medium following by an initial transfection with
50 nM (f.c) of CEBPA-51 whilst the cells were still in suspension
(reverse transfection). The cells were then allowed to form a
monolayer for 5 hours before the plating media was replaced with
maintenance media. 24 hours following reverse transfection, a
second (forward) transfection was carried out with 50 nM of
CEBPA-51. Fresh maintenance media was replaced every 24 hour until
harvest point at 72 hours following reverse transfection where
total RNA extracted from the cells were screened for target gene
expression.
ELISA
[0518] The culture media at the 72-hour time point of this study
was collected for an ELISA using human specific anti-Albumin
(Bethyl Laboratories, A80-129A) immobilized onto the wells of a 96
well plate. A standard curve of known albumin amounts (Bethyl
Laboratories, RS10-110-4) was added at 10 .mu.g/ml; 400 ng/ml; 200
ng/ml; 100 ng/ml; 50 ng/ml; 25 ng/ml; 12.5 ng/ml and 6.25 ng/ml.
The samples and the known control amounts were left to incubated on
the ELISA plates on a rotating plate for 3 hours at room
temperature (20-25.degree. C.). After the appropriate number of
washes as detailed in the manufacturer's protocol, HRP detection
antibody (Bethyl Laboratories, A80-129P) was added at a
concentration of 1:150,000) and incubated for 1 hour on a rotating
plate for 3 hours at room temperature (20-25.degree. C.). After 5
washes, the TMB substrate was added and allowed to incubate at room
temperature until the enzymatic color reaction developed. The
reaction was stopped by the addition of the ELISA Stop solution
where the absorbance at optical density of 450 nm was measured on a
plate reader.
Data Evaluation
Relative Expression
[0519] Real-time PCR results were analysed using the Livak method
(2-.DELTA..DELTA.CT) (Livak K & Schmittgen T D, 2001). Ct
values are determined using SDS software (Life Technologies) and
relative quantities are calculated by normalization to
untransfected cells. The housekeeping gene GAPDH is served as an
internal control. The transfection experiment was conducted in
triplicate; determination of qPCR was performed in triplicates.
Statistical significance was determined using a non-parametric
t-test with Welch's correction.
ELISA
[0520] A standard curve to determine the amount of human albumin
the unknown samples were prepared as the average absorbance value
minus the blank value for each standard concentration on the
vertical (Y) axis versus the corresponding human albumin
concentration on the horizontal (X) axis using a curve-fitting
software (Excel). The amount of human albumin concentration in the
unknown samples was calculated using the human albumin
concentration (X axis) that correlated with the absorbance value (Y
axis) obtained for the unknown sample.
Results and Discussion
[0521] Transfection of CEBPA-51 to primary human hepatocytes
induces a significant 2.5 fold increase in CEBPA transcript levels
as well as albumin, FIGS. 14A and 14B. To confirm efficient
transfection efficiency, Aha1-siRNA was used as a control and
demonstrated a 7 fold reduction in target transcript, FIG. 14C.
Biological Effect of CEBPA-51 in Normal Human Primary
Hepatocyte
[0522] After confirming increase in endogenous expression levels of
CEBPA transcript following CEBPA-51 transfection, the cultured
media from the cells were measured for levels of secreted albumin.
An ELISA assay using human specific anti-Albumin antibody confirmed
a significant 1.3 fold increase in secretion of albumin from the
hepatocytes (FIG. 15).
[0523] To assess if increased CEBPA and Albumin transcript levels
also mirrored a positive regulation in factors important for liver
function; the expression levels of the followed transcripts were
assessed in the primary hepatocytes transfected with CEBPA-51: FIG.
16A Alanine-glyoxylate aminotransferase (AGXT) increased 1.4 fold;
FIG. 16B albumin increased 1.5 fold; FIG. 16C Cytochrome P450 3A4
(CYP3A4) increased 1.5 fold; FIG. 16D Ornithine transcarbamylase
(OTC) increased 2.3 fold; FIG. 16E Hepatocyte nuclear factor
4-alpha (HNF4A) increased 1.5 fold; and FIG. 16F CEBPA increased
1.6 fold.
Conclusion
[0524] CEBPA is recognised as an important liver enriched
transcription factor. Its biological function becomes more evident
in knock out and knock-in transgenic animal studies. This study
demonstrates the transcriptional response of CEBPA-51 induced
upregulation of CEBPA and its down-stream effectors that were more
relevant for hepatocyte function in normal human primary
hepatocytes. It is found that normal primary hepatocytes respond
favorably to CEBPA-51 transfection with a significant increase in
albumin secretion and a significant upregulation of detoxification
enzymes.
Example 8. CEBPA-saRNA saRNA Activity in Cynomogus Fibroblasts
Cell Lines
[0525] Primary cynomolgus hepatocytes were obtained from Primacyt
Cell Culture Technology (Schwerin, Germany). CYNOM-K1 cynomolgus
embryonic fibroblast cells (Public Health England, Salisbury, UK)
were maintained in MEM medium supplemented with 10% Fetal Bovine
Serum, 1% non-essential amino acids (Life Technologies), and
1.times. L-glutamine-penicillin-streptomycin solution
(Sigma-Aldrich, St. Louis, Mo.) in an incubator maintained at
37.degree. C. with 5% CO2. The ability of CEBPA-51 to upregulate
CEBPA mRNA in cynomolgus cells was assessed to confirm
cross-reactivity. First, the cynomolgus genomic sequence at the
CEBPA-51 target site was verified. The sequence was accessed from
publically available databases as well as verified by direct
sequencing of gDNA-derived PCR products. CEBPA51 target sequence
was used as a query search on the Macaca fascicularis (cynomolgus
monkey) genome using BLAST. The query mapped to the genomic
location of CEBPA and there were no mismatches between the sequence
of CEBPA-51 and the genomic target site. To verify this sequence
information, gDNA was isolated from primary cynomolgus hepatocytes
and a PCR product of the target site was generated for direct
sequencing. The resulting sequence aligns with no mismatches to the
published cynomolgus genomic sequence and the CEBPA-51 target
site.
[0526] After confirming the cynomolgus genomic target sequence,
CEBPA-51 was transfected into cynomolgus fibroblasts to determine
if CEBPA-51 is cross-reactive and able to upregulate CEBPA mRNA in
other cells than hepatocytes. mRNA levels in untransfected cells,
siFLUC transfected cells and CEBPA51 transfected cells were
measured. The experiment was performed in triplicate. CYNOM-K1
cells were seeded in 24-well dishes at 100,000 cells per well and
were reverse transfected with 20 nM (f.c.) of each test item using
Lipofectamine 2000. After an incubation period of 24 hours an
additional forward transfection step was conducted with 20 nM
(f.c.) of each test item using Lipofectamine 2000. Preliminary
experiments determined that maximal saRNA activity is observed
after a second transfection. Twenty-four hours after the second
transfection, cells were lysed and collected to determine the CEBPA
mRNA levels by quantitative reverse transcription-PCR (qRT-PCR). As
shown in FIG. 17, compared to untransfected cells, a 2-fold CEBPA
mRNA upregulation was observed 24 hours after cells were
transfected the second time with CEBPA-51, while no upregulation
was seen with siFLUC. This upregulation was statistically
significant at p<0.05.
[0527] Therefore, cross-reactivity of CEBPA-saRNA was confirmed in
cynomogus cell line. It was demonstrated that the genomic sequence
of cynomolgus contains no mismatches with the CEBPA-51 target
sequence according to the BLAST database. This was further verified
by sequencing of primary cynomolgus gDNA. Cross-reactivity of
CEBPA-51 was then confirmed by transfection in cynomolgus
fibroblasts and the observation of CEBPA gene activation.
Example 9. In Vitro Stability Analysis in Rat, Cynomolgus Monkey
and Human Serum
[0528] This study is an in vitro stability analysis investigating
the stability of CEBPA-51 and the liposomal-formulated MTL-CEBPA in
rat, cynomolgus monkey and human plasma anticoagulated with EDTA-K2
over 120 min at 37.degree. C.
[0529] 3 .mu.L of a 50 uM CEBPA-51 solution in PBS or 3 .mu.L of
the MTL-CEBPA solution were mixed with 30 .mu.L of plasma and
incubated in plasma for 0, 5, 10, 20, 30, 60 and 120 min at
37.degree. C. Incubation of 3 .mu.L CEBPA-51 solution in PBS or 3
.mu.L MTL-CEBPA solution in 30 .mu.L PBS served as control for
unspecific degradation. Incubation was done in sealed 96-well PCR
plates in an Eppendorf Mastercycler. Incubation was stopped at the
indicated time points by a proteinase K treatment to digest all
present nucleases in the plasma samples. After proteinse K
treatment, CEBPA-51 is stable in the samples and in the lysis
buffer containing SDS CEBPA-51 is released from the LNP formulation
in MTL-CEBPA.
[0530] Samples were subsequently analysed by a generic AEX-HPLC
method under denaturing conditions at elevated pH (11) and
40.degree. C. on a ThermoFisher DNA Pac PA200 column (4.times.250
mm). A sodium bromide gradient from 250 to 620 mM in 18 min at a
flow rate of 1 mL/min was used to separate and elute the RNA
strands from the HPLC column. Detection was conducted with a UV
detector at 260 nm.
[0531] Under these conditions the two single strands of the
CEBPA-51 alone or CEBPA-51 released from MTL-CEBPA were separated
from each other and from the degradation products and could be
evaluated as distinct peaks. As no reference single strands were
available and AEX-HPLC could not be combined with mass
spectrometry, an assignment of the two single strands was not
possible. Therefore, the two strands were labelled 1.sup.st and
2.sup.nd strand depended on the retention time during gradient
elution. For data evaluation, only the peak area of the two single
strands of CEBPA-51 alone and CEBPA-51 in MTL-CEBPA were evaluated.
Peak area at T=0 was set to 100% and all other time points were
normalized to peak area at T=0 for plasma of each species. The data
were then reported as % intact strand normalized to T=0.
Results
[0532] CEBPA-51 with No Formulation:
[0533] CEBPA-51 is relatively stable in rat plasma anticoagulated
with EDTA and 15% degradation of the first and 8% of the second
peak was observed (see FIG. 18A). CEBPA-51 is degraded by
.about.50% over 2 hours in human plasma (see FIG. 18B). CEBPA-51 is
least stable in cynomolgus monkey plasma with .about.85% of both
strands degraded within 2 hours (see FIG. 18C).
[0534] Data demonstrate that CEBPA-51 alone was less stable in
human and cynomolgus monkey plasma, but relatively stable over two
hours in rat plasma. Not willing to be bound to any theory, in rat
plasma, degradation of RNA is mainly induced by 3'-exonuclaese that
depends on divalent cations. Therefore, the use of EDTA as
anticoagulant blocks this degradation pathway efficiently and
CEBPA-51 is relatively stable. In contrast, the main degradation
pathway in human and cynomolgus monkey plasma is dependent on RNase
A. The activity of this endonuclease is independent from divalent
cations and therefore the CEBPA-51 without protection by liposomal
formulation is degraded in plasma of these species.
Liposomal Formulated CEBPA-51:
[0535] CEBPA-51 in MTL-CEBPA was stable over 2 hours in plasma of
all species and no significant degradation was observed (see FIGS.
19A, 19B, and 19C). This indicates that the LNP formulation in
MTL-CEBPA is stable over 2 hours and completely protects CEBPA-51
from degradation in plasma.
[0536] From the results, it can be concluded that MTL-CEBPA
formulation is intact over at least 2 hours in plasma.
Example 10. In Vivo Pharmacokinetic Study in Rat
[0537] This study is a PK study investigating CEBPA-51 and the
liposomal-formulated MTL-CEBPA in rat plasma samples after one
single IV application of 2.175 mg/kg MTL-CEBPA in Group 1 and 1.5
mg/kg CEBPA-51 in Group 2. Each group comprised of 3 male rats.
Blood was collected after 0.25, 0.5, 1, 2, 3, 6, 12, 24 and 48 hr
for both groups.
[0538] An aliquot of the plasma was homogenized by a proteinase K
treatment in an SDS containing buffer system. After proteinase K
digestion, the SDS was precipitated with 3M KCl and removed by
centrifugation. The supernatant was heated in presence of a
complementary 15-mer fluorescently labelled peptide nucleic acid
(PNA)-probe to specifically form stable duplexes between the PNA
and the antisense strand of CEBPA-51 alone or CEBPA-51 released
from liposomal formulated MTL-CEBPA. PNA formed duplexes between
CEBPA-51 (referred to in this example as the parent compound), but
also with metabolites or impurities from the synthesis. The formed
duplexes were then analysed by non-denaturing Anion Exchange-High
Performance Liquid Chromatography (AEX-HPLC) coupled to a
fluorescence detector. The metabolites or synthetic impurities were
separated from the main compound by AEX-HPLC.
[0539] The concentrations of CEBPA-51 were calculated using
external calibration curves generated from known concentrations of
the parent compound spiked into untreated plasma lysates. The total
metabolite/synthetic impurity level for each sample was determined
by subtraction of the peak area for the parent compound from the
total peak area. The resulting peak area was then quantified
against the external calibration curve.
Results
[0540] CEBPA-51 with No Formulation:
[0541] A high degradation of the parent compound was observed in
plasma obtained from rats treated with CEBPA-51. The parent
compound was only detected at the first sampling time-point (15
minutes post administration, see FIGS. 20A and 20B). Metabolites
were detected up to 60 minutes, but below detection limit (BDL) 2
hours post administration.
Liposomal Formulated CEBPA-51:
[0542] The MTL-CEBPA formulation showed a high stability in plasma.
The parent compound was found in all plasma lysates obtained from
rats treated with MTL-CEBPA. The ratio between parent compound and
metabolites was about 9:1, i.e. approx. 88% of intact parent in the
liposomal formulated groups. At later time points, the relative
content of parent compound increased over the
"metabolites/impurities", as the signals were below detection limit
for most of the minor site peaks in the chromatograms (see FIG.
21A). 48 hours post administration CEBPA-51 was still detectable in
rat plasma (see FIGS. 21A and 21B).
[0543] This study suggests that the observed metabolites after IV
administration of MTL-CEBPA, which account for about 10% of
detected RNA, might originate from the RNA synthesis process rather
than from strong metabolism. However, as comparison to the
un-encapsulated compound (CEBPA-51) is not feasible due to plasma
instability and extremely fast clearance of the unformulated
CEBPA-51, metabolic conversion of the dsRNA cannot be excluded.
Example 11. In Vivo Studies of CCL4 Induced Liver Failure/Fibrosis
with CEBPA-saRNA
[0544] Liver fibrosis is the pathologic result of chronic
inflammatory liver diseases such as chronic viral hepatitis (e.g.
hepatitis B and C), alcohol abuse, drug overload/toxicity,
cholestatic liver injury, congenital abnormalities or autoimmune
attack of hepatocytes. It is characterized by hepatic stellate cell
(HSC) proliferation and differentiation into myofibroblast-like
cells which results in the deposition of extracellular matrix (ECM)
and collagen. Carbon tetrachloride (CCL4) induced hepatic fibrosis
is a well-established and widely accepted experimental model in
rodents for the study of liver fibrosis and cirrhosis. Chronic
administration of carbon tetrachloride to rats induces severe
disturbances of hepatic function together with histologically
observable liver fibrosis.
[0545] A 10-week long study was carried out with CEBPA51 formulated
with amphoteric liposomes (NOV340 Smarticle liposomes provided by
Marina Biotech), referred to as MTL-CEBPA. CEBPA51 (XD-03934) has
the same sequence as AW51, but with 2'O-Me and 5' inverted abasic
modifications on sense strand. Lipids in NOV340 include Mochol,
Chems, DOPE, and POPC. Liver failure in Sprague Dawley rats was
induced by i.p. injection of carbon tetrachloride (CCL4) twice
weekly. Male Sprague Dawley rats with a starting body weight of
120-150 g were used. The animals were administered intraperitoneal
(i.p.) injection of CCL4: Olive oil (1:1 ratio) twice a week with 2
ml/kg for 2 weeks, followed by 1 ml/kg i.p, twice weekly for 8
weeks. The animals were weighed twice weekly and maintained in a
controlled environment with 22.+-.3.degree. C. temperature,
50.+-.20% humidity, a light/dark cycle of 12 hours each and 15-20
fresh air changes per hour. Animals were housed group wise (3
animals/cage), autoclaved corncob was used as a bedding material
and were fed, ad libitum, with certified Irradiated Laboratory
Rodent Diet (Nutrilab brand, Tetragon Chemie Pvt. Ltd, Bangalore)
during the study period.
TABLE-US-00025 CEBPA51 GACCAGUGAC SEQ antisense (X11283)
AAUGACCGCuu ID No. 109 CEBPA51 sense (invabasic) SEQ (X11273)
GcGgUCAUUgU ID CAcUGGUCuu No. (lower case 110 stands for 2'O-Me
modifications)
[0546] The rats were randomized based on bilirubin, body weight and
AST. They were grouped into Group 1: Sham control; Group 2: Path
control-1; Group 3: Path control-2; Group 4: Test compound -0.3
mg/kg; Group 5: Test compound -1 mg/kg; Group 6: Test compound -3
mg/kg. Test compound=MTL-CEBPA. Rats in Groups 4-6 were treated
with Test compound starting from week 8 for 2 weeks via tail vein
injection up to 3 mg/kg together with continued injection of CCL4.
Rats in Group 3 were administered NOV340/siFLUC at 3 mg/kg. Test
compound i.v. injections happened at week 8, week 8.5, week 9, and
week 9.5.
TABLE-US-00026 Gr. Duration of CCL4 Dosing Euthanized No Group
administration Treatment Route Regimen n at 1 Sham -- -- -- -- 5
week 10 2 Path CCL4 administered for -- -- -- 9 week 10 Control-1 8
weeks 3 Path CCL4 administered for Vehicle i.v. week 8, 8.5, 9 9
week 10 Control-2 10 weeks and 9.5 4 Test CCL4 administered for 0.3
mg/kg i.v. week 8, 8.5, 9 9 week 10 Compound 10 weeks and 9.5 5
Test CCL4 administered for 1 mg/kg i.v. week 8, 8.5, 9 9 week 10
Compound 10 weeks and 9.5 6 Test CCL4 administered for 3 mg/kg i.v.
week 8, 8.5, 9 9 week 10 Compound 10 weeks and 9.5
[0547] Liver function tests, hydroxyproline levels and
histopathology in Path Control -1 group were done to assess
fibrosis status after 8 weeks of CCL4 administration. Liver
function tests, hydroxyproline levels and histopathology in Path
Control -2 group were done to assess fibrosis status after 10 weeks
of CCL4 administration. The efficacy of Test compounds was assessed
by its ability to limit progression of disease or reverse fibrosis.
Parameters assessed were body weight (once every three days), liver
function test (day 0, day 42 (week 6), day 56 (week 8), day 63
(week 9) and day 70 (week 10)), histopathology at the end of the
study and hydroxyl proline assay at the end of the study (week 8
for Path control -1 and week 10 for rest of the groups).
Histopathology included H&E staining, Mason Trichrome staining,
and Sirius red staining done for all the animals at the end of the
study. Liver function test included Alanine aminotransferase (ALT),
Aspartate aminotransferase (AST), Alkaline phosphatase (ALP),
Gamma-glutamyl transfertase (GGT), total bilirubin (TBIL), Total
protein (TP), Albumin, Globulin and Albumin/Globulin ratio measured
at week 0, 4, 6, 8, 9 and 10. Parameters at week 8, week 9 and week
10 shown in FIG. 22A-22K demonstrated reversal and near
normalisation of clinically relevant parameters including bilirubin
(75% decrease, FIG. 22F), circulating alanine and aspartate
aminotransferase (60% decreases, FIG. 22B and FIG. 22C) and
prothrombin time (20% decrease, FIG. 22I). In addition, there were
significant increases in serum albumin (FIG. 22H) and total protein
(FIG. 22G) and significant decreases in alkaline phosphatase (ALP)
(FIG. 22D) and gamma-glutamyl-transpeptidase (GGT) (FIG. 22E).
Liver hydroxyproline was significantly decreased in a
dose-dependent manner (FIG. 22K). A significant increase in body
weight was observed with no associated toxicity (FIG. 22A).
[0548] Pathology results were shown in FIG. 23. Naive animals had
healthy livers are brownish pink and are smooth with sharp borders.
Livers of animals treated with CCL4 and vehicle control had paler
firmer livers with multiple cirrhotic nodules spreading over the
surface. Livers of animals treated with CCL4 and MTL-CEBPA (0.3
mg/kg) had pale firmer livers with small nodules spreading over the
surface. The liver color was darker than the liver color of the
CCL4 and vehicle control group. Livers of animals treated with CCL4
and MTL-CEBPA (3 mg/kg) had a normal brownish color with uneven
surface and very mild nodulation.
[0549] Histology staining results were shown in FIG. 24A-24C. FIG.
24A is sham control. FIG. 24B is CCL4-treated rats that received
NOV340/siFluc treatment (negative control). FIG. 24C is
CCL4-treated rats that received MTL-CEBPA treatment. MTL-CEBPA
treated animals had reduced fibrous tissue and pseudolobule
formation than the animals in the control groups.
[0550] Data discussed above showed that CEBPA-saRNA reversed liver
failure across all clinical relevant parameters. Many parameters
were reversed to normal. Serum albumin level was even better than
normal.
Example 12. In Vivo Studies of Treating Acute Liver Failure
[0551] Acute liver failure (ALF) is a clinical condition with high
mortality rate. Acute liver failure (ALF) is a condition
characterized by rapid and severe deterioration of hepatocyte
function in patients without known prior liver disease. Hepatotoxic
drug thioacetamide (TAA) was used to induce ALF in this study. To
establish thioacetamide (TAA) induced acute liver failure model in
SD rats, the following parameters were measured: a) survival rate;
b) liver function test (LFT) and biochemical parameters.
Test System
[0552] Test species: Rattus norvegicus.
[0553] Strain: Sprague Dawley rat (SD rats).
[0554] Sex: Male.
[0555] Body weight/Age: 150-200 g/7-8 weeks.
[0556] No. of groups: 4. No. of animals/group: 8.
[0557] Source: Harlan Laboratories.
[0558] Study period: 7 days.
[0559] Male SD rats, 6-7 weeks of age were procured from Harlan.
Animals were maintained in a controlled environment with
22.+-.3.degree. C. temperature, 50.+-.20% humidity, a light/dark
cycle of 12 h each and 15-20 fresh air changes per hour. Animals
were housed group wise (3 animals per cage) and autoclaved corncob
was used as bedding material. Upon receipt, animals were kept in
quarantine for one week. The animals were assigned a temporary
number at the base of tail using an indelible marker pen. After
quarantine, animals were transferred to the experimental room and
kept for acclimatization for a period of one week before initiation
of the experiment.
[0560] Equipments: EM-360 clinical chemistry analyzer (Erba
Mannheim, Germany).
Disease Induction
[0561] All animals were randomized into four groups based on basal
body weight, bilirubin and AST on .about.2 day with consideration
of less than 10% intergroup variation for basal parameters. Cages
were identified by cage cards indicating the study number, study
code, group number, sex, dose, cage number, number of animals and
animal number details. Groups 1-4: All study animals were
administered single intraperitoneal (i.p) injection of TAA in
saline at the dose of 350 mg/kg (mpk), volume 5 ml/kg on day 0.
