U.S. patent application number 16/049212 was filed with the patent office on 2019-03-28 for alternative nucleic acid molecules containing reduced uracil content and uses thereof.
The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Stephen G. HOGE, William Joseph ISSA, Edward John MIRACCO, Jennifer NELSON, John REYNDERS, Matthew STANTON.
Application Number | 20190092828 16/049212 |
Document ID | / |
Family ID | 55954873 |
Filed Date | 2019-03-28 |
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United States Patent
Application |
20190092828 |
Kind Code |
A1 |
HOGE; Stephen G. ; et
al. |
March 28, 2019 |
ALTERNATIVE NUCLEIC ACID MOLECULES CONTAINING REDUCED URACIL
CONTENT AND USES THEREOF
Abstract
The present disclosure provides alternative nucleosides,
nucleotides, and nucleic acids, and methods of using them. In some
aspects, the disclosure provides mRNA wherein the uracil content
has been modified and which may be particularly effective for use
in therapeutic compositions, because they may benefit from both
high expression levels and limited induction of the innate immune
response. In some aspects, the disclosure provides methods for the
production of pharmaceutical compositions including mRNA without
reverse phase chromatography.
Inventors: |
HOGE; Stephen G.;
(Brookline, MA) ; ISSA; William Joseph;
(Roslindale, MA) ; MIRACCO; Edward John;
(Cambridge, MA) ; NELSON; Jennifer; (Brookline,
MA) ; REYNDERS; John; (Newton, MA) ; STANTON;
Matthew; (Marlton, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
55954873 |
Appl. No.: |
16/049212 |
Filed: |
July 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15656740 |
Jul 21, 2017 |
10072057 |
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16049212 |
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14960031 |
Dec 4, 2015 |
9751925 |
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15656740 |
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PCT/US2015/059112 |
Nov 4, 2015 |
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14960031 |
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62077871 |
Nov 10, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 113/12007 20130101;
A61K 48/005 20130101; C07K 14/535 20130101; C12N 9/0069 20130101;
C07K 14/505 20130101; C12P 19/34 20130101; C12P 21/02 20130101 |
International
Class: |
C07K 14/535 20060101
C07K014/535; C07K 14/505 20060101 C07K014/505; C12P 19/34 20060101
C12P019/34; A61K 48/00 20060101 A61K048/00; C12N 9/02 20060101
C12N009/02; C12P 21/02 20060101 C12P021/02 |
Claims
1. An mRNA encoding a polypeptide comprising: (i) at least one
5'-cap structure; (ii) a 5'-UTR; (iii) an open reading frame
encoding the polypeptide and consisting of nucleotides that contain
5-methoxy-uracil, cytosine, adenine, or guanine, wherein the open
reading frame has uracil content between the theoretical minimum
and 200% of the theoretical minimum and at least one codon is a low
frequency codon; (iv) a 3'-UTR; and (v) a poly-A region, wherein,
upon administration to a mammalian cell, the mRNA has increased
expression of the encoded polypeptide relative to a corresponding
mRNA wherein all of the codons are high frequency codons.
2. The mRNA of claim 1, wherein the mRNA does not contain more than
four consecutive uracils.
3. The mRNA of claim 1, wherein the open reading frame has the
theoretical minimum uracil content.
4. The mRNA of claim 1, wherein the uracil content of the open
reading frame is less than 20% of the total nucleotide content in
the open reading frame.
5. The mRNA of claim 1, wherein the mRNA contains fewer than ten
uracil pairs.
6. The mRNA of claim 1, wherein the uracil content within any 20
nucleotide window within the open reading frame does not exceed
50%.
7. The mRNA of claim 1, wherein the open reading frame contains at
least one of the following codons: GCG, GGG, CCG, AGG, ACG, CUC,
CGC, UCC, and GUC.
8. The mRNA of claim 1, wherein the uracil content of the 5'-UTR
and/or the 3'-UTR is between the theoretical minimum and 200% of
the theoretical minimum.
9. The mRNA of claim 8, wherein the 5'-UTR and/or the 3'-UTR has
the theoretical minimum uracil content.
10. The mRNA of claim 1, wherein the at least one 5'-cap structure
is cap0, cap1, or ARCA.
11. The mRNA of claim 1, wherein the 3'-UTR is an alpha-globin
3'-UTR.
12. The mRNA of claim 1, wherein the poly-A region is at least 160
nucleotides in length.
13. The mRNA of claim 1, wherein, upon administration to a
mammalian cell, the mRNA induces a detectably lower level of
IFN-.beta. relative to a corresponding mRNA having uracil content
of greater than 200% of the theoretical minimum.
14. The mRNA of claim 1, wherein, upon administration to a
mammalian cell, the mRNA has a longer half-life or greater area
under the curve of protein expression relative to a corresponding
mRNA having uracil content of greater than 200% of the theoretical
minimum.
15. A method of producing a codon-modified mRNA comprising an open
reading frame encoding a polypeptide, the method comprising: (a)
providing a parent sequence of an open reading frame encoding the
polypeptide; (b) modifying the parent sequence to produce a second
sequence of an open reading frame having uracil content between the
theoretical minimum and 200% of the theoretical minimum, by
replacing codons containing uracils with codons having the lowest
number of uracils, wherein at least one replacement codon is a low
frequency codon; and (c) producing the codon-modified mRNA having
the second sequence consisting of nucleotides including
5-methoxy-uracil as the uracil source, cytosine, adenine, and
guanine, wherein the open reading frame has uracil content between
the theoretical minimum and 200% of the theoretical minimum and at
least one codon is a low frequency codon.
16-27. (canceled)
Description
REFERENCE TO A SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] There are multiple problems with prior methodologies of
effecting protein expression. For example, heterologous DNA
introduced into a cell can be inherited by daughter cells (whether
or not the heterologous DNA has integrated into the chromosome) or
by offspring. Introduced DNA can integrate into host cell genomic
DNA at some frequency, resulting in alterations and/or damage to
the host cell genomic DNA. In addition, multiple steps must occur
before a protein is made. Once inside the cell, DNA must be
transported into the nucleus where it is transcribed into RNA. The
RNA transcribed from DNA must then enter the cytoplasm where it is
translated into protein. This need for multiple processing steps
creates lag times before the generation of a protein of interest.
Further, it is difficult to obtain DNA expression in cells;
frequently DNA enters cells but is not exprfressed or not expressed
at reasonable rates or concentrations. This can be a particular
problem when DNA is introduced into cells such as primary cells or
modified cell lines.
[0003] Naturally occurring RNAs are synthesized from four basic
ribonucleotides: ATP, CTP, UTP and GTP, but may contain
post-transcriptionally modified nucleotides. Further, approximately
one hundred different nucleoside alterations have been identified
in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA
Modification Database: 1999 update. Nucl Acids Res 27:
196-197).
[0004] There is a need in the art for biological modalities to
address the modulation of intracellular translation of nucleic
acids. The present invention solves this problem by providing new
mRNA molecules incorporating chemical alterations which impart
properties which are advantageous to therapeutic development.
SUMMARY OF THE INVENTION
[0005] The present disclosure provides, inter alia, alternative
nucleosides, alternative nucleotides, and alternative nucleic acids
including an alternative nucleobase, sugar, or backbone.
[0006] In a first aspect, the invention features an mRNA encoding a
polypeptide of interest and including an open reading frame,
wherein (a) the uracil content of the mRNA is less than 20% of the
total nucleotide content in the open reading frame; and (b) at
least 90% (e.g., at least 95%, at least 99%, or 100%) of the
uracils in the open reading frame are 5-methoxy-uracil. In
preferred embodiments, the open reading frame consists of
nucleotides including 5-methoxy-uracil and/or uracil, cytosine,
adenine, and guanine.
[0007] In some embodiments, the uracil content of the mRNA is
different than the uracil content of a corresponding wild-type
sequence. In other embodiments, the percentage of uracil of the
total nucleotide content in the open reading frame is different
relative to the corresponding wild-type sequence. In certain
embodiments, the percentage of uracils of the total nucleotide
content in one or more subsequences (e.g., a subsequence 5 to 40
nucleotides in length) of the open reading frame is different
relative to the corresponding wild-type sequence. In some
embodiments, the uracil distribution within the open reading frame
is different relative to the corresponding wild-type sequence. In
other embodiments, the percentage of uracils of the total
nucleotide content in the open reading frame is unchanged relative
to the corresponding wild-type sequence. In certain embodiments,
the number of uracil clusters or the size of one or more uracil
clusters in the open reading frame is different relative to the
corresponding wild-type sequence. In other embodiments, the
distribution of uracil clusters in the open reading frame is
different relative to the corresponding wild-type sequence. In
certain embodiments, the distance between the uracil clusters or
the location of one or more of the uracil clusters in the open
reading frame is different relative to the corresponding wild-type
sequence.
[0008] In some embodiments, the mRNA does not contain more than
four consecutive uracils.
[0009] In some embodiments, the uracil content of the open reading
frame is between a theoretical minimum and 200% of the theoretical
minimum (e.g., between the theoretical minimum and 125% of the
theoretical minimum, between the theoretical minimum and 150% of
the theoretical minimum, or between the theoretical minimum and
175% of the theoretical minimum).
[0010] In other embodiments, the uracil content within any 20
nucleotide window within the open reading frame does not exceed 50%
(e.g., does not exceed 40%, does not exceed 30%, does not exceed
20%, or does not exceed the theorectical minimum).
[0011] In certain embodiments, the guanine content of the open
reading frame is maximized for at least 90% (e.g., at least 95%, at
least 99%, or 100%) of the codons.
[0012] In some embodiments, the cytosine content of the open
reading frame is maximized for at least 90% (e.g., at least 95%, at
least 99%, or 100%) of the codons.
[0013] In another aspect, the invention provides an mRNA encoding a
polypeptide of interest and including an open reading frame,
wherein (a) at least 90% (e.g., at least 95%, at least 99%, or
100%) of the uracils in the open reading frame are 5-methoxy-uracil
and (b) at least 50% (e.g., at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, at least 99%, or 100%) of the
codons in the open reading frame are guanine and/or cytosine
maximized codons, wherein the open reading frame includes at least
one low frequency (i.e., a codon that is not the highest frequency
codon) guanine and/or cytosine maximized codon. In some
embodiments, at least 50% (e.g., at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 99%, or 100%) of
the codons in the open reading frame are guanine maximized codons,
wherein the open reading frame comprises at least one low frequency
guanine maximized codon. In other embodiments, at least 50% (e.g.,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 99%, or 100%) of the codons in the open reading frame
are cytosine maximized codons, wherein the open reading frame
comprises at least one low frequency cytosine maximized codon. In
certain embodiments, at least 50% (e.g., at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 99%, or
100%) of the codons in the open reading frame are guanine and
cytosine maximized codons, wherein the open reading frame comprises
at least one low frequency guanine and/or cytosine maximized
codon.
[0014] In another aspect, the invention features an mRNA encoding a
polypeptide of interest and including an open reading frame,
wherein (a) at least 90% (e.g., at least 95%, at least 99%, or
100%) of the uracils in the open reading frame are 5-methoxy-uracil
and (b) the open reading frame includes at least one of the
following codons: GCG, GGG, CCG, AGG, ACG, CUC, CGC, UCC, and GUC.
In some embodiments, the open reading frame comprises at least one
of the following codons: GCG, GGG, CCG, AGG, and ACG. In other
embodiments, the open reading frame comprises at least one of the
following codons: CUC, CGC, UCC, and GUC. In certain embodiments,
the open reading frame comprises (i) at least one of the following
codons: GCG, GGG, CCG, AGG, and ACG and (ii) at least one of the
following codons CUC, CGC, UCC, and GUC.
[0015] In other embodiments of any of the foregoing mRNAs, the
sequence of the mRNA has at least 55% (e.g., at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95%) identity to the corresponding wild-type
sequence. In some embodiments of any of the foregoing mRNAs, the
sequence of the mRNA has 60-80% (e.g., 65-75%, 60-65%, 65-70%,
70-75%, or 75-80%) identity to the corresponding wild-type
sequence.
[0016] In certain embodiments of any of the foregoing mRNA, the
mRNA further includes:
[0017] (i) at least one 5'-cap structure;
[0018] (ii) a 5'-UTR; and
[0019] (iii) a `3`-UTR.
[0020] In some embodiments, the at least one 5'-cap structure is
Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,
2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.
[0021] In other embodiments of any of the foregoing mRNA, the mRNA
further includes a poly-A tail.
[0022] In certain embodiments of any of the foregoing mRNA, the
mRNA is purified.
[0023] In another aspect, the invention features a pharmaceutical
composition including any of the foregoing mRNA and a
pharmaceutically acceptable excipient.
[0024] In another aspect, the invention features any of the
foregoing mRNA or pharmaceutical compositions, for use in
therapy.
[0025] In some embodiments of any of the foregoing mRNA, the mRA
induces a detectably lower innate immune response relative to the
corresponding wild-type mRNA.
[0026] In other embodiments of any of the foregoing mRNA, the mRNA
exhibits enhanced ability to produce the encoded protein of
interest in a mammalian cell compared to the corresponding
wild-type mRNA.
[0027] In some embodiments of any of the foregoing mRNA, the mRNA
exhibits increased stability. For example, in some embodiments, the
mRNA exhibits increased stability in a cell into which it is
introduced, relative to a corresponding wild-type mRNA. In some
embodiments of any of the foregoing mRNA, the mRNA exhibits
increased stability including resistance to nucleases, thermal
stability, and/or increased stabilization of secondary structure.
In some embodiments of any of the foregoing mRNA, increased
stability exhibited by the mRNA is measured by determining the half
life of the mRNA (e.g., in a plasma, cell, or tissue sample) and/or
determining the area under the curve (AUC) of the protein
expression by the mRNA over time (e.g., in vitro or in vivo). An
mRNA is identified as having increased stability if the half life
and/or the AUC is greater than the corresponding wild-type
mRNA.
[0028] In some embodiments of any of the foregoing mRNA, the mRNA
exhibits enhanced ability to translate or to produce the encoded
protein of interest, exhibits increased stability, and/or induces a
detectably lower immune response (e.g., innate or acquired)
relative to a corresponding wild-type mRNA and/or an mRNA including
one or more different alternative nucleic acids of the wild-type
mRNA which have been altered in a different manner (e.g., an
alternative nucleic acid including an alternative nucleosides other
than 5-methoxy-uridine or an alternative nucleic acid for which
uridine content has not been reduced) in a cell such as in a
mammalian cell.
[0029] In another aspect, the invention features a method of
expressing a polypeptide of interest in a mammalian cell, the
method including the steps of:
[0030] (i) providing any of the foregoing mRNA; and
[0031] (ii) introducing the mRNA to a mammalian cell under
conditions that permit the expression of the polypeptide of
interest by the mammalian cell.
[0032] In another aspect, the invention features a composition
including:
[0033] a) a DNA template;
[0034] b) an RNA polymerase;
[0035] c) ATP, GTP, CTP, and 5-methoxy-UTP; and
[0036] d) one or more copies of mRNA produced by the RNA
polymerase, and
[0037] e) less than 70% (e.g., less than 60%, less than 50%, less
than 40%, less than 30%, less than 20%, less than 10%, less than
5%, less than 4%, less than 3%, less than 2%, less than 1%, between
0.01 and 5%, between 1% and 10%, between 5% and 20%, between 10%
and 30%, between 15% and 40%, between 20% and 50%, between 30% and
60%, between 40% and 70%) of aberrant transcription products
relative to full length mRNA (e.g., as measured by moles of
aberrant transcription products/moles of aberrant transcription
products and moles full length mRNA).
[0038] In another aspect, the invention features the use of
5-methoxy-uridine in the production of a medicament including an
mRNA, wherein the production does not include reverse phase
purification, e.g. wherein the reverse phase purification is not
needed to remove aberrant transcription products.
[0039] In another aspect, the invention features a method of
producing a pharmaceutical composition including mRNA molecules,
the method including:
[0040] a) performing in vitro synthesis to produce a composition
including mRNA molecules; and
[0041] b) determining the level of aberrant transcription products
in the composition.
[0042] In another aspect, the invention features a method of
producing a pharmaceutical composition including mRNA molecules,
the method including:
[0043] a) performing in vitro transcription with an RNA polymerase
and a DNA template to produce a composition including mRNA
molecules; and
[0044] b) determining the level of aberrant transcription products
in the composition.
[0045] In some embodiments, the method further includes c)
purifying the composition if the level of aberrant transcription
products in the composition is greater than a predetermined level
(e.g., 70%). In some embodiments, purifying includes reverse phase
chromatography.
[0046] In another aspect, the invention features a method of
producing mRNA, the method includes a purification step including
removal of aberrant transcription products without reverse phase
chromatography including for example affinity chromatography,
precipitation, and/or membrane purification such as tangential flow
filtration (TFF) methods as are known in the art.
[0047] In another aspect, the invention features a method of
producing a pharmaceutical composition including mRNA, the method
including determining the level of mRNA including aberrant
transcription products in the composition.
[0048] In another aspect, the invention features a pharmaceutical
composition including mRNA, wherein the pharmaceutical composition
has been determined to include less than less than 70% (e.g., less
than 60%, less than 50%, less than 40%, less than 30%, less than
20%, less than 10%, less than 5%, less than 4%, less than 3%, less
than 2%, less than 1%, between 0.01 and 5%, between 1% and 10%,
between 5% and 20%, between 10% and 30%, between 15% and 40%,
between 20% and 50%, between 30% and 60%, between 40% and 70%)
aberrant transcription products relative to full length mRNA (e.g.,
as measured by moles of aberrant transcription products/moles of
aberrant transcription products and moles full length mRNA).
[0049] In some embodiments of the foregoing compositions, uses, and
methods, a composition including a lower amount of aberrant
transcription products exhibits decreased immunogenicity relative
to a composition including a higher amount of mRNA including
aberrant transcription products. In some embodiments of the
foregoing compositions, uses, and methods, the mRNA in the
composition includes 5-methoxy-uridine.
[0050] In another aspect, the invention features a method of
producing a pharmaceutical composition including an mRNA comprising
5-methoxy-uridine, the method including: (a) producing (e.g.,
directing the production of) a composition including in vitro
synthesized mRNA comprising 5-methoxy-uridine; and (b) purifying
(e.g., directing the purification of) the composition without
reverse phase chromatography, thereby producing a pharmaceutical
composition including an mRNA comprising 5-methoxy-uridine.
[0051] In some embodiments, step (a) includes in vitro
transcription (e.g., with ATP, GTP, CTP, and 5-methoxy-UTP, an RNA
polymerase such as T7 RNA polymerase and a DNA template such as
cDNA).
[0052] In some embodiments, the method further includes (c)
formulating the composition for administration. In some
embodiments, step (c) includes formulating the composition in unit
dosage form. In some embodiments, formulating includes includes one
or more of: processing the composition into a drug product;
combining the composition with a second component, e.g., an
excipient and/or diluent; changing the concentration of the mRNA in
the composition; lyophilizing the composition; combining a first
and second aliquot of the composition to provide a third, larger,
aliquot; dividing the composition into smaller aliquots; disposing
the composition into a container, e.g., a gas or liquid tight
container; packaging the composition; and/or associating a
container including the composition with a label (e.g.,
labeling).
[0053] In some embodiments, the invention features a method of
producing a pharmaceutical composition including an mRNA, wherein
the mRNA comprises 5-methoxy-uridine, the method including: (a)
providing a composition including in vitro synthesized mRNA; and
(b) purifying (e.g., directing the purification of) the composition
without reverse phase chromatography, thereby producing a
pharmaceutical composition including mRNA.
[0054] In some embodiments, the in vitro synthesized mRNA is
produced by in vitro transcription including an RNA polymerase
(e.g., T7 RNA polymerase) and a DNA template (e.g., cDNA).
[0055] In some embodiments, the method further includes (c)
formulating the composition for administration. In some
embodiments, step (c) includes formulating the composition in unit
dosage form. In some embodiments, formulating includes includes one
or more of: processing the composition into a drug product;
combining the composition with a second component, e.g., an
excipient or diluent; changing the concentration of the mRNA in the
composition; lyophilizing the composition; combining a first and
second aliquot of the composition to provide a third, larger,
aliquot; dividing the composition into smaller aliquots; disposing
the composition into a container, e.g., a gas or liquid tight
container; packaging the composition; and/or associating a
container including the composition with a label (e.g.,
labeling).
[0056] In some embodiments, the composition exhibits decreased
immunogenicity relative to a composition including an mRNA that
does not comprise 5-methoxy-uridine and is produced by the same
method. In some embodiments, the composition exhibits increased
protein expression relative to a composition including an mRNA that
does not comprise 5-methoxy-uridine and is produced by the same
method. In some embodiments, the mRNA comprising 5-methoxy-uridine
exhibits increased stability relative to an mRNA that does not
comprise 5-methoxy-uridine and is produced by the same method. In
some embodiments, the composition does not exhibit decreased
immunogenicity relative to a composition including an mRNA that
does not comprise 5-methoxy-uridine and is produced by a method
including reverse phase purification. In some embodiments, the
composition includes fewer RNA impurities resulting from aberrant
transcription products (e.g., short RNAs resulting from abortive
transcription, double stranded RNA resulting from RNA dependent RNA
polymerase activity, and/or RNA including 3' extension region)
relative to a composition including an mRNA that does not comprise
5-methoxy-uridine and is produced by the same method.
[0057] In another aspect, the invention features a pharmaceutical
composition including an mRNA comprising 5-methoxy-uridine produced
by performing in vitro transcription (e.g., with an RNA polymerase
such as T7 RNA polymerase and a DNA template such as cDNA), to
produce a composition including the mRNA and purifying the
composition without reverse phase chromatography.
[0058] In some embodiments, the composition is formulated for
administration. In some embodiments, the composition is formulated
in unit dosage form. In some embodiments, formulating the
composition includes one or more of: processing the composition
into a drug product; combining the composition with a second
component, e.g., an excipient or diluent; changing the
concentration of the mRNA in the composition; lyophilizing the
composition; combining a first and second aliquot of the
composition to provide a third, larger, aliquot; dividing the
composition into smaller aliquots; disposing the composition into a
container, e.g., a gas or liquid tight container; packaging the
composition; and/or associating a container including the
composition with a label (e.g., labeling).
[0059] In some embodiments, the composition exhibits decreased
immunogenicity relative to a composition including mRNA that does
not comprise 5-methoxy-uridine produced with a method including
purification without reverse phase chromatography. In some
embodiments, the composition exhibits increased protein expression
relative to a composition including mRNA that does not comprise
5-methoxy-uridine. In some embodiments, the mRNA exhibits increased
stability relative to mRNA that does not comprise
5-methoxy-uridine. In some embodiments, the composition does not
exhibit decreased immunogenicity relative to a composition
including an mRNA that does not comprise 5-methoxy-uridine and that
is purified by reverse phase chromatography. In some embodiments,
the composition includes fewer RNA impurities such as aberrant
transcription products (e.g., short RNAs resulting from abortive
transcription, double stranded RNA resulting from RNA dependent RNA
polymerase activity, and/or RNA including a 3' extension region)
relative to a composition including an mRNA that does not comprise
5-methoxy-uridine and is produced with a method including
purification without reverse phase chromatography.
[0060] In some embodiments of any of the foregoing methods or
compositions, the aberrant transcription products include short
RNAs as a result of abortive transcription initiation events. In
some embodiments of any of the foregoing methods or compositions,
the aberrant transcription products include double stranded
(ds)RNAs generated by RNA dependent RNA polymerase activity. In
some embodiments of any of the foregoing methods or compositions,
the aberrant transcription products include RNA-primed
transcription from RNA templates. In some embodiments of any of the
foregoing methods or compositions, the aberrant transcription
products include RNA comprising a self-complementary 3' extension
region.
[0061] In some embodiments of any of the foregoing methods or
compositions, the level of RNA impurities and/or aberrant
transcription products may be determined by any method known in the
art (e.g., liquid chromatography such as HPLC, UPLC, or LC-MS
analysis or capillary electrophoresis).
[0062] In some embodiments of any of the foregoing methods or
compositions, the mRNA comprises 5-methoxy-uridine. In some
embodiments of any of the foregoing methods or compositions at
least 5% (e.g., at least 10%, at least 15%, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 99%, or 100%) of
the uridines in the m RNA are 5-methoxy-uridine.
BRIEF DESCRIPTION OF THE FIGURES
[0063] FIG. 1 is a graph of protein expression by uridine-minimized
mRNA relative to the corresponding wild-type mRNA.
[0064] FIG. 2 is a graph of protein expression by uridine-minimized
mRNA relative to the corresponding wild-type mRNA.
[0065] FIG. 3 is a graph of the induction of INF.beta. by mRNA
produced by IVT at different temperatures.
[0066] FIG. 4 is a graph of the induction of INF.beta. by mRNA with
3'-terminal poly-U feature.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The present disclosure provides, inter a/ia, alternative
nucleosides, alternative nucleotides, and alternative nucleic acids
that exhibit improved therapeutic properties including, but not
limited to, increased expression and/or a reduced innate immune
response when introduced into a population of cells.
[0068] As there remains a need in the art for therapeutic
modalities to address the myriad barriers surrounding the
efficacious modulation of intracellular translation and processing
of nucleic acids encoding polypeptides or fragments thereof, the
inventors have shown that certain alternative mRNA sequences have
the potential as therapeutics with benefits beyond just evading,
avoiding or diminishing the immune response.
[0069] The present invention addresses this need by providing
nucleic acid based compounds or polynucleotides (e.g., alternative
mRNAs) which encode a polypeptide of interest and which have
structural and/or chemical features that avoid one or more of the
problems in the art, for example, features which are useful for
optimizing nucleic acid-based therapeutics while retaining
structural and functional integrity, overcoming the threshold of
expression, improving expression rates, half life and/or protein
concentrations, optimizing protein localization, and avoiding
deleterious bio-responses such as the immune response and/or
degradation pathways.
[0070] In particular, the inventors have identified that mRNA
wherein the uracil content has been modified may be particularly
effective for use in therapeutic compositions, because they may
benefit from both high expression levels and limited induction of
the innate immune response, as shown in the Examples (in
particular, high performance may be observed across the assays in
Examples 6-9). In the invention, a percentage of the uracils in the
open reading frame (and optionally other components of an mRNA) are
5-methoxy-uracil. Preferably, at least 90%, e.g., at least 95% or
100%, of the uracils are 5-methoxy-uracil. Thus, as is apparent
from the context, the term uracils can refer to 5-methoxy-uracil
and naturally occurring uracil.
[0071] Polypeptides of interest, according to the present
invention, may be selected from any of those disclosed in US
2013/0259924, US 2013/0259923, WO 2013/151663, WO 2013/151669, WO
2013/151670, WO 2013/151664, WO 2013/151665, WO 2013/151736, U.S.
Provisional Patent Application No. 61/618,862, U.S. Provisional
Patent Application No. 61/681,645, U.S. Provisional Patent
Application No. 61/618,873, U.S. Provisional Patent Application No.
61/681,650, U.S. Provisional Patent Application No. 61/618,878,
U.S. Provisional Patent Application No. 61/681,654, U.S.
Provisional Patent Application No. 61/618,885, U.S. Provisional
Patent Application No. 61/681,658, U.S. Provisional Patent
Application No. 61/618,911, U.S. Provisional Patent Application No.
61/681,667, U.S. Provisional Patent Application No. 61/618,922,
U.S. Provisional Patent Application No. 61/681,675, U.S.
Provisional Patent Application No. 61/618,935, U.S. Provisional
Patent Application No. 61/681,687, U.S. Provisional Patent
Application No. 61/618,945, U.S. Provisional Patent Application No.
61/681,696, U.S. Provisional Patent Application No. 61/618,953, and
U.S. Provisional Patent Application No. 61/681,704, the
polypeptides of each of which are incorporated herein by
reference.
[0072] Provided herein, in part, are polynucleotides encoding
polypeptides of interest which have been chemically modified to
improve one or more of the stability and/or clearance in tissues,
receptor uptake and/or kinetics, cellular access by the
compositions, engagement with translational machinery, mRNA
half-life, translation efficiency, immune evasion, protein
production capacity, secretion efficiency (when applicable),
accessibility to circulation, protein half-life and/or modulation
of a cell's status, function and/or activity.
[0073] The alternative polynucleotides of the invention, including
the combination of alterations taught herein, have superior
properties making them more suitable as therapeutic modalities.
[0074] In one aspect of the invention, methods of determining the
effectiveness of an alternative mRNA as compared to wild-type
involves the measure and analysis of one or more cytokines whose
expression is triggered by the administration of the exogenous
nucleic acid of the invention. These values are compared to
administration of an unaltered nucleic acid or to a standard metric
such as cytokine response, PolyIC, R-848 or other standard known in
the art.
[0075] One example of a standard metric herein is the measure of
the ratio of the level or amount of encoded polypeptide (protein)
produced in the cell, tissue or organism to the level or amount of
one or more (or a panel) of cytokines whose expression is triggered
in the cell, tissue or organism as a result of administration or
contact with the alternative nucleic acid. Such ratios are referred
to herein as the Protein:Cytokine Ratio or "PC" Ratio. The higher
the PC ratio, the more efficacious the alternative nucleic acid
(polynucleotide encoding the protein measured). Preferred PC
Ratios, by cytokine, of the present invention may be greater than
1, greater than 10, greater than 100, greater than 1000, greater
than 10,000 or more. Alternative nucleic acids having higher PC
Ratios than an alternative nucleic acid of a different or unaltered
construct are preferred.
[0076] The PC ratio may be further qualified by the percent
alteration present in the polynucleotide. For example, normalized
to a 100% alternative nucleic acid, the protein production as a
function of cytokine (or risk) or cytokine profile can be
determined.
[0077] Preferably, the alternative mRNAs are substantially non
toxic and non mutagenic.
[0078] The compositions and methods described herein can be used,
in vivo and in vitro, both extracellularly and intracellularly, as
well as in assays such as cell free assays.
[0079] In another aspect, the present disclosure provides chemical
alterations located on the sugar moiety of the nucleotide.
[0080] In another aspect, the present disclosure provides chemical
alterations located on the phosphate backbone of the nucleic
acid.
[0081] In another aspect, the present disclosure provides
nucleotides that contain chemical alterations, wherein the
nucleotide reduces the cellular innate immune response, as compared
to the cellular innate immune induced by a corresponding unaltered
nucleic acid.
[0082] In another aspect, the present disclosure provides
compositions comprising a compound as described herein. In some
embodiments, the composition is a reaction mixture. In some
embodiments, the composition is a pharmaceutical composition. In
some embodiments, the composition is a cell culture. In some
embodiments, the composition further comprises an RNA polymerase
and a cDNA template. In some embodiments, the composition further
comprises a nucleotide having a nucleobase selected from the group
consisting of adenine, cytosine, guanine, 5-methoxy-uracil and/or
uracil.
[0083] In a further aspect, the present disclosure provides methods
of making a pharmaceutical formulation comprising a physiologically
active secreted protein, comprising transfecting a first population
of human cells with the pharmaceutical nucleic acid made by the
methods described herein, wherein the secreted protein is active
upon a second population of human cells.
[0084] In some embodiments, the secreted protein is capable of
interacting with a receptor on the surface of at least one cell
present in the second population.
[0085] In certain embodiments, provided herein are combination
therapeutics containing one or more alternative nucleic acids
containing translatable regions that encode for a protein or
proteins that boost a mammalian subject's immunity along with a
protein that induces antibody-dependent cellular toxicity.
[0086] In one embodiment, it is intended that the compounds of the
present disclosure are stable. It is further appreciated that
certain features of the present disclosure, which are, for clarity,
described in the context of separate embodiments, can also be
provided in combination in a single embodiment. Conversely, various
features of the present disclosure which are, for brevity,
described in the context of a single embodiment, can also be
provided separately or in any suitable subcombination.
[0087] Uracil Content
[0088] The present disclosure provides nucleic acids wherein with
altered uracil content at least one codon in the wild-type sequence
has been replaced with an alternative codon to generate a
uracil-altered sequence. Altered uracil sequences can have at least
one of the following properties:
[0089] (i) an increase or decrease in global uracil content (i.e.,
the percentage of uracil of the total nucleotide content in the
nucleic acid of a section of the nucleic acid, e.g., the open
reading frame); or,
[0090] (ii) an increase or decrease in local uracil content (i.e.,
changes in uracil content are limited to specific subsequences);
or,
[0091] (iii) a change in uracil distribution without a change in
the global uracil content; or,
[0092] (iv) a change in uracil clustering (e.g., number of
clusters, location of clusters, or distance between clusters);
or,
[0093] (v) combinations thereof.
[0094] In some aspects, the percentage of uracil nucleobases in the
nucleic acid sequence is reduced with respect to the percentage of
uracil nucleobases in the wild-type nucleic acid sequence. For
example, 30% of nucleobases may be uracils in the wild-type
sequence and 10% in the nucleic acid sequence of the invention. The
percentage uracil content can be determined by dividing the number
of uracils in a sequence by the total number of nucleotides and
multiplying by 100.
[0095] In other aspects, the percentage of uracil nucleobases in a
subsequence of the nucleic acid sequence is reduced with respect to
the percentage of uracil nucleobases in the corresponding
subsequence of the wild-type sequence. For example, the wild-type
sequence may have a 5'-end region (e.g., 30 codons) with a local
uracil content of 30%, and the uracil content in that same region
could be reduced to 10% in the nucleic acid sequence of the
invention.
[0096] In specific aspects, codons in the nucleic acid sequence of
the invention reduce or modify, for example, the number, size,
location, or distribution of uracil clusters that could have
deleterious effects on protein translation. Although as a general
rule lower uracil content is desirable, in certain aspects, the
uracil content, and in particular the local uracil content, of some
subsequences of the wild-type sequence can be greater than the
wild-type sequence and still maintain beneficial features (e.g.,
increased expression).
[0097] In some aspects, the uracil-modified sequence induces a
lower Toll-Like Receptor (TLR) response when compared to the
wild-type sequence. Several TLRs recognize and respond to nucleic
acids. Double-stranded (ds)RNA, a frequent viral constituent, has
been shown to activate TLR3. See Alexopoulou et al. (2001) Nature,
413:732-738 and Wang et al. (2004) Nat. Med., 10:1366-1373.
Single-stranded (ss)RNA activates TLR7. See Diebold et al. (2004)
Science 303:1529-1531. RNA oligonucleotides, for example RNA with
phosphorothioate internucleotide linkages, are ligands of human
TLR8. See Heil et al. (2004) Science 303:1526-1529. DNA containing
unmethylated CpG motifs, characteristic of bacterial and viral DNA,
activate TLR9. See Hemmi et al. (2000) Nature, 408: 740-745. See
also, Kariko et al. (2005) Immunity 23:165-175, which is herein
incorporated by reference in its entirety.
[0098] As used herein, the term "TLR response" is defined as the
recognition of single-stranded RNA by a TLR7 receptor, and in some
aspects encompasses the degradation of the RNA and/or physiological
responses caused by the recognition of the single-stranded RNA by
the receptor. Methods to determine and quantify the binding of an
RNA to a TLR7 are known in the art. Similarly, methods to determine
whether an RNA has triggered a TLR7-mediated physiological response
(e.g., cytokine secretion) are well known in the art. In some
aspects, a TLR response can be mediated by TLR3, TLR8, or TLR9
instead of TLR7.
[0099] Suppression of TLR7-mediated response can be accomplished
via nucleoside modification. RNA undergoes over a hundred different
nucleoside modifications in nature (see the RNA Modification
Database, available at mods.rna.albany.edu). Human rRNA, for
example, has ten times more pseudouracil (.psi.) and 25 times more
2'-O-methylated nucleosides than bacterial rRNA. Bacterial mRNA
contains no nucleoside modifications, whereas mammalian mRNAs have
modified nucleosides such as 5-methylcytidine (m5C),
N6-methyladenosine (m6A), inosine and many 2'-O-methylated
nucleosides in addition to N7-methylguanosine (m7G).
[0100] Uracil and ribose, the two defining features of RNA, are
both necessary and sufficient for TLR7 stimulation, and short
single-stranded RNA (ssRNA) act as TLR7 agonists in a
sequence-independent manner as long as they contain several uracils
in close proximity. See Diebold et al. (2006) Eur. J. Immunol.
36:3256-3267, which is herein incorporated by reference in its
entirety. Accordingly, a nucleic acid sequence of the invention may
have reduced uracil content (locally and/or locally) and/or reduced
or altered uracil clustering to reduce or to suppress a
TLR7-mediated response.
[0101] In some aspects, the TLR response (e.g., a response mediated
by TLR7) caused by the uracil-modified sequence is at least about
10%, at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or at least 100% lower than the TLR response caused by
the wild-type nucleic acid sequence.
[0102] In some aspects, the TLR response caused by the wild-type
nucleic acid is at least about 1-fold, at least about 1.1-fold, at
least about 1.2-fold, at least about 1.3-fold, at least about
1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at
least about 1.7-fold, at least about 1.8-fold, at least about
1.9-fold, at least about 2-fold, at least about 3-fold, at least
about 4-fold, at least about 5-fold, at least about 6-fold, at
least about 7-fold, at least about 8-fold, at least about 9-fold,
or at least about 10-fold higher than the TLR response caused by
the uracil-modified sequence.
[0103] In other aspects, the uracil content of the uracil-altered
sequence is lower than the uracil content of the wild-type nucleic
acid sequence. Accordingly, in some aspects, the sequence of the
invention contains at least about 5%, at least about 10%, at least
about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 100% less uracil than the wild-type nucleic acid
sequence.
