U.S. patent application number 14/406424 was filed with the patent office on 2015-09-24 for nuclease resistant polynucleotides and uses thereof.
The applicant listed for this patent is SHIRE HUMAN GENETIC THERAPIES, INC.. Invention is credited to Frank DeRosa, Braydon Charles Guild, Michael Heartlein.
Application Number | 20150267192 14/406424 |
Document ID | / |
Family ID | 49712695 |
Filed Date | 2015-09-24 |
United States Patent
Application |
20150267192 |
Kind Code |
A1 |
Heartlein; Michael ; et
al. |
September 24, 2015 |
NUCLEASE RESISTANT POLYNUCLEOTIDES AND USES THEREOF
Abstract
The invention provides, among other things, methods of mRNA
stabilizing mRNA and nuclease resistant mRNA prepared in accordance
with such methods. In certain embodiments, the nuclease resistant
mRNA encodes a functional protein, such as enzyme, and is
characterized by its resistance to nuclease digestion, increased
half-life and/or its ability to produce increased amounts of the
functional protein (e.g., enzyme) encoded thereby.
Inventors: |
Heartlein; Michael;
(Boxborough, MA) ; Guild; Braydon Charles;
(Concord, MA) ; DeRosa; Frank; (Chelmsford,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIRE HUMAN GENETIC THERAPIES, INC. |
Lexington |
MA |
US |
|
|
Family ID: |
49712695 |
Appl. No.: |
14/406424 |
Filed: |
June 7, 2013 |
PCT Filed: |
June 7, 2013 |
PCT NO: |
PCT/US13/44769 |
371 Date: |
December 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61657465 |
Jun 8, 2012 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
536/23.1; 536/23.51 |
Current CPC
Class: |
C07K 14/505 20130101;
C12N 2310/3519 20130101; C12N 2320/51 20130101; C07H 21/02
20130101; C07H 21/00 20130101; C12N 15/111 20130101; C12P 21/02
20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C07K 14/505 20060101 C07K014/505 |
Claims
1. A method of modulating the nuclease resistance of a
polynucleotide having a coding region and a non-coding region, the
method comprising a step of contacting the polynucleotide with a
stabilizing oligonucleotide, thereby modulating the nuclease
resistance of the polynucleotide, wherein the stabilizing
oligonucleotide is complementary to the non-coding region of the
polynucleotide and comprises at least one modified nucleobase.
2. (canceled)
3. The method of claim 1, wherein the RNA is mRNA.
4. The method of claim 1, wherein the non-coding region of the
polynucleotide is selected from the group of regions consisting of
a 3' untranslated region (UTR), a 5' untranslated region (UTR), a
poly-A tail, a terminal cap, and combination thereof.
5. The method of claim 1, wherein the non-coding region of the
polynucleotide comprises a poly(A) tail.
6. The method of claim 5, wherein the stabilizing oligonucleotide
comprises a poly-U sequence.
7. (canceled)
8. The method of claim 1, wherein the stabilizing oligonucleotide
is about 1 to about 50 nucleotides in length.
9-32. (canceled)
33. The method of claim 3, wherein the mRNA encodes a protein
selected from the group consisting of erythropoietin, human growth
hormone, cystic fibrosis transmembrane conductance regulator
(CFTR), alpha-galactosidase A, alpha-L-iduronidase,
iduronate-2-sulfatase, N-acetylglucosamine-1-phosphate transferase,
N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase,
N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase,
beta-glucosidase, galactose-6-sulfate sulfatase,
beta-galactosidase, beta-glucuronidase, glucocerebrosidase, heparan
sulfamidase, hyaluronidase, galactocerebrosidase, ornithine
transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1),
argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL),
and arginase 1 (ARG1).
34. The method of claim 1, wherein the stabilizing oligonucleotide
and polynucleotide are contacted at a ratio ranging between about
0.01:1 and about 100:1.
35-39. (canceled)
40. A nuclease resistant mRNA comprising mRNA having a coding
region and a non-coding region and a complementary stabilizing
oligonucleotide hybridized to at least a portion of the non-coding
region of the mRNA, wherein the stabilizing oligonucleotide
comprises at least one modified nucleobase, and wherein the
nuclease resistant mRNA is more resistant to nuclease degradation
relative to the un-hybridized mRNA.
41-64. (canceled)
65. A method of increasing translation of polypeptide from an mRNA
transcript having a coding region and a non-coding region, the
method comprising a step of hybridizing a stabilizing
oligonucleotide to a portion of the non-coding region of the mRNA
transcript thereby increasing amount of the polypeptide translated
from the mRNA transcript; and wherein the stabilizing
oligonucleotide comprises at least one modified nucleobase.
66. The method of claim 65, wherein the non-coding region of the
mRNA transcript is selected from the group of regions consisting of
a 3' untranslated region (UTR), a 5' untranslated region (UTR), a
poly-A tail and a terminal cap.
67-86. (canceled)
87. A method of increasing translation of an exogenous mRNA
transcript having a coding region and a non-coding region, the
method comprising a step of co-administering the exogenous mRNA
transcript with a stabilizing oligonucleotide into a cell; wherein
the stabilizing oligonucleotide is complementary to the non-coding
region of the mRNA transcript; wherein the co-administering results
in increased translation of the exogenous mRNA transcript; and
wherein the stabilizing oligonucleotide comprises at least one
modified nucleobase.
88-119. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/657,465, filed on Jun. 8, 2012, the disclosure
of which is incorporated herein by reference.
BACKGROUND
[0002] The administration of exogenous nucleic acids and
polynucleotides, for example DNA vectors and plasmids, to a subject
for the treatment of protein or enzyme deficiencies represents a
significant advance in the treatment of such deficiencies however,
the administration of such exogenous nucleic acids to a subject
remains especially challenging. For example, gene therapies that
rely on viruses to carry and deliver exogenous polynucleotides
(e.g., DNA) to host cells and that cause the integration of such
polynucleotides into the host cells' genome are capable of
eliciting serious immunological and inflammatory responses.
Furthermore, in certain instances the integration of such exogenous
polynucleotides into the host cells' genome has the potential of
misregulating the expression of the host's endogenous genes and
unpredictably impacting cellular activity.
[0003] Similarly, plasmid vector expression systems have
represented an attractive alternative approach for gene therapy
because of their ease of preparation, stability, and relative
safety compared to viral vectors. Such plasmids are however,
frequently characterized as having highly inefficient cellular
uptake in vivo.
[0004] To date, the treatment of protein (e.g., enzyme)
deficiencies have primarily involved the administration of
recombinantly-prepared proteins (e.g., enzymes) to the affected
subject. While in some instances, the use of recombinant proteins
and enzymes may provide a means of ameliorating the symptoms of the
underlying deficiency, the utility of such therapies are often
limited and are not considered curative. Furthermore, recombinant
proteins or enzymes are often prepared using non-human cell lines
and may lack certain post-translational modifications (e.g., human
glycosylation) relative to their endogenously produced
counterparts. Such differences may contribute to the lower efficacy
of such recombinantly-prepared proteins or enzymes and/or may
contribute to their immunogenicity and the incidence of adverse
reactions (e.g., infusion-related reactions such as fever,
pruritus, edema, hives and other allergic-like symptoms).
Recombinant protein and enzyme replacement therapies are also
associated with great financial expense. For example, the average
cost for enzyme replacement therapy in the United States may
approach $200,000-$300,000 USD per year depending on the subject's
weight and prescribed dose. (Brady, R O., Annual Review of Medicine
(2006), 57: 283-296.) Since replacement therapies are not curative,
the costs associated with, for example, enzyme replacement
therapies impose a significant burden on the already taxed
healthcare system. Further contributing to the costs associated
with such therapies, such therapies often require the
administration of multiple weekly or monthly doses, with each such
dose being administered by a qualified healthcare professional.
[0005] The administration of polynucleotides such as RNA (e.g.,
mRNA) that do not have to be transcribed may also represent a
suitable alternative to protein or enzyme replacement therapies.
While the development of exogenous therapeutic mRNA polynucleotides
encoding functional proteins or enzymes represents a promising
advancement, in practice the utility of such treatments may be
limited by the poor stability of such polynucleotides in vivo. In
particular, the poor stability of exogenous polynucleotides may
result in the inefficient expression (e.g., translation) of such
polynucleotides, further resulting in a poor production of the
protein or enzyme encoded thereby. Especially detrimental to the
ability of mRNA polynucleotides to be efficiently translated into a
functional protein or enzyme is the environment to which such
polynucleotides are exposed in vivo. Following the administration
of a polynucleotide, the polynucleotide may undergo degradation,
for example upon exposure to one or more nucleases in vivo.
Ribonucleases (e.g., endoribonucleases and exoribonucleases)
represent a class of nuclease enzymes that are capable of
catalyzing the degradation of RNA polynucleotides into smaller
components and thereby render the polynucleotide ineffective.
Nuclease enzymes (e.g., RNase) are therefore capable of shortening
the circulatory half-life (t.sub.1/2) of, for example, exogenous or
recombinantly-prepared mRNA polynucleotides. As a result, the
polynucleotide is not translated, the polynucleotide is prevented
from exerting an intended therapeutic benefit and its efficacy
significantly reduced.
[0006] Previous efforts to stabilize polynucleotides have focused
on complexing the polynucleotide with, for example, a liposomal
delivery vehicle. While such means may positively impact the
stability of the encapsulated polynucleotides, many lipids used as
a component of such liposomal vehicles (e.g., cationic lipids) may
be associated with toxicity. Other efforts have been directed
towards the modification of one or more nucleotides that comprise
the polynucleotide.
[0007] Novel, cost effective and therapeutically efficient
approaches and therapies are still needed for the treatment of
protein and enzyme deficiencies. Particularly needed are strategies
and therapies which overcome the challenges and limitations
associated with the administration of exogenous mRNA
polynucleotides, including for example, novel methods and
compositions relating to the stabilization of exogenous
polynucleotides. Also needed are polynucleotides (e.g., RNA) and
compositions that exhibit enhanced stability (e.g., increased
half-life in vivo) and nuclease resistance and which facilitate the
efficient expression or production of functional proteins or
enzymes. The development of such stable and/or nuclease resistant
compositions are necessary to overcome the limitations of
conventional gene therapy and could provide viable treatments or
even cures for diseases associated with the aberrant production of
proteins or enzymes.
SUMMARY OF THE INVENTION
[0008] Disclosed herein are nuclease resistant polynucleotides and
related compositions and methods. Such polynucleotides and
compositions generally encode functional polypeptides, proteins
and/or enzymes (e.g., an mRNA polynucleotide may encode a
functional urea cycle enzyme). In certain embodiments, such
compositions are characterized as being more resistant to nuclease
degradation relative to their unmodified or native
counterparts.
[0009] Disclosed herein are methods of stabilizing or modulating
(e.g., increasing or otherwise improving) the nuclease resistance
of a polynucleotide (e.g., an RNA polynucleotide). The
polynucleotides that are the subject of the present inventions
preferably encode a functional expression product (e.g., a protein
or enzyme) and may be generally characterized as comprising both a
coding region and a non-coding region. In some embodiments, the
methods disclosed herein generally comprise a step of contacting
the non-coding region of the polynucleotide (e.g., the poly-A tail
of an mRNA polynucleotide) with a complementary (e.g., a perfectly
complementary) stabilizing oligonucleotide under suitable
conditions, thereby causing the stabilizing oligonucleotide to
hybridize to the non-coding region of the polynucleotide. Upon
hybridizing of the stabilizing oligonucleotide (e.g., a 15-mer
poly-U oligonucleotide) to the polynucleotide, the polynucleotide
is rendered more resistant to nuclease degradation. For example, in
certain embodiments, provided herein are methods of increasing the
nuclease resistance of an mRNA polynucleotide comprising a poly-A
tail by contacting the poly-A tail of such polynucleotide with a
complementary poly-U stabilizing oligonucleotide. Upon hybridizing
to the non-coding region of the polynucleotide (e.g., the poly-A
tail) to form a duplexed or double-stranded region, nuclease
degradation of the polynucleotide may be reduced, delayed or
otherwise prevented. Without wishing to be bound by any particular
theories, it is believed that the observed stability and nuclease
resistance of the polynucleotides disclosed herein is due in part
to the single-stranded specificity of some nuclease enzymes (e.g.,
ribonucleases).
[0010] In certain embodiments, the stabilizing oligonucleotides
disclosed herein may hybridize to the non-coding region of the
polynucleotide (e.g., the 5' or 3' non-coding regions of an mRNA
polynucleotide) so as not to interfere with the message encoded by
the coding region of such polynucleotide. Stabilizing
oligonucleotides may be prepared such that they are perfectly
complementary to a fragment of the non-coding region (e.g.,
perfectly complementary to a fragment of the poly-A tail of an mRNA
polynucleotide). For example, the stabilizing oligonucleotide may
be complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97%, 98%, 99% or 100%
complementary) to one or more non-coding regions of the
polynucleotide selected from the group of regions consisting of the
3' untranslated region (UTR), the 5' untranslated region (UTR), the
poly-A tail and a terminal cap. Similarly, the stabilizing
oligonucleotide may be complementary (e.g., perfectly
complementary) to a region spanning discreet structures within the
non-coding region. For example, a stabilizing oligonucleotide may
be prepared such that it is perfectly complementary to a region (or
fragment of a region) that spans either the 3' UTR and the poly-A
tail or alternatively the 5' UTR and a 5' cap structure.
[0011] While certain embodiments described herein contemplate the
hybridization of the stabilizing oligonucleotide to the non-coding
region of the polynucleotide, the present inventions are not
limited to such embodiments. Rather, also contemplated are methods
and compositions in which the stabilizing oligonucleotide
hybridizes to a region spanning or comprising both a fragment of
the coding region as well as a fragment of the non-coding region of
the polynucleotide. In such embodiments (particularly where the
non-coding region comprises the poly-A tail of the polynucleotide)
hybridization to a region of the polynucleotide comprising
fragments of both the coding and non-coding regions may provide a
means to direct the hybridization of the stabilizing
oligonucleotide to a specific region of the polynucleotide.
[0012] Also contemplated by the present invention is the
administration of exogenous stabilizing oligonucleotides to a
subject, for example, to treat a disease or condition associated
with the aberrant expression or under-expression or production of a
protein or enzyme. The foregoing may be particularly suitable for
the treatment of diseases or conditions characterized as having a
suboptimal or sub-therapeutic endogenous production of a protein or
enzyme. In such embodiments, an exogenous stabilizing
oligonucleotide that is complementary (e.g., perfectly
complementary) to a region of the under expressed endogenous
polynucleotide (e.g., one or more of the 5' and/or 3' UTR) is
administered to a subject. Following administration of the
exogenous oligonucleotide, such oligonucleotide may hybridize to
the one or more endogenous polynucleotides (e.g., mRNA) encoding an
under-expressed polypeptide, protein or enzyme such that the
stability (e.g., the nuclease resistance) of the endogenous
polynucleotide is modulated (e.g., enhanced or otherwise
increased). The stabilized or nuclease resistant endogenous
polynucleotide (e.g., mRNA) may be characterized as having an
increased circulatory half-life (t.sub.1/2) and/or an increased
translational efficiency relative to its native counterpart,
generally causing the amount of the expression product (e.g., a
lysosomal enzyme) encoded by such endogenous polynucleotide to be
enhanced or otherwise increased. In certain embodiments, the
stabilizing oligonucleotide is delivered or administered in a
suitable pharmaceutical carrier or composition (e.g., encapsulated
in a lipid nanoparticle vehicle).
[0013] In some embodiments, the present invention is directed to
stable or nuclease resistant polynucleotides (e.g., mRNA) and
methods of their preparation. Such polynucleotides (e.g.,
recombinantly-prepared mRNA) may be prepared by hybridizing one or
more complementary (e.g., perfectly complementary) stabilizing
oligonucleotides to the coding and/or non-coding regions of the
polynucleotide. The polynucleotides disclosed herein may encode a
functional polypeptide, protein or enzyme. For example, the
polynucleotide (e.g., mRNA) may encode a protein or enzyme selected
from the group consisting of erythropoietin, human growth hormone,
cystic fibrosis transmembrane conductance regulator (CFTR),
alpha-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase,
N-acetylglucosamine-1-phosphate transferase,
N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase,
N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase,
beta-glucosidase, galactose-6-sulfate sulfatase,
beta-galactosidase, beta-glucuronidase, glucocerebrosidase, heparan
sulfamidase, hyaluronidase, galactocerebrosidase, ornithine
transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1),
argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL),
and arginase 1 (ARG1).
[0014] Also disclosed herein are methods of treating one or more
diseases or conditions associated with a protein or enzyme
deficiency or the aberrant expression of one or more nucleic acids.
Such methods comprise a step of administering a composition (e.g.,
a liposomal vehicle) comprising one or more of the nuclease
resistant polynucleotides (e.g., mRNA) of the present invention to
a subject affected by such disease or condition. Following the
administration of such compositions to a subject, one or more
targeted host cells are transfected and the contents of such
composition delivered intracellularly where it may be translated
and the expression product (e.g., a polypeptide, protein or enzyme)
produced. In certain instances, the expression product (e.g., a
translated protein or enzyme) may be excreted extracellularly by
the one or more targeted host cells (e.g., hepatocytes).
[0015] Also disclosed herein are stabilized or nuclease resistant
polynucleotides (e.g., mRNA) that comprise a complementary
stabilizing oligonucleotide hybridized to the coding and/or
non-coding regions of such polynucleotide. In certain embodiments,
the stabilizing oligonucleotide and/or the polynucleotide (e.g.,
mRNA) comprise at least one modification. The modification of one
or both of the polynucleotide (e.g., mRNA) and/or the stabilizing
oligonucleotide to incorporate one or more modifications may be
used as a means of further modulating (e.g., enhancing or
increasing) the nuclease resistance of the polynucleotide. Without
wishing to be bound by a particular theory, it is believed that the
incorporation of modifications (e.g., 2'-O-alkyl sugar
modifications) to either the stabilizing oligonucleotide and/or the
polynucleotide act to sterically block or delay nuclease
degradation of the polynucleotide and thereby improve stability.
Accordingly, in certain embodiments, the polynucleotide and/or the
stabilizing oligonucleotide (e.g., a poly-U oligonucleotide)
comprise at least one (e.g., two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or
more) modified nucleobase.
[0016] Contemplated modifications may include, for example, sugar
modifications or substitutions (e.g., one or more of a 2'-O-alkyl
modification, a locked polynucleotide (LNA) or a peptide
polynucleotide (PNA).) In embodiments where the sugar modification
is a 2'-O-alkyl modification, such modification may include, but
are not limited to a 2'-deoxy-2'-fluoro modification, a 2'-O-methyl
modification, a 2'-O-methoxyethyl modification and a 2'-deoxy
modification. In certain embodiments where the modification is a
nucleobase modification, such modification may be selected from the
group consisting of a 5-methyl cytidine, pseudouridine, 2-thio
uridine, 5-methylcytosine, isocytosine, pseudoisocytosine,
5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine,
inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, and
combinations thereof.
[0017] In certain embodiments, the contemplated modification may
involve the inter-nucleosidic bonds that comprise the stabilizing
oligonucleotide and/or the polynucleotides. For example,
contemplated modifications introduced to one or both of the
stabilizing oligonucleotide and/or the polynucleotide may include
one or more phosphorothioate bonds. In one embodiment, all of the
inter-nucleosidic bonds of one or both of the stabilizing
oligonucleotide and the polynucleotide are phosphorothioate
bonds.
[0018] The nuclease resistance of the polynucleotides disclosed
herein may be characterized relative to the native or unmodified
counterpart polynucleotides (e.g., relative to an un-hybridized
polynucleotide that has not been contacted or treated with a
stabilizing oligonucleotide). For example, the nuclease resistant
polynucleotides disclosed herein may be at least about two, three,
four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty,
twenty-five, thirty, fifty, one hundred times more stable in vivo
relative to their native or un-hybridized counterparts. In certain
embodiments, the circulatory half-life (t.sub.1/2) of the
polynucleotide in vivo is indicative of such polynucleotide's
stability. In other embodiments, the relative amount of expression
product (e.g., a polypeptide, protein or enzyme) expressed (e.g.,
translated) from the polynucleotide is indicative of its
stability.
[0019] In some embodiments, the present invention relates to
methods of increasing the quantity of an expression product (e.g.,
a functional protein or enzyme) that is or may be expressed (e.g.,
translated) from a polynucleotide transcript. For example, such
methods may generally comprise a step of contacting a portion of
the coding and/or non-coding regions of an mRNA polynucleotide
transcript with a stabilizing oligonucleotide such that the
stabilizing oligonucleotide hybridizes to the mRNA transcript. In
certain embodiments, the stabilizing oligonucleotide and the mRNA
polynucleotide transcript are contacted at about a 0.1:1 ratio. In
other embodiments, the stabilizing oligonucleotide and the mRNA
polynucleotide transcript are contacted at about a 0.25:1 ratio. In
yet other embodiments, the stabilizing oligonucleotide and the mRNA
polynucleotide transcript are contacted at about a 0.5:1 ratio. In
still other embodiments, the stabilizing oligonucleotide and mRNA
polynucleotide transcript are contacted at about a 1:1 ratio. In
certain embodiments, the stabilizing oligonucleotide and the mRNA
polynucleotide transcript are contacted at about a 2:1, 5:1, a
10:1, a 100:1 or a 1,000:1 ratio.
[0020] Upon contacting the mRNA polynucleotide transcript with a
complementary stabilizing oligonucleotide, the stabilizing
oligonucleotide will hybridize to the mRNA polynucleotide (e.g., at
a region of complementarity). Upon hybridizing to the mRNA, the
stabilizing oligonucleotide will form a duplexed region with, for
example, the non-coding region of the mRNA polynucleotide and
thereby render the mRNA polynucleotide more resistant to nuclease
degradation. As a result of being rendered more resistant to
nuclease (e.g., endonuclease) degradation, the amount of the
expression product (e.g., a polypeptide) translated from the mRNA
polynucleotide transcript may be increased (e.g., increased by at
least about 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 33%, 36%, 40%,
50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 110%, 120%, 125%,
150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 750%, 800%,
900%, 1,000% or more). In certain embodiments, one or both of the
mRNA transcript or the stabilizing oligonucleotide may comprise at
least one modification (e.g., one or more chemically modified
nucleobases or modified inter-nucleotide bonds).
[0021] Also provided herein are methods of increasing the
translational efficiency of an exogenous mRNA transcript. Such
methods may facilitate, for example, an increase in the production
of an expression product produced following translation of the mRNA
polynucleotides or transcripts of the present inventions.
Generally, such methods comprise a step of contacting the mRNA
polynucleotide transcript with a stabilizing oligonucleotide that
is complementary to the coding and/or non-coding region of the mRNA
transcript under suitable conditions (e.g., high stringency
conditions), thereby causing the mRNA polynucleotide transcript and
the stabilizing oligonucleotide to hybridize to each other. Such
methods may be employed to render the mRNA transcript more
resistant to nuclease (e.g., exonuclease) degradation while
modulating (e.g., increasing) the translational efficiency of the
exogenous mRNA transcript by one or more target cells. In certain
embodiments, the stabilizing oligonucleotide comprises at least one
modified nucleobase. In certain embodiments, the mRNA transcript
also comprises one or more modifications (e.g., one or more
chemical modifications and/or phosphorothioate inter-nucleosidic
bonds).
[0022] The above discussed and many other features and attendant
advantages of the present invention will become better understood
by reference to the following detailed description of the invention
when taken in conjunction with the accompanying examples. The
various embodiments described herein are complimentary and can be
combined or used together in a manner understood by the skilled
person in view of the teachings contained herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 illustrates one embodiment of the present invention
whereby an mRNA polynucleotide transcript (as indicated by ) having
a poly-A tail located downstream (3') of the coding region is
contacted with a 15-mer poly(2'O-Me-uracil) stabilizing
oligonucleotide having a phosphorothioate backbone. As illustrated,
upon contacting the poly-A tail of the mRNA polynucleotide with the
fully complementary stabilizing oligonucleotide a duplexed region
is formed and thereby stabilizes the mRNA polynucleotide by
rendering it more resistant to nuclease degradation. Because the
stabilizing oligonucleotide is fully complementary to multiple
regions of the depicted poly-A tail, there exist several possible
duplexed constructions, only four of which are illustrated in the
depicted embodiment.
[0024] FIG. 2 depicts the cumulative amount of erythropoietin
protein (EPO) produced in vitro over a seventy-two hour period by
293T cells transfected with various nuclease resistant
polynucleotides of the present invention. Non-denatured human EPO
mRNA was hybridized with a 15-mer of fully phosphorothioated
2-OMe-uridine oligonucleotides in the ratios listed (oligo:mRNA).
The depicted plot is represented as a percentage of the protein
produced from the unhybridized native EPO mRNA. As illustrated in
FIG. 2 relative to the untreated EPO mRNA polynucleotide
(designated "Unhybridized"), the stabilized EPO polynucleotide mRNA
transcripts generally demonstrated an increase in the amount of EPO
protein expressed by the 293T cells that were transfected with the
stabilized mRNA transcript, and in certain instances an
approximately 160% increase in the amount of EPO protein translated
and produced was observed relative to the Unhybridized control.
[0025] FIG. 3 illustrates the quantification of cumulative human
erythropoietin (EPO) protein produced in vitro over a seventy-two
hour period by 293T cells transfected with various stabilized mRNA
transcripts of the present invention. Denatured human EPO mRNA was
hybridized with a 15-mer of fully phosphorothioated 2-OMe-uridine
oligonucleotides in the ratios listed (oligo:mRNA). The depicted
plot is represented as a percentage of the protein produced from
the unhybridized denatured EPO mRNA. As illustrated in FIG. 3, the
stabilized EPO polynucleotide transcripts generally yielded higher
cumulative amounts of EPO protein translated and produced by the
293T cells transfected with the stabilized mRNA transcripts
disclosed herein.
[0026] FIG. 4 depicts the cumulative amount of erythropoietin (EPO)
protein produced in vitro by 293T cells transfected with various
nuclease resistant polynucleotides of the present invention at
different time points over a ninety-six hour period. Human EPO mRNA
was hybridized with a 30-mer of fully phosphorothioated
2-OMe-uridine oligonucleotides in the ratios listed (oligo:mRNA).
The depicted plot is represented as a percentage of the protein
produced from the respective unhybridized native EPO mRNA. As
illustrated in FIG. 4, relative to the untreated EPO mRNA
polynucleotide (designated "Unhybridized"), the stabilized or
nuclease resistant polynucleotide mRNA transcripts generally
demonstrated an increase in the amount of EPO protein expressed by
the 293T cells transfected with such stabilized mRNA transcripts.
In particular, those stabilized or nuclease resistant mRNA
transcripts that were prepared by exposure of the mRNA transcript
to lower concentrations of stabilizing oligonucleotide (e.g., 0.1
and 0.5) demonstrated higher translational efficiencies relative to
their unmodified counterparts.
DETAILED DESCRIPTION
[0027] The present inventions are directed to stabilized or
nuclease resistant polynucleotides and compositions (e.g., mRNA
polynucleotides) and related methods of their use and preparation.
In certain embodiments the polynucleotides and compositions
disclosed herein encode one or more functional expression products
(e.g., polypeptides, proteins and/or enzymes) and are not subject
to some of the limitations that are generally associated with
conventional gene or enzyme replacement therapies. For example, in
embodiments where the polynucleotide transcripts disclosed herein
comprise mRNA, such polynucleotides need not integrate into a host
cells' genome to exert their therapeutic effect. Similarly, in
certain embodiments, the exogenous polynucleotide transcripts are
translated by the host cells and accordingly are characterized by
the native post-translational modifications that are present in the
native expression product.
[0028] While the administration of exogenous polynucleotides (e.g.,
DNA or RNA) represents a meaningful advancement in the treatment
diseases, the administration of such exogenous polynucleotides is
often hampered by the limited stability of such polynucleotides,
particularly following their in vivo administration. For example,
following their administration to a subject, many polynucleotides
may be subject to nuclease (e.g., exonuclease and/or endonuclease)
degradation. Nuclease degradation may negatively influence the
capability of an mRNA polynucleotide transcript to reach a target
cell or to be translated, the result of which is to preclude the
exogenous polynucleotide from exerting an intended therapeutic
effect.
[0029] Nucleases represent a class of enzymes that are responsible
for the cleavage or hydrolysis of the phosphodiester bonds that
hold the nucleotides of DNA or RNA together. Those nuclease enzymes
that cleave or hydrolyze the phosphodiester bonds of DNA are called
deoxyribonucleases, while the nuclease enzymes that cleave the
phosphodiester bonds of RNA are called ribonucleases. As generally
used herein, the term "nuclease" refers to an enzyme with the
capability to degrade or otherwise digest polynucleotides or
nucleic acid molecules (e.g., DNA or RNA). Representative examples
of nucleases include ribonucleases (RNase) which digests RNA, and
deoxyribonuclease (DNase) which digests DNA. Unless otherwise
specified, the term "nuclease" generally encompasses nuclease
enzymes that are capable of degrading single-stranded
polynucleotides (e.g., mRNA) and/or double stranded polynucleotides
(e.g., DNA).
[0030] In certain aspects, the present invention is directed to
methods and strategies for stabilizing polynucleotides from
nuclease degradation or for improving the resistance of one or more
polynucleotides (e.g., mRNA) to nuclease degradation. It should be
noted that in certain embodiments, improvements in the stability
and/or nuclease resistance of the polynucleotides disclosed herein
may be made with reference to a native or unmodified
polynucleotide. For example, in certain embodiments, the stability
and/or nuclease resistance of a polynucleotide (e.g., an mRNA
transcript) is increased by at least about 105%, 110%, 115%, 120%,
125%, 130%, 135%, 140%, 150%, 160%, 170%, 175%, 180%, 190%, 200%,
250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%,
or more relative to the native or unmodified polynucleotide
transcript.
[0031] As used herein to characterize a polynucleotide (e.g., an
mRNA transcript encoding a functional urea cycle enzyme), the term
"stable" generally refers to a reduced susceptibility to
degradation or destruction (e.g., a reduced susceptibility to
nuclease cleavage in vivo). For example, the term "stable" may be
used to refer to a reduction in the rate of nuclease degradation of
a polynucleotide in vivo. In certain embodiments, the half-life
(t1/2) of a polynucleotide represents an objective measurement of
its stability. Similarly, in certain embodiments, the amount or
mass of an expression product that is produced following the
expression (e.g., translation) of a stable or nuclease resistant
polynucleotide represents an objective measurement of its
stability. Preferred are modifications made or otherwise introduced
into a polynucleotide that serve to enhance (e.g., increase) the
half-life or translational efficiency of such polynucleotide in
vivo relative to its unmodified counterpart. For example, in
certain embodiments, the t1/2 of a nuclease resistant
polynucleotide (e.g., an mRNA transcript) is increased by at least
about 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%, 160%,
170%, 175%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%,
600%, 700%, 800%, 900%, 1,000%, or more relative to its native or
unmodified polynucleotide counterpart. In certain embodiments, the
stability of hybridized mRNA may be in part due to the inherent
single strand specificity of some nuclease enzymes, and in
particular RNase enzymes.
[0032] The methods disclosed herein generally comprise a step of
contacting the non-coding region of the polynucleotide (e.g., the
poly-A tail of an mRNA polynucleotide) with a complementary (e.g.,
a perfectly complementary) stabilizing oligonucleotide under
suitable conditions, thereby causing the stabilizing
oligonucleotide to hybridize to the non-coding region of the
polynucleotide. As used herein, the terms "contact" and
"contacting" generally refer to bringing two or more moieties
together or within close proximity of one another such that the
moieties may react. For example, in certain embodiments of the
present invention, a polynucleotide (e.g., an mRNA transcript) may
be contacted with one or more stabilizing oligonucleotides (e.g., a
stabilizing oligonucleotide that is perfectly complementary to a
region or fragment of the polynucleotide) such that the
polynucleotide and stabilizing oligonucleotide would be expected to
react (e.g., hybridize to one another) under suitable
conditions.
[0033] Upon hybridizing of the stabilizing oligonucleotide (e.g., a
15-mer poly(2'-O-Me-uracil) oligonucleotide) to the polynucleotide,
the polynucleotide is rendered more resistant to nuclease
degradation. For example, in certain embodiments, provided herein
are methods of increasing the nuclease resistance of an mRNA
polynucleotide comprising a poly-A tail by contacting the poly-A
tail of such polynucleotide with a complementary poly-U stabilizing
oligonucleotide. Upon hybridizing to the non-coding region of the
polynucleotide (e.g., the poly-A tail) to form a duplexed or
double-stranded region, nuclease degradation of the polynucleotide
may be reduced, delayed or otherwise prevented. Without wishing to
be bound by any particular theories, it is believed that the
observed stability and nuclease resistance of the polynucleotides
disclosed herein is due in part to the single-stranded specificity
of some nuclease enzymes (e.g., ribonucleases). In those
embodiments where one or both of the stabilizing oligonucleotide
and/or the polynucleotide comprise a modification (e.g., a
chemically-modified nucleobases and/or a phosphorothioate backbone)
such modifications may serve to further stabilize the
polynucleotide by sterically interfering with nuclease
degradation.
[0034] It should be noted that while the terms "polynucleotide" and
"oligonucleotide" may be generally understood by those of ordinary
skill in the art to generally be synonymous with each other, such
terms are used herein for convenience to distinguish the targeted
sense nucleic acid transcripts (e.g., mRNA) from the shorter (e.g.,
about 15-50 mer) complementary or anti-sense nucleic acids that are
used to modulate the stability of a targeted sense nucleic acid
transcript in accordance with the teachings of the present
inventions. In particular, the phrase "stabilizing oligonucleotide"
is used herein to describe a nucleic acid sequence that is
generally complementary or anti-sense to a region or fragment of a
polynucleotide sequence encoding a functional expression product.
While such stabilizing oligonucleotides may generally be of any
length, in certain embodiments the stabilizing oligonucleotides are
less than 500 nucleotides, less than 400 nucleotides, less than 300
nucleotides, less than 250 nucleotides, less than 200 nucleotides,
less than 100 nucleotides, or more preferably less than 50
nucleotides, less than 40 nucleotides, less than 30 nucleotides,
less than 25 nucleotides, less than 20 nucleotides, less than 19
nucleotides, less than 18 nucleotides, less than 17 nucleotides,
less than 16 nucleotides or less than 15 nucleotides in length.
[0035] In certain embodiments, the stabilizing oligonucleotides
(e.g., a 15-mer poly-U stabilizing oligonucleotide) disclosed
herein comprise one or more modifications (e.g., modifications such
as 2'-O-alkyl sugar substitutions). For example, in some
embodiments the stabilizing oligonucleotide comprises one or more
chemical modifications, such as one or more 2'-O-alkyl modified or
substituted nucleobases or the inclusion of one or more
phosphorothioate inter-nucleobase linkages. Such modifications may
further improve the ability of the stabilizing oligonucleotide to
hybridize to a complementary polynucleotide or may improve the
stability or nuclease resistance of such polynucleotide (e.g., by
interfering with recognition of such polynucleotide by nuclease
enzymes).
[0036] The present inventors have surprisingly discovered that
stabilized mRNA polynucleotides that were prepared by exposure of
the mRNA polynucleotides to higher concentrations of stabilizing
oligonucleotides resulted in the production of lower quantities of
the encoded expression product (e.g., erythropoietin protein) by
cells transfected with such polynucleotides. Without wishing to be
bound by a particular theory, it is suspected that higher degrees
of hybridization of the stabilizing oligonucleotides to the
polynucleotide may interfere with the ability of the resulting
duplexed (i.e., hybridized or stabilized) polynucleotide to form
secondary or even tertiary structures (e.g., hairpin loops, bulges,
and internal loops) that may also contribute to the stability of
such polynucleotide. For example, while higher degrees of
hybridization of the poly-A tail region of an mRNA polynucleotide
transcript may improve the nuclease resistance of such mRNA
transcript, the longer duplexed regions may also interfere with the
ability of the duplexed mRNA transcript to properly fold. In
certain instances where the proper folding of such mRNA transcript
contributes to its stability (e.g., nuclease resistance), it is
expected that interference with the ability of such transcript to
properly fold may be associated with a corresponding reduction in
its stability. Accordingly, in certain embodiments, shorter
stabilizing oligonucleotides (e.g., about 75, 70, 60, 65, 50, 45,
40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5 nucleotides or less) are preferred.
Similarly, in certain embodiments the hybridization of a
stabilizing oligonucleotide to a polynucleotide does not materially
interfere with the ability of the resulting nuclease resistant
polynucleotide to form secondary or tertiary structures.
[0037] The degree to which the nuclease resistant polynucleotides
disclosed herein hybridize may be a direct function of the manner
in which such nuclease resistant polynucleotides were prepared. As
depicted in FIG. 1, to the extent that an mRNA polynucleotide is
contacted with a high concentration of a complementary stabilizing
oligonucleotide, the stabilizing oligonucleotide may hybridize to
the mRNA polynucleotide at multiple regions. In certain
embodiments, the extent to which a polynucleotide hybridizes with a
complementary stabilizing oligonucleotide may be manipulated or
otherwise controlled by modifying the relative concentrations of
stabilizing oligonucleotide to which the polynucleotide is exposed.
For example, in certain preferred embodiments, the stabilizing
oligonucleotide and the mRNA polynucleotide transcript are
contacted at about a 0.1:1 ratio. In other embodiments, the
stabilizing oligonucleotide and the mRNA polynucleotide transcript
are contacted at about a 0.25:1 ratio. In yet other embodiments,
the stabilizing oligonucleotide and the mRNA polynucleotide
transcript are contacted at about a 0.5:1 ratio. In still other
embodiments, the stabilizing oligonucleotide and mRNA
polynucleotide transcript are contacted at about a 1:1 ratio. In
certain embodiments, the stabilizing oligonucleotide and the mRNA
polynucleotide transcript are contacted at about a 5:1, a 10:1, a
100:1 or a 1,000:1 ratio.
[0038] As used herein, the term "polynucleotide" is generally used
to refer to a nucleic acid (e.g., DNA or RNA) to be stabilized or
rendered more nuclease resistant in accordance with the teachings
of the present invention. In certain embodiments, the
polynucleotides disclosed herein (or particular regions or
fragments thereof) represent the nucleic acid target to which the
stabilizing oligonucleotides may hybridize. The polynucleotides
(e.g., an mRNA polynucleotide) disclosed herein may also comprise
one or more modifications. For example, in some embodiments the
mRNA polynucleotide transcripts disclosed herein comprise one or
more chemical modifications, which in certain instances may further
improve the stability or nuclease resistance of such polynucleotide
transcript (e.g., by sterically hindering or otherwise interfering
with nuclease degradation).
[0039] The polynucleotides may comprise both coding and non-coding
regions and in certain embodiments described herein, the
stabilizing oligonucleotides hybridize to the non-coding region of
the polynucleotide. As used herein, the phrase "non-coding region"
generally refers to that portion or region of the polynucleotide or
a gene that is not a coding region and that is not expressed,
transcribed, translated or otherwise processed into an expression
product such as an amino acid, polypeptide, protein or enzyme. In
the context of DNA polynucleotides, the non-coding region may
comprise intron sequences or other sequences located 5' or 3'
(e.g., upstream or downstream) of the coding region (e.g.,
promoters, enhancers, silencers). In the context of RNA
polynucleotides, the non-coding region may comprise sequences
located 5' or 3' (e.g., upstream or downstream) of the coding
region (e.g., 3' untranslated region (UTR), a 5' untranslated
region (UTR), a poly-A tail and a terminal cap). In certain
embodiments, the targeted non-coding region may comprise two
distinct, but overlapping regions. For example, as briefly depicted
below a stabilizing oligonucleotide may be prepared such that it is
perfectly complementary to a region of a polynucleotide comprising
or spanning a fragment of the 3' untranslated region (UTR) and a
fragment of the poly-A tail.
TABLE-US-00001 mRNA Polynucleotide Fragment: 5'- . . .
AUGGCACAUCCUGUAAAAAAAAAAAAAAAAAAAAA . . . -3' |||||||||||||||||
Stabilizing Oligonucleotide: 3'- CAUUUUUUUUUUUUUUU -5'
[0040] Similarly, a stabilizing oligonucleotide may be prepared
such that it is complementary to a region of a polynucleotide
comprising or spanning a fragment of a 5' cap structure and a
fragment of the 5' UTR. For example, the stabilizing
oligonucleotide may be complementary (e.g., at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97%,
98%, 99% or 100% complementary) to one or more non-coding regions
of the polynucleotide selected from the group of regions consisting
of the 5' UTR, a 5' terminal cap, the 3' UTR and the poly-A tail.
In certain embodiments, the hybridization of a complementary
stabilizing oligonucleotide to the non-coding region of an mRNA
polynucleotide is preferred, in part due to concerns relating to
the ability of the resultant duplexed region (i.e., the hybridized
polynucleotide and stabilizing oligonucleotide) to interfere with
the translation of the coding region.
[0041] As used herein, the phrase "coding region" generally refers
to that portion or region of the polynucleotide or a gene that when
expressed, transcribed, translated or otherwise processed results
in the production of an expression product, such as an amino acid,
polypeptide, protein or enzyme. It should be understood that while
certain embodiments disclosed herein contemplate the hybridization
of complementary stabilizing oligonucleotides to the non-coding
region of a polynucleotide transcript, the present invention need
not be limited to such embodiments. Rather, the present invention
also contemplates the hybridization of the complementary
stabilizing oligonucleotides to regions of the polynucleotide
transcript (e.g., mRNA) comprising or spanning both the coding and
non-coding regions. For example, a stabilizing oligonucleotide may
be prepared such that it targets and/or is complementary (e.g.,
perfectly complementary) to a fragment of the coding region of an
mRNA polynucleotide transcript and a fragment of the non-coding 3'
UTR located downstream of the coding region. The foregoing
therefore provides a means of specifically targeting a particular
region of the polynucleotide, such as the region located
immediately downstream of the coding region. Additionally, the
foregoing also provides means of controlling or otherwise affecting
the degree to which a stabilizing oligonucleotide hybridizes to a
complementary region of the polynucleotide. In certain embodiments
where the stabilizing oligonucleotide targets the coding region (or
a fragment thereof) preferably the hybridization of the stabilizing
oligonucleotide to such coding region (or fragment thereof) does
not interfere with the expression (e.g., transcription or
translation) of such polynucleotide. Similarly, in embodiments
where the stabilizing oligonucleotide targets the coding region (or
a fragment thereof) preferably the hybridization of the stabilizing
oligonucleotide to such coding region (or fragment thereof) does
not substantially interfere with the expression (e.g.,
transcription or translation) of such polynucleotide.
[0042] In the context of the present invention the term
"expression" is used in its broadest sense to refer to either the
transcription of a specific polynucleotide (e.g., a gene or nucleic
acid) into an RNA transcript, or the translation of at least one
mRNA polynucleotide into a polypeptide, protein or enzyme. For
example, disclosed herein are compositions which comprise one or
more mRNA polynucleotides that encode functional expression
products (e.g., proteins or enzymes), and in the context of such
mRNA polynucleotides, the term expression refers to the translation
of such mRNA polynucleotides to produce a polypeptide, protein or
enzyme encoded thereby. Similarly, the phrase "expression product"
is used herein in its broadest sense to generally refer to an RNA
transcription product that is transcribed from a DNA
polynucleotide, or alternatively to a polypeptide, protein or
enzyme that is the natural translation product of an mRNA
polynucleotide. In certain embodiments, the expression product of
the polynucleotide is a functional enzyme (e.g., a urea cycle
enzyme). In certain embodiments, the expression product of the
polynucleotide is a functional protein (e.g., hormone) or enzyme.
In those instances where the polynucleotide is DNA, following
expression (i.e., transcription) of such DNA the encoded expression
product (i.e., RNA) may be produced. Similarly, in those
embodiments where the polynucleotide is mRNA, following expression
(i.e., translation) of such mRNA the encoded expression product
(e.g., a polypeptide, protein or enzyme) may be produced and/or
excreted.
[0043] In some embodiments, the present inventions are directed to
methods of modulating (e.g., increasing, improving or otherwise
enhancing) the translational efficiency of one or more mRNA
polynucleotides in a target cell. As used herein, the phrase
"translational efficiency" refers to the rate at which an mRNA
polynucleotide is translated and the corresponding expression
product produced. In certain instances, the stable or nuclease
resistant mRNA polynucleotides disclosed herein may be
characterized by their increased translational efficiency,
resulting in a corresponding increase in the production of the
expression product encoded by such mRNA polynucleotide. Such
methods generally comprise an initial step of contacting an mRNA
polynucleotide with a complementary (e.g., perfectly or partially
complementary) stabilizing oligonucleotide under suitable
conditions (e.g., high stringency conditions), thereby causing the
mRNA polynucleotide and one or more stabilizing oligonucleotides to
hybridize to each other. As a result, the mRNA polynucleotide is
rendered more resistant to nuclease degradation and the
translational efficiency of such polynucleotide in one or more
target cells increased. In certain embodiments, one or both of the
stabilizing oligonucleotide and/or the mRNA polynucleotide may
comprise at least one modified nucleobase (e.g., a 2'-O-alkyl sugar
substitution). In certain embodiments, one or both of the
stabilizing oligonucleotide and the mRNA polynucleotide also
comprise one or more modifications (e.g., one or more nucleobases
linked by phosphorothioate bonds).
[0044] In certain instances, the nuclease resistant polynucleotides
disclosed herein may be recombinantly-prepared (e.g., a
recombinantly-prepared codon-optimized mRNA polynucleotide). In
such embodiments, such polynucleotides (e.g., a
recombinantly-prepared mRNA polynucleotide) may be contacted with a
complementary stabilizing oligonucleotide prior to being
administered to a subject in a suitable carrier or vehicle (e.g., a
lipid nanoparticle).
[0045] Also contemplated by the present invention is the direct
administration of an exogenous stabilizing oligonucleotide to a
subject (e.g., for the treatment of a disease or condition
associated with the suboptimal or sub-therapeutic production of an
expression product, such as a protein or enzyme). In such
embodiments, the present inventions provide a means of modulating
(e.g., increasing or otherwise enhancing) the expression,
production and/or secretion of an endogenous expression product.
For example, the present inventions contemplate the administration
of a stabilizing oligonucleotide to a subject, wherein the
stabilizing oligonucleotide is complementary (e.g., perfectly- or
partially-complementary) to an endogenous polynucleotide (e.g.,
mRNA). In such embodiments, an exogenously-prepared stabilizing
oligonucleotide that is complementary (e.g., perfectly
complementary) to a region of an endogenous polynucleotide (e.g.,
the non-coding region of an endogenous mRNA polynucleotide) is
administered to a subject. Following the administration of the
exogenous stabilizing oligonucleotide, such oligonucleotide
hybridizes to the one or more endogenous polynucleotides (e.g.,
mRNA) encoding an under-expressed expression product such that the
stability or the nuclease resistance of the endogenous
polynucleotide is modulated (e.g., enhanced or otherwise increased)
and/or its translational efficiency increased. The resulting
stabilized or nuclease resistant endogenous polynucleotide (e.g.,
mRNA) may be characterized as having an increase circulatory
half-life (t.sub.1/2) relative to its native counterpart and, in
certain instances an improved translational efficiency. As a
result, the amount of the expression product (e.g., a lysosomal
enzyme) encoded by such endogenous polynucleotides may be enhanced
or otherwise increased and an underlying condition (e.g., a protein
or enzyme deficiency) or its symptoms thereby treated or mitigated.
The foregoing therefore provides a means of increasing the
expression of sub-optimally expressed endogenous mRNA
polynucleotides by rendering such polynucleotides more nuclease
resistant relative to their native (and under-expressed)
counterparts. It should be understood that while the foregoing
embodiments (i.e., the direct administration of stabilizing
oligonucleotides to a subject) may generally relate to traditional
anti-sense or RNAi mechanisms of targeting endogenous nucleic acids
(e.g., mRNA), the observed effect of such targeting is an increase,
rather than a decrease, in the production of the expression product
encoded by the targeted polynucleotide. In certain embodiments, the
stabilizing oligonucleotide is delivered or administered to a
subject in a suitable pharmaceutical carrier, vehicle or
composition (e.g., encapsulated in a lipid nanoparticle
vehicle).
[0046] The polynucleotides provided herein, and in particular the
mRNA polynucleotides provided herein, preferably retain at least
some ability to be expressed or translated, to thereby produce a
functional expression product (e.g., a protein or enzyme) within a
target cell. Accordingly, the present invention also relates to the
administration of stabilized or duplexed polynucleotides to a
subject (e.g., mRNA which has been stabilized against in vivo
nuclease digestion or degradation). In a preferred embodiment of
the present invention, the therapeutic activity of the nuclease
resistant polynucleotide is prolonged or otherwise evident over an
extended period of time (e.g., at least about twelve hours,
twenty-four hours, thirty-six hours, seventy-two hours, four days,
five days, 1 week, ten days, two weeks, three weeks, four weeks,
six weeks, eight weeks, ten weeks, twelve weeks or longer). For
example, the therapeutic activity of the nuclease resistant
polynucleotides may be prolonged such that the compositions of the
present invention are administered to a subject on a semi-weekly or
bi-weekly basis, or more preferably on a monthly, bi-monthly,
quarterly or even on an annual basis. The extended or prolonged
activity of the compositions of the present invention, and in
particular of the nuclease resistant mRNA polynucleotides comprised
therein, is directly related to the translational efficiency of
such polynucleotide and the quantity of the expression product
(e.g., a functional protein or enzyme) that can be translated from
such mRNA.
[0047] In certain embodiments the translational efficiency and the
in vivo activity of the nuclease resistant polynucleotides and
compositions of the present invention may be further extended or
prolonged by the introduction of one or more modifications to such
polynucleotides to improve or enhance their half-life (VA). For
example, the Kozac consensus sequence plays a role in the
initiation of protein translation, and the inclusion of such a
Kozac consensus sequence in the mRNA polynucleotides of the present
invention may further extend or prolong the activity or
translational efficiency of such mRNA polynucleotides. Furthermore,
the quantity of functional protein or enzyme translated by the
target cell is a function of the quantity of polynucleotide (e.g.,
mRNA) delivered to the target cells and the stability of such
polynucleotide. To the extent that the stability and/or half-life
of the nuclease resistant polynucleotides of the present invention
may be improved or enhanced, the therapeutic activity of the
translated protein or enzyme and/or the dosing frequency of the
composition may be further extended.
[0048] Accordingly, in a preferred embodiment, one or both of the
polynucleotides and/or the stabilizing oligonucleotides disclosed
herein comprise at least one modification. As used herein, the
terms "modification" and "modified" as they relate to the
polynucleotides and/or stabilizing oligonucleotides provided
herein, refer to at least one alteration or chemical modification
introduced into such polynucleotides and/or stabilizing
oligonucleotides and which preferably renders them more stable
(e.g., resistant to nuclease digestion) than the wild-type or
naturally occurring version of the polynucleotide. For example, the
introduction of chemical modifications into one or more of the
polynucleotide and the stabilizing oligonucleotide may interfere
with, sterically hinder or otherwise delay their recognition and/or
degradation by one or more nuclease enzymes (e.g., RNase).
Increased stability can include, for example, less sensitivity to
hydrolysis or other destruction by endogenous enzymes (e.g.,
endonucleases or exonucleases) or conditions within the target cell
or tissue, thereby increasing or enhancing the circulatory
half-life or residence time of such polynucleotides in the target
cell, tissue, subject and/or cytoplasm. The stabilized or nuclease
resistant polynucleotides provided herein may demonstrate longer
half-lives relative to their naturally occurring or un-hybridized
counterparts (e.g. the wild-type version of the polynucleotide).
Also contemplated by the terms "modification" and "modified", as
such terms relate to mRNA polynucleotides and/or stabilizing
oligonucleotides of the present invention, are alterations which
improve or enhance the translational efficiency of such mRNA
polynucleotides, including for example, the inclusion of sequences
which affect the initiation of protein translation (e.g., the Kozac
consensus sequence). (See, Kozak, M., Nucleic Acids Res. (1987); 15
(20): 8125-48).
[0049] Exemplary modifications to a polynucleotide may also include
the depletion of a base (e.g., by deletion or by the substitution
of one nucleotide for another) or modification of a base, for
example, the chemical modification of a base. The phrase "chemical
modifications" as used herein, includes modifications which
introduce chemistries that differ from those observed in naturally
occurring polynucleotides, for example, covalent modifications such
as the introduction of modified bases (e.g., nucleotide analogs, or
the inclusion of pendant groups which are not naturally found in
such polynucleotides). In certain embodiments, exemplary chemical
modifications that may be introduced into one or both of the
polynucleotide and the stabilizing oligonucleotide include
pseudouridine, 2-thiouracil, 5-methyl cytidine, 5-methylcytosine,
isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,
6-aminopurine, 2-aminopurine, inosine, diaminopurine and
2-chloro-6-aminopurine cytosine.
[0050] In addition, suitable modifications may include alterations
in one or more nucleotides of a codon such that the codon encodes
the same amino acid but is more stable relative to the wild-type
codon of the polynucleotide found in nature. For example, an
inverse relationship between the stability of RNA and a higher
number cytidines (C) and/or uridines (U) residues has been
demonstrated, and RNA lacking C and U residues have been found to
be stable to most RNases. (Heidenreich, et al. J Biol Chem 269,
2131-8 (1994)). In some embodiments, the number of C and/or U
residues in an mRNA sequence is reduced. In other embodiments, the
number of C and/or U residues is reduced by substitution of one
codon encoding a particular amino acid for another codon encoding
the same or a related amino acid. Contemplated modifications to the
mRNA polynucleotides of the present invention also include the
incorporation of pseudouridines. The incorporation of
pseudouridines into the mRNA polynucleotides of the present
invention may enhance their stability and translational capacity,
as well as diminish their immunogenicity in vivo. (See, e.g.,
Kariko, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008)).
Substitutions and modifications to the polynucleotides of the
present invention may be performed by methods readily known to one
or ordinary skill in the art.
[0051] The constraints on reducing the number of C and U residues
in a sequence will likely be greater within the coding region of an
mRNA polynucleotide, compared to its untranslated region, (i.e., it
will likely not be possible to eliminate all of the C and U
residues present in the coding region while still retaining the
ability of the message to encode the desired amino acid sequence).
The degeneracy of the genetic code, however presents an opportunity
to allow the number of C and/or U residues that are present in the
sequence to be reduced, while maintaining the same coding capacity
(i.e., depending on which amino acid is encoded by a codon, several
different possibilities for modification of RNA sequences may be
possible). For example, the codons for Gly can be altered to GGA or
GGG instead of GGU or GGC.
[0052] As previously mentioned, the term modification also
includes, for example, the incorporation of non-nucleotide linkages
or modified nucleotides into the polynucleotides and/or stabilizing
oligonucleotides of the present invention. Such modifications
include the addition of bases to a polynucleotide sequence (e.g.,
the inclusion of a poly-A tail or the lengthening of the poly-A
tail), the alteration of the 3' UTR or the 5' UTR, and the
inclusion of elements which change the structure of a
polynucleotide and/or stabilizing oligonucleotide (e.g., elements
which modulate the ability of such polynucleotides or their
expression products to form secondary structures).
[0053] In certain embodiments the poly-A tail and the region
immediately upstream represent suitable regions of a polynucleotide
that the stabilizing oligonucleotides (e.g., a 15-mer poly-U
stabilizing oligonucleotide) disclosed herein may target and/or
hybridize to. The poly-A tail is thought to naturally stabilize
natural mRNA polynucleotides and synthetic sense RNA. Therefore, in
certain embodiments a long poly-A tail can be added to an mRNA
polynucleotide and thus render the mRNA more stable. In other
embodiments, the poly-A tail or a particular region thereof may be
contacted under suitable condition (e.g., high stringency
conditions) with a complementary stabilizing oligonucleotide (e.g.,
a poly-U stabilizing oligonucleotide) and thereby render the
polynucleotide more nuclease resistant. Poly-A tails can be added
using a variety of art-recognized techniques. For example, long
poly-A tails can be added to synthetic or in vitro transcribed RNA
using poly-A polymerase. (Yokoe, et al. Nature Biotechnology. 1996;
14: 1252-1256). In addition, poly-A tails can be added by
transcription directly from PCR products or may be ligated to the
3' end of an mRNA polynucleotide with RNA ligase. (See, e.g.,
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991
edition)). In certain embodiments, the length of the poly-A tail is
at least about 20, 40, 50, 75, 90, 100, 150, 200, 250, 300, 350,
400, 450 or at least 500 nucleotides. In certain embodiments, the
length of the poly-A tail is adjusted to control the stability of
an mRNA polynucleotide of the invention. For example, since the
length of the poly-A tail can influence the half-life of an mRNA
polynucleotide, the length of the poly-A tail can be adjusted to
modify the level of resistance of the mRNA to nucleases and thereby
control its translational efficiency in a target cell. In certain
embodiments, the stabilized or nuclease resistant polynucleotides
are sufficiently resistant to in vivo degradation (e.g., by
nucleases), such that they may be delivered to the target cell
without a carrier.
[0054] In certain embodiments, a polynucleotide can be modified by
the incorporation 3' and/or 5' untranslated (UTR) sequences which
are not naturally found in the wild-type polynucleotide. In certain
embodiments, 3' and/or 5' flanking sequences which naturally flank
an mRNA and encode a second, unrelated protein can be incorporated
into the nucleotide sequence of an mRNA polynucleotide in order to
further enhance its translational efficiency. For example, 3' or 5'
sequences from mRNA polynucleotides which are stable (e.g., globin,
actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) can
be incorporated into the 3' and/or 5' region of a sense mRNA
polynucleotide to increase its stability. To the extent such
modifications are incorporated into a polynucleotide, in certain
embodiments the regions of the polynucleotide including such
modifications (e.g., a 3' UTR) may also represent a suitable target
to which the stabilizing oligonucleotides disclosed herein may
hybridize to in an effort to further stabilize such modified
polynucleotide.
[0055] The present inventions also contemplate modifications to the
5' end of the polynucleotides (e.g., mRNA) to include a partial
sequence of a CMV immediate-early 1 (IE1) gene, or a fragment
thereof (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) to improve the
nuclease resistance and/or improve the half-life of the
polynucleotide. In addition to increasing the stability of the mRNA
polynucleotide sequence, it has been surprisingly discovered that
the inclusion of a partial sequence of a CMV immediate-early 1
(IE1) gene enhances the translation of the mRNA and the expression
of the functional protein or enzyme. Also contemplated is the
inclusion of a sequence encoding human growth hormone (hGH), or a
fragment thereof (e.g., SEQ ID NO: 3) to one or both of the 3' and
5' ends of the polynucleotide (e.g., mRNA) to further stabilize the
polynucleotide. Generally, preferred modifications improve the
stability, translational efficiency, nuclease resistance and/or
pharmacokinetic properties (e.g., half-life) of the polynucleotide
relative to its unmodified counterpart, and include, for example
modifications made to improve such polynucleotide's resistance to
in vivo nuclease digestion.
[0056] The administration of the compositions, stabilized
polynucleotides and stabilizing oligonucleotides disclosed herein
may be facilitated by formulating such compositions in a suitable
carrier (e.g., a lipid nanoparticle). As used herein, the term
"carrier" includes any of the standard pharmaceutical carriers,
vehicles, diluents, excipients and the like which are generally
intended for use in connection with the administration of
biologically active agents, including polynucleotides. The
compositions and in particular the carriers described herein are
capable of delivering polynucleotides and/or stabilizing
oligonucleotides of varying sizes to their target cells or tissues.
In certain embodiments of the present invention, the carriers of
the present invention are capable of delivering large
polynucleotide sequences (e.g., polynucleotides of at least 1 kb,
1.5 kb, 2 kb, 2.5 kb, 5 kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30
kb, 35 kb, 40 kb, 45 kb, 50 kb, or more). The polynucleotides can
be formulated with one or more acceptable reagents to facilitate
the delivery of such polynucleotides to target cells. Appropriate
reagents are generally selected with regards to a number of
factors, which include, among other things, the biological or
chemical properties of the polynucleotides (e.g., charge), the
intended route of administration, the anticipated biological
environment to which such polynucleotides will be exposed and the
specific properties of the intended target cells. In some
embodiments, carriers, such as liposomes or synthetically-prepared
exosomes, encapsulate the polynucleotides. In some embodiments, the
carrier demonstrates preferential and/or substantial binding to a
target cell relative to non-target cells. In a preferred
embodiment, the carrier delivers its contents to the target cell
such that the polynucleotides are delivered to the appropriate
subcellular compartment, such as the cytoplasm.
[0057] In certain embodiments, the carriers disclosed herein
comprise a liposomal vesicle, or other means to facilitate the
transfer of a polynucleotide to target cells and tissues. Suitable
carriers include, but are not limited to, liposomes, nanoliposomes,
ceramide-containing nanoliposomes, proteoliposomes, both natural
and synthetically-derived exosomes, natural, synthetic and
semi-synthetic lamellar bodies, nanoparticulates, calcium
phosphor-silicate nanoparticulates, calcium phosphate
nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline
particulates, semiconductor nanoparticulates, poly(D-arginine),
nanodendrimers, starch-based delivery systems, micelles, emulsions,
niosomes, plasmids, viruses, calcium phosphate nucleotides,
aptamers, peptides and other vectorial tags. Also contemplated is
the use of bionanocapsules and other viral capsid proteins
assemblies as a suitable carrier. (Hum. Gene Ther. 2008 September;
19(9):887-95).
[0058] In a preferred embodiment of the present invention, the
carrier is formulated as a lipid nanoparticle. As used herein, the
phrase "lipid nanoparticle" refers to a carrier comprising one or
more lipids (e.g., cationic and/or non-cationic lipids).
Preferably, the lipid nanoparticles are formulated to deliver one
or more polynucleotides (e.g., mRNA) to one or more target cells or
tissues. The use of lipids, either alone or as a component of the
carrier, and in particular lipid nanoparticles, is preferred.
Examples of suitable lipids include, for example, the phosphatidyl
compounds (e.g., phosphatidylglycerol, phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, sphingolipids,
cerebrosides, and gangliosides). Also contemplated is the use of
polymers as carriers, whether alone or in combination with other
carriers. Suitable polymers may include, for example,
polyacrylates, polyalkycyanoacrylates, polylactide,
polylactide-polyglycolide copolymers, polycaprolactones, dextran,
albumin, gelatin, alginate, collagen, chitosan, cyclodextrins and
polyethylenimine. In certain embodiments, the carrier is selected
based upon its ability to facilitate the transfection of a target
cell with one or more polynucleotides.
[0059] In certain embodiments of the present invention, the carrier
may be selected and/or prepared to optimize delivery of the
polynucleotide to the target cell, tissue or organ. For example, if
the target cell is a pneumocyte the properties of the carrier
(e.g., size, charge and/or pH) may be optimized to effectively
deliver such carrier to the target cell or organ, reduce immune
clearance and/or promote retention in that target organ.
Alternatively, if the target tissue is the central nervous system
(e.g., to facilitate delivery of mRNA polynucleotides to targeted
brain or spinal tissue) selection and preparation of the carrier
must consider penetration of, and retention within the blood brain
barrier and/or the use of alternate means of directly delivering
such carrier to such target tissue. In certain embodiments, the
compositions of the present invention may be combined with agents
that facilitate the transfer of exogenous polynucleotides from the
local tissues or organs into which such compositions were
administered to one or more peripheral target organs or
tissues.
[0060] The use of liposomal carriers to facilitate the delivery of
polynucleotides to target cells is contemplated by the present
invention. Liposomes (e.g., liposomal lipid nanoparticles) are
generally useful in a variety of applications in research,
industry, and medicine, particularly for their use as carriers of
diagnostic or therapeutic compounds in vivo (Lasic, Trends
Biotechnol., 16: 307-321, 1998; Drummond et al., Pharmacol. Rev.,
51: 691-743, 1999) and are usually characterized as microscopic
vesicles having an interior aqua space sequestered from an outer
medium by a membrane of one or more bilayers. Bilayer membranes of
liposomes are typically formed by amphiphilic molecules, such as
lipids of synthetic or natural origin that comprise spatially
separated hydrophilic and hydrophobic domains (Lasic, Trends
Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes
can also be formed by amphiphilic polymers and surfactants (e.g.,
polymerosomes, niosomes, etc.).
[0061] In the context of the present invention, a liposomal carrier
typically serves to transport the polynucleotide and/or stabilizing
oligonucleotide to a target cell. For the purposes of the present
invention, the liposomal carriers are prepared to contain the
desired polynucleotides. The process of incorporating a desired
compound (e.g., a stabilized or nuclease resistant polynucleotide
and/or a stabilizing oligonucleotide) into a liposome is often
referred to as "loading" (Lasic, et al., FEBS Lett., 312: 255-258,
1992). The liposome-incorporated polynucleotides may be completely
or partially located in the interior space of the liposome, within
the bilayer membrane of the liposome, or associated with the
exterior surface of the liposome membrane. The incorporation of a
polynucleotide into liposomes is also referred to herein as
"encapsulation" wherein the polynucleotide is entirely contained
within the interior space of the liposome.
[0062] One primary purpose of incorporating a polynucleotide into a
carrier, such as a liposome, is to protect the polynucleotide from
an environment which may contain enzymes (e.g., nuclease enzymes)
or chemicals that degrade or otherwise negatively influence the
stability of the polynucleotides encapsulated therein. Accordingly,
in a preferred embodiment of the present invention, the selected
carrier is capable of further enhancing the stability of the
nuclease resistant polynucleotides (e.g., a nuclease resistant mRNA
polynucleotide encoding a functional protein) contained therein.
For example, a liposomal carrier may allow the encapsulated
polynucleotide to reach the target cell and/or may preferentially
allow the encapsulated polynucleotide to reach the target cell, or
alternatively limit the delivery of such polynucleotides to other
sites or cells where the presence of the administered
polynucleotide may be useless or undesirable. Furthermore,
incorporating the polynucleotides into a carrier, such as for
example, a cationic liposome, also facilitates the delivery of such
polynucleotides into a target cell.
[0063] Ideally, liposomal carriers are prepared to encapsulate one
or more desired polynucleotides (e.g., a nuclease resistant mRNA
polynucleotide encoding a urea cycle enzyme) such that the
compositions demonstrate a high transfection efficiency, enhanced
stability and improved translational efficiency. While liposomes
can facilitate the introduction of polynucleotides into target
cells, the addition of polycations (e.g., poly L-lysine and
protamine), as a copolymer can further facilitate, and in some
instances markedly enhance the transfection efficiency of several
types of cationic liposomes by 2-28 fold in a number of cell lines
both in vitro and in vivo. (See N. J. Caplen, et al., Gene Ther.
1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.)
[0064] The present invention contemplates the use of cationic
lipids and liposomes to encapsulate and/or enhance the delivery of
the nuclease resistant polynucleotides and/or stabilizing
oligonucleotides disclosed herein into their target cells and
tissues. As used herein, the phrase "cationic lipid" refers to any
of a number of lipid species that carry a net positive charge at a
selected pH, such as physiological pH. The contemplated liposomal
carriers and lipid nanoparticles may be prepared by including
multi-component lipid mixtures of varying ratios employing one or
more cationic lipids, non-cationic lipids and PEG-modified lipids.
Several cationic lipids have been described in the literature, many
of which are commercially available. In some embodiments, the
cationic lipid
N-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium chloride or
"DOTMA" is used. (Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413
(1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or
can be combined with a neutral lipid, such as, e.g.,
dioleoylphosphatidylethanolamine or "DOPE" or other cationic or
non-cationic lipids into a liposomal carrier or a lipid
nanoparticle, and such liposomes can be used to enhance the
delivery of polynucleotides into target cells. Particularly
suitable cationic lipids for use in the compositions and methods of
the invention include those described in international patent
publication WO 2010/053572, incorporated herein by reference, and
most particularly, C12-200 described at paragraph [00225] of WO
2010/053572. Another particularly suitable cationic lipid for use
in connection with the invention is
2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimeth-
ylethanamine or "DLin-KC2-DMA" (See, WO 2010/042877; Semple et al.,
nature Biotech. 28:172-176 (2010). Other suitable cationic lipids
include, for example, 5-carboxyspermylglycinedioctadecylamide or
"DOGS,"
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium or "DOSPA" (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982
(1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761),
1,2-Dioleoyl-3-Dimethylammonium-Propane or "DODAP",
1,2-Dioleoyl-3-Trimethylammonium-Propane or "DOTAP". Contemplated
cationic lipids also include
1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA",
1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA",
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA",
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA",
N-dioleyl-N,N-dimethylammonium chloride or "DODAC",
N,N-distearyl-N,N-dimethylammonium bromide or "DDAB",
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide or "DMRIE",
3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane or "CLinDMA",
2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy
1-(cis,cis-9', 1-2'-octadecadienoxy)propane or "CpLinDMA",
N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA",
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP",
2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP",
1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane or
"DLincarbDAP", 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or
"DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or
"DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
or "DLin-K-XTC2-DMA", or mixtures thereof (Heyes, J., et al., J
Controlled Release 107: 276-287 (2005); Morrissey, D V., et al.,
Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication
W02005/121348A1).
[0065] The use of cholesterol-based cationic lipids is also
contemplated by the present invention. Such cholesterol-based
cationic lipids can be used, either alone or in combination with
other cationic or non-cationic lipids. Suitable cholesterol-based
cationic lipids include, for example, DC-Chol
(N,N-dimethyl-N-ethylcarboxamidocholesterol),
1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem.
Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23,
139 (1997); U.S. Pat. No. 5,744,335).
[0066] In addition, several reagents are commercially available to
enhance transfection efficacy. Suitable examples include LIPOFECTIN
(DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE
(DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000. (Invitrogen), FUGENE,
TRANSFECTAM (DOGS), and EFFECTENE.
[0067] Also contemplated are cationic lipids such as the
dialkylamino-based, imidazole-based, and guanidinium-based lipids.
For example, certain embodiments are directed to a composition
comprising one or more imidazole-based cationic lipids, for
example, the imidazole cholesterol ester or "ICE" lipid
(3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,-
10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
3-(1H-imidazol-4-yl)propanoate. In a preferred embodiment, a
carrier (e.g., a lipid nanoparticle) for delivery of RNA (e.g.,
mRNA) or protein (e.g., an enzyme), for example a therapeutic
amount of RNA or protein, may comprise one or more imidazole-based
cationic lipids, for example, the imidazole cholesterol ester or
"ICE" lipid
(3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,-
10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
3-(1H-imidazol-4-yl)propanoate. The imidazole-based cationic lipids
are also characterized by their reduced toxicity relative to other
cationic lipids. The imidazole-based cationic lipids (e.g., ICE)
may be used as the sole cationic lipid in the carrier or lipid
nanoparticle, or alternatively may be combined with traditional
cationic lipids (e.g., DOPE, DC-Chol), non-cationic lipids,
PEG-modified lipids and/or helper lipids. The cationic lipid may
comprise a molar ratio of about 1% to about 90%, about 2% to about
70%, about 5% to about 50%, about 10% to about 40% of the total
lipid present in the carrier, or preferably about 20% to about 70%
of the total lipid present in the carrier.
[0068] Similarly, certain embodiments are directed to lipid
nanoparticles comprising the HGT4003 cationic lipid
2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-di-
methylethanamine, as further described in U.S. Provisional
Application No. 61/494,882 filed Jun. 8, 2011, the entire teachings
of which are incorporated herein by reference in their entirety. In
other embodiments the compositions and methods described herein are
directed to lipid nanoparticles comprising one or more ionizable
cationic lipids, such as, for example, one or more of the cationic
lipids or compounds (e.g., HGT5001, HGT5002 and HGT5003), as
further described in U.S. Provisional Application No. 61/617,468,
incorporated herein by reference in their entirety.
[0069] In other embodiments the compositions and methods described
herein are directed to lipid nanoparticles comprising one or more
cleavable lipids, such as, for example, one or more cationic lipids
or compounds that comprise a cleavable disulfide (S--S) functional
group (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as
further described in U.S. Provisional Application No. 61/494,882,
incorporated herein by reference in their entirety.
[0070] The use of polyethylene glycol (PEG)-modified phospholipids
and derivatized lipids such as derivatized cerarmides (PEG-CER),
including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene
Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the
present invention, either alone or preferably in combination with
other lipid formulations together which comprise the carrier (e.g.,
a lipid nanoparticle). Contemplated PEG-modified lipids include,
but is not limited to, a polyethylene glycol chain of up to 5 kDa
in length covalently attached to a lipid with alkyl chain(s) of
C.sub.6-C.sub.20 length. The addition of such components may
prevent complex aggregation and may also provide a means for
increasing circulation lifetime and increasing the delivery of the
lipid-polynucleotide composition to the target tissues, (Klibanov
et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be
selected to rapidly exchange out of the formulation in vivo (see
U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids
are PEG-ceramides having shorter acyl chains (e.g., C.sub.14 or
C.sub.18). The PEG-modified phospholipid and derivatized lipids of
the present invention may comprise a molar ratio from about 0% to
about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4%
to about 10%, or about 2% of the total lipid present in the
liposomal carrier. In some embodiments, the PEG-modified lipid
employed in the compositions and methods of the invention is
1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW
PEG) (DMG-PEG2000).
[0071] The present invention also contemplates the use of
non-cationic lipids to facilitate delivery of the nuclease
resistant polynucleotides or stabilizing oligonucleotides to one or
more target cells, organs or tissues. As used herein, the phrase
"non-cationic lipid" refers to any neutral, zwitterionic or anionic
lipid. As used herein, the phrase "anionic lipid" refers to any of
a number of lipid species that carry a net negative charge at a
selected pH, such as physiological pH. Non-cationic lipids include,
but are not limited to, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof. Such non-cationic lipids may be used alone, but
are preferably used in combination with other excipients, for
example, cationic lipids. When used in combination with a cationic
lipid, the non-cationic lipid may comprise a molar ratio of 5% to
about 90%, or preferably about 10% to about 70% of the total lipid
present in the carrier.
[0072] Preferably, the carrier (e.g., a lipid nanoparticle) is
prepared by combining multiple lipid and/or polymer components. For
example, a carrier may be prepared using
DSPC/CHOL/DODAP/C8-PEG-5000 ceramide in a molar ratio of about 1 to
50:5 to 65:5 to 90:1 to 25, respectively. A carrier may be
comprised of additional lipid combinations in various ratios,
including for example, DSPC/CHOL/DODAP/mPEG-5000 (e.g., combined at
a molar ratio of about 33:40:25:2), DSPC/CHOL/DODAP/C8 PEG-2000-Cer
(e.g., combined at a molar ratio of about 31:40:25:4),
POPC/DODAP/C8-PEG-2000-Cer (e.g., combined at a molar ratio of
about 75-87:3-14:10) or DSPC/CHOL/DOTAP/C8 PEG-2000-Cer (e.g.,
combined at a molar ratio of about 31:40:25:4). The selection of
cationic lipids, non-cationic lipids and/or PEG-modified lipids
which comprise the liposomal carrier or lipid nanoparticle, as well
as the relative molar ratio of such lipids to each other, is based
upon the characteristics of the selected lipid(s), the nature of
the intended target cells or tissues and the characteristics of the
polynucleotides to be delivered by the liposomal carrier.
Additional considerations include, for example, the saturation of
the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity
and toxicity of the selected lipid(s).
[0073] The liposomal carriers for use in the present invention can
be prepared by various techniques which are presently known in the
art. Multi-lamellar vesicles (MLV) may be prepared by conventional
techniques, for example, by depositing a selected lipid on the
inside wall of a suitable container or vessel by dissolving the
lipid in an appropriate solvent, and then evaporating the solvent
to leave a thin film on the inside of the vessel or by spray
drying. An aqueous phase may then added to the vessel with a
vortexing motion which results in the formation of MLVs.
Unilamellar vesicles (ULV) can then be formed by homogenization,
sonication or extrusion of the multi-lamellar vesicles. In
addition, unilamellar vesicles can be formed by detergent removal
techniques.
[0074] In certain embodiments of this invention, the compositions
comprise a carrier wherein a nuclease resistant polynucleotide
(e.g., mRNA encoding OTC) is associated on both the surface of the
carrier (e.g., a liposome) and encapsulated within the same
carrier. For example, during preparation of the compositions of the
present invention, cationic liposomal carriers may associate with
the polynucleotides (e.g., mRNA) through electrostatic interactions
with such therapeutic mRNA.
[0075] In certain embodiments, the compositions or polynucleotides
of the present invention may comprise or be loaded with a
diagnostic radionuclide, fluorescent material or other material
that is detectable in both in vitro and in vivo applications. For
example, suitable diagnostic materials for use in the present
invention may include Rhodamine-dioleoylphosphatidylethanolamine
(Rh-PE), Green Fluorescent Protein mRNA (GFP mRNA), Renilla
Luciferase mRNA and Firefly Luciferase mRNA.
[0076] During the preparation of liposomal carriers, water soluble
carrier agents may be encapsulated in the aqueous interior by
including them in the hydrating solution, and lipophilic molecules
may be incorporated into the lipid bilayer by inclusion in the
lipid formulation. In the case of certain molecules (e.g., cationic
or anionic lipophilic polynucleotides), loading of the
polynucleotide into preformed liposomes may be accomplished, for
example, by the methods described in U.S. Pat. No. 4,946,683, the
disclosure of which is incorporated herein by reference. Following
encapsulation of the polynucleotide, the liposomes may be processed
to remove un-encapsulated mRNA through processes such as gel
chromatography, diafiltration or ultrafiltration. For example, if
it is desirous to remove externally bound polynucleotide from the
surface of the liposomal carrier formulation, such liposomes may be
subject to a Diethylaminoethyl SEPHACEL column.
[0077] In addition to the encapsulated nuclease resistant
polynucleotide, one or more secondary therapeutic or diagnostic
agents may be included in the carrier. For example, such additional
therapeutic agents may be associated with the surface of the
liposome, can be incorporated into the lipid bilayer of a liposome
by inclusion in the lipid formulation or loading into preformed
liposomes. (See, e.g., U.S. Pat. Nos. 5,194,654 and 5,223,263,
which are incorporated by reference herein).
[0078] There are several methods for reducing the size, or
"sizing", of liposomal carriers, and any of these methods may
generally be employed when sizing is used as part of the invention.
The extrusion method is a preferred method of liposome sizing.
(Hope, M J et al. Reduction of Liposome Size and Preparation of
Unilamellar Vesicles by Extrusion Techniques. In: Liposome
Technology (G. Gregoriadis, Ed.) Vol. 1. p 123 (1993)). The method
comprises a step of extruding liposomes through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane to reduce
liposome sizes to a relatively well-defined size distribution.
Typically, the suspension is cycled through the membrane one or
more times until the desired liposome size distribution is
achieved. The liposomes may be extruded through successively
smaller pore membranes to achieve gradual reduction in liposome
size.
[0079] A variety of alternative methods known in the art are
available for sizing of a population of liposomal carriers. One
such sizing method is described in U.S. Pat. No. 4,737,323, the
entire teachings of which are incorporated herein by reference.
Sonicating a liposome suspension either by bath or probe sonication
produces a progressive size reduction down to small ULV less than
about 0.05 microns in diameter. Homogenization is another method
that relies on shearing energy to fragment large liposomes into
smaller ones. In a typical homogenization procedure, MLV are
recirculated through a standard emulsion homogenizer until selected
liposome sizes, typically between about 0.1 and 0.5 microns, are
observed. The size of the liposomal vesicles may be determined by
quasi-electric light scattering (QELS) as described in Bloomfield,
Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein
by reference. Average liposome diameter may be reduced by
sonication of formed liposomes. Intermittent sonication cycles may
be alternated with QELS assessment to guide efficient liposome
synthesis.
[0080] Selection of the appropriate size of a carrier must take
into consideration the site of the target cell or tissue and to
some extent the application for which the liposome is being made.
For example, to the extent that the compositions are intended for
pulmonary administration (e.g., as an inhalable liquid or solid
carrier), the ability of the carrier to distribute into the tissues
of the lung may be influenced by the size of the carrier particles
that comprise such composition. Accordingly, in certain
embodiments, it may be desirable to enhance the distribution of
such compositions to certain cells or tissues of the lung by
appropriately sizing such compositions such that upon
administration (e.g., by inhalation), such compositions distribute
to one or more targeted cells and tissues.
[0081] In some embodiments, the compositions provided herein are
generally administered via the pulmonary route of administration.
Accordingly, in certain embodiments the carriers and/or
compositions disclosed herein are prepared for pulmonary
administration. For example, a pulmonary surfactant may be added as
an excipient component of a carrier formulation (e.g., a lipid
nanoparticle comprising one or more cationic lipids, neutral lipids
and pulmonary surfactants). Alternatively, in certain embodiments,
the compositions disclosed herein may comprise one or more
pulmonary surfactants that may be formulated independently of the
carrier. The inclusion of pulmonary surfactants (e.g., lamellar
bodies) in the compositions disclosed herein may also serve to
loosen, break-up or otherwise facilitate the elimination of mucous
from the lungs of the subject, thereby improving the distribution
of the compositions into the tissues of the lung. In certain
embodiments, such lamellar bodies may also function as a carrier to
facilitate the delivery or distribution of one or more
polynucleotides to target cells, tissues and/or organs. For
example, such lamellar body carriers may also be loaded or
otherwise prepared such that they also comprise one or more
polynucleotides (e.g., mRNA encoding a functional protein or
enzyme). In other embodiments, the compositions disclosed herein
may comprise synthetically- or naturally-prepared lamellar bodies
and lipid nanoparticles.
[0082] Where the compositions disclosed herein comprise lamellar
bodies, such lamellar bodies may comprise one or more of
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylinositol (PI),
phosphatidylglycerol (PG), sphingomyelin (SM), cholesterol (CHOL)
and dipalmitoylphosphatidylcholine (DPPC).
[0083] In certain embodiments, the compositions and/or carriers
disclosed herein may also comprise one or more exosomes. Exosomes
are small micro-vesicles that are shed from the surface membranes
of most cell types (e.g., mammalian cell types) and that have been
implicated as playing a pivotal role in cell-to-cell communications
(e.g., as a vehicle for transferring various bioactive molecules).
(See, e.g., Camussi, et al., Kidney Int. (2010); 78(9): 838-48, the
contents of which are incorporated herein by reference in their
entirety.)
[0084] In certain embodiments, the liver represents an important
peripheral target organ for the compositions of the present
invention in part due to its central role in metabolism and
production of proteins and accordingly diseases which are caused by
defects in liver-specific gene products (e.g., the urea cycle
disorders) may benefit from specific targeting of cells (e.g.,
hepatocytes). Accordingly, in certain embodiments of the present
invention, the structural characteristics of the target tissue may
be exploited to direct the distribution of the liposomal carrier
and its polynucleotide payload to such target tissues. For example,
to target hepatocytes a liposomal carrier may be sized such that
its dimensions are smaller than the fenestrations of the
endothelial layer lining hepatic sinusoids in the liver;
accordingly the liposomal carrier can readily penetrate such
endothelial fenestrations to reach the target hepatocytes.
Alternatively, a liposomal carrier may be sized such that the
dimensions of the liposome are of a sufficient diameter to limit or
expressly avoid distribution into certain cells or tissues (e.g.,
peripheral cells and tissues). For example, a liposomal carrier may
be sized such that its dimensions are larger than the fenestrations
of the endothelial layer lining hepatic sinusoids to thereby limit
distribution of the liposomal carrier to hepatocytes. In such an
embodiment, large liposomal carriers will not easily penetrate the
endothelial fenestrations, and would instead be cleared by the
macrophage Kupffer cells that line the liver sinusoids. Generally,
the size of the carrier is within the range of about 25 to 250 nm,
preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm,
75 nm, 50 nm, 25 nm or 10 nm.
[0085] Similarly, the compositions of the present invention may be
prepared to preferentially distribute to other local and/or
peripheral target tissues, cells or organs, such as the brain,
cerebrospinal fluid, muscle, heart, lungs, kidneys and/or spleen.
For example, the carriers of the present invention may be prepared
to achieve enhanced delivery to the target cells and tissues.
Accordingly, the compositions of the present invention may be
enriched with additional cationic, non-cationic and PEG-modified
lipids to further target tissues or cells.
[0086] In certain embodiments, one or more peripheral target cells
and tissues may function as a biological reservoir or depot capable
of expressing or otherwise producing and systemically excreting a
functional protein or enzyme, as disclosed for example, in
International Application No. PCT/US2010/058457 and in U.S.
Provisional Application No. 61/494,881, the teachings of which are
both incorporated herein by reference in their entirety.
Accordingly, in certain embodiments of the present invention the
liposomal carrier may target cells and/or preferentially distribute
to one or more target cells and tissues (e.g., target cells and
tissues of the liver) following their delivery to a subject.
Following transfection of the target cells (e.g., local endothelial
cells of the lung), the nuclease resistant mRNA polynucleotides
loaded in the carrier are translated and a functional expression
product expressed, excreted and systemically distributed.
[0087] In some embodiments, the compositions of the present
invention comprise one or more additional molecules (e.g.,
proteins, peptides, aptamers or oliogonucleotides) which facilitate
the transfer of the polynucleotides (e.g., mRNA, miRNA, snRNA and
snoRNA) from the carrier into an intracellular compartment of the
target cell. In certain embodiments, the additional molecule
facilitates the delivery of the polynucleotides into, for example,
the cytosol, the lysosome, the mitochondrion, the nucleus, the
nucleolae or the proteasome of a target cell. Such agents may
facilitate the transport of the translated protein of interest from
the cytoplasm to its normal intercellular location (e.g., in the
mitochondrion) to treat deficiencies in that organelle. In some
embodiments, the agent is selected from the group consisting of a
protein, a peptide, an aptamer, and an oligonucleotide. Similarly,
in certain embodiments where such agents may exploit the presence
of one or more endogenous receptors or mechanisms to actively
transport such expressed proteins or enzymes into the plasma. In
other embodiments, the compositions described herein may comprise
one or more excipients that facilitate the distribution of such
compositions into the plasma, where such compositions may further
distribute to one or more additional target organs, tissues or
cells.
[0088] In certain embodiments, the compositions of the present
invention facilitate a subject's endogenous production of one or
more functional proteins and/or enzymes. The endogenous production
or translation of exogenous nuclease resistant mRNA polynucleotides
by a subject to produce one or more expression products (e.g.,
proteins and/or enzymes) may, in certain instances demonstrate less
immunogenicity relative to their recombinantly-prepared
counterparts that often lack native post-translational
modifications (e.g., glycosylation). Similarly, the endogenously
produced or translated proteins and/or enzymes may demonstrate more
biological activity relative to their recombinantly-prepared
counterparts. In a preferred embodiment of the present invention,
the carriers comprise nuclease resistant mRNA polynucleotides which
encode a deficient expression product (e.g., a protein or enzyme).
The administration of an mRNA polynucleotide (e.g., a nuclease
resistant mRNA polynucleotide) encoding a deficient protein or
enzyme avoids the need to deliver the polynucleotides to specific
organelles within a target cell (e.g., mitochondria). Rather, upon
transfection of a target cell and delivery of the polynucleotides
to the cytoplasm of the target cell, the mRNA polynucleotide
contents of a carrier may be translated and a functional protein or
enzyme expressed.
[0089] The present invention also contemplates the discriminatory
targeting of target cells and tissues by both passive and active
targeting means. The phenomenon of passive targeting exploits the
natural distributions patterns of a carrier in vivo without relying
upon the use of additional excipients or means to enhance
recognition of the carrier by target cells. For example, carriers
which are subject to phagocytosis by the cells of the
reticulo-endothelial system are likely to accumulate in the liver
or spleen, and accordingly may provide means to passively direct
the delivery of the compositions to such target cells.
[0090] The present invention also contemplates active targeting,
which involves the use of additional excipients, referred to herein
as "targeting ligands" that may be bound (either covalently or
non-covalently) to the carrier to encourage localization of such
carrier at certain target cells or target tissues. For example,
targeting may be mediated by the inclusion of one or more
endogenous targeting ligands (e.g., apolipoprotein E) in or on the
carrier to encourage distribution to the target cells or tissues.
Recognition of the targeting ligand by the target tissues actively
facilitates tissue distribution and cellular uptake of the carrier
and/or its polynucleotide contents in the target cells and tissues
(e.g., the inclusion of an apolipoprotein-E targeting ligand in or
on the carrier may encourage recognition and binding of the carrier
to endogenous low density lipoprotein receptors expressed by
hepatocytes). As provided herein, the composition can comprise a
ligand capable of enhancing affinity of the composition to the
target cell. Targeting ligands may be linked to the outer bilayer
of the lipid particle during formulation or post-formulation. These
methods are well known in the art. In addition, some lipid particle
formulations may employ fusogenic polymers such as PEAA,
hemagluttinin, other lipopeptides (see U.S. patent application Ser.
Nos. 08/835,281, and 60/083,294, which are incorporated herein by
reference) and other features useful for in vivo and/or
intracellular delivery. In other some embodiments, the compositions
of the present invention demonstrate improved transfection
efficacies, and/or demonstrate enhanced selectivity towards target
cells or tissues of interest. Contemplated therefore are
compositions which comprise one or more ligands (e.g., peptides,
aptamers, oligonucleotides, a vitamin or other molecules) that are
capable of enhancing the affinity of the compositions and their
polynucleotide contents for the target cells or tissues. Suitable
ligands may optionally be bound or linked to the surface of the
carrier. In some embodiments, the targeting ligand may span the
surface of a carrier or be encapsulated within the carrier.
Suitable ligands and are selected based upon their physical,
chemical or biological properties (e.g., selective affinity and/or
recognition of target cell surface markers or features.)
Cell-specific target sites and their corresponding targeting ligand
can vary widely. Suitable targeting ligands are selected such that
the unique characteristics of a target cell are exploited, thus
allowing the composition to discriminate between target and
non-target cells. For example, compositions of the present
invention may bear surface markers (e.g., apolipoprotein-B or
apolipoprotein-E) that selectively enhance recognition of, or
affinity to hepatocytes (e.g., by receptor-mediated recognition of
and binding to such surface markers). Additionally, the use of
galactose as a targeting ligand would be expected to direct the
compositions of the present invention to parenchymal hepatocytes,
or alternatively the use of mannose containing sugar residues as a
targeting ligand would be expected to direct the compositions of
the present invention to liver endothelial cells (e.g., mannose
containing sugar residues that may bind preferentially to the
asialoglycoprotein receptor present in hepatocytes). (See Hillery A
M, et al. "Drug Delivery and Targeting: For Pharmacists and
Pharmaceutical Scientists" (2002) Taylor & Francis, Inc.) The
presentation of such targeting ligands that have been conjugated to
moieties present in the carrier (e.g., a lipid nanoparticle)
therefore facilitate recognition and uptake of the compositions of
the present invention in target cells and tissues. Examples of
suitable targeting ligands include one or more peptides, proteins,
aptamers, vitamins and oligonucleotides.
[0091] In certain embodiments, the carriers disclosed herein may
also comprise one or more opsonization-inhibiting moieties, which
are typically large hydrophilic polymers that are chemically or
physically bound to a carrier or vehicle such as a lipid
nanoparticle (e.g., by the intercalation of a lipid-soluble anchor
into the membrane itself, or by binding directly to active groups
of membrane lipids). These opsonization-inhibiting hydrophilic
polymers form a protective surface layer which significantly
decreases the uptake of the pharmaceutical carrier or vehicle
(e.g., liposomes) by the macrophage-monocyte system and
reticulo-endothelial system, as described for example, in U.S. Pat.
No. 4,920,016, the entire disclosure of which is herein
incorporated by reference. Carriers modified with
opsonization-inhibition moieties thus remain in the circulation
much longer than their unmodified counterparts.
[0092] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, to which the compositions and
methods of the present invention are administered. Typically, the
terms "subject" and "patient" are used interchangeably herein in
reference to a human subject.
[0093] As used herein, the term "target cell" refers to a cell to
which a composition, nuclease resistant polynucleotide and/or
stabilizing oligonucleotide of the invention are to be directed or
targeted. In some embodiments, the target cells are deficient in a
protein or enzyme of interest. In some embodiments, cells are
targeted based on their ability to secrete one or more expression
products extracellularly. The compositions and methods of the
present invention may be prepared to preferentially target a
variety of target cells, which include, but are not limited to,
pulmonary epithelial cells (e.g., Type I and II pneumocytes),
alveolar cells, hepatocytes, hematopoietic cells, epithelial cells,
endothelial cells, lung cells, bone cells, stem cells, mesenchymal
cells, neural cells (e.g., meninges, astrocytes, motor neurons,
cells of the dorsal root ganglia and anterior horn motor neurons),
photoreceptor cells (e.g., rods and cones), retinal pigmented
epithelial cells, secretory cells, cardiac cells, adipocytes,
vascular smooth muscle cells, cardiomyocytes, skeletal muscle
cells, beta cells, pituitary cells, synovial lining cells, ovarian
cells, testicular cells, fibroblasts, B cells, T cells,
reticulocytes, leukocytes, granulocytes and tumor cells. In certain
embodiments, the target cells comprise Type I pneumocytes, Type II
pneumocytes, alveolar cells and combinations thereof. Following
transfection of one or more target cells by the compositions and
nuclease resistant polynucleotides of the present invention,
expression of the polypeptide, protein or enzyme encoded by such
polynucleotide may be preferably stimulated and the capability of
such target cells to express the protein of interest enhanced. For
example, transfection of a target cell with a stabilized or
duplexed mRNA polynucleotide encoding the OTC enzyme may facilitate
the enhanced expression of the corresponding expression product
(OTC) following translation of the mRNA polynucleotide.
[0094] Also contemplated by the present inventions are methods of
treating a subject having or otherwise affected by a protein or
enzyme deficiency. Such methods generally comprise administering to
the subject (e.g., parenterally) a composition comprising a
nuclease resistant mRNA polynucleotide and a suitable carrier,
wherein the mRNA encodes an enzyme or protein in which the subject
is deficient.
[0095] The compositions and methods of the present invention may be
suitable for the treatment of diseases or disorders relating to the
deficiency of proteins and/or enzymes. In certain embodiments, the
stabilized or nuclease resistant polynucleotides of the present
invention encode functional proteins or enzymes that are excreted
or secreted by the target cell into the surrounding extracellular
fluid (e.g., mRNA encoding hormones and neurotransmitters).
Alternatively, in other embodiments, the polynucleotides (e.g.,
mRNA encoding urea cycle metabolic disorders) of the present
invention encode functional proteins or enzymes that remain in the
cytosol of the target cell. Other disorders for which the present
invention are useful include disorders such as Duchenne muscular
dystrophy, blood clotting disorders, such as e.g., hemophelia,
SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral
sclerosis (ALS); GALT-related galactosemia; Cystic Fibrosis (CF);
SLC3A1-related disorders including cystinuria; COL4A5-related
disorders including Alport syndrome; galactocerebrosidase
deficiencies; X-linked adrenoleukodystrophy and
adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher
disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilippo B
syndrome (MPS IIIB); CTNS-related cystinosis; the FMR1-related
disorders which include Fragile X syndrome, Fragile X-Associated
Tremor/Ataxia Syndrome and Fragile X Premature Ovarian Failure
Syndrome; Prader-Willi syndrome; hereditary hemorrhagic
telangiectasia (AT); Niemann-Pick disease Type C1; the neuronal
ceroid lipofuscinoses-related diseases including Juvenile Neuronal
Ceroid Lipofuscinosis (JNCL), Juvenile Batten disease,
Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1
and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and
EIF2B5-related childhood ataxia with central nervous system
hypomyelination/vanishing white matter; CACNA1A and CACNB4-related
Episodic Ataxia Type 2; the MECP2-related disorders including
Classic Rett Syndrome, MECP2-related Severe Neonatal Encephalopathy
and PPM-X Syndrome; CDKL5-related Atypical Rett Syndrome; Kennedy's
disease (SBMA); Notch-3 related cerebral autosomal dominant
arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL); SCN1A and SCN1B-related seizure disorders; the
Polymerase G-related disorders which include Alpers-Huttenlocher
syndrome, POLG-related sensory ataxic neuropathy, dysarthria, and
ophthalmoparesis, and autosomal dominant and recessive progressive
external ophthalmoplegia with mitochondrial DNA deletions; X-Linked
adrenal hypoplasia; X-linked agammaglobulinemia; and Wilson's
disease. In certain embodiments, the polynucleotides, and in
particular mRNA, of the present invention may encode functional
proteins or enzymes. For example, the compositions of the present
invention may include mRNA encoding erythropoietin, al-antitrypsin,
carboxypeptidase N, human growth hormone, Factor VII, Factor III,
Factor IX, or cystic fibrosis transmembrane conductance regulator
(CFTR).
[0096] Alternatively the nuclease resistant polynucleotides
disclosed herein may encode full length antibodies or smaller
antibodies (e.g., both heavy and light chains) to confer immunity
to a subject. While certain embodiments of the present invention
relate to methods and compositions useful for conferring immunity
to a subject (e.g., via the translation of mRNA polynucleotides
encoding functional antibodies), the inventions disclosed herein
and contemplated hereby are broadly applicable. In an alternative
embodiment the compositions of the present invention encode
antibodies that may be used to transiently or chronically affect a
functional response in subjects. For example, the nuclease
resistant mRNA polynucleotides of the present invention may encode
a functional monoclonal or polyclonal antibody, which upon
translation (and as applicable, systemic excretion from the target
cells) may be useful for targeting and/or inactivating a biological
target (e.g., a stimulatory cytokine such as tumor necrosis
factor). Similarly, the nuclease resistant mRNA polynucleotides of
the present invention may encode, for example, functional
anti-nephritic factor antibodies useful for the treatment of
membranoproliferative glomerulonephritis type II or acute hemolytic
uremic syndrome, or alternatively may encode anti-vascular
endothelial growth factor (VEGF) antibodies useful for the
treatment of VEGF-mediated diseases, such as cancer.
[0097] The compositions of the present invention may be
administered and dosed in accordance with current medical practice,
taking into account the clinical condition of the subject, the site
and method of administration, the scheduling of administration, the
subject's age, sex, body weight and other factors relevant to
clinicians of ordinary skill in the art. The "effective amount" for
the purposes herein may be determined by such relevant
considerations as are known to those of ordinary skill in
experimental clinical research, pharmacological, clinical and
medical arts. In some embodiments, the amount administered is
effective to achieve at least some stabilization, improvement or
elimination of symptoms and other indicators as are selected as
appropriate measures of disease progress, regression or improvement
by those of skill in the art. For example, a suitable amount and
dosing regimen is one that causes at least transient expression of
the stable or nuclease resistant polynucleotide in the target
cell.
[0098] Suitable routes of administration of the compositions
disclosed herein may include, for example, pulmonary, oral, rectal,
vaginal, transmucosal, or intestinal administration; parenteral
delivery, including intramuscular, subcutaneous, intramedullary
injections, as well as intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections.
[0099] In certain embodiments, the compositions of the present
invention are formulated such that they are suitable for
extended-release of the stabilized or nuclease resistant
polynucleotides contained therein. Such extended-release
compositions may be conveniently administered to a subject at
extended dosing intervals. For example, in certain embodiments, the
compositions of the present invention are administered to a subject
twice day, daily or every other day. In a preferred embodiment, the
compositions of the present invention are administered to a subject
twice a week, once a week, every ten days, every two weeks, every
three weeks, or more preferably every four weeks, once a month,
every six weeks, every eight weeks, every other month, every three
months, every four months, every six months, every eight months,
every nine months or annually. Also contemplated are compositions
and liposomal carriers which are formulated for depot
administration (e.g., intramuscularly, subcutaneously,
intravitreally) to either deliver or release a polynucleotides
(e.g., mRNA) over extended periods of time. Preferably, the
extended-release means employed are combined with modifications
made to the polynucleotide to enhance stability.
[0100] Also contemplated herein are lyophilized pharmaceutical
compositions comprising one or more of the compounds disclosed
herein and related methods for the use of such lyophilized
compositions as disclosed for example, in U.S. Provisional
Application No. 61/494,882 filed Jun. 8, 2011, the teachings of
which are incorporated herein by reference in their entirety. For
example, the lyophilized pharmaceutical compositions according to
the invention may be reconstituted prior to their administration to
a subject (e.g., reconstituted using purified water or normal
saline and inhaled by a subject using a device such as a
nebulizer). In certain embodiments, the lyophilized compositions
can be reconstituted in vivo, for example by lyophilizing such
composition in an appropriate dosage form (e.g., an intradermal
dosage form such as a disk, rod or membrane) and administering such
composition such that it is rehydrated over time in vivo by the
individual's bodily fluids.
[0101] While certain compounds, compositions and methods of the
present invention have been described with specificity in
accordance with certain embodiments, the following examples serve
only to illustrate the compounds of the invention and are not
intended to limit the same. Each of the publications, reference
materials, accession numbers and the like referenced herein to
describe the background of the invention and to provide additional
detail regarding its practice are hereby incorporated by reference
in their entirety.
[0102] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
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 also includes embodiments in
which more than one, or the entire group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where elements are presented as lists, (e.g., in
Markush group or similar format) it is to be understood that each
subgroup of the elements is also disclosed, and any element(s) can
be removed from the group. It should be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not in every case
been specifically set forth in so many words herein. It should also
be understood that any embodiment or aspect of the invention can be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification. The
publications and other reference materials referenced herein to
describe the background of the invention and to provide additional
detail regarding its practice are hereby incorporated by
reference.
EXAMPLES
Example 1
[0103] The present example illustrates the ability of stabilizing
oligonucleotides of the present invention to enhance the production
of protein when co-administered with non-denatured in vitro
transcribed mRNA. Without wishing to be bound by any theory, it is
contemplated that the stabilizing oligonucleotides modulate the
nuclease resistance and increases the translational efficiency of
mRNA polynucleotide transcripts.
[0104] To perform the instant studies, a 15-mer (2'O-Me-uracil)
stabilizing oligonucleotide having a phosphorothioate backbone
(MW=4965.8 g/mol) was prepared and which was designed to be
complementary to the poly-A tail of an mRNA polynucleotide
(MW=299605 g/mol) encoding human erythropoietin (EPO) protein. The
EPO mRNA transcript was contacted with the stabilizing
oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 1:1, 10:1 and
100:1 parts stabilizing oligonucleotide to mRNA polynucleotide. The
resultant stabilized mRNA transcripts (designated "0.001", "0.01",
"0.1", "0.25", "1", "10" or "100") or the untreated, non-denatured
EPO polynucleotide control transcript (designated "Unhybridized")
were then transiently transfected into 293T cells. The cumulative
amounts of EPO protein produced and expressed by the transfected
293T cells were then measured at 6, 24 and 72 hour intervals.
[0105] As illustrated in FIG. 2 and Table 1, with the exception of
the stabilized EPO mRNA transcript prepared using 100:1 parts
stabilizing oligonucleotide to mRNA (designated "100"), the
cumulative amount of EPO protein produced and secreted by the 293T
cells that were transfected with the stabilized mRNA transcripts
exceeded the cumulative amount of EPO protein produced by the cells
transfected with the Unhybridized mRNA transcript. In particular,
the stabilized EPO mRNA transcripts designated 0.001, 0.01, 0.1,
0.25, 1 and 10 each resulted in the production of more EPO protein
relative to the Unhybridized EPO control and, in certain instances
exceeded the amount of EPO protein produced by the control by over
160% at the 6 hour time point.
TABLE-US-00002 TABLE 1 Cumulative Amount EPO Produced (%) 6 hr 24
hr 72 hr Unhybridized 100 100 100 100 66.5257 57.06707 54.53366 10
153.1145 132.3777 125.6762 1 161.0291 130.1917 122.7891 0.25
163.0904 139.4457 133.4223 0.1 157.012 127.0178 122.2577 0.01
146.0027 121.2735 114.2745 0.001 152.7725 150.0675 145.8507 Blank 0
0 0 Lipofectamine 0 0 0
Example 2
[0106] The present example further illustrates the ability of the
stabilizing oligonucleotides of the present invention to enhance
the protein production by first hybridizing to a denatured
single-stranded mRNA to form a stabilized mRNA before administering
into cells for protein production
[0107] As described in Example 1 above, a 15-mer (2'O-Me-uracil)
stabilizing oligonucleotide having a phosphorothioate backbone was
prepared and which was designed to be complementary to the poly-A
tail of an mRNA polynucleotide encoding human erythropoietin (EPO)
protein. The EPO mRNA transcript was first denatured at 65.degree.
C. for 10 minutes, and then contacted with the stabilizing
oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 1:1, 10:1 and
100:1 parts stabilizing oligonucleotide to mRNA polynucleotide. The
resultant stabilized mRNA transcripts (designated "0.001", "0.01",
"0.1", "0.25", "1", "10" or "100") or the untreated, denatured EPO
polynucleotide control transcript (designated "Unhybridized") were
then transiently transfected into 293T cells. The cumulative
amounts of EPO protein produced and expressed by the transfected
293T cells were then measured at 6, 24 and 72 hour intervals.
[0108] As illustrated in FIG. 3 and in Table 2 below, relative to
the denatured Unhybridized control mRNA, the percentage of the
cumulative amount of EPO protein produced and secreted by the 293T
cells transfected with the stabilized mRNA polynucleotide
consistently exceeded the percentage of the cumulative amount of
EPO protein produced and secreted by the Unhybridized mRNA
polynucleotide at each time point evaluated.
TABLE-US-00003 TABLE 2 Cumulative Amount EPO Produced (%) 6 hr 24
hr 72 hr Unhybridized 100 100 100 100 351.0201 398.7672 383.0498 10
482.7039 586.7506 555.2077 1 633.1448 685.7419 656.1553 0.25
597.5827 598.5531 572.3968 0.1 512.6893 587.4839 554.1234 0.01
697.0948 1062.025 1003.248 0.001 281.3981 314.8646 296.2899
[0109] For example, the stabilized EPO transcript designated 0.01
demonstrated an approximately 700% increase in the cumulative
amount of EPO protein produced relative to the Unhybridized control
transcript at the 6 hour time point and in excess of 1,000% at both
the 24 hour and 72 hour time points. Each of the stabilized mRNA
transcripts evaluated were characterized by an increase in the
cumulative amount of EPO protein produced relative to the
Unhybridized control.
Example 3
[0110] The instant study was performed to investigate optimal
length of the stabilizing oligonucleotides of the present
invention.
[0111] A 30-mer (2'O-Me-uracil) stabilizing oligonucleotide having
a phosphorothioate backbone was prepared and which was designed to
be complementary to the poly-A tail of an mRNA polynucleotide
encoding human erythropoietin (EPO) protein. A non-denatured EPO
mRNA transcript was contacted with the stabilizing oligonucleotide
at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1 and 2:1 parts
stabilizing oligonucleotide to mRNA polynucleotide. The resultant
stabilized mRNA transcripts (designated "0.001", "0.01", "0.1",
"0.25", "0.5", "1" or "2") or the untreated, non-denatured EPO
polynucleotide control transcript (designated "Unhybridized") were
then transiently transfected into 293T cells. The cumulative
amounts of EPO protein produced and expressed by the transfected
293T cells were then measured at 24, 48, 72 and 96 hour
intervals.
[0112] As illustrated in FIG. 4, those stabilized mRNA
polynucleotides prepared using 0.1:1 and 0.5:1 parts stabilizing
oligonucleotide to mRNA polynucleotide (designated "0.1" and
"0.5"), cumulatively produced and secreted more EPO protein
relative to the Unhybridized control polynucleotide. Interestingly,
an approximately 10% reduction of the cumulative amount of EPO
protein produced relative to the Unhybridized control
polynucleotide was observed with several of the stabilized mRNA
transcripts evaluated (e.g., the stabilized mRNA transcript
designated "0.25"). In general, the cumulative amount of EPO
protein produced using the 30-mer stabilizing oligonucleotide
appeared to be less than that observed using shorter stabilizing
oligonucleotides (e.g., a 15-mer stabilizing oligonucleotide).
Without wishing to be bound by any particular theory, such
reduction may be due in part to the greater degree of hybridization
observed with longer stabilizing oligonucleotides, or the
interference with the ability of the mRNA transcript to form stable
secondary structures.
[0113] The foregoing examples demonstrate that the stabilized mRNA
transcripts that were prepared by exposure of the mRNA
polynucleotides to stabilizing oligonucleotides produced more
protein and demonstrated improved translational efficiencies
relative to those stabilized mRNA transcripts that were prepared by
exposure to the highest ratios of stabilizing oligonucleotide to
mRNA polynucleotide. In particular, those stabilized mRNA
polynucleotides prepared by exposure to about 0.001:1, 0.01:1,
0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 10:1 parts stabilizing
oligonucleotide to mRNA polynucleotide appeared result in more
protein being produced and secreted by the transfected cells
relative to the native or un-stabilized mRNA transcript.
[0114] Without wishing to be bound by any particular theories, it
is believed that a greater degree of hybridization of the
stabilizing oligonucleotide to the mRNA transcript may interfere
(e.g., sterically interfere) with the ability of the mRNA
transcript to form secondary structures (e.g., hairpin loops) that
may serve to further protect and stabilize the mRNA transcript from
nuclease degradation. Similarly, a greater degree of hybridization
of the mRNA transcript may negatively impacting endogenous cellular
function, for example, by interfering with the ability of cells or
of organelles within such cells to translate the mRNA
polynucleotide transcript. The present inventors have also observed
that hybridization of the stabilizing oligonucleotides to the mRNA
polynucleotide transcript at lower concentrations (in particular at
0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 10:1 parts stabilizing
oligonucleotide to mRNA polynucleotide) appear to have stabilized
the mRNA polynucleotide from nuclease degradation, while not
materially impacting or negatively interfering with the ability of
such stabilized mRNA transcript to form secondary structures. The
exposure of an mRNA transcript to lower concentrations or ratios of
the stabilizing oligonucleotide (e.g., about 0.01:1, 0.1:1, 0.25:1,
0.5:1, 1:1, 2:1, 10:1 parts stabilizing oligonucleotide to mRNA
polynucleotide) therefore appears to provide optimum stabilization
of mRNA polynucleotide transcript. Similarly, in certain
embodiments, upon hybridizing to an mRNA transcript, the
stabilizing oligonucleotides of shorter lengths (e.g., about
15-mer) appear to demonstrate optimal stabilization of the mRNA
transcript. Accordingly, the foregoing evidences the methods of
modulating the nuclease resistance of polynucleotides and the
improved translational efficiencies observed when polynucleotides
are stabilized with one or more stabilizing oligonucleotides.
Sequence CWU 1
1
31140RNAArtificial SequenceCMV Polynucleotide Sequence 1ggacagaucg
ccuggagacg ccauccacgc uguuuugacc uccauagaag acaccgggac 60cgauccagcc
uccgcggccg ggaacggugc auuggaacgc ggauuccccg ugccaagagu
120gacucaccgu ccuugacacg 1402157RNAArtificial SequenceCMV
Polynucleotide Sequence 2uaauacgacu cacuauagga cagaucgccu
ggagacgcca uccacgcugu uuugaccucc 60auagaagaca ccgggaccga uccagccucc
gcggccggga acggugcauu ggaacgcgga 120uuccccgugc caagagugac
ucaccguccu ugacacg 1573100RNAHomo sapiens 3cggguggcau cccugugacc
ccuccccagu gccucuccug gcccuggaag uugccacucc 60agugcccacc agccuugucc
uaauaaaauu aaguugcauc 100
* * * * *