Group 1 did not receive any treatment and served as pathological
control. The MTL-CEBPA was injected intravenously to Group 2 at -24
h, Group 3 at 0 h and Group 4 at 24 h after TAA injection. Animals
were assessed for LFT and biochemical parameters such as Alanine
aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline
phosphatase (ALP), Gamma-glutamyl transferase (GGT), total
bilirubin (TBIL), Total protein (TP), Albumin (ALB) and ammonia at
days 1, 2, 3, 4 and 5. At end of the study, all the available
animals were euthanized by CO2 asphyxiation and plasma was
collected and stored at -80.degree. C.
Experimental Groups:
TABLE-US-00027 [0562] Description of No. of Test Groups groups
Induction Treatment animals dose Group 1 TAA control TAA 350 NA 8
NA (G1) mpk i.p. Group 2 MTL-CEBPA TAA 350 MTL-CEBPA 8 3 (G2) (-24
h) mpk i.p. (24 h before TAA) mpk Group 3 MTL-CEBPA TAA 350
MTL-CEBPA 8 3 (G3) (0 h) mpk i.p. (0 h before TAA) mpk Group 4
MTL-CEBPA TAA 350 MTL-CEBPA 8 3 (G4) (24 h) mpk i.p. (24 h after
TAA) mpk
Administration of MTL-CEBPA
[0563] The test item MTL-CEBPA was administered intravenously
through tail vein with appropriate disposable syringe and needle.
The animals in group 2, 3 and 4 were administered with MTL-CEBPA.
All the doses were administered at a dose volume of 1 ml/kg of
animal body weight.
Observation
Body Weight and Animal Mortality
[0564] Initial body weight was recorded individually for all
animals and daily once thereafter for the entire study period of 7
days. General health observation was done on a daily basis at the
same time of the day. This includes alertness, hair texture, cage
movement and presence of any discharge from nose, eyes, mouth and
ears. All the group animals were monitored daily for mortality due
to TAA administration.
Biochemical Analysis-LFT (for Randomization)
[0565] At day -2, blood samples were collected by retro-orbital
puncture method under light isoflurane anesthesia and plasma was
separated for estimating ALT, AST, ALP, GGT, TBIL, TP, ALB, and
ammonia by fully automated random access clinical chemistry
analyzer (EM-360, Make: Erba Mannheim, Germany). Animals were then
randomized based on total bilirubin, body weight and AST.
Assessment of Biochemical Parameter, LFT after TAA Injection
[0566] Blood samples were collected by retro-orbital puncture
method under light Isoflurane anesthesia from all the animals (from
day 1 after TAA injection till end of study) and plasma was
separated for estimating ALT, AST, ALP, GGT, TBIL, TP, ALB and
ammonia.
Statistical Analysis
[0567] Statistical analysis was performed using one way or two way
analysis of variance (ANOVA), followed by Dunnett's multiple
comparison test wherever applicable. p<0.05 was considered to be
statistically significant. Data expressed as Mean.+-.SEM.
Results
Liver Functional Parameters and Body Weight
[0568] At day -2, all animals were randomized into four groups
based on total bilirubin (TB), body weight and AST level for TAA
injection to induce ALF and for MTL-CEBPA prophylactic, concurrent
and preventive treatment.
[0569] Randomization of Groups Based on Bilirubin, Body Weight and
AST:
TABLE-US-00028 Total bilirubin Body AST Groups n (mg/dl) weight (g)
(U/L) 1. TAA control 8 0.09 .+-. 0.01 263.8 .+-. 2.5 91.5 .+-. 7.6
2. MTL-CEBPA 8 0.08 .+-. 0.01 267.0 .+-. 4.4 92.9 .+-. 4.0 (-24 h)
3. MTL-CEBPA 8 0.09 .+-. 0.01 266.5 .+-. 4.2 91.2 .+-. 4.6 (0 h) 4.
MTL-CEBPA 8 0.09 .+-. 0.01 266.5 .+-. 6.4 90.7 .+-. 4.6 (24 h)
Data Expressed as Mean.+-.SEM
[0570] Pathological control (TAA control) group showed a
significant reduction in body weight on days 2, 3, 4 (p<0.01)
& 5 (p<0.05) compared to its basal body weight. Intravenous
administration of MTL-CEBPA in Group 2, Group 3 and Group 4 at
different time intervals showed no significant changes in body
weight from day 1 to day 5 when compared with TAA control group at
different days.
[0571] There was no mortality observed during the entire duration
of study (7 days) in TAA and test item treatment groups. Animals
were regularly monitored for local (site of injection) and general
clinical signs. All animals were found lethargic on day 1 after TAA
injection, and recovered to normal on subsequent days.
[0572] Daily Mean Body Weight of Animals:
TABLE-US-00029 Gr. No. n Day -2 Day 1 Day 2 Day 3 Day 4 Day 5 1.
TAA control 8 263.8 253.3 245.3 246.8 246.7 248.6 2. MTL- 8 267.0
254.6 248.6 253.3 257.1 266.4 CEBPA (-24 h) 3. MTL- 8 266.5 249.7
244.6 248.6 252.7 260.1 CEBPA (0 h) 4. MTL- 8 266.5 253.4 244.3
246.2 250.3 262.0 CEBPA (24 h)
Biochemical Analysis
[0573] Single i.p, injection of TAA resulted insignificant increase
in most of the liver function parameters such as ALT on day 1
(p<0.05) and day 2 (p<0.001), AST on day 1 and day 2
(p<0.001), GGT on day 3 (p<0.05), bilirubin on day 3
(p<0.05) and significant decrease in other parameters such as
albumin on day 1 (p<0.01), day 2 (p<0.001), day 3 (p<0.05)
and ammonia on day 1 and day 2 (p<0.001) when compared with its
basal readings.
[0574] Prophylactic, concurrent but not preventive MTL-CEBPA
injection showed significant improvement in the liver function
parameters such as ALT (p<0.05), AST (p<0.05), ALP
(p<0.001), GGT (p<0.05), bilirubin (p<0.05) and other
parameters such as total protein (p<0.01), albumin (p<0.01)
and ammonia (p<0.01) when compared with TAA control on different
days after treatment (FIG. 25A-25H).
Discussion and Conclusion
[0575] In this study, acute liver failure model was established in
SD rats by administration of TAA injection intraperitoneally. No
mortality was observed in any of the groups after TAA injection.
TAA injection resulted in significant changes in the liver
biochemical parameters such as, AST, ALT, Bilirubin, GGT, Total
Protein, Albumin, and Ammonia when compared to its basal levels as
observed in pathological control (TAA control) animals. Significant
decrease in body weight was also observed in TAA control animals.
MTL-CEBPA treatment showed significant improvement in the LFT
parameters and ammonia when it was injected prophylactically (-24
hrs) and concurrent (0 hrs) of TAA injection.
Example 13. In Vivo Studies of Treating Diabetes
[0576] Given the role of CEBPA in the regulation of glucose
metabolism, a study was conducted in a rat model of diabetes to
determine if CEBPA activation could improve clinically relevant
blood parameters. Type II diabetes was induced in six wistar rats
by high fat diet. The rats were then treated with a total of 4.35
mg/kg CEBPA-saRNA (AW1-50) or non-targeting FLUC siRNA formulated
in a NOV340 liposome. 6 days after last treatment, blood was drawn
and the animals were sacrificed to assess changes in serum
chemistry and weight. Experimental
Groups and Doses:
TABLE-US-00030 [0577] Group Test article Dose Control NOV340-siFLUC
1.45 mg/kg, 3 doses Treatment NOV340-CEBPA 1.45 mg/kg, 3 doses
(i.e., MTL-CEBPA)
[0578] As shown in FIG. 26A-26N, compared to control, rats treated
with NOV340-CEBPA showed significant decreases in liver
cholesterol, serum AST, fasting glucose, and the ratio of
triglycerides to HDL-c. They also had a significant reduction in
body weight as well as the ratio of liver to body weight (FIG. 26L
and FIG. 26N). Insulin level increases with CEBPA-saRNA treatment
(FIG. 26K). The results indicate that CEBPA upregulation with
CEBPA-saRNA may be beneficial for the management of diabetes.
CEBPA-saRNA may also be used for treating fatty liver disease and
insulin resistance.
Example 14. In Vivo Studies of Treating NASH
[0579] Nonalcoholic steatohepatitis (NASH) is liver inflammation
and damage that may be caused by a buildup of fat in the liver. It
is part of a group of conditions called nonalcoholic fatty liver
disease. NASH may cause scarring of the liver, which may lead to
cirrhosis. The effect of MTL-CEBPA in treating NASH is studied
using animals fed with methionine choline deficient (MCD) diet. The
MCD diet results in liver injury similar to NASH.
[0580] In this study, CEBPA-saRNA was used to treat MCD-induced
NASH in C57BL/6 mice. The length of the study was 6 weeks. Male 7-8
weeks-old C57B/L6 mice were randomized based on body weight on Day
0. Group 1 had normal diet for 4 weeks, and Group 2 for 6 weeks.
Group 3 had MCD diet for 4 weeks, and Groups 4-8 for 6 weeks. At
week 4, treatment groups (Groups 4-8) were randomized based
bilirubin, body weight and ALT levels. Groups 4-8 received PBS
treatment or therapeutic treatment via i.v. injection twice weekly
(week 4, 4.5, 5 and 5.5).
[0581] At week 4, Group 1 and 3 were terminated. At week 6, Group 2
and Groups 4-8 were terminated. Liver function tests (LFT) (ALT,
AST, ALP, albumin, total bilirubin & liver triglyceride (TG))
and histopathology of liver (H&E stain, oil red O staining, and
Masson trichrome) were conducted. Serum cytokines/markers
(IL1.beta., IL6 and TNF-.alpha.) & .alpha.2 Macroglobulin were
measured.
[0582] Studies groups and description of treatment were summarized
below:
TABLE-US-00031 No. of GroupNo. Groups animals Treatment Dose
Regimen/ROA G1 Normal diet control (4 5 -- N/A weeks), terminated
at week 4. G2 Normal diet control (6 5 -- N/A weeks), terminated at
week 6. G3 MCD diet control (4 10 -- N/A weeks), terminated at week
4. G4 MCD diet control (6 10 PBS Treatment Twice weekly, i.v
injection, weeks) + PBS treatment, from week 4 to week 6 G5 MCD +
NOV340/siFLUC 10 NOV340/siFLUC Twice weekly, i.v injection, control
3 mpk from week 4 to week 6 3 mpk therapeutic. G6 MCD + MTL-CEBPA
10 MTL-CEBPA Twice weekly, i.v injection, 0.3 mpk Therapeutic. 0.3
mpk from week 4 to week 6 G7 MCD + MTL-CEBPA 10 MTL-CEBPA Twice
weekly, i.v injection, 1 mpk Therapeutic. 1 mpk from week 4 to week
6 G8 MCD + MTL-CEBPA 10 MTL-CEBPA Twice weekly, i.v injection, 3
mpk Therapeutic. 3 mpk from week 4 to week 6
[0583] FIG. 27A-27B showed body weight and feed consumption
changes. As expected, MCD diet treatment showed significant
reduction in body weight and feed consumption throughout the study.
Treatment groups (G5-G8) showed no significant changes in body
weight and feed intake compared to MCD diet control. Hence,
MTL-CEBPA treatment did not change body weight and feed
consumption. FIG. 27C-27G showed LFT results including ALT, AST,
ALP, bilirubin, and albumin level changes. Animals fed with MCD
diet showed significant increase in LFT parameters such as ALT,
AST, bilirubin and reduction in protein levels when compared with
normal diet control. Treatment with MTL-CEBPA showed significant
reduction in ALT and AST levels. Reduction was also observed in ALP
& bilirubin with MTL-CEBPA treatment. FIG. 27H showed liver
triglyceride (TG) level changes. Liver TG was significantly
increased in the MCD diet control group. Treatment with MTL-CEBPA
showed significant reduction in liver TG levels, reversing liver
TGs to normal levels.
[0584] Therefore, CEBPA-saRNA treatment may be used to for treating
NASH.
Example 15. Other In Vivo Studies with CEBPA-saRNA--Evaluation of
MTL-CEBPA Efficacy in a Rat Model of DEN-Induced HCC
[0585] The purpose of this study was to investigate if activation
of CEBPA by treatment with MTL-CEBPA would improve clinical
parameters in a rat model of HCC.
[0586] Experimental Design: Male Wistar rats were treated with DEN
to induce HCC. Briefly, the animals were treated for 9 weeks with
DEN followed by 3 treatment-free weeks. Animals were then
randomized into three groups according to body weight (6 to 7
males/group). Group 1 was sacrificed on Day 1 to serve as the
pre-treatment control and groups 2 and 3 were treated i.v. 3 times
(Day 1, 3, and 5) with either a non-targeting dsRNA formulated in
NOV340 (siFLUC) or MTL-CEBPA at a dose of 4 mg/kg. On Day 12, blood
was drawn and all animals were sacrificed. Tumour and liver weights
were measured and sections of liver tissue were immediately
flash-frozen for mRNA analysis. CEBPA and albumin mRNA levels were
determined by qRT-PCR (housekeeping gene: GAPDH; measured in
triplicates)
[0587] Results: As shown in FIG. 28, compared to NOV340/siFLUC
(non-targeting liposome control, vehicle-control), animals treated
with MTL-CEBPA showed a significant increase in CEBPA mRNA
expression in the liver. A trend for increased albumin mRNA
expression was observed, this was not statistically
significant.
[0588] As shown in FIG. 29A-29I, compared to the NOV340/siFLUC
control, rats treated with MTL-CEBPA showed a significant decrease
in serum ammonia (p<0.05), as well as changes in body weight
(bw), tumour volume, cholesterol, and haemoglobin that were
trending towards statistical significance (bw: p=0.063; tumour
volume: p=0.10; cholesterol: p=0.08; hemoglobin: p=0.05). Compared
to pretreatment control, rats treated with MTL-CEBPA showed a
significant decrease in AST, ALT, and bilirubin (p<0.05).
[0589] Conclusion: In the DEN-induced rat model of HCC, MTL-CEBPA
treatment resulted in target engagement (upregulation of CEBPA mRNA
in the liver) and an improvement in several disease markers,
including haemoglobin, ammonia, and cholesterol, when compared to
NOV340/siFLUC control. Although not statistically significant due
to small animal numbers, the group treated with MTL-CEBPA showed a
trend toward tumour growth inhibition with a mean tumour size
approximately 80% smaller than the NOV340/siFLUC control. As seen
in comparison to the pretreatment disease control, MTL-CEBPA not
only stabilized disease symptoms but reversed some liver toxicity
serum markers, including AST, ALT and bilirubin, consistent with
the benefits seen in these markers in the CCL4 fibrosis model.
Taken together, these results indicate that MTL-CEBPA can improve
liver function and reduce tumour growth in the widely used rat
model of DEN-induced liver fibrosis and HCC.
Example 16. Studies of CEBPA-saRNA Interactions with Ago
Proteins
[0590] HepG2 cells were transfected with Biotinylated antisense
strand (AS) and sense strand (ss)-CEBPA51 and compared to
untransfected or scramble-biotin control. At harvest point (72 hr)
Biotinylated conjugates were cross-linked with 1% formaldehyde
followed by immobilization on streptavidin agarose beads.
Co-immunoprecipitation (Co-IP) with anti-Ago1, Ago2, Ago3 and Ago4
was then performed. Isotype IgG was used as a negative control.
Co-immunoprecipitated conjugates were then immobilized with
Dynabead-Protein-G. The pulldown immune complex was washed and
eluted on a magnetic column. Samples were then separated on
SDS-PAGE and transferred onto PVDF for Western blotting against the
respective ARgonaute antibody.
[0591] As shown in FIG. 30A, Ago2 appears strongly on the AS-biotin
strand compared to the SS-biotin strand. Ago1, 3 and 4 do not
appear to be present on either strand. This indicates anti-sense
strand of CEBPA-saRNA associates with Ago2 and not the other
Argonautes.
[0592] In a further study, Ago2 was knocked out in mouse embryonic
fibroblasts (MEF) cells. Wild-type and Ago2 knock-out cells were
seeded in 24 well plates at 9.8.times.10.sup.5 per well. 20 nM of
CEBPA51 and Fluc were transfected as previously described
(forward+reverse). RNA was harvested to determine activity of saRNA
at 48 hour time point. As shown in FIG. 30B, CEBPA transcript
levels increased 2-fold in CEBPA51 transfected wild type cells vs
Fluc. FIG. 30C showed that p21 transcript levels increased 4-fold
in CEBPA51 transfected wild type cells vs Fluc. However, no CEBPA
or p21 induction was measured in Ago2 knock-out cells. It is
demonstrated that Ago2 is required for gene activation by
saRNA.
Example 17. Formulation of CEBPA-saRNA
[0593] CEBPA-51 saRNA is encapsulated into liposomes. The delivery
technology used is the NOV340 SMARTICLES.RTM. technology owned by
Marina Biotech. The lipid components of these nanoparticles are
comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
cholesteryl-hemi succinate (CHEMS), and
4-(2-aminoethyl)-morpholino-cholesterol hemi succinate (MOCHOL).
NOV340 consists of POPC, DOPE, CHEMS and MOCHOL in the molar ratio
of 6:24:23:47. The nanoparticles are anionic at physiological pH,
and their specific lipid ratio imparts a "pH-tunable" character and
a charge to the liposomes, which changes depending upon the
surrounding pH of the microenvironment to facilitate movement
across physiologic membranes. SMARTICLES.RTM. nanoparticles are
sized to avoid extensive immediate hepatic sequestration, with an
average diameter of approximately about 50-about 150 nm, or about
100-about 120 nm, facilitating more prolonged systemic distribution
and improved serum stability after i.v. injection leading to
broader tissue distribution with high levels in liver, spleen and
bone marrow reported.
[0594] Sequence of CEBPA-51, Sense and Antisense (also shown in
FIG. 39):
TABLE-US-00032 saRNA name CEBPA-51 Total base: 21 mer, including
base modifications mer 3 6 9 12 15 18 21 Sense strand bmGmCG mGUC
AUU mGUC AmCU GGU CmUmU 5` .fwdarw. 3` (SEQ ID No. 128)
Complementary mUmUC GCC AGU AAC AGU GAC CAG antisense strand 3`
.fwdarw. 5` (SEQ ID No. 129) Definition of symbols: A, U, G, C are
2`-OH ribonucleotides, mU, mG, mC are 2`-O-methyl ribonucleotides,
b = inverted abasic sugar cap (additional modification).
[0595] Each strand of CEBPA-51 is synthesized on a solid support by
coupling phosphoramidite monomers sequentially. The synthesis is
performed on an automatic synthesizer such as an Akta Oligopilot
100 (GE Healthcare) and a Technikrom synthesizer (Asahi Kasei Bio)
that delivers specified volumes of reagents and solvents to and
from the synthesis reactor (column type) packed with solid support.
The process begins with charging reagents to the designated
reservoirs connected to the reactor and packing of the reactor
vessel with the appropriate solid support. The flow of reagent and
solvents is regulated by a series of computer-controlled valves and
pumps with automatic recording of flow rate and pressure. The
solid-phase approach enables efficient separation of reaction
products as coupled to the solid phase from reagents in solution
phase at each step in the synthesis by washing of the solid support
with solvent.
[0596] General overview of CEBPA-51 synthesis is shown in FIG.
40A.
[0597] A detailed flow chart of CEBPA-51 synthesis is shown in FIG.
40B.
[0598] The general size and purity of CEBPA-51 present in solution
is determined by size exclusion chromatography (SE-HPLC), mainly to
differentiate between double and single strand versions. The
melting point of the CEBPA-51 double strand is sequence specific.
It is determined as the inflection point in the UV (at 260 nm)
versus T (.degree. C.) curve created during heat induced `melting`
(dehybridisation) of the duplex. This T.sub.m value was determined
to be at 81.3.degree. C., in connection with the increased
absorption at 260 nm (hyperchrome effect). Extinction coefficient
has been determined in PBS at 260 nm and 25.degree. C., based on
.gtoreq.90% content oligonucleotide as sodium salt. Molecular mass
is determined for both single strands by LC-MS during the
manufacturing process. For the release test, the duplex was
separated into single strands and each peak was analyzed by MS
which is performed by a combination of IPRP-HPLC with ESI-MS.
Impurities
[0599] Product-Related Impurities:
[0600] Potential product-related impurities are multimers,
aggregates, as well as extended or truncated/degraded forms. These
are controlled by SE-HPLC.
[0601] Furthermore and as a result of incomplete or inefficient
synthesis, polymeric by-products can occur which differ by lacking
e.g. n-1 or n-2 nucleotides (with "n=21" i.e. 20-mers, or 19-mers
instead of 21-mers for full chain length of sense and antisense
strands). Also sequence extension by 1 or 2 nucleotides can occur,
resulting in n+1 or n+2 oligonucleotides (22- or 23-mers). The
latter however with lower probability. These variants cannot be
identified by SE-HPLC due to limited resolution, but can be
determined by IPRP-HPLC.
[0602] Furthermore, also mis-incorporation or modification of
ribonucleotides may occur also leading to product-related
impurities. The latter are either detected by ion pair
reversed-phase high-pressure chromatography (IPRP-HPLC) MS or
MS/MS-sequencing.
[0603] Process-Related Impurities:
[0604] Potential process-related impurities include residual
reagents, reactants and solvents from chemical synthesis. Based on
the given RNA synthesis on solid-phase and reagents used in the
production process the following process related impurities can be
expected (Table 14):
TABLE-US-00033 TABLE 14 Process-related Impurities of CEBPA-51
Production Residual Solvent Origin Actual Results Acetonitrile
(class 2) synthesis n.d.* (<410 ppm) DMSO--Dimethylsulfoxide
(class 3) synthesis n.d.* (<5000 ppm) Toluene (class 2)
synthesis n.d.* (<890 ppm) TEA, Triethylamine synthesis (<320
ppm) *n.d. = "not detected" (below LoQ)
[0605] Formulations
[0606] The required amount of CEBPA-51 is dissolved at ambient
temperature in sodium acetate/sucrose buffer pH 4.0 and the
required amounts of lipids are dissolved in absolute ethanol at
55.degree. C. Liposomes are prepared by crossflow ethanol injection
technology. Immediately after liposome formation, the suspension is
online diluted with sodium chloride/phosphate buffer pH 9.0. The
collected intermediate product is extruded through polycarbonate
membranes with a pore size of 0.2 .mu.m. The target saRNA
concentration is achieved by ultrafiltration. Non-encapsulated drug
substance and residual ethanol is removed by subsequent
diafiltration with sucrose/phosphate buffer pH 7.5. Thereafter, the
concentrated liposome suspension is 0.2 .mu.m filtrated and stored
at 5.+-.3.degree. C. Finally, the bulk product is formulated, 0.2
.mu.m filtrated and filled in 20 ml vials.
[0607] MTL-CEBPA is presented as a concentrate solution for
infusion. Each vial contains 50 mg of CEBPA-51 (saRNA) in 20 ml of
sucrose/phosphate buffer pH about 7.5.
[0608] The composition of MTL-CEBPA is provided in Table 15
below.
TABLE-US-00034 TABLE 15 Qualitative and quantitative composition of
MTL-CEBPA (2.5 mg/ml) Quantity Name of Ingredient Function
Reference (per ml) CEBPA-51 (saRNA) Active pharmaceutical
Manufacturer's 2.5 mg/ml ingredient specifications
1-palmitoyl-2-oleoyl-sn-glycero-3- Membrane forming lipid
Manufacturer's 4.65 mg/ml phosphocholine (POPC) specifications
1,2-dioleoyl-sn-glycero-3- Membrane forming Manufacturer's 18.0
mg/ml phosphoethanolamine (DOPE) fusogenic lipid specifications
Cholesteryl hemisuccinate (CHEMS) Anionic ampotheric lipid
Manufacturer's 11.3 mg/ml specifications Cholesteryl-4-[[2-(4-
Cationic amphoteric lipid Manufacturer's 27.0 mg/ml
morpholinyl)ethyl]amino]-4-oxobutanoate specifications (MOCHOL)
Sucrose Cryoprotectant, BP, JP, NF, EP 92.4 mg/ml osmolality
control Disodium hydrogen phosphate, dihydrate Buffer pH adjustment
BP, USP, EP 1.44 mg/ml Potassium dihydrogen phosphate Buffer pH
adjustment EP, BP, NF 0.2 mg/ml Potassium chloride (KCl) Ionic
strength adjuster EP, BP, USP 0.2 mg/ml Water for injection (WFI)
Solvent WFI (USP, EP) qs 1 ml
[0609] MTL-CEBPA is supplied in the form of a suspension and will
be packaged in 20 mL glass vials with stopper. To ensure that 20 ml
can be withdrawn from the primary container by syringe, there is an
overfill of 20.6 ml (equivalent to 21.4 g). There is no
manufacturing overage. The formulation is:
TABLE-US-00035 Quantity per mL Quantity per vial MTL-CEBPA 2.5 mg
50 mg
[0610] Excipients
[0611] The excipients in MTL-CEBPA can be categorized into two
groups: the liposome-forming lipid excipients
(NOV340-Smarticles.RTM. technology owned by Marina Biotech) and the
buffer forming excipients sucrose and phosphate-salts (also refer
to Table 15). The development of the liposomes and their
composition is described by Andreakos E. et al., Arthritis Rheum,
vol. 60(4): 994-1005 (2009), the contents of which are incorporated
herein by reference in their entirety. The used
sucrose-phosphate-buffer, pH 7.5, is known to have good
compatibility with the excipients and the drug substance.
[0612] The liposme-forming lipid excipients are comprised of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine (DOPE),
cholesteryl-hemi succinate (CHEMS), and 4-(2-aminoethyl)
morpholino-cholesterol hemisuccinate (MOCHOL) in a molar ratio of:
6:24:23:47 as shown in Table 16 below.
TABLE-US-00036 TABLE 16 Lipid Components of NOV340 Molecular Mass
Abbr. Name Chemical Name Formula [g/mol] POPC 1-Palmitoyl
-2-oleoyl-sn- C.sub.42H.sub.82NO.sub.8P 760.08
glycero-3-phosphocholine DOPE 1,2-Dioleoyl-sn-glycero-3-
C.sub.41H.sub.78NO.sub.8P 743.55 phosphoethanolamine CHEMS
3.beta.-Hydroxy-5-cholestene3- C.sub.31H.sub.50O.sub.4 486.37
hemisuccinale 5- cholesten.cndot.3.beta.-ol 3-hemisuccinate MOCHOL
4-(2-Aminoethyl) morpholino- C.sub.37H.sub.62N2O.sub.4 598.90
cholesterol hemisuccinate Solvent: Ethanol, lipid ratios:
POPC:DOPE:CHEMS:MOCHOL in a molar ratio of: 6:24:23:47
[0613] A general overview of MTL-CEBPA production process is shown
in FIG. 31. A detailed process of MTL-CEBPA production (Steps 1-9)
is shown in FIG. 41.
[0614] MTL-CEBPA was prepared by first dissolving the lipids in
ethanol and dissolving CEBPA-51 separately in sodium
acetate/sucrose buffer pH 4.0 (Step 1a and Step 1b).
[0615] The two solutions are then combined through an injection
process to form the primary vesicles, after which the ethanol
concentration is rapidly decreased and the pH rapidly increased by
the addition of the pH adjustment buffer (sodium chloride/phosphate
buffer pH 9.0) (Step 2).
[0616] The intermediate liposome mixture is then extruded through a
polycarbonate membrane to reduce the size of large particles and
aggregates (Step 3).
[0617] The bulk mixture is then concentrated by ultrafiltration,
and the buffer is exchanged against at least 7 volumes of a
sucrose/phosphate buffer pH 7.5 to reduce the concentration of
ethanol and salts (Step 4). This bulk product is then passed
through a 0.2 .mu.m filter for bioburden reduction (Step 5). The
product is adjusted to the final target concentration and
sterilized by 0.2 .mu.m sterile filtration (Step 6). Subsequently
the bulk product is filled into sterile vials (Step 7). The filled
vials are tested for final release (Step 8). Current in-process
controls are depicted in the respective figures below. Final
release of the drug product according to the specifications defined
in this document is performed on the drug product.
[0618] After completion of the process, including final vialling
and release, the entire DP vials are stored at or below -20.degree.
C. (Step 9).
[0619] Lipid to drug ratio at the point of liposome formation is of
major importance for the encapsulation efficiency of RNA within
liposomes, as CEBPA-51 solubilzed in a pH 4.0 buffer interacts with
MOCHOL, which is positively charged in this environment. To
optimize the encapsulation yield, lipid concentration in the EtOH
solution was kept constant and the concentration of CEBPA-51 in the
solution was altered ranging from 1.06 up to 3.44 mg/ml. The
obtained results clearly showed a trend indicating that by
decreasing the CEBPA-51 concentration in the solution, there is a
slight increase in the encapsulation efficiency.
[0620] Container Closure System
[0621] The container closure system selected for MTL-CEBPA is a
standard 20 ml serum vial configuration with 20 mm closure. The
glass vials are made from clear USP Type I borosilicate glass,
which provides for good protection of the product with minimal
potential for leaching. This type of glass also has good thermal
stability, which is critical for storing frozen product. The
stopper is made from a standard chlorobutyl rubber compound, and
product-contacting surfaces are coated with a fluoropolymer which
minimizes the potential for product adsorption. Finally, the
aluminum crimp cap provides for a secure seal of the stopper to the
vial, and protects the external interface of the rubber septum from
potential contamination until the time of use.
[0622] Microbiological Attributes
[0623] MTL-CEBPA is provided as a sterile, single use vial. No
preservative is added to inhibit microbial growth. The standard
glass serum vial with rubber stopper and aluminum crimp cap
provides a well-proven barrier to prevent microbial
contamination.
[0624] The bioburden of MTL-CEBPA is reduced by filtration through
a 0.2 .mu.m filter. Immediately prior to filling into sterile
vials, the product is sterilized by passing through a
sterilization-grade 0.2 .mu.m filter (Step 6, DP). All packaging
components are provided sterile "ready to use". They are handled
under aseptic conditions in an ISO class 5 environment without
additional treatment.
[0625] MTL-CEBPA is stored frozen at -20.degree. C..+-.5.degree. C.
and protected from light. Shipping is performed in a cooler packed
with dry ice. MTL-CEBPA is stable when stored at -20.degree. C. for
up to 6 months, showing no trend for slight decrease or changes
(within the range of analytical variability). A shelf life of 12
months for material stored at -20.degree. C. is proposed since a
rapid decrease of material quality under frozen conditions is
considered very unlikely.
[0626] MTL-CEBPA is administered by i.v. infusion (250 mL).
MTL-CEBPA at a concentration of 2.5 mg/mL saRNA is thawed at room
temperature before diluting the suspension in 0.9% Normal Saline
for Intravenous Use in order to obtain a volume of 250 mL
regardless of the concentration.
[0627] The test item and diluent are mixed together by manual
inversion of the infusion bag (to avoid foaming).
[0628] MTL-CEBPA is administered at a constant rate over 60 minutes
into a vein (peripheral or central) using an infusion pump.
[0629] Storage of prepared solution: Room temperature (15 to
25.degree. C.) with a maximum expected in-use shelf life of at
least 6 hours.
[0630] The properties of CEBPA-51 saRNA and MTL-CEBPA are shown in
Table 17-1 and Table 17-2: Generally, MTL-CEBPA is a milky white
suspension. saRNA encapsulation measured by fluorescence detection
is .gtoreq.75%. Particle size measured by dynamic light scattering
is between about 50 nm to about 150 nm or about 100 to about 140
nm. Polydispersity index measured by dynamic light scattering is
.ltoreq.0.200. Zeta potential measured by dynamic light scattering
is .ltoreq.-30.0 mV at pH 7.2-7.8. pH value measured by
potentiometric is between about 7.2 to about 7.8. Osmolality
measured by freezing point depression is between about 280 to about
400 mOsmol/kg. Impurities saRNA measured by RP-HPLC is
.ltoreq.15%.
[0631] saRNA encapsulation: The total and "external" free saRNA
contents are quantitated by measuring fluorescence intensity from
RiboGreen and saRNA complexation to determine the % encapsulation.
The drug product sample solutions are analyzed under two different
conditions, untreated samples for external saRNA and samples
treated with Triton X-100 for total saRNA. The content of RNA is
determined using a calibration curve generated from a standard with
known concentration.
[0632] The percent content of encapsulated saRNA is calculated:
E ( % ) = C T - C F C T 100 E ( % ) = ( C T - C F ) 100 / C T
##EQU00001## E ( % ) % encapsulated saRNA ##EQU00001.2## C T total
content of saRNA ##EQU00001.3## C F content free ( external ) saRNA
##EQU00001.4##
[0633] Particle size and polydispersity index: The size of
liposomes and PDI are determined by Photon Correlation Spectroscopy
(PCS) using a Zetasizer Nano ZS instrument (Malvern).
[0634] Zeta potential: The surface potential is determined by Laser
Doppler Velocity/Laser Doppler Anemometry (LDV/LDA) using a
Zetasizer Nano ZS instrument (Malvern).
[0635] pH: The pH of liposomal drug product is measured at
20-25.degree. C. using a glass electrode.
[0636] Osmolality: The determination of osmolality is based on the
principle of freezing point depression in comparison to pure water
using an Osmomat 030 (Gonotec).
[0637] Residual ethanol: Residual ethanol in liposomal curcumin
drug product is quantified by head-space gas chromatography using a
flame ionization detector (GC/FID).
[0638] Impurities saRNA: The content (area %) of saRNA impurites is
quantified by ion pair-reversed phase (IPRP) HPLC using a
Waters)(Bridge C18 column (4.6.times.100 mm, 3.5 .mu.m particle
size). Nanoparticles are disrupted with 2% Triton X-100 buffer and
released saRNA is separated on the HPLC column using a a gradient
of 100 mM hexafluoroisopropanol (HFIP)/7 mM triethylamine (TEA) in
water and 100% methanol. RNA is detected at 260 nm. The content of
impurities (area %) is determined by subtracting the peak areas (%)
of the main strands (anti-sense, sense strand) from the total peak
area (100 area %).
TABLE-US-00037 TABLE 17-1 Properties of CEBPA-51 Specifications
Analytical Tests Single Strand Duplex Results Appearance A white to
pale Conforms yellow powder Identification (LC-MS) Sense 6920 .+-.
3 Da 6920 .+-. 3 Da 6920 Da Antisense 6723 .+-. 3 Da 6723 .+-. 3 Da
6722 Da Purity (RP-IP HPLC, area %) Sense NLT 90% 96.90% Antisense
NLT 90% 90.50% Duplex NLT 90% 92.60% Purity (/EX HPLC, area %)
Sense NLT 85% 94.60% Antisense NLT 85% 92.20% Duplex NLT 85% 94.30%
Purity (SEC NLT 90% 99.5% HPLC, area %) Bio burden NMT 100 30 CFU/g
CFU/g Bacterial NMT 1 EU/mg <0.25 EU/mg Endotoxins Water
contents (Karl-Fisher) NMT 10% 5% Sodium content Report 5.6% w/w
(/GP-MS) (Anhydrous Basis) Oligonucleotide Report 1040 .mu.g/mg
Content by UV (Anhydrous Basis)
TABLE-US-00038 TABLE 17-2 Properties of MTL-CEBPA Test Method
Result Appearance milky, white Total saRNA 2.50-2.56 mg/ml saRNA
encapsulation 83%-85% Content MoChol 24.9-26.9 mg/ml Content DOPE
17.3-18.4 mg/ml Content CHEMS 11.0-11.5 mg/ml Content POPC 4.7-4.8
mg/ml Content cholesterol 0.9-1.0 mg/ml Particle size (z-average)
107 nm-112 nm Polydispersity index 0.160-0.169 Zeta potential
-35.6--39.1 mV saRNA impurities 5.9%-6% Residual ethanol
.ltoreq.0.5% mV pH 7.4-7.6 Osmolality 349 mOsmol/kg Subvisible
particles .gtoreq.10 .mu.m: 2 particles/container .gtoreq.25 .mu.m:
<1 particle/container Extractable volume pass Endotoxin <0.5
EU/ml Sterility Pass/no growth
Example 18. Immunosafety Study of CEBPA-51 in Primary Human
PBMCs
[0639] Aim of Study: The objective of this study was to assess the
immunosafety of CEBPA-51, as measured by the activation of TLR
pathways ex vivo in human blood cells.
[0640] Experimental Design: The induction of cytokines by CEBPA-51
was tested with peripheral blood mononuclear cells isolated from
two human donors (huPBMCs).
[0641] TNF-.alpha.: The huPBMCs were transfected in triplicates
with 133 nM of CEBPA-51 or control sequence RD-01010 (positive
control) and RD-01011 (negative control) using Dotap as a
transfection reagent. Transfection reagent alone was used as mock
control. In addition, controls ODN2216 (CpG-oligonucleotide) and
RD-01002 (cholesterol-conjugated siRNA) were added directly at a
concentration of 500 nM without transfection. After 20 hours of
incubation the supernatants from the triplicate transfections were
pooled and TNF-.alpha. secretion was measured using a commercial
human TNF-.alpha. ELISA assay (samples measured in duplicates).
[0642] IFN-.alpha.: HuPBMCs were transfected in triplicates with
133 nM CEBPA-51 or control sequences RD-01010 (positive control)
and RD-01011 (negative control) using Geneporter-2 as a
transfection reagent. Transfection reagent alone was used as mock
control. In addition, controls ODN2216 (CpG-oligonucleotide) and
RD-01002 (cholesterol-conjugated siRNA) were added directly at a
concentration of 500 nM without transfection. After 20 hours of
incubation the supernatants from the triplicate transfections were
pooled and IFN-.alpha. secretion was measured using a commercial
human IFN-.alpha. ELISA assay (sample measured in duplicate.
[0643] Results: The secretion of cytokines TNF-.alpha. and
IFN-.alpha. by human PBMCs was measured after transfection with
CEBPA-51 or control oligos. CEBPA-51 elicited no significant
secretion of either cytokine into the cell culture media after
incubation for 20 hours whereas the positive controls triggered the
expected cytokine release (see FIGS. 32A and 32B).
[0644] Conclusion: The CEBPA-51 did not trigger activation of the
TLR-8 or TLR7/9 pathways in human PBMCs, as indicated by the lack
of cytokine TNF-.alpha. and IFN-.alpha. release following
transfection of CEBPA-51. These results suggest that the chemically
modified saRNA does not have immune-stimulatory activity.
Example 19. Phase I Study and Selection of a Safe Starting Dose
[0645] Proposed First-in-Human Clinical Trial
[0646] The FIH study will be a multi-centre, open-label, Phase I
clinical study with RNA oligonucleotide MTL-CEBPA to investigate
its safety and tolerability in patients with advanced liver
cancer.
[0647] Indication
[0648] Treatment of patients with histologically advanced cancer
characterised by hepatocellular carcinoma or advanced stage cancer
presenting with secondary liver tumours derived from extra hepatic
primary cancer types who are considered unfit for any therapy or
surgery, or are progressing following loco-regional therapy and
sorafenib.
[0649] Study Objectives
[0650] Primary Objective:
[0651] To determine the safety and tolerability of weekly
administration of MTL-CEBPA for 3 weeks to participants with
histologically advanced cancer characterised by hepatocellular
carcinoma or advanced stage cancer presenting with secondary liver
tumours derived from extra hepatic primary cancer types.
[0652] Secondary Objectives:
[0653] To determine the Recommended Phase 2 Dose (RP2D) of
MTL-CEBPA; to characterise the pharmacokinetics (PK) parameters of
MTL-CEBPA; to assess the pharmacodynamic (PD) process of MTL-CEBPA
notably the characterisation of MTL-CEBPA effect on serum albumin
and bilirubin; to assess changes in health-related quality of life
in HCC patients following administration of MTL-CEBPA.
[0654] Biomarkers
Biomarker Strategy
[0655] Predictive Biomarkers
Inclusion/Exclusion Biomarkers
[0656] To date, no predictive biomarkers or gene signatures have
been identified for MTL-CEBPA. Therefore, patient eligibility
criteria for the study do not include such biomarkers.
Exploratory Predictive Biomarker
[0657] Preclinical data with CEBPA-51 and tool compounds and the
scientific literature suggest multiple biomarker hypotheses for HCC
response to saRNA against CEBPA. For instance, tumour cell growth
arrest in response to CEBPA saRNA may depend on the basal level of
CEBPA expression. Certain regulatory mechanisms that inactivate
C/EBP-.alpha. protein may result in resistance to CEBPA saRNA,
including for instance dephosphorylation of Ser193 as a consequence
of PI3K-AKT pathway activation or overexpression of
dominant-negative forms of C/EBP-.beta..
[0658] The study does not include prospective exploratory
biomarkers of tumour response. However, retrospective analyses of
biomarkers of tumour sensitivity/resistance may be conducted based
on archived biopsies.
[0659] No definitive biomarker hypotheses have been formulated to
date for responsiveness of fibrotic or cirrhotic liver to
MTL-CEBPA, although certain endocrine loops, e.g. via insulin and
TNF-.alpha., are known to modulate CEBPA activity. No prospective
analyses of predictive biomarkers are currently envisioned;
however, retrospective analyses may be conducted on archived
tissues.
[0660] Response Biomarkers
Target Engagement
[0661] The molecular target of CEBPA-51 saRNA is the CEBPA
promoter. Upregulation of CEBPA transcription can be measured in
tissue samples via qRT-PCR of CEBPA mRNA or via immunostaining for
C/EBP-.alpha. protein.
[0662] Several exploratory approaches will be pursued to measure
changes in CEBPA expression and establish proof-of-mechanism.
During the expansion phase, circulating tumour cells (CTC) will be
collected pre/post-treatment for immunostaining of C/EBP-.alpha.
protein levels. If we obtain sufficient tissue from any matched
pre/post-treatment biopsies we would aim to look at CEBPA mRNA
levels by the qRT-PCR method used in the preclinical models, as
well as at C/EBP-.alpha. protein levels by immunostaining. The use
of surrogate tissues such as WBCs has not been validated to date
but will be explored as another potential option for demonstrating
target engagement that may be introduced once the MTD is
established.
PD Biomarkers
[0663] PD biomarkers include liver-specific genes, and their
respective protein products, that are under direct transcriptional
control of C/EBP-.alpha. (proximal biomarkers), as well as
downstream gene targets and proteins that are markers of
C/EBP-.alpha.-dependent differentiation or proliferation programs
(distal biomarkers). Several proximal biomarkers of C/EBP-.alpha.
are secreted proteins that can be monitored in serum including, for
instance, albumin, AFP, transferrin, and coagulation factors.
[0664] Serum albumin was selected as the primary liver-specific PD
biomarker because of the ease of sampling and the availability of
validated clinical assays. Other serum-based biomarkers are under
investigation as additional exploratory PD biomarkers.
[0665] Depending on availability of pre/post-treatment tumour
biopsies, changes in the cell cycle regulatory protein p21 will be
assessed in tumour sections by IHC, as a marker of tumour cell
growth arrest.
Surrogate Efficacy Biomarkers
[0666] Serum albumin and total bilirubin levels are secondary
endpoints in this study. Albumin and bilirubin are validated
biomarkers of overall liver function status. In addition, a
combined measure of albumin and bilirubin serum levels (ALBI grade)
has been shown to correlate with survival in patients with advanced
HCC and liver disease. Both, serum albumin and bilirubin have been
shown to respond to MTL-CEBPA treatment in preclinical models of
liver disease, with improvements in albumin compared to control
groups and near normalization of bilirubin levels.
[0667] CTCs and circulating DNA will be collected during the
expansion phase as exploratory biomarker of tumour response. Serum
alpha-fetoprotein (AFP) is a validated marker of HCC burden.
However, the AFP gene is also under the control of C/EBP-.alpha. in
normal liver, with MTL-CEBPA treatment expected to increase AFP
serum levels. Because of this opposite response in liver and
tumour, AFP levels will need to be interpreted with caution.
Efficacy Biomarkers
[0668] Liver function status will be assessed by serum chemistry
including, albumin, total protein, bilirubin, ALP, GGT, ALT, AST
and ammonia levels. Tumour responses to MTL-CEBPA treatment will be
monitored by CT or Mill and assessed using standard criteria
(RECIST).
Drug PK and Biodistribution
[0669] ADME monitoring of MTL-CEBPA will be limited to
determination of total API levels in plasma. Because of the rapid
metabolism and clearance of free dsRNA, total API levels will
primarily represent encapsulated CEBPA-51. The nanoparticle and its
lipid components will not be monitored separately. It is assumed
that the levels of intact nanoparticles in circulation are
proportional to the levels of total API. Tissue biodistribution and
metabolites of CEBPA-51 will not be measured.
Biomarker Assays
[0670] All serum biomarkers will be assessed by local accredited
laboratories. Gene expression in biopsies will be measured by
validated Immunohistochemistry (IHC) assay.
[0671] A program-specific PK assay was developed to measure the
total API in plasma of participants. The assay is based on
heat-denaturing of CEBPA-51 to separate the two RNA strands,
followed by hybridization of the antisense strand to an immobilized
complementary PNA probe. As such the assay measures the
concentration of the antisense strand (and any of its metabolites
capable of hybridizing to the PNA probe), rather than the RNA
duplex itself. The assay was developed and validated for
non-clinical and clinical use by Axolabs GmbH, Germany.
[0672] Study Outcome Measures
[0673] Primary Outcome Measures
[0674] Continuous measurement of vital signs (incl. blood pressure,
pulse, body temperature, respiratory rate), ECG (12 lead) and,
safety laboratory data (incl. haematology, coagulation, clinical
chemistry, clotting and activation fragment of complement factor B
(Bb) and complement component 3a (C3a)) as well as the description
of both participant and investigator assessment of tolerability
will be collected.
[0675] All adverse events documented following the first MTL-CEBPA
infusion will be graded for seriousness, expectedness and
relationship to study drug as `unrelated`, `possible`, `probable`
or `definite`.
[0676] Safety and tolerability of MTL-CEBPA will be evaluated in
terms of frequency of adverse events graded according to toxicity
criteria (NCI CTCAE v 4.03) and categorised by body system and
diagnosis.
[0677] Secondary Outcome Measures
[0678] The plasma concentration of MTL-CEBPA will be analysed at
defined time points using hybridization-based HPLC-assay in order
to determine the Pharmacokinetic (PK) properties of MTL-CEBPA in
plasma after intravenous administration.
[0679] This protocol plans to collect measurements of surrogate PD
biomarkers including albumin, bilirubin, liver enzyme levels,
chemokines and tumour markers. Gene and protein expression levels
in tumour tissue will also support the determination of the
pharmacodynamics characteristics of MTL-CEBPA in participants with
liver tumours after intravenous administration.
[0680] Health-related quality of life questionnaire data will be
collected on Day 1, Day 15, week 8 and at EOS of part 1b using the
self-administered FACT-Hep questionnaire.
[0681] Study Design
[0682] This study is a multi-centre, open-label, first-in-human,
phase 1 clinical study in two parts: dose escalation followed by
dose expansion.
[0683] Part 1a--Dose Escalation
[0684] The dose escalation part of the study follows a standard 3+3
design. Doses are between about 20 to about 160 mg/m.sup.2.
Participants with advanced HCC or participants with secondary liver
tumours who meet the eligibility criteria will be recruited into 6
cohorts of 3 participants each at the following doses: 28, 47, 70,
98, 130, 160 mg/m.sup.2 until there is either development of drug
related Grade 3 toxicities (NCI-CTCAE version 4.03) or the maximum
improvement in serum albumin has been observed as defined above.
Dose escalation procedure is described above. Dose and schedule
might be modified depending on data arising from the study.
[0685] In the first dose cohort the first participant receives
MTL-CEBPA treatment at the study's starting dose. MTL-CEBPA is
administered by intravenous infusion over 60 minutes once a week
for 3 weeks followed by a rest period of 1 week; this defines a
4-week cycle. The determination of the starting dose of MTL-CEBPA
was based on GLP toxicity studies in rodents and cynomolgus
monkeys. Based on these data, a starting dose of MTL-CEBPA 28
mg/m.sup.2 was considered to be the safe starting dose in
humans.
[0686] Participants in Part 1a of the study who obtain clinical
benefit will be offered further cycles. Participants may also
continue to receive MTL-CEBPA on compassionate grounds; the
investigator must discuss with the sponsor on case by case basis
before the participant can continue on treatment.
[0687] An additional cohort of three HCC only participants may be
added after completion of Part 1a and before commencing Part 1b to
confirm the RP2D in this group of patients. The cohort will only be
considered if deemed appropriate by the PIs and Sponsor's Safety
Committee after review of clinical data and recruitment of HCC
participants to Part 1a of the study.
[0688] The RP2D will be defined by the safety review committee
(SRC) as the most appropriate dose to maximise a favourable
risk/benefit reward for the participant population in the dose
expansion part of the study.
[0689] Part 1b--Dose Expansion
[0690] Once the RP2D is obtained, an additional group of 12-15
eligible participants with advanced HCC will be recruited
sequentially. Each participant will be enrolled for 2 cycles and
administered with MTL-CEBPA at the RP2D until the participant
withdraw from the study.
[0691] Participants in Part 1b of the study who obtain clinical
benefit will be offered further cycles. Participants may also
continue to receive MTL-CEBPA on compassionate grounds. The
investigator must discuss this with the sponsor on case by case
basis before the participant can continue on treatment.
[0692] Study Endpoints
[0693] Primary Endpoint
[0694] In Part 1a, the primary endpoint will be dose limiting
toxicity (DLT) defined as: Any drug related toxicity grade greater
than or equal to 3 according to the Common Terminology Criteria for
Adverse Events (CTCAE) v4.03, including: Grade .gtoreq.3 nausea,
vomiting and diarrhoea despite adequate treatment for more than 3
days; Decrease in the participant's performance status .gtoreq.2
points compared to baseline; Grade .gtoreq.3 fatigue for more than
7 days; Grade .gtoreq.3 haemoglobin, platelets or neutrophils
abnormal laboratory value, myelosuppression for more than 5 days;
Grade .gtoreq.3 bilirubin abnormal laboratory value
(>3.0.times.ULN); Grade 4 AST and/or AST abnormal laboratory
value (>20.0.times.ULN).
[0695] Evaluation of any potential DLT will be performed during the
first 28 days (i.e. first cycle). All patients on the cohort should
clear the DLT period before any dose escalation can take place; to
this end an interval of not less than 7 days after the third dose
administered to the final participant in the previous cohort during
Part 1a of the study to assess for possible treatment related
side-effects is mandatory.
[0696] In Part 1b, safety and tolerability of MTL-CEBPA will be
evaluated in terms of frequency of adverse events graded according
to toxicity criteria (NCI CTCAE v 4.03) and categorised by body
system and diagnosis.
[0697] Secondary Endpoints
[0698] PK parameters will be defined by the maximum plasma
concentration (Cmax), time to maximum plasma concentration (Tmax),
area under the plasma concentration curve (AUC) and the half-life
(t1/2) of MTL-CEBPA after intravenous administration.
[0699] This protocol plans to evaluate the clinical efficacy (PD)
of MTL-CEBPA in participants with advanced HCC or participants
presenting with secondary liver tumours using descriptive analysis
of changes from baseline of surrogate biomarkers including albumin,
bilirubin, liver enzyme levels, chemokines, CTCs, tumour markers
and gene and protein expression levels in tumour tissue.
Health-related quality of life will be assessed in participants
with advanced HCC using descriptive analysis of changes from
baseline of FACT-Hep score.
[0700] Study Enrolment and Withdrawal
[0701] During the screening phase, after the participant has signed
the ICF, the following criteria will be assessed; each participant
should meet all of the inclusion criteria and none of the exclusion
criteria for this study. Under no circumstances can there be
exceptions to this rule and no waiver will be approved by the
sponsor as it is considered to be inappropriate and non-compliant
to GCP.
[0702] Participants will be assigned with a unique participant
trial ID sequentially in order of their recruitment (e.g. 001, 002,
etc.)
[0703] Inclusion Criteria
[0704] Participants should meet all of the following inclusion to
be eligible to participate in the study.
[0705] Inclusion Criteria for Dose Escalation (Part 1a)
[0706] The dose escalation part of the study will focus on
recruiting patients with either advanced HCC or with secondary
liver tumours. Recruitment will depend on observed toxicity.
Nevertheless, the protocol aims to recruit a maximum of 30
participants in Part 1a.
[0707] Inclusion Criteria for Patients with Advanced HCC:
Histologically confirmed advanced HCC; Patients who are considered
ineligible for surgery, or any other treatment, who are progressing
following loco-regional therapy and/or sorafenib (naive sorafenib
patients are eligible); At least one measurable lesion with target
lesion size .gtoreq.1.0 cm as measured by MRI or CT; Child-Pugh A
or Class B7 disease; Platelets .gtoreq.75.times.10.sup.9/L; Serum
albumin >28 g/L and <35 g/L; ALT and AST
.ltoreq.5.times.ULN
[0708] Inclusion Criteria for Patients with Secondary Liver Cancer:
Histologically confirmed advanced extra-hepatic solid tumour and
incurable liver tumours refractory to prior standard therapies or
for whom no standard therapy exists; At least one measurable lesion
with target lesion size .gtoreq.1.0 cm as measured by MM or CT
located in the liver; Platelets .gtoreq.100.times.109/L; Serum
albumin >25 g/L; ALT and AST .ltoreq.3.times.ULN.
[0709] Other Inclusion Criteria: Written informed consent obtained
prior to any trial specific procedure; Male or female aged
.gtoreq.16 years; ECOG performance status 0 and 1; Available
archival tumour tissue or ability and willingness to perform a
pre-treatment tumour biopsy; Acceptable laboratory parameters, as
demonstrated by: Bilirubin .ltoreq.50 .mu.mol/L, WBC
.gtoreq.2.0.times.10.sup.9/L, Absolute neutrophil count
.gtoreq.1.5.times.10.sup.9/L, Haemoglobin .gtoreq.9.0 g/dL or
Prothrombin time (PT)<20 seconds; Acceptable renal function as
demonstrated by: Serum creatinine .ltoreq.1.5.times.ULN or
Calculated creatinine clearance .gtoreq.60 mL/min/1.73 m2
(estimated using the CKD-EPI formula); Negative blood pregnancy
test for women of childbearing potential; Safe contraception in
females of childbearing potential during the entire study, using an
established treatment with hormonal contraceptives for at least 2
months prior to start of screening: For females of child bearing
potential (without using hormonal contraceptives for at least 2
months prior to start of screening) a double contraception method
is required during the entire study meeting the criteria for an
effective method of birth control, i.e. at least two effective
birth control methods such as condoms, diaphragms or intra-uterine
devices must be used; Male participants with partners of child
bearing potential are required to use barrier contraception in
addition to having their partner use another method of
contraception during the trial and for 3 months after the last
dose. Male participants will also be advised to abstain from sexual
intercourse with pregnant or lactating women, or to use condoms;
Willingness and ability to comply with all protocol requirements
including scheduled visits, treatment plans, laboratory tests and
other study procedures.
[0710] Inclusion Criteria for Dose Expansion (Part 1b)
[0711] This protocol aims to recruit 12 to 15 participants with
advanced HCC:
[0712] Histologically proven advanced HCC; Patients who are
considered not eligible for surgery, or any other treatment, who
are progressing following loco-regional therapy and sorafenib
(naive sorafenib patients are eligible); At least one measurable
lesion with target lesion(s) size .gtoreq.1.0 cm as measured by MRI
or CT; Child-Pugh A or B7 disease; Platelets
.gtoreq.75.times.10.sup.9/L; Liver dysfunction with serum albumin
>28 g/L and <35 g/L; ALT and AST .ltoreq.5 times upper limit
of normal range; Bilirubin .ltoreq.50 .mu.mol/L; Written informed
consent obtained prior to any trial specific procedure; Male or
female aged .gtoreq.16 years; ECOG performance status 0 and 1;
Available archival tumour tissue or ability and willingness to
perform a pre-treatment tumour biopsy; Acceptable laboratory
parameters, as demonstrated by: WBC .gtoreq.2.0.times.10.sup.9/L,
Absolute neutrophil count .gtoreq.1.5.times.10.sup.9/L, Haemoglobin
.gtoreq.9.0 g/dL, or Prothrombin time (PT)<20 seconds;
Acceptable renal function as demonstrated by: Serum creatinine
.ltoreq.1.5.times.ULN, Calculated creatinine clearance .gtoreq.60
mL/min/1.73 m2 (CKD-EPI formula), or Negative blood pregnancy test
for women of childbearing potential; Safe contraception in females
of childbearing potential during the entire study, using an
established treatment with hormonal contraceptives for at least 2
months prior to start of screening: For females of child bearing
potential (without using hormonal contraceptives for at least 2
months prior to start of screening) a double contraception method
is requested during the entire study meeting the criteria for an
effective method of birth control, i.e. at least two effective
birth control methods such as condoms, diaphragms or intra-uterine
devices must be used. Male participants with partners of child
bearing potential are requested to use barrier contraception in
addition to having their partner use another method of
contraception during the trial and for 3 months after the last
dose. Male participants will also be advised to abstain from sexual
intercourse with pregnant or lactating women, or to use condoms;
Willingness and ability to comply with all protocol requirements
including scheduled visits, treatment plans, laboratory tests and
other study procedures.
[0713] Participant Exclusion Criteria
[0714] Patients should not enter the study if any of the following
exclusion criteria are fulfilled.
[0715] Exclusion Criteria for Advanced HCC Patients: Child-Pugh
classes B8, B9 or C;
[0716] Patients who have been treated with TACE, sorafenib or
chemotherapy within the last 28 days
[0717] Other Exclusion Criteria: Prior systemic cancer-directed
treatments within 15 days or investigational drugs within the last
30 days; Grade >1 treatment-related toxicities (excluding
alopaecia) at the time of screening; Patients with clinically
significant cancer ascites; Any episode of bleeding from
oesophageal varices or other uncontrolled bleeding within the last
3 months; Patients administered with serum albumin within the last
7 days prior to the first MTL-CEBPA injection; Known infection with
human immunodeficiency virus (HIV); Patient with central nervous
system (CNS) metastasis; Signs and symptoms of heart failure
characterised as greater than the New York Heart Association (NYHA)
Class I; Patient presenting with a prolonged corrected QT (QTc)
interval defined as .gtoreq.450 ms (males) and .gtoreq.460 ms
(females) using Fridericia's correction formula; or other
clinically significant cardiac abnormalities; Major surgery within
the last 30 days; Patients with sepsis, obstructive jaundice or
encephalopathy; Evidence of spontaneous bacterial peritonitis or
renal failure or allergic reactions to the agent or excipient;
Pregnant or lactating women; Any other condition (e.g., known or
suspected poor compliance, etc.) that, in the judgment of the
investigator, may affect the participant's ability to follow the
protocol specific procedures.
[0718] Treatment Assignment Procedures
[0719] Part 1a--Dose Escalation
[0720] Participants will be enrolled for 2 cycles. The dose
escalation phase of the study will follow a standard 3+3 design as
shown in FIG. 33. Six cohorts of 3 eligible participants are
planned at the following doses: 28, 47, 70, 98, 130, 160
mg/m.sup.2. Individual dose will be based on the participant's most
recent height and weight using the DuBois & DuBois (133) body
surface area (BSA) calculation.
[0721] At each dose cohort the first participant receives MTL-CEBPA
treatment at the study starting dose. MTL-CEBPA is administered
once a week for 3 weeks on Day 1, Day 8 and Day 15 by intravenous
infusion over 60 minutes followed by a week of rest. This defines a
cycle.
[0722] Subsequent participants will be recruited not less than 7
days after the first dose of the first participant in order allow
the assessment of treatment related side-effects.
[0723] Evaluation of any potential DLT as defined in above will be
performed during the first 28 days (i.e. first cycle).
[0724] A gap of not less than 7 days should lapse between the last
dose of the final participant in the previous cohort to allow for
adverse events or toxicities to become apparent. A decision to
progress to the next cohort will require a review of the previous
cohort safety and clinical data for all Participants by the SRC.
Following the review, the next cohort may be initiated. The
decision will be documented in writing, and a record will be
retained in the Trial Master File (TMF).
[0725] If there is no occurrence of toxicities qualifying as DLT in
3 participants of a dose cohort, dose escalation to the next dose
level may be performed. If there is a DLT in one of three
participants in a dose cohort, a further 3 participants will be
enrolled at this dose. If there are no further DLT occurrences in
these additional 3 participants, escalation to the next dose level
may be performed. If, however, 2 or more of those 6 participants
(3+3) present with a DLT there will no further dose escalation step
and the dose level will be considered as the maximum tolerated dose
(MTD). Additionally, if 2 participants present with a DLT in a
single cohort then there will be no further dose escalation step
and the dose level will be considered as the MTD. All dose
escalation decisions will be based on the judgement of the SRC.
[0726] An additional cohort of three HCC only participants may be
added after completion of Part 1a and before commencing Part 1b and
may be administered with an intermediate dose of MTL-CEBPA using
the same dosing regimen as in Part 1a. The cohort will only be
considered if deemed appropriate by the PIs and Sponsor s Safety
Committee after review of clinical data and recruitment of HCC
participants to Part 1a of the study.
[0727] The RP2D will be defined by the SRC as the most appropriate
dose to maximise a favourable risk/benefit reward for the
participant population in the dose expansion part of the study.
FIG. 33 is a flowchart for dose escalation.
[0728] Part 1b--Dose Expansion
[0729] Following completion of the escalation part of the study,
12-15 eligible participants presenting with advanced HCC will be
enrolled for 2 cycles sequentially onto the dose expansion part of
the study and will be administered MTL-CEBPA at the RP2D.
[0730] Reasons for Withdrawal
[0731] A participant may be discontinued from the study for the
following reasons:
[0732] Patient decision: The participant is at any time free to
withdraw his/her participation in the study, without prejudice;
Grade .gtoreq.3 infusion related allergic reaction to the study
medication not controlled with prophylactic procedure as
described); Any clinical adverse event (AE), laboratory
abnormality, intercurrent illness, or other medical condition or
situation occurs or worsens such that continued participation in
the study would not be in the best interest of the participants;
Confirmed disease progression, unless the participant is receiving
clinical benefit in the opinion of the investigator; Severe
non-compliance to this protocol as judged by the investigator; The
participant becomes pregnant; The participant dies.
[0733] Study-specific discontinuation criteria: Presence of benefit
with an increase in serum albumin .gtoreq.45 mg/L; the participant
may be advised by the investigator to discontinue from the study
medication if this is in the participant's best interest, or if the
participant's response is positive and allows the participant to
receive another conventional therapy that was inappropriate at the
start of the study (i.e. stage migration), such as surgery, RFA,
TACE or sorafenib.
[0734] Termination of Study
[0735] Patients are free at any time to withdraw from the study (IP
and assessments), without prejudice to further treatment
(withdrawal of consent). Such participants will always be asked for
the reason(s) and the presence of any AEs. If possible, they will
be seen and assessed by an Investigator. AEs will be followed
up.
[0736] Survival, based on publicly available sources or contact
with the participant medical care may be investigated at the
scheduled study end and in cases where participants have withdrawn
consent. These data will be collected in the eCRF.
[0737] To prevent participants being lost to follow-up, their
contact details, including next of kin contacts should be collected
initially and updated regularly by the site staff or
representative.
[0738] Dosing Scheme
[0739] Administration of MTL-CEBPA will be once a week for 3 weeks
followed by a rest period of 1 week [3 plus 1 week=4 weeks=one
cycle]. Other schedules and dosage may be explored depending on the
results of ongoing preclinical experiments or from data arising
from the study.
[0740] Six cohorts of 3 eligible participants are planned at the
following doses: 28, 47, 70, 98, 130, 160 mg/m.sup.2.
[0741] An extended treatment is allowed for those patients who
clinically benefit from treatment in the planned FIE Phase 1 study
in patients with primary or secondary liver tumours for whom no
further approved treatment options exist, i.e., treatment may
continue as long as the clinical benefit persists.
[0742] Considerations on Calculation of Human Starting Dose
[0743] MTL-CEBPA was efficacious in two liver disease models. In
the CCL4 model, biweekly doses as low as 0.3 mg/kg showed reduction
or reversal of a subset of disease symptoms. However, the highest
dose of 3 mg/kg was required for maximal impact on some biomarkers
considered disease relevant, including serum albumin and liver
hydroxyproline. Therefore, the maximally effective dose in this
model is likely 3 mg/kg or higher, for a 2-week biw regimen. Only a
single dose level (3 doses of 4 mg/kg) was evaluated in the DEN
model. It is therefore not clear if lower doses would have activity
or whether higher doses would further improve the observed impact
on tumours and other disease metrics. The effective dose for a
single week of treatment is therefore estimated at 4 mg/kg.
[0744] Based on the rat PK data with MTL-CEBPA, the biw and tiw
schedules in the CCL4 and DEN models should not have led to drug
accumulation in circulation, thus allowing human dose estimations
based on single doses. Taken together, and considering the very
short treatment period in the DEN model, repeat doses of 3-4 mg/kg
may be sufficient for meaningful anti-tumour efficacy and doses of
0.3 to 3 mg/kg for improvement in liver function.
[0745] The non-clinical toxicity program included repeat-dose
toxicity testing in rats and cynomolgus monkeys, including
toxicokinetic profiling and local tolerance evaluation.
[0746] MTL-CEBPA, given daily for 3 consecutive days by the
intravenous route (1-hour infusion) at 7.5 mg/kg for 4 weeks (total
of 12 administrations) to cynomologus monkeys was clinically
well-tolerated and only induced transient non-adverse changes in
body weight, food consumption, clinical laboratory parameters as
well as decreases in platelet count and activation of the
alternative and common complement pathways. The 7.5 mg/kg
administered 3 times weekly for the duration of the 1 month study
was defined as a NOAEL in cynomologus monkeys. MTL-CEBPA
administered to rats given daily for 3 consecutive days by the
intravenous route (1-hour infusion) at 7.5 mg/kg for 4 weeks (total
of 12 administrations) induced lower body weight gain and food
intake, clinical signs in a few animals, various changes in
haematological, coagulation and serum clinical chemistry parameters
as well as local reactions at the infusion sites. Because of their
small magnitude and reversibility, these clinical pathology changes
were not considered adverse. Histologically, the main finding was
macrophage vacuolation in several organs or tissues, which could
reflect clearance of the particulate test item and this was not
considered adverse. The 7.5 mg/kg administered 3 times weekly for
the duration of the 1 month study was defined as a HNSTD in
rats.
[0747] There were no MTL-CEBPA-related ophthalmological or
cardiovascular findings in monkeys or rats at the end of the
treatment period.
[0748] An in vitro immunogenicity assay was performed with primary
human peripheral blood mononuclear cells (PBMCs) transfected
CEBPA-51. The assessment of TNF-.alpha. and IFN-.alpha. showed no
induction and thus no immune-stimulatory activity in terms of
toll-like receptor (TLR) pathway induction.
[0749] As above 7.5 mg/kg administered 3 times weekly for the
duration of the 1 month study was defined as a HNSTD in rats and a
NOAEL for MTL-CEBPA in monkeys. Although dose extrapolation from
animals to humans has traditionally been based on body surface area
(BSA)-related scaling or similar mathematical paradigms, these
conventions were derived from studies performed with small-molecule
anticancer agents and are very unlikely to be relevant for dose
extrapolation with a product that consists of an RNA in liposomal
or other lipid particle delivery system, such as MTL-CEBPA. The
original impetus for BSA-based cross-species scaling stemmed from
reports that direct extrapolation from the body weight-relative MTD
under-predicted human sensitivity to cytotoxic anticancer agents,
and better correlation of the MTD across species was obtained when
doses were expressed per body surface area. The primary reason for
the lesser sensitivity of smaller species such as rodents vs.
larger species to small-molecule anticancer agents is that smaller
species tend to metabolize such molecules via the hepatic
cytochrome P450 system faster than higher species and/or exhibit
faster clearance from the blood compartment, which collectively
contributes to more rapid or more extensive detoxification than in
higher species, including humans.
[0750] For many of the lipid-formulated oligonucleotides advanced
through nonclinical development, the MTDs in rodents tend to be
similar to or lower than in monkeys or other non-rodent species,
which is the case for MTL-CEBPA. This pattern is not consistent
with the fundamental principle of BSA-based scaling, which would
predict a higher MTD in rodents vs. a larger species like monkey.
It is not surprising that formulated oligonucleotide products
behave differently than small-molecule anticancer drugs, as neither
the nucleic acid payload nor the excipients have been shown to (or
would be expected to) interact significantly with the hepatic
cytochrome P450 system, and the formulation traverses the
bloodstream in particulate form, exhibiting unique pharmacokinetics
and clearance pathways unlike small-molecule drugs.
[0751] Although the plasma AUC for the active saRNA ingredient
(CEBPA-51) was substantially lower in rats than in monkeys at
similar dose levels, the toxicity produced by MTL-CEBPA is
unrelated to the amount of drug in circulation, as none of the
toxicities stem from interaction with blood components, and the
primary effect observed (i.e., vacuolation of macrophages in
various tissues) would be expected to correlate with tissue, not
blood, concentrations. In fact, the faster clearance of MTL-CEBPA
(CEBPA-51) from circulation in rats very likely reflects more rapid
uptake by macrophages, which could result in greater activation of
those cells and more pronounced downstream sequelae from such
activation, which would account for the greater severity of
toxicity in rats vs. monkeys. Thus, although the lesser plasma
exposure in rats vs. monkeys at the same mg/kg dose levels may
appear to be consistent with conventional BSA-based scaling, this
difference certainly does not correlate with a lesser degree of
toxicity in rats, and the faster clearance of the particles from
the blood compartment in rats may actually underlie the greater
toxicity. In other words, for this type of drug product, when
comparing exposure across species, faster clearance, reflected by
lower AUC values, should not be construed to imply lesser
sensitivity, as has been seen with cytotoxic anticancer agents.
[0752] Therefore, BSA-based scaling is not applicable to
calculation of the human-equivalent dose (HED) from the cynomolgus
monkey NOAEL and the rat HNSTD. It is also viewed that that monkey
may be a better predictor of human sensitivity for MTL-CEBPA, but
this cannot be proven at this time. Thus, in the interest of trying
to identify an appropriate starting dose level for the initial
clinical trial that achieves a sufficient safety margin, while not
falling so conservatively low that pharmacologic activity and
clinical efficacy is undermined, it is believed that a dose level
10-fold below the HNSTD of 7.5 mg/kg/adm. (3.times. weekly dosing
for 4 weeks) in rats and a NOAEL in monkeys i.e., 0.75 mg/kg, is an
appropriate choice. This proposed starting dose level is even more
conservative when one considers that, at the HNSTD in the 4-week
rat and monkey studies, doses were given for 3 consecutive days
each week, as opposed to the once-weekly dosing intended for the
initial trial. Thus it is concluded that MTL-CEBPA is expected to
be safe and well-tolerated with no unusual or alarming signs of
toxicity that would preclude the use in humans at the intended
initial dose of 0.75 mg/kg (28 mg/m.sup.2) administered as a
60-minute intravenous (i.v.) infusion once weekly. Based on the
pharmacology we might expect to see liver function benefits from
0.3-3.0 mg/kg and tumour benefit at approximately 4 mg/kg. Starting
at a dose of 0.75 mg/kg thus gives the initial patients the
potential opportunity to benefit from liver improvements although
dose escalation may be required to achieve direct anti-tumour
activity.
[0753] Dosage, Preparation and Administration of Investigational
Product
[0754] During the dose escalation and dose expansion, the dosage
will follow the schedule. The dose will be based on the
participant's most recent height and weight using the DuBois &
DuBois (133) body surface area (BSA) calculation:
BSA (m2)=0.007184.times.Height (cm).sup.0.725.times.Weight
(kg).sup.0.425
[0755] MTL-CEBPA is thawed at room temperature before diluting the
drug product suspension in 0.9% Normal Saline for Intravenous Use.
The volume of the prepared infusion bag should be 250 mL regardless
of the concentration and administered at a constant rate over 60
minutes into a vein (peripheral or central) using an infusion pump
with no filter (refer to the Pharmacy manual for more details on
instruction for handling IMP).
[0756] The preparation should be kept at room temperature
(25.degree. C.) with a maximum in-use shelf-life of 6 h.
[0757] Compatibility issue between the IMP and diluent and/or
infusion devices is not expected.
[0758] Modification of Investigational Product Dosing for a
Participant
[0759] Other schedules and dosage may be explored depending on the
results of ongoing preclinical experiments or from data arising
from the study.
[0760] In the event of a grade .gtoreq.3 infusion reaction (e.g.
drop in blood pressure, facial flushing, chest tightness, back or
abdominal pain, elevated heart rate, sweating), the infusion should
be stopped immediately until the symptoms subside; then the
infusion can be restarted. If the symptoms reappear the
investigator should stop the infusion. The volume of infusion
administered at this point will be capture in the CRF. The
investigator should discuss with the medical monitor any dose
modification plan prior implementation. This may result in dividing
the remaining weekly dose equally over the two following days. The
following administration would follow a 3 days administration
schedule as described.
[0761] If, despite modification of dosing, the symptoms persist the
treatment should be discontinued and the participant advised to
withdraw from the study.
[0762] Concomitant Medications/Treatments
[0763] Information on any treatment in the 4 weeks prior to
starting study treatment and all concomitant treatments given
during the study, with reasons for the treatment, will be recorded
in the eCRF.
[0764] Prohibited Medications and Procedures
[0765] The following medications are prohibited during the
participants' participation in the trial: Other investigational
agents; Anti-neoplastic agents.
[0766] Prophylactic Medications and Procedures
[0767] All participants will be premedicated (unless
contraindication) prior to dosing with MTL-CEBPA to reduce the
potential for an infusion reaction. Premedication should be
administered 30 to 60 minutes prior to the start of the infusion as
follow: Steroid single dose (i.e. dexamethasone oral 8 mg or
intravenous 10 mg); Oral H2 blocker single dose (i.e. Ranitidine
150 mg or famotidine 20 mg or equivalent other H2 blocker dose);
Oral H1 blocker single dose, 10 mg cetirizine (hydroxyzine 25 mg or
fexofenadine may be substituted if participant dose not tolerate
cetirizine).
[0768] Overdose of Investigational Medicinal Product
[0769] MTL-CEBPA is an investigational agent and is contraindicated
for all conditions other than those mentioned in this protocol.
[0770] Should an overdose occur, there is no known antidote.
Symptoms and signs attributed to the overdose should be treated
symptomatically. Any participant who inadvertently receives a
higher dose than intended should be monitored closely, managed with
appropriate supportive care until recovery and followed up
expectantly.
[0771] Such overdoses should be recorded as follows: An overdose
with associated AEs/SAEs is recorded as the AE diagnosis/symptoms
on the relevant AE/SAE modules in the eCRF and on the overdose eCRF
module; An overdose with no associated symptoms is only reported on
the overdose eCRF module.
[0772] If an overdose occurs in the course of the study, site
personnel must inform the PI within one day, i.e. immediately, but
no later than the end of the next business day of when he or she
becomes aware of it. An overdose will be reported by the PI to the
sponsor.
[0773] For overdoses associated with an SAE, standard reporting
timelines apply. For other overdoses, reporting should be done
within 30 days.
[0774] Pregnancy and Maternal Exposure
[0775] As MTL-CEBPA is an investigational agent it is
contraindicated for pregnant women and as such they are excluded
from participating in this study. For all women of child bearing
potential, barrier contraception should be used and should be
continued for at least three months following the end of treatment
with MTL-CEBPA. However, should a participant become pregnant
whilst on study, despite using barrier contraception as mandated,
immediate discontinuation of study is required.
[0776] It is not known if the study medicine will affect sperm or
semen and therefore men are advised to use a reliable barrier form
of contraception during the treatment phase and for at least three
months following the final treatment.
[0777] Study Schedule
[0778] The study schedule applies to Part 1a and Part 1b of the
study. Each treatment cycle consists of 3 weeks of treatment on
Days 1, 8, and 15 followed by 1 week of rest.
[0779] Screening Visit (Day -21-Day -1): The following data will be
collected at enrolment and recorded in the appropriate sections of
the CRF: Date of signed ICF; Demographic data, full medical
history, physical examination, recording of vital signs,
performance score, weight (kg), height (cm) and girth measurement
(cm). Evaluation against inclusion and exclusion criteria.
Recording of baseline symptoms and causality. Prior and concomitant
medication. Blood pregnancy test for women of childbearing
potential. 6-hour fasting blood sampling for assessment of
haematology, clotting profile, clinical biochemistry (including
LFT, renal profile), lipid profile, appropriate tumour marker(s),
cytokine profile and complement activation factors Bb and C3a. 12
lead ECG. Chest X-Ray. MRI or CT scan of liver and abdomen with
RECIST report. (Note: Scan should not be repeated if a previous
scan available within 1 month prior to the start of study
treatment). Fibroscan will be performed in HCC participants only.
FDG-PET scan (Part 1b only). Radiological guided liver biopsy of
tumour tissue will be performed in participants in whom archival
material is unavailable. The tumour tissue will be a
formalin-fixed, paraffin-embedded (FFPE) sample.
[0780] Participants who fail their first screening visit due to
serum albumin level being outside the inclusion range can be
re-screened 14 days later. A third screening is not acceptable.
[0781] Days 1, 8 and 15 Visit (+\-2 days): Unless otherwise
specified, procedures and assessments should be undertaken
pre-dose. Standard physical examination, weight and girth
measurements. Recording of new symptoms and new medications since
previous visit, performance score, 12 lead ECG. FACT-Hep quality of
life questionnaire administration at day 1 pre-dose and post-dose
at Day 15, week 8 and at the end of study visit (participant in
Part 1b only). Place cannula into a vein (peripheral or central).
6-hour fasting blood sampling assessment of haematology, clotting
profile and clinical biochemistry (including LFT and renal function
tests) and complement activation factors Bb and C3a. Day 8 and 15
only: Blood sampling for appropriate tumour markers and cytokine
profile. If required administer pre-medication via the cannula 30
minutes pre MTL-CEBPA infusion. Administer MTL-CEBPA intravenously
via the cannula over 60 minutes as per dosing schedule. Vital signs
(other than weight and girth measurement) recording pre-dose, and
at 15 minute, 30 minute, 1 hour and 2 hour time points post
administration. PK samples pre and post infusion (Part 1a
participants on Days 1 and 8 only) at the following time points:
pre-dose, immediately post infusion, 0.25 hour, 1 hour, 3 hours and
6 hours timed from the completion of the infusion. FDG-PET scan at
Day 15 post-dose (participants in Part 1b only) (Note: All
pre-treatment blood samples, except for PK, may be taken and
analysed the day prior to administration of MTL-CEBPA.)
[0782] Days 2, 9 and 16 Visit: Recording of new symptoms and
medications, vital signs and performance score. 6-hour fasting
blood sampling assessment of haematology, clotting profile and
clinical biochemistry (including LFT and renal function tests) and
complement activation factors Bb and C3a. PK (Part 1a participants
on Days 2 and 9 only) at 24 hours after completion of the
infusion.
[0783] Days 3 and 10 Visit: 48 hour PK sampling for participants in
Part 1a only
[0784] Days 4 and 11 Visit: 72 hour PK sampling for participants in
Part 1a only
[0785] Day 22 Visit (+\-2 days): Standard physical examination.
Recording of new symptoms and medications, vital signs and
performance score. Blood pregnancy test for women of childbearing
potential. 6-hour fasting blood sampling for assessment of
haematology, clotting profile, clinical biochemistry (including
LFT, renal profile), lipid profile, appropriate tumour marker(s),
cytokine profile and complement activation factors Bb and C3a.
FACT-Hep quality of life questionnaire administration (Part 1b
only). Chest X-Ray.
[0786] Week 8-Day 22 of Cycle 2 Visit (+\-2 days): In addition to
the Day 22 investigations and procedures listed above, the
following imaging procedures should be carried out: MRI/CT scan
will be performed and every 8 weeks thereafter; FDG-PET scan will
be performed at week 8 only for participants in Part 1b and will
not be repeated thereafter.
[0787] End of Study Visit (Day 29 or 14 days+/-7 days after last
dose): Standard physical examination, weight and girth measurement.
Recording of new symptoms and new medications since previous visit,
vital signs and performance score. FACT-Hep quality of life
questionnaire administration (Part 1b only). Blood pregnancy test
for women of childbearing potential. 6-hour fasting blood sampling
assessment of haematology, clotting profile and clinical
biochemistry (including LFT and renal function tests) and
complement activation factors Bb and C3a. For premature withdrawal
participants only (i.e. if not done at Day 22) blood sampling for
fasting lipid profile, appropriate tumour marker(s) and cytokine
profile. Fibroscan (in participants with HCC only). Tumour biopsy;
a post treatment biopsy is highly desirable and will be performed
on investigator judgement.
[0788] Early Termination Visit: Should the participant withdraw
from the study the assessments described above should be
undertaken.
[0789] Unscheduled Visit: Unscheduled visits or phone contacts may
be performed for adverse event follow-up.
[0790] Pregnancy Visit: In the event of a participant becoming
pregnant during the study, the participant should be advised to
stop study treatment immediately. The reason for withdrawal will be
recorded in the eCRF and the participant will be seen and assessed
by an Investigator(s) in order to complete all assessments for an
End of Study Visit to assess the safety of the study drug as
described above.
[0791] Treatment Plan for Responders: Participants in Part 1a and
Part 1b are initially enrolled for 2 cycles. At the end of the
first cycle, participants may receive additional cycle(s) of
MTL-CEBPA on the same treatment regimen basis (excluding PK
sampling) should participants display clinical benefits (e.g.
improvements in liver function) and agree to continue in the study.
At the end of the second cycle, tumour response will be assessed;
participants who do not show tumour progression will be offered a
further 2 additional treatment cycles.
[0792] At this point participants may be advised by the
investigator to exit from the study if this is in the participant's
best interest, or if the participant's response is positive, which
allows the participant to receive another conventional therapy that
was inappropriate at the start of the study (i.e. stage migration),
such as surgery, RFA, TACE or sorafenib.
[0793] Participants will be given the opportunity to receive
further cycles of MTL-CEBPA as long as the response to treatment
lasts.
[0794] Study Procedures/Evaluations--Patient Reported Outcome
[0795] The Functional Assessment of Cancer Therapy-Hepatobilliary
(FACT-Hep) version 4 questionnaire will be used to assess changes
in health-related quality of life for participants following the
administration of MTL-CEBPA at the RP2D level (Part 1b) on, Day 1
and 15 and week 8.
[0796] FACT-Hep Questionnaire
[0797] The Functional Assessment of Cancer Therapy-Hepatobilliary
(FACT-Hep) is a validated health-related quality of life
questionnaire and is a combination of the FACT-General and the
18-item module specifically designed to measure symptoms and side
effects of treatment associated with hepatobiliary carcinoma. The
FACT-G is a multidimensional 27-item instrument that measures four
dimensions of quality of life including physical, social/family,
emotional, and functional well-being. The FACT-Hep also includes an
18-item module assessing the symptoms of hepatobiliary carcinoma
and side effects of treatment (Additional Concerns).
[0798] Method of Assessment
[0799] The FACTHep will be self-administered using a paper version
of the questionnaire at scheduled visits. The questionnaire will be
assessed at Day 1 pre-dose and post dose at Day 15 and then at Day
22 (week 8).
[0800] Administration of PRO Questionnaires
[0801] The FACIT scales are designed for participant
self-administration, but can also be administered by interview
format. For self-administration, participants should be instructed
to read the brief directions at the top of the page. After the
participant's correct understanding has been confirmed, he/she
should be encouraged to complete every item in order without
skipping any. Patients should be encouraged to circle the response
that is most applicable. If, for example, a participant is not
currently receiving any treatment, the participant should circle
"not at all" to the question "I am bothered by side effects of
treatment." Interview administration is considered appropriate
given adequate training of interviewers so as to elicit non-biased
participant responses.
[0802] Scoring
[0803] The FACT-Hep includes 5 dimensions: "physical well-being",
"Social/family well-being, "emotional well-being", "Functional
well-being" and "Additional concerns". Each dimension has 5 levels:
"Not at all", "A little bit", "Somewhat", "Quite a bit" and "Very
much". The participant rates his/her current health state on the
FACT-Hep by circling or marking one number per line to indicate
his/her response as it applies to the past 7 days to the statement
on the FACT-Hep. This is the FACT-Hep score.
[0804] Laboratory Safety Evaluations
[0805] Sample collection times are included in the study schedule
of event. Details of methodology and reference ranges will be
stored in the TMF. Laboratory values that have changed
significantly from baseline and are considered to be of clinical
concern must be recorded as an adverse event and followed up as
appropriate.
[0806] The estimated blood volumes to be collected from each
participant over 1 cycle are presented below:
TABLE-US-00039 Sample No. of Total volume Assessment volume (mL)
Samples (mL) Safety Biochemistry [a] 3.5 9 31.5 Plasma ammonia [a]
4 9 36 Tumour markers [a] 2.5 5 12.5 Fasting glucose 2 9 18
Haematology 3 9 27 Coagulation 4 9 36 Complement (C3a and Bb) 2 9
18 Serum pregnancy test [b] 2 2 4 Pharmacokinetic 5 18 90 Plasma
MTL-CEBPA Pharmacodynamics 3 6 18 Cytokines Total: Male 287 Female
291 [a] Total protein, albumin, total bilirubin, ALT, AST, plasma
ammonia, tumour markers total cholesterol, HDL-C and triglycerides
will be assessed to allow review of PD biomarkers. [b] For women of
childbearing potential.
[0807] Less than 50 mL will be taken from each participant on each
visit and a total of less than 300 mL per 4-week cycle.
[0808] Local Laboratory Tests
[0809] All samples for laboratory safety assessment will be
collected at each investigational site according to local practices
and analysed at the local laboratory using standard methods for
routine tests. All blood samples should be collected pre-MTL-CEBPA
infusion at screening and on Days 1, 2, 8, 9, 15, 16, 22 and EOS
(with the exception of lipid profile).
[0810] Clinical biochemistry and haematology parameters will be
measured. Clinical biochemistry samples including liver function,
ammonia and glucose as well as the lipid profile should be
collected in a fasting state (6 hours before blood sample).
TABLE-US-00040 Serum Biochemistry Parameters Sodium Total bilimbin
[a] Potassium ALP [a] Calcium Alanine transaminase (ALT) [a]
Phosphate Aspartate transaminase (AST) [a] Urea (BUN) Gamma
glutamyl transferase (GGT) [a] Fasting glucose Plasma ammonia [a]
Total protein [a] Total cholesterol [b] Creatinine HDL-C [b]
Albumin [a] Triglycerides [b] [a] Total protein, albumin, total
bilirubin, ALT, AST and plasma ammonia will be assessed to allow
review of eligibility at screening and allow for review of PD
biomarkers. [b] Total cholesterol, HDL-C and triglycerides will be
assessed at screening, at the end of each cycle (day 22) and EOS
(for premature withdrawal participants) to allow review of
exploratory PD biomarkers.
TABLE-US-00041 Haematology Parameters White blood cells (WBC)
Lymphocytes absolute and % Red blood cells (RBC) Monocytes absolute
and % Haemoglobin (Hb) Eosinophils absolute and % Glycosylated
haemoglobin (HbA1c) Basophils absolute and % Neutrophils absolute
and % Platelets
TABLE-US-00042 Coagulation Parameters International normalised
ratio (INR) Prothrombin time (PT) Activated partial thromboplastin
time (aPTT)
TABLE-US-00043 Complement Activation Parameters Activation fragment
of complement factor B (Bb) Complement component 3a (C3a)
[0811] Pregnancy Test (Blood)
[0812] Human chorionic gonadotrophin (hCG) will be measured for
women of childbearing potential at screening, Day 22 and every 8
weeks thereafter (if the participant is receiving further cycles of
treatment).
[0813] Tumour Marker(s)
[0814] The most suitable marker(s) among the list below will be
selected on the basis of the cancer history and will be performed
at screening and on Days 8, 15, 22 and EOS (for premature
withdrawal participants).
TABLE-US-00044 Tumour Markers Alpha-fetoprotein (AFP)
Carcinoembryonic antigen (CEA) Cancer antigen 125 (CA125) Cancer
Antigen 15-3 Calbohydmte antigen 19-9 (CA 19-9) (CA15-3)
[0815] Imaging
[0816] Chest X-ray: Chest X-ray will be performed during screening
and Week 4. A further X-ray will be performed at Week 12 and
thereafter according to standard of care if the participant is
receiving further cycles of MTL-CEBPA.
[0817] MRI or CT Scans: Mill or CT scan of the chest and abdomen
will be performed during screening (within 1 month prior to the
first dose of the study drug) and at week 8. Mill is the preferred
method, but CT scans are allowed; whether MRI or CT scan is used,
it is important to maintain consistency of assessment method for
each participant.
[0818] MRI/CT scans will then be performed every 8 weeks.
[0819] Fibroscan: Fibroscan will be performed only in participants
with HCC at screening and at the end of the study in order to
assess the fibrotic characteristics of the liver prior and post
treatment with MTL-CEBPA.
[0820] FDG-PET Scan: Metabolism of the liver and the tumour will be
assessed using FDG-PET scan in participants enrolled in Part 1b
only. FDG-PET scan will be performed during screening, at Day 15
post MTL-CEBPA infusion and at the time of restaging CT (i.e. week
8).
[0821] Patients are not allowed to consume any food or sugar for at
least 6 h prior to the start of the PET study (i.e. with respect to
time of injection of FDG). In practice, this means that patients
scheduled to undergo the PET study in the morning should not eat
after midnight and preferably have a light meal (no alcohol) during
the evening prior to the PET study. Those scheduled for an
afternoon PET study may have a light breakfast before 8.00 a.m.
(i.e. up to two sandwiches, no sugars or sugar containing sandwich
filling). Medication can be taken as prescribed.
[0822] Liver Biopsy
[0823] If available, an archival tissue sample in the form of a
formalin fixed paraffin embedded (FFPE) tumour block will be
collected for each participant. If it is not possible to obtain the
tumour block or it does not exist, the participant must have agreed
to a biopsy at screening as part of the informed consent
process.
[0824] Archival tumour tissue should be requested by the research
team and should be sent as described. Archival tumour blocks will
be returned to source at the end of the study or, upon request,
earlier if required.
[0825] A post treatment liver tumour biopsy is highly desirable at
the EOS visit.
[0826] Administration of fresh frozen plasma and platelets to
correct any coagulation abnormalities should be administered as
necessary.
[0827] Pharmacokinetics (PK)
[0828] PK blood samples, 5 mL of whole blood, be will be collected
in an EDTA tube from each participant in each of the 6 cohorts of
the dose escalation part of the study on Days 1, 2, 3 and 4, 8, 9,
10 11 of the first cycle and at the following time points: pre-dose
of study drug, immediately after completion of the infusion, then
at 0.25 hour, 1 hour, 3 hours, 6 hours, 24 hours, 48 hours and 72
hours time points from completion of the infusion.
[0829] Clinical staff is encouraged to take the blood samples for
PK analysis at the scheduled time point. However, deviations from
the scheduled sample times are not considered protocol deviations.
The exact time and date of the blood draw must be recorded using an
unambiguous format.
[0830] Plasma concentration of MTL-CEBPA will be analysed centrally
at the defined time points using a hybridization-based
HPLC-assay.
[0831] Instructions for specimen preparation, handling, and storage
are described below.
[0832] Pharmacodynamics (PD)
[0833] Liver function tests, AFP tumour marker (for HCC
participants), cytokine profile and CEBPA gene expression have been
identified as surrogate biomarkers of the pharmacological effect of
MTL-CEBPA.
[0834] Liver function tests: alanine transaminase (ALT), serum
albumin, plasma ammonia, aspartate transaminase (AST), and total
bilirubin will be taken on Day 1 (pre-dose), Day 2, Day 8
(pre-dose), Day 15 (pre-dose), Day 22 and at EOS visits. These
samples will be analysed at the local laboratory.
[0835] AFP tumour marker: AFP will be tested on Day 1 (pre-dose),
Day 8 (pre-dose) Day 15 (pre-dose), Day 22 and at EOS visits (for
premature withdrawal participants). These samples will be analysed
at the local laboratory.
[0836] Lipid profile: Total cholesterol, HDL-C and triglycerides
will be assessed on days 1, 8, 15, 22 of each cycle and EOS (for
premature withdrawal participants) to allow review of exploratory
PD biomarkers
[0837] Cytokine profile: IL-2, IL-6, TNF-a, IFN-g, IL-4, IL-17a,
IL-10 will be tested at screening, Day 8 (pre-dose), Day 15
(pre-dose), Day 22 visits and at EOS visits (for premature
withdrawal participants). Samples will be analysed at a central
laboratory.
[0838] Gene expression: CEBPA gene expression will be studied using
CEBP.alpha. and p21 protein expression using immunochemistry
staining assay on formalin-fixed, paraffin-embedded (FFPE) tumour
biopsy samples obtained from archival tumour tissue and/or from new
biopsy tissue at screening visit and EOS visit.
Specimen Preparation, Handling, and Shipping
Instructions for Specimen Preparation, Handling, and Storage
[0839] All samples with the exception of PK samples and cytokine
profile samples will be collected by the sites according to local
practices and analysed at the local laboratory using standard
methods for routine tests.
[0840] A Laboratory Manual for Investigators giving detailed
instructions will be provided to each study site prior to the start
of the study. The investigator and delegated site personnel should
follow the procedures defined in the Laboratory Manual.
[0841] For PK samples, 5 mL of whole blood is collected into
EDTA-treated tubes. The EDTA tube will be processed and centrifuged
using a refrigerated centrifuge (4.degree. C.) immediately after
sampling to generate plasma. Once obtained, the plasma will be
divided into at least two aliquots of 100 .mu.L while on ice.
Aliquots will be centrifuged following the same protocol mentioned
above and snap frozen using liquid nitrogen or dry ice before
storage at -80.degree. C. The recommended time from blood
collection to plasma storage is 30 min.
[0842] For the cytokines profile, 3 mL will be collected into
EDTA-treated tubes. The tubes will be centrifuged in order to
generate plasma. The samples will be stored frozen at -80.degree.
C. on site before to be sent to the central laboratory (see section
below).
[0843] An archival tumour block, if available, should be requested
from the relevant pathology department. Newly obtain tumour tissue
samples should be formalin fixed paraffin embedded (FFPE). Block
(preferably) or slides will be sent to the central laboratory
contracted to undertake IHC staining testing (see section
below).
[0844] Adverse Events (AE)
[0845] An adverse event (AE) is defined as any untoward medical
occurrence (including deterioration of a pre-existing medical
condition) in a participant or clinical investigation participants
administered a pharmaceutical product regardless of its causal
relationship to the study treatment. An AE can therefore be any
unfavourable and unintended sign including abnormal results of an
investigation (e.g. laboratory finding, electrocardiogram),
symptom(s) (e.g. nausea, chest pain), signs (e.g. tachycardia,
enlarged liver) or disease temporally associated with the use of
the investigational medicinal product (IMP).
[0846] AEs will be collected throughout the study, from informed
consent until the end of study visit. All AEs including local and
systemic reactions not meeting the criteria for "serious adverse
events" should be captured on the appropriate eCRF.
[0847] All AEs spontaneously reported by the participant or
reported in response to the open question from the study personnel
or revealed by observation will be collected and recorded in the
CRF. When collecting AEs, recording a diagnosis is preferred (when
possible) to recording a list of signs and symptoms. However, if a
diagnosis is known and there are other signs or symptoms that are
not generally part of the diagnosis, the diagnosis and each sign or
symptom will be recorded separately.
[0848] Any medical condition that is present at the time when the
informed consent is signed should be considered as medical history
and not reported as an AE. However, if it deteriorates at any time
after the signed ICF, it should be recorded as an AE.
[0849] AE Variables
[0850] The following variables will be collected for each AE:
Diagnosis/symptoms (verbatim). Date when the AE started (date of
onset) and stopped (date of resolution). NCI-CTCAE maximum
severity. Whether or not the AE is serious (seriousness).
Investigator causality rating against the investigational product.
Action taken with regard to the IMP. Whether or not AE caused
participant's withdrawal from IP. Outcome.
[0851] In addition, the following variables will be collected for
SAEs: Date AE met criteria for serious AE. Date Investigator became
aware of serious AE. Reason for classification as serious. Date of
hospitalisation. Date of discharge. Reason for hospitalisation.
Probable cause of death. Date of death. Post mortem performed.
Causality assessment in relation to study procedure(s). Causality
assessment in relation to other medication. Causality assessment in
relation to additional study drug. Description of AE.
[0852] All AEs occurring while on study must be documented
appropriately regardless of relationship. All AEs will be followed
up according to local practice until the event has stabilised or
adequate resolution.
[0853] All AEs must be graded for severity and relationship to
study drug.
[0854] Modification of MTL-CEBPA Administration for a
Participant
[0855] Alternative or intermediate doses and schedules maybe
required depending on arising clinical data from the study. This
may include rescheduling, dividing, reducing or de-escalating the
required dose.
[0856] Schedule Modification for a Participant
[0857] Dosing visits should be planned within the allowable time
window as specified in this protocol. However, in the event of the
participant not being able to attend the scheduled dosing day (e.g.
participant feeling unwell), the dosing may be rescheduled and the
timing of subsequent study visits should be altered
accordingly.
[0858] Administration of MTL-CEBPA may be delayed up to 1 week.
Should the participant not be able to attend the next dosing date,
the participant should be withdrawn from the study and an EOS study
visit should be schedule.
[0859] Dose and Schedule Modifications for a Participant
[0860] In the event of grade .gtoreq.3 infusion reaction the
procedure mentioned above should be followed. Following this
procedure the initial dose may be divided over 3 consecutive
days.
[0861] An example of divided dose schedule is shown below.
TABLE-US-00045 Starting Dose 28 mg/m.sup.2 days 1, 8, 15 Divided
dosing over 14 mg/m.sup.2 days 1, 2, 3, 8, 9 10, 15, 16, 17 3
consecutive days
[0862] In the event of divided dosing over three days, the PK
samples should be taken as planned at Day 1 and Day 8 and then
pre-dose at Day 2, 3 and at Day 9, 10.
[0863] In any case, a dose and schedule modification should be
discussed with the medical monitor prior implementation.
[0864] Dose Modifications for a Participant
[0865] Decision to de-escalate (e.g. if the starting dose on a
particular schedule results in toxicities such that the MTD is
exceeded) or to reduce the dose will be advised by the SRC.
Example 20. Formulation Optimiztion Study
[0866] NOV340 is a well-established liposomal formulation used for
encapsulation of oligonucleotides. Table 18 shows the lipid
composition of the formulation in molar ratio.
TABLE-US-00046 TABLE 18 Composition of NOV340 Molar Lipid ratio
POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) 6 DOPE
(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 24 Chems
(cholesteryl hemisuccinate) 23 MoChol
(Cholesteryl-4-[[2-(4-morpholinyl)ethyl]amino]- 47
4-oxobutanoate)
[0867] The present optimization study was done to improve the
encapsulation efficiency of CEBPA-51 into liposomes. Based on the
in-house experience in process development and optimization of
oligonucleotides formulated in NOV340 liposomes, the most critical
parameter to be optimized for CEBPA-51 is the encapsulation
efficiency. Encapsulation efficiency (%) is the amount of the
liposome-associated oligonucleotide (encapsulated and membrane
bound) divided by the total amount dissolved in the API solution
intended for encapsulation, excluding water content and impurities.
High process losses when preparing at small scale, should be also
taken under consideration when encapsulation efficiency is
calculated. Those losses are due to sampling and dead volumes in
filter units and tubing. Process losses will be reduced when the
production scale is increasing. Therefore, encapsulation efficiency
based on lipid recovery is also calculated. To improve
encapsulation efficiency, maximization of the interaction between
RNA and lipid was the most reasonable direction to go for. To
achieve this, two different parameters were varied and evaluated.
The first was lipid to drug ratio at the point of liposome
formation and the second was the pH of the buffer used to dissolve
the CEBPA-51. The interaction between RNA and lipid formulation
occurs primarily between MoChol and the oligonucleotide. MoChol is
an amphiphilic lipid with a pKa of 6.5 which is positively charged
in acidic medium. The interaction between the positively charged
MoChol with the negatively charged RNA results in increased
encapsulation of RNA into liposomes. Therefore, our hypothesis was
that decreasing the pH of the API buffer would result to an
increased MoChol charge, thus leading to increased interaction
between MoChol and CEBPA-51 and increased encapsulation efficiency.
The second option was to identify the optimal lipid to drug ratio,
or simpler the "saturation point" (maximum amount of CEBPA-51 that
can interact with MoChol). Therefore, various lipid to drug ratios
were tested in the course of liposome preparation experiments.
Lipid to drug ratio was changed by decreasing the concentration of
CEBPA-51 in the API solution. Seven different CEBPA-51
concentrations were tested.
[0868] Methods
[0869] Liposome Preparation
[0870] Liposomes were prepared by crossflow ethanol injection
method. In brief, lipids (POPC, Chems and DOPE) are dissolved in
absolute EtOH at 55.degree. C. After complete solubilisation, the
solution is quantitatively transferred into another bottle
containing pre-weighed MoChol. Selection of absolute EtOH and
solubilization of lipids in two steps have been identified to be
necessary to minimize the degradation of MoChol to Chol. After
complete dissolution of the lipids, the ethanolic lipid solution is
filtered through a 0.2 .mu.m filter and transferred quantitatively
in the injector which is as tempered at 55.degree. C. in a heating
cabinet. In the meantime, the oligonucleotide is dissolved in
Na-Acetate/Sucrose buffer and filtered through a 0.2 .mu.m filter
into the API buffer bottle at RT. The standard pH of the API buffer
for NOV340 formulations is at pH 4 but in the present optimization
study buffers with pH 3.5 and pH 4.0 were used. Liposomes are
forming when lipid solution is mixed with the API buffer in the
injection module. Immediately after liposome formation there is an
online dilution step with the dilution buffer
(NaCl/Na.sub.2HPO.sub.4 pH 9.0 at RT) in order to neutralize the pH
of the liposomal formulation. The liposome formulated
oligonucleotide is collected in the IV bottle and stirred at room
temperature for 30 minutes before extrusion. This intermediate
suspension is then extruded through 0.2 .mu.m polycarbonate
membrane to refine the size and PdI of liposomes. After extrusion,
free RNA and EtOH are removed by diafiltration and concentrated to
the target saRNA concentration by ultrafiltration. Before filling
into vials, the liposomal product is sterile filtered through a 0.2
.mu.m filter.
[0871] Size Measurements of Liposomes
[0872] Measurements for the determination of liposome size were
performed by Dynamic-Laser-Light-Scattering (DLS) using a Malvern
Nano ZS (224/SOP/002). This system is equipped with a 4 mW
Helium/Neon Laser at 633 nm wavelength and measures the liposome
samples with the non-invasive backscatter technology at a detection
angle of 173.degree.. Liposomes were diluted in aqueous phase to
reach optimal liposome concentration and the experiments were
carried out at 25.degree. C. The results are presented in an
average diameter of the liposome suspension (z-average mean) with
the polydispersity index to determine liposome homogeneity.
[0873] Zeta Potential Measurement of Liposomes
[0874] Zeta potential of liposomes was measured from the final bulk
product using a Malvern Nano ZS according to 224/SOP/012.
[0875] Quantification of RNA-Extinction Coefficient
[0876] Quantification of RNA was done by spectrophotometer at OD
260 nm according to 221/SOP/012. RNA was quantified in API solution
and in the final bulk product. In the very early stage of CEBPA-51
formulation development, the extinction coefficient of each single
strand of saRNA was measured. Therefore, quantification of the
saRNA was done using the average of extinction coefficient values
of each single strand. Due to hyperchromicity effect,
quantification of saRNA using the average extinction coefficient
resulted in decreased encapsulation efficiency values in the low 30
percent range. During development phase, the correct extinction
coefficient value was determined by STPharm resulted in the correct
saRNA quantification and in optimized encapsulation efficiency. The
first extinction coefficient used for quantification of CEBPA-51
was: 30.17 (L/(molecm)) and the updated value was: 20.68
(L/(molecm)).
[0877] Quantification of Lipids
[0878] Lipid concentration in the samples was measured from the
bulk volume using HPLC according to 222/SOP/004.
[0879] Results & Discussion
[0880] Comparison Between Old and New Extinction Coefficient
[0881] Table 19 lists the derived encapsulation efficiencies as
calculated by both extinction coefficient values for the samples
prepared before starting process optimization of the formulation.
From the derived values it is clear that the updated extinction
coefficient improves markedly the encapsulation efficiency of
CEBPA-51 into liposomes. However, further optimization studies were
conducted to maximize the yield of the saRNA in the final
formulation. Indeed, from an initial encapsulation efficiency of
almost 30% the optimized formulation resulted in encapsulation
efficiency of almost 60%.
TABLE-US-00047 TABLE 19 Encapsulation efficiency of CEBPA-51 into
liposomes calculated by old and new extinction coefficient values
Encapsulation efficiency (%) Calculated with old Calculated with
new Ext. coef. Ext. coef. Sample name 30.17 (L/(mole cm)) 20.68
(L/(mole cm)) IV/MIT/120315/1 24.7 36.0 IV/MIT/120315/2 33.7 49.1
IV/MIT/120315/3 27.3 39.8 IV/MIT/250315/1 39.2 57.3 IV/MIT/250315/2
24.6 35.9 IV/MIT/250315/3 27.8 40.5 IVMIT/300315/1 29.7 43.3
IV/MIT/300315/2 23.5 34.2
[0882] Liposome Preparation with API Solubilized in pH 3.5
Buffer
[0883] To study the effect of API solution's pH on the
encapsulation efficiency, the pH of the API solution was reduced
from pH 4.0 to pH 3.5. In addition, three different CEBPA-51
concentrations in the API solution were used, to study the effect
of lipid to drug ratio on the encapsulation efficiency. Table 20
lists all the formulations prepared with API solution of pH 3.5 and
their characteristics. Tables 21, 22 and 23 show the formulations
prepared with API solution of pH 3.5 in comparison with their
respective formulations prepared with API solution of pH 4.0. FIG.
34 presents the results shown in Tables 22, 23 and 24 as graph.
From the obtained results, it is obvious that changing the pH of
the API buffer results in slightly increased encapsulation
efficiency just at low concentration of RNA in the API solution
(1.85 mg/mL). With regard to the other two tested concentrations
(2.25 and 2.65 mg/mL), no obvious trend towards this direction
could be observed. Additionally, reduction of the pH resulted in
the formation of liposomes with increased size and PdI. Such an
increase in size and PdI would require an additional extrusion
cycle, which is not recommended as it would increase RNA losses and
process duration. Consequently, reduction of the injection buffer
pH from 4.0 to 3.5 is not an option. With regard to alteration of
the lipid to drug ratio, it was noticed that by decreasing the RNA
concentration in the API buffer, there is an increase on the
encapsulation efficiency. This trend seems to be similar for both
pH values of the API solution.
TABLE-US-00048 TABLE 20 Prepared liposomal samples using buffer
with pH 3.5 IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/ Sample name
250315/3 150415/1 150415/4 150415/3 150415/2 [CEBPA-51] in the 2.65
2.65 2.65 2.25 1.85 API solution (mg/mL) [CEBPA-51] in the 1.014
1.667 1.981 1.791 2.009 final product (mg/mL) Encapsulation 40.53
32.72 37.8 41.47 56 efficiency (%) [POPC] in the final 1.67 3.36
4.21 3.82 4.01 product (mg/mL) [DOPE] in the final 6.68 13.38 16.5
14.96 16.10 product (mg/mL) [Chems] in the final 4.05 8.30 10.25
9.34 9.90 product (mg/mL) [MoChol] in the final 10.42 20.13 24.13
22.96 23.87 product (mg/mL) Total [lipid] in the final 22.82 45.17
55.09 51.08 53.88 product (mg/mL) Encapsulation 52.47 42.91 41.38
47.87 62.03 efficiency based on lipid recovery (%) Size/PdI of the
final 125.5/0.176 124.7/0.184 125.6/0.185 116.1/0.190 109.6/0.195
product (nm/PdI) Maximum feasible 2.65 2.28 2.36 2.22 2.42
concentration of CEBPA-51 in the final product (mg/mL)
TABLE-US-00049 TABLE 21 Comparison between samples prepared with pH
3.5 and pH 4.0 buffer. CEBPA-51 concentration in the injection
buffer is 2.65 mg/mL. pH of API pH 3.5 pH 4.0 solution [CEBPA-51]
2.65 in the API solution (mg/mL) Sample IV/MIT/ IV/MIT/ IV/MIT/
IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/ name 250315/3 300315/2 150415/1
150415/4 120315/1 300315/1 160415/1 [CEBPA-51] 1.017 1 1.667 1.981
1.104 1.486 2.03 in the final product (mg/mL) Encapsulation 40.53
34.2 32.72 37.80 36.0 43.3 41.89 efficiency (%) Encapsulation 52.47
44.33 42.91 41.38 40.80 54.31 51.02 efficiency based on lipid
recovery (%) Size and PdI 159.0/0.321 144.4/0.372 158.8/0.511
128.2/0.398 136.8/0.220 148.5/0.250 151.1/0.259 of the IV liposomes
(nm/PdI) Size and PdI 124.7/0.184 128.2/0.207 125.6/0.185
116.1/0.190 128.0/0.149 132/0.154 n.a. of the final product
(nm/PdI)
TABLE-US-00050 TABLE 22 Comparison between samples prepared with pH
3.5 and pH 4.0 buffer. CEBPA-51 concentration in the injection
buffer is 2.25 mg/mL. pH of API solution pH 3.5 pH4.0 [CEBPA-51] in
the API solution (mg/mL) 2.25 Sample name IV/MIT/250315/3
IV/MIT/140415/2 IV/MIT/050515/2 [CEBPA-51] in the final product
(mg/mL) 1.79 2.23 2.62 Encapsulation efficiency (%) 41.47 51.65
47.66 Encapsulation efficiency based on lipid recovery 47.87 56.12
52.71 (%) Size and PdI of the IV liposomes (nm/PdI) 128.2/0.398
130.6/0.220 140.5/0.249 Size and PdI of the final product (nm/PdI)
116.1/0.190 127/0.152 131.3/0.166
TABLE-US-00051 TABLE 23 Comparison between samples prepared with pH
3.5 and pH 4.0 buffer. CEBPA-51 concentration in the injection
buffer is 1.85 mg/mL. pH of API solution pH3.5 pH4.0 [CEBPA-51] in
the API solution (mg/mL) 1.85 Sample name IV/MIT/150415/2
IV/MIT/120315/2 [CEBPA-51] in the final product 2.01 1.13 (mg/mL)
Encapsulation efficiency (%) 56.01 49.14 Encapsulation efficiency
based 62.03 52.64 on lipid recovery (%) Size and PdI of the IV
112.2/0.294 132.9/0.233 liposomes (nm/PdI) Size and PdI of the
final 109.6/0.195 124.6/0.150 product (nm/PdI)
[0884] Optimization of Lipid to Drug Ratio.
[0885] Lipid to drug ratio at the point of liposome formation is of
major importance for the encapsulation efficiency of RNA within
liposomes. To optimize this, lipid concentration in the EtOH
solution was kept the same and the concentration of CEBPA-51 in the
API solution was altered ranging from 1.06 up to 3.44 mg/mL. All
other process parameters remained constant and the pH of the API
solution was maintained at pH 4.0. Table 24 lists all the
formulations prepared and their characteristics. In FIG. 35 the
same results are plotted as graph. From the obtained results it is
clear that there is a slight trend indicating that by decreasing
the CEBPA-51 concentration in the API solution, there is a slight
increase in the encapsulation efficiency. At this point it is
important to mention that lipid concentration in the final product
is a key result which should be taken under consideration when
trying to achieve the maximum yield of saRNA. On the other hand,
increased lipid concentration in the final product can become
critical at the final 0.2 .mu.m filtration, as at some point, the
filter membranes might block upon too much lipids exposed to filter
membrane area. Consequently, the aim is not solely to minimize RNA
losses, but also to achieve the optimal lipid concentration which
would allow increased CEBPA-51 concentration in the final
product.
TABLE-US-00052 TABLE 24 Prepared liposomal samples using buffer
with pH 4.0. Sample IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/ IV/MIT/
name 120315/3 120315/1 300315/1 290415/1 290415/2 170415/1
[CEBPA-51] in the 3.44 2.65 2.65 2.52 2.52 2.38 API solution
(mg/mL) [CEBPA-51] in the 1.781 1.10 1.486 2.27 1.94 1.69 final
product (mg/mL) Encapsulation 39.76 35.99 43.28 46.74 45.41 50.37
efficiency (%) [POPC] in the final 2.75 2.27 2.30 3.88 3.43 2.75
product (mg/mL) [DOP7.06E] in the 11.39 9.48 9.35 15.30 13.28 10.96
final product (mg/mL) [Chems] in the final 6.61 5.61 5.47 9.55 8.47
7.06 product (mg/mL) [MoChol] in the final 17.17 14.55 15.16 22.92
19.92 16.72 product (mg/mL) Total [lipid] in the 37.94 31.92 32.27
51.66 45.10 37.49 final product (mg/mL) Encapsulation 42.71 40.80
54.31 54.10 52.38 58.17 efficiency based on lipid recovery (%)
Size/PdI of the final 139.2/0.135 128.0/0.149 132.0/0.154
128.1/0.149 130.8/0.155 127.2/0.150 product (nm/PdI) Maximum
feasible 3.17 2.25 2.70 2.76 2.72 2.80 concentration of CEBPA-51 in
the final product (mg/mL)
[0886] Confirmation of the Optimization Study.
[0887] After conducting all the experiments towards optimization of
encapsulation efficiency of saRNA into liposomes, two final batches
of larger volume were prepared to confirm the results. Table 25
shows the derived data, which is also graphically presented
together with the results of all other prepared batches in FIG. 35.
The obtained data, clearly confirms the decision made to set the
concentration of CEBPA-51 in the injection buffer to 2.38 mg/mL as
it results both in higher encapsulation efficiency of saRNA and in
higher concentration of CEBPA-51 in the final liposomal
product.
TABLE-US-00053 TABLE 25 Liposomal samples prepared for confirmation
of the derived results. Sample name IV/MIT/ IV/MIT/ 270515/1
280515/1 [CEBPA-51] in the API solution 2.52 2.38 (mg/mL)
[CEBPA-51] in the final product 2.29 2.65 (mg/mL) Encapsulation
efficiency (%) 44.62 51.0 [POPC] in the final product (mg/mL) 4.53
4.79 [DOP7.06E] in the final product (mg/mL) 17.38 18.46 [Chems] in
the final product (mg/mL) 11.00 11.71 [MoChol] in the final product
(mg/mL) 25.88 27.91 Total [lipid] in the final product (mg/mL)
58.79 62.88 Encapsulation efficiency based on lipid 47.18 53.81
recovery (%) Size/PdI of the final product (nm/PdI) 130.8/0.143
124.6/0.141 Maximum feasible concentration of 2.59 2.80 CEBPA-51 in
the final product (mg/mL)
[0888] Conclusion
[0889] This study optimized encapsulation efficiency of CEBPA-51
into liposomes. The results derived indicate that production of
liposomes should be done with API buffer of pH 4.0 and CEBPA-51
concentration in the API solution should be set at around 2.38
mg/mL. Those settings should result in a liposomal formulation with
a yield in the area of higher 50 percent which is the typical yield
derived when formulating oligonucleotides with NOV340.
Example 21. In-Use Stability Study
[0890] 51.80 to 296.00 mg MTL-CEBPA was diluted to a total infusion
volume of 250 ml resulting in a final concentration of 0.21 to 1.18
mg/ml in infusion bags. One type of infusion bag was used (Baxter
VIAFLO 250 ml Sodium Chloride 0.9% Intravenous Infusion BP) and 2
representative, typical space lines (PVC and PVC-free) were
selected for evaluation. The lowest dose of 0.21 mg/ml
(corresponding to a clinical dose level of 28 mg/m.sup.2) was
selected for this in-use study as worst case scenario.
Material
[0891] 5 vials MTL-CEBPA drug product, lot MIT1215-A, saRNA
content: 2.5 mg/ml; nominal volume 20 ml; Infusion bags:
4.times.Baxter VIAFLO 250 ml Sodium Chloride 0.9% Intravenous
Infusion BP (ref: FE1322); Infusion space lines: 1). 2.times.Braun
Infusomat.RTM. Space line-Neutrapur (polyurethane) PVC-free (ref:
8700110SP); 2). 2.times.Braun Infusomat.RTM. Space line-PVC
(DEHP-free) (ref: 8700036SP); Needles: e.g. BD Mirolance (ref:
301300); Syringes: e.g. BD Syringe (ref: 309658); Incubator
QC-GTBS01MM (23-27.degree. C.).
Preparation of Dosing Solutions, Sampling and Storage
[0892] MIT1215-A (2.5 mg/ml) was diluted in infusion bags by
removal of approximately 21 ml 0.9% normal saline and replacement
with approximately 21 ml of drug product. Duplicate bags were
prepared for each space line resulting in a total of 4 infusion
bags. Bags were weighed before and after removal of saline and
after addition of drug product. A density of 1.04 g/ml was used for
calculation of added amount of drug product. Two bags (#1 and #2)
were connected with PVC-free space lines, the remaining two bags
(#3 and #4) with PVC space lines. Bags were stored at
25.+-.2.degree. C. for 24 hours.
TABLE-US-00054 TABLE 26 Preparation of dosing solutions Weight
Weight Removed Weight Added bag bag after saline after drug drug
before saline volume product product Bag saline removal
(calculated) addition (calculated) ID removal [g] [ml] .sup.1) [g]
[ml] .sup.2) #1 285.47 264.47 21.00 285.71 20.42 #2 285.21 264.83
20.38 286.26 20.61 #3 281.36 260.73 20.63 282.47 20.90 #4 284.43
263.44 20.99 285.02 20.75 .sup.1) density saline: 1.00 g/ml .sup.2)
density DP: 1.04 g/ml
[0893] Samples (4.times.0.5 ml per time point and bag) were
collected via space lines immediately after bag preparation (time
point 0 hours), after 8 hours and 24 hours and stored at
-20.+-.5.degree. C. At time points 8 and 24 hours the lines were
purged with approximately 30 ml before sampling to ensure that
sample material from infusion bags was collected rather than
material incubated in the space lines. The analyses of all samples
were performed within a single analytical run of SEC-HPLC (content)
and RP-HPLC (lipids).
Tests and Acceptance Criteria
Overview
TABLE-US-00055 [0894] Acceptance Acceptance criterion DP Test/
criterion DP (post-dilution; target Analysis Method (prior to
dilution) concentration) Content total SEC-HPLC 2.5 .+-. 0.5 mg/ml
0.21 .+-. 0.04 mg/ml saRNA 222/SOP/013 Content RP-HPLC 3.5-5.8
mg/ml 0.29-0.49 mg/ml POPC 222/SOP/018 Content RP-HPLC 13.5-22.6
mg/ml 1.13-1.90 mg/ml DOPE 222/SOP/018 Content RP-HPLC 8.5-14.1
mg/ml 0.71-1.18 mg/ml CHEMS 222/SOP/018 Content RP-HPLC 20.3-33.8
mg/ml 1.71-2.84 mg/ml MoChol 222/SOP/018 Content RP-HPLC
.ltoreq.2.0 mg/ml .ltoreq.0.17 mg/ml cholesterol 222/SOP/018
[0895] Acceptance criteria were applied based on Drug Product (DP)
specifications under consideration of respective dilution factor of
about 12-fold upon preparation of the suspension for infusion. i.e.
diluting saRNA content from 2.5.+-.0.5 mg/ml to 0.21.+-.0.04
mg/ml.
Results
[0896] Content saRNA
[0897] The content of total saRNA was measured by RP-HPLC with UV
detection according 222/SOP/013. All samples were analysed within
the same HPLC sequence. Results are listed in Table 27. All bags
contained saRNA concentrations close to the target value and met
the acceptance criterion of 0.21.+-.0.04 mg/ml throughout the
observation period.
TABLE-US-00056 TABLE 27 saRNA contents (mg/ml) in infusion bags Bag
#1 Bag #2 Bag #3 Bag #4 (PVC free) (PVC free) (PVC) (PVC) 0 hours
0.20 0.20 0.20 0.20 8 hours 0.19 0.19 0.20 0.20 24 hours 0.20 0.19
0.20 0.20 Acceptance criterion met met met met 0.21 .+-. 0.04
mg/ml
Content POPC
[0898] The content of total POPC was measured by RP-HPLC with CAD
detection according 222/SOP/018. All samples were analysed within
the same HPLC sequence. Results are listed in Table 28. All bags
contained POPC concentrations close to the target value and met the
acceptance criterion of 0.29-0.49 mg/ml throughout the observation
period.
TABLE-US-00057 TABLE 28 POPC contents (mg/ml) in infusion bags Bag
#1 Bag #2 Bag #3 Bag #4 Time point (PVC free) (PVC free) (PVC)
(PVC) 0 hours 0.32 0.40 0.40 0.37 8 hours 0.39 0.36 0.36 0.36 24
hours 0.34 0.30 0.34 0.32 Acceptance criterion met met met met
0.29-0.49 mg/ml
Content DOPE
[0899] The content of total DOPE was measured by RP-HPLC with CAD
detection according 222/SOP/018. All samples were analysed within
the same HPLC sequence. Results are listed in Table 29. All bags
contained DOPE concentrations close to the target value and met the
acceptance criterion of 1.13-1.90 mg/ml throughout the observation
period.
TABLE-US-00058 TABLE 29 DOPE contents (mg/ml) in infusion bags Bag
#1 Bag #2 Bag #3 Bag #4 Time point (PVC free) (PVC free) (PVC)
(PVC) 0 hours 1.27 1.51 1.52 1.45 8 hours 1.50 1.40 1.38 1.41 24
hours 1.31 1.17 1.30 1.24 Acceptance criterion met met met met
1.13-1.90 mg/ml
Content CHEMS
[0900] The content of total CHEMS was measured by RP-HPLC with CAD
detection according 222/SOP/018. All samples were analysed within
the same HPLC sequence. Results are listed in Table 30.
TABLE-US-00059 TABLE 30 CHEMS contents (mg/ml) in infusion bags Bag
#1 Bag #2 Bag #3 Bag #4 Time point (PVC free) (PVC free) (PVC)
(PVC) 0 hours 0.73 0.81 0.86 0.81 8 hours 0.83 0.77 0.75 0.78 24
hours 0.72 0.66 0.73 0.70 Acceptance criterion met met at 0, 8 h
met met at 0, 8 h 0.71-1.18 mg/ml failed at 24 h failed at 24 h
Content MoChol
[0901] The content of total MoChol was measured by RP-HPLC with CAD
detection according 222/SOP/018. All samples were analysed within
the same HPLC sequence. Results are listed in Table 31. All bags
contained MoChol concentrations close to the target value and met
the acceptance criterion of 1.71-2.84 mg/ml throughout the
observation period.
TABLE-US-00060 TABLE 31 MoChol contents (mg/ml) in infusion bags
Bag #1 Bag #2 Bag #3 Bag #4 Time point (PVC free) (PVC free) (PVC)
(PVC) 0 hours 1.75 2.02 2.06 1.95 8 hours 2.00 1.86 1.84 1.89 24
hours 1.72 1.55 1.75 1.64 Acceptance criterion met met at 0, 8 h
met met at 0, 8h 1.71-2.84 mg/ml failed at 24 h failed at 24 h
Content Cholesterol
[0902] The content of total cholesterol was measured by RP-HPLC
with CAD detection according 222/SOP/018. All samples were analysed
within the same HPLC sequence. Results are listed in Table 32. All
bags contained cholesterol concentrations lower than the maximally
allowed limit and met the acceptance criterion of .ltoreq.0.17
mg/ml throughout the observation period.
TABLE-US-00061 TABLE 32 Cholesterol contents (mg/ml) in infusion
bags Bag #1 Bag #2 Bag #3 Bag #4 Time point (PVC free) (PVC free)
(PVC) (PVC) 0 hours 0.06 0.08 0.08 0.08 8 hours 0.09 0.08 0.08 0.09
24 hours 0.09 0.09 0.10 0.09 Acceptance criterion met met met met
.ltoreq.0.17 mg/ml
Conclusions
[0903] The in-use study was conducted to confirm stability of
MTL-CEBPA in infusion bags at room temperature over a 24 hour
period and compatibility with the intended infusion lines.
[0904] Results confirm that the drug product is stable and
compatible over at least 8 hours at the lowest intended clinical
dose of 0.21 mg/ml.
[0905] Thus it can be concluded that the selected materials
(infusion bags and space lines) are compatible with the drug
product and can be used in clinical studies of MTL-CEBPA within a
time period of at least 8 hours.
Example 22. Stability Studies for MTL-CEBPA
[0906] Long term storage was performed at -20.+-.5.degree. C.
Stability under accelerated conditions was investigated at
2-8.degree. C. Stress test studies for identification of stability
indicating parameters were performed by storage at 25.+-.2.degree.
C. and 40.+-.2.degree. C. Table 33 provides an overview of batches
tested in those stability studies including duration, condition and
currently available data.
[0907] The analytical procedures used in the stability programme
included tests for appearance, pH, assay, purity, lipid content and
particle characteristics.
TABLE-US-00062 TABLE 33 Stability Studies for MTL-CEBPA Drug
Product Date of Stability Batches Tested Manufacture Study Start
Duration Conditions Available Data MIT0615-A June 2015 25 Jun. 2015
0-36 M -20 .+-. 5.degree. C. 0-3 months 2 Nov. 2015 0-72 h 25 .+-.
2.degree. C. 0-72 hours 2 Nov. 2015 0-72 h 40 .+-. 2.degree. C.
0-72 hours MIT1215-A December 2015 3 Dec. 2015 0-24 M -20 .+-.
5.degree. C. 0-1 month 3 Dec. 2015 0-6 M 5 .+-. 3.degree. C. 0-1
month 18 Jan. 2016 0-24 h 25 .+-. 2.degree. C. 0-24 hours
Stability Summary and Conclusion
[0908] Stability data were available for one batch of MTL-CEBPA
(MIT0615-A) stored at long-term storage conditions
(-20.+-.5.degree. C.). Stress test data were available for the same
batch stored at 25.+-.2.degree. C. and 40.+-.2.degree. C.
[0909] No change was observed for MIT0615-A stored at
-20.+-.5.degree. C. for up to 6 months. Samples stored under stress
conditions (25.+-.2.degree. C. and 40.+-.2.degree. C.) showed a
decrease in MOCHOL by degradation of MOCHOL and conversion into
cholesterol upon storage. This degradation was more pronounced at
40.+-.2.degree. C. compared to 25.+-.2.degree. C.
[0910] Under stress testing at 25.degree. C. for three days (72 h),
content of the key lipid, morpholinoethaneamine cholesterol
(MOCHOL) decreased from 33.2 to 27.8 mg/ml (a 16% decrease), while
after three days at 40.degree. C. the content of MOCHOL decreased
from 30.1 to 22.5 mg/ml (a decrease of 25%). This reduction in
MOCHOL corresponds to the formation of the degradation product
cholesterol, which increased from 1.1 to 2.2 mg/ml after three days
at 25.degree. C. and from 1.0 to 8.2 mg/ml after three days at
40.degree. C. The other lipid excipients did not exhibit
significant changes under accelerated conditions. No significant
changes were observed for the other lipids, total saRNA content and
its impurity. Tables 35-38 shows stability and stress test results
of MTL-CEBPA at different conditions.
TABLE-US-00063 TABLE 34 Long-term Stability at -20 .+-. 5.degree.
C. Batch MIT1215-A (long-term, -20 .+-. 5.degree. C.) Parameter
Acceptance Criterion 0 month 1 month 3 months 6 months Appearance
milky white suspension pass pass pass pass Content total saRNA 2.5
.+-. 0.5 mg/ml 2.6 2.6 2.6 2.6 saRNA .gtoreq.75% 83 81 80 78
encapsulation Content POPC 3.5-5.80 mg/ml 4.8 4.2 4.8 4.6 Content
DOPE 13.5-22.6 mg/ml 18.4 17.0 18.9 16.9 Content CHEMS 8.5-14.1
mg/ml 11.5 10.4 11.6 10.8 Content MOCHOL 20.3-33.8 mg/ml 26.9 24.6
27.2 24.2 Content Cholesterol .ltoreq.2.0 mg/ml 1.0 0.8 1.0 1.1
Particle size 100-140 nm 107 108 108 108 Polydispersity index
.ltoreq.0.200 0.169 0.159 0.163 0.167 Zeta potential .ltoreq.-30.0
mV at pH 7.2-7.8 -39.1 -38.5 -36.6 -35.2 pH 7.2-7.8 7.4 -- -- --
Osmolality 280-400 mOsmol/kg 349 -- -- -- Impurities saRNA
.ltoreq.15 % 6 7 5 7 Sub-visible particles part. .gtoreq.10 .mu.m:
.ltoreq.3000/vial 2 -- -- -- part. .gtoreq.25 .mu.m: <300/vial
<1 Endotoxin .ltoreq.5.0 EU/ml <0.5 -- -- -- Sterility no
growth pass -- -- --
TABLE-US-00064 TABLE 35 Accelerated Stability at 5 .+-. 3.degree.
C. of Batch MIT1215-A Parameter Acceptance Criterion 0 month 1
month Appearance milky white suspension pass pass Total saRNA 2.5
.+-. 0.5 mg/ml 2.5 2.6 saRNA .gtoreq.75% 85 82 encapsulation
Content POPC 3.5-5.80 mg/ml 4.7 4.4 Content DOPE 13.5-22.6 mg/ml
17.3 16.0 Content CHEMS 8.5-14.1 mg/ml 11.0 10.2 Content MOCHOL
20.3-33.8 mg/ml 24.9 22.3 Content Cholesterol .ltoreq.2.0 mg/ml 0.9
1.9 Particle size 100-140 nm 112 113 Polydispersity index
.ltoreq.0.200 0.160 0.140 Zeta potential .ltoreq.-30.0 mV at pH
7.2-7.8 -35.6 -36.1 pH 7.2-7.8 7.6 7.4 Osmolality 280-400 mOsmol/kg
334 341 Impurities saRNA .ltoreq.15% 6 7
TABLE-US-00065 TABLE 36 Stress test data at 25 .+-. 2.degree. C. of
Batch MIT0615-A Time points [hours] Parameter Acceptance Criterion
0 8 16 24 48 72 Total saRNA report result-[mg/ml] 2.6 2.6 2.7 2.6
2.6 2.6 Content POPC report result-[mg/ml] 5.5 5.2 5.5 5.3 5.3 4.9
Content DOPE report result-[mg/ml] 22.6 21.2 22.4 21.8 21.7 20.3
Content CHEMS report result-[mg/ml] 13.8 13.7 13.1 13.3 13.4 12.7
Content report result-[mg/ml] 33.2 31.0 32.4 31.5 30.4 27.8 MOCHOL
Content report result-[mg/ml] 1.1 1.2 1.4 1.5 1.9 2.2 Cholesterol
Impurities saRNA report result-[%] 8 8 8 8 7 5
TABLE-US-00066 TABLE 37 Stress test data at 40 .+-. 2.degree. C. of
Batch MIT0615-A Acceptance Time points [hours] Parameter Criterion
0 8 16 24 48 72 Total saRNA report result-[mg/ml] 2.6 2.6 2.7 2.6
2.5 2.6 Content POPC report result-[mg/ml] 5.0 5.4 5.3 4.3 5.6 5.8
Content DOPE report result-[mg/ml] 20.6 22.1 21.5 17.7 22.5 23.3
Content CHEMS report result-[mg/ml] 12.5 13.6 12.8 10.7 13.7 14.2
Content report result-[mg/ml] 30.1 31.1 28.0 22.1 24.4 22.5 MOCHOL
Content report result-[mg/ml] 1.0 1.9 2.6 2.8 5.8 8.2 Cholesterol
Impurities saRNA report result-[%] 6 7 7 7 5 6
[0911] MTL-CEBPA is stable when stored at long-term storage
condition (-20.+-.5.degree. C.) for at least 6 months, showing no
trend for decrease or changes other than derived from analytical
variability. It is stable for at least 1 month under accelerated
conditions at 2-8.degree. C. (5.+-.3.degree. C.).
Example 23. In Vivo Studies of CCL4 Induced Liver Cirrhosis with
CEBPA-saRNA
[0912] This study was a repeat of Example 11 with ascites and
survival exploring delayed administration. Carbon tetrachloride
(CCL4) induced hepatic fibrosis is a well-established and widely
accepted experimental model in rodents for the study of liver
fibrosis and cirrhosis. Chronic administration of carbon
tetrachloride to rats induces severe disturbances of hepatic
function together with histologically observable liver
fibrosis.
[0913] Liver cirrhosis in Sprague Dawley rats was induced by i.p.
injection of CCL4. Male Sprague Dawley rats with a starting body
weight of 120-150 g were used. CCL4 treated animals showed
significant reduction in body weight throughout the study. CCL4
treated animals showed significant increase in liver function test
(LFT) parameters such as Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), Alkaline phosphatase (ALP), Prothrombin
time (PT), and Bilirubin, but not GGT up to 8 weeks (first
randomization). A significant increase in Ammonia and a significant
reduction in total protein and in path control animals up to 8
weeks.
[0914] Treatment with MTL-CEBPA (1 mpk) at week 8 showed
significant improvement in body weight of animals. LFT and ammonia
levels were significantly reversed till 13 weeks. Total protein
levels were significantly increased. Treatment with MTL-CEBPA (1
mpk) at week 11 also showed significant reversal in LFT and ammonia
levels till 13 weeks. FIG. 36 showed that MTL-CEBPA treatment from
week 8 reversed hyper-ammonaemia.
[0915] FIG. 37A and FIG. 37B showed that MTL-CEBPA treatment
attenuated ascites. Ascites was assessed on a scale from 0 to 3
based on visual/physical examination. Absence of ascites was
recorded as 0; barely palpable ascites as 1; gross ascites with
expansion of the flanks as 2 and tense ascites at 3. Ascites was
noticeable from week 9 onwards and attained high score at week
13.
[0916] MTL-CEBPA treatment resulted in significant improvement in
survival as shown in survival graphs in FIG. 38A and FIG. 38B.
Example 24.CEBPA-51 Cross-Reactivity in Mouse and Rat
[0917] Aim of Study: The purpose of this study was to investigate
if rat and mouse are appropriate rodent species for preclinical
liver disease models and for non-clinical toxicology studies with
MTL-CEBPA/CEBPA-51.
[0918] Experimental Design: The study comprised 3 parts, 1)
sequence match from database searches, 2) sequencing of rat and
mouse genomic DNA (gDNA), and 3) confirming upregulation of CEBPA
in rat and mouse cell lines.
[0919] First, the sequence homology of CEBPA was assessed with
publically available databases (BLAST search). The CEBPA-51 target
sequence was used as a query search on the Rattus norvegicus
(Wistar strain) and Mus musculus (C57BL/6J strain) genomes using
BLAST.
[0920] Additionally, genomic DNA was isolated from rat and mouse
liver lobes and PCR products of the target sites were generated for
direct sequencing. The resulting sequences were then compared with
the published rat and mouse genome sequences via BLAST.
[0921] Functional cross-reactivity was then assessed by
transfecting CEBPA-51 into rat liver clone-9 cells and mouse liver
AML12 cells and measuring upregulation of CEBPA mRNA. Cells were
reverse transfected with 20 nM (f.c.) of each test item (CEBPA-51
or siFLUC, an untargeted siRNA) using Lipofectamine 2000, followed
by an additional forward transfection step (20 nM f.c.) of each
test item using Lipofectamine 2000. 24 hours after the second
transfection CEBPA mRNA levels were determined by qRT-PCR
(housekeeping gene: GAPDH; measured in triplicates).
[0922] Results: The BLAST search confirmed the absence of
mismatches between the sequence of CEBPA-51 and the rat and mouse
target sites (genomic location of the CEBPA gene). In addition, no
mismatches were found comparing CEBPA-51 and the amplified sequence
derived from gDNA from rat and mouse liver.
[0923] Transfection of CEBPA-51 into rat clone-9 cells and mouse
AML12 cells resulted in a 2-fold (n=1) and 1.7-fold (n=2) CEBPA
mRNA upregulation, respectively, while no upregulation was observed
with the untargeted control RNA duplex (siFLUC).
[0924] Conclusion: The genomic sequences of rat and mouse CEBPA are
identical to the CEBPA-51 target sequence according to the BLAST
database. This was further verified by sequencing of rat and mouse
liver gDNA. Functional cross-reactivity of CEBPA-51 was verified by
demonstration of CEBPA gene upregulation in initial studies in rat
and mouse liver cell lines. Rat and mouse models of liver cancer
and disease are therefore considered appropriate for preclinical
assessment of MTL-CEBPA activity, and rat is considered an
appropriate rodent species for non-clinical toxicology testing of
MTL-CEBPA.
Other Embodiments
[0925] It is to be understood that while the present disclosure has
been described in conjunction with the detailed description
thereof, the foregoing description is intended to illustrate and
not limit the scope of the present disclosure, which is defined by
the scope of the appended claims. Other aspects, advantages, and
modifications are within the scope of the following claims.
Sequence CWU 1
1
129119RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1cggucauugu cacugguca 19219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2ugaccaguga caaugaccg 19319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3agcugaaagg auucauccu 19419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4aggaugaauc cuuccagcu 19519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5acauaguccc agugauuaa 19619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6uuaaucacug ggacuaugu 19719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7gaauaagacu uuguccaau 19819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8auuggacaaa gucuuauuc 19919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9gcgcggauuc ucuuucaaa 191019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10uuugaaagag aauccgcgc 191119RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11ccaggaacuc gucguugaa 191219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12uucaacgacg aguuccugg 191319RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13agaaguuggc cacuuccau 191419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14auggagucgg ccgacuucu 191519RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15aagaggucgg agaggaagu 191619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16aguuccuggc cgaccuguu 191719RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17uuguacucgu cgcugugcu 191819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18agaacagcaa cgaguaccg 191919RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19uacucgucgc ugugcuugu 192019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20acaagaacag caacgagua 192125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 21agaaguuggc cacuuccaug gggga 252227DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 22tcccccaugg agucggccga cuucuac 272325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 23aagaggucgg agaggaaguc gucgt 252427DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 24acgacgaguu ccuggccgac cuguucc 272525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 25uuguacucgu cgcugugcuu gucca 252627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 26tggacaagaa cagcaacgag uaccggg 272725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 27uacucgucgc ugugcuuguc caccg 252827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 28cgguggacaa gaacagcaac gaguacc 272921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29cugaguaauc gcuuaaagau u 213021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30ucuuuaagcg auuacucagu u 213121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31gaaacuuuag cgagucagau u 213221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32ucugacucgc uaaaguuucu u 213321RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33acuacugagu gacaguagau u 213419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 17 of SEQ ID NO 67
34gguauacauc cucagagcu 193519RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 34 35agcucugagg auguauacc 193619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 46 of SEQ ID NO 67
36cuagcuuucu ggugugacu 193719RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 36 37agucacacca gaaagcuag 193819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 305 of SEQ ID NO
67 38cgggcuuguc gggaucuca 193919RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 37 39ugagaucccg acaagcccg 194019RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 457 of SEQ ID NO
67 40gcauuggagc ggugaguuu 194119RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 40 41aaacucaccg cuccaaugc 194219RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 486 of SEQ ID NO
67 42ggcacaaggu uauccuaaa 194319RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 42 43uuuaggauaa ccuugugcc 194419RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 487 of SEQ ID NO
67 44gcacaagguu auccuaaau 194519RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 44 45auuuaggaua accuugugc 194619RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 883 of SEQ ID NO
67 46cggucauugu cacugguca 194719RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 46 47ugaccaguga caaugaccg 194819RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSense strand starting at position 1616 of SEQ ID NO
67 48ccaggaacuc gucguugaa 194919RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideAntisense strand to
SEQ ID NO 48 49uucaacgacg aguuccugg 195021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50cggucauugu cacuggucau u 215121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 51ugaccaguga caaugaccgu u 215221RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(21)2'O-Me modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
52cggucauugu cacuggucau u 215321RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(21)2'O-Me modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
53ugaccaguga caaugaccgu u 215420DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemisc_feature(1)..(2)Phosphorothioate
linkagemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(20)Inverted
nucleotidemodified_base(20)..(20)Deoxy-nucleotide 54cggucauugu
cacuggucat 205521RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemisc_feature(1)..(2)Phosphorothioate
linkagemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(21)2'O-Me modified
nucleotidemisc_feature(19)..(21)Phosphorothioate linkage
55ugaccaguga caaugaccgu u 215621DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(1)Deoxy-nucleotidemodified_base(2)..(3)2'O-M-
e modified nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(21)Inverted
nucleotidemodified_base(21)..(21)Deoxy-nucleotide 56tcggucauug
ucacugguca
t 215721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(1)Deoxy-nucleotidemodified_base(2)..(2)2'F
modified
nucleotidemodified_base(3)..(4)Deoxy-nucleotidemodified_base(5).-
.(6)2'F modified
nucleotidemodified_base(7)..(7)Deoxy-nucleotidemodified_base(8)..(9)2'F
modified
nucleotidemodified_base(10)..(10)Deoxy-nucleotidemodified_base(1-
1)..(12)2'F modified
nucleotidemodified_base(13)..(13)Deoxy-nucleotidemodified_base(14)..(15)2-
'F modified
nucleotidemodified_base(16)..(17)Deoxy-nucleotidemodified_base(18)..(19)2-
'F modified
nucleotidemodified_base(20)..(21)Deoxy-nucleotidemodified_base(21)..(21)I-
nverted nucleotide 57tcggucauug ucacugguca t 215821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)2'F modified
nucleotidemodified_base(2)..(3)2'O-Me modified
nucleotidemodified_base(4)..(5)2'F modified
nucleotidemodified_base(6)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(16)2'O-Me modified
nucleotidemodified_base(17)..(18)2'F modified
nucleotidemodified_base(19)..(21)2'O-Me modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
58ugaccaguga caaugaccgu u 215923RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(20)2'O-Me modified
nucleotidemodified_base(21)..(21)2'F modified
nucleotidemodified_base(22)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
59gcggucauug ucacuggucu uuu 236023RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
60aagaccagug acaaugaccg cuu 236122DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemisc_feature(1)..(2)Phosphorothioate
linkagemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(20)2'O-Me modified
nucleotidemodified_base(21)..(21)2'F modified
nucleotidemodified_base(22)..(22)Inverted
nucleotidemodified_base(22)..(22)Deoxy-nucleotide 61gcggucauug
ucacuggucu ut 226223RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemisc_feature(20)..(21)Phosphorothioate
linkagemodified_base(21)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
62aagaccagug acaaugaccg cuu 236323DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(1)Deoxy-nucleotidemodified_base(2)..(3)2'O-M-
e modified nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(21)2'O-Me modified
nucleotidemodified_base(22)..(22)2'F modified
nucleotidemodified_base(23)..(23)Inverted
nucleotidemodified_base(23)..(23)Deoxy-nucleotide 63tgcggucauu
gucacugguc uut 236423DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(2)Deoxy-nucleotidemodified_base(3)..(3)2'F
modified
nucleotidemodified_base(4)..(5)Deoxy-nucleotidemodified_base(6).-
.(7)2'F modified
nucleotidemodified_base(8)..(8)Deoxy-nucleotidemodified_base(9)..(10)2'F
modified
nucleotidemodified_base(11)..(11)Deoxy-nucleotidemodified_base(1-
2)..(13)2'F modified
nucleotidemodified_base(14)..(14)Deoxy-nucleotidemodified_base(15)..(16)2-
'F modified
nucleotidemodified_base(17)..(18)Deoxy-nucleotidemodified_base(19)..(22)2-
'F modified nucleotidemodified_base(23)..(23)Inverted
nucleotidemodified_base(23)..(23)Deoxy-nucleotide 64tgcggucauu
gucacugguc uut 236523RNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemodified_base(3)..(4)2'F modified
nucleotidemodified_base(5)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(15)2'O-Me modified
nucleotidemodified_base(16)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(21)2'F modified
nucleotidemodified_base(22)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
65gaccagugac aaugaccgcu uuu 236623RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(20)2'O-Me modified
nucleotidemodified_base(21)..(21)2'F modified
nucleotidemodified_base(22)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
66ugaaaggauu cauccuccuu uuu 236723RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
67aaaggaggau gaauccuuuc auu 236822DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(2)2'O-Me modified
nucleotidemisc_feature(1)..(2)Phosphorothioate
linkagemodified_base(3)..(3)2'F modified
nucleotidemodified_base(4)..(4)2'O-Me modified
nucleotidemodified_base(5)..(5)2'F modified
nucleotidemodified_base(6)..(6)2'O-Me modified
nucleotidemodified_base(7)..(7)2'F modified
nucleotidemodified_base(8)..(8)2'O-Me modified
nucleotidemodified_base(9)..(9)2'F modified
nucleotidemodified_base(10)..(10)2'O-Me modified
nucleotidemodified_base(11)..(11)2'F modified
nucleotidemodified_base(12)..(12)2'O-Me modified
nucleotidemodified_base(13)..(13)2'F modified
nucleotidemodified_base(14)..(14)2'O-Me modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(16)..(16)2'O-Me modified
nucleotidemodified_base(17)..(17)2'F modified
nucleotidemodified_base(18)..(18)2'O-Me modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemodified_base(20)..(20)2'O-Me modified
nucleotidemodified_base(21)..(21)2'F modified
nucleotidemodified_base(22)..(22)Inverted
nucleotidemodified_base(22)..(22)Deoxy-nucleotide 68ugaaaggauu
cauccuccuu ut 226923RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(1)..(2)2'F modified
nucleotidemodified_base(3)..(3)2'O-Me modified
nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(23)2'O-Me modified
nucleotidemisc_feature(21)..(23)Phosphorothioate linkage
69aaaggaggau gaauccuuuc auu 237023DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(1)Deoxy-nucleotidemodified_base(2)..(3)2'O-M-
e modified nucleotidemodified_base(4)..(4)2'F modified
nucleotidemodified_base(5)..(5)2'O-Me modified
nucleotidemodified_base(6)..(6)2'F modified
nucleotidemodified_base(7)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(9)2'O-Me modified
nucleotidemodified_base(10)..(10)2'F modified
nucleotidemodified_base(11)..(11)2'O-Me modified
nucleotidemodified_base(12)..(12)2'F modified
nucleotidemodified_base(13)..(13)2'O-Me modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(15)..(15)2'O-Me modified
nucleotidemodified_base(16)..(16)2'F modified
nucleotidemodified_base(17)..(17)2'O-Me modified
nucleotidemodified_base(18)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(20)2'F modified
nucleotidemodified_base(21)..(21)2'O-Me modified
nucleotidemodified_base(22)..(22)2'F modified
nucleotidemodified_base(23)..(23)Inverted
nucleotidemodified_base(23)..(23)Deoxy-nucleotide 70tugaaaggau
ucauccuccu uut 237123DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(1)..(1)Inverted
nucleotidemodified_base(1)..(1)Deoxy-nucleotidemodified_base(2)..(2)2'F
modified
nucleotidemodified_base(3)..(9)Deoxy-nucleotidemodified_base(10)-
..(12)2'F modified
nucleotidemodified_base(13)..(13)Deoxy-nucleotidemodified_base(14)..(22)2-
'F modified nucleotidemodified_base(23)..(23)Inverted
nucleotidemodified_base(23)..(23)Deoxy-nucleotide 71tugaaaggau
ucauccuccu uut 237223RNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(7)2'O-Me modified
nucleotidemodified_base(8)..(8)2'F modified
nucleotidemodified_base(9)..(11)2'O-Me modified
nucleotidemodified_base(12)..(18)2'F modified
nucleotidemodified_base(19)..(19)2'O-Me modified
nucleotidemodified_base(20)..(21)2'F modified
nucleotidemodified_base(22)..(23)2'O-Me modified
nucleotidemisc_feature(22)..(23)Phosphorothioate linkage
72aggaggauga auccuuucau uuu 237321RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 73gcggucauug
ucacuggucu u 217421RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 74ugaaaggauu cauccuccuu u
217521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 75gaccagugac aaugaccgcu u
217621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 76aggaggauga auccuuucau u 21774001DNAHomo
sapiens 77tccctctccc accaggggta tacatcctca gagctgaccc acgacctagc
tttctggtgt 60gactcggggt gggggctccc actggtcacc tggtgacccc catcgcagtg
agttccgccc 120caaggggaag cccagcctat agcaggctgg ggtggggtgt
gtgcggaggg aggtgggaga 180ggcgtggaac tagagaccct ccaccttcat
gtagaactag gggaacaacc ttaggttcca 240agccccaagt ccctatgttt
ccaccccttt ctaaggacag gcgtggagga gcggctgggg 300ctggcgggct
tgtcgggatc tcagctccct gagccctcct cctgccacgg gcctgctccc
360ctccttctct catgggggtc tgctgtagcc tcgggaagga ggcaggaaac
ctccaaataa 420aatgacaagg cacgatttgc tccccctact cagtaggcat
tggagcggtg agtttgcatt 480tccaaggcac aaggttatcc taaatactag
agttgccggg ctcccagctc agccccaaga 540attctcccct cctcgcaggg
agaagccacc gcctggcccc ctcatcttag acgcaccaag 600tccggcgcag
aggaagggag gggacacgcg gagcaggcca ggctttcagg aggcaccgga
660atctcctagt cctggctcgc acggctcggg caagcctcga gatccggcga
ccccaaacca 720ctccctgggt ccccgccgga ggctggccca gggcggtccc
acagccgcgc gcctcacgcg 780cagttgccca tggccttgac caaggagctc
tctggcagct ggcggaagat gccccgcagc 840gtgtccagtt cgcggctcag
ctgttccacc cgcttgcgca ggcggtcatt gtcactggtc 900agctccagca
ccttctgctg cgtctccacg ttgcgctgct tggccttgtc gcggctcttg
960cgcaccgcga tgttgttgcg ctcgcgccgc acccggtact cgttgctgtt
cttgtccacc 1020gacttcttgg ccttgcccgc gccgctgccg ccactcgcgc
ggaggtcggg gtgcgcggcg 1080cccagcccct tgagcgcgct gccagggccc
ggcaggccgg cggcaccgag cgcgggcgcg 1140gggtgcgggc tgggcacggg
cgtgggcggc ggcgtggggt gaccgggctg caggtgcatg 1200gtggtctggc
cgcagtgcgc gatctggaac tgcaggtgcg gggcggccag gtgcgcgggc
1260ggcgggtgcg ggtgcgggtg cgagggcggc ggcggcggcg gcggctggta
agggaagagg 1320ccggccagcg ccagctgctt ggcttcatcc tcctcgcggg
gctcctgctt gatcaccagc 1380ggccgcagcg ccggcgcccc gacgcgctcg
tacaggggct ccagcctgcc gtccaggtag 1440ccggcggccg cgcagccgta
gccgggcggg ggcccgtgcg ctcccccggg catgacggcg 1500ccgccggggc
ccgcgggcgc gcccgggtag tcaaagtcgc cgccgccgcc gccgcccgtg
1560gggcccacgg ccgccttggc cttctcctgc tgccggctgt gctggaacag
gtcggccagg 1620aactcgtcgt tgaaggcggc cgggtcgatg taggcgctga
tgtcgatgga cgtctcgtgc 1680tcgcagatgc cgcccagcgg ctccggggcg
gcaggtgggg cgggaggctg cgcggggccc 1740gcgccccggg gaaagccgaa
ggcggcgctg ctgggcgcgt gcggggggct ctgcaggtgg 1800ctgctcatcg
ggggccgcgg ctccgcctcg tagaagtcgg ccgactccat gggggagtta
1860gagttctccc ggcatggcga gcctcggcgg cctccagcct gcgcggggcg
tcgccgccgc 1920ccacccggag accctgctcg cccgcgcccg cgcacctccg
ggtcgcgaat ggcccggccc 1980gcgccggccc agcttttata cccggcaggc
cgcgtcgccc cctagagtcc gaggcggcct 2040ctgtccccgg gctgcggcgg
cgcggcgcct gctgggtcct agcgcgcggc cggcatgggg 2100cggcgaacca
gcgcggcaca gcgccgcgct ccccaggcag gccgcggcgc aacgcccacc
2160gcctccagcg cgcccagcag agccgcggcg ctcgctccaa gctccgcccc
cggcccggcc 2220gtcgcccccg cgcccacgtg gtcggtagcg ggggccccct
cctcctgcct gccctaggcg 2280cccgtatcca gccacggccg ggagcccagg
agtatcccga ggctgcacgg ggtaggggtg 2340gggggcggag ggcgagtctt
ggtcttgagc tgctggggcg cggattctct ttcaaagcca 2400gaaccaggcc
tgtcccggac ccgcgtcccg gggaggctgc agcgcagagc agcggggctg
2460gggccggtgg ggggccgttt gggacgcgcg gagaggtcct gagcgcggtg
gctctgcgtc 2520tcctagctct gatctccagg ctacccctgt gattccgcgc
agaggtacct ctcggaggac 2580gccggggtcc catgggcggc gccgcgcagg
gcgctaggac cccgcgggga gcggaggcgg 2640cctcggcccg ggagcctgga
ggacctggcc ggtcgatccg cccgggctgg aaaactttct 2700ttataattac
ttctccaggt cggagcgcgc ggcttgctag gcgcgcgggg ccggcgctgt
2760tacccggcgt ggagtcgccg attttttttc ctgcgggacc gcggggcccc
ccagactagc 2820ggagctggac gccggggcga gcacggggag gggcgcaccg
agggaggaga caaacttaac 2880tctggggccg ggattccgag gcgggggccg
cagccctcga ggcccgaagc caccgcttcc 2940tcccccgcct ccccattcag
gtgggcgcca acggcgggag cgagggtgtc caggccgccg 3000ggctgccagg
tccgagcacg cacagggaga actctgccca gtggttcgcc gggcgctgta
3060gtccccggga tcctagggac cgaggcggcc aggccctggg gccccttgag
tgcggcagct 3120aatgctctca ccgcggcggg ggaaggagct tgccaccgag
acccccagcc acgtgcgtcc 3180ctcgcattct ttaccggggc cggggtggcg
gctacggacc gtcagctggg cccagatgga 3240gtcttgggag ccctcaagtg
tctcctgtcc ttgcccgcgc cgcccctcgc cactggcgct 3300gaggcctgac
gccgcctgcg tcccggctag aggcgcgctt gcctacaggt gagggaagac
3360ccccttcacc gacagtggcc ttaggcctgg caaggcgcca cgacccgccc
aggagccccg 3420gagggggcac agctaaaaac accgctggag agccccgagc
ttccacgacg atcgcagtaa 3480agaagcagtt tcatctgggc aacgcacact
gcgctttaat caagttccta ttcaacatag 3540tcccagtgat taatagccca
actgcttcgt tttcggtcca gagctcataa acaagatatt 3600tttagcttga
cgcttttgga cgggagggag taaaaaccag atacgttaaa taaatatccc
3660gatgtgagcc ggagagctgc ttgctgagcc aaatgcagga cccattcata
tagcattcac 3720ctgtggaggg agacctggac ggaaatcaaa aagcaccaag
agcgatttgc gtttttttct 3780gcggtgctaa aactaatggc ttttcctacc
taggaacaaa gaaacgccac tgtacatgca 3840cggttcccgg cctgtggagt
tgtgggagga aggcgatgtc tggccttttt tgcacagctg 3900ctgttgcctg
cccagagatc gggaactctg ccccgtagga ctggaagaaa cctcagtaat
3960gggaataaga ctttgtccaa tagggggctg atgaatgtgt g
40017821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(2)..(2)2'-OMe modified
nucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemisc_feature(19)..(21)Phosphorothioate
linkagesmodified_base(20)..(21)2'-OMe modified nucleotide
78cuuacgcuga guacuucgau u 217921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(9)..(9)2'-OMe modified
nucleotidemodified_base(15)..(16)2'-OMe modified
nucleotidemodified_base(19)..(19)2'-OMe modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkages
79ggaugaagug gagauuagut t 218021DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(4)..(5)2'F modified
nucleotidemodified_base(7)..(10)2'F modified
nucleotidemodified_base(14)..(17)2'F modified
nucleotidemodified_base(19)..(19)2'F modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
80ggaucaucuc aagucuuact t 218121RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 81ucuacuguca
cucaguaguu u 218221RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemodified_base(2)..(2)2'-OMe
modified nucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(9)..(9)2'-OMe modified
nucleotidemodified_base(14)..(14)2'-OMe modified
nucleotidemisc_feature(19)..(21)Phosphorothioate
linkagesmodified_base(19)..(21)2'-OMe modified nucleotide
82ucgaaguacu cagcguaagu u 218321DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(3)..(3)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(15)..(15)2'-OMe modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkages
83acuaaucucc acuucaucct t 218421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(2)..(2)2'F modified
nucleotidemodified_base(7)..(9)2'F modified
nucleotidemodified_base(14)..(14)2'F modified
nucleotidemodified_base(17)..(19)2'F modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
84guaagacuug agaugaucct t 218521RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 85gcggucauug
ucacuggucu u 218621RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 86gaccagugac aaugaccgcu u
218721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 87ugaaaggauu cauccuccuu u
218821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 88aggaggauga auccuuucau u
218921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemisc_feature(20)..(21)Phosphorothioate
linkage 89ggaugaagug gagauuagut t 219021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 90acuaaucucc acuucaucct t 219121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(3)2'-OMe modified
nucleotidemodified_base(5)..(5)2'-OMe modified
nucleotidemodified_base(7)..(8)2'-OMe modified
nucleotidemodified_base(12)..(12)2'-OMe modified
nucleotidemodified_base(14)..(17)2'-OMe modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
91cuuacgcuga guacuucgat t 219221DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(11)..(11)2'-OMe modified
nucleotidemodified_base(16)..(16)2'-OMe modified
nucleotidemisc_feature(20)..(21)Phosphorothioate linkage
92ucgaaguacu cagcguaagt t 219321RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 93gaccagugac
aaugaccgcu u 219421RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 94gcggucauug ucacuggucu u
219521RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 95acuacugagu gacaguagau u
219621RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 96ucuacuguca cucaguaguu u
219721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97gcggucauug ucacuggucu u
219821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 98gaccagugac aaugaccgcu u
219921RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(1)Inverted abasic
modification 99gcggucauug ucacuggucu u 2110021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)Inverted abasic modification
100gaccagugac aaugaccgcu u 2110121RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 101gcggucauac
acacuggucu u 2110221RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 102gaccagugug
uaugaccgcu u 2110321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotidemodified_base(1)..(3)2'-OMe
modified nucleotidemodified_base(5)..(5)2'-OMe modified
nucleotidemodified_base(7)..(8)2'-OMe modified
nucleotidemodified_base(12)..(12)2'-OMe modified
nucleotidemodified_base(14)..(17)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
103cuuacgcuga guacuucgat t 2110421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(11)..(11)2'-OMe modified
nucleotidemodified_base(16)..(16)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
104ucgaaguacu cagcguaagt t 2110521DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(9)..(9)2'-OMe modified
nucleotidemodified_base(15)..(16)2'-OMe modified
nucleotidemodified_base(19)..(19)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
105ggaugaagug gagauuagut t 2110621DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(3)..(3)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(15)..(15)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
106acuaaucucc acuucaucct t 2110721RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)Inverted abasic
modificationmodified_base(1)..(2)2'-OMe modified
nucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(14)..(14)2'-OMe modified
nucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
107gcggucauug ucacuggucu u 2110821RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
108gaccagugac aaugaccgcu u 2110921RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
109gaccagugac aaugaccgcu u 2111021RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)Inverted abasic
modificationmodified_base(1)..(2)2'-OMe modified
nucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(14)..(14)2'-OMe modified
nucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
110gcggucauug ucacuggucu u 2111123RNAArtificial SequenceSynthetic
oligonucleotidemodified_base(2)..(2)2'-OMe modified
nucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(20)..(23)2'-OMe modified nucleotide
111cuuacgcuga guacuucgas usu 2311221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(9)..(9)2'-OMe modified
nucleotidemodified_base(15)..(16)2'-OMe modified
nucleotidemodified_base(19)..(19)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
112ggaugaagug gagauuagut t 2111321DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(5)..(6)2'F modified
nucleotidemodified_base(8)..(11)2'F modified
nucleotidemodified_base(15)..(18)2'F modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkage
113ggaucaucuc aagucuuact t 2111421RNAArtificial SequenceSynthetic
oligonucleotide 114ucuacuguca cucaguaguu u 2111523RNAArtificial
SequenceSynthetic oligonucleotidemodified_base(2)..(2)2'-OMe
modified nucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(9)..(9)2'-OMe modified
nucleotidemodified_base(14)..(14)2'-OMe modified
nucleotidemodified_base(19)..(23)2'-OMe modified nucleotide
115ucgaaguacu cagcguaags usu 2311621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(3)..(3)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(15)..(15)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkages
116acuaaucucc acuucaucct t 2111721DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(3)..(3)2'F modified
nucleotidemodified_base(8)..(10)2'F modified
nucleotidemodified_base(15)..(15)2'F modified
nucleotidemodified_base(18)..(19)2'F modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkages
117guaagacuug agaugaucct t 2111821RNAArtificial SequenceSynthetic
oligonucleotide 118cggucauugu cacuggucau u 2111921RNAArtificial
SequenceSynthetic oligonucleotide 119ugaccaguga caaugaccgu u
2112021RNAArtificial SequenceSynthetic oligonucleotide
120gcggucauug ucacuggucu u 2112121RNAArtificial SequenceSynthetic
oligonucleotide 121gaccagugac aaugaccgcu u 2112221RNAArtificial
SequenceSynthetic oligonucleotide 122ugaaaggauu cauccuccuu u
2112321RNAArtificial SequenceSynthetic oligonucleotide
123aggaggauga auccuuucau u 2112421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(20)..(21)Phosphorothioate linkages
124ggaugaagug gagauuagut t 2112521DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 125acuaaucucc
acuucaucct t 2112621DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotidemodified_base(1)..(3)2'-OMe
modified nucleotidemodified_base(5)..(5)2'-OMe modified
nucleotidemodified_base(7)..(8)2'-OMe modified
nucleotidemodified_base(12)..(12)2'-OMe modified
nucleotidemodified_base(14)..(17)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkages
126cuuacgcuga guacuucgat t 2112721DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic
oligonucleotidemodified_base(7)..(7)2'-OMe modified
nucleotidemodified_base(11)..(11)2'-OMe modified
nucleotidemodified_base(16)..(16)2'-OMe modified
nucleotidemodified_base(20)..(21)Phosphorothioate linkages
127ucgaaguacu cagcguaagt t 2112821RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)Inverted abasic
modificationmodified_base(1)..(2)2'-OMe modified
nucleotidemodified_base(4)..(4)2'-OMe modified
nucleotidemodified_base(10)..(10)2'-OMe modified
nucleotidemodified_base(14)..(14)2'-OMe modified
nucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
128gcggucauug ucacuggucu u 2112921RNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotidemodified_base(20)..(21)2'-OMe modified nucleotide
129gaccagugac aaugaccgcu u 21
* * * * *