[0104] In some aspects, the uracil content is less than 50%, 49%,
48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%,
35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,
22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total nucleobases in
the sequence of the invention. In some aspects, the uracil content
of the sequence of the invention is between about 5% and about 25%.
In some particular aspects, the uracil content of the sequence of
the invention is between about 15% and about 25%.
[0105] In some aspects, the uracil content of the wild-type nucleic
acid sequence can be measured using a sliding window. In some
aspects, the length of the sliding window is 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleobases. In
some aspects, the sliding window is over 40 nucleobases in length.
In a preferred aspect, the sliding window is 20 nucleobases in
length. Based on the uracil content measured with a sliding window,
it is possible to generate a histogram representing the uracil
content throughout the length of the wild-type nucleic acid
sequence and nucleic acid sequence of the invention. In some
aspects, the nucleic acid sequence of the invention has fewer peaks
in the representation that are above a certain percentage value
relative to the candidate sequence. In some aspects, the nucleic
acid sequence of the invention does not have peaks in the
sliding-window representation which are above 65%, 60%, 55%, 50%,
45%, 40%, 35%, or 30% uracil. In a preferred aspect, the nucleic
acid sequence of the invention has no peaks over 30% uracil, as
measured using a 20 nucleobase sliding window. In some aspects, the
nucleic acid sequence of the invention has no more than a
predetermined number of peaks, as measured using a 20 nucleobase
sliding window, above a certain threshold value. For example, in
some aspects, the nucleic acid sequence of the invention has no
peaks or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peaks in the
above 10%, 15%, 20%, 25% or 30% uracil. In a preferred aspect, the
nucleic acid sequence of the invention contains between 0 peaks and
2 peaks with uracil content 30% or higher.
[0106] In some aspects, the nucleic acid sequence has reduced
consecutive uracils. For example, two consecutive leucines could be
encoded by the sequence CUUUUG, which would include a four uracil
cluster. Such a subsequence could be substituted with CUGCUC, which
would effectively remove the uracil cluster. Accordingly, a nucleic
sequence may have reduced or no uracil pairs (UU), uracil triplets
(UUU) or uracil quadruplets (UUUU), relative to the wild-type
nucleic acid sequence. In some aspects, the nucleic acid sequence
does not include uracil pairs (UU) and/or uracil triplets (UUU)
and/or uracil quadruplets (UUUU). In other aspects, the nucleic
acid sequence does not include uracil pairs (UU) and/or uracil
triplets (UUU) and/or uracil quadruplets (UUUU) above a certain
threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
occurrences in the nucleic acid sequence. In a particular aspect,
the nucleic acid sequence contains fewer than 5, 4, 3, 2, or 1
uracil pairs. In another particular aspect, the nucleic acid
sequence contains no uracil pairs.
[0107] In some aspects, the wild-type nucleic acid sequence can
comprise uracil clusters which due to their number, size, location,
distribution or combinations thereof have negative effects on
translation. As used herein, the term "uracil cluster" refers to a
subsequence in a nucleic acid sequence that contains a uracil
content (usually described as a percentage) which is above a
certain threshold. Thus, in certain aspects, if a subsequence
comprises more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60% or 65% uracil content, such subsequence would be
considered a uracil cluster.
[0108] The negative effects of uracil clusters can be, for example,
eliciting a TLR7 response. Thus, in some embodiments, the nucleic
acid of the invention has a reduced number of clusters, size of
clusters, location of clusters (e.g., close to the 5' and/or 3' end
of a nucleic acid sequence), distance between clusters, or
distribution of uracil clusters (e.g., a certain pattern of
clusters along a nucleic acid sequence, distribution of clusters
with respect to secondary structure elements in the expressed
product, or distribution of clusters with respect to the secondary
structure of an mRNA) relative to wild-type.
[0109] In some aspects, the wild-type sequence comprises at least
one uracil cluster, wherein said uracil cluster is a subsequence of
the wild-type nucleic acid sequence wherein the percentage of total
uracil nucleobases in said subsequence is above a predetermined
threshold. In some aspects, the length of the subsequence is at
least about 10, at least about 15, at least about 20, at least
about 25, at least about 30, at least about 35, at least about 40,
at least about 45, at least about 50, at least about 55, at least
about 60, at least about 65, at least about 70, at least about 75,
at least about 80, at least about 85, at least about 90, at least
about 95, or at least about 100 nucleobases. In some aspects, the
subsequence is longer than 100 nucleobases. In some aspects, the
threshold is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%
uracil content. In some aspects, the threshold is above 25%.
[0110] For example, an amino acid sequence such as ADGSR could be
encoded by the nucleic acid sequence GCU GAU GGU AGU CGU. Although
such a sequence does not contain any uracil pairs, triplets, or
quadruplets, one third of the nucleobases would be uracils. Such a
uracil cluster could be removed by using alternative codons, for
exemple, by using the coding sequence GCC GAC GGC AGC CGC, which
would contain no uracils.
[0111] In other aspects, the wild-type sequence comprises at least
one uracil cluster, wherein said uracil cluster is a subsequence of
the wild-type nucleic acid sequence wherein the percentage of
uracil nucleobases of said subsequence as measured using a sliding
window is above a predetermined threshold. In some aspects, the
length of the sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleobases. In some aspects,
the sliding window is over 40 nucleobases in length. In some
aspects, the threshold is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,
24% or 25% uracil content. In some aspects, the threshold is above
25%.
[0112] In some aspects, the wild-type nucleic acid sequence
comprises at least two uracil clusters. In some aspects, the
sequence of the invention contains fewer uracil-rich clusters than
the wild-type nucleic acid sequence. In some aspects, the sequence
of the invention contains more uracil-rich clusters than the
wild-type nucleic acid sequence. In some aspects, the sequence of
the invention contains uracil-rich clusters which are shorter in
length than corresponding uracil-rich clusters in the wilde type
nucleic acid sequence. In other aspects, the sequence of the
invention contains uracil-rich clusters which are longer in length
that corresponding uracil-rich cluster in the wild-type nucleic
acid sequence.
[0113] Alternative Nucleotides, Nucleosides and Polynucleotides of
the Invention
[0114] Herein, in a nucleotide, nucleoside or polynucleotide (such
as the nucleic acids of the invention, e.g., mRNA molecule), the
terms "alteration" or, as appropriate, "alternative" refer to
alteration with respect to A, G, U or C ribonucleotides. Generally,
herein, these terms are not intended to refer to the ribonucleotide
alterations in naturally occurring 5'-terminal mRNA cap moieties.
In a polypeptide, the term "alteration" refers to an alteration as
compared to the canonical set of 20 amino acids, moiety)
[0115] The alterations may be various distinct alterations. In some
embodiments, where the nucleic acid is an mRNA, the coding region,
the flanking regions and/or the terminal regions may contain one,
two, or more (optionally different) nucleoside or nucleotide
alterations. In some embodiments, an alternative polynucleotide
introduced to a cell may exhibit reduced degradation in the cell,
as compared to an unaltered polynucleotide.
[0116] The polynucleotides can include any useful alteration, such
as to the sugar or the internucleoside linkage (e.g., to a linking
phosphate/to a phosphodiester linkage/to the phosphodiester
backbone). In certain embodiments, alterations (e.g., one or more
alterations) are present in each of the sugar and the
internucleoside linkage. Alterations according to the present
invention may be alterations of ribonucleic acids (RNAs) to
deoxyribonucleic acids (DNAs), e.g., the substitution of the 2'OH
of the ribofuranosyl ring to 2'H, threose nucleic acids (TNAs),
glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked
nucleic acids (LNAs) or hybrids thereof). Additional alterations
are described herein.
[0117] As described herein, the polynucleotides of the invention do
not substantially induce an innate immune response of a cell into
which the polynucleotide (e.g., mRNA) is introduced. Features of an
induced innate immune response include 1) increased expression of
pro-inflammatory cytokines, 2) activation of intracellular PRRs
(RIG-I, MDA5, etc), and/or 3) termination or reduction in protein
translation.
[0118] In certain embodiments, it may desirable for an alternative
nucleic acid molecule introduced into the cell to be degraded
intracellularly. For example, degradation of an alternative nucleic
acid molecule may be preferable if precise timing of protein
production is desired. Thus, in some embodiments, the invention
provides an alternative nucleic acid molecule containing a
degradation domain, which is capable of being acted on in a
directed manner within a cell.
[0119] The polynucleotides can optionally include other agents
(e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs,
antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce
triple helix formation, aptamers, vectors, etc.). In some
embodiments, the polynucleotides may include one or more messenger
RNAs (mRNAs) having one or more alternative nucleoside or
nucleotides (i.e., alternative mRNA molecules). Details for these
polynucleotides follow.
[0120] Polynucleotides
[0121] The polynucleotides of the invention typically include a
first region of linked nucleosides encoding a polypeptide of
interest, a first flanking region located at the 5' terminus of the
first region, and a second flanking region located at the 3'
terminus of the first region.
[0122] Alterations on the Sugar
[0123] The alternative nucleosides and nucleotides, which may be
incorporated into a polynucleotide (e.g., RNA or mRNA, as described
herein), can be altered on the sugar of the ribonucleic acid. For
example, the 2' hydroxyl group (OH) can be modified or replaced
with a number of different substituents. Exemplary substitutions at
the 2'-position include, but are not limited to, H, halo,
optionally substituted C.sub.1-6 alkyl; optionally substituted
C.sub.1-6 alkoxy; optionally substituted C.sub.6-10 aryloxy;
optionally substituted C.sub.3-8 cycloalkyl; optionally substituted
C.sub.3-8 cycloalkoxy; optionally substituted C.sub.6-10 aryloxy;
optionally substituted C.sub.6-10 aryl-C.sub.1-6 alkoxy, optionally
substituted C.sub.1-12 (heterocyclyl)oxy; a sugar (e.g., ribose,
pentose, or any described herein); a polyethyleneglycol (PEG),
--O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR, where R is H or
optionally substituted alkyl, and n is an integer from 0 to 20
(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1
to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2
to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4
to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked"
nucleic acids (LNA) in which the 2'-hydroxyl is connected by a
C.sub.1-6 alkylene or C.sub.1-6 heteroalkylene bridge to the
4'-carbon of the same ribose sugar, where exemplary bridges
included methylene, propylene, ether, or amino bridges; aminoalkyl;
aminoalkoxy; amino; and amino acid.
[0124] Generally, RNA includes the sugar group ribose, which is a
5-membered ring having an oxygen. Exemplary, non-limiting
alternative nucleotides include replacement of the oxygen in ribose
(e.g., with S, Se, or alkylene, such as methylene or ethylene);
addition of a double bond (e.g., to replace ribose with
cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g.,
to form a 4-membered ring of cyclobutane or oxetane); ring
expansion of ribose (e.g., to form a 6- or 7-membered ring having
an additional carbon or heteroatom, such as anhydrohexitol,
altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that
also has a phosphoramidate backbone); multicyclic forms (e.g.,
tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA)
(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units
attached to phosphodiester bonds), threose nucleic acid (TNA, where
ribose is replace with .alpha.-L-threofuranosyl-(3'2')), and
peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages
replace the ribose and phosphodiester backbone). The sugar group
can also contain one or more carbons that possess the opposite
stereochemical configuration than that of the corresponding carbon
in ribose. Thus, a polynucleotide molecule can include nucleotides
containing, e.g., arabinose, as the sugar.
[0125] Alterations on the Internucleoside Linkage
[0126] The alternative nucleotides, which may be incorporated into
a polynucleotide molecule, can be altered on the internucleoside
linkage (e.g., phosphate backbone). Herein, in the context of the
polynucleotide backbone, the phrases "phosphate" and
"phosphodiester" are used interchangeably. Backbone phosphate
groups can be altered by replacing one or more of the oxygen atoms
with a different substituent.
[0127] The alternative nucleosides and nucleotides can include the
wholesale replacement of an unaltered phosphate moiety with another
internucleoside linkage as described herein. Examples of
alternative phosphate groups include, but are not limited to,
phosphorothioate, phosphoroselenates, boranophosphates,
boranophosphate esters, hydrogen phosphonates, phosphoramidates,
phosphorodiamidates, alkyl or aryl phosphonates, and
phosphotriesters. Phosphorodithioates have both non-linking oxygens
replaced by sulfur. The phosphate linker can also be altered by the
replacement of a linking oxygen with nitrogen (bridged
phosphoramidates), sulfur (bridged phosphorothioates), and carbon
(bridged methylene-phosphonates).
[0128] The alternative nucleosides and nucleotides can include the
replacement of one or more of the non-bridging oxygens with a
borane moiety (BH.sub.3), sulfur (thio), methyl, ethyl and/or
methoxy. As a non-limiting example, two non-bridging oxygens at the
same position (e.g., the alpha (.alpha.), beta (.beta.) or gamma
(.gamma.) position) can be replaced with a sulfur (thio) and a
methoxy.
[0129] The replacement of one or more of the oxygen atoms at the a
position of the phosphate moiety (e.g., .alpha.-thio phosphate) is
provided to confer stability (such as against exonucleases and
endonucleases) to RNA and DNA through the unnatural
phosphorothioate backbone linkages. Phosphorothioate DNA and RNA
have increased nuclease resistance and subsequently a longer
half-life in a cellular environment. While not wishing to be bound
by theory, phosphorothioate linked polynucleotide molecules are
expected to also reduce the innate immune response through weaker
binding/activation of cellular innate immune molecules.
[0130] Other internucleoside linkages that may be employed
according to the present invention, including internucleoside
linkages which do not contain a phosphorous atom, are described
herein below.
[0131] Synthesis of Polynucleotide Molecules
[0132] The polynucleotide molecules for use in accordance with the
invention may be prepared according to any useful technique, as
described herein. The alternative nucleosides and nucleotides used
in the synthesis of polynucleotide molecules disclosed herein can
be prepared from readily available starting materials using the
following general methods and procedures. Where typical or
preferred process conditions (e.g., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are provided,
a skilled artisan would be able to optimize and develop additional
process conditions. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0133] The processes described herein can be monitored according to
any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or
mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
[0134] Preparation of polynucleotide molecules of the present
invention can involve the protection and deprotection of various
chemical groups. The need for protection and deprotection, and the
selection of appropriate protecting groups can be readily
determined by one skilled in the art. The chemistry of protecting
groups can be found, for example, in Greene, et al., Protective
Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which
is incorporated herein by reference in its entirety.
[0135] The reactions of the processes described herein can be
carried out in suitable solvents, which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, i.e., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected.
[0136] Resolution of racemic mixtures of alternative
polynucleotides or nucleic acids (e.g., polynucleotides or
alternative mRNA molecules) can be carried out by any of numerous
methods known in the art. An example method includes fractional
recrystallization using a "chiral resolving acid" which is an
optically active, salt-forming organic acid. Suitable resolving
agents for fractional recrystallization methods are, for example,
optically active acids, such as the D and L forms of tartaric acid,
diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic
acid, lactic acid or the various optically active camphorsulfonic
acids. Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0137] Alternative nucleosides and nucleotides (e.g., building
block molecules) can be prepared according to the synthetic methods
described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009);
Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et
al., Biochemistry, 1(4): 563-568 (1962); and Xu et al.,
Tetrahedron, 48(9): 1729-1740 (1992), each of which are
incorporated by reference in their entirety.
[0138] The polynucleotides of the invention may or may not be
uniformly altered 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 altered in a polynucleotide of the invention, or in a
given predetermined sequence region thereof. In some embodiments,
all nucleotides X in a polynucleotide of the invention (or in a
given sequence region thereof) are altered, wherein X may 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.
[0139] Different sugar alterationsand/or internucleoside linkages
(e.g., backbone structures) may exist at various positions in the
polynucleotide. One of ordinary skill in the art will appreciate
that the nucleotide analogs or other alteration(s) may be located
at any position(s) of a polynucleotide such that the function of
the polynucleotide is not substantially decreased. An alteration
may also be a 5' or 3' terminal alteration. The polynucleotide may
contain from about 1% to about 100% alternative 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%). It will be understood that any remaining percentage is
accounted for by the presence of A, G, U, or C.
[0140] Alternative Nucleic Acids
[0141] The present disclosure provides nucleic acids (or
polynucleotides), including RNAs such as mRNAs that contain one or
more alternative nucleosides (termed "alternative nucleic acids")
or nucleotides as described herein, which have useful properties
including the lack of a substantial induction of the innate immune
response of a cell into which the mRNA is introduced. Because these
alternative nucleic acids enhance the efficiency of protein
production, intracellular retention of nucleic acids, and viability
of contacted cells, as well as possess reduced immunogenicity,
these nucleic acids having these properties are also termed
"enhanced nucleic acids" herein.
[0142] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. In this context, the term nucleic acid is
used synonymously with polynucleotide. Exemplary nucleic acids for
use in accordance with the present disclosure include, but are not
limited to, one or more of DNA, RNA including messenger mRNA
(mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs,
shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that
induce triple helix formation, aptamers, and vectors.
[0143] Provided are alternative nucleic acids containing a
translatable region and one, two, or more than two different
nucleoside alterations. In some embodiments, the alternative
nucleic acid exhibits reduced degradation in a cell into which the
nucleic acid is introduced, relative to a corresponding unaltered
nucleic acid. Exemplary nucleic acids include ribonucleic acids
(RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids
(TNAs), glycol nucleic acids (GNAs), or a hybrid thereof. In
preferred embodiments, the alternative nucleic acid includes
messenger RNAs (mRNAs). As described herein, the nucleic acids of
the present disclosure do not substantially induce an innate immune
response of a cell into which the mRNA is introduced.
[0144] In certain embodiments, it is desirable to intracellularly
degrade an alternative nucleic acid introduced into the cell, for
example if precise timing of protein production is desired. Thus,
the present disclosure provides an alternative nucleic acid
containing a degradation domain, which is capable of being acted on
in a directed manner within a cell.
[0145] Other components of nucleic acid are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'-UTR are provided, wherein either or both
may independently contain one or more different nucleoside
alterations. In such embodiments, nucleoside alterations may also
be present in the translatable region. Also provided are nucleic
acids containing a Kozak sequence.
[0146] Additionally, provided are nucleic acids containing one or
more intronic nucleotide sequences capable of being excised from
the nucleic acid.
[0147] Further, provided are nucleic acids containing an internal
ribosome entry site (IRES). An IRES may act as the sole ribosome
binding site, or may serve as one of multiple ribosome binding
sites of an mRNA. An mRNA containing more than one functional
ribosome binding site may encode several peptides or polypeptides
that are translated independently by the ribosomes ("multicistronic
mRNA"). When nucleic acids are provided with an IRES, further
optionally provided is a second translatable region. Examples of
IRES sequences that can be used according to the present disclosure
include without limitation, those from picornaviruses (e.g. FMDV),
pest viruses (CFFV), polio viruses (PV), encephalomyocarditis
viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C
viruses (HCV), classical swine fever viruses (CSFV), murine
leukemia virus (MLV), simian immune deficiency viruses (SIV) or
cricket paralysis viruses (CrPV).
[0148] Major Groove Interacting Partners
[0149] As described herein, the phrase "major groove interacting
partner" refers to RNA recognition receptors that detect and
respond to RNA ligands through interactions, e.g. binding, with the
major groove face of a nucleotide or nucleic acid. As such, RNA
ligands comprising alternative nucleotides or nucleic acids as
described herein decrease interactions with major groove binding
partners, and therefore decrease an innate immune response, or
expression and secretion of pro-inflammatory cytokines, or
both.
[0150] Example major groove interacting, e.g. binding, partners
include, but are not limited to the following nucleases and
helicases. Within membranes, TLRs (Toll-like Receptors) 3, 7, and 8
can respond to single- and double-stranded RNAs. Within the
cytoplasm, members of the superfamily 2 class of DEX(D/H) helicases
and ATPases can sense RNAs to initiate antiviral responses. These
helicases include the RIG-I (retinoic acid-inducible gene I) and
MDA5 (melanoma differentiation-associated gene 5). Other examples
include laboratory of genetics and physiology 2 (LGP2), HIN-200
domain containing proteins, or Helicase-domain containing
proteins.
[0151] Prevention or Reduction of Innate Cellular Immune
Response
[0152] The term "innate immune response" includes a cellular
response to exogenous single stranded nucleic acids, generally of
viral or bacterial origin, which involves the induction of cytokine
expression and release, particularly the interferons, and cell
death. Protein synthesis is also reduced during the innate cellular
immune response. While it is advantageous to eliminate the innate
immune response triggered by introduction of exogenous nucleic
acids in a cell, the present disclosure provides alternative
nucleic acids such as mRNAs that substantially reduce the immune
response, including interferon signaling, without entirely
eliminating such a response. In some embodiments, the immune
response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 99%, 99.9%, or greater than 99.9% as compared to the immune
response induced by a corresponding unaltered nucleic acid. Such a
reduction can be measured by expression or activity level of Type 1
interferons or the expression of interferon-regulated genes such as
the toll-like receptors (e.g., TLR7 and TLR8). Reduction or lack of
induction of innate immune response can also be measured by
decreased cell death following one or more administrations of
alternative RNAs to a cell population; e.g., cell death is 10%,
25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death
frequency observed with a corresponding unaltered nucleic acid.
Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%,
5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the
alternative nucleic acids.
[0153] In some embodiments, the alternative nucleic acids,
including polynucleotides and/or mRNA molecules are alternative in
such a way as to not induce, or induce only minimally, an immune
response by the recipient cell or organism. Such evasion or
avoidance of an immune response trigger or activation is a novel
feature of the alternative polynucleotides of the present
invention.
[0154] The present disclosure provides for the repeated
introduction (e.g., transfection) of alternative nucleic acids into
a target cell population, e.g., in vitro, ex vivo, or in vivo. The
step of contacting the cell population may be repeated one or more
times (such as two, three, four, five or more than five times). In
some embodiments, the step of contacting the cell population with
the alternative nucleic acids is repeated a number of times
sufficient such that a predetermined efficiency of protein
translation in the cell population is achieved. Given the reduced
cytotoxicity of the target cell population provided by the nucleic
acid alterations, such repeated transfections are achievable in a
diverse array of cell types in vitro and/or in vivo.
[0155] Minimization of RNA Impurities to Decrease Innate Immune
Response
[0156] In some embodiments, RNA impurities (e.g., aberrant
transcription products) in a composition including mRNA induce an
immune response. The RNA impurities such as, short RNAs as a result
of abortive transcription initiation events, double stranded RNA
generated by RNA dependent RNA polymerase activity, RNA-primed
transcription from RNA templates, and/or RNA comprising a
self-complementary 3' extension region, may be removed by
purification, including purification by reverse phase
chromatography. It may be advantageous to eliminate the need for
purification by reverse phase chromatography during production of a
composition including RNA; therefore, there is a need for
strategies to minimizing RNA impurities without reverse phase
purification.
[0157] In some embodiments, RNA impurities may be minimized by
using 5-methoxy-uridine as the uridine source for in vitro
synthesis, e.g., in vitro transcription with an RNA polymerase
(e.g., T7 RNA polymerase). In some embodiments, a composition
including mRNA including 5-methoxy-uridine has fewer RNA
impurities. In some embodiments, RNA impurities may be minimized
with reverse phase by performing affinity chromatography,
precipitation, membrane purification, or tangential flow filtration
(TFF) to remove the aberrant transcription products. In some
embodiments, the level of aberrant transcription products in a
composition may be determined, and purification of the composition,
e.g., by reverse phase chromatography performed if the level of
aberrant transcription products is greater than a predetermined
value.
[0158] Polypeptide Variants
[0159] Provided are nucleic acids that encode variant polypeptides,
which have a certain identity with a reference polypeptide
sequence. The term "identity" as known in the art, refers to a
relationship between the sequences of two or more peptides, as
determined by comparing the sequences. In the art, "identity" also
means the degree of sequence relatedness between peptides, as
determined by the number of matches between strings of two or more
amino acid residues. "Identity" measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (i.e., "algorithms"). Identity of related peptides
can be readily calculated by known methods. Such methods include,
but are not limited to, 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; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0160] In some embodiments, the polypeptide variant has the same or
a similar activity as the reference polypeptide. Alternatively, the
variant has an altered activity (e.g., increased or decreased)
relative to a reference polypeptide. Generally, variants of a
particular polynucleotide or polypeptide of the present disclosure
will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to that particular reference polynucleotide or
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the
art.
[0161] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of this present disclosure.
For example, provided herein is any protein fragment of a reference
protein (meaning a polypeptide sequence at least one amino acid
residue shorter than a reference polypeptide sequence but otherwise
identical) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than
100 amino acids in length In another example, any protein that
includes a stretch of about 20, about 30, about 40, about 50, or
about 100 amino acids which are about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95%, or about 100% identical
to any of the sequences described herein can be utilized in
accordance with the present disclosure. In certain embodiments, a
protein sequence to be utilized in accordance with the present
disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations
as shown in any of the sequences provided or referenced herein.
[0162] Erythropoietin (EPO) and granulocyte colony-stimulating
factor (GCSF) are exemplary polypeptides.
[0163] Polypeptide Libraries
[0164] Also provided are polynucleotide libraries containing
nucleoside alterations, wherein the polynucleotides individually
contain a first nucleic acid sequence encoding a polypeptide, such
as an antibody, protein binding partner, scaffold protein, and
other polypeptides known in the art. Preferably, the
polynucleotides are mRNA in a form suitable for direct introduction
into a target cell host, which in turn synthesizes the encoded
polypeptide.
[0165] In certain embodiments, multiple variants of a protein, each
with different amino acid alteration(s), are produced and tested to
determine the best variant in terms of pharmacokinetics, stability,
biocompatibility, and/or biological activity, or a biophysical
property such as expression level. Such a library may contain 10,
10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, or over 10.sup.9 possible variants (including
substitutions, deletions of one or more residues, and insertion of
one or more residues).
[0166] Polypeptide-Nucleic Acid Complexes
[0167] Proper protein translation involves the physical aggregation
of a number of polypeptides and nucleic acids associated with the
mRNA. Provided by the present disclosure are protein-nucleic acid
complexes, containing a translatable mRNA having one or more
nucleoside alterations (e.g., at least two different nucleoside
alterations) and one or more polypeptides bound to the mRNA.
Generally, the proteins are provided in an amount effective to
prevent or reduce an innate immune response of a cell into which
the complex is introduced.
[0168] Synthesis of Alternative Nucleic Acids
[0169] Nucleic acids for use in accordance with the present
disclosure may be prepared according to any available technique
including, but not limited to chemical synthesis, enzymatic
synthesis, which is generally termed in vitro transcription,
enzymatic or chemical cleavage of a longer precursor, etc. Methods
of synthesizing RNAs are known in the art (see, e.g., Gait, M. J.
(ed.) Oligonucleotide synthesis: a practical approach, Oxford
[Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.
(ed.) Oligonucleotide synthesis: methods and applications, Methods
in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana
Press, 2005; both of which are incorporated herein by
reference).
[0170] In certain embodiments, a method for producing an mRNA
encoding a polypeptide of interest comprises contacting a cDNA that
encodes the protein of interest with an RNA polymerase in the
presence of a nucleotide triphosphate mix, wherein at least 90%
(e.g., at least 95% or 100%) of the uracils are 5-methoxy-uracil.
The invention also provides mRNA produced by such methods. The
methods may include additional steps, such as capping (e.g. the
addition of a 5'-cap structure), addition of a poly-A tail and/or
formulation into a pharmaceutical composition. The RNA polymerase
may be T7 RNA polymerase. The in vitro transcription reaction
mixture may include a transcription buffer (such as 400 mM Tris-HCl
pH 8.0, or an equivalent) and may include MgCl.sub.2, DTT,
Spermidine (or equivalents). An RNase inhibitor may be included.
The remaining reaction volume is generally made up with dH.sub.2O.
The reaction may be incubated at approximately 37.degree. C. (such
as between 30 and 40.degree. C.) and may be incubated for 3 hr-5
hrs (such as 31/2 hr-41/2 hr, or about 4 hr). The RNA may then be
cleaned using DNase and a purification kit.
[0171] Alternative nucleic acids need not be uniformly present
along the entire length of the molecule. Different nucleotide
alterations and/or backbone structures may exist at various
positions in the nucleic acid. One of ordinary skill in the art
will appreciate that the nucleotide analogs or other alteration(s)
may be located at any position(s) of a nucleic acid such that the
function of the nucleic acid is not substantially decreased. An
alteration may also be a 5' or 3' terminal alteration. The nucleic
acids may contain at a minimum one and at maximum 100% alternative
nucleotides, or any intervening percentage, such as at least 5%
alternative nucleotides, at least 10% alternative nucleotides, at
least 25% alternative nucleotides, at least 50% alternative
nucleotides, at least 80% alternative nucleotides, or at least 90%
alternative nucleotides. For example, the nucleic acids may contain
an alternative pyrimidine such as uracil or cytosine. In some
embodiments, at least 5%, at least 10%, at least 25%, at least 50%,
at least 80%, at least 90% or 100% of the uracil in the nucleic
acid is replaced with an alternative uracil. The alternative uracil
can be replaced by a compound having a single unique structure, or
can be replaced by a plurality of compounds having different
structures (e.g., 2, 3, 4 or more unique structures). In some
embodiments, at least 5%, at least 10%, at least 25%, at least 50%,
at least 80%, at least 90% or 100% of the cytosine in the nucleic
acid is replaced with an alternative cytosine. The alternative
cytosine can be replaced by a compound having a single unique
structure, or can be replaced by a plurality of compounds having
different structures (e.g., 2, 3, 4 or more unique structures).
[0172] Generally, the shortest length of an alternative mRNA of the
present disclosure can be the length of an mRNA sequence that is
sufficient to encode for a dipeptide. In another embodiment, the
length of the mRNA sequence is sufficient to encode for a
tripeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a tetrapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
pentapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a hexapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
heptapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for an octapeptide. In another embodiment,
the length of an mRNA sequence is sufficient to encode for a
nonapeptide. In another embodiment, the length of an mRNA sequence
is sufficient to encode for a decapeptide.
[0173] Examples of dipeptides that the alternative nucleic acid
sequences can encode for include, but are not limited to, carnosine
and anserine.
[0174] In a further embodiment, the mRNA is greater than 30
nucleotides in length. In another embodiment, the RNA molecule is
greater than 35 nucleotides in length. In another embodiment, the
length is at least 40 nucleotides. In another embodiment, the
length is at least 45 nucleotides. In another embodiment, the
length is at least 55 nucleotides. In another embodiment, the
length is at least 50 nucleotides. In another embodiment, the
length is at least 60 nucleotides. In another embodiment, the
length is at least 80 nucleotides. In another embodiment, the
length is at least 90 nucleotides. In another embodiment, the
length is at least 100 nucleotides. In another embodiment, the
length is at least 120 nucleotides. In another embodiment, the
length is at least 140 nucleotides. In another embodiment, the
length is at least 160 nucleotides. In another embodiment, the
length is at least 180 nucleotides. In another embodiment, the
length is at least 200 nucleotides. In another embodiment, the
length is at least 250 nucleotides. In another embodiment, the
length is at least 300 nucleotides. In another embodiment, the
length is at least 350 nucleotides. In another embodiment, the
length is at least 400 nucleotides. In another embodiment, the
length is at least 450 nucleotides. In another embodiment, the
length is at least 500 nucleotides. In another embodiment, the
length is at least 600 nucleotides. In another embodiment, the
length is at least 700 nucleotides. In another embodiment, the
length is at least 800 nucleotides. In another embodiment, the
length is at least 900 nucleotides. In another embodiment, the
length is at least 1000 nucleotides. In another embodiment, the
length is at least 1100 nucleotides. In another embodiment, the
length is at least 1200 nucleotides. In another embodiment, the
length is at least 1300 nucleotides. In another embodiment, the
length is at least 1400 nucleotides. In another embodiment, the
length is at least 1500 nucleotides. In another embodiment, the
length is at least 1600 nucleotides. In another embodiment, the
length is at least 1800 nucleotides. In another embodiment, the
length is at least 2000 nucleotides. In another embodiment, the
length is at least 2500 nucleotides. In another embodiment, the
length is at least 3000 nucleotides. In another embodiment, the
length is at least 4000 nucleotides. In another embodiment, the
length is at least 5000 nucleotides, or greater than 5000
nucleotides.
[0175] For example, the alternative nucleic acids described herein
can be prepared using methods that are known to those skilled in
the art of nucleic acid synthesis.
[0176] In some embodiments, the present disclosure provides for
methods of synthesizing a pharmaceutical nucleic acid, comprising
the steps of:
[0177] a) providing a complementary deoxyribonucleic acid (cDNA)
that encodes a pharmaceutical protein of interest;
[0178] b) selecting a nucleotide and
[0179] c) contacting the provided cDNA and the selected nucleotide
with an RNA polymerase, under conditions such that the
pharmaceutical nucleic acid is synthesized.
[0180] In further embodiments, the pharmaceutical nucleic acid is a
ribonucleic acid (RNA).
[0181] In still a further aspect of the present disclosure, the
alternative nucleic acids can be prepared using solid phase
synthesis methods.
[0182] 5'-Capping
[0183] The 5'-cap structure of an mRNA is involved in nuclear
export, increasing mRNA stability and binds the mRNA Cap Binding
Protein (CBP), which is responsible for mRNA stability in the cell
and translation competency through the association of CBP with
poly(A) binding protein to form the mature cyclic mRNA species. The
cap further assists the removal of 5' proximal introns removal
during mRNA splicing.
[0184] Endogenous mRNA molecules may be 5'-end capped generating a
5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and the 5'-terminal transcribed sense nucleotide of the
mRNA. This 5'-guanylate cap may then be methylated to generate an
N7-methyl-guanylate residue. The ribose sugars of the terminal
and/or anteterminal transcribed nucleotides of the 5' end of the
mRNA may optionally also be 2'-O-methylated. 5'-decapping through
hydrolysis and cleavage of the guanylate cap structure may target a
nucleic acid molecule, such as an mRNA molecule, for
degradation.
[0185] Alterations to the nucleic acids of the present invention
may generate a non-hydrolyzable cap structure preventing decapping
and thus increasing mRNA half-life. Because cap structure
hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester
linkages, alternative nucleotides may be used during the capping
reaction. For example, a Vaccinia Capping Enzyme from New England
Biolabs (Ipswich, Mass.) may be used with .alpha.-thio-guanosine
nucleotides according to the manufacturer's instructions to create
a phosphorothioate linkage in the 5'-ppp-5' cap. Additional
alternative guanosine nucleotides may be used such as
.alpha.-methyl-phosphonate and seleno-phosphate nucleotides.
[0186] Additional alterations include, but are not limited to,
2'-O-methylation of the ribose sugars of 5'-terminal and/or
5'-anteterminal nucleotides of the mRNA (as mentioned above) on the
2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap
structures can be used to generate the 5'-cap of a nucleic acid
molecule, such as an mRNA molecule.
[0187] 5'-cap structures include those described in International
Patent Publication Nos. WO2008127688, WO 2008016473, and WO
2011015347, each of which is incorporated herein by reference in
its entirety.
[0188] Cap analogs, which herein are also referred to as synthetic
cap analogs, chemical caps, chemical cap analogs, or structural or
functional cap analogs, differ from natural (i.e. endogenous,
wild-type or physiological) 5'-caps in their chemical structure,
while retaining cap function. Cap analogs may be chemically (i.e.
non-enzymatically) or enzymatically synthesized and/linked to a
nucleic acid molecule.
[0189] For example, the Anti-Reverse Cap Analog (ARCA) cap contains
two guanosines linked by a 5'-5'-triphosphate group, wherein one
guanosine contains an N7 methyl group as well as a 3'-O-methyl
group (i.e.,
N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine
(m.sup.7G-3'mppp-G; which may equivalently be designated
3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other, unaltered,
guanosine becomes linked to the 5'-terminal nucleotide of the
capped nucleic acid molecule (e.g. an mRNA or mmRNA). The N7- and
3'-O-methlyated guanosine provides the terminal moiety of the
capped nucleic acid molecule (e.g. mRNA or mmRNA).
[0190] Another exemplary cap is mCAP, which is similar to ARCA but
has a 2'-O-methyl group on guanosine (i.e.,
N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine,
mtm-ppp-G).
[0191] In one embodiment, the cap is a dinucleotide cap analog. As
a non-limiting example, the dinucleotide cap analog may be modified
at different phosphate positions with a boranophosphate group or a
phophoroselenoate group such as the dinucleotide cap analogs
described in U.S. Pat. No. 8,519,110, the contents of which are
herein incorporated by reference in its entirety.
[0192] In another embodiment, the cap analog is a
N7-(4-chlorophenoxyethyl) substituted dicnucleotide form of a cap
analog known in the art and/or described herein. Non-limiting
examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide
form of a cap analog include a
N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a
N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analog (See e.g.,
the various cap analogs and the methods of synthesizing cap analogs
described in Kore et al. Bioorganic & Medicinal Chemistry 2013
21:4570-4574; the contents of which are herein incorporated by
reference in its entirety). In another embodiment, a cap analog of
the present invention is a 4-chloro/bromophenoxyethyl analog.
[0193] While cap analogs allow for the concomitant capping of a
nucleic acid molecule in an in vitro transcription reaction, up to
20% of transcripts remain uncapped. This, as well as the structural
differences of a cap analog from endogenous 5'-cap structures of
nucleic acids produced by the endogenous, cellular transcription
machinery, may lead to reduced translational competency and reduced
cellular stability.
[0194] Alternative nucleic acids of the invention may also be
capped post-transcriptionally, using enzymes, in order to generate
more authentic 5'-cap structures. As used herein, the phrase "more
authentic" refers to a feature that closely mirrors or mimics,
either structurally or functionally, an endogenous or wild-type
feature. That is, a "more authentic" feature is better
representative of an endogenous, wild-type, natural or
physiological cellular function and/or structure as compared to
synthetic features or analogs, etc., of the prior art, or which
outperforms the corresponding endogenous, wild-type, natural or
physiological feature in one or more respects. Non-limiting
examples of more authentic 5'-cap structures of the present
invention are those which, among other things, have enhanced
binding of cap binding proteins, increased half life, reduced
susceptibility to 5' endonucleases and/or reduced 5' decapping, as
compared to synthetic 5'-cap structures known in the art (or to a
wild-type, natural or physiological 5'-cap structure). For example,
recombinant Vaccinia Virus Capping Enzyme and recombinant
2'-O-methyltransferase enzyme can create a canonical
5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an
mRNA and a guanosine cap nucleotide wherein the cap guanosine
contains an N7 methylation and the 5'-terminal nucleotide of the
mRNA contains a 2'-O-methyl. Such a structure is termed the Cap1
structure. This cap results in a higher translational-competency
and cellular stability and a reduced activation of cellular
pro-inflammatory cytokines, as compared, e.g., to other 5'cap
analog structures known in the art. Cap structures include
7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NImpNp (cap 1),
7mG(5')-ppp(5')NImpN2mp (cap 2) and
m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).
[0195] Because the alternative nucleic acids may be capped
post-transcriptionally, and because this process is more efficient,
nearly 100% of the alternative nucleic acids may be capped. This is
in contrast to .about.80% when a cap analog is linked to an mRNA in
the course of an in vitro transcription reaction.
[0196] According to the present invention, 5' terminal caps may
include endogenous caps or cap analogs. According to the present
invention, a 5' terminal cap may comprise a guanosine analog.
Useful guanosine analogs include inosine, N1-methyl-guanosine,
2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
[0197] In one embodiment, the nucleic acids described herein may
contain a modified 5'-cap. A modification on the 5'-cap may
increase the stability of mRNA, increase the half-life of the mRNA,
and could increase the mRNA translational efficiency. The modified
5'-cap may include, but is not limited to, one or more of the
following modifications: modification at the 2' and/or 3' position
of a capped guanosine triphosphate (GTP), a replacement of the
sugar ring oxygen (that produced the carbocyclic ring) with a
methylene moiety (CH.sub.2), a modification at the triphosphate
bridge moiety of the cap structure, or a modification at the
nucleobase (G) moiety.
[0198] The 5'-cap structure that may be modified includes, but is
not limited to, the caps described herein such as Cap0 having the
substrate structure for cap dependent translation of:
##STR00001##
or Cap1 having the substrate structure for cap dependent
translation of:
##STR00002##
[0199] As a non-limiting example, the modified 5'-cap may have the
substrate structure for cap dependent translation of:
##STR00003## ##STR00004## ##STR00005## ##STR00006##
##STR00007##
where R.sub.1 and R.sub.2 are defined in Table 5:
TABLE-US-00001 TABLE 5 R.sub.1 and R.sub.2 groups for CAP-022 to
CAP096. Cap Structure Number R.sub.1 R.sub.2 CAP-022 C.sub.2H.sub.5
(Ethyl) H CAP-023 H C.sub.2H.sub.5 (Ethyl) CAP-024 C.sub.2H.sub.5
(Ethyl) C.sub.2H.sub.5 (Ethyl) CAP-025 C.sub.3H.sub.7 (Propyl) H
CAP-026 H C.sub.3H.sub.7 (Propyl) CAP-027 C.sub.3H.sub.7 (Propyl)
C.sub.3H.sub.7 (Propyl) CAP-028 C.sub.4H.sub.9 (Butyl) H CAP-029 H
C.sub.4H.sub.9 (Butyl) CAP-030 C.sub.4H.sub.9 (Butyl)
C.sub.4H.sub.9 (Butyl) CAP-031 C.sub.5H.sub.11 (Pentyl) H CAP-032 H
C.sub.5H.sub.11 (Pentyl) CAP-033 C.sub.5H.sub.11 (Pentyl)
C.sub.5H.sub.11 (Pentyl) CAP-034 H.sub.2C--C.ident.CH (Propargyl) H
CAP-035 H H.sub.2C--C.ident.CH (Propargyl) CAP-036
H.sub.2C--C.ident.CH (Propargyl) H.sub.2C--C.ident.CH (Propargyl)
CAP-037 CH.sub.2CH.dbd.CH.sub.2 (Allyl) H CAP-038 H
CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-039 CH.sub.2CH.dbd.CH.sub.2
(Allyl) CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-040 CH.sub.2OCH.sub.3
(MOM) H CAP-041 H CH.sub.2OCH.sub.3 (MOM) CAP-042 CH.sub.2OCH.sub.3
(MOM) CH.sub.2OCH.sub.3 (MOM) CAP-043
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) H CAP-044 H
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-045
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM)
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-046 CH.sub.2SCH.sub.3
(MTM) H CAP-047 H CH.sub.2SCH.sub.3 (MTM) CAP-048 CH.sub.2SCH.sub.3
(MTM) CH.sub.2SCH.sub.3 (MTM) CAP-049 CH.sub.2C.sub.6H.sub.5
(Benzyl) H CAP-050 H CH.sub.2C.sub.6H.sub.5 (Benzyl) CAP-051
CH.sub.2C.sub.6H.sub.5 (Benzyl) CH.sub.2C.sub.6H.sub.5 (Benzyl)
CAP-052 CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) H CAP-053 H
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-054
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM)
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-055
CH.sub.2C.sub.6H.sub.4--OMe (p-Methoxybenzyl) H CAP-056 H
CH.sub.2C.sub.6H.sub.4--OMe (p-Methoxybenzyl) CAP-057
CH.sub.2C.sub.6H.sub.4--OMe (p-Methoxybenzyl)
CH.sub.2C.sub.6H.sub.4--OMe (p-Methoxybenzyl) CAP-058
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) H CAP-059 H
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) CAP-060
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl)
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) CAP-061
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where H X.dbd.F, Cl, Br or
I CAP-062 H CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X.dbd.F,
Cl, Br or I CAP-063 CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X.dbd.F, Cl, Br or I
X.dbd.F, Cl, Br or I CAP-064 CH.sub.2C.sub.6H.sub.4--N.sub.3
(p-Azidobenzyl) H CAP-065 H CH.sub.2C.sub.6H.sub.4--N.sub.3
(p-Azidobenzyl) CAP-066 CH.sub.2C.sub.6H.sub.4--N.sub.3
(p-Azidobenzyl) CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl)
CAP-067 CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- H
Trifluoromethylbenzyl) CAP-068 H CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- Trifluoromethylbenzyl) CAP-069 CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- Trifluoromethylbenzyl)
Trifluoromethylbenzyl) CAP-070 CH.sub.2C.sub.6H.sub.4--OCF.sub.3
(p- H Trifluoromethoxylbenzyl) CAP-071 H
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
CAP-072 CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p-
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
Trifluoromethoxylbenzyl) CAP-073
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4- H
bis(Trifluoromethyl)benzyl] CAP-074 H
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] CAP-075
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-076
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t- H Butyldiphenylsilyl)
CAP-077 H Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9
(t-Butyldiphenylsilyl) CAP-078
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-Butyldiphenylsilyl)
Butyldiphenylsilyl) CAP-079 CH.sub.2CH.sub.2CH.dbd.CH.sub.2
(Homoallyl) H CAP-080 H CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CAP-081 CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl) CAP-082 P(O)(OH).sub.2
(MP) H CAP-083 H P(O)(OH).sub.2 (MP) CAP-084 P(O)(OH).sub.2 (MP)
P(O)(OH).sub.2 (MP) CAP-085 P(S)(OH).sub.2 (Thio-MP) H CAP-086 H
P(S)(OH).sub.2 (Thio-MP) CAP-087 P(S)(OH).sub.2 (Thio-MP)
P(S)(OH).sub.2 (Thio-MP) CAP-088 P(O)(CH.sub.3)(OH) H
(Methylphophonate) CAP-089 H P(O)(CH.sub.3)(OH) (Methylphophonate)
CAP-090 P(O)(CH.sub.3)(OH) P(O)(CH.sub.3)(OH) (Methylphophonate)
(Methylphophonate) CAP-091 PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN)
H (Phosporamidite) CAP-092 H
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite) CAP-093
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN)
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite)
(Phosporamidite) CAP-094 SO.sub.2CH.sub.3 (Methanesulfonic acid) H
CAP-095 H SO.sub.2CH.sub.3 (Methanesulfonic acid) CAP-096
SO.sub.2CH.sub.3 (Methanesulfonic acid) SO.sub.2CH.sub.3
(Methanesulfonic acid)
##STR00008##
or where R.sub.1 and R.sub.2 are defined in Table 6:
TABLE-US-00002 TABLE 6 R.sub.1 and R.sub.2 groups for CAP-097 to
CAP111. Cap Structure Number R.sub.1 R.sub.2 CAP-097 NH.sub.2
(amino) H CAP-098 H NH.sub.2 (amino) CAP-099 NH.sub.2 (amino)
NH.sub.2 (amino) CAP-100 N.sub.3 (Azido) H CAP-101 H N.sub.3
(Azido) CAP-102 N.sub.3 (Azido) N.sub.3 (Azido) CAP-103 X (Halo: F,
Cl, Br, I) H CAP-104 H X (Halo: F, Cl, Br, I) CAP-105 X (Halo: F,
Cl, Br, I) X (Halo: F, Cl, Br, I) CAP-106 SH (Thiol) H CAP-107 H SH
(Thiol) CAP-108 SH (Thiol) SH (Thiol) CAP-109 SCH.sub.3
(Thiomethyl) H CAP-110 H SCH.sub.3 (Thiomethyl) CAP-111 SCH.sub.3
(Thiomethyl) SCH.sub.3 (Thiomethyl)
[0200] In Table 5, "MOM" stands for methoxymethyl, "MEM" stands for
methoxyethoxymethyl, "MTM" stands for methylthiomethyl, "BOM"
stands for benzyloxymethyl and "MP" stands for monophosphonate.
[0201] In a non-limiting example, the modified 5'-cap may have the
substrate structure for vaccinia mRNA capping enzyme of:
##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013##
where R.sub.1 and R.sub.2 are defined in Table 7:
TABLE-US-00003 TABLE 7 R.sub.1 and R.sub.2 groups for CAP-136 to
CAP-210. Cap Structure Number R.sub.1 R.sub.2 CAP-136
C.sub.2H.sub.5 (Ethyl) H CAP-137 H C.sub.2H.sub.5 (Ethyl) CAP-138
C.sub.2H.sub.5 (Ethyl) C.sub.2H.sub.5 (Ethyl) CAP-139
C.sub.3H.sub.7 (Propyl) H CAP-140 H C.sub.3H.sub.7 (Propyl) CAP-141
C.sub.3H.sub.7 (Propyl) C.sub.3H.sub.7 (Propyl) CAP-142
C.sub.4H.sub.9 (Butyl) H CAP-143 H C.sub.4H.sub.9 (Butyl) CAP-144
C.sub.4H.sub.9 (Butyl) C.sub.4H.sub.9 (Butyl) CAP-145
C.sub.5H.sub.11 (Pentyl) H CAP-146 H C.sub.5H.sub.11 (Pentyl)
CAP-147 C.sub.5H.sub.11 (Pentyl) C.sub.5H.sub.11 (Pentyl) CAP-148
H.sub.2C--C.ident.CH (Propargyl) H CAP-149 H H.sub.2C--C.ident.CH
(Propargyl) CAP-150 H.sub.2C--C.ident.CH (Propargyl)
H.sub.2C--C.ident.CH (Propargyl) CAP-151 CH.sub.2CH.dbd.CH.sub.2
(Allyl) H CAP-152 H CH.sub.2CH.dbd.CH.sub.2 (Allyl) CAP-153
CH.sub.2CH.dbd.CH.sub.2 (Allyl) CH.sub.2CH.dbd.CH.sub.2 (Allyl)
CAP-154 CH.sub.2OCH.sub.3 (MOM) H CAP-155 H CH.sub.2OCH.sub.3 (MOM)
CAP-156 CH.sub.2OCH.sub.3 (MOM) CH.sub.2OCH.sub.3 (MOM) CAP-157
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) H CAP-158 H
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-159
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM)
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3 (MEM) CAP-160 CH.sub.2SCH.sub.3
(MTM) H CAP-161 H CH.sub.2SCH.sub.3 (MTM) CAP-162 CH.sub.2SCH.sub.3
(MTM) CH.sub.2SCH.sub.3 (MTM) CAP-163 CH.sub.2C.sub.6H.sub.5
(Benzyl) H CAP-164 H CH.sub.2C.sub.6H.sub.5 (Benzyl) CAP-165
CH.sub.2C.sub.6H.sub.5 (Benzyl) CH.sub.2C.sub.6H.sub.5 (Benzyl)
CAP-166 CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) H CAP-167 H
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-168
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM)
CH.sub.2OCH.sub.2C.sub.6H.sub.5 (BOM) CAP-169
CH.sub.2C.sub.6H.sub.4--OMe (p- H Methoxybenzyl) CAP-170 H
CH.sub.2C.sub.6H.sub.4--OMe (p-Methoxybenzyl) CAP-171
CH.sub.2C.sub.6H.sub.4--OMe (p- CH.sub.2C.sub.6H.sub.4--OMe
(p-Methoxybenzyl) Methoxybenzyl) CAP-172
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) H CAP-173 H
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) CAP-174
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl)
CH.sub.2C.sub.6H.sub.4--NO.sub.2 (p-Nitrobenzyl) CAP-175
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) H where X.dbd.F, Cl, Br or
I CAP-176 H CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X.dbd.F,
Cl, Br or I CAP-177 CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl)
CH.sub.2C.sub.6H.sub.4--X (p-Halobenzyl) where X.dbd.F, where
X.dbd.F, Cl, Br or I Cl, Br or I CAP-178
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl) H CAP-179 H
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl) CAP-180
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl)
CH.sub.2C.sub.6H.sub.4--N.sub.3 (p-Azidobenzyl) CAP-181
CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p- H Trifluoromethylbenzyl)
CAP-182 H CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p-Trifluoromethylbenzyl) CAP-183 CH.sub.2C.sub.6H.sub.4--CF.sub.3
(p- CH.sub.2C.sub.6H.sub.4--CF.sub.3 (p-Trifluoromethylbenzyl)
Trifluoromethylbenzyl) CAP-184 CH.sub.2C.sub.6H.sub.4--OCF.sub.3
(p- H Trifluoromethoxylbenzyl) CAP-185 H
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
CAP-186 CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p-
CH.sub.2C.sub.6H.sub.4--OCF.sub.3 (p- Trifluoromethoxylbenzyl)
Trifluoromethoxylbenzyl) CAP-187
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4- H
bis(Trifluoromethyl)benzyl] CAP-188 H
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] CAP-189
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
CH.sub.2C.sub.6H.sub.3--(CF.sub.3).sub.2 [2,4-
bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-190
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t- H Butyldiphenylsilyl)
CAP-191 H Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9
(t-Butyldiphenylsilyl) CAP-192
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-
Si(C.sub.6H.sub.5).sub.2C.sub.4H.sub.9 (t-Butyldiphenylsilyl)
Butyldiphenylsilyl) CAP-193 CH.sub.2CH.sub.2CH.dbd.CH.sub.2
(Homoallyl) H CAP-194 H CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CAP-195 CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl)
CH.sub.2CH.sub.2CH.dbd.CH.sub.2 (Homoallyl) CAP-196 P(O)(OH).sub.2
(MP) H CAP-197 H P(O)(OH).sub.2 (MP) CAP-198 P(O)(OH).sub.2 (MP)
P(O)(OH).sub.2 (MP) CAP-199 P(S)(OH).sub.2 (Thio-MP) H CAP-200 H
P(S)(OH).sub.2 (Thio-MP) CAP-201 P(S)(OH).sub.2 (Thio-MP)
P(S)(OH).sub.2 (Thio-MP) CAP-202 P(O)(CH.sub.3)(OH) H
(Methylphophonate) CAP-203 H P(O)(CH.sub.3)(OH) (Methylphophonate)
CAP-204 P(O)(CH.sub.3)(OH) P(O)(CH.sub.3)(OH) (Methylphophonate)
(Methylphophonate) CAP-205 PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN)
H (Phosporamidite) CAP-206 H
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite) CAP-207
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN)
PN(.sup.iPr).sub.2(OCH.sub.2CH.sub.2CN) (Phosporamidite)
(Phosporamidite) CAP-208 SO.sub.2CH.sub.3 (Methanesulfonic H acid)
CAP-209 H SO.sub.2CH.sub.3 (Methanesulfonic acid) CAP-210
SO.sub.2CH.sub.3 (Methanesulfonic SO.sub.2CH.sub.3 (Methanesulfonic
acid) acid)
##STR00014##
or where R.sub.1 and R.sub.2 are defined in Table 8:
TABLE-US-00004 TABLE 8 R.sub.1 and R.sub.2 groups for CAP-211 to
225. Cap Structure Number R.sub.1 R.sub.2 CAP-211 NH.sub.2 (amino)
H CAP-212 H NH.sub.2 (amino) CAP-213 NH.sub.2 (amino) NH.sub.2
(amino) CAP-214 N.sub.3 (Azido) H CAP-215 H N.sub.3 (Azido) CAP-216
N.sub.3 (Azido) N.sub.3 (Azido) CAP-217 X (Halo: F, Cl, Br, I) H
CAP-218 H X (Halo: F, Cl, Br, I) CAP-219 X (Halo: F, Cl, Br, I) X
(Halo: F, Cl, Br, I) CAP-220 SH (Thiol) H CAP-221 H SH (Thiol)
CAP-222 SH (Thiol) SH (Thiol) CAP-223 SCH.sub.3 (Thiomethyl) H
CAP-224 H SCH.sub.3 (Thiomethyl) CAP-225 SCH.sub.3 (Thiomethyl)
SCH.sub.3 (Thiomethyl)
[0202] In Table 7, "MOM" stands for methoxymethyl, "MEM" stands for
methoxyethoxymethyl, "MTM" stands for methylthiomethyl, "BOM"
stands for benzyloxymethyl and "MP" stands for monophosphonate.
[0203] In another non-limiting example, of the modified capping
structure substrates CAP-112-CAP-225 could be added in the presence
of vaccinia capping enzyme with a component to create enzymatic
activity such as, but not limited to, S-adenosylmethionine
(AdoMet), to form a modified cap for mRNA.
[0204] In one embodiment, the replacement of the sugar ring oxygen
(that produced the carbocyclic ring) with a methylene moiety
(CH.sub.2) could create greater stability to the C--N bond against
phosphorylases as the C--N bond is resitant to acid or enzymatic
hydrolysis. The methylene moiety may also increase the stability of
the triphosphate bridge moiety and thus increasing the stability of
the mRNA. As a non-limiting example, the cap substrate structure
for cap dependent translation may have the structure such as, but
not limited to, CAP-014 and CAP-015 and/or the cap substrate
structure for vaccinia mRNA capping enzyme such as, but not limited
to, CAP-123 and CAP-124. In another example, CAP-112-CAP-122 and/or
CAP-125-CAP-225, can be modified by replacing the sugar ring oxygen
(that produced the carbocyclic ring) with a methylene moiety
(CH.sub.2).
[0205] In another embodiment, the triphophosphate bridge may be
modified by the replacement of at least one oxygen with sulfur
(thio), a borane (BH.sub.3) moiety, a methyl group, an ethyl group,
a methoxy group and/or combinations thereof. This modification
could increase the stability of the mRNA towards decapping enzymes.
As a non-limiting example, the cap substrate structure for cap
dependent translation may have the structure such as, but not
limited to, CAP-016-CAP-021 and/or the cap substrate structure for
vaccinia mRNA capping enzyme such as, but not limited to,
CAP-125-CAP-130. In another example, CAP-003-CAP-015,
CAP-022-CAP-124 and/or CAP-131-CAP-225, can be modified on the
triphosphate bridge by replacing at least one of the triphosphate
bridge oxygens with sulfur (thio), a borane (BH.sub.3) moiety, a
methyl group, an ethyl group, a methoxy group and/or combinations
thereof.
[0206] In one embodiment, CAP-001-134 and/or CAP-136-CAP-225 may be
modified to be a thioguanosine analog similar to CAP-135. The
thioguanosine analog may comprise additional modifications such as,
but not limited to, a modification at the triphosphate moiety
(e.g., thio, BH.sub.3, CH.sub.3, C.sub.2H.sub.5, OCH.sub.3, S and S
with OCH.sub.3), a modification at the 2' and/or 3' positions of
6-thio guanosine as described herein and/or a replacement of the
sugar ring oxygen (that produced the carbocyclic ring) as described
herein.
[0207] In one embodiment, CAP-001-121 and/or CAP-123-CAP-225 may be
modified to be a modified 5'-cap similar to CAP-122. The modified
5'-cap may comprise additional modifications such as, but not
limited to, a modification at the triphosphate moiety (e.g., thio,
BH.sub.3, CH.sub.3, C.sub.2H.sub.5, OCH.sub.3, S and S with
OCH.sub.3), a modification at the 2' and/or 3' positions of 6-thio
guanosine as described herein and/or a replacement of the sugar
ring oxygen (that produced the carbocyclic ring) as described
herein.
[0208] In one embodiment, the 5'-cap modification may be the
attachment of biotin or conjugation at the 2' or 3' position of a
GTP.
[0209] In another embodiment, the 5'-cap modification may include a
CF.sub.2 modified triphosphate moiety.
[0210] In another embodiment, the triphosphate bridge of any of the
cap structures described herein may be replaced with a
tetraphosphate or pentaphosphate bridge. Examples of tetraphosphate
and pentaphosphate containing bridges and other cap modifications
are described in Jemielity, J. et al. RNA 2003 9:1108-1122;
Grudzien-Nogalska, E. et al. Methods Mol. Biol. 2013 969:55-72; and
Grudzien, E. et al. RNA, 2004 10:1479-1487, each of which is
incorporated herein by reference in its entirety.
[0211] Terminal Architecture Alterations: Stem Loop
[0212] In one embodiment, the nucleic acids of the present
invention may include a stem loop such as, but not limited to, a
histone stem loop. The stem loop may be a nucleotide sequence that
is about 25 or about 26 nucleotides in length such as, but not
limited to, SEQ ID NOs: 7-17 as described in International Patent
Publication No. WO2013103659, incorporated herein by reference in
its entirety. The histone stem loop may be located 3' relative to
the coding region (e.g., at the 3' terminus of the coding region).
As a non-limiting example, the stem loop may be located at the 3'
end of a nucleic acid described herein.
[0213] In one embodiment, the stem loop may be located in the
second terminal region. As a non-limiting example, the stem loop
may be located within an untranslated region (e.g., 3'3'-UTR) in
the second terminal region.
[0214] In one embodiment, the nucleic acid such as, but not limited
to mRNA, which comprises the histone stem loop may be stabilized by
the addition of at least one chain terminating nucleoside. Not
wishing to be bound by theory, the addition of at least one chain
terminating nucleoside may slow the degradation of a nucleic acid
and thus can increase the half-life of the nucleic acid.
[0215] In one embodiment, the chain terminating nucleoside may be,
but is not limited to, those described in International Patent
Publication No. WO2013103659, incorporated herein by reference in
its entirety. In another embodiment, the chain terminating
nucleosides which may be used with the present invention includes,
but is not limited to, 3'-deoxyadenosine (cordycepin),
3'-deoxyuridine, 3'-deoxycytidine, 3'-deoxyguanosine,
3'-deoxythymidine, 2',3'-dideoxynucleosides, such as
2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytidine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymidine, a 2'-deoxynucleoside, or a 2' or
3'-O-methylnucleoside.
[0216] In another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprises the histone stem loop may be
stabilized by an alteration to the 3'-region of the nucleic acid
that can prevent and/or inhibit the addition of oligio(U) (see
e.g., International Patent Publication No. WO2013103659,
incorporated herein by reference in its entirety).
[0217] In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprises the histone stem loop may be
stabilized by the addition of an oligonucleotide that terminates in
a 3'-deoxynucleoside, 2',3'-dideoxynucleoside 3'-O-methyl
nucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other
alternative nucleosides known in the art and/or described
herein.
[0218] In one embodiment, the nucleic acids of the present
invention may include a histone stem loop, a polyA tail sequence
and/or a 5'-cap structure. The histone stem loop may be before
and/or after the polyA tail sequence. The nucleic acids comprising
the histone stem loop and a polyA tail sequence may include a chain
terminating nucleoside described herein.
[0219] In another embodiment, the nucleic acids of the present
invention may include a histone stem loop and a 5'-cap structure.
The 5'-cap structure may include, but is not limited to, those
described herein and/or known in the art.
[0220] In one embodiment, the conserved stem loop region may
comprise a miR sequence described herein. As a non-limiting
example, the stem loop region may comprise the seed sequence of a
miR sequence described herein. In another non-limiting example, the
stem loop region may comprise a miR-122 seed sequence.
[0221] In another embodiment, the conserved stem loop region may
comprise a miR sequence described herein and may also include a TEE
sequence.
[0222] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or decrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'-UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
[0223] In one embodiment, the alternative nucleic acids described
herein may comprise at least one histone stem-loop and a polyA
sequence or polyadenylation signal. Non-limiting examples of
nucleic acid sequences encoding for at least one histone stem-loop
and a polyA sequence or a polyadenylation signal are described in
International Patent Publication No. WO2013120497, WO2013120629,
WO2013120500, WO2013120627, WO2013120498, WO2013120626,
WO2013120499 and WO2013120628, the contents of each of which are
incorporated herein by reference in their entirety. In one
embodiment, the nucleic acid encoding for a histone stem loop and a
polyA sequence or a polyadenylation signal may code for a pathogen
antigen or fragment thereof such as the nucleic acid sequences
described in International Patent Publication No WO2013120499 and
WO2013120628, the contents of both of which are incorporated herein
by reference in their entirety. In another embodiment, the nucleic
acid encoding for a histone stem loop and a polyA sequence or a
polyadenylation signal may code for a therapeutic protein such as
the nucleic acid sequences described in International Patent
Publication No WO2013120497 and WO2013120629, the contents of both
of which are incorporated herein by reference in their entirety. In
one embodiment, the nucleic acid encoding for a histone stem loop
and a polyA sequence or a polyadenylation signal may code for a
tumor antigen or fragment thereof such as the nucleic acid
sequences described in International Patent Publication No
WO2013120500 and WO2013120627, the contents of both of which are
incorporated herein by reference in their entirety. In another
embodiment, the nucleic acid encoding for a histone stem loop and a
polyA sequence or a polyadenylation signal may code for an
allergenic antigen or an autoimmune self-antigen such as the
nucleic acid sequences described in International Patent
Publication No WO2013120498 and WO2013120626, the contents of both
of which are incorporated herein by reference in their
entirety.
[0224] Terminal Architecture Alterations: 3'-UTR and Triple
Helices
[0225] In one embodiment, nucleic acids of the present invention
may include a triple helix on the 3' end of the alternative nucleic
acid, enhanced alternative RNA or ribonucleic acid. The 3' end of
the nucleic acids of the present invention may include a triple
helix alone or in combination with a Poly-A tail.
[0226] In one embodiment, the nucleic acid of the present invention
may comprise at least a first and a second U-rich region, a
conserved stem loop region between the first and second region and
an A-rich region. The first and second U-rich region and the A-rich
region may associate to form a triple helix on the 3' end of the
nucleic acid. This triple helix may stabilize the nucleic acid,
enhance the translational efficiency of the nucleic acid and/or
protect the 3' end from degradation. Exemplary triple helices
include, but are not limited to, the triple helix sequence of
metastasis-associated lung adenocarcinoma transcript 1 (MALAT1),
MEN-.beta. and polyadenylated nuclear (PAN) RNA (See Wilusz et al.,
Genes & Development 2012 26:2392-2407; herein incorporated by
reference in its entirety). In one embodiment, the 3' end of the
alternative nucleic acids, enhanced alternative RNA or ribonucleic
acids of the present invention comprises a first U-rich region
comprising TTTTTCTTTT (SEQ ID NO: 1), a second U-rich region
comprising TTTTGCTTTTT (SEQ ID NO: 2) or TTTTGCTTTT (SEQ ID NO: 3),
an A-rich region comprising AAAAAGCAAAA (SEQ ID NO: 4). In another
embodiment, the 3' end of the nucleic acids of the present
invention comprises a triple helix formation structure comprising a
first U-rich region, a conserved region, a second U-rich region and
an A-rich region.
[0227] In one embodiment, the triple helix may be formed from the
cleavage of a MALAT1 sequence prior to the cloverleaf structure.
While not meaning to be bound by theory, MALAT1 is a long
non-coding RNA which, when cleaved, forms a triple helix and a
tRNA-like cloverleaf structure. The MALAT1 transcript then
localizes to nuclear speckles and the tRNA-like cloverleaf
localizes to the cytoplasm (Wilusz et al. Cell 2008 135(5):
919-932; incorporated herein by reference in its entirety).
[0228] As a non-limiting example, the terminal end of the nucleic
acid of the present invention comprising the MALAT1 sequence can
then form a triple helix structure, after RNaseP cleavage from the
cloverleaf structure, which stabilizes the nucleic acid (Peart et
al. Non-mRNA 3' end formation: how the other half lives; WIREs RNA
2013; incorporated herein by reference in its entirety).
[0229] In one embodiment, the nucleic acids or mRNA described
herein comprise a MALAT1 sequence. In another embodiment, the
nucleic acids or mRNA may be polyadenylated. In yet another
embodiment, the nucleic acids or mRNA is not polyadenylated but has
an increased resistance to degradation compared to unaltered
nucleic acids or mRNA.
[0230] In one embodiment, the nucleic acids of the present
invention may comprise a MALAT1 sequence in the second flanking
region (e.g., the 3'-UTR). As a non-limiting example, the MALAT1
sequence may be human or mouse.
[0231] In another embodiment, the cloverleaf structure of the
MALAT1 sequence may also undergo processing by RNaseZ and CCA
adding enzyme to form a tRNA-like structure called mascRNA
(MALAT1-associated small cytoplasmic RNA). As a non-limiting
example, the mascRNA may encode a protein or a fragment thereof
and/or may comprise a microRNA sequence. The mascRNA may comprise
at least one chemical alteration described herein.
[0232] Terminal Architecture Alterations: Poly-A Tails
[0233] During RNA processing, a long chain of adenosine nucleotides
(poly-A tail) is normally added to a messenger RNA (mRNA) molecules
to increase the stability of the molecule. Immediately after
transcription, the 3' end of the transcript is cleaved to free a 3'
hydroxyl. Then poly-A polymerase adds a chain of adenosine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that is between 100 and 250 residues long.
[0234] Methods for the stabilization of RNA by incorporation of
chain-terminating nucleosides at the 3'-terminus include those
described in International Patent Publication No. WO2013103659,
incorporated herein in its entirety.
[0235] Unique poly-A tail lengths may provide certain advantages to
the alternative RNAs of the present invention.
[0236] Generally, the length of a poly-A tail of the present
invention is greater than 10 nucleotides. In some embodiments, the
poly-A tail is greater than 20 nucleotides. In some embodiments,
the poly-A tail is greater than 30 nucleotides in length. In
another embodiment, the poly-A tail is greater than 35 nucleotides
in length. In another embodiment, the length of the poly-A tail is
at least 40 nucleotides. In another embodiment, the length of the
poly-A tail is at least 45 nucleotides. In another embodiment, the
length of the poly-A tail is at least 55 nucleotides. In another
embodiment, the length of the poly-A tail is at least 60
nucleotides. In another embodiment, the length of the poly-A tail
is at least 70 nucleotides. In another embodiment, the length of
the poly-A tail is at least 80 nucleotides. In another embodiment,
the length of the poly-A tail is at least 90 nucleotides. In
another embodiment, the length of the poly-A tail is at least 100
nucleotides. In another embodiment, the length of the poly-A tail
is at least 120 nucleotides. In another embodiment, the length of
the poly-A tail is at least 140 nucleotides. In another embodiment,
the length of the poly-A tail is at least 160 nucleotides. In
another embodiment, the length of the poly-A tail is at least 180
nucleotides. In another embodiment, the length of the poly-A tail
is at least 200 nucleotides. In another embodiment, the length of
the poly-A tail is at least 250 nucleotides. In another embodiment,
the length of the poly-A tail is at least 300 nucleotides.
[0237] In another embodiment, the length of the mRNA is at least
350 nucleotides. In another embodiment, the length of the mRNA is
at least 400 nucleotides. In another embodiment, the length of the
mRNA is at least 450 nucleotides. In another embodiment, the length
of the mRNA is at least 500 nucleotides. In another embodiment, the
length of the mRNA is at least 600 nucleotides. In another
embodiment, the length of the mRNA is at least 700 nucleotides. In
another embodiment, the length of the mRNA is at least 800
nucleotides. In another embodiment, the length of the mRNA is at
least 900 nucleotides. In another embodiment, the length of the
mRNA is at least 1000 nucleotides. In another embodiment, the
length of the mRNA is at least 1100 nucleotides. In another
embodiment, the length of the mRNA is at least 1200 nucleotides. In
another embodiment, the length of the mRNA is at least 1300
nucleotides. In another embodiment, the length of the mRNA is at
least 1400 nucleotides. In another embodiment, the length of the
mRNA is at least 1500 nucleotides. In another embodiment, the
length of the mRNA is at least 1600 nucleotides. In another
embodiment, the length of the mRNA is at least 1700 nucleotides. In
another embodiment, the length of the mRNA is at least 1800
nucleotides. In another embodiment, the length of the mRNA is at
least 1900 nucleotides. In another embodiment, the length of the
mRNA is at least 2000 nucleotides. In another embodiment, the
length of the mRNA is at least 2500 nucleotides. In another
embodiment, the length of the mRNA is at least 3000
nucleotides.
[0238] In one embodiment, the poly-A tail may be 80 nucleotides,
120 nucleotides, 160 nucleotides in length on an alternative RNA
molecule described herein.
[0239] In another embodiment, the poly-A tail may be 20, 40, 80,
100, 120, 140 or 160 nucleotides in length on an alternative RNA
molecule described herein.
[0240] In one embodiment, the poly-A tail is designed relative to
the length of the overall alternative RNA molecule. This design may
be based on the length of the coding region of the alternative RNA,
the length of a particular feature or region of the alternative RNA
(such as the mRNA), or based on the length of the ultimate product
expressed from the alternative RNA. When relative to any additional
feature of the alternative RNA (e.g., other than the mRNA portion
which includes the poly-A tail) the poly-A tail may be 10, 20, 30,
40, 50, 60, 70, 80, 90 or 100% greater in length than the
additional feature. The poly-A tail may also be designed as a
fraction of the alternative RNA to which it belongs. In this
context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or
90% or more of the total length of the construct or the total
length of the construct minus the poly-A tail.
[0241] In one embodiment, engineered binding sites and/or the
conjugation of nucleic acids or mRNA for Poly-A binding protein may
be used to enhance expression. The engineered binding sites may be
sensor sequences which can operate as binding sites for ligands of
the local microenvironment of the nucleic acids and/or mRNA. As a
non-limiting example, the nucleic acids and/or mRNA may comprise at
least one engineered binding site to alter the binding affinity of
Poly-A binding protein (PABP) and analogs thereof. The
incorporation of at least one engineered binding site may increase
the binding affinity of the PABP and analogs thereof.
[0242] Additionally, multiple distinct nucleic acids or mRNA may be
linked together to the PABP (Poly-A binding protein) through the
3'-end using alternative nucleotides at the 3'-terminus of the
poly-A tail. Transfection experiments can be conducted in relevant
cell lines at and protein production can be assayed by ELISA at 12
hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection. As a
non-limiting example, the transfection experiments may be used to
evaluate the effect on PABP or analogs thereof binding affinity as
a result of the addition of at least one engineered binding
site.
[0243] In one embodiment, a polyA tail may be used to modulate
translation initiation. While not wishing to be bound by theory,
the polyA tail recruits PABP which in turn can interact with
translation initiation complex and thus may be essential for
protein synthesis.
[0244] In another embodiment, a polyA tail may also be used in the
present invention to protect against 3'-5' exonuclease
digestion.
[0245] In one embodiment, the nucleic acids or mRNA of the present
invention are designed to include a polyA-G Quartet. The G-quartet
is a cyclic hydrogen bonded array of four guanosine nucleotides
that can be formed by G-rich sequences in both DNA and RNA. In this
embodiment, the G-quartet is incorporated at the end of the poly-A
tail. The resultant nucleic acid or mRNA may be assayed for
stability, protein production and other parameters including
half-life at various time points. It has been discovered that the
polyA-G quartet results in protein production equivalent to at
least 75% of that seen using a poly-A tail of 120 nucleotides
alone.
[0246] In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a polyA tail and may be stabilized by the
addition of a chain terminating nucleoside. The nucleic acids
and/or mRNA with a polyA tail may further comprise a 5'-cap
structure.
[0247] In another embodiment, the nucleic acids or mRNA of the
present invention may comprise a polyA-G Quartet. The nucleic acids
and/or mRNA with a polyA-G Quartet may further comprise a 5'-cap
structure.
[0248] In one embodiment, the chain terminating nucleoside which
may be used to stabilize the nucleic acid or mRNA comprising a
polyA tail or polyA-G Quartet may be, but is not limited to, those
described in International Patent Publication No. WO2013103659,
incorporated herein by reference in its entirety. In another
embodiment, the chain terminating nucleosides which may be used
with the present invention includes, but is not limited to,
3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytidine,
3'-deoxyguanosine, 3'-deoxythymidine, 2',3'-dideoxynucleosides,
such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytidine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymidine, a 2'-deoxynucleoside, or a 2' or
3'-O-methylnucleoside.
[0249] In another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprise a polyA tail or a polyA-G Quartet
may be stabilized by an alteration to the 3'-region of the nucleic
acid that can prevent and/or inhibit the addition of oligio(U) (see
e.g., International Patent Publication No. WO2013103659,
incorporated herein by reference in its entirety).
[0250] In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprise a polyA tail or a polyA-G Quartet
may be stabilized by the addition of an oligonucleotide that
terminates in a 3'-deoxynucleoside, 2',3'-dideoxynucleoside
3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and
other alternative nucleosides known in the art and/or described
herein.
[0251] 5'-UTR, 3'-UTR and Translation Enhancer Elements (TEEs)
[0252] In one embodiment, the 5'-UTR of the polynucleotides,
primary constructs, alternative nucleic acids and/or mmRNA may
include at least one translational enhancer polynucleotide,
translation enhancer element, translational enhancer elements
(collectively referred to as "TEE"s). As a non-limiting example,
the TEE may be located between the transcription promoter and the
start codon. The polynucleotides, primary constructs, alternative
nucleic acids and/or mmRNA with at least one TEE in the 5'-UTR may
include a cap at the 5'-UTR. Further, at least one TEE may be
located in the 5'-UTR of polynucleotides, primary constructs,
alternative nucleic acids and/or mmRNA undergoing cap-dependent or
cap-independent translation.
[0253] The term "translational enhancer element" or "translation
enhancer element" (herein collectively referred to as "TEE") refers
to sequences that increase the amount of polypeptide or protein
produced from an mRNA.
[0254] In one aspect, TEEs are conserved elements in the UTR which
can promote translational activity of a nucleic acid such as, but
not limited to, cap-dependent or cap-independent translation. The
conservation of these sequences has been previously shown by Panek
et al (Nucleic Acids Research, 2013, 1-10; incorporated herein by
reference in its entirety) across 14 species including humans.
[0255] In one non-limiting example, the TEEs known may be in the
5'-leader of the Gtx homeodomain protein (Chappell et al., Proc.
Natl. Acad. Sci. USA 101:9590-9594, 2004, incorporated herein by
reference in their entirety).
[0256] In another non-limiting example, TEEs are disclosed as SEQ
ID NOs: 1-35 in US Patent Publication No. US20090226470, SEQ ID
NOs: 1-35 in US Patent Publication US20130177581, SEQ ID NOs: 1-35
in International Patent Publication No. WO2009075886, SEQ ID NOs:
1-5, and 7-645 in International Patent Publication No.
WO2012009644, SEQ ID NO: 1 in International Patent Publication No.
WO1999024595, SEQ ID NO: 1 in U.S. Pat. No. 6,310,197, and SEQ ID
NO: 1 in U.S. Pat. No. 6,849,405, each of which is incorporated
herein by reference in its entirety.
[0257] In yet another non-limiting example, the TEE may be an
internal ribosome entry site (IRES), HCV-IRES or an IRES element
such as, but not limited to, those described in U.S. Pat. No.
7,468,275, US Patent Publication Nos. US20070048776 and
US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2001055369, each of which is incorporated herein
by reference in its entirety. The IRES elements may include, but
are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt,
Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci. USA
101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) and
in US Patent Publication Nos. US20070048776 and US20110124100 and
International Patent Publication No. WO2007025008, each of which is
incorporated herein by reference in its entirety.
[0258] "Translational enhancer polynucleotides" or "translation
enhancer polynucleotide sequences" are polynucleotides which
include one or more of the specific TEE exemplified herein and/or
disclosed in the art (see e.g., U.S. Pat. Nos. 6,310,197,
6,849,405, 7,456,273, 7,183,395, US20090226470, US20070048776,
U520110124100, US20090093049, US20130177581, WO2009075886,
WO2007025008, WO2012009644, WO2001055371 WO1999024595, and
EP2610341A1 and EP2610340A1; each of which is incorporated herein
by reference in its entirety) or their variants, homologs or
functional derivatives. One or multiple copies of a specific TEE
can be present in the polynucleotides, primary constructs,
alternative nucleic acids and/or mm RNA. The TEEs in the
translational enhancer polynucleotides can be organized in one or
more sequence segments. A sequence segment can harbor one or more
of the specific TEEs exemplified herein, with each TEE being
present in one or more copies. When multiple sequence segments are
present in a translational enhancer polynucleotide, they can be
homogenous or heterogeneous. Thus, the multiple sequence segments
in a translational enhancer polynucleotide can harbor identical or
different types of the specific TEEs exemplified herein, identical
or different number of copies of each of the specific TEEs, and/or
identical or different organization of the TEEs within each
sequence segment.
[0259] In one embodiment, the polynucleotides, primary constructs,
alternative nucleic acids and/or mm RNA may include at least one
TEE that is described in International Patent Publication No.
WO1999024595, WO2012009644, WO2009075886, WO2007025008,
WO1999024595, European Patent Publication No. EP2610341A1 and
EP2610340A1, U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273,
7,183,395, US Patent Publication No. US20090226470, US20110124100,
US20070048776, US20090093049, and US20130177581 each of which is
incorporated herein by reference in its entirety. The TEE may be
located in the 5'-UTR of the polynucleotides, primary constructs,
alternative nucleic acids and/or mm RNA.
[0260] In another embodiment, the polynucleotides, primary
constructs, alternative nucleic acids and/or mmRNA may include at
least one TEE that has at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95% or at least 99% identity with the TEEs
described in US Patent Publication Nos. US20090226470,
US20070048776, US20130177581 and US20110124100, International
Patent Publication No. WO1999024595, WO2012009644, WO2009075886 and
WO2007025008, European Patent Publication No. EP2610341A1 and
EP2610340A1, U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273,
7,183,395, each of which is incorporated herein by reference in its
entirety.
[0261] In one embodiment, the 5'-UTR of the polynucleotides,
primary constructs, alternative nucleic acids and/or mmRNA may
include at least 1, at least 2, at least 3, at least 4, at least 5,
at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18 at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55 or more than 60 TEE sequences. The TEE sequences in the
5'-UTR of the polynucleotides, primary constructs, alternative
nucleic acids and/or mmRNA of the present invention may be the same
or different TEE sequences. The TEE sequences may be in a pattern
such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof
repeated once, twice, or more than three times. In these patterns,
each letter, A, B, or C represent a different TEE sequence at the
nucleotide level.
[0262] In one embodiment, the 5'-UTR may include a spacer to
separate two TEE sequences. As a non-limiting example, the spacer
may be a 15 nucleotide spacer and/or other spacers known in the
art. As another non-limiting example, the 5'-UTR may include a TEE
sequence-spacer module repeated at least once, at least twice, at
least 3 times, at least 4 times, at least 5 times, at least 6
times, at least 7 times, at least 8 times and at least 9 times or
more than 9 times in the 5'-UTR.
[0263] In another embodiment, the spacer separating two TEE
sequences may include other sequences known in the art which may
regulate the translation of the polynucleotides, primary
constructs, alternative nucleic acids and/or mmRNA of the present
invention such as, but not limited to, miR sequences described
herein (e.g., miR binding sites and miR seeds). As a non-limiting
example, each spacer used to separate two TEE sequences may include
a different miR sequence or component of a miR sequence (e.g., miR
seed sequence).
[0264] In one embodiment, the TEE in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mmRNA of the present invention may include at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%
or more than 99% of the TEE sequences disclosed in US Patent
Publication Nos. US20090226470, US20070048776, US20130177581 and
US20110124100, International Patent Publication No. WO1999024595,
WO2012009644, WO2009075886 and WO2007025008, European Patent
Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. Nos.
6,310,197, 6,849,405, 7,456,273, and 7,183,395 each of which is
incorporated herein by reference in its entirety. In another
embodiment, the TEE in the 5'-UTR of the polynucleotides, primary
constructs, alternative nucleic acids and/or mmRNA of the present
invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide
fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a
5-10 nucleotide fragment of the TEE sequences disclosed in US
Patent Publication Nos. US20090226470, US20070048776, US20130177581
and U520110124100, International Patent Publication No.
WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European
Patent Publication No. EP2610341A1 and EP2610340A1, U.S. Pat. Nos.
6,310,197, 6,849,405, 7,456,273, and 7,183,395; each of which is
incorporated herein by reference in its entirety.
[0265] In one embodiment, the TEE in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mmRNA of the present invention may include at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%
or more than 99% of the TEE sequences disclosed in Chappell et al.
(Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al.
(PNAS 102:6273-6278, 2005), in Supplemental Table 1 and in
Supplemental Table 2 disclosed by Wellensiek et al (Genome-wide
profiling of human cap-independent translation-enhancing elements,
Nature Methods, 2013; DOI:10.1038/NMETH.2522); each of which is
herein incorporated by reference in its entirety. In another
embodiment, the TEE in the 5'-UTR of the polynucleotides, primary
constructs, alternative nucleic acids and/or mmRNA of the present
invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide
fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a
5-10 nucleotide fragment of the TEE sequences disclosed in Chappell
et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et
al. (PNAS 102:6273-6278, 2005), in Supplemental Table 1 and in
Supplemental Table 2 disclosed by Wellensiek et al (Genome-wide
profiling of human cap-independent translation-enhancing elements,
Nature Methods, 2013; DOI:10.1038/NMETH.2522); each of which is
incorporated herein by reference in its entirety.
[0266] In one embodiment, the TEE used in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mm RNA of the present invention is an IRES sequence such as,
but not limited to, those described in U.S. Pat. No. 7,468,275 and
International Patent Publication No. WO2001055369, each of which is
incorporated herein by reference in its entirety.
[0267] In one embodiment, the TEEs used in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mm RNA of the present invention may be identified by the
methods described in US Patent Publication No. US20070048776 and
US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2012009644, each of which is incorporated herein
by reference in its entirety.
[0268] In another embodiment, the TEEs used in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mm RNA of the present invention may be a transcription
regulatory element described in U.S. Pat. Nos. 7,456,273 and
7,183,395, US Patent Publication No. US20090093049, and
International Publication No. WO2001055371, each of which is
incorporated herein by reference in its entirety. The transcription
regulatory elements may be identified by methods known in the art,
such as, but not limited to, the methods described in U.S. Pat.
Nos. 7,456,273 and 7,183,395, US Patent Publication No.
US20090093049, and International Publication No. WO2001055371, each
of which is incorporated herein by reference in its entirety.
[0269] In yet another embodiment, the TEE used in the 5'-UTR of the
polynucleotides, primary constructs, alternative nucleic acids
and/or mm RNA of the present invention is an oligonucleotide or
portion thereof as described in U.S. Pat. Nos. 7,456,273 and
7,183,395, US Patent Publication No. US20090093049, and
International Publication No. WO2001055371, each of which is
incorporated herein by reference in its entirety.
[0270] The 5' UTR comprising at least one TEE described herein may
be incorporated in a monocistronic sequence such as, but not
limited to, a vector system or a nucleic acid vector. As a
non-limiting example, the vector systems and nucleic acid vectors
may include those described in US Patent Nos. 7456273 and U.S. Pat.
No. 7,183,395, US Patent Publication No. US20070048776,
US20090093049 and US20110124100 and International Patent
Publication Nos. WO2007025008 and WO2001055371, each of which is
incorporated herein by reference in its entirety.
[0271] In one embodiment, the TEEs described herein may be located
in the 5'-UTR and/or the 3'-UTR of the polynucleotides, primary
constructs, alternative nucleic acids and/or mmRNA. The TEEs
located in the 3'-UTR may be the same and/or different than the
TEEs located in and/or described for incorporation in the
5'-UTR.
[0272] In one embodiment, the 3'-UTR of the polynucleotides,
primary constructs, alternative nucleic acids and/or mmRNA may
include at least 1, at least 2, at least 3, at least 4, at least 5,
at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18 at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55 or more than 60 TEE sequences. The TEE sequences in the
3'-UTR of the polynucleotides, primary constructs, alternative
nucleic acids and/or mmRNA of the present invention may be the same
or different TEE sequences. The TEE sequences may be in a pattern
such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof
repeated once, twice, or more than three times. In these patterns,
each letter, A, B, or C represent a different TEE sequence at the
nucleotide level.
[0273] In one embodiment, the 3'-UTR may include a spacer to
separate two TEE sequences. As a non-limiting example, the spacer
may be a 15 nucleotide spacer and/or other spacers known in the
art. As another non-limiting example, the 3'-UTR may include a TEE
sequence-spacer module repeated at least once, at least twice, at
least 3 times, at least 4 times, at least 5 times, at least 6
times, at least 7 times, at least 8 times and at least 9 times or
more than 9 times in the 3'-UTR.
[0274] In another embodiment, the spacer separating two TEE
sequences may include other sequences known in the art which may
regulate the translation of the polynucleotides, primary
constructs, alternative nucleic acids and/or mm RNA of the present
invention such as, but not limited to, miR sequences described
herein (e.g., miR binding sites and miR seeds). As a non-limiting
example, each spacer used to separate two TEE sequences may include
a different miR sequence or component of a miR sequence (e.g., miR
seed sequence).
[0275] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or decrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'-UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
[0276] Heterologous 5'-UTRs
[0277] A 5' UTR may be provided as a flanking region to the
alternative nucleic acids (mRNA), enhanced alternative RNA or
ribonucleic acids of the invention. 5'-UTR may be homologous or
heterologous to the coding region found in the alternative nucleic
acids (mRNA), enhanced alternative RNA or ribonucleic acids of the
invention. Multiple 5' UTRs may be included in the flanking region
and may be the same or of different sequences. Any portion of the
flanking regions, including none, may be codon optimized and any
may independently contain one or more different structural or
chemical alterations, before and/or after codon optimization.
[0278] Shown in Lengthy Table 21 in U.S. Provisional Application
No. 61/775,509, and in Lengthy Table 21 and in Table 22 in U.S.
Provisional Application No. 61/829,372, the contents of each of
which are incorporated herein by reference in their entirety, is a
listing of the start and stop site of the alternative nucleic acids
(mRNA), enhanced alternative RNA or ribonucleic acids of the
invention. In Table 21 each 5'-UTR (5'-UTR-005 to 5'-UTR 68511) is
identified by its start and stop site relative to its native or
wild-type (homologous) transcript (ENST; the identifier used in the
ENSEMBL database).
[0279] To alter one or more properties of the polynucleotides,
primary constructs or mmRNA of the invention, 5'-UTRs which are
heterologous to the coding region of the alternative nucleic acids
(mRNA), enhanced alternative RNA or ribonucleic acids of the
invention are engineered into compounds of the invention. The
alternative nucleic acids (mRNA), enhanced alternative RNA or
ribonucleic acids are then administered to cells, tissue or
organisms and outcomes such as protein level, localization and/or
half-life are measured to evaluate the beneficial effects the
heterologous 5'-UTR may have on the alternative nucleic acids
(mRNA), enhanced alternative RNA or ribonucleic acids of the
invention. Variants of the 5' UTRs may be utilized wherein one or
more nucleotides are added or removed to the termini, including A,
T, C or G. 5'-UTRs may also be codon-optimized or altered in any
manner described herein.
[0280] Incorporating microRNA Binding Sites
[0281] In one embodiment, alternative nucleic acids (mRNA),
enhanced alternative RNA or ribonucleic acids of the invention
would not only encode a polypeptide but also a sensor sequence.
Sensor sequences include, for example, microRNA binding sites,
transcription factor binding sites, structured mRNA sequences
and/or motifs, artificial binding sites engineered to act as
pseudo-receptors for endogenous nucleic acid binding molecules.
Non-limiting examples, of polynucleotides comprising at least one
sensor sequence are described in co-pending and co-owned U.S.
Provisional Patent Application No. U.S. 61/753,661, filed Jan. 17,
2013, U.S. Provisional Patent Application No. U.S. 61/754,159,
filed Jan. 18, 2013, U.S. Provisional Patent Application No. U.S.
61/781,097, filed Mar. 14, 2013, U.S. Provisional Patent
Application No. U.S. 61/829,334, filed May 31, 2013, U.S.
Provisional Patent Application No. U.S. 61/839,893, filed Jun. 27,
2013, U.S. Provisional Patent Application No. U.S. 61/842,733,
filed Jul. 3, 2013, and US Provisional Patent Application No. U.S.
61/857,304, filed Jul. 23, 2013, the contents of each of which are
incorporated herein by reference in their entirety.
[0282] In one embodiment, microRNA (miRNA) profiling of the target
cells or tissues is conducted to determine the presence or absence
of miRNA in the cells or tissues.
[0283] microRNAs (or miRNA) are 19-25 nucleotide long noncoding
RNAs that bind to the 3'UTR of nucleic acid molecules and
down-regulate gene expression either by reducing nucleic acid
molecule stability or by inhibiting translation. The alternative
nucleic acids (mRNA), enhanced alternative RNA or ribonucleic acids
of the invention may comprise one or more microRNA target
sequences, microRNA sequences, or microRNA seeds. Such sequences
may correspond to any known microRNA such as those taught in US
Publication US2005/0261218 and US Publication US2005/0059005, the
contents of which are incorporated herein by reference in their
entirety.
[0284] A microRNA sequence comprises a "seed" region, i.e., a
sequence in the region of positions 2-8 of the mature microRNA,
which sequence has perfect Watson-Crick complementarity to the
miRNA target sequence. A microRNA seed may comprise positions 2-8
or 2-7 of the mature microRNA. In some embodiments, a microRNA seed
may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature
microRNA), wherein the seed-complementary site in the corresponding
miRNA target is flanked by an adenosine (A) opposed to microRNA
position 1. In some embodiments, a microRNA seed may comprise 6
nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein
the seed-complementary site in the corresponding miRNA target is
flanked by an adenosine (A) opposed to microRNA position 1. See for
example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L
P, Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of
the microRNA seed have complete complementarity with the target
sequence. By engineering microRNA target sequences into the 3'UTR
of nucleic acids or mRNA of the invention one can target the
molecule for degradation or reduced translation, provided the
microRNA in question is available. This process will reduce the
hazard of off target effects upon nucleic acid molecule delivery.
Identification of microRNA, microRNA target regions, and their
expression patterns and role in biology have been reported (Bonauer
et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr
Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012
26:404-413 (2011 Dec. 20. doi: 10.1038/Ieu.2011.356); Bartel Cell
2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner
and Naldini, Tissue Antigens. 2012 80:393-403 and all references
therein; each of which is incorporated herein by reference in its
entirety).
[0285] For example, if the mRNA is not intended to be delivered to
the liver but ends up there, then miR-122, a microRNA abundant in
liver, can inhibit the expression of the gene of interest if one or
multiple target sites of miR-122 are engineered into the 3'UTR of
the alternative nucleic acids, enhanced alternative RNA or
ribonucleic acids. Introduction of one or multiple binding sites
for different microRNA can be engineered to further decrease the
longevity, stability, and protein translation of an alternative
nucleic acids, enhanced alternative RNA or ribonucleic acids. As
used herein, the term "microRNA site" refers to a microRNA target
site or a microRNA recognition site, or any nucleotide sequence to
which a microRNA binds or associates. It should be understood that
"binding" may follow traditional Watson-Crick hybridization rules
or may reflect any stable association of the microRNA with the
target sequence at or adjacent to the microRNA site.
[0286] Conversely, for the purposes of the alternative nucleic
acids, enhanced alternative RNA or ribonucleic acids of the present
invention, microRNA binding sites can be engineered out of (i.e.
removed from) sequences in which they naturally occur in order to
increase protein expression in specific tissues. For example,
miR-122 binding sites may be removed to improve protein expression
in the liver.
[0287] In one embodiment, the alternative nucleic acids, enhanced
alternative RNA or ribonucleic acids of the present invention may
include at least one miRNA-binding site in the 3'-UTR in order to
direct cytotoxic or cytoprotective mRNA therapeutics to specific
cells such as, but not limited to, normal and/or cancerous cells
(e.g., HEP3B or SNU449).
[0288] In another embodiment, the alternative nucleic acids,
enhanced alternative RNA or ribonucleic acids of the present
invention may include three miRNA-binding sites in the 3'-UTR in
order to direct cytotoxic or cytoprotective mRNA therapeutics to
specific cells such as, but not limited to, normal and/or cancerous
cells (e.g., HEP3B or SNU449).
[0289] Regulation of expression in multiple tissues can be
accomplished through introduction or removal or one or several
microRNA binding sites. The decision of removal or insertion of
microRNA binding sites, or any combination, is dependent on
microRNA expression patterns and their profilings in diseases.
[0290] Examples of tissues where microRNA are known to regulate
mRNA, and thereby protein expression, include, but are not limited
to, liver (miR-122), muscle (miR-133, miR-206, miR-208),
endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p,
miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose
tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192,
miR-194, miR-204), and lung epithelial cells (let-7, miR-133,
miR-126).
[0291] Specifically, microRNAs are known to be differentially
expressed in immune cells (also called hematopoietic cells), such
as antigen presenting cells (APCs) (e.g. dendritic cells and
macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes,
granuocytes, natural killer cells, etc. Immune cell specific
microRNAs are involved in immunogenicity, autoimmunity, the
immune-response to infection, inflammation, as well as unwanted
immune response after gene therapy and tissue/organ
transplantation. Immune cells specific microRNAs also regulate many
aspects of development, proliferation, differentiation and
apoptosis of hematopoietic cells (immune cells). For example,
miR-142 and miR-146 are exclusively expressed in the immune cells,
particularly abundant in myeloid dendritic cells. It was
demonstrated in the art that the immune response to exogenous
nucleic acid molecules was shut-off by adding miR-142 binding sites
to the 3'-UTR of the delivered gene construct, enabling more stable
gene transfer in tissues and cells. miR-142 efficiently degrades
the exogenous mRNA in antigen presenting cells and suppresses
cytotoxic elimination of transduced cells (Annoni A et al., blood,
2009, 114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5),
585-591; Brown B D, et al., blood, 2007, 110(13): 4144-4152, each
of which is incorporated herein by reference in its entirety).
[0292] An antigen-mediated immune response can refer to an immune
response triggered by foreign antigens, which, when entering an
organism, are processed by the antigen presenting cells and
displayed on the surface of the antigen presenting cells. T cells
can recognize the presented antigen and induce a cytotoxic
elimination of cells that express the antigen.
[0293] Introducing the miR-142 binding site into the 3'-UTR of a
polypeptide of the present invention can selectively repress the
gene expression in the antigen presenting cells through miR-142
mediated mRNA degradation, limiting antigen presentation in APCs
(e.g. dendritic cells) and thereby preventing antigen-mediated
immune response after the delivery of the polynucleotides. The
polynucleotides are therefore stably expressed in target tissues or
cells without triggering cytotoxic elimination.
[0294] In one embodiment, microRNAs binding sites that are known to
be expressed in immune cells, in particular, the antigen presenting
cells, can be engineered into the polynucleotide to suppress the
expression of the sensor-signal polynucleotide in APCs through
microRNA mediated RNA degradation, subduing the antigen-mediated
immune response, while the expression of the polynucleotide is
maintained in non-immune cells where the immune cell specific
microRNAs are not expressed. For example, to prevent the
immunogenic reaction caused by a liver specific protein expression,
the miR-122 binding site can be removed and the miR-142 (and/or
mirR-146) binding sites can be engineered into the 3-UTR of the
polynucleotide.
[0295] To further drive the selective degradation and suppression
of mRNA in APCs and macrophage, the polynucleotide may include
another negative regulatory element in the 3-UTR, either alone or
in combination with mir-142 and/or mir-146 binding sites. As a
non-limiting example, one regulatory element is the Constitutive
Decay Elements (CDEs).
[0296] Immune cells specific microRNAs include, but are not limited
to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c,
hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p,
hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184,
hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p,
miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p,
miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p,
miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p,
miR-147a, miR-147b, miR-148a-5p, miR-148a-3p, miR-150-3p,
miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a-3p,
miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p, miR-16-2-3p,
miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p,
miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p,
miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,
miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,
miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p,
miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p,
miR-29b-1-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p,
miR-30e-5p, miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p,
miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, miR-363-3p,
miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p,
miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j,
miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p,
miR-99a-5p, miR-99b-3p and miR-99b-5p. Furthermore, novel miroRNAs
are discovered in the immune cells in the art through micro-array
hybridization and microtome analysis (Jima D D et al, Blood, 2010,
116:e118-el 27; Vaz C et al., BMC Genomics, 2010, 11,288, the
content of each of which is incorporated herein by reference in its
entirety.)
[0297] MicroRNAs that are known to be expressed in the liver
include, but are not limited to, miR-107, miR-122-3p, miR-122-5p,
miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303,
miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p,
miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p,
miR-557, miR-581, miR-939-3p, miR-939-5p. MicroRNA binding sites
from any liver specific microRNA can be introduced to or removed
from the polynucleotides to regulate the expression of the
polynucleotides in the liver. Liver specific microRNAs binding
sites can be engineered alone or further in combination with immune
cells (e.g. APCs) microRNA binding sites in order to prevent immune
reaction against protein expression in the liver.
[0298] MicroRNAs that are known to be expressed in the lung
include, but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p,
miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p,
miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134,
miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p,
miR-337-3p, miR-337-5p, miR-381-3p, miR-381-5p. MicroRNA binding
sites from any lung specific microRNA can be introduced to or
removed from the polynucleotide to regulate the expression of the
polynucleotide in the lung. Lung specific microRNAs binding sites
can be engineered alone or further in combination with immune cells
(e.g. APCs) microRNA binding sites in order to prevent an immune
reaction against protein expression in the lung.
[0299] MicroRNAs that are known to be expressed in the heart
include, but are not limited to, miR-1, miR-133a, miR-133b,
miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b,
miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p,
miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p,
miR-92b-3p and miR-92b-5p. MicroRNA binding sites from any heart
specific microRNA can be introduced to or removed from the
polynucleotides to regulate the expression of the polynucleotides
in the heart. Heart specific microRNAs binding sites can be
engineered alone or further in combination with immune cells (e.g.
APCs) microRNA binding sites to prevent an immune reaction against
protein expression in the heart.
[0300] MicroRNAs that are known to be expressed in the nervous
system include, but are not limited to, miR-124-5p, miR-125a-3p,
miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p,
miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p,
miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p,
miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,
miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,
miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,
miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,
miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329,
miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383,
miR-410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483,
miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571,
miR-7-1-3p, miR-7-2-3p, miR-7-5p, miR-802, miR-922, miR-9-3p and
miR-9-5p. MicroRNAs enriched in the nervous system further include
those specifically expressed in neurons, including, but not limited
to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p,
miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e,
miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328,
miR-922 and those specifically expressed in glial cells, including,
but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,
miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,
miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657. MicroRNA
binding sites from any CNS specific microRNA can be introduced to
or removed from the polynucleotides to regulate the expression of
the polynucleotide in the nervous system. Nervous system specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites in
order to prevent immune reaction against protein expression in the
nervous system.
[0301] MicroRNAs that are known to be expressed in the pancreas
include, but are not limited to, miR-105-3p, miR-105-5p, miR-184,
miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p,
miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p,
miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p
and miR-944. MicroRNA binding sites from any pancreas specific
microRNA can be introduced to or removed from the polynucleotide to
regulate the expression of the polynucleotide in the pancreas.
Pancreas specific microRNAs binding sites can be engineered alone
or further in combination with immune cells (e.g. APCs) microRNA
binding sites in order to prevent an immune reaction against
protein expression in the pancreas.
[0302] MicroRNAs that are known to be expressed in the kidney
further include, but are not limited to, miR-122-3p, miR-145-5p,
miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p,
miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210,
miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,
miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,
miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p and
miR-562. MicroRNA binding sites from any kidney specific microRNA
can be introduced to or removed from the polynucleotide to regulate
the expression of the polynucleotide in the kidney. Kidney specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites to
prevent an immune reaction against protein expression in the
kidney.
[0303] MicroRNAs that are known to be expressed in the muscle
further include, but are not limited to, let-7g-3p, let-7g-5p,
miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p,
miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p,
miR-206, miR-208a, miR-208b, miR-25-3p and miR-25-5p. MicroRNA
binding sites from any muscle specific microRNA can be introduced
to or removed from the polynucleotide to regulate the expression of
the polynucleotide in the muscle. Muscle specific microRNAs binding
sites can be engineered alone or further in combination with immune
cells (e.g. APCs) microRNA binding sites to prevent an immune
reaction against protein expression in the muscle.
[0304] MicroRNAs are differentially expressed in different types of
cells, such as endothelial cells, epithelial cells and adipocytes.
For example, microRNAs that are expressed in endothelial cells
include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p,
miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p,
miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p,
miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p,
miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p,
miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p,
miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p,
miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,
miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p and
miR-92b-5p. Many novel microRNAs are discovered in endothelial
cells from deep-sequencing analysis (Voellenkle C et al., RNA,
2012, 18, 472-484, herein incorporated by reference in its
entirety) microRNA binding sites from any endothelial cell specific
microRNA can be introduced to or removed from the polynucleotide to
modulate the expression of the polynucleotide in the endothelial
cells in various conditions.
[0305] For further example, microRNAs that are expressed in
epithelial cells include, but are not limited to, let-7b-3p,
let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p,
miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429,
miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p,
miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in
respiratory ciliated epithelial cells; let-7 family, miR-133a,
miR-133b, miR-126 specific in lung epithelial cells; miR-382-3p,
miR-382-5p specific in renal epithelial cells and miR-762 specific
in corneal epithelial cells. MicroRNA binding sites from any
epithelial cell specific MicroRNA can be introduced to or removed
from the polynucleotide to modulate the expression of the
polynucleotide in the epithelial cells in various conditions.
[0306] In addition, a large group of microRNAs are enriched in
embryonic stem cells, controlling stem cell self-renewal as well as
the development and/or differentiation of various cell lineages,
such as neural cells, cardiac, hematopoietic cells, skin cells,
osteogenic cells and muscle cells (Kuppusamy K T et al., Curr. Mol
Med, 2013, 13(5), 757-764; Vidigal J A and Ventura A, Semin Cancer
Biol. 2012, 22(5-6), 428-436; Goff L A et al., PLoS One, 2009,
4:e7192; Morin R D et al., Genome Res, 2008, 18, 610-621; Yoo J K
et al., Stem Cells Dev. 2012, 21(11), 2049-2057, each of which is
herein incorporated by reference in its entirety). MicroRNAs
abundant in embryonic stem cells include, but are not limited to,
let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,
miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,
miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,
miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p,
miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p,
miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e,
miR-367-3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371,
miR-373, miR-380-5p, miR-423-3p, miR-423-5p, miR-486-5p,
miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR-548g-5p,
miR-548i, miR-548k, miR-548l, miR-548m, miR-548n, miR-5480-3p,
miR-5480-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p,
miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p, miR-885-5p,
miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p, miR-99b-3p and
miR-99b-5p. Many predicted novel microRNAs are discovered by deep
sequencing in human embryonic stem cells (Morin R D et al., Genome
Res,2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;
Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each
of which is incorporated herein by references in its entirety).
[0307] In one embodiment, the binding sites of embryonic stem cell
specific microRNAs can be included in or removed from the 3-UTR of
the polynucleotide to modulate the development and/or
differentiation of embryonic stem cells, to inhibit the senescence
of stem cells in a degenerative condition (e.g. degenerative
diseases), or to stimulate the senescence and apoptosis of stem
cells in a disease condition (e.g. cancer stem cells).
[0308] Many microRNA expression studies are conducted in the art to
profile the differential expression of microRNAs in various cancer
cells/tissues and other diseases. Some microRNAs are abnormally
over-expressed in certain cancer cells and others are
under-expressed. For example, microRNAs are differentially
expressed in cancer cells (WO2008/154098, US2013/0059015,
US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);
pancreatic cancers and diseases (US2009/0131348, US2011/0171646,
US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation
(U.S. Pat. No. 8,415,096); prostate cancer (US2013/0053264);
hepatocellular carcinoma (WO2012/151212, US2012/0329672,
WO2008/054828, U.S. Pat. No. 8,252,538); lung cancer cells
(WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357);
cutaneous T cell lymphoma (WO2013/011378); colorectal cancer cells
(WO2011/0281756, WO2011/076142); cancer positive lymph nodes
(WO2009/100430, US2009/0263803); nasopharyngeal carcinoma (EP21
12235); chronic obstructive pulmonary disease (US2012/0264626,
US2013/0053263); thyroid cancer (WO2013/066678); ovarian cancer
cells US2012/0309645, WO2011/095623); breast cancer cells
(WO2008/154098, WO2007/081740, US2012/0214699), leukemia and
lymphoma (WO2008/073915, US2009/0092974, US2012/0316081,
US2012/0283310, WO2010/018563, the content of each of which is
incorporated herein by reference in its entirety.)
[0309] As a non-limiting example, microRNA sites that are
over-expressed in certain cancer and/or tumor cells can be removed
from the 3-UTR of the polynucleotide encoding the polypeptide of
interest, restoring the expression suppressed by the over-expressed
microRNAs in cancer cells, thus ameliorating the corresponsive
biological function, for instance, transcription stimulation and/or
repression, cell cycle arrest, apoptosis and cell death. Normal
cells and tissues, wherein microRNAs expression is not
up-regulated, will remain unaffected.
[0310] MicroRNA can also regulate complex biological processes such
as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011
18:171-176). In the alternative nucleic acids, enhanced alternative
RNA or ribonucleic acids of the invention, binding sites for
microRNAs that are involved in such processes may be removed or
introduced, in order to tailor the expression of the alternative
nucleic acids, enhanced alternative RNA or ribonucleic acids
expression to biologically relevant cell types or to the context of
relevant biological processes. In this context, the mRNA are
defined as auxotrophic mRNA.
[0311] MicroRNA gene regulation may be influenced by the sequence
surrounding the microRNA such as, but not limited to, the species
of the surrounding sequence, the type of sequence (e.g.,
heterologous, homologous and artificial), regulatory elements in
the surrounding sequence and/or structural elements in the
surrounding sequence. The microRNA may be influenced by the 5'-UTR
and/or the 3'-UTR. As a non-limiting example, a non-human 3'-UTR
may increase the regulatory effect of the microRNA sequence on the
expression of a polypeptide of interest compared to a human 3'-UTR
of the same sequence type.
[0312] In one embodiment, other regulatory elements and/or
structural elements of the 5'-UTR can influence microRNA mediated
gene regulation. One example of a regulatory element and/or
structural element is a structured IRES (Internal Ribosome Entry
Site) in the 5'-UTR, which is necessary for the binding of
translational elongation factors to initiate protein translation.
EIF4A2 binding to this secondarily structured element in the 5'-UTR
is necessary for microRNA mediated gene expression (Meijer H A et
al., Science, 2013, 340, 82-85, herein incorporated by reference in
its entirety). The alternative nucleic acids, enhanced alternative
RNA or ribonucleic acids of the invention can further be
alternative to include this structured 5'-UTR in order to enhance
microRNA mediated gene regulation.
[0313] At least one microRNA site can be engineered into the 3' UTR
of the alternative nucleic acids, enhanced alternative RNA or
ribonucleic acids of the present invention. In this context, at
least two, at least three, at least four, at least five, at least
six, at least seven, at least eight, at least nine, at least ten or
more microRNA sites may be engineered into the 3' UTR of the
ribonucleic acids of the present invention. In one embodiment, the
microRNA sites incorporated into the alternative nucleic acids,
enhanced alternative RNA or ribonucleic acids may be the same or
may be different microRNA sites. In another embodiment, the
microRNA sites incorporated into the alternative nucleic acids,
enhanced alternative RNA or ribonucleic acids may target the same
or different tissues in the body. As a non-limiting example,
through the introduction of tissue-, cell-type-, or
disease-specific microRNA binding sites in the 3' UTR of an
alternative nucleic acid mRNA, the degree of expression in specific
cell types (e.g. hepatocytes, myeloid cells, endothelial cells,
cancer cells, etc.) can be reduced.
[0314] In one embodiment, a microRNA site can be engineered near
the 5' terminus of the 3'-UTR, about halfway between the 5'
terminus and 3'terminus of the 3'-UTR and/or near the 3'terminus of
the 3'-UTR. As a non-limiting example, a microRNA site may be
engineered near the 5' terminus of the 3'-UTR and about halfway
between the 5' terminus and 3'terminus of the 3'-UTR. As another
non-limiting example, a microRNA site may be engineered near the
3'terminus of the 3'-UTR and about halfway between the 5' terminus
and 3'terminus of the 3'-UTR. As yet another non-limiting example,
a microRNA site may be engineered near the 5' terminus of the
3'-UTR and near the 3' terminus of the 3'-UTR.
[0315] In another embodiment, a 3'-UTR can comprise 4 microRNA
sites. The microRNA sites may be complete microRNA binding sites,
microRNA seed sequences and/or microRNA binding site sequences
without the seed sequence.
[0316] In one embodiment, a nucleic acid of the invention may be
engineered to include at least one microRNA in order to dampen the
antigen presentation by antigen presenting cells. The microRNA may
be the complete microRNA sequence, the microRNA seed sequence, the
microRNA sequence without the seed or a combination thereof. As a
non-limiting example, the microRNA incorporated into the nucleic
acid may be specific to the hematopoietic system. As another
non-limiting example, the microRNA incorporated into the nucleic
acid of the invention to dampen antigen presentation is
miR-142-3p.
[0317] In one embodiment, a nucleic acid may be engineered to
include microRNA sites which are expressed in different tissues of
a subject. As a non-limiting example, an alternative nucleic acid,
enhanced alternative RNA or ribonucleic acid of the present
invention may be engineered to include miR-192 and miR-122 to
regulate expression of the alternative nucleic acid, enhanced
alternative RNA or ribonucleic acid in the liver and kidneys of a
subject. In another embodiment, an alternative nucleic acid,
enhanced alternative RNA or ribonucleic acid may be engineered to
include more than one microRNA sites for the same tissue. For
example, an alternative nucleic acid, enhanced alternative RNA or
ribonucleic acid of the present invention may be engineered to
include miR-17-92 and miR-126 to regulate expression of the
alternative nucleic acid, enhanced alternative RNA or ribonucleic
acid in endothelial cells of a subject.
[0318] In one embodiment, the therapeutic window and or
differential expression associated with the target polypeptide
encoded by the alternative nucleic acid, enhanced alternative RNA
or ribonucleic acid encoding a signal (also referred to herein as a
polynucleotide) of the invention may be altered. For example,
polynucleotides may be designed whereby a death signal is more
highly expressed in cancer cells (or a survival signal in a normal
cell) by virtue of the miRNA signature of those cells. Where a
cancer cell expresses a lower level of a particular miRNA, the
polynucleotide encoding the binding site for that miRNA (or miRNAs)
would be more highly expressed. Hence, the target polypeptide
encoded by the polynucleotide is selected as a protein which
triggers or induces cell death. Neighboring noncancer cells,
harboring a higher expression of the same miRNA would be less
affected by the encoded death signal as the polynucleotide would be
expressed at a lower level due to the effects of the miRNA binding
to the binding site or "sensor" encoded in the 3'-UTR. Conversely,
cell survival or cytoprotective signals may be delivered to tissues
containing cancer and non-cancerous cells where a miRNA has a
higher expression in the cancer cells--the result being a lower
survival signal to the cancer cell and a larger survival signature
to the normal cell. Multiple polynucleotides may be designed and
administered having different signals according to the previous
paradigm.
[0319] In one embodiment, the expression of a nucleic acid may be
controlled by incorporating at least one sensor sequence in the
nucleic acid and formulating the nucleic acid. As a non-limiting
example, a nucleic acid may be targeted to an orthotopic tumor by
having a nucleic acid incorporating a miR-122 binding site and
formulated in a lipid nanoparticle comprising a cationic lipid such
as DLin-MC3-DMA.
[0320] According to the present invention, the polynucleotides may
be altered as to avoid the deficiencies of other
polypeptide-encoding molecules of the art. Hence, in this
embodiment the polynucleotides are referred to as alternative
polynucleotides.
[0321] Through an understanding of the expression patterns of
microRNA in different cell types, alternative nucleic acids,
enhanced alternative RNA or ribonucleic acids such as
polynucleotides can be engineered for more targeted expression in
specific cell types or only under specific biological conditions.
Through introduction of tissue-specific microRNA binding sites,
alternative nucleic acids, enhanced alternative RNA or ribonucleic
acids, could be designed that would be optimal for protein
expression in a tissue or in the context of a biological
condition.
[0322] Transfection experiments can be conducted in relevant cell
lines, using engineered alternative nucleic acids, enhanced
alternative RNA or ribonucleic acids and protein production can be
assayed at various time points post-transfection. For example,
cells can be transfected with different microRNA binding
site-engineering nucleic acids or mRNA and by using an ELISA kit to
the relevant protein and assaying protein produced at 6 hr, 12 hr,
24 hr, 48 hr, 72 hr and 7 days post-transfection. In vivo
experiments can also be conducted using microRNA-binding
site-engineered molecules to examine changes in tissue-specific
expression of formulated alternative nucleic acids, enhanced
alternative RNA or ribonucleic acids.
[0323] Non-limiting examples of cell lines which may be useful in
these investigations include those from ATCC (Manassas, Va.)
including MRC-5, A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688],
WI-38, WI-38 VA-13 subline 2RA, WI-26 VA4, C3A [HepG2/C3A,
derivative of Hep G2 (ATCC HB-8065)], THLE-3, H69AR, NCI-H292
[H292], CFPAC-1, NTERA-2 cI.D1 [NT2/D1], DMS 79, DMS 53, DMS 153,
DMS 114, MSTO-211H, SW 1573 [SW-1573, SW1573], SW 1271 [SW-1271,
SW1271], SHP-77, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387,
SNU-423, NL20, NL20-TA [NL20T-A], THLE-2, HBE135-E6E7, HCC827,
HCC4006, NCI-H23 [H23], NCI-H1299, NCI-H187 [H187], NCI-H358
[H-358, H358], NCI-H378 [H378], NCI-H522 [H522], NCI-H526 [H526],
NCI-H727 [H727], NCI-H810 [H810], NCI-H889 [H889], NCI-H1155
[H1155], NCI-H1404 [H1404], NCI-N87 [N87], NCI-H196 [H196],
NCI-H211 [H211], NCI-H220 [H220], NCI-H250 [H250], NCI-H524 [H524],
NCI-H647 [H647], NCI-H650 [H650], NCI-H711 [H711], NCI-H719 [H719],
NCI-H740 [H740], NCI-H748 [H748], NCI-H774 [H774], NCI-H838 [H838],
NCI-H841 [H841], NCI-H847 [H847], NCI-H865 [H865], NCI-H920 [H920],
NCI-H1048 [H1048], NCI-H1092 [H1092], NCI-H1105 [H1105], NCI-H1184
[H1184], NCI-H1238 [H1238], NCI-H1341 [H1341], NCI-H1385 [H1385],
NCI-H1417 [H1417], NCI-H1435 [H1435], NCI-H1436 [H1436], NCI-H1437
[H1437], NCI-H1522 [H1522], NCI-H1563 [H1563], NCI-H1568 [H1568],
NCI-H1573 [H1573], NCI-H1581 [H1581], NCI-H1618 [H1618], NCI-H1623
[H1623], NCI-H1650 [H-1650, H1650], NCI-H1651 [H1651], NCI-H1666
[H-1666, H1666], NCI-H1672 [H1672], NCI-H1693 [H1693], NCI-H1694
[H1694], NCI-H1703 [H1703], NCI-H1734 [H-1734, H1734], NCI-H1755
[H1755], NCI-H1755 [H1755], NCI-H1770 [H1770], NCI-H1793 [H1793],
NCI-H1836 [H1836], NCI-H1838 [H1838], NCI-H1869 [H1869], NCI-H1876
[H1876], NCI-H1882 [H1882], NCI-H1915 [H1915], NCI-H1930 [H1930],
NCI-H1944 [H1944], NCI-H1975 [H-1975, H1975], NCI-H1993 [H1993],
NCI-H2023 [H2023], NCI-H2029 [H2029], NCI-H2030 [H2030], NCI-H2066
[H2066], NCI-H2073 [H2073], NCI-H2081 [H2081], NCI-H2085 [H2085],
NCI-H2087 [H2087], NCI-H2106 [H2106], NCI-H2110 [H2110], NCI-H2135
[H2135], NCI-H2141 [H2141], NCI-H2171 [H2171], NCI-H2172 [H2172],
NCI-H2195 [H2195], NCI-H2196 [H2196], NCI-H2198 [H2198], NCI-H2227
[H2227], NCI-H2228 [H2228], NCI-H2286 [H2286], NCI-H2291 [H2291],
NCI-H2330 [H2330], NCI-H2342 [H2342], NCI-H2347 [H2347], NCI-H2405
[H2405], NCI-H2444 [H2444], UMC-11, NCI-H64 [H64], NCI-H735 [H735],
NCI-H735 [H735], NCI-H1963 [H1963], NCI-H2107 [H2107], NCI-H2108
[H2108], NCI-H2122 [H2122], Hs 573.T, Hs 573.Lu, PLC/PRF/5,
BEAS-2B, Hep G2, Tera-1, Tera-2, NCI-H69 [H69], NCI-H128 [H128],
ChaGo-K-1, NCI-H446 [H446], NCI-H209 [H209], NCI-H146 [H146],
NCI-H441 [H441], NCI-H82 [H82], NCI-H460 [H460], NCI-H596 [H596],
NCI-H676B [H67613], NCI-H345 [H345], NCI-H820 [H820], NCI-H520
[H520], NCI-H661 [H661], NCI-H510A [H510A, NCI-H510], SK-HEP-1,
A-427, Calu-1, Calu-3, Calu-6, SK-LU-1, SK-MES-1, SW 900 [SW-900,
SW900], Malme-3M, and Capan-1.
[0324] In some embodiments, alternative messenger RNA can be
designed to incorporate microRNA binding region sites that either
have 100% identity to known seed sequences or have less than 100%
identity to seed sequences. The seed sequence can be partially
mutated to decrease microRNA binding affinity and as such result in
reduced downmodulation of that mRNA transcript. In essence, the
degree of match or mis-match between the target mRNA and the
microRNA seed can act as a rheostat to more finely tune the ability
of the microRNA to modulate protein expression. In addition,
mutation in the non-seed region of a microRNA binding site may also
impact the ability of a microRNA to modulate protein
expression.
[0325] In one embodiment, a miR sequence may be incorporated into
the loop of a stem loop.
[0326] In another embodiment, a miR seed sequence may be
incorporated in the loop of a stem loop and a miR binding site may
be incorporated into the 5' or 3' stem of the stem loop.
[0327] In one embodiment, a TEE may be incorporated on the 5'end of
the stem of a stem loop and a miR seed may be incorporated into the
stem of the stem loop. In another embodiment, a TEE may be
incorporated on the 5'end of the stem of a stem loop, a miR seed
may be incorporated into the stem of the stem loop and a miR
binding site may be incorporated into the 3'end of the stem or the
sequence after the stem loop. The miR seed and the miR binding site
may be for the same and/or different miR sequences.
[0328] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or decrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'-UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010,
incorporated herein by reference in its entirety).
[0329] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or decrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'-UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010,
incorporated herein by reference in its entirety).
[0330] In one embodiment, the 5'-UTR may comprise at least one
microRNA sequence. The microRNA sequence may be, but is not limited
to, a 19 or 22 nucleotide sequence and/or a microRNA sequence
without the seed.
[0331] In one embodiment the microRNA sequence in the 5'-UTR may be
used to stabilize the nucleic acid and/or mRNA described
herein.
[0332] In another embodiment, a microRNA sequence in the 5'-UTR may
be used to decrease the accessibility of the site of translation
initiation such as, but not limited to a start codon. Matsuda et al
(PLoS One. 2010 11(5):e15057; incorporated herein by reference in
its entirety) used antisense locked nucleic acid (LNA)
oligonucleotides and exon-junction complexes (EJCs) around a start
codon (-4 to +37 where the A of the AUG codons is +1) in order to
decrease the accessibility to the first start codon (AUG). Matsuda
showed that altering the sequence around the start codon with an
LNA or EJC the efficiency, length and structural stability of the
nucleic acid or mRNA is affected. The nucleic acids or mRNA of the
present invention may comprise a microRNA sequence, instead of the
LNA or EJC sequence described by Matsuda et al, near the site of
translation initiation in order to decrease the accessibility to
the site of translation initiation. The site of translation
initiation may be prior to, after or within the microRNA sequence.
As a non-limiting example, the site of translation initiation may
be located within a microRNA sequence such as a seed sequence or
binding site. As another non-limiting example, the site of
translation initiation may be located within a miR-122 sequence
such as the seed sequence or the mir-122 binding site.
[0333] In one embodiment, the nucleic acids or mRNA of the present
invention may include at least one microRNA in order to dampen the
antigen presentation by antigen presenting cells. The microRNA may
be the complete microRNA sequence, the microRNA seed sequence, the
microRNA sequence without the seed or a combination thereof. As a
non-limiting example, the microRNA incorporated into the nucleic
acids or mRNA of the present invention may be specific to the
hematopoietic system. As another non-limiting example, the microRNA
incorporated into the nucleic acids or mRNA of the present
invention to dampen antigen presentation is miR-142-3p.
[0334] In one embodiment, the nucleic acids or mRNA of the present
invention may include at least one microRNA in order to dampen
expression of the encoded polypeptide in a cell of interest. As a
non-limiting example, the nucleic acids or mRNA of the present
invention may include at least one miR-122 binding site in order to
dampen expression of an encoded polypeptide of interest in the
liver. As another non-limiting example, the nucleic acids or mRNA
of the present invention may include at least one miR-142-3p
binding site, miR-142-3p seed sequence, miR-142-3p binding site
without the seed, miR-142-5p binding site, miR-142-5p seed
sequence, miR-142-5p binding site without the seed, miR-146 binding
site, miR-146 seed sequence and/or miR-146 binding site without the
seed sequence.
[0335] In one embodiment, the nucleic acids or mRNA of the present
invention may comprise at least one microRNA binding site in the
3'-UTR in order to selectively degrade mRNA therapeutics in the
immune cells to subdue unwanted immunogenic reactions caused by
therapeutic delivery. As a non-limiting example, the microRNA
binding site may be the alternative nucleic acids more unstable in
antigen presenting cells. Non-limiting examples of these microRNA
include mir-142-5p, mir-142-3p, mir-146a-5p and mir-146-3p.
[0336] In one embodiment, the nucleic acids or mRNA of the present
invention comprises at least one microRNA sequence in a region of
the nucleic acid or mRNA which may interact with a RNA binding
protein.
[0337] RNA Motifs for RNA Binding Proteins (RBPs)
[0338] RNA binding proteins (RBPs) can regulate numerous aspects of
co- and post-transcription gene expression such as, but not limited
to, RNA splicing, localization, translation, turnover,
polyadenylation, capping, alteration, export and localization.
RNA-binding domains (RBDs), such as, but not limited to, RNA
recognition motif (RR) and hnRNP K-homology (KH) domains, typically
regulate the sequence association between RBPs and their RNA
targets (Ray et al. Nature 2013. 499:172-177; incorporated herein
by reference in its entirety). In one embodiment, the canonical
RBDs can bind short RNA sequences. In another embodiment, the
canonical RBDs can recognize structure RNAs.
[0339] In one embodiment, to increase the stability of the mRNA of
interest, an mRNA encoding HuR can be co-transfected or co-injected
along with the mRNA of interest into the cells or into the tissue.
These proteins can also be tethered to the mRNA of interest in
vitro and then administered to the cells together. Poly A tail
binding protein, PABP interacts with eukaryotic translation
initiation factor elF4G to stimulate translational initiation.
Co-administration of mRNAs encoding these RBPs along with the mRNA
drug and/or tethering these proteins to the mRNA drug in vitro and
administering the protein-bound mRNA into the cells can increase
the translational efficiency of the mRNA. The same concept can be
extended to co-administration of mRNA along with mRNAs encoding
various translation factors and facilitators as well as with the
proteins themselves to influence RNA stability and/or translational
efficiency.
[0340] In one embodiment, the nucleic acids and/or mRNA may
comprise at least one RNA-binding motif such as, but not limited to
a RNA-binding domain (RBD).
[0341] In one embodiment, the RBD may be any of the RBDs, fragments
or variants thereof descried by Ray et al. (Nature 2013.
499:172-177; incorporated herein by reference in its entirety).
[0342] In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a sequence for at least one RNA-binding
domain (RBDs). When the nucleic acids or mRNA of the present
invention comprise more than one RBD, the RBDs do not need to be
from the same species or even the same structural class.
[0343] In one embodiment, at least one flanking region (e.g., the
5'-UTR and/or the 3'-UTR) may comprise at least one RBD. In another
embodiment, the first flanking region and the second flanking
region may both comprise at least one RBD. The RBD may be the same
or each of the RBDs may have at least 60% sequence identity to the
other RBD. As a non-limiting example, at least on RBD may be
located before, after and/or within the 3'-UTR of the nucleic acid
or mRNA of the present invention. As another non-limiting example,
at least one RBD may be located before or within the first 300
nucleosides of the 3'-UTR.
[0344] In another embodiment, the nucleic acids and/or mRNA of the
present invention may comprise at least one RBD in the first region
of linked nucleosides. The RBD may be located before, after or
within a coding region (e.g., the ORF).
[0345] In yet another embodiment, the first region of linked
nucleosides and/or at least one flanking region may comprise at
least on RBD. As a non-limiting example, the first region of linked
nucleosides may comprise a RBD related to splicing factors and at
least one flanking region may comprise a RBD for stability and/or
translation factors.
[0346] In one embodiment, the nucleic acids and/or mRNA of the
present invention may comprise at least one RBD located in a coding
and/or non-coding region of the nucleic acids and/or mRNA.
[0347] In one embodiment, at least one RBD may be incorporated into
at least one flanking region to increase the stability of the
nucleic acid and/or mRNA of the present invention.
[0348] In one embodiment, a microRNA sequence in a RNA binding
protein motif may be used to decrease the accessibility of the site
of translation initiation such as, but not limited to a start
codon. The nucleic acids or mRNA of the present invention may
comprise a microRNA sequence, instead of the LNA or EJC sequence
described by Matsuda et al, near the site of translation initiation
in order to decrease the accessibility to the site of translation
initiation. The site of translation initiation may be prior to,
after or within the microRNA sequence. As a non-limiting example,
the site of translation initiation may be located within a microRNA
sequence such as a seed sequence or binding site. As another
non-limiting example, the site of translation initiation may be
located within a miR-122 sequence such as the seed sequence or the
mir-122 binding site.
[0349] In another embodiment, an antisense locked nucleic acid
(LNA) oligonucleotides and exon-junction complexes (EJCs) may be
used in the RNA binding protein motif. The LNA and EJCs may be used
around a start codon (-4 to +37 where the A of the AUG codons is
+1) in order to decrease the accessibility to the first start codon
(AUG).
[0350] Codon Optimization
[0351] The polynucleotides of the invention, their regions or parts
or subregions may be codon optimized. Codon optimization methods
are known in the art and may be useful in efforts to achieve one or
more of several goals. These goals include to match codon
frequencies in target and host organisms to ensure proper folding,
bias GC content to increase mRNA stability or reduce secondary
structures, minimize tandem repeat codons or base runs that may
impair gene construction or expression, customize transcriptional
and translational control regions, insert or remove protein
trafficking sequences, remove/add post translation modification
sites in encoded protein (e.g., glycosylation sites), add, remove
or shuffle protein domains, insert or delete restriction sites,
modify ribosome binding sites and mRNA degradation sites, to adjust
translational rates to allow the various domains of the protein to
fold properly, or to reduce or eliminate problem secondary
structures within the polynucleotide. Codon optimization tools,
algorithms and services are known in the art, non-limiting examples
include services from GeneArt (Life Technologies), DNA2.0 (Menlo
Park Calif.) and/or proprietary methods. In one embodiment, the ORF
sequence is optimized using optimization algorithms. Codon options
for each amino acid are given in Table 9.
TABLE-US-00005 TABLE 9 Codon Options. Single Letter Amino Acid Code
Codon Options Isoleucine I AUU, AUC, AUA Leucine L CUU, CUC, CUA,
CUG, UUA, UUG Valine V GUU, GUC, GUA, GUG Phenylalanine F UUU, UUC
Methionine M AUG Cysteine C UGU, UGC Alanine A GCU, GCC, GCA, GCG
Glycine G GGU, GGC, GGA, GGG Proline P CCU, CCC, CCA, CCG Threonine
T ACU, ACC, ACA, ACG Serine S UCU, UCC, UCA, UCG, AGU, AGC Tyrosine
Y UAU, UAC Tryptophan W UGG Glutamine Q CAA, CAG Asparagine N AAU,
AAC Histidine H CAU, CAC Glutamic acid E GAA, GAG Aspartic acid D
GAU, GAC Lysine K AAA, AAG Arginine R CGU, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocystein
insertion element (SECIS) Stop codons Stop UAA, UAG, UGA
[0352] "Codon optimized" refers to the modification of a starting
nucleotide sequence by replacing at least one codon of the starting
nucleotide sequence with another codon encoding the same amino acid
(e.g., to increase in vivo expression). Table 10 contains the codon
usage frequency for humans (Codon usage database:
[[www.]]kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606&aa=1&style=-
N).
TABLE-US-00006 TABLE 10 Codon usage frequency table for humans.
Amino Amino Amino Amino Codon Acid % Codon Acid % Codon Acid %
Codon Acid % UUU F (2) 46 UCU S (3) 19 UAU Y (2) 44 UGU C (2) 46
UUC F (1) 54 UCC S (2) 22 UAC Y (1) 56 UGC C (1) 54 UUA L (5) 8 UCA
S (4) 15 UAA * 30 UGA * 47 UUG L (4) 13 UCG S (6) 5 UAG * 24 UGG W
(1) 100 CUU L (3) 13 CCU P (2) 29 CAU H (2) 42 CGU R (6) 8 CUC L
(2) 20 CCC P (1) 32 CAC H (1) 58 CGC R (4) 18 CUA L (6) 7 CCA P (3)
28 CAA Q (2) 27 CGA R (5) 11 CUG L (1) 40 CCG P (4) 11 CAG Q (1) 73
CGG R (3) 20 AUU I (2) 36 ACU T (3) 25 AAU N (2) 47 AGU S (5) 15
AUC I (1) 47 ACC T (1) 36 AAC N (1) 53 AGC S (1) 24 AUA I (3) 17
ACA T (2) 28 AAA K (2) 43 AGA R (2) 21 AUG M (1) 100 ACG T (4) 11
AAG K (1) 57 AGG R (1) 21 GUU V (3) 18 GCU A (2) 27 GAU D (2) 46
GGU G (4) 16 GUC V (2) 24 GCC A (1) 40 GAC D (1) 54 GGC G (1) 34
GUA V (4) 12 GCA A (3) 23 GAA E (2) 42 GGA G (2) 25 GUG V (1) 46
GCG A (4) 11 GAG E (1) 58 GGG G (3) 25
[0353] In Table 10, the number in parentheses after the one letter
amino acid code indicates the frequency of that codon relative to
other codons encoding the same amino acid, where "1" is the highest
frequency and higher integers indicate less frequent codons.
[0354] A guanine maximized codon is a codon having the highest
number of guanines possible for a specified amino acid. A cytosine
maximized codon is a codon having the highest number of cytosines
possible for a specified amino acid. A guanine/cytosine maximized
codon refers to a codon having the highest number of guanines,
cytosines, or combination of guanines and cytosines for a specified
amino acid. When two or more codons have the same number of
guanines, cytosines, or combination thereof for a specified amino
acid, a low frequency maximized codon is a codon that is not the
highest frequency codon.
[0355] In one embodiment, after a nucleotide sequence has been
codon optimized it may be further evaluated for regions containing
restriction sites. At least one nucleotide within the restriction
site regions may be replaced with another nucleotide in order to
remove the restriction site from the sequence, but the replacement
of nucleotides does not alter the amino acid sequence which is
encoded by the codon optimized nucleotide sequence.
[0356] Features, which may be considered beneficial in some
embodiments of the present invention, may be encoded by regions of
the polynucleotide and such regions may be upstream (5') or
downstream (3') to a region which encodes a polypeptide. These
regions may be incorporated into the polynucleotide before and/or
after codon optimization of the protein encoding region or open
reading frame (ORF). It is not required that a polynucleotide
contain both a 5' and 3' flanking region. Examples of such features
include, but are not limited to, untranslated regions (UTRs), Kozak
sequences, an oligo(dT) sequence, and detectable tags and may
include multiple cloning sites which may have XbaI recognition.
[0357] In some embodiments, a 5' UTR and/or a 3' UTR region may be
provided as flanking regions. Multiple 5' or 3' UTRs may be
included in the flanking regions and may be the same or of
different sequences. Any portion of the flanking regions, including
none, may be codon optimized and any may independently contain one
or more different structural or chemical alterations, before and/or
after codon optimization.
[0358] After optimization (if desired), the polynucleotides
components are reconstituted and transformed into a vector such as,
but not limited to, plasmids, viruses, cosmids, and artificial
chromosomes. For example, the optimized polynucleotide may be
reconstituted and transformed into chemically competent E. coli,
yeast, neurospora, maize, drosophila, etc. where high copy
plasmid-like or chromosome structures occur by methods described
herein.
[0359] Uses of Alternative Nucleic Acids
[0360] Therapeutic Agents
[0361] The alternative nucleic acids described herein can be used
as therapeutic agents. For example, an alternative nucleic acid
described herein can be administered to an animal or subject,
wherein the alternative nucleic acid is translated in vivo to
produce a therapeutic peptide in the animal or subject.
Accordingly, provided herein are mRNA, compositions (such as
pharmaceutical compositions), methods, kits, and reagents for
treatment or prevention of disease or conditions in humans and
other mammals. The active therapeutic agents of the present
disclosure include alternative nucleic acids, cells containing
alternative nucleic acids or polypeptides translated from the
alternative nucleic acids, polypeptides translated from alternative
nucleic acids, cells contacted with cells containing alternative
nucleic acids or polypeptides translated from the alternative
nucleic acids, tissues containing cells containing alternative
nucleic acids and organs containing tissues containing cells
containing alternative nucleic acids.
[0362] Provided are methods of inducing translation of a synthetic
or recombinant polynucleotide to produce a polypeptide in a cell
population using the alternative nucleic acids described herein.
Such translation can be in vivo, ex vivo, in culture, or in vitro.
The cell population is contacted with an effective amount of a
composition containing a nucleic acid that has at least one
nucleoside alteration, and a translatable region encoding the
polypeptide. The population is contacted under conditions such that
the nucleic acid is localized into one or more cells of the cell
population and the recombinant polypeptide is translated in the
cell from the nucleic acid.
[0363] An effective amount of the composition is provided based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the nucleic acid (e.g.,
size, and extent of alternative nucleosides), and other
determinants. In general, an effective amount of the composition
provides efficient protein production in the cell, preferably more
efficient than a composition containing a corresponding unaltered
nucleic acid. Increased efficiency may be demonstrated by increased
cell transfection (i.e., the percentage of cells transfected with
the nucleic acid), increased protein translation from the nucleic
acid, decreased nucleic acid degradation (as demonstrated, e.g., by
increased duration of protein translation from a modified nucleic
acid), or reduced innate immune response of the host cell or
improve therapeutic utility.
[0364] Aspects of the present disclosure are directed to methods of
inducing in vivo translation of a recombinant polypeptide in a
mammalian subject in need thereof. Therein, an effective amount of
a composition containing a nucleic acid that has at least one
nucleoside alteration and a translatable region encoding the
polypeptide is administered to the subject using the delivery
methods described herein. The nucleic acid is provided in an amount
and under other conditions such that the nucleic acid is localized
into a cell or cells of the subject and the recombinant polypeptide
is translated in the cell from the nucleic acid. The cell in which
the nucleic acid is localized, or the tissue in which the cell is
present, may be targeted with one or more than one rounds of
nucleic acid administration.
[0365] Other aspects of the present disclosure relate to
transplantation of cells containing alternative nucleic acids to a
mammalian subject. Administration of cells to mammalian subjects is
known to those of ordinary skill in the art, such as local
implantation (e.g., topical or subcutaneous administration), organ
delivery or systemic injection (e.g., intravenous injection or
inhalation), as is the formulation of cells in pharmaceutically
acceptable carrier. Compositions containing alternative nucleic
acids are formulated for administration intramuscularly,
transarterially, intraperitoneally, intravenously, intranasally,
subcutaneously, endoscopically, transdermally, or intrathecally. In
some embodiments, the composition is formulated for extended
release.
[0366] The subject to whom the therapeutic agent is administered
suffers from or is at risk of developing a disease, disorder, or
deleterious condition. Provided are methods of identifying,
diagnosing, and classifying subjects on these bases, which may
include clinical diagnosis, biomarker levels, genome-wide
association studies (GWAS), and other methods known in the art.
[0367] In certain embodiments, the administered alternative nucleic
acid directs production of one or more recombinant polypeptides
that provide a functional activity which is substantially absent in
the cell in which the recombinant polypeptide is translated. For
example, the missing functional activity may be enzymatic,
structural, or gene regulatory in nature.
[0368] In other embodiments, the administered alternative nucleic
acid directs production of one or more recombinant polypeptides
that replace a polypeptide (or multiple polypeptides) that is
substantially absent in the cell in which the recombinant
polypeptide is translated. Such absence may be due to genetic
mutation of the encoding gene or regulatory pathway thereof. In
other embodiments, the administered alternative nucleic acid
directs production of one or more recombinant polypeptides to
supplement the amount of polypeptide (or multiple polypeptides)
that is present in the cell in which the recombinant polypeptide is
translated. Alternatively, the recombinant polypeptide functions to
antagonize the activity of an endogenous protein present in, on the
surface of, or secreted from the cell. Usually, the activity of the
endogenous protein is deleterious to the subject, for example, due
to mutation of the endogenous protein resulting in altered activity
or localization. Additionally, the recombinant polypeptide
antagonizes, directly or indirectly, the activity of a biological
moiety present in, on the surface of, or secreted from the cell.
Examples of antagonized biological moieties include lipids (e.g.,
cholesterol), a lipoprotein (e.g., low density lipoprotein), a
nucleic acid, a carbohydrate, or a small molecule toxin.
[0369] The recombinant proteins described herein are engineered for
localization within the cell, potentially within a specific
compartment such as the nucleus, or are engineered for secretion
from the cell or translocation to the plasma membrane of the
cell.
[0370] As described herein, a useful feature of the alternative
nucleic acids of the present disclosure is the capacity to reduce,
evade, avoid or eliminate the innate immune response of a cell to
an exogenous nucleic acid. Provided are methods for performing the
titration, reduction or elimination of the immune response in a
cell or a population of cells. In some embodiments, the cell is
contacted with a first composition that contains a first dose of a
first exogenous nucleic acid including a translatable region and at
least one nucleoside alteration, and the level of the innate immune
response of the cell to the first exogenous nucleic acid is
determined. Subsequently, the cell is contacted with a second
composition, which includes a second dose of the first exogenous
nucleic acid, the second dose containing a lesser amount of the
first exogenous nucleic acid as compared to the first dose.
Alternatively, the cell is contacted with a first dose of a second
exogenous nucleic acid. The second exogenous nucleic acid may
contain one or more alternative nucleosides, which may be the same
or different from the first exogenous nucleic acid or,
alternatively, the second exogenous nucleic acid may not contain
alternative nucleosides. The steps of contacting the cell with the
first composition and/or the second composition may be repeated one
or more times. Additionally, efficiency of protein production
(e.g., protein translation) in the cell is optionally determined,
and the cell may be re-transfected with the first and/or second
composition repeatedly until a target protein production efficiency
is achieved.
[0371] Therapeutics for Diseases and Conditions
[0372] Provided are methods for treating or preventing a symptom of
diseases characterized by missing or aberrant protein activity, by
replacing the missing protein activity or overcoming the aberrant
protein activity. Because of the rapid initiation of protein
production following introduction of alternative mRNAs, as compared
to viral DNA vectors, the compounds of the present disclosure are
particularly advantageous in treating acute diseases such as
sepsis, stroke, and myocardial infarction. Moreover, the lack of
transcriptional regulation of the alternative mRNAs of the present
disclosure is advantageous in that accurate titration of protein
production is achievable. Multiple diseases are characterized by
missing (or substantially diminished such that proper protein
function does not occur) protein activity. Such proteins may not be
present, are present in very low quantities or are essentially
non-functional. The present disclosure provides a method for
treating such conditions or diseases in a subject by introducing
nucleic acid or cell-based therapeutics containing the alternative
nucleic acids provided herein, wherein the alternative nucleic
acids encode for a protein that replaces the protein activity
missing from the target cells of the subject.
[0373] Diseases characterized by dysfunctional or aberrant protein
activity include, but not limited to, cancer and proliferative
diseases, genetic diseases (e.g., cystic fibrosis), autoimmune
diseases, diabetes, neurodegenerative diseases, cardiovascular
diseases, and metabolic diseases. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing nucleic acid or cell-based therapeutics containing the
alternative nucleic acids provided herein, wherein the alternative
nucleic acids encode for a protein that antagonizes or otherwise
overcomes the aberrant protein activity present in the cell of the
subject.
[0374] Specific examples of a dysfunctional protein are the
missense or nonsense mutation variants of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which produce a
dysfunctional or nonfunctional, respectively, protein variant of
CFTR protein, which causes cystic fibrosis.
[0375] Thus, provided are methods of treating cystic fibrosis in a
mammalian subject by contacting a cell of the subject with an
alternative nucleic acid having a translatable region that encodes
a functional CFTR polypeptide, under conditions such that an
effective amount of the CTFR polypeptide is present in the cell.
Preferred target cells are epithelial cells, such as the lung, and
methods of administration are determined in view of the target
tissue; i.e., for lung delivery, the RNA molecules are formulated
for administration by inhalation. Therefore, in certain
embodiments, the polypeptide of interest encoded by the mRNA of the
invention is the CTFR polypeptide and the mRNA or pharmaceutical
composition of the invention is for use in treating cystic
fibrosis.
[0376] In another embodiment, the present disclosure provides a
method for treating hyperlipidemia in a subject, by introducing
into a cell population of the subject with an alternative mRNA
molecule encoding Sortilin, a protein recently characterized by
genomic studies, thereby ameliorating the hyperlipidemia in a
subject. The SORT1 gene encodes a trans-Golgi network (TGN)
transmembrane protein called Sortilin. Genetic studies have shown
that one of five individuals has a single nucleotide polymorphism,
rs12740374, in the 1p13 locus of the SORT1 gene that predisposes
them to having low levels of low-density lipoprotein (LDL) and
very-low-density lipoprotein (VLDL). Each copy of the minor allele,
present in about 30% of people, alters LDL cholesterol by 8 mg/dL,
while two copies of the minor allele, present in about 5% of the
population, lowers LDL cholesterol 16 mg/dL. Carriers of the minor
allele have also been shown to have a 40% decreased risk of
myocardial infarction. Functional in vivo studies in mice describes
that overexpression of SORT1 in mouse liver tissue led to
significantly lower LDL-cholesterol levels, as much as 80% lower,
and that silencing SORT1 increased LDL cholesterol approximately
200% (Musunuru K et al. From noncoding variant to phenotype via
SORT1 at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).
Therefore, in certain embodiments, the polypeptide of interest
encoded by the mRNA of the invention is Sortilin and the mRNA or
pharmaceutical composition of the invention is for use in treating
hyperlipidemia.
[0377] In certain embodiments, the polypeptide of interest encoded
by the mRNA of the invention is granulocyte colony-stimulating
factor (GCSF), and the mRNA or pharmaceutical composition of the
invention is for use in treating a neurological disease such as
cerebral ischemia, or treating neutropenia, or for use in
increasing the number of hematopoietic stem cells in the blood
(e.g. before collection by leukapheresis for use in hematopoietic
stem cell transplantation).
[0378] In certain embodiments, the polypeptide of interest encoded
by the mRNA of the invention is erythropoietin (EPO), and the mRNA
or pharmaceutical composition of the invention is for use in
treating anemia, inflammatory bowel disease (such as Crohn's
disease and/or ulcer colitis) or myelodysplasia.
[0379] Methods of Cellular Nucleic Acid Delivery
[0380] Methods of the present disclosure enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside alteration and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unaltered nucleic acid. The
retention of the enhanced nucleic acid is greater than the
retention of the unaltered nucleic acid. In some embodiments, it is
at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than
200% greater than the retention of the unaltered nucleic acid. Such
retention advantage may be achieved by one round of transfection
with the enhanced nucleic acid, or may be obtained following
repeated rounds of transfection.
[0381] In some embodiments, the enhanced nucleic acid is delivered
to a target cell population with one or more additional nucleic
acids. Such delivery may be at the same time, or the enhanced
nucleic acid is delivered prior to delivery of the one or more
additional nucleic acids. The additional one or more nucleic acids
may be alternative nucleic acids or unaltered nucleic acids. It is
understood that the initial presence of the enhanced nucleic acids
does not substantially induce an innate immune response of the cell
population and, moreover, that the innate immune response will not
be activated by the later presence of the unaltered nucleic acids.
In this regard, the enhanced nucleic acid may not itself contain a
translatable region, if the protein desired to be present in the
target cell population is translated from the unaltered nucleic
acids.
[0382] Targeting Moieties
[0383] In embodiments of the present disclosure, alternative
nucleic acids are provided to express a protein-binding partner or
a receptor on the surface of the cell, which functions to target
the cell to a specific tissue space or to interact with a specific
moiety, either in vivo or in vitro. Suitable protein-binding
partners include antibodies and functional fragments thereof,
scaffold proteins, or peptides. Additionally, alternative nucleic
acids can be employed to direct the synthesis and extracellular
localization of lipids, carbohydrates, or other biological
moieties.
[0384] Permanent Gene Expression Silencing
[0385] A method for epigenetically silencing gene expression in a
mammalian subject, comprising a nucleic acid where the translatable
region encodes a polypeptide or polypeptides capable of directing
sequence-specific histone H3 methylation to initiate
heterochromatin formation and reduce gene transcription around
specific genes for the purpose of silencing the gene. For example,
a gain-of-function mutation in the Janus Kinase 2 gene is
responsible for the family of Myeloproliferative Diseases.
[0386] Pharmaceutical Compositions
[0387] Pharmaceutical compositions may optionally comprise one or
more additional therapeutically active substances. In accordance
with some embodiments, a method of administering pharmaceutical
compositions comprising an alternative nucleic acid encoding one or
more proteins to be delivered to a subject in need thereof is
provided. In some embodiments, compositions are administered to
humans
[0388] 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 animals of all sorts.
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 chickens, ducks, geese, and/or turkeys.
[0389] 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, shaping and/or packaging
the product into a desired single- or multi-dose unit.
[0390] A pharmaceutical composition in accordance with the present
disclosure 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.
[0391] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure 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% (w/w)
active ingredient.
[0392] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes 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, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional excipient
medium is 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, its use is contemplated to be
within the scope of this present disclosure.
[0393] In some embodiments, a pharmaceutically acceptable excipient
is at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% pure. In some embodiments, an excipient is approved
for use in humans and for veterinary use. In some embodiments, an
excipient is approved by United States Food and Drug
Administration. In some embodiments, an excipient is pharmaceutical
grade. In some embodiments, an excipient meets the standards of the
United States Pharmacopoeia (USP), the European Pharmacopoeia (EP),
the British Pharmacopoeia, and/or the International
Pharmacopoeia.
[0394] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, dispersing and/or granulating agents,
surface active agents and/or emulsifiers, disintegrating agents,
binding agents, preservatives, buffering agents, lubricating
agents, and/or oils. Such excipients may optionally be included in
pharmaceutical formulations. Excipients such as cocoa butter and
suppository waxes, coloring agents, coating agents, sweetening,
flavoring, and/or perfuming agents can be present in the
composition, according to the judgment of the formulator.
[0395] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium carbonate, calcium phosphate, dicalcium
phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose, cellulose, microcrystalline cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch,
cornstarch, powdered sugar, etc., and/or combinations thereof.
[0396] Exemplary granulating and/or dispersing agents include, but
are not limited to, potato starch, corn starch, tapioca starch,
sodium starch glycolate, clays, alginic acid, guar gum, citrus
pulp, agar, bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds,
etc., and/or combinations thereof.
[0397] Exemplary surface active agents and/or emulsifiers include,
but are not limited to, natural emulsifiers (e.g. acacia, agar,
alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,
xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol,
wax, and lecithin), colloidal clays (e.g. bentonite [aluminum
silicate] and Veegum.RTM. [magnesium aluminum silicate]), long
chain amino acid derivatives, high molecular weight alcohols (e.g.
stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate, ethylene glycol distearate, glyceryl monostearate,
and propylene glycol monostearate, polyvinyl alcohol), carbomers
(e.g. carboxy polymethylene, polyacrylic acid, acrylic acid
polymer, and carboxyvinyl polymer), carrageenan, cellulosic
derivatives (e.g. carboxymethylcellulose sodium, powdered
cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate
[Tween.RTM.20], polyoxyethylene sorbitan [Tween.RTM.60],
polyoxyethylene sorbitan monooleate [Tween.RTM.80], sorbitan
monopalmitate [Span.RTM.40], sorbitan monostearate [Span.RTM.60],
sorbitan tristearate [Span.RTM.65], glyceryl monooleate, sorbitan
monooleate [Span.RTM.80]), polyoxyethylene esters (e.g.
polyoxyethylene monostearate [Myrj.RTM.45], polyoxyethylene
hydrogenated castor oil, polyethoxylated castor oil,
polyoxymethylene stearate, and Solutol.RTM.), sucrose fatty acid
esters, polyethylene glycol fatty acid esters (e.g.
Cremophor.RTM.), polyoxyethylene ethers, (e.g. polyoxyethylene
lauryl ether [Brij.RTM.30]), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM.F 68, Poloxamer.RTM.188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof.
[0398] Exemplary binding agents include, but are not limited to,
starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol,
and mannitol); natural and synthetic gums (e.g. acacia, sodium
alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage
of isapol husks, carboxymethylcellulose, methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, microcrystalline cellulose,
cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum
silicate (Veegum.RTM.), and larch arabogalactan); alginates;
polyethylene oxide; polyethylene glycol; inorganic calcium salts;
silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and
combinations thereof.
[0399] Exemplary preservatives may include, but are not limited to,
antioxidants, chelating agents, antimicrobial preservatives,
antifungal preservatives, alcohol preservatives, acidic
preservatives, and/or other preservatives. Exemplary antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid,
acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and/or sodium sulfite. Exemplary chelating
agents include ethylenediaminetetraacetic acid (EDTA), citric acid
monohydrate, disodium edetate, dipotassium edetate, edetic acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric
acid, and/or trisodium edetate. Exemplary antimicrobial
preservatives include, but are not limited to, benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and/or thimerosal.
Exemplary antifungal preservatives include, but are not limited to,
butyl paraben, methyl paraben, ethyl paraben, propyl paraben,
benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium
sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
Exemplary alcohol preservatives include, but are not limited to,
ethanol, polyethylene glycol, phenol, phenolic compounds,
bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl
alcohol. Exemplary acidic preservatives include, but are not
limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric
acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid,
and/or phytic acid. Other preservatives include, but are not
limited to, tocopherol, tocopherol acetate, deteroxime mesylate,
cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened
(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl
ether sulfate (SLES), sodium bisulfite, sodium metabisulfite,
potassium sulfite, potassium metabisulfite, Glydant Plus.RTM.,
Phenonip.RTM., methylparaben, Germall.RTM.115, Germaben.RTM.II,
Neolone.TM., Kathon.TM., and/or Euxyl.RTM..
[0400] Exemplary buffering agents include, but are not limited to,
citrate buffer solutions, acetate buffer solutions, phosphate
buffer solutions, ammonium chloride, calcium carbonate, calcium
chloride, calcium citrate, calcium glubionate, calcium gluceptate,
calcium gluconate, d-gluconic acid, calcium glycerophosphate,
calcium lactate, propanoic acid, calcium levulinate, pentanoic
acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium
phosphate, calcium hydroxide phosphate, potassium acetate,
potassium chloride, potassium gluconate, potassium mixtures,
dibasic potassium phosphate, monobasic potassium phosphate,
potassium phosphate mixtures, sodium acetate, sodium bicarbonate,
sodium chloride, sodium citrate, sodium lactate, dibasic sodium
phosphate, monobasic sodium phosphate, sodium phosphate mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic
acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl
alcohol, etc., and/or combinations thereof.
[0401] Exemplary lubricating agents include, but are not limited
to, magnesium stearate, calcium stearate, stearic acid, silica,
talc, malt, glyceryl behanate, hydrogenated vegetable oils,
polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate,
etc., and combinations thereof.
[0402] Exemplary oils include, but are not limited to, almond,
apricot kernel, avocado, babassu, bergamot, black current seed,
borage, cade, camomile, canola, caraway, carnauba, castor,
cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton
seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba,
kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary oils include, but are not limited
to, butyl stearate, caprylic triglyceride, capric triglyceride,
cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl
myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone
oil, and/or combinations thereof.
[0403] Liquid dosage forms for oral and parenteral administration
include, but are not limited to, pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups, and/or
elixirs. In addition to active ingredients, liquid dosage forms may
comprise inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, oral compositions can include adjuvants
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, and/or perfuming agents. In certain
embodiments for parenteral administration, compositions are mixed
with solubilizing agents such as Cremophor.RTM., alcohols, oils,
modified oils, glycols, polysorbates, cyclodextrins, polymers,
and/or combinations thereof.
[0404] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing agents, wetting agents,
and/or suspending agents. Sterile injectable preparations may be
sterile injectable solutions, suspensions, and/or emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P., and isotonic sodium chloride solution. Sterile,
fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. Fatty acids such as
oleic acid can be used in the preparation of injectables.
[0405] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0406] In order to prolong the effect of an active ingredient, it
is often desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
[0407] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing
compositions with suitable non-irritating excipients such as cocoa
butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
ingredient.
[0408] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
an active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient such as sodium citrate or
dicalcium phosphate and/or fillers or extenders (e.g., starches,
lactose, sucrose, glucose, mannitol, and silicic acid), binders
(e.g., carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.,
glycerol), disintegrating agents (e.g., agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate), solution retarding agents (e.g., paraffin),
absorption accelerators (e.g., quaternary ammonium compounds),
wetting agents (e.g., cetyl alcohol and glycerol monostearate),
absorbents (e.g., kaolin and bentonite clay), and lubricants (e.g.,
talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate), and mixtures thereof. In the case
of capsules, tablets and pills, the dosage form may comprise
buffering agents.
[0409] Solid compositions of a similar type may be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. Solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally comprise opacifying agents and can be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type may be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polyethylene glycols and the
like.
[0410] Dosage forms for topical and/or transdermal administration
of a composition may include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants and/or patches.
Generally, an active ingredient is admixed under sterile conditions
with a pharmaceutically acceptable excipient and/or any needed
preservatives and/or buffers as may be required. Additionally, the
present disclosure contemplates the use of transdermal patches,
which often have the added advantage of providing controlled
delivery of a compound to the body. Such dosage forms may be
prepared, for example, by dissolving and/or dispensing the compound
in the proper medium. Alternatively or additionally, rate may be
controlled by either providing a rate controlling membrane and/or
by dispersing the compound in a polymer matrix and/or gel.
[0411] Suitable devices for use in delivering intradermal
pharmaceutical compositions described herein include short needle
devices such as those described in U.S. Pat. Nos. 4,886,499;
5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496;
and 5,417,662. Intradermal compositions may be administered by
devices which limit the effective penetration length of a needle
into the skin, such as those described in PCT publication WO
99/34850 and functional equivalents thereof. Jet injection devices
which deliver liquid compositions to the dermis via a liquid jet
injector and/or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis are suitable. Jet injection
devices are described, for example, in U.S. Pat. Nos. 5,480,381;
5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;
5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;
5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460;
and PCT publications WO 97/37705 and WO 97/13537. Ballistic
powder/particle delivery devices which use compressed gas to
accelerate vaccine in powder form through the outer layers of the
skin to the dermis are suitable. Alternatively or additionally,
conventional syringes may be used in the classical mantoux method
of intradermal administration.
[0412] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi liquid preparations such
as liniments, lotions, oil in water and/or water in oil emulsions
such as creams, ointments and/or pastes, and/or solutions and/or
suspensions. Topically-administrable formulations may, for example,
comprise from about 1% to about 10% (w/w) active ingredient,
although the concentration of active ingredient may be as high as
the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0413] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for pulmonary administration
via the buccal cavity. Such a formulation may comprise dry
particles which comprise the active ingredient and which have a
diameter in the range from about 0.5 nm to about 7 nm or from about
1 nm to about 6 nm. Such compositions are conveniently in the form
of dry powders for administration using a device comprising a dry
powder reservoir to which a stream of propellant may be directed to
disperse the powder and/or using a self propelling solvent/powder
dispensing container such as a device comprising the active
ingredient dissolved and/or suspended in a low-boiling propellant
in a sealed container. Such powders comprise particles wherein at
least 98% of the particles by weight have a diameter greater than
0.5 nm and at least 95% of the particles by number have a diameter
less than 7 nm. Alternatively, at least 95% of the particles by
weight have a diameter greater than 1 nm and at least 90% of the
particles by number have a diameter less than 6 nm. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0414] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50% to 99.9%
(w/w) of the composition, and active ingredient may constitute 0.1%
to 20% (w/w) of the composition. A propellant may further comprise
additional ingredients such as a liquid non-ionic and/or solid
anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0415] Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of droplets
of a solution and/or suspension. Such formulations may be prepared,
packaged, and/or sold as aqueous and/or dilute alcoholic solutions
and/or suspensions, optionally sterile, comprising active
ingredient, and may conveniently be administered using any
nebulization and/or atomization device. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, a flavoring agent such as saccharin sodium, a
volatile oil, a buffering agent, a surface active agent, and/or a
preservative such as methylhydroxybenzoate. Droplets provided by
this route of administration may have an average diameter in the
range from about 0.1 nm to about 200 nm.
[0416] Formulations described herein as being useful for pulmonary
delivery are useful for intranasal delivery of a pharmaceutical
composition. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient
and having an average particle from about 0.2 .mu.m to 500 .mu.m.
Such a formulation is administered in the manner in which snuff is
taken, i.e., by rapid inhalation through the nasal passage from a
container of the powder held close to the nose.
[0417] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of active ingredient, and may comprise one or more of
the additional ingredients described herein. A pharmaceutical
composition may be prepared, packaged, and/or sold in a formulation
suitable for buccal administration. Such formulations may, for
example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, 0.1% to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable
and/or degradable composition and, optionally, one or more of the
additional ingredients described herein. Alternately, formulations
suitable for buccal administration may comprise a powder and/or an
aerosolized and/or atomized solution and/or suspension comprising
active ingredient. Such powdered, aerosolized, and/or aerosolized
formulations, when dispersed, may have an average particle and/or
droplet size in the range from about 0.1 nm to about 200 nm, and
may further comprise one or more of any additional ingredients
described herein.
[0418] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form
of eye drops including, for example, a 0.1/1.0% (w/w) solution
and/or suspension of the active ingredient in an aqueous or oily
liquid excipient. Such drops may further comprise buffering agents,
salts, and/or one or more other of any additional ingredients
described herein. Other opthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form and/or in a liposomal preparation. Ear
drops and/or eye drops are contemplated as being within the scope
of this present disclosure.
[0419] General considerations in the formulation and/or manufacture
of pharmaceutical agents may be found, for example, in Remington's
The Science and Practice of Pharmacy, 21.sup.st Edition, A. R.
Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006;
incorporated herein by reference).
[0420] Administration
[0421] The present disclosure provides methods comprising
administering mRNA in accordance with the present disclosure to a
subject in need thereof. mRNA, or pharmaceutical, imaging,
diagnostic, or prophylactic compositions thereof, may be
administered to a subject using any amount and any route of
administration effective for preventing, treating, diagnosing, or
imaging a disease, disorder, and/or condition (e.g., a disease,
disorder, and/or condition relating to working memory deficits).
The exact amount required will vary from subject to subject,
depending on the species, age, and general condition of the
subject, the severity of the disease, the particular composition,
its mode of administration, its mode of activity, and the like.
Compositions in accordance with the present disclosure are
typically formulated in dosage unit form for ease of administration
and uniformity of dosage. It will be understood, however, that the
total daily usage of the compositions of the present disclosure
will be decided by the attending physician within the scope of
sound medical judgment. The specific therapeutically effective,
prophylactically effective, or appropriate imaging dose level for
any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts.
[0422] mRNAs to be delivered and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof may be administered to
animals, such as mammals (e.g., humans, domesticated animals, cats,
dogs, mice, rats, etc.). In some embodiments, pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof are
administered to humans.
[0423] mRNAs to be delivered and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof in accordance with the
present disclosure may be administered by any route. In some
embodiments, mRNAs and/or pharmaceutical, prophylactic, diagnostic,
or imaging compositions thereof, are administered by one or more of
a variety of routes, including oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, subcutaneous,
intraventricular, transdermal, interdermal, rectal, intravaginal,
intraperitoneal, topical (e.g., by powders, ointments, creams,
gels, lotions, and/or drops), mucosal, nasal, buccal, enteral,
vitreal, intratumoral, sublingual; by intratracheal instillation,
bronchial instillation, and/or inhalation; as an oral spray, nasal
spray, and/or aerosol, and/or through a portal vein catheter. In
some embodiments, mRNAs, and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof, are administered by
systemic intravenous injection. In specific embodiments, mRNAs
and/or pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof may be administered intravenously and/or
orally. In specific embodiments, mRNAs, and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof, may be
administered in a way which allows the mRNA to cross the
blood-brain barrier, vascular barrier, or other epithelial
barrier.
[0424] However, the present disclosure encompasses the delivery of
mRNAs, and/or pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof, by any appropriate route taking into
consideration likely advances in the sciences of drug delivery.
[0425] In general the most appropriate route of administration will
depend upon a variety of factors including the nature of the mRNA
associated with at least one agent to be delivered (e.g., its
stability in the environment of the gastrointestinal tract,
bloodstream, etc.), the condition of the patient (e.g., whether the
patient is able to tolerate particular routes of administration),
etc. The present disclosure encompasses the delivery of the
pharmaceutical, prophylactic, diagnostic, or imaging compositions
by any appropriate route taking into consideration likely advances
in the sciences of drug delivery.
[0426] In certain embodiments, compositions in accordance with the
present disclosure may be administered at dosage levels sufficient
to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40
mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01
mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or
from about 1 mg/kg to about 25 mg/kg, of subject body weight per
day, one or more times a day, to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect. The desired dosage may
be delivered three times a day, two times a day, once a day, every
other day, every third day, every week, every two weeks, every
three weeks, or every four weeks. In certain embodiments, the
desired dosage may be delivered using multiple administrations
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, or more administrations).
[0427] mRNAs may be used in combination with one or more other
therapeutic, prophylactic, diagnostic, or imaging agents. By "in
combination with," it is not intended to imply that the agents must
be administered at the same time and/or formulated for delivery
together, although these methods of delivery are within the scope
of the present disclosure. Compositions can be administered
concurrently with, prior to, or subsequent to, one or more other
desired therapeutics or medical procedures. In general, each agent
will be administered at a dose and/or on a time schedule determined
for that agent. In some embodiments, the present disclosure
encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or imaging compositions in combination with agents that
improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body.
[0428] It will further be appreciated that therapeutically,
prophylactically, diagnostically, or imaging active agents utilized
in combination may be administered together in a single composition
or administered separately in different compositions. In general,
it is expected that agents utilized in combination with be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0429] The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will take into
account compatibility of the desired therapeutics and/or procedures
and the desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, a composition useful for
treating cancer in accordance with the present disclosure may be
administered concurrently with a chemotherapeutic agent), or they
may achieve different effects (e.g., control of any adverse
effects).
[0430] Kits
[0431] The present disclosure provides a variety of kits for
conveniently and/or effectively carrying out methods of the present
disclosure. 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.
[0432] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleic acid alteration, wherein the
nucleic acid is capable of evading or avoiding induction of an
innate immune response of a cell into which the first isolated
nucleic acid is introduced, and packaging and instructions.
[0433] In one aspect, the disclosure provides kits for protein
production, comprising: a first isolated alternative nucleic acid
comprising a translatable region, provided in an amount effective
to produce a desired amount of a protein encoded by the
translatable region when introduced into a target cell; a second
nucleic acid comprising an inhibitory nucleic acid, provided in an
amount effective to substantially inhibit the innate immune
response of the cell; and packaging and instructions.
[0434] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and a nucleoside alteration, wherein the
nucleic acid exhibits reduced degradation by a cellular nuclease,
and packaging and instructions.
[0435] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least two different nucleoside
alterations, wherein the nucleic acid exhibits reduced degradation
by a cellular nuclease, and packaging and instructions.
[0436] In one aspect, the disclosure provides kits for protein
production, comprising a first isolated nucleic acid comprising a
translatable region and at least one nucleoside alteration, wherein
the nucleic acid exhibits reduced degradation by a cellular
nuclease; a second nucleic acid comprising an inhibitory nucleic
acid; and packaging and instructions.
[0437] In another aspect, the disclosure provides compositions for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and a nucleoside alteration,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease, and a mammalian cell suitable for translation of the
translatable region of the first nucleic acid.
Definitions
[0438] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges. For example, the term "C.sub.1-5 alkyl" is
specifically intended to individually disclose methyl, ethyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0439] Aberrant transcription product: As used herein, the term
"aberrant transcription product" refers to any contaminating
transcription product or impurity that differs from the intended
high fidelity RNA transcript that is encoded by a given DNA
template. Such aberrant transcription products can include short
RNAs as a result of abortive transcription initiation events
(Milligan et al., 1987, Nucleic Acids Res 15:8783-8798) and double
stranded (ds)RNAs generated by RNA dependent RNA polymerase
activity (Arnaud-Barbe et al, 1998, Nucleic Acids Res
26:3550-3554), RNA-primed transcription from RNA templates (Nacheva
and Berzal-Herranz, 2003, Eur J Biochem 270: 1458-1465), and
self-complementary 3' extension (Triana-Alonso et al., 1995, J Biol
Chem 270:6298-6307), i.e. a "3'-transcript extension region".
[0440] About: As used herein, the term "about" when used in the
context of the amount of an alternative nucleobase or nucleoside in
a polynucleic acid means+/-10% of the recited value. For example, a
polynucleotide containing about 25% of an alternative uracil
includes between 22.5-27.5% of the alternative uracil.
[0441] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents 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 closely together such that a combinatorial
(e.g., a synergistic) effect is achieved.
[0442] Altered: As used herein "altered" refers to a changed state
or structure of a molecule of the invention. Molecules may be
altered in many ways including chemically, structurally, and
functionally. In one embodiment, the mRNA molecules of the present
invention are altered by the introduction of non-natural
nucleosides and/or nucleotides, e.g., as it relates to the natural
ribonucleotides A, U, G, and C. Noncanonical nucleotides such as
the cap structures are not considered "altered" although they
differ from the chemical structure of the A, C, G, U
ribonucleotides.
[0443] 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.
[0444] Antigens of interest or desired antigens: As used herein,
the terms "antigens of interest" or "desired antigens" include
those proteins and other biomolecules provided herein that are
immunospecifically bound by the antibodies and fragments, mutants,
variants, and alterations thereof described herein. Examples of
antigens of interest include, but are not limited to, insulin,
insulin-like growth factor, hGH, tPA, cytokines, such as
interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or
IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF
beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
[0445] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest other than
the amount of an alternative nucleobase or nucleoside in a
polynucleic acid, 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).
[0446] 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.
[0447] 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.
[0448] Biodegradable: As used herein, the term "biodegradable"
means capable of being broken down into innocuous products by the
action of living things.
[0449] 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, a polynucleotide of
the present invention may be considered biologically active if even
a portion of the polynucleotide is biologically active or mimics an
activity considered biologically relevant.
[0450] Compound: As used herein, the term "compound," is meant to
include all stereoisomers, geometric isomers, tautomers, and
isotopes of the structures depicted.
[0451] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present disclosure that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds of the present disclosure are described
and may be isolated as a mixture of isomers or as separated
isomeric forms.
[0452] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond and the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Examples prototropic tautomers include ketone--enol
pairs, amide--imidic acid pairs, lactam--lactim pairs,
amide--imidic acid pairs, enamine--imine pairs, and annular forms
where a proton can occupy two or more positions of a heterocyclic
system, such as, 1H- and 3H-imidazole, 1H-, 2H- and
4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.
Tautomeric forms can be in equilibrium or sterically locked into
one form by appropriate substitution.
[0453] Compounds of the present disclosure also include all of the
isotopes of the atoms occurring in the intermediate or final
compounds. "Isotopes" refers to atoms having the same atomic number
but different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium.
[0454] The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to form
solvates and hydrates by routine methods.
[0455] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[0456] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence may apply to the entire length of an oligonucleotide or
polypeptide or may apply to a portion, region or feature
thereof.
[0457] Cyclic or Cyclized: As used herein, the term "cyclic" refers
to the presence of a continuous loop. Cyclic molecules need not be
circular, only joined to form an unbroken chain of subunits. Cyclic
molecules such as the mRNA of the present invention may be single
units or multimers or comprise one or more components of a complex
or higher order structure.
[0458] 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.
[0459] 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.
[0460] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound, substance, entity, moiety, cargo
or payload.
[0461] Delivery Agent: As used herein, "delivery agent" refers to
any substance which facilitates, at least in part, the in vivo
delivery of a polynucleotide to targeted cells.
[0462] 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.
[0463] 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 or
proteins disclosed herein. They may be within the amino acids, the
peptides, or proteins, or located at the N- or C-termini.
[0464] Digest: As used herein, the term "digest" means to break
apart into smaller pieces or components. When referring to
polypeptides or proteins, digestion results in the production of
peptides.
[0465] Distal: As used herein, the term "distal" means situated
away from the center or away from a point or region of
interest.
[0466] Encoded protein cleavage signal: As used herein, "encoded
protein cleavage signal" refers to the nucleotide sequence which
encodes a protein cleavage signal.
[0467] 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.
[0468] 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.
[0469] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element.
[0470] Formulation: As used herein, a "formulation" includes at
least a polynucleotide and a delivery agent.
[0471] 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.
[0472] 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.
[0473] 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.
[0474] 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 FASTAn
altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0475] 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.
[0476] 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).
[0477] In vitro synthesis: As used herein, the term "in vitro
synthesis" refers to an extracellular method of synthesis of
mRNA.
[0478] 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).
[0479] 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.
[0480] 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 an alternative 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 multimers (e.g., through linkage of two or
more polynucleotides) or 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, 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.
[0481] Maximized codons: As used herein the term "maximized codon"
refers to a codon with the highest number of a nucleotide. For
example, a "guanine maximized codon" is the codon for a particular
amino acid that has the highest number of guanines.
[0482] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[0483] Non-human vertebrate: As used herein, a "non human
vertebrate" includes all vertebrates except Homo sapiens, including
wild and domesticated species. Examples of non-human vertebrates
include, but are not limited to, mammals, such as alpaca, banteng,
bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama, mule, pig, rabbit, reindeer, sheep water
buffalo, and yak.
[0484] Off-target: As used herein, "off target" refers to any
unintended effect on any one or more target, gene, or cellular
transcript.
[0485] Open reading frame: As used herein, "open reading frame" or
"ORF" refers to a sequence which does not contain a stop codon in a
given reading frame.
[0486] Operably linked: As used herein, the phrase "operably
linked" refers to a functional connection between two or more
molecules, constructs, transcripts, entities, moieties or the
like.
[0487] Paratope: As used herein, a "paratope" refers to the
antigen-binding site of an antibody.
[0488] 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.
[0489] Optionally substituted: Herein a phrase of the form
"optionally substituted X" (e.g., optionally substituted alkyl) is
intended to be equivalent to "X, wherein X is optionally
substituted" (e.g., "alkyl, wherein said alkyl is optionally
substituted"). It is not intended to mean that the feature "X"
(e.g. alkyl) per se is optional.
[0490] 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.
[0491] 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.
[0492] 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.
[0493] 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 altered 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 The Science and Practice of Pharmacy,
21.sup.st Edition, A. R. Gennaro (Lippincott, Williams &
Wilkins, Baltimore, Md., 2006); 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.
[0494] Pharmacokinetic: As used herein, "pharmacokinetic" refers to
any one or more properties of a molecule or compound as it relates
to the determination of the fate of substances administered to a
living organism. Pharmacokinetics is divided into several areas
including the extent and rate of absorption, distribution,
metabolism and excretion. This is commonly referred to as ADME
where: (A) Absorption is the process of a substance entering the
blood circulation; (D) Distribution is the dispersion or
dissemination of substances throughout the fluids and tissues of
the body; (M) Metabolism (or Biotransformation) is the irreversible
transformation of parent compounds into daughter metabolites; and
(E) Excretion (or Elimination) refers to the elimination of the
substances from the body. In rare cases, some drugs irreversibly
accumulate in body tissue.
[0495] 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."
[0496] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[0497] 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.
[0498] 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.
[0499] 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.
[0500] Protein cleavage site: As used herein, "protein cleavage
site" refers to a site where controlled cleavage of the amino acid
chain can be accomplished by chemical, enzymatic or photochemical
means.
[0501] Protein cleavage signal: As used herein "protein cleavage
signal" refers to at least one amino acid that flags or marks a
polypeptide for cleavage.
[0502] Protein of interest: As used herein, the terms "proteins of
interest" or "desired proteins" include those provided herein and
fragments, mutants, variants, and alterations thereof.
[0503] Proximal: As used herein, the term "proximal" means situated
nearer to the center or to a point or region of interest.
[0504] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection.
[0505] 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.
[0506] Signal Sequences: As used herein, the phrase "signal
sequences" refers to a sequence which can direct the transport or
localization of a protein.
[0507] Significant or Significantly: As used herein, the terms
"significant" or "significantly" are used synonymously with the
term "substantially."
[0508] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administed in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[0509] 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.
[0510] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0511] 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.
[0512] Stabilized: As used herein, the term "stabilize",
"stabilized," "stabilized region" means to make or become
stable.
[0513] 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.
[0514] 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.
[0515] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0516] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term means within 2 seconds.
[0517] 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.
[0518] 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.
[0519] 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.
[0520] 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.
[0521] Theoretical Minimum: The term "theoretical minimum" refers
to a nucleotide sequence with all of the codons in the open reading
frame replaced to minimize the number of uracils in the
sequence.
[0522] 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.
[0523] 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.
[0524] 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.
[0525] 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.
[0526] 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.
[0527] 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.
[0528] Unaltered: As used herein, "unaltered" refers to any
substance, compound or molecule prior to being changed in any way.
Unaltered may, but does not always, refer to the wild-type or
native form of a biomolecule. Molecules may undergo a series of
alterations whereby each alternative molecule may serve as the
"unaltered" starting molecule for a subsequent alteration.
[0529] Uracil Content: As used herein, "uracil content" refers to
the number and/or distribution of uracils in a particular sequence,
e.g., an open reading frame.
[0530] Wild-type Sequence: As used herein, a "wild-type sequence"
is the sequence of the naturally occurring mRNA that encodes the
polypeptide of interest.
[0531] Equivalents and Scope
[0532] 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.
[0533] 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.
[0534] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0535] 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.
[0536] 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.
EXAMPLES
[0537] The present disclosure is further described in the following
examples, which do not limit the scope of the disclosure described
in the claims.
Example 1: PCR for cDNA Production
[0538] PCR procedures for the preparation of cDNA are performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times.KAPA ReadyMix12.5
.mu.l; Forward Primer (10 uM) 0.75 .mu.l; Reverse Primer (10 uM)
0.75 .mu.l; Template cDNA 100 ng; and dH.sub.2O diluted to 25.0
.mu.l. The reaction conditions are at 95.degree. C. for 5 min. and
25 cycles of 98.degree. C. for 20 sec, then 58.degree. C. for 15
sec, then 72.degree. C. for 45 sec, then 72.degree. C. for 5 min.
then 4.degree. C. to termination.
[0539] The reverse primer of the instant invention incorporates a
poly-T.sub.120 for a poly-A.sub.120 in the mRNA. Other reverse
primers with longer or shorter poly-T tracts can be used to adjust
the length of the poly-A tail in the mRNA.
[0540] The reaction is cleaned up using Invitrogen's PURELINK.TM.
PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions
(up to 5 .mu.g). Larger reactions will require a cleanup using a
product with a larger capacity. Following the cleanup, the cDNA is
quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the cDNA is the expected size. The cDNA
is then submitted for sequencing analysis before proceeding to the
in vitro transcription reaction.
Example 2. In Vitro Transcription (IVT)
[0541] A. Materials and Methods
[0542] Alternative mRNAs according to the invention are made using
standard laboratory methods and materials for in vitro
transcription with the exception that the nucleotide mix contains
alternative nucleotides. The open reading frame (ORF) of the gene
of interest may be flanked by a 5' untranslated region (UTR)
containing a strong Kozak translational initiation signal and an
alpha-globin 3' UTR terminating with an oligo(dT) sequence for
templated addition of a polyA tail for mRNAs not incorporating
adenosine analogs. Adenosine-containing mRNAs are synthesized
without an oligo (dT) sequence to allow for post-transcription poly
(A) polymerase poly-(A) tailing.
[0543] The ORF may also include various upstream or downstream
additions (such as, but not limited to, .beta.-globin, tags, etc.)
may be ordered from an optimization service such as, but limited
to, DNA2.0 (Menlo Park, Calif.) and may contain multiple cloning
sites which may have XbaI recognition. Upon receipt of the
construct, it may be reconstituted and transformed into chemically
competent E. coli.
[0544] For the present invention, NEB DH5-alpha Competent E. coli
may be used. Transformations are performed according to NEB
instructions using 100 ng of plasmid. The protocol is as
follows:
[0545] Thaw a tube of NEB 5-alpha Competent E. coli cells on ice
for 10 minutes.
[0546] Add 1-5 .mu.l containing 1 pg-100 ng of plasmid DNA to the
cell mixture. Carefully flick the tube 4-5 times to mix cells and
DNA. Do not vortex.
[0547] Place the mixture on ice for 30 minutes. Do not mix.
[0548] Heat shock at 42.degree. C. for exactly 30 seconds. Do not
mix.
[0549] Place on ice for 5 minutes. Do not mix.
[0550] Pipette 950 .mu.l of room temperature SOC into the
mixture.
[0551] Place at 37.degree. C. for 60 minutes. Shake vigorously (250
rpm) or rotate.
[0552] Warm selection plates to 37.degree. C.
[0553] Mix the cells thoroughly by flicking the tube and
inverting.
[0554] Spread 50-100 .mu.l of each dilution onto a selection plate
and incubate overnight at 37.degree. C. Alternatively, incubate at
30.degree. C. for 24-36 hours or 25.degree. C. for 48 hours.
[0555] A single colony is then used to inoculate 5 ml of LB growth
media using the appropriate antibiotic and then allowed to grow
(250 RPM, 37.degree. C.) for 5 hours. This is then used to
inoculate a 200 ml culture medium and allowed to grow overnight
under the same conditions.
[0556] To isolate the plasmid (up to 850 .mu.g), a maxi prep is
performed using the Invitrogen PURELINK.TM. HiPure Maxiprep Kit
(Carlsbad, Calif.), following the manufacturer's instructions.
[0557] In order to generate cDNA for In Vitro Transcription (IVT),
the plasmid is first linearized using a restriction enzyme such as
XbaI. A typical restriction digest with XbaI will comprise the
following: Plasmid 1.0 .mu.g; 10.times. Buffer 1.0 .mu.l; XbaI 1.5
.mu.l; dH.sub.2O up to 10 .mu.l; incubated at 37.degree. C. for 1
hr. If performing at lab scale (<5 .mu.g), the reaction is
cleaned up using Invitrogen's PURELINK.TM. PCR Micro Kit (Carlsbad,
Calif.) per manufacturer's instructions. Larger scale purifications
may need to be done with a product that has a larger load capacity
such as Invitrogen's standard PURELINK.TM. PCR Kit (Carlsbad,
Calif.). Following the cleanup, the linearized vector is quantified
using the NanoDrop and analyzed to confirm linearization using
agarose gel electrophoresis.
[0558] IVT Reaction
[0559] The in vitro transcription reaction generates mRNA
containing alternative nucleotides or alternative RNA. The input
nucleotide triphosphate (NTP) mix is made in-house using natural
and unnatural NTPs.
[0560] A typical in vitro transcription reaction includes the
following:
TABLE-US-00007 Template cDNA 1.0 .mu.g 10.times. transcription
buffer (400 mM Tris-HCl 2.0 .mu.l pH 8.0, 190 mM MgCl.sub.2, 50 mM
DTT, 10 mM Spermidine) Custom NTPs (25 mM each 7.2 .mu.l RNase
Inhibitor 20 U T7 RNA polymerase 3000 U dH.sub.2O up to 20.0 .mu.l
Incubation at 37.degree. C. for 3 hr-5 hrs.
[0561] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase is then used to
digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA is purified using Ambion's MEGACLEAR.TM.
Kit (Austin, Tex.) following the manufacturer's instructions. This
kit can purify up to 500 .mu.g of RNA. Following the cleanup, the
RNA is quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred.
[0562] The T7 RNA polymerase may be selected from, T7 RNA
polymerase, T3 RNA polymerase and mutant polymerases such as, but
not limited to, the novel polymerases able to incorporate
alternative NTPs as well as those polymerases described by Liu
(Esvelt et al. (Nature (2011) 472(7344):499-503 and U.S.
Publication No. 20110177495) which recognize alternate promoters,
Ellington (Chelliserrykattil and Ellington, Nature Biotechnology
(2004) 22(9):1155-1160) describing a T7 RNA polymerase variant to
transcribe 2'-O-methyl RNA and Sousa (Padilla and Sousa, Nucleic
Acids Research (2002) 30(24): e128) describing a T7 RNA polymerase
double mutant; herein incorporated by reference in their
entireties.
[0563] B. Agarose Gel Electrophoresis of Alternative mRNA
[0564] Individual alternative mRNAs (200-400 ng in a 20 .mu.l
volume) are loaded into a well on a non-denaturing 1.2% Agarose
E-Gel (Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes
according to the manufacturer protocol.
[0565] C. Agarose Gel Electrophoresis of RT-PCR Products
[0566] Individual reverse transcribed-PCR products (200-400 ng) are
loaded into a well of a non-denaturing 1.2% Agarose E-Gel
(Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes according
to the manufacturer protocol.
[0567] D. Nanodrop Alternative mRNA Quantification and UV Spectral
Data
[0568] Alternative mRNAs in TE buffer (1 .mu.l) are used for
Nanodrop UV absorbance readings to quantitate the yield of each
alternative mRNA from an in vitro transcription reaction (UV
absorbance traces are not shown).
Example 3. Enzymatic Capping of mRNA
[0569] Capping of the mRNA is performed as follows where the
mixture includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.2O up to 72
.mu.l. The mixture is incubated at 65.degree. C. for 5 minutes to
denature RNA, and then is transferred immediately to ice.
[0570] The protocol then involves the mixing of 10.times. Capping
Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl.sub.2)
(10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl Methionine
(2.5 .mu.l); RNase Inhibitor (100 U); 2'-O-Methyltransferase (400
U); Vaccinia capping enzyme (Guanylyl transferase) (40 U);
dH.sub.2O (Up to 28 .mu.l); and incubation at 37.degree. C. for 30
minutes for 60 .mu.g RNA or up to 2 hours for 180 .mu.g of RNA.
[0571] The mRNA is then purified using Ambion's MEGACLEAR.TM. Kit
(Austin, Tex.) following the manufacturer's instructions. Following
the cleanup, the RNA is quantified using the NANODROP.TM.
(ThermoFisher, Waltham, Mass.) and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred. The RNA product may also be
sequenced by running a reverse-transcription-PCR to generate the
cDNA for sequencing.
Example 4. 5'-Guanosine Capping
[0572] A. Materials and Methods
[0573] The cloning, gene synthesis and vector sequencing may be
performed by DNA2.0 Inc. (Menlo Park, Calif.). The ORF is
restriction digested using XbaI and used for cDNA synthesis using
tailed- or tail-less-PCR. The tailed-PCR cDNA product is used as
the template for the alternative mRNA synthesis reaction using 25
mM each alternative nucleotide mix (all alternative nucleotides may
be custom synthesized or purchased from TriLink Biotech, San Diego,
Calif. except pyrrolo-C triphosphate which may be purchased from
Glen Research, Sterling Va.; unmodifed nucleotides are purchased
from Epicenter Biotechnologies, Madison, Wis.) and CellScript
MEGASCRIPT.TM. (Epicenter Biotechnologies, Madison, Wis.) complete
mRNA synthesis kit.
[0574] The in vitro transcription reaction is run for 4 hours at
37.degree. C. Alternative mRNAs incorporating adenosine analogs are
poly (A) tailed using yeast Poly (A) Polymerase (Affymetrix, Santa
Clara, Calif.). The PCR reaction uses HiFi PCR 2.times. MASTER
MIX.TM. (Kapa Biosystems, Woburn, Mass.). Alternative mRNAs are
post-transcriptionally capped using recombinant Vaccinia Virus
Capping Enzyme (New England BioLabs, Ipswich, Mass.) and a
recombinant 2'-O-methyltransferase (Epicenter Biotechnologies,
Madison, Wis.) to generate the 5'-guanosine Cap1 structure. Cap 2
structure and Cap 2 structures may be generated using additional
2'-O-methyltransferases. The In vitro transcribed mRNA product is
run on an agarose gel and visualized. Alternative mRNA may be
purified with Ambion/Applied Biosystems (Austin, Tex.) MEGAClear
RNA.TM. purification kit. The PCR uses PURELINK.TM. PCR
purification kit (Invitrogen, Carlsbad, Calif.). The product is
quantified on NANODROP.TM. UV Absorbance (ThermoFisher, Waltham,
Mass.). Quality, UV absorbance quality and visualization of the
product was performed on an 1.2% agarose gel. The product is
resuspended in TE buffer.
[0575] B. 5'-Capping Alternative Nucleic Acid (mRNA) Structure
[0576] 5'-capping of alternative mRNA may be completed
concomitantly during the in vitro-transcription reaction using the
following chemical RNA cap analogs to generate the 5'-guanosine cap
structure according to manufacturer protocols:
3'-O-Me-m.sup.7G(5')ppp(5')G (the ARCA cap); G(5')ppp(5')A;
G(5')ppp(5')G; m.sup.7G(5')ppp(5')A; m.sup.7G(5')ppp(5')G (New
England BioLabs, Ipswich, Mass.). 5'-capping of alternative mRNA
may be completed post-transcriptionally using a Vaccinia Virus
Capping Enzyme to generate the "Cap 0" structure:
m.sup.7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes are preferably derived from a
recombinant source.
[0577] When transfected into mammalian cells, the alternative mRNAs
have a stability of 12-18 hours or more than 18 hours, e.g., 24,
36, 48, 60, 72 or greater than 72 hours.
Example 5. PolyA Tailing Reaction
[0578] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This is done by
mixing Capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2)(12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.2O up to 123.5 .mu.l and incubation at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction may be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 .mu.g). Poly-A Polymerase is preferably a recombinant
enzyme expressed in yeast.
[0579] For studies performed and described herein, the poly-A tail
is encoded in the IVT template to comprise 160 nucleotides in
length. However, it should be understood that the processivity or
integrity of the poly-A tailing reaction may not always result in
exactly 160 nucleotides. Hence poly-A tails of approximately 160
nucleotides, acid about 150-165, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164 or 165 are within the scope of the invention.
Example 6. In Vivo Expression of Selected Sequences
[0580] FIG. 1 shows in vivo expression data corresponding to
control expression of Target Protein 2 compared to the expression
data for constructs generated using 4 novel codon sets (CO1, CO2,
CO3 and CO4), after intravenous administration of 0.05 mg/kg of
each construct in MC3-LNP to mice. Similarly, FIG. 2 shows in vivo
expression data corresponding to control expression of Target
Protein 2 compared to the expression data for constructs generated
using 6 novel codon sets (CO5, CO6, CO7, CO8, CO9 and CO10).
TABLE-US-00008 TABLE 11 Uracil Content of Selected Sequences
Sequence Uracil Content CO1 23% uracil CO2 27% uracil CO3 13%
uracil + only 4 uracil pairs CO4 17% uracil CO7 13% uracil + only 4
uracil pairs CO9 14.7% uracil + only 4 uracil pairs
Example 7. Method of Screening for Protein Expression
[0581] A. Electrospray Ionization
[0582] A biological sample which may contain proteins encoded by
alternative RNA administered to the subject is prepared and
analyzed according to the manufacturer protocol for electrospray
ionization (ESI) using 1, 2, 3 or 4 mass analyzers. A biologic
sample may also be analyzed using a tandem ESI mass spectrometry
system.
[0583] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
[0584] B. Matrix-Assisted Laser Desorption/Ionization
[0585] A biological sample which may contain proteins encoded by
alternative RNA administered to the subject is prepared and
analyzed according to the manufacturer protocol for matrix-assisted
laser desorption/ionization (MALDI).
[0586] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
[0587] C. Liquid Chromatography-Mass Spectrometry-Mass
Spectrometry
[0588] A biological sample, which may contain proteins encoded by
alternative RNA, may be treated with a trypsin enzyme to digest the
proteins contained within. The resulting peptides are analyzed by
liquid chromatography-mass spectrometry-mass spectrometry
(LC/MS/MS). The peptides are fragmented in the mass spectrometer to
yield diagnostic patterns that can be matched to protein sequence
databases via computer algorithms. The digested sample may be
diluted to achieve 1 ng or less starting material for a given
protein. Biological samples containing a simple buffer background
(e.g., water or volatile salts) are amenable to direct in-solution
digest; more complex backgrounds (e.g., detergent, non-volatile
salts, glycerol) require an additional clean-up step to facilitate
the sample analysis.
[0589] Patterns of protein fragments, or whole proteins, are
compared to known controls for a given protein and identity is
determined by comparison.
Example 8. Transfection
[0590] A. Reverse Transfection
[0591] For experiments performed in a 24-well collagen-coated
tissue culture plate, Keratinocytes or other cells are seeded at a
cell density of 1.times.10.sup.5. For experiments performed in a
96-well collagen-coated tissue culture plate, Keratinocytes are
seeded at a cell density of 0.5.times.10.sup.5. For each
alternative mRNA to be transfected, alternative mRNA: RNAIMAX.TM.
are prepared as described and mixed with the cells in the
multi-well plate within 6 hours of cell seeding before cells had
adhered to the tissue culture plate.
[0592] B. Forward Transfection
[0593] In a 24-well collagen-coated tissue culture plate, cells are
seeded at a cell density of 0.7.times.10.sup.5. For experiments
performed in a 96-well collagen-coated tissue culture plate,
keratinocytes, if used, are seeded at a cell density of
0.3.times.10.sup.5. Cells are then grown to a confluency of >70%
for over 24 hours. For each alternative mRNA to be transfected,
alternative mRNA: RNAIMAX.TM. are prepared as described and
transfected onto the cells in the multi-well plate over 24 hours
after cell seeding and adherence to the tissue culture plate.
[0594] C. Translation Screen: ELISA
[0595] Cells are grown in EpiLife medium with Supplement S7 from
Invitrogen at a confluence of >70%. Cells are reverse
transfected with 300 ng of the indicated chemically alternative
mRNA complexed with RNAIMAX.TM. from Invitrogen. Alternatively,
cells are forward transfected with 300 ng alternative mRNA
complexed with RNAIMAX.TM. from Invitrogen. The RNA: RNAIMAX.TM.
complex is formed by first incubating the RNA with Supplement-free
EPILIFE.RTM. media in a 5.times. volumetric dilution for 10 minutes
at room temperature.
[0596] In a second vial, RNAIMAX.TM. reagent is incubated with
Supplement-free EPILIFE.RTM. Media in a 10.times. volumetric
dilution for 10 minutes at room temperature. The RNA vial is then
mixed with the RNAIMAX.TM. vial and incubated for 20-30 at room
temperature before being added to the cells in a drop-wise fashion.
Secreted polypeptide concentration in the culture medium is
measured at 18 hours post-transfection for each of the chemically
alternative mRNAs in triplicate. Secretion of the polypeptide of
interest from transfected human cells is quantified using an ELISA
kit from Invitrogen or R&D Systems (Minneapolis, Minn.)
following the manufacturer's recommended instructions.
[0597] D. Dose and Duration: ELISA
[0598] Cells are grown in EPILIFE.RTM. medium with Supplement S7
from Invitrogen at a confluence of >70%. Cells are reverse
transfected with Ong, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750
ng, or 1500 ng alternative mRNA complexed with RNAIMAX.TM. from
Invitrogen. The alternative mRNA: RNAIMAX.TM. complex is formed as
described. Secreted polypeptide concentration in the culture medium
is measured at 0, 6, 12, 24, and 48 hours post-transfection for
each concentration of each alternative mRNA in triplicate.
Secretion of the polypeptide of interest from transfected human
cells is quantified using an ELISA kit from Invitrogen or R&D
Systems following the manufacturers recommended instructions.
Example 9. Cellular Innate Immune Response: IFN-Beta ELISA and
TNF-Alpha ELISA
[0599] An enzyme-linked immunosorbent assay (ELISA) for Human Tumor
Necrosis Factor-.alpha. (TNF-.alpha.), Human Interferon-.beta.
(IFN-.beta.) and Human Granulocyte-Colony Stimulating Factor
(G-CSF) secreted from in vitro-transfected Human Keratinocyte cells
is tested for the detection of a cellular innate immune
response.
[0600] Cells are grown in EPILIFE.RTM. medium with Human Growth
Supplement in the absence of hydrocortisone from Invitrogen at a
confluence of >70%. Cells are reverse transfected with Ong,
93.75 ng, 187.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of the
indicated chemically alternative mRNA complexed with RNAIMAX.TM.
from Invitrogen as described in triplicate. Secreted TNF-.alpha. in
the culture medium is measured 24 hours post-transfection for each
of the chemically alternative mRNAs using an ELISA kit from
Invitrogen according to the manufacturer protocols.
[0601] Secreted IFN-.beta. is measured 24 hours post-transfection
for each of the alternative mRNAs using an ELISA kit from
Invitrogen according to the manufacturer protocols. Secreted
hu-G-CSF concentration is measured at 24 hours post-transfection
for each of the alternative mRNAs. Secretion of the polypeptide of
interest from transfected human cells is quantified using an ELISA
kit from Invitrogen or R&D Systems (Minneapolis, Minn.)
following the manufacturers recommended instructions. These data
indicate which alternative mRNA are capable eliciting a reduced
cellular innate immune response in comparison to natural and other
alternative polynucleotides or reference compounds by measuring
exemplary type 1 cytokines such as TNF-alpha and IFN-beta.
Example 10. Cytotoxicity and Apoptosis
[0602] This experiment demonstrates cellular viability, cytotoxity
and apoptosis for distinct alternative mRNA-in vitro transfected
Human Keratinocyte cells. Keratinocytes are grown in EPILIFE.RTM.
medium with Human Keratinocyte Growth Supplement in the absence of
hydrocortisone from Invitrogen at a confluence of >70%.
Keratinocytes are reverse transfected with Ong, 46.875 ng, 93.75
ng, 187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of
alternative mRNA complexed with RNAIMAX.TM. from Invitrogen. The
alternative mRNA: RNAIMAX.TM. complex is formed. Secreted huG-CSF
concentration in the culture medium is measured at 0, 6, 12, 24,
and 48 hours post-transfection for each concentration of each
alternative mRNA in triplicate. Secretion of the polypeptide of
interest from transfected human keratinocytes is quantified using
an ELISA kit from Invitrogen or R&D Systems following the
manufacturers recommended instructions. Cellular viability,
cytotoxicity and apoptosis is measured at 0, 12, 48, 96, and 192
hours post-transfection using the APOTOX-GLO.TM. kit from Promega
(Madison, Wis.) according to manufacturer instructions.
Example 11. In Vivo Assays with Human EPO Containing Alternative
Nucleotides
[0603] Formulation
[0604] Alternative hEPO mRNAs were formulated in lipid
nanoparticles (LNPs) comprising DLin-KC2-DMA, DSPC, Cholesterol,
and PEG-DMG at 50:10:38.5:1.5 mol % respectively (Table 12). The
LNPs were made by direct injection utilizing nanoprecipitation of
ethanol solubilized lipids into a pH 4.0 50 mM citrate mRNA
solution. The EPO LNP particle size distributions were
characterized by DLS. Encapsulation efficiency (EE) was determined
using a Ribogreen.TM. fluorescence-based assay for detection and
quantification of nucleic acids.
TABLE-US-00009 TABLE 12 Formulation Conditions Ionizable Lipid PEG
Lipid 2-(2,2-di((9Z,12Z)- Phospholipid 1,2-Dimyristoyl-sn-
octadeca-9,12-dien-1yl)- 1,2-distearoyl-sn- glycerol,
1,3-diocolan-4-yl)-N,N- glycero-3- Cholesterol methoxypolyethylene
dimethylethanamine phosphocholine cholest-5-en-3 -ol Glycol
(Lipid/Mol %) (Lipid/Mol %) (Lipid/Mol %) (Lipid/Mol %)
DLin-KC2-DMA DSPC Cholesterol PEG-DMG 50 10 38.5 1.5
[0605] Methods and Data
[0606] Female Balb/c mice (n=5) were administered 0.05 mg/kg IM (50
.mu.l in the quadriceps) or IV (100 .mu.l in the tail vein) of
human EPO mRNA. At time 8 hours after the injection mice were
euthanized and blood was collected in serum separator tubes. The
samples were spun, and serum samples were then run on an EPO ELISA
following the kit protocol (Stem Cell Technologies Catalog
#01630).
Example 12. mRNA Sequences for Constructs Used to Screen Compounds
of the Invention
TABLE-US-00010 [0607] hEPO DNA2.0 sequence (SEQ ID NO: 5):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGGAGTGCACGAGTGTCCCGCGTGGTTGTGGTTGCTGCT
GTCGCTCTTGAGCCTCCCACTGGGACTGCCTGTGCTGGGGGCACCACCCAGATTGATCTGCGACTC
ACGGGTACTTGAGAGGTACCTTCTTGAAGCCAAAGAAGCCGAAAACATCACAACCGGATGCGCCGA
GCACTGCTCCCTCAATGAGAACATTACTGTACCGGATACAAAGGTCAATTTCTATGCATGGAAGAGA
ATGGAAGTAGGACAGCAGGCCGTCGAAGTGTGGCAGGGGCTCGCGCTTTTGTCGGAGGCGGTGTT
GCGGGGTCAGGCCCTCCTCGTCAACTCATCACAGCCGTGGGAGCCCCTCCAACTTCATGTCGATAA
AGCGGTGTCGGGGCTCCGCAGCTTGACGACGTTGCTTCGGGCTCTGGGCGCACAAAAGGAGGCTA
TTTCGCCGCCTGACGCGGCCTCCGCGGCACCCCTCCGAACGATCACCGCGGACACGTTTAGGAAG
CTTTTTAGAGTGTACAGCAATTTCCTCCGCGGAAAGCTGAAATTGTATACTGGTGAAGCGTGTAGGA
CAGGGGATCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCC
AGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC
TAGA hEPO CO9 sequence (SEQ ID NO: 6):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCTGCTGCT
GAGCCTGCTGAGCCTGCCCCTGGGCCTGCCCGTGCTGGGCGCCCCCCCCCGCCTCATCTGCGACT
CCCGCGTCCTCGAGCGCTACCTCCTCGAGGCCAAGGAGGCCGAGAACATCACCACCGGCTGCGCC
GAGCACTGCTCCCTCAACGAGAACATCACCGTCCCCGACACCAAGGTCAACTTCTACGCCTGGAAG
CGCATGGAGGTCGGCCAGCAGGCCGTCGAGGTCTGGCAGGGCCTCGCCCTCCTCTCCGAGGCCGT
CCTCCGCGGCCAGGCCCTCCTCGTCAACTCCTCCCAGCCCTGGGAGCCCCTCCAGCTCCACGTCG
ACAAGGCCGTCTCCGGCCTCCGCTCCCTCACCACCCTCCTCCGCGCCCTCGGCGCCCAGAAGGAG
GCCATCTCCCCCCCCGACGCCGCCTCCGCCGCCCCCCTCCGCACCATCACCGCCGACACCTTCCG
CAAGCTCTTCCGCGTCTACTCCAACTTCCTCCGCGGCAAGCTCAAGCTCTACACCGGCGAGGCCTG
CCGCACCGGCGACCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTC
CCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGC
GGC GCSF DNA2.0 sequence (SEQ ID NO: 7):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGC
CCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACACCTTTAGGACCTG
CTTCTTCTTTACCTCAATCTTTTTTATTAAAATGTTTAGAACAAGTTAGAAAAATTCAAGGAGATGGAG
CTGCTTTACAAGAAAAATTATGTGCTACATATAAATTATGTCATCCTGAAGAATTAGTTTTATTAGGAC
ATTCTTTAGGAATTCCTTGGGCTCCTTTATCTTCTTGTCCTTCTCAAGCTTTACAATTAGCTGGATGTT
TATCTCAATTACATTCTGGATTATTTTTATATCAAGGATTATTACAAGCTTTAGAAGGAATTTCTCCTGA
ATTAGGACCTACATTAGATACATTACAATTAGATGTTGCTGATTTTGCTACAACAATTTGGCAACAAAT
GGAAGAATTAGGAATGGCTCCTGCTTTACAACCTACACAAGGAGCTATGCCTGCTTTTGCTTCTGCTT
TTCAAAGAAGAGCTGGAGGAGTTTTAGTTGCTTCTCATTTACAATCTTTTTTAGAAGTTTCTTATAGAG
TTTTAAGACATTTAGCTCAACCTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTG
GGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA
GTGGGCGGC GCSF CO3 sequence (SEQ ID NO: 8):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGC
CCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACCCCCCTGGGCCCCG
CCAGCAGCCTGCCCCAGAGCTTCCTGCTGAAGTGCCTGGAGCAGGTGCGGAAGATCCAGGGCGAC
GGCGCCGCCCTGCAGGAGAAGCTGTGCGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCT
GCTGGGCCACAGCCTGGGCATCCCCTGGGCCCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAG
CTGGCCGGCTGCCTGAGCCAGCTGCACAGCGGCCTGTTCCTGTACCAGGGCCTGCTGCAGGCCCT
GGAGGGCATCAGCCCCGAGCTGGGCCCCACCCTGGACACCCTGCAGCTGGACGTGGCCGACTTCG
CCACCACCATCTGGCAGCAGATGGAGGAGCTGGGCATGGCCCCCGCCCTGCAGCCCACCCAGGGC
GCCATGCCCGCCTTCGCCAGCGCCTTCCAGCGGCGGGCCGGCGGCGTGCTGGTGGCCAGCCACC
TGCAGAGCTTCCTGGAGGTGAGCTACCGGGTGCTGCGGCACCTGGCCCAGCCCTGATAATAGGCT
GGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCAC
CCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC GCSF CO7 sequence (SEQ ID
NO: 9):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGC
CCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACGCCGCTGGGGCCG
GCGAGCAGCCTGCCGCAGAGCTTCCTGCTGAAGTGCCTGGAGCAGGTGAGGAAGATCCAGGGGGA
CGGGGCGGCGCTGCAGGAGAAGCTGTGCGCGACGTACAAGCTGTGCCACCCGGAGGAGCTGGTG
CTGCTGGGGCACAGCCTGGGGATCCCGTGGGCGCCGCTGAGCAGCTGCCCGAGCCAGGCGCTGC
AGCTGGCGGGGTGCCTGAGCCAGCTGCACAGCGGGCTGTTCCTGTACCAGGGGCTGCTGCAGGCG
CTGGAGGGGATCAGCCCGGAGCTGGGGCCGACGCTGGACACGCTGCAGCTGGACGTGGCGGACT
TCGCGACGACGATCTGGCAGCAGATGGAGGAGCTGGGGATGGCGCCGGCGCTGCAGCCGACGCA
GGGGGCGATGCCGGCGTTCGCGAGCGCGTTCCAGAGGAGGGCGGGGGGGGTGCTGGTGGCGAG
CCACCTGCAGAGCTTCCTGGAGGTGAGCTACAGGGTGCTGAGGCACCTGGCGCAGCCGTGATAATA
GGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCT
GCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC GCSF CO9 sequence
(SEQ ID NO: 10):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGC
CCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACCCCCCTCGGCCCCG
CCTCCTCCCTCCCCCAGTCCTTCCTCCTCAAGTGCCTCGAGCAGGTCCGCAAGATCCAGGGCGACG
GCGCCGCCCTCCAGGAGAAGCTCTGCGCCACCTACAAGCTCTGCCACCCCGAGGAGCTCGTCCTC
CTCGGCCACTCCCTCGGCATCCCCTGGGCCCCCCTCTCCTCCTGCCCCTCCCAGGCCCTCCAGCTC
GCCGGCTGCCTCTCCCAGCTCCACTCCGGCCTCTTCCTCTACCAGGGCCTCCTCCAGGCCCTCGAG
GGCATCTCCCCCGAGCTCGGCCCCACCCTCGACACCCTCCAGCTCGACGTCGCCGACTTCGCCAC
CACCATCTGGCAGCAGATGGAGGAGCTCGGCATGGCCCCCGCCCTCCAGCCCACCCAGGGCGCCA
TGCCCGCCTTCGCCTCCGCCTTCCAGCGCCGCGCCGGCGGCGTCCTCGTCGCCTCCCACCTCCAG
TCCTTCCTCGAGGTCTCCTACCGCGTCCTCCGCCACCTCGCCCAGCCCTGATAATAGGCTGGAGCC
TCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTAC
CCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Luc DNA2.0 sequence (SEQ ID NO:
11):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGAAGATGCGAAGAACATCAAGAAGGGACCTGCCCCGTTT
TACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATGAAACGCTACGCCCTGGTC
CCCGGAACGATTGCGTTTACCGATGCACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAA
TGTCGGTGAGGCTGGCGGAAGCGATGAAGAGATATGGTCTTAACACTAATCACCGCATCGTGGTGT
GTTCGGAGAACTCATTGCAGTTTTTCATGCCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGC
GCCAGCGAACGACATCTACAATGAGCGGGAACTCTTGAATAGCATGGGAATCTCCCAGCCGACGGT
CGTGTTTGTCTCCAAAAAGGGGCTGCAGAAAATCCTCAACGTGCAGAAGAAGCTCCCCATTATTCAA
AAGATCATCATTATGGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATGTATACCTTTGTGACATC
GCATTTGCCGCCAGGGTTTAACGAGTATGACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATC
GCGCTGATTATGAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTGGCGTTGCCCCACCGCACT
GCTTGTGTGCGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAA
TCCTGTCCGTGGTACCTTTTCATCACGGTTTTGGCATGTTCACGACTCTCGGCTATTTGATTTGCGGT
TTCAGGGTCGTACTTATGTATCGGTTCGAGGAAGAACTGTTTTTGAGATCCTTGCAAGATTACAAGAT
CCAGTCGGCCCTCCTTGTGCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATG
ACCTTTCCAATCTGCATGAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAG
TGGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGA
TCCTTATCACGCCCGAGGGTGACGATAAGCCGGGAGCCGTCGGAAAAGTGGTCCCCTTCTTTGAAG
CCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTGAACCAGAGGGGCGAGCTCTGCGTG
AGAGGGCCGATGATCATGTCAGGTTACGTGAATAACCCTGAAGCGACGAATGCGCTGATCGACAAG
GATGGGTGGTTGCATTCGGGAGACATTGCCTATTGGGATGAGGATGAGCACTTCTTTATCGTAGATC
GACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTGCCGAGCTCGAGTCAATCCTGCT
CCAGCACCCCAACATTTTCGACGCCGGAGTGGCCGGGTTGCCCGATGACGACGCGGGTGAGCTGC
CAGCGGCCGTGGTAGTCCTCGAACATGGGAAAACAATGACCGAAAAGGAGATCGTGGACTACGTAG
CATCACAAGTGACGACTGCGAAGAAACTGAGGGGAGGGGTAGTCTTTGTGGACGAGGTCCCGAAAG
GCTTGACTGGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTGATTAAGGCAAAGAAAGGCGGGA
AAATCGCTGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCC
AGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
Luc CO3 sequence (SEQ ID NO: 12):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGAGGACGCCAAGAACATCAAGAAGGGCCCCGCCCCCTT
CTACCCCCTGGAGGACGGCACCGCCGGCGAGCAGCTGCACAAGGCCATGAAGCGGTACGCCCTGG
TGCCCGGCACCATCGCCTTCACCGACGCCCACATCGAGGTGGACATCACCTACGCCGAGTACTTCG
AGATGAGCGTGCGGCTGGCCGAGGCCATGAAGCGGTACGGCCTGAACACCAACCACCGGATCGTG
GTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGCGCCCTGTTCATCGGCGTGGCC
GTGGCCCCCGCCAACGACATCTACAACGAGCGGGAGCTGCTGAACAGCATGGGCATCAGCCAGCC
CACCGTGGTGTTCGTGAGCAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCCAT
CATCCAGAAGATCATCATCATGGACAGCAAGACCGACTACCAGGGCTTCCAGAGCATGTACACCTTC
GTGACCAGCCACCTGCCCCCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGA
CAAGACCATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCCAAGGGCGTGGCCCTGC
CCCACCGGACCGCCTGCGTGCGGTTCAGCCACGCCCGGGACCCCATCTTCGGCAACCAGATCATC
CCCGACACCGCCATCCTGAGCGTGGTGCCCTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGC
TACCTGATCTGCGGCTTCCGGGTGGTGCTGATGTACCGGTTCGAGGAGGAGCTGTTCCTGCGGAGC
CTGCAGGACTACAAGATCCAGAGCGCCCTGCTGGTGCCCACCCTGTTCAGCTTCTTCGCCAAGAGC
ACCCTGATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCAGCGGCGGCGCCCCCCTGAG
CAAGGAGGTGGGCGAGGCCGTGGCCAAGCGGTTCCACCTGCCCGGCATCCGGCAGGGCTACGGC
CTGACCGAGACCACCAGCGCCATCCTGATCACCCCCGAGGGCGACGACAAGCCCGGCGCCGTGGG
CAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGCGTGA
ACCAGCGGGGCGAGCTGTGCGTGCGGGGCCCCATGATCATGAGCGGCTACGTGAACAACCCCGAG
GCCACCAACGCCCTGATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGA
GGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAGTACAAGGGCTACCAGGTGGC
CCCCGCCGAGCTGGAGAGCATCCTGCTGCAGCACCCCAACATCTTCGACGCCGGCGTGGCCGGCC
TGCCCGACGACGACGCCGGCGAGCTGCCCGCCGCCGTGGTGGTGCTGGAGCACGGCAAGACCAT
GACCGAGAAGGAGATCGTGGACTACGTGGCCAGCCAGGTGACCACCGCCAAGAAGCTGCGGGGCG
GCGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGGAAGATCCG
GGAGATCCTGATCAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTGATAATAGGCTGGAGCCTCGG
TGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC
GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Luc CO7 sequence (SEQ ID NO: 13):
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGAGGACGCCAAGAACATCAAGAAGGGCCCCGCCCCCTT
CTACCCCCTGGAGGACGGCACCGCCGGCGAGCAGCTGCACAAGGCCATGAAGAGGTACGCGCTGG
TGCCGGGGACGATCGCGTTCACGGACGCGCACATCGAGGTGGACATCACGTACGCGGAGTACTTC
GAGATGAGCGTGAGGCTGGCGGAGGCGATGAAGAGGTACGGGCTGAACACGAACCACAGGATCGT
GGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCGGTGCTGGGGGCGCTGTTCATCGGGGTGG
CGGTGGCGCCGGCGAACGACATCTACAACGAGAGGGAGCTGCTGAACAGCATGGGGATCAGCCAG
CCGACGGTGGTGTTCGTGAGCAAGAAGGGGCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCC
GATCATCCAGAAGATCATCATCATGGACAGCAAGACGGACTACCAGGGGTTCCAGAGCATGTACAC
GTTCGTGACGAGCCACCTGCCGCCGGGGTTCAACGAGTACGACTTCGTGCCGGAGAGCTTCGACA
GGGACAAGACGATCGCGCTGATCATGAACAGCAGCGGGAGCACGGGGCTGCCGAAGGGGGTGGC
GCTGCCGCACAGGACGGCGTGCGTGAGGTTCAGCCACGCGAGGGACCCGATCTTCGGGAACCAGA
TCATCCCGGACACGGCGATCCTGAGCGTGGTGCCGTTCCACCACGGGTTCGGGATGTTCACGACG
CTGGGGTACCTGATCTGCGGGTTCAGGGTGGTGCTGATGTACAGGTTCGAGGAGGAGCTGTTCCTG
AGGAGCCTGCAGGACTACAAGATCCAGAGCGCGCTGCTGGTGCCGACGCTGTTCAGCTTCTTCGCG
AAGAGCACGCTGATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCGAGCGGGGGGGCGCC
GCTGAGCAAGGAGGTGGGGGAGGCGGTGGCGAAGAGGTTCCACCTGCCGGGGATCAGGCAGGGG
TACGGGCTGACGGAGACGACGAGCGCGATCCTGATCACGCCGGAGGGGGACGACAAGCCGGGGG
CGGTGGGGAAGGTGGTGCCGTTCTTCGAGGCGAAGGTGGTGGACCTGGACACGGGGAAGACGCT
GGGGGTGAACCAGAGGGGGGAGCTGTGCGTGAGGGGGCCGATGATCATGAGCGGGTACGTGAAC
AACCCGGAGGCGACGAACGCGCTGATCGACAAGGACGGGTGGCTGCACAGCGGGGACATCGCGTA
CTGGGACGAGGACGAGCACTTCTTCATCGTGGACAGGCTGAAGAGCCTGATCAAGTACAAGGGGTA
CCAGGTGGCGCCGGCGGAGCTGGAGAGCATCCTGCTGCAGCACCCGAACATCTTCGACGCGGGG
GTGGCGGGGCTGCCGGACGACGACGCGGGGGAGCTGCCGGCGGCGGTGGTGGTGCTGGAGCAC
GGGAAGACGATGACGGAGAAGGAGATCGTGGACTACGTGGCGAGCCAGGTGACGACGGCGAAGAA
GCTGAGGGGGGGGGTGGTGTTCGTGGACGAGGTGCCGAAGGGGCTGACGGGGAAGCTGGACGCG
AGGAAGATCAGGGAGATCCTGATCAAGGCGAAGAAGGGGGGGAAGATCGCGGTGTGATAATAGGC
TGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCA
CCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC mCherry wild-type (SEQ
ID NO: 14)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGTATCCAAGGGGGAGGAGGACAACATGGCGATCATCAA
GGAGTTCATGCGATTCAAGGTGCACATGGAAGGTTCGGTCAACGGACACGAATTTGAAATCGAAGG
AGAGGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGCGAAACTCAAGGTCACGAAAGGGGGAC
CACTTCCTTTCGCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTCCAAAGCATATGTGAAGCAT
CCCGCCGATATTCCTGACTATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGGAGCGGGTCATGA
ACTTTGAGGACGGGGGTGTAGTCACCGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTCATCT
ACAAGGTCAAACTGCGGGGGACTAACTTTCCGTCGGATGGGCCGGTGATGCAGAAGAAAACGATGG
GATGGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATGGTGCATTGAAGGGGGAGATCAAGCAG
AGACTGAAGTTGAAAGATGGGGGACATTATGATGCCGAGGTGAAAACGACATACAAAGCGAAAAAG
CCGGTGCAGCTTCCCGGAGCGTATAATGTGAATATCAAGTTGGATATTACTTCACACAATGAGGACT
ACACAATTGTCGAACAGTACGAACGCGCTGAGGGTAGACACTCGACGGGAGGCATGGACGAGTTGT
ACAAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCC
TCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC mCherry
CO3 sequence (SEQ ID NO: 15)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAA
GGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGG
GCGAGGGCGAGGGCCGGCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGG
CCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAA
GCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGGG
TGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTGCAGGACGGCGAG
TTCATCTACAAGGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAG
ACCATGGGCTGGGAGGCCAGCAGCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGA
TCAAGCAGCGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAG
GCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCAC
AACGAGGACTACACCATCGTGGAGCAGTACGAGCGGGCCGAGGGCCGGCACAGCACCGGCGGCA
TGGACGAGCTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCT
CCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGG
CGGC mCherry CO7 sequence (SEQ ID NO: 16)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAG
AGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAA
GGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGG
GGGAGGGGGAGGGGAGGCCGTACGAGGGGACGCAGACGGCGAAGCTGAAGGTGACGAAGGGGG
GGCCGCTGCCGTTCGCGTGGGACATCCTGAGCCCGCAGTTCATGTACGGGAGCAAGGCGTACGTG
AAGCACCCGGCGGACATCCCGGACTACCTGAAGCTGAGCTTCCCGGAGGGGTTCAAGTGGGAGAG
GGTGATGAACTTCGAGGACGGGGGGGTGGTGACGGTGACGCAGGACAGCAGCCTGCAGGACGGG
GAGTTCATCTACAAGGTGAAGCTGAGGGGGACGAACTTCCCGAGCGACGGGCCGGTGATGCAGAA
GAAGACGATGGGGTGGGAGGCGAGCAGCGAGAGGATGTACCCGGAGGACGGGGCGCTGAAGGGG
GAGATCAAGCAGAGGCTGAAGCTGAAGGACGGGGGGCACTACGACGCGGAGGTGAAGACGACGTA
CAAGGCGAAGAAGCCGGTGCAGCTGCCGGGGGCGTACAACGTGAACATCAAGCTGGACATCACGA
GCCACAACGAGGACTACACGATCGTGGAGCAGTACGAGAGGGCGGAGGGGAGGCACAGCACGGG
GGGGATGGACGAGCTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTG
GGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA
GTGGGCGGC mCherry CO9 sequence (SEQ ID NO: 17)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGA
AGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATC
ATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAG
ATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTCAAGGTCAC
CAAGGGCGGCCCCCTCCCCTTCGCCTGGGACATCCTCTCCCCCCAGTTCATGTACGGCTCCAA
GGCCTACGTCAAGCACCCCGCCGACATCCCCGACTACCTCAAGCTCTCCTTCCCCGAGGGCTT
CAAGTGGGAGCGCGTCATGAACTTCGAGGACGGCGGCGTCGTCACCGTCACCCAGGACTCCTC
CCTCCAGGACGGCGAGTTCATCTACAAGGTCAAGCTCCGCGGCACCAACTTCCCCTCCGACGG
CCCCGTCATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGCATGTACCCCGAGGA
CGGCGCCCTCAAGGGCGAGATCAAGCAGCGCCTCAAGCTCAAGGACGGCGGCCACTACGACG
CCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTCCAGCTCCCCGGCGCCTACAACGTCA
ACATCAAGCTCGACATCACCTCCCACAACGAGGACTACACCATCGTCGAGCAGTACGAGCGCG
CCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTCTACAAGTGATAATAGGCTGGAGCCT
CGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTA
CCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC
Example 13. Transfection in HeLa Cells
[0608] The day before transfection, 20,000 HeLa cells (ATCC no.
CCL-2; Manassas, Va.) were harvested by treatment with Trypsin-EDTA
solution (LifeTechnologies, Grand Island, N.Y.) and seeded in a
total volume of 100 .mu.l EMEM medium (supplemented with 10% FCS
and 1.times. Glutamax) per well in a 96-well cell culture plate
(Corning, Manassas, Va.). The cells were grown at 37.degree. C. in
5% CO.sub.2 atmosphere overnight. Next day, 83 ng of Luciferase
modRNA or 250 ng of human GCSF modRNA, harboring chemical
alterations on the bases or the ribose units, were diluted in 10
.mu.L final volume of OPTI-MEM (LifeTechnologies, Grand Island,
N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)
was used as transfection reagent and 0.2 .mu.L were diluted in 10
.mu.L final volume of OPTI-MEM. After 5 min incubation at room
temperature, both solutions were combined and incubated additional
15 min at room temperature. Then the 20 .mu.L were added to the 100
.mu.L cell culture medium containing the HeLa cells. The plates
were then incubated as described before. For transfection with
mCherry or nanoLuc, a mixture of mRNA expressing mCherry or nanoLuc
is mixed with 0.5 .mu.L of Lipofectamine2000 (Life Technologies;
cat#11668019) and OptiMem (Life Tehnologies; cat#31985062). A final
volume of 20 .mu.L of this mixture is added to 100 .mu.L of cells.
The final amount of human EPO, G-CSF, Firefly Luciferase, mCherry
and nanoLuc mRNA used per well is 250 ng except for nanoLuc mRNA
which we used at 25 ng per well, respectively.
[0609] After 18 h to 22 h incubation, cells expressing luciferase
were lysed with 100 .mu.l Passive Lysis Buffer (Promega, Madison,
Wis.) according to manufacturer instructions. Aliquots of the
lysates were transferred to white opaque polystyrene 96-well plates
(Corning, Manassas, Va.) and combined with 100 .mu.L complete
luciferase assay solution (Promega, Madison, Wis.). The lysate
volumes were adjusted or diluted until no more than 2 mio relative
light units per well were detected for the strongest signal
producing samples. The background signal of the plates without
reagent was about 200 relative light units per well. The plate
reader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The results
are shown in Table 13.
[0610] For the cells transfected with mCherry, mCherry fluorescence
reading was measured directly of the cells at excitation of 585 nm
and emission of 615 nm wavelength. The results are shown in Table
14.
[0611] After 18 h to 22 h incubation, cell culture supernatants of
cells expressing human EPO were collected and centrifuged at 10,000
rcf for 2 min. The cleared supernatants were transferred and
analyzed with a human GCSF-specific or EPO ELISA kit (both from
R&D Systems, Minneapolis, Minn.; Cat. #s SCS50, DEPOO,
respectively) according to the manufacturer instructions. All
samples were diluted until the determined values were within the
linear range of the human EPO ELISA standard curve. The results are
shown in Table 15.
TABLE-US-00011 TABLE 13 Expression of FFLuc in HeLa cells Construct
FFLuc expression (RLU; 24 hrs) 1-methyl pseudo U (DNA2.0) 78300
1-methyl pseudo U (CO7) 115150 5-methoxy-uridine (CO3) 162900
5-methoxy-uridine (CO7) 68550 5-methoxy-uridine (CO9) 93150
TABLE-US-00012 TABLE 14 Expression of mCherry in HeLa cells
Construct mCherry expression (FLU; 24 hrs) 1-methyl pseudo U (WT)
1137 1-methyl pseudo U (CO7) 2229 5-methoxy-uridine (CO3) 1464
5-methoxy-uridine (CO7) 3007 5-methoxy-uridine (CO9) 4344
TABLE-US-00013 TABLE 15 Expression of hEPO in HeLa cells hEPO
expression in HeLa Construct (mIU/mL; 24 hours) 1-methyl pseudo U
(DNA2.0) 250537 5-methoxy-uridine (CO3) 253718 5-methoxy-uridine
(CO7) 290925 5-methoxy-uridine (CO9) 123977
Example 14. Transfection in BJ Fibroblasts
[0612] At 2 or 3 days prior to transfection, 100,000 BJ fibroblast
cells (ATCC no. CRL-2522; Manassas, Va.) were harvested by
treatment with trypsin-EDTA solution (LifeTechnologies, Grand
Island, N.Y.) and seeded in a total volume of 500 .mu.L EMEM medium
(supplemented with 10% FCS and 10% Glutamax, both LifeTechnologies,
Grand Island, N.Y.) per well in 24-well cell culture plates
(Corning, Manassas, Va.). The cells were grown at 37.degree. C. in
a 5% CO.sub.2 atmosphere overnight. On the next day, 500 ng modRNA,
harboring chemical alterations on the bases or the ribose units,
were diluted in 25 .mu.L final volume of OPTI-MEM
(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000
(LifeTechnologies, Grand Island, N.Y.) was used as transfection
reagent and 1.0 .mu.L was diluted in 25 .mu.L final volume of
OPTI-MEM. After 5 min incubation at room temperature, both
solutions were combined and incubated an additional 15 min at room
temperature. The 50 .mu.L were added to the 500 .mu.L cell culture
medium containing the BJ fibroblast cells. The plates were then
incubated as described above.
[0613] After 18 h to 22 h incubation, cell culture supernatants of
cells expressing human GCSF or human EPO were collected and
centrifuged at 10,000 rcf for 2 min. The cleared supernatants were
transferred and analyzed with a human GCSF-specific or EPO ELISA
kit (both from R&D Systems, Minneapolis, Minn.; Cat. #s SCS50,
DEPOO, respectively) according to the manufacturer instructions.
All samples were diluted until the determined values were within
the linear range of the human GCSF or EPO ELISA standard curve. The
results are shown in Tables 16, 17, and 18.
TABLE-US-00014 TABLE 16 Expression of hEPO in BJ Fibroblast cells
hEPO expression in BJ Construct (mIU/mL; 48 hours) 1-methyl pseudo
U (DNA2.0) 153713 5-methoxy-uridine (CO3) 146050 5-methoxy-uridine
(CO7) 158195 5-methoxy-uridine (CO9) 68986
TABLE-US-00015 TABLE 17 Expression of GCSF in BJ Fibroblast cells
(25 ng/well) Construct GCSF expression in BJ (pg/mL; 48 hours)
1-methyl pseudo U (DNA2.0) 153172 5-methoxy-uridine (CO3) 366060
5-methoxy-uridine (CO7) 190776 5-methoxy-uridine (CO9) 119084
TABLE-US-00016 TABLE 18 Expression of GCSF in BJ Fibroblast cells
(15 ng/well) Construct GCSF expression in BJ (pg/mL; 48 hours)
1-methyl pseudo U (DNA2.0) 357902 5-methoxy-uridine (CO3) 766241
5-methoxy-uridine (CO7) 555814 5-methoxy-uridine (CO9) 330441
Example 15. Cytokine Screen in BJ Fibroblast Cells
[0614] At 2 or 3 days prior to transfection, 100,000 BJ fibroblast
cells (ATCC no. CRL-2522; Manassas, Va.) were harvested by
treatment with trypsin-EDTA solution (LifeTechnologies, Grand
Island, N.Y.) and seeded in a total volume of 500 ul EMEM medium
(supplemented with 10% FCS and 10% Glutamax, both LifeTechnologies,
Grand Island, N.Y.) per well in 24-well cell culture plates
(Corning, Manassas, Va.). The cells were grown at 37.degree. C. in
a 5% CO.sub.2 atmosphere overnight. On the next day, 500 ng modRNA,
harboring chemical alterations on the bases or the ribose units,
were diluted in 25 .mu.L final volume of OPTI-MEM
(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000
(LifeTechnologies, Grand Island, N.Y.) was used as transfection
reagent and 1.0 .mu.L was diluted in 25 .mu.L final volume of
OPTI-MEM. After 5 min incubation at room temperature, both
solutions were combined and incubated an additional 15 min at room
temperature. The 50 .mu.L were added to the 500 .mu.L cell culture
medium containing the BJ fibroblast cells. The plates were then
incubated as described above.
[0615] After 18 h to 22 h incubation, cell culture supernatants
were collected and centrifuged at 10,000 rcf for 2 min. The cleared
supernatants were transferred and analyzed with a human IFN-beta
ELISA (R&D Systems, Minneapolis, Minn.; Cat. #s 41410-2) and
human CCL-5/RANTES ELISA (R&D Systems, Minneapolis, Minn.; Cat.
#s SRNOOB) according to the manufacturer instructions. All samples
were diluted until the determined values were within the linear
range of the ELISA standard curves using a BioTek Synergy H1 plate
reader (BioTek, Winooski, Vt.). The results are shown in Tables 19,
20, and 21.
TABLE-US-00017 TABLE 19 INF.beta. expression in BJ Fibroblast cells
by GCSF mRNA Construct IFN-b expression in BJ (pg/mL; 48 hours)
1-methyl pseudo U (DNA2.0) 20 5-methoxy-uridine (CO3) 0
5-methoxy-uridine (CO7) 0 5-methoxy-uridine (CO9) 0
TABLE-US-00018 TABLE 20 INF.beta. expression in BJ Fibroblast cells
by FFLuc mRNA Construct IFN-b expression in BJ (pg/mL; 48 hours)
1-methyl pseudo U (CO7) 194 5-methoxy-uridine (CO3) 0
5-methoxy-uridine (CO7) 0 5-methoxy-uridine (CO9) 0
TABLE-US-00019 TABLE 21 INF.beta. expression in BJ Fibroblast cells
by mCherry mRNA Construct IFN-b expression in BJ (pg/mL; 48 hours)
1-methyl pseudo U (CO7) 309 5-methoxy-uridine (CO3) 0
5-methoxy-uridine (CO7) 0 5-methoxy-uridine (CO9) 0
Example 16. In Vivo Expression of mRNA
[0616] Using the method described in Example 11, in vivo expression
of the alternative mRNA of Example 12 was studied. Female CD-1 mice
were administered the mRNAs intravenously at 0.05 mg/kg. The
results are shown in Tables 22, 23, and 24.
TABLE-US-00020 TABLE 22 In vivo expression of GCSF Dose group (0.05
mg/kg) GCSF @ 6 hours (ng/mL) 1-methyl pseudo (WT) 71.4
5-methoxy-uridine (CO3) 32.5 5-methoxy-uridine (CO7) 8.6
5-methoxy-uridine (CO9) 30.7
TABLE-US-00021 TABLE 23 In vivo expression of Luciferase Dose group
(0.05 mg/kg) Total flux @ 6 hours (RLU) 1-methyl pseudo (DNA2.0)
2.38 .times. 10.sup.8 1-methyl pseudo (CO7) 1.40 .times. 10.sup.9
5-methoxy-uridine (CO3) 5.26 .times. 10.sup.8 5-methoxy-uridine
(CO7) 1.86 .times. 10.sup.8
TABLE-US-00022 TABLE 24 In vivo expression of GCSF GCSF @ GCSF @ 3
hours GCSF @ 6 hours 24 hours Dose group (0.05 mg/kg) (ng/mL)
(ng/mL) (ng/mL) 1-methyl pseudoU 409.6 546.0 168.2 (DNA2.0)
1-methyl pseudoU 517.9 637.4 274.0 (CO3) 1-methyl pseudoU 355.8
473.5 220.2 (CO7) 1-methyl pseudoU 547.7 726.3 124.5 (CO9)
5-methoxy-uridine (CO3) 234.1 277.8 64.6 5-methoxy-uridine (CO7)
308.6 341.9 83.3 5-methoxy-uridine (CO9) 253.9 285.3 51.9
Example 17. In Vivo Expression of mRNA in Non-Human Primates
[0617] Cynomolgus monkeys are administered a standard MC3
formulation including an mRNA encoding hEPO. The expression of hEPO
was measured using an ELISA method before and 2, 6, 24, 48, 72, 96,
and 120 hours after administration. Male monkeys were administered
the formulation at a dose rate of 5 mL/kg/h for 1 hour.
TABLE-US-00023 TABLE 25 In vivo expression of hEPO Dose group (0.05
mg/kg) hEPO Cmax (ng/mL) AUC (hr * pg/mL) 1-methyl pseudo (DNA2.0)
70.0 954954 5-methoxy-uridine (CO9) 50.6 984832
TABLE-US-00024 TABLE 26 In vivo expression of hEPO hEPO @ Dose hEPO
@ 6 hours hEPO @ 12 hours 24 hours group (0.05 mg/kg) (ng/mL)
(ng/mL) (ng/mL) 1-methyl pseudoU 72.7 14.7 2.1 (DNA2.0)
5-methoxy-uridine 87.1 62.6 18.9 (CO9)
Example 18. mRNA-Templated In Vitro Transcription
[0618] Human Epo 1-methylpseudouridine-containing mRNA was produced
by run-off in vitro transcription using standard 4 h plasmid-based
IVT reaction conditions. The material was subjected to reverse
phase purification, and the INF-.beta. clear fractions were pooled.
From this pooled material, a standard
1-methylpseudouridine-containing and 5-methoxy-uridine-containing 4
h plasmid-based IVT reaction was run but in place of DNA template,
INF-.beta. clear mRNA was added to a final concentration of 1
mg/mL. After 4 h, the reaction was split in two, with part being
used for LC analysis and part to be transfected into BJ fibroblasts
using L-2000 according to the manufacturer suggested protocol.
After 48 hours, the presence of INF-.beta. was determined by ELISA.
LC analysis showed the presence of n+1 polymers to be in much
higher abundance in the samples incubated with
1-methylpseudouridine-containing nucleotides compared to
5-methoxy-uridine-containing nucleotides. Additionally, the
1-methylpseudouridine-containing sample showed significantly more
INF-.beta. response compared to the 5-methoxy-uridine-containing
sample.
Example 19. In Vitro Transcription Temperature Dependence
[0619] Human Epo 1-methylpseudouridine-containing and
5-methoxy-uridine-containing mRNA was produced by run-off in vitro
transcription using our standard 4 h plasmid-based IVT reaction
conditions. The mRNA was split and part was subjected to oligo dT
purification whereas the other part was crude reaction mixture.
Both were transfected into BJ fibroblasts using L-2000, and
INF-.beta. levels were determined by ELISA. The dT purified and
crude 5-methoxy-uridine-containing mRNA showed marginal INF-.beta.
whether the IVT was performed at 25.degree. C. or 37.degree. C.,
whereas the 1-methylpseudouridine-containing mRNA showed
significant increases in INF-8 induction at 25.degree. C. compared
to 37.degree. C. The results are shown in FIG. 3.
Example 20. Production of mRNA with a 20 Consecutive Uridine
Tail
[0620] A reverse PCR primer was designed to code for an mRNA with a
tail structure of 100A20U-3'. PCR was completed as previously
described, and run-off IVT was performed according to PCR-templated
4 h IVT conditions under either 1-methylpseudouridine-containing
mRNA or 5-methoxy-uridine-containing mRNA conditions. The IVT was
reverse-phase purified, the fractions were diafiltered into water,
and transfected into BJ fibroblasts using L-2000. After 48 hours,
INF-8 levels were determined by ELISA. The results are shown in
FIG. 4.
OTHER EMBODIMENTS
[0621] 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
alterations are within the scope of the following claims.
Sequence CWU 1
1
17110DNAArtificial Sequence3'-U Rich Region 1tttttctttt
10211DNAArtificial Sequence3'-U Rich Region 2ttttgctttt t
11310DNAArtificial Sequence3'-U Rich Region 3ttttgctttt
10411DNAArtificial Sequence3'-A Rich Region 4aaaaagcaaa a
115796DNAArtificial SequenceSynthetic Construct 5tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatgggagt gcacgagtgt cccgcgtggt
120tgtggttgct gctgtcgctc ttgagcctcc cactgggact gcctgtgctg
ggggcaccac 180ccagattgat ctgcgactca cgggtacttg agaggtacct
tcttgaagcc aaagaagccg 240aaaacatcac aaccggatgc gccgagcact
gctccctcaa tgagaacatt actgtaccgg 300atacaaaggt caatttctat
gcatggaaga gaatggaagt aggacagcag gccgtcgaag 360tgtggcaggg
gctcgcgctt ttgtcggagg cggtgttgcg gggtcaggcc ctcctcgtca
420actcatcaca gccgtgggag cccctccaac ttcatgtcga taaagcggtg
tcggggctcc 480gcagcttgac gacgttgctt cgggctctgg gcgcacaaaa
ggaggctatt tcgccgcctg 540acgcggcctc cgcggcaccc ctccgaacga
tcaccgcgga cacgtttagg aagcttttta 600gagtgtacag caatttcctc
cgcggaaagc tgaaattgta tactggtgaa gcgtgtagga 660caggggatcg
ctgataatag gctggagcct cggtggccat gcttcttgcc ccttgggcct
720ccccccagcc cctcctcccc ttcctgcacc cgtacccccg tggtctttga
ataaagtctg 780agtgggcggc tctaga 7966790DNAArtificial
SequenceSynthetic Construct 6tcaagctttt ggaccctcgt acagaagcta
atacgactca ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca
ccatgggcgt gcacgagtgc cccgcctggc 120tgtggctgct gctgagcctg
ctgagcctgc ccctgggcct gcccgtgctg ggcgcccccc 180cccgcctcat
ctgcgactcc cgcgtcctcg agcgctacct cctcgaggcc aaggaggccg
240agaacatcac caccggctgc gccgagcact gctccctcaa cgagaacatc
accgtccccg 300acaccaaggt caacttctac gcctggaagc gcatggaggt
cggccagcag gccgtcgagg 360tctggcaggg cctcgccctc ctctccgagg
ccgtcctccg cggccaggcc ctcctcgtca 420actcctccca gccctgggag
cccctccagc tccacgtcga caaggccgtc tccggcctcc 480gctccctcac
caccctcctc cgcgccctcg gcgcccagaa ggaggccatc tccccccccg
540acgccgcctc cgccgccccc ctccgcacca tcaccgccga caccttccgc
aagctcttcc 600gcgtctactc caacttcctc cgcggcaagc tcaagctcta
caccggcgag gcctgccgca 660ccggcgaccg ctgataatag gctggagcct
cggtggccat gcttcttgcc ccttgggcct 720ccccccagcc cctcctcccc
ttcctgcacc cgtacccccg tggtctttga ataaagtctg 780agtgggcggc
7907823DNAArtificial SequenceSynthetic Construct 7tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggccgg ccccgccacc cagagcccca
120tgaagctgat ggccctgcag ctgctgctgt ggcacagcgc cctgtggacc
gtgcaggagg 180ccacaccttt aggacctgct tcttctttac ctcaatcttt
tttattaaaa tgtttagaac 240aagttagaaa aattcaagga gatggagctg
ctttacaaga aaaattatgt gctacatata 300aattatgtca tcctgaagaa
ttagttttat taggacattc tttaggaatt ccttgggctc 360ctttatcttc
ttgtccttct caagctttac aattagctgg atgtttatct caattacatt
420ctggattatt tttatatcaa ggattattac aagctttaga aggaatttct
cctgaattag 480gacctacatt agatacatta caattagatg ttgctgattt
tgctacaaca atttggcaac 540aaatggaaga attaggaatg gctcctgctt
tacaacctac acaaggagct atgcctgctt 600ttgcttctgc ttttcaaaga
agagctggag gagttttagt tgcttctcat ttacaatctt 660ttttagaagt
ttcttataga gttttaagac atttagctca accttgataa taggctggag
720cctcggtggc catgcttctt gccccttggg cctcccccca gcccctcctc
cccttcctgc 780acccgtaccc ccgtggtctt tgaataaagt ctgagtgggc ggc
8238823DNAArtificial SequenceSynthetic Construct 8tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggccgg ccccgccacc cagagcccca
120tgaagctgat ggccctgcag ctgctgctgt ggcacagcgc cctgtggacc
gtgcaggagg 180ccacccccct gggccccgcc agcagcctgc cccagagctt
cctgctgaag tgcctggagc 240aggtgcggaa gatccagggc gacggcgccg
ccctgcagga gaagctgtgc gccacctaca 300agctgtgcca ccccgaggag
ctggtgctgc tgggccacag cctgggcatc ccctgggccc 360ccctgagcag
ctgccccagc caggccctgc agctggccgg ctgcctgagc cagctgcaca
420gcggcctgtt cctgtaccag ggcctgctgc aggccctgga gggcatcagc
cccgagctgg 480gccccaccct ggacaccctg cagctggacg tggccgactt
cgccaccacc atctggcagc 540agatggagga gctgggcatg gcccccgccc
tgcagcccac ccagggcgcc atgcccgcct 600tcgccagcgc cttccagcgg
cgggccggcg gcgtgctggt ggccagccac ctgcagagct 660tcctggaggt
gagctaccgg gtgctgcggc acctggccca gccctgataa taggctggag
720cctcggtggc catgcttctt gccccttggg cctcccccca gcccctcctc
cccttcctgc 780acccgtaccc ccgtggtctt tgaataaagt ctgagtgggc ggc
8239823DNAArtificial SequenceSynthetic Construct 9tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggccgg ccccgccacc cagagcccca
120tgaagctgat ggccctgcag ctgctgctgt ggcacagcgc cctgtggacc
gtgcaggagg 180ccacgccgct ggggccggcg agcagcctgc cgcagagctt
cctgctgaag tgcctggagc 240aggtgaggaa gatccagggg gacggggcgg
cgctgcagga gaagctgtgc gcgacgtaca 300agctgtgcca cccggaggag
ctggtgctgc tggggcacag cctggggatc ccgtgggcgc 360cgctgagcag
ctgcccgagc caggcgctgc agctggcggg gtgcctgagc cagctgcaca
420gcgggctgtt cctgtaccag gggctgctgc aggcgctgga ggggatcagc
ccggagctgg 480ggccgacgct ggacacgctg cagctggacg tggcggactt
cgcgacgacg atctggcagc 540agatggagga gctggggatg gcgccggcgc
tgcagccgac gcagggggcg atgccggcgt 600tcgcgagcgc gttccagagg
agggcggggg gggtgctggt ggcgagccac ctgcagagct 660tcctggaggt
gagctacagg gtgctgaggc acctggcgca gccgtgataa taggctggag
720cctcggtggc catgcttctt gccccttggg cctcccccca gcccctcctc
cccttcctgc 780acccgtaccc ccgtggtctt tgaataaagt ctgagtgggc ggc
82310823DNAArtificial SequenceSynthetic Construct 10tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggccgg ccccgccacc cagagcccca
120tgaagctgat ggccctgcag ctgctgctgt ggcacagcgc cctgtggacc
gtgcaggagg 180ccacccccct cggccccgcc tcctccctcc cccagtcctt
cctcctcaag tgcctcgagc 240aggtccgcaa gatccagggc gacggcgccg
ccctccagga gaagctctgc gccacctaca 300agctctgcca ccccgaggag
ctcgtcctcc tcggccactc cctcggcatc ccctgggccc 360ccctctcctc
ctgcccctcc caggccctcc agctcgccgg ctgcctctcc cagctccact
420ccggcctctt cctctaccag ggcctcctcc aggccctcga gggcatctcc
cccgagctcg 480gccccaccct cgacaccctc cagctcgacg tcgccgactt
cgccaccacc atctggcagc 540agatggagga gctcggcatg gcccccgccc
tccagcccac ccagggcgcc atgcccgcct 600tcgcctccgc cttccagcgc
cgcgccggcg gcgtcctcgt cgcctcccac ctccagtcct 660tcctcgaggt
ctcctaccgc gtcctccgcc acctcgccca gccctgataa taggctggag
720cctcggtggc catgcttctt gccccttggg cctcccccca gcccctcctc
cccttcctgc 780acccgtaccc ccgtggtctt tgaataaagt ctgagtgggc ggc
823111861DNAArtificial SequenceSynthetic Construct 11tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggaaga tgcgaagaac atcaagaagg
120gacctgcccc gttttaccct ttggaggacg gtacagcagg agaacagctc
cacaaggcga 180tgaaacgcta cgccctggtc cccggaacga ttgcgtttac
cgatgcacat attgaggtag 240acatcacata cgcagaatac ttcgaaatgt
cggtgaggct ggcggaagcg atgaagagat 300atggtcttaa cactaatcac
cgcatcgtgg tgtgttcgga gaactcattg cagtttttca 360tgccggtcct
tggagcactt ttcatcgggg tcgcagtcgc gccagcgaac gacatctaca
420atgagcggga actcttgaat agcatgggaa tctcccagcc gacggtcgtg
tttgtctcca 480aaaaggggct gcagaaaatc ctcaacgtgc agaagaagct
ccccattatt caaaagatca 540tcattatgga tagcaagaca gattaccaag
ggttccagtc gatgtatacc tttgtgacat 600cgcatttgcc gccagggttt
aacgagtatg acttcgtccc cgagtcattt gacagagata 660aaaccatcgc
gctgattatg aattcctcgg gtagcaccgg tttgccaaag ggggtggcgt
720tgccccaccg cactgcttgt gtgcggttct cgcacgctag ggatcctatc
tttggtaatc 780agatcattcc cgacacagca atcctgtccg tggtaccttt
tcatcacggt tttggcatgt 840tcacgactct cggctatttg atttgcggtt
tcagggtcgt acttatgtat cggttcgagg 900aagaactgtt tttgagatcc
ttgcaagatt acaagatcca gtcggccctc cttgtgccaa 960cgcttttctc
attctttgcg aaatcgacac ttattgataa gtatgacctt tccaatctgc
1020atgagattgc ctcaggggga gcgccgctta gcaaggaagt cggggaggca
gtggccaagc 1080gcttccacct tcccggaatt cggcagggat acgggctcac
ggagacaaca tccgcgatcc 1140ttatcacgcc cgagggtgac gataagccgg
gagccgtcgg aaaagtggtc cccttctttg 1200aagccaaggt cgtagacctc
gacacgggaa aaaccctcgg agtgaaccag aggggcgagc 1260tctgcgtgag
agggccgatg atcatgtcag gttacgtgaa taaccctgaa gcgacgaatg
1320cgctgatcga caaggatggg tggttgcatt cgggagacat tgcctattgg
gatgaggatg 1380agcacttctt tatcgtagat cgacttaaga gcttgatcaa
atacaaaggc tatcaggtag 1440cgcctgccga gctcgagtca atcctgctcc
agcaccccaa cattttcgac gccggagtgg 1500ccgggttgcc cgatgacgac
gcgggtgagc tgccagcggc cgtggtagtc ctcgaacatg 1560ggaaaacaat
gaccgaaaag gagatcgtgg actacgtagc atcacaagtg acgactgcga
1620agaaactgag gggaggggta gtctttgtgg acgaggtccc gaaaggcttg
actgggaagc 1680ttgacgctcg caaaatccgg gaaatcctga ttaaggcaaa
gaaaggcggg aaaatcgctg 1740tctgataata ggctggagcc tcggtggcca
tgcttcttgc cccttgggcc tccccccagc 1800ccctcctccc cttcctgcac
ccgtaccccc gtggtctttg aataaagtct gagtgggcgg 1860c
1861121861DNAArtificial SequenceSynthetic Construct 12tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggagga cgccaagaac atcaagaagg
120gccccgcccc cttctacccc ctggaggacg gcaccgccgg cgagcagctg
cacaaggcca 180tgaagcggta cgccctggtg cccggcacca tcgccttcac
cgacgcccac atcgaggtgg 240acatcaccta cgccgagtac ttcgagatga
gcgtgcggct ggccgaggcc atgaagcggt 300acggcctgaa caccaaccac
cggatcgtgg tgtgcagcga gaacagcctg cagttcttca 360tgcccgtgct
gggcgccctg ttcatcggcg tggccgtggc ccccgccaac gacatctaca
420acgagcggga gctgctgaac agcatgggca tcagccagcc caccgtggtg
ttcgtgagca 480agaagggcct gcagaagatc ctgaacgtgc agaagaagct
gcccatcatc cagaagatca 540tcatcatgga cagcaagacc gactaccagg
gcttccagag catgtacacc ttcgtgacca 600gccacctgcc ccccggcttc
aacgagtacg acttcgtgcc cgagagcttc gaccgggaca 660agaccatcgc
cctgatcatg aacagcagcg gcagcaccgg cctgcccaag ggcgtggccc
720tgccccaccg gaccgcctgc gtgcggttca gccacgcccg ggaccccatc
ttcggcaacc 780agatcatccc cgacaccgcc atcctgagcg tggtgccctt
ccaccacggc ttcggcatgt 840tcaccaccct gggctacctg atctgcggct
tccgggtggt gctgatgtac cggttcgagg 900aggagctgtt cctgcggagc
ctgcaggact acaagatcca gagcgccctg ctggtgccca 960ccctgttcag
cttcttcgcc aagagcaccc tgatcgacaa gtacgacctg agcaacctgc
1020acgagatcgc cagcggcggc gcccccctga gcaaggaggt gggcgaggcc
gtggccaagc 1080ggttccacct gcccggcatc cggcagggct acggcctgac
cgagaccacc agcgccatcc 1140tgatcacccc cgagggcgac gacaagcccg
gcgccgtggg caaggtggtg cccttcttcg 1200aggccaaggt ggtggacctg
gacaccggca agaccctggg cgtgaaccag cggggcgagc 1260tgtgcgtgcg
gggccccatg atcatgagcg gctacgtgaa caaccccgag gccaccaacg
1320ccctgatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg
gacgaggacg 1380agcacttctt catcgtggac cggctgaaga gcctgatcaa
gtacaagggc taccaggtgg 1440cccccgccga gctggagagc atcctgctgc
agcaccccaa catcttcgac gccggcgtgg 1500ccggcctgcc cgacgacgac
gccggcgagc tgcccgccgc cgtggtggtg ctggagcacg 1560gcaagaccat
gaccgagaag gagatcgtgg actacgtggc cagccaggtg accaccgcca
1620agaagctgcg gggcggcgtg gtgttcgtgg acgaggtgcc caagggcctg
accggcaagc 1680tggacgcccg gaagatccgg gagatcctga tcaaggccaa
gaagggcggc aagatcgccg 1740tgtgataata ggctggagcc tcggtggcca
tgcttcttgc cccttgggcc tccccccagc 1800ccctcctccc cttcctgcac
ccgtaccccc gtggtctttg aataaagtct gagtgggcgg 1860c
1861131861DNAArtificial SequenceSynthetic Construct 13tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggagga cgccaagaac atcaagaagg
120gccccgcccc cttctacccc ctggaggacg gcaccgccgg cgagcagctg
cacaaggcca 180tgaagaggta cgcgctggtg ccggggacga tcgcgttcac
ggacgcgcac atcgaggtgg 240acatcacgta cgcggagtac ttcgagatga
gcgtgaggct ggcggaggcg atgaagaggt 300acgggctgaa cacgaaccac
aggatcgtgg tgtgcagcga gaacagcctg cagttcttca 360tgccggtgct
gggggcgctg ttcatcgggg tggcggtggc gccggcgaac gacatctaca
420acgagaggga gctgctgaac agcatgggga tcagccagcc gacggtggtg
ttcgtgagca 480agaaggggct gcagaagatc ctgaacgtgc agaagaagct
gccgatcatc cagaagatca 540tcatcatgga cagcaagacg gactaccagg
ggttccagag catgtacacg ttcgtgacga 600gccacctgcc gccggggttc
aacgagtacg acttcgtgcc ggagagcttc gacagggaca 660agacgatcgc
gctgatcatg aacagcagcg ggagcacggg gctgccgaag ggggtggcgc
720tgccgcacag gacggcgtgc gtgaggttca gccacgcgag ggacccgatc
ttcgggaacc 780agatcatccc ggacacggcg atcctgagcg tggtgccgtt
ccaccacggg ttcgggatgt 840tcacgacgct ggggtacctg atctgcgggt
tcagggtggt gctgatgtac aggttcgagg 900aggagctgtt cctgaggagc
ctgcaggact acaagatcca gagcgcgctg ctggtgccga 960cgctgttcag
cttcttcgcg aagagcacgc tgatcgacaa gtacgacctg agcaacctgc
1020acgagatcgc gagcgggggg gcgccgctga gcaaggaggt gggggaggcg
gtggcgaaga 1080ggttccacct gccggggatc aggcaggggt acgggctgac
ggagacgacg agcgcgatcc 1140tgatcacgcc ggagggggac gacaagccgg
gggcggtggg gaaggtggtg ccgttcttcg 1200aggcgaaggt ggtggacctg
gacacgggga agacgctggg ggtgaaccag aggggggagc 1260tgtgcgtgag
ggggccgatg atcatgagcg ggtacgtgaa caacccggag gcgacgaacg
1320cgctgatcga caaggacggg tggctgcaca gcggggacat cgcgtactgg
gacgaggacg 1380agcacttctt catcgtggac aggctgaaga gcctgatcaa
gtacaagggg taccaggtgg 1440cgccggcgga gctggagagc atcctgctgc
agcacccgaa catcttcgac gcgggggtgg 1500cggggctgcc ggacgacgac
gcgggggagc tgccggcggc ggtggtggtg ctggagcacg 1560ggaagacgat
gacggagaag gagatcgtgg actacgtggc gagccaggtg acgacggcga
1620agaagctgag ggggggggtg gtgttcgtgg acgaggtgcc gaaggggctg
acggggaagc 1680tggacgcgag gaagatcagg gagatcctga tcaaggcgaa
gaaggggggg aagatcgcgg 1740tgtgataata ggctggagcc tcggtggcca
tgcttcttgc cccttgggcc tccccccagc 1800ccctcctccc cttcctgcac
ccgtaccccc gtggtctttg aataaagtct gagtgggcgg 1860c
186114919DNAArtificial SequenceSynthetic Construct 14tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggtatc caagggggag gaggacaaca
120tggcgatcat caaggagttc atgcgattca aggtgcacat ggaaggttcg
gtcaacggac 180acgaatttga aatcgaagga gagggtgaag gaaggcccta
tgaagggaca cagaccgcga 240aactcaaggt cacgaaaggg ggaccacttc
ctttcgcctg ggacattctt tcgccccagt 300ttatgtacgg gtccaaagca
tatgtgaagc atcccgccga tattcctgac tatctgaaac 360tcagctttcc
cgagggattc aagtgggagc gggtcatgaa ctttgaggac gggggtgtag
420tcaccgtaac ccaagactca agcctccaag acggcgagtt catctacaag
gtcaaactgc 480gggggactaa ctttccgtcg gatgggccgg tgatgcagaa
gaaaacgatg ggatgggaag 540cgtcatcgga gaggatgtac ccagaagatg
gtgcattgaa gggggagatc aagcagagac 600tgaagttgaa agatggggga
cattatgatg ccgaggtgaa aacgacatac aaagcgaaaa 660agccggtgca
gcttcccgga gcgtataatg tgaatatcaa gttggatatt acttcacaca
720atgaggacta cacaattgtc gaacagtacg aacgcgctga gggtagacac
tcgacgggag 780gcatggacga gttgtacaaa tgataatagg ctggagcctc
ggtggccatg cttcttgccc 840cttgggcctc cccccagccc ctcctcccct
tcctgcaccc gtacccccgt ggtctttgaa 900taaagtctga gtgggcggc
91915919DNAArtificial SequenceSynthetic Construct 15tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggtgag caagggcgag gaggacaaca
120tggccatcat caaggagttc atgcggttca aggtgcacat ggagggcagc
gtgaacggcc 180acgagttcga gatcgagggc gagggcgagg gccggcccta
cgagggcacc cagaccgcca 240agctgaaggt gaccaagggc ggccccctgc
ccttcgcctg ggacatcctg agcccccagt 300tcatgtacgg cagcaaggcc
tacgtgaagc accccgccga catccccgac tacctgaagc 360tgagcttccc
cgagggcttc aagtgggagc gggtgatgaa cttcgaggac ggcggcgtgg
420tgaccgtgac ccaggacagc agcctgcagg acggcgagtt catctacaag
gtgaagctgc 480ggggcaccaa cttccccagc gacggccccg tgatgcagaa
gaagaccatg ggctgggagg 540ccagcagcga gcggatgtac cccgaggacg
gcgccctgaa gggcgagatc aagcagcggc 600tgaagctgaa ggacggcggc
cactacgacg ccgaggtgaa gaccacctac aaggccaaga 660agcccgtgca
gctgcccggc gcctacaacg tgaacatcaa gctggacatc accagccaca
720acgaggacta caccatcgtg gagcagtacg agcgggccga gggccggcac
agcaccggcg 780gcatggacga gctgtacaag tgataatagg ctggagcctc
ggtggccatg cttcttgccc 840cttgggcctc cccccagccc ctcctcccct
tcctgcaccc gtacccccgt ggtctttgaa 900taaagtctga gtgggcggc
91916919DNAArtificial SequenceSynthetic Construct 16tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggtgag caagggcgag gaggacaaca
120tggccatcat caaggagttc atgcggttca aggtgcacat ggagggcagc
gtgaacggcc 180acgagttcga gatcgagggg gagggggagg ggaggccgta
cgaggggacg cagacggcga 240agctgaaggt gacgaagggg gggccgctgc
cgttcgcgtg ggacatcctg agcccgcagt 300tcatgtacgg gagcaaggcg
tacgtgaagc acccggcgga catcccggac tacctgaagc 360tgagcttccc
ggaggggttc aagtgggaga gggtgatgaa cttcgaggac gggggggtgg
420tgacggtgac gcaggacagc agcctgcagg acggggagtt catctacaag
gtgaagctga 480gggggacgaa cttcccgagc gacgggccgg tgatgcagaa
gaagacgatg gggtgggagg 540cgagcagcga gaggatgtac ccggaggacg
gggcgctgaa gggggagatc aagcagaggc 600tgaagctgaa ggacgggggg
cactacgacg cggaggtgaa gacgacgtac aaggcgaaga 660agccggtgca
gctgccgggg gcgtacaacg tgaacatcaa gctggacatc acgagccaca
720acgaggacta cacgatcgtg gagcagtacg agagggcgga ggggaggcac
agcacggggg 780ggatggacga gctgtacaag tgataatagg ctggagcctc
ggtggccatg cttcttgccc 840cttgggcctc cccccagccc ctcctcccct
tcctgcaccc gtacccccgt ggtctttgaa 900taaagtctga gtgggcggc
91917919DNAArtificial SequenceSynthetic Construct 17tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggtgag caagggcgag gaggacaaca
120tggccatcat caaggagttc atgcggttca aggtgcacat ggagggcagc
gtgaacggcc 180acgagttcga gatcgagggc gagggcgagg gccgccccta
cgagggcacc cagaccgcca 240agctcaaggt caccaagggc ggccccctcc
ccttcgcctg ggacatcctc tccccccagt 300tcatgtacgg ctccaaggcc
tacgtcaagc accccgccga catccccgac tacctcaagc 360tctccttccc
cgagggcttc aagtgggagc gcgtcatgaa cttcgaggac ggcggcgtcg
420tcaccgtcac ccaggactcc tccctccagg acggcgagtt catctacaag
gtcaagctcc 480gcggcaccaa cttcccctcc gacggccccg tcatgcagaa
gaagaccatg ggctgggagg 540cctcctccga gcgcatgtac cccgaggacg
gcgccctcaa gggcgagatc aagcagcgcc 600tcaagctcaa ggacggcggc
cactacgacg ccgaggtcaa gaccacctac aaggccaaga 660agcccgtcca
gctccccggc gcctacaacg tcaacatcaa gctcgacatc acctcccaca
720acgaggacta caccatcgtc gagcagtacg agcgcgccga gggccgccac
tccaccggcg 780gcatggacga gctctacaag tgataatagg ctggagcctc
ggtggccatg cttcttgccc 840cttgggcctc cccccagccc ctcctcccct
tcctgcaccc gtacccccgt ggtctttgaa 900taaagtctga gtgggcggc 919
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