U.S. patent application number 16/286423 was filed with the patent office on 2019-06-27 for liver specific delivery of messenger rna.
The applicant listed for this patent is Translate Bio, Inc.. Invention is credited to Frank DeRosa, Braydon Charles Guild, Michael Heartlein.
Application Number | 20190192690 16/286423 |
Document ID | / |
Family ID | 44115246 |
Filed Date | 2019-06-27 |
United States Patent
Application |
20190192690 |
Kind Code |
A1 |
Guild; Braydon Charles ; et
al. |
June 27, 2019 |
LIVER SPECIFIC DELIVERY OF MESSENGER RNA
Abstract
Disclosed herein are compositions and methods of modulating the
expression of gene or the production of a protein by transfecting
target cells with nucleic acids. The compositions disclosed herein
demonstrate a high transfection efficacy and are capable of
ameliorating diseases associated with protein or enzyme
deficiencies.
Inventors: |
Guild; Braydon Charles;
(Lexington, MA) ; DeRosa; Frank; (Lexington,
MA) ; Heartlein; Michael; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Translate Bio, Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
44115246 |
Appl. No.: |
16/286423 |
Filed: |
February 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15092226 |
Apr 6, 2016 |
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16286423 |
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13800501 |
Mar 13, 2013 |
10143758 |
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15092226 |
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12957340 |
Nov 30, 2010 |
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13800501 |
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61265653 |
Dec 1, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/0033 20130101;
A61K 48/005 20130101; C07J 43/003 20130101; A61K 9/1271 20130101;
A61K 9/1272 20130101; A61P 1/16 20180101; A61K 48/0008 20130101;
A61K 9/0019 20130101; C12N 15/67 20130101; A61K 31/7105
20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07J 43/00 20060101 C07J043/00; C12N 15/67 20060101
C12N015/67; A61K 9/127 20060101 A61K009/127; A61K 9/00 20060101
A61K009/00; A61K 31/7105 20060101 A61K031/7105 |
Claims
1.-126. (canceled)
127. A method of delivery of messenger RNA (mRNA) for in vivo
production of protein, comprising administering systemically to a
subject in need of delivery a composition comprising an mRNA
encoding a protein, encapsulated within a liposome such that the
administering of the composition results in the prolonged stable
expression of the protein encoded by the mRNA in the liver; wherein
the protein encoded by the mRNA is an enzyme; and wherein the
liposome comprises one or more cationic lipids, one or more
non-cationic lipids, one or more cholesterol-based lipids and one
or more PEG-modified lipids and has a size less than about 100
nm.
128. The method of claim 127, wherein the one or more non-cationic
lipids comprise DOPE.
129. The method of claim 127, wherein the one or more cationic
lipids comprise DLinDMA, CLinDMA, or DLin-K-XTC2-DMA.
130. The method of claim 127, wherein the one or more PEG-modified
lipids comprise DMG-PEG-2000 or C8-PEG-2000.
131. The method of claim 127, wherein the mRNA is of at least 30
kDa.
132. The method of claim 127, wherein the enzyme is a
liver-specific enzyme.
133. The method of claim 132, wherein the enzyme is selected from
ornithine transcarbamylase (OTC), carbamyl phosphate synthetase
(CPS), argininosuccinate synthetase 1 (ASS1) argininosuccinate
lyase (ASL), and arginase (ARG).
134. The method of claim 127, wherein the mRNA is modified to
enhance stability.
135. The method of claim 127, wherein the mRNA is modified to
include a modified nucleotide, an alteration to the 5' or 3'
untranslated region, a cap structure or a poly A tail.
136. The method of claim 135, wherein the modified nucleotide is
pseudouridine.
137. The method of claim 127, wherein the mRNA is unmodified.
138. The method of claim 127, wherein the protein is retained
within the cytosol of the liver cells after expression.
139. The method of claim 127, wherein the protein is secreted
extracellularly after expression.
140. The method of claim 127, wherein the protein is systemically
distributed.
141. The method of claim 127, wherein the composition is
administered intravenously.
142. The method of claim 127, wherein the expression of the protein
encoded by the mRNA in the liver is detectable at least 4 hours
post administration.
143. The method of claim 141, wherein the expression of the protein
encoded by the mRNA in the liver is detectable at least 4 hours
post administration.
144. The method of claim 127, wherein the one or more cationic
lipids carry a net positive charge at physiological pH.
145. The method of claim 144, wherein the one or more cationic
lipids constitute about 20-70% mol % of the total lipids present in
the liposome.
146. The method of claim 145, wherein the one or more PEG-modified
lipids comprise 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.
147. The method of claim 146, wherein the one or more PEG-modified
lipids constitute up to about 20% by molar of the total lipids
present in the liposome.
148. The method of claim 127, wherein the subject is suffering from
or susceptible to a metabolic disorder resulting from deficiency of
the enzyme encoded by the mRNA.
149. The method of claim 148, wherein the composition is
administered bi-weekly.
150. The method of claim 148, wherein the composition is
administered monthly.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/265,653, filed Dec. 1, 2009 (Attorney
Docket No. SHIR-004-001), the entire teachings of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Novel approaches and therapies are still needed for the
treatment of protein and enzyme deficiencies, particularly
strategies and therapies which overcome the challenges and
limitations associated with the administration of nucleic acids and
the transfection of target cells. Additional approaches which
modulate or supplement the expression of a deficient protein or
enzyme and thus ameliorate the underlying deficiency would be
useful in the development of appropriate therapies for associated
disorders.
[0003] For example, the urea cycle metabolic disorders represent
protein and enzyme deficiencies for which there are no currently
available cures. The urea cycle is a series of biochemical
reactions which occurs in many animals that produce urea
((NH.sub.2).sub.2CO) from ammonia (NH.sub.3) and, in mammals, takes
place only in the liver. Specifically, the urea cycle consists of a
series of five biochemical reactions and serves two primary
functions: the elimination of nitrogen as urea and the synthesis of
arginine. Defects in the urea cycle result in the accumulation of
ammonia and its precursor amino acids (glutamine, glutamic acid,
aspartic acid, and glycine). The resulting high levels of ammonia
are neurotoxic, and the triad of hyperammonemia, encephalopathy,
and respiratory alkalosis characterizes the urea cycle
disorders.
[0004] Ornithine transcarbamylase (OTC) deficiency represents one
such urea cycle genetic disorder. Typically, a subject with OTC
deficiency has a reduced level of the enzyme OTC. In the classic
severe form of OTC deficiency, within the first days of life
patients present with lethargy, convulsions, coma and severe
hyperammonemia that quickly lead to a deteriorating and fatal
outcome absent appropriate medical intervention. If left untreated,
complications from OTC deficiency may include developmental delay,
mental retardation and/or death.
[0005] Treatment of OTC deficient patients primarily involves the
regulation of serum ammonia and hemodialysis remains the only
effective means to rapidly lower serum ammonia levels. Generally,
the treatment goal of urea cycle metabolic disorders is to provide
sufficient protein and arginine for growth, development, and energy
while preventing the development of hyperammonemia and
hyperglutaminemia. Therapeutic approaches that are currently
available for the therapeutic management of urea cycle metabolic
disorders such as OTC deficiency rely heavily upon dietary
management. There are no currently available long-term treatments
or cures for urea cycle metabolic disorders. Novel therapies that
increase the level or production of an affected protein or enzyme
in target cells, such as hepatocytes, or that modulate the
expression of nucleic acids encoding the affected protein or enzyme
could provide a treatment or even a cure for metabolic disorders,
including metabolic disorders such as OTC deficiency.
SUMMARY OF THE INVENTION
[0006] Disclosed are methods of intracellular delivery of nucleic
acids that are capable of correcting existing genetic defects
and/or providing beneficial functions to one or more target cells.
Following successful delivery to target tissues and cells, the
compositions and nucleic acids of the present invention transfect
that target cell and the nucleic acids (e.g., mRNA) can be
translated into the gene product of interest (e.g., a functional
protein or enzyme) or can otherwise modulate or regulate the
presence or expression of the gene product of interest.
[0007] The compositions and methods provided herein are useful in
the management and treatment of a large number of diseases, in
particular diseases which result from protein and/or enzyme
deficiencies. Individuals suffering from such diseases may have
underlying genetic defects that lead to the compromised expression
of a protein or enzyme, including, for example, the non-synthesis
of the protein, the reduced synthesis of the protein, or synthesis
of a protein lacking or having diminished biological activity. In
particular, the methods and compositions provided herein are useful
for the treatment of the urea cycle metabolic disorders that occur
as a result of one or more defects in the biosynthesis of enzymes
involved in the urea cycle. The methods and compositions provided
herein are also useful in various in vitro and in vivo applications
in which the delivery of a nucleic acid (e.g., mRNA) to a target
cell and transfection of that target cell are desired.
[0008] In one embodiment, the compositions provided herein may
comprise a nucleic acid, a transfer vehicle and an agent to
facilitate contact with, and subsequent transfection of a target
cell. The nucleic acid can encode a clinically useful gene product
or protein. For example, the nucleic acid may encode a functional
urea cycle enzyme. In preferred embodiments, the nucleic acid is
RNA, or more preferably mRNA encoding a functional protein or
enzyme.
[0009] In some embodiments, compositions and methods for increasing
expression of a functional protein or enzyme in a target cell are
provided. For example, the compositions and methods provided herein
may be used to increase the expression of a urea cycle enzyme
(e.g., OTC, CPS1, ASS1, ASL or ARG1). In some embodiments, the
composition comprises an mRNA and a transfer vehicle. In some
embodiments, the mRNA encodes a urea cycle enzyme. In some
embodiments the mRNA can comprise one or more modifications that
confer stability to the mRNA (e.g., compared to a wild-type or
native version of the mRNA) and may also comprise one or more
modifications relative to the wild-type which correct a defect
implicated in the associated aberrant expression of the protein.
For example, the nucleic acids of the present invention may
comprise modifications to one or both the 5' and 3' untranslated
regions. Such modifications may include, but are not limited to,
the inclusion of a partial sequence of a cytomegalovirus (CMV)
immediate-early 1 (IE1) gene, a poly A tail, a Cap1 structure or a
sequence encoding human growth hormone (hGH)).
[0010] Methods of treating a subject, wherein the subject has a
protein or enzyme deficiency are also provided. The methods can
comprise administering a composition provided herein. For example,
methods of treating or preventing conditions in which production of
a particular protein and/or utilization of a particular protein is
inadequate or compromised are provided. In one embodiment, the
methods provided herein can be used to treat a subject having a
deficiency in one or more urea cycle enzymes. The method can
comprise contacting and transfecting target cells or tissues (such
as hepatocytes that are deficient in one or more urea cycle
enzymes) with a composition provided herein, wherein the nucleic
acid encodes the deficient urea cycle enzyme. In this manner, the
expression of the deficient enzyme in the target cell is increased,
which in turn is expected to ameliorate the effects of the
underlying enzyme deficiency. The protein or enzyme expressed by
the target cell from the translated mRNA may be retained within the
cytosol of the target cell or alternatively may be secreted
extracellularly. In some embodiments, the nucleic acid is an mRNA.
In some embodiments, the mRNA comprises a modification that confers
stability to the mRNA code (e.g., when compared to the wild-type or
native version of the mRNA). For example, the mRNA encoding a
functional enzyme may comprise one or more modifications to one or
both the 5' and 3' untranslated regions.
[0011] In a preferred embodiment, the nucleic acids (e.g., mRNA)
provided herein are formulated in a lipid or liposomal transfer
vehicle to facilitate delivery to the target cells and/or to
stabilize the nucleic acids contained therein. Contemplated
transfer vehicles may comprise one or more cationic lipids,
non-cationic lipids, and/or PEG-modified lipids. For example, the
transfer vehicle may comprise a mixture of the lipids CHOL, DOPE,
DLinDMA and DMG-PEG-2000. In another embodiment, the transfer
vehicle may comprise the lipids ICE, DOPE and DMG-PEG-2000. In
still another embodiment the transfer vehicle may comprise one or
more lipids selected from the group consisting of ICE, DSPC, CHOL,
DODAP, DOTAP and C8-PEG-2000 ceramide. In a preferred embodiment,
the transfer vehicle is a liposome or a lipid nanoparticle which is
capable of preferentially distributing to the target cells and
tissues in vivo.
[0012] Methods of expressing a functional protein or enzyme (e.g.,
a urea cycle enzyme) in a target cell are also provided. In some
embodiments, the target cell is deficient in a urea cycle enzyme.
The methods comprise contacting the target cell with a composition
comprising an mRNA and a transfer vehicle. Following expression of
the protein or enzyme encoded by the mRNA, the expressed protein or
enzyme may be retained within the cytosol of the target cell or
alternatively may be secreted extracellularly. In some embodiments,
the mRNA encodes a urea cycle enzyme. In some embodiments the mRNA
can comprise one or more modifications that confer stability to the
mRNA and may also comprise one or more modifications relative to
the wild-type that correct a defect implicated in the associated
aberrant expression of the protein. In some embodiments, the
compositions and methods of the present invention rely on the
target cells to express the functional protein or enzyme encoded by
the exogenously administered nucleic acid (e.g., mRNA). Because the
protein or enzyme encoded by the exogenous mRNA are translated by
the target cell, the proteins and enzymes expressed may be
characterized as being less immunogenic relative to their
recombinantly prepared counterparts.
[0013] Also provided are compositions and methods useful for
facilitating the transfection and delivery of one or more nucleic
acids (e.g., mRNA) to target cells. For example, the compositions
and methods of the present invention contemplate the use of
targeting ligands capable of enhancing the affinity of the
composition to one or more target cells. In one embodiment, the
targeting ligand is apolipoprotein-B or apolipoprotein-E and
corresponding target cells express low-density lipoprotein
receptors, thereby facilitating recognition of the targeting
ligand. The methods and compositions of the present invention may
be used to preferentially target a vast number of target cells. For
example, contemplated target cells include, but are not limited to,
hepatocytes, epithelial cells, hematopoietic cells, epithelial
cells, endothelial cells, lung cells, bone cells, stem cells,
mesenchymal cells, neural 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.
[0014] 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
[0015] FIG. 1 illustrates the synthesis of the imidazole
cholesterol ester lipid ICE.
[0016] FIG. 2 illustrates the presence of firefly, luciferase
activity produced from the delivery of exogenous mRNA in the livers
and spleens of treated and untreated CD-1 mice.
[0017] FIG. 3 illustrates codon-optimized firefly luciferase mRNA
in situ hybridization in control and treated (B1 and B2) mouse
livers observed on x-ray film under low (2.times.) magnification.
(A) represents cresyl violet staining of control (Ct) and treated
liver sections B1 and B2 mice; (B) represents X-ray film
autoradiography detection by antisense probes of CO-FF luciferase
mRNA in B1 and B2 mouse livers; and (C) represents control (sense)
hybridization. The abbreviations "cv", "as" and "s" correspond to
cresyl violet, antisense, and sense, respectively.
[0018] FIG. 4 illustrates codon-optimized firefly luciferase mRNA
labeling in treated (B1) and control livers. (A) represents
emulsion autoradiography detection of CO-FF luciferase mRNA in a B1
liver section seen as bright labeling under darkfield illumination;
(B) represents the same region as (A) seen under brightfield
illumination using cresyl violet as a counter-stain; (C) represents
B1 liver section treated with the CO-FF luciferase control (sense)
riboprobe establishing the level of non-specific labeling; (D)
represents the same region as (C) seen under brightfield
illumination; (E) represents untreated control liver section
treated with CO-FF luciferase antisense probe, no signal was
detected; (F) represents the same region as (E) seen under
brightfield illumination; (G) represents control liver section
treated with the CO-FF luciferase control (sense) riboprobe
establishing the level of non-specific labeling; and (H) represents
the same region as (G) seen under brightfield illumination. The
abbreviations "BD", "HA", "H", "PV", "as" and "s" correspond to
bile duct, hepatic artery, hepatocyte, portal vein, antisense and
sense respectively. Magnification: 100.times..
[0019] FIG. 5 illustrates immunohistochemical staining of mouse
livers for the detection of firefly luciferase protein. (A)
represents negative luciferase staining for control liver of mouse
treated with 1x PBS (20.times.); (B) represents positive luciferase
protein detection via immunohistochemical fluorescence-based
methods, demonstrating that firefly luciferase protein is observed
in the hepatocytes (20.times.), as well as a small number of
sinusoidal endothelial cells that were positive for luciferase
protein as well; (C) represents a positive firefly luciferase
protein staining shown at higher magnification (40.times.).
Luciferase protein is observed throughout the cytoplasm of the
hepatocytes. The abbreviations (S) and (H) correspond to sinusoidal
cells and hepatocytes, respectively.
[0020] FIG. 6 shows the nucleotide sequence of CO-FF luciferase
mRNA (SEQ ID NO: 1).
[0021] FIG. 7 shows the nucleotide sequences of a 5' CMV sequence
(SEQ ID NO: 2) and a 3' hGH sequence (SEQ ID NO; 3) which may be
used to flank an mRNA sequence of interest.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Disclosed herein are compositions that facilitate the
delivery of nucleic acids to, and the subsequent transfection of,
target cells. In particular, the compositions provided herein are
useful for the treatment of diseases which result from the
deficient production of proteins and/or enzymes, For example,
suitable diseases that may be treated are those in which a genetic
mutation in a particular gene causes affected cells to not express,
have reduced expression of, or to express a non-functional product
of that gene. Contacting such target cells with the compositions of
the present invention such that the target cells are transfected
with a nucleic acid encoding a functional version of the gene
product allows the production of a functional protein or enzyme
product this is useful in the treatment of such deficiency.
[0023] Provided herein are compositions for modulating the
expression of a protein in a target cell, In some embodiments, the
composition comprises an RNA molecule and a transfer vehicle.
Compositions for increasing expression of a urea cycle enzyme in a
target cell are also provided, The compositions comprise, for
example, an mRNA and a transfer vehicle, The mRNA encodes, for
example, a functional urea cycle enzyme. In some embodiments, the
mRNA of the composition can be modified to impart enhanced
stability (e.g., relative to the wild-type version of the mRNA
and/or the version of the mRNA found endogenously in the target
cell). For example, the mRNA of the composition can include a
modification compared to a wild-type version of the mRNA, wherein
the modification confers stability to the mRNA of the
composition.
[0024] Methods of expressing a urea cycle enzyme in a target cell
are provided. In some embodiments, the target cell is deficient in
a urea cycle enzyme. The methods provided herein comprise
contacting the target cell with a composition comprising an mRNA
and a transfer vehicle, wherein the mRNA encodes one or more urea
cycle enzymes. In some embodiments, the mRNA of the composition is
more stable than the wild-type version of the mRNA and/or more
stable than the version of the mRNA found endogenously in the
target cell.
[0025] Methods of treating a subject with a urea cycle deficiency
are provided. The methods comprise administering a composition
comprising an mRNA and a transfer vehicle, wherein the mRNA encodes
a urea cycle enzyme. In some embodiments, the mRNA of the
composition is more stable than the wild-type version of the mRNA
and/or more stable than the version of the mRNA found endogenously
in the target.
[0026] Provided herein are methods of and compositions for
modulating the level of mRNA and/or the expression of proteins. In
some embodiments, the compositions provided herein are capable of
modulating the expression of a particular protein by decreasing
expression of mRNA encoding that protein in a target cell or
tissue. For example, in one embodiment, the composition comprises a
miRNA or a nucleic acid encoding miRNA where the miRNA is capable
of reducing or eliminating expression of a particular mRNA in a
target cell. In some embodiments, the nucleic acid of the
composition is more stable (e.g., limited nuclease susceptibility)
compared to a wild-type and/or endogenous version of the nucleic
acid.
[0027] As used herein, the term "nucleic acid" refers to genetic
material (e.g., oligonucleotides or polynucleotides comprising DNA
or RNA). In some embodiments, the nucleic acid of the compositions
is RNA. Suitable RNA includes mRNA, siRNA, miRNA, snRNA and snoRNA.
Contemplated nucleic acids also include large intergenic non-coding
RNA (lineRNA), which generally do not encode proteins, but rather
function, for example, in immune signaling, stem cell biology and
the development of disease. (See, e.g., Guttman, et al., 458:
223-227 (2009); and Ng, et al., Nature Genetics 42: 1035-1036
(2010), the contents of which are incorporated herein by
reference). In a preferred embodiment, the nucleic acids of the
invention include RNA or stabilized RNA encoding a protein or
enzyme. The present invention contemplates the use of such nucleic
acids (and in particular RNA or stabilized RNA) as a therapeutic
capable of facilitating the expression of a functional enzyme or
protein, and preferably the expression of a functional enzyme of
protein in which a subject is deficient (e.g., a urea cycle
enzyme). The term "functional", as used herein to qualify a protein
or enzyme, means that the protein or enzyme has biological
activity, or alternatively is able to perform the same, or a
similar function as the native or normally-functioning protein or
enzyme. The subject nucleic acid compositions of the present
invention are useful for the treatment of a various metabolic or
genetic disorders, and in particular those genetic or metabolic
disorders which involve the non-expression, misexpression or
deficiency of a protein or enzyme.
[0028] 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 gene or nucleic acid into at least one
mRNA transcript, or the translation of at least one mRNA or nucleic
acid into a protein or enzyme. For example, contemplated by the
present invention are compositions which comprise one or more mRNA
nucleic acids which encode functional proteins or enzymes, and in
the context of such mRNA nucleic acids, the term expression refers
to the translation of such mRNA to produce the protein or enzyme
encoded thereby.
[0029] The nucleic acids provided herein can be introduced into
cells or tissues of interest. In some embodiments, the nucleic acid
is capable of being expressed (e.g., the transcription of mRNA from
a gene), translated (e.g., the translation of the encoded protein
or enzyme from a synthetic or exogenous mRNA transcript) or
otherwise capable of conferring a beneficial property to the target
cells or tissues (e.g., reducing the expression of a target nucleic
acid or gene). The nucleic acid may encode, for example, a hormone,
enzyme, receptor, polypeptide, peptide or other protein of
interest. A nucleic acid may also encode a small interfering RNA
(siRNA) or antisense RNA for the purpose of decreasing or
eliminating expression of an endogenous nucleic acid or gene. In
one embodiment of the present invention, the nucleic acid (e.g.,
mRNA encoding a deficient protein or enzyme) may optionally have
chemical or biological modifications which, for example, improve
the stability and/or half-life of such nucleic acid or which
improve or otherwise facilitate translation.
[0030] The nucleic acids of the present invention may be natural or
recombinant in nature and may exert their therapeutic activity
using either sense or antisense mechanisms of action.
[0031] Also contemplated by the present invention is the
co-delivery of one or more unique nucleic acids to target cells,
for example, by combining two unique nucleic acids into a single
transfer vehicle. In one embodiment of the present invention, a
therapeutic first nucleic acid, such as mRNA encoding
galactose-1-phosphate uridyltransferase (GALT), and a therapeutic
second nucleic acid, such as mRNA encoding galatokinase (GALK), may
be formulated in a single transfer vehicle and administered (e.g.,
for the treatment of galactosemia). The present invention also
contemplates co-delivery and/or co-administration of a therapeutic
first nucleic acid and a second nucleic acid to facilitate and/or
enhance the function or delivery of the therapeutic first nucleic
acid. For example, such a second nucleic acid (e.g., exogenous or
synthetic mRNA) may encode a membrane transporter protein that upon
expression (e.g., translation of the exogenous or synthetic mRNA)
facilitates the delivery or enhances the biological activity of the
first nucleic acid. Alternatively, the therapeutic first nucleic
acid may be administered with a second nucleic acid that functions
as a "chaperone" for example, to direct the folding of either the
therapeutic first nucleic acid or endogenous nucleic acids.
[0032] Also contemplated is the delivery of one or more therapeutic
nucleic acids to treat a single disorder or deficiency, wherein
each such therapeutic nucleic acid functions by a different
mechanism of action. For example, the compositions of the present
invention may comprise a therapeutic first nucleic acid which, for
example, is administered to correct an endogenous protein or enzyme
deficiency, and which is accompanied by a second nucleic acid,
which is administered to deactivate or "knock-down" a
malfunctioning endogenous nucleic acid and its protein or enzyme
product. Such nucleic acids may encode, for example mRNA and
siRNA.
[0033] While in vitro transcribed nucleic acids (e.g., mRNA) may be
transfected into target cells, such nucleic acids are readily and
efficiently degraded by the cell in vivo, thus rendering such
nucleic acids ineffective. Moreover, some nucleic acids are
unstable in bodily fluids (particularly human serum) and can be
degraded even before reaching a target cell. In addition, within a
cell, a natural mRNA can decay with a half-life of between 30
minutes and several days.
[0034] The nucleic acids provided herein, and in particular the
mRNA nucleic acids provided herein, preferably retain at least some
ability to be translated, thereby producing a functional protein or
enzyme within a target cell. Accordingly, the present invention
relates to the administration of a stabilized nucleic acid (e.g.,
mRNA which has been stabilized against in vivo nuclease digestion
or degradation) to modulate the expression of a gene or the
translation of a functional enzyme or protein within a target cell.
In a preferred embodiment of the present invention, the activity of
the nucleic acid (e.g., mRNA encoding a functional protein or
enzyme) is prolonged over an extended period of time. For example,
the activity of the nucleic acids 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 an annual basis. The extended or
prolonged activity of the compositions of the present invention,
and in particular of the mRNA comprised therein, is directly
related to the quantity of functional protein or enzyme translated
from such mRNA. Similarly, the activity of the compositions of the
present invention may be further extended or prolonged by
modifications made to improve or enhance translation of the mRNA
nucleic acids. 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 nucleic acids of the
present invention may further extend or prolong the activity of the
mRNA nucleic acids. Furthermore, the quantity of functional protein
or enzyme translated by the target cell is a function of the
quantity of nucleic acid (e.g., mRNA) delivered to the target cells
and the stability of such nucleic acid. To the extent that the
stability of the nucleic acids of the present invention may be
improved or enhanced, the half-life, the activity of the translated
protein or enzyme and the dosing frequency of the composition may
be further extended.
[0035] Accordingly, in a preferred embodiment, the nucleic acids
provided herein comprise at least one modification which confers
increased or enhanced stability to the nucleic acid, including, for
example, improved resistance to nuclease digestion in vivo. As used
herein, the terms "modification" and "modified" as such terms
relate to the nucleic acids provided herein, include at least one
alteration which preferably enhances stability and renders the
nucleic acid more stable (e.g., resistant to nuclease digestion)
than the wild-type or naturally occurring version of the nucleic
acid. As used herein, the terms "stable" and "stability" as such
terms relate to the nucleic acids of the present invention, and
particularly with respect to the mRNA, refer to increased or
enhanced resistance to degradation by, for example nucleases (i.e.,
endonucleases or exonucleases) which are normally capable of
degrading such RNA. 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
residence of such nucleic acids in the target cell, tissue, subject
and/or cytoplasm. The stabilized nucleic acid molecules provided
herein demonstrate longer half-lives relative to their naturally
occurring, unmodified counterparts (e.g. the wild-type version of
the nucleic acid). Also contemplated by the terms "modification"
and "modified" as such terms related to the nucleic acids of the
present invention are alterations which improve or enhance
translation of mRNA nucleic acids, including for example, the
inclusion of sequences which function in the initiation of protein
translation (e.g., the Kozac consensus sequence). (Kozak, M.,
Nucleic Acids Res 15 (20): 8125-48 (1987)).
[0036] In some embodiments, the nucleic acids of the present
invention have undergone a chemical or biological modification to
render them more stable. Exemplary modifications to a nucleic acid
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 which differ from those seen in
naturally occurring nucleic acids, for example, covalent
modifications such as the introduction of modified nucleotides,
(e.g., nucleotide analogs, or the inclusion of pendant groups which
are not naturally found in such nucleic acid molecules).
[0037] In addition, suitable modifications include alterations in
one or more nucleotides of a codon such that the codon encodes the
same amino acid but is more stable than the codon found in the
wild-type version of the nucleic acid. For example, an inverse
relationship between the stability of RNA and a higher number
cytidines (C's) and/or uridines (U's) residues has been
demonstrated, and RNA devoid of 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 a another embodiment,
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 nucleic acids of the present invention also include the
incorporation of pseudouridines. The incorporation of
pseudouridines into the mRNA nucleic acids of the present invention
may enhance stability and translational capacity, as well as
diminishing immunogenicity in vivo. (See, e.g., Kariko, K., et al.,
Molecular Therapy 16 (11): 1833-1840 (2008)). Substitutions and
modifications to the nucleic acids of the present invention may be
performed by methods readily known to one or ordinary skill in the
art.
[0038] 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, compared to an untranslated region, (i.e., it will likely not
be possible to eliminate all of the C and U residues present in the
message 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.
[0039] The term modification also includes, for example, the
incorporation of non-nucleotide linkages or modified nucleotides
into the nucleic acid sequences of the present invention (e.g.,
modifications to one or both the 3' and 5' ends of an mRNA molecule
encoding a functional protein or enzyme). Such modifications
include the addition of bases to a nucleic acid sequence (e.g., the
inclusion of a poly A tail or a longer poly A tail), the alteration
of the 3' UTR or the 5' UTR, complexing the nucleic acid with an
agent (e.g., a protein or a complementary nucleic acid molecule),
and inclusion of elements which change the structure of a nucleic
acid molecule (e.g., which form secondary structures).
[0040] The poly A tail is thought to stabilize natural messengers
and synthetic sense RNA. Therefore, in one embodiment a long poly A
tail can be added to an mRNA molecule thus rendering the RNA more
stable. 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). A
transcription vector can also encode long poly A tails. In
addition, poly A tails can be added by transcription directly from
PCR products. Poly A may also be ligated to the 3' end of a sense
RNA 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 one embodiment, the
length of the poly A tail is at least about 90, 200, 300, 400 at
least 500 nucleotides. In one embodiment, the length of the poly A
tail is adjusted to control the stability of a modified sense mRNA
molecule of the invention and, thus, the transcription of protein.
For example, since the length of the poly A tail can influence the
half-life of a sense mRNA molecule, the length of the poly A tail
can be adjusted to modify the level of resistance of the mRNA to
nucleases and thereby control the time course of protein expression
in a cell. In one embodiment, the stabilized nucleic acid molecules
are sufficiently resistant to in viva degradation (e.g., by
nucleases), such that they may be delivered to the target cell
without a transfer vehicle.
[0041] In one embodiment, a nucleic acid encoding a protein can be
modified by the incorporation 3' and/or 5' untranslated (UTR)
sequences which are not naturally found in the wild-type nucleic
acid. In one embodiment, 3' and/or 5' flanking sequence which
naturally flanks an mRNA and encodes a second, unrelated protein
can be incorporated into the nucleotide sequence of an mRNA
molecule encoding a therapeutic or functional protein in order to
modify it. For example, 3' or 5' sequences from mRNA molecules
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 nucleic acid molecule to increase the
stability of the sense mRNA molecule.
[0042] Also contemplated by the present invention are modifications
to the nucleic acid sequences made to one or both of the 3' and 5'
ends of the nucleic acid. For example, the present invention
contemplates modifications to the 5' end of the nucleic acids
(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: 2) to improve
the nuclease resistance and/or improve the half life of the nucleic
acid. In addition to increasing the stability of the mRNA nucleic
acid sequence, it has been surprisingly discovered 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 nucleic
acid (e.g., mRNA) to further stabilize the nucleic acid. Generally,
preferred modifications improve the stability and/or
pharmacokinetic properties (e.g., half-life) of the nucleic acid
relative to their unmodified counterparts, and include, for example
modifications made to improve such nucleic acid's resistance to in
viva nuclease digestion.
[0043] In some embodiments, the composition can comprise a
stabilizing reagent. The compositions can include one or more
formulation reagents that bind directly or indirectly to, and
stabilize the nucleic acid, thereby enhancing residence time in the
cytoplasm of a target cell. Such reagents preferably lead to an
improved half-life of a nucleic acid in the target cells. For
example, the stability of an mRNA and efficiency of translation may
be increased by the incorporation of "stabilizing reagents" that
form complexes with the nucleic acids (e.g., mRNA) that naturally
occur within a cell (see e.g., U.S. Pat. No. 5,677,124).
Incorporation of a stabilizing reagent can be accomplished for
example, by combining the poly A and a protein with the mRNA to be
stabilized in vitro before loading or encapsulating the mRNA within
a transfer vehicle. Exemplary stabilizing reagents include one or
more proteins, peptides, aptamers, translational accessory protein,
mRNA binding proteins, and/or translation initiation factors.
[0044] Stabilization of the compositions may also be improved by
the use of opsonization-inhibiting moieties, which are typically
large hydrophilic polymers that are chemically or physically bound
to the transfer vehicle (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
liposomes by the macrophage-monocyte system and
reticulo-endothelial system (e.g., as described in U.S. Pat. No.
4,920,016, the entire disclosure of which is herein incorporated by
reference). Transfer vehicles modified with opsonization-inhibition
moieties thus remain in the circulation much longer than their
unmodified counterparts.
[0045] When RNA is hybridized to a complementary nucleic acid
molecule (e.g., DNA or RNA) it may be protected from nucleases.
(Krieg, et al. Melton, Methods in Enzymology. 1987; 155, 397-415).
The stability of hybridized mRNA is likely due to the inherent
single strand specificity of most RNases. In some embodiments, the
stabilizing reagent selected to complex a nucleic acid is a
eukaryotic protein, (e.g., a mammalian protein). In yet another
embodiment, the nucleic acid molecule (e.g., mRNA) for use in sense
therapy can be modified by hybridization to a second nucleic acid
molecule. If an entire mRNA molecule were hybridized to a
complementary nucleic acid molecule translation initiation may be
reduced. In some embodiments the 5' untranslated region and the AUG
start region of the mRNA molecule may optionally be left
unhybridized. Following translation initiation, the unwinding
activity of the ribosome complex can function even on high affinity
duplexes so that translation can proceed. (Liebhaber. J. Mol. Biol.
1992; 226: 2-13; Monia, et al. J Biol Chem. 1993; 268:
14514-22.)
[0046] It will be understood that any of the above described
methods for enhancing the stability of nucleic acids may be used
either alone or in combination with one or more of any of the other
above-described methods and/or compositions.
[0047] In one embodiment, the compositions of the present invention
facilitate the delivery of nucleic acids to target cells. In some
embodiments, facilitating delivery to target cells includes
increasing the amount of nucleic acid that comes in contact with
the target cells. In some embodiments, facilitating delivery to
target cells includes reducing the amount of nucleic acid that
comes into contact with non-target cells. In some embodiments,
facilitating delivery to target cells includes allowing the
transfection of at least some target cells with the nucleic acid.
In some embodiments, the level of expression of the product encoded
by the delivered nucleic acid is increased in target cells.
[0048] The nucleic acids of the present invention may be optionally
combined with a reporter gene (e.g., upstream or downstream of the
coding region of the nucleic acid) which, for example, facilitates
the determination of nucleic acid delivery to the target cells or
tissues. Suitable reporter genes may include, for example, Green
Fluorescent Protein mRNA (GFP mRNA), Renilla Luciferase mRNA
(Luciferase mRNA), Firefly Luciferase mRNA, or any combinations
thereof. For example, GFP mRNA may be fused with a nucleic acid
encoding OTC mRNA to facilitate confirmation of mRNA localization
in the target cells, tissues or organs.
[0049] As used herein, the terms "transfect" or "transfection" mean
the intracellular introduction of a nucleic acid into a cell, or
preferably into a target cell. The introduced nucleic acid may be
stably or transiently maintained in the target cell. The term
"transfection efficiency" refers to the relative amount of nucleic
acid up-taken by the target cell which is subject to transfection.
In practice, transfection efficiency is estimated by the amount of
a reporter nucleic acid product expressed by the target cells
following transfection. Preferred are compositions with high
transfection efficacies and in particular those compositions that
minimize adverse effects which are mediated by transfection of
non-target cells and tissues. The compositions of the present
invention that demonstrate high transfection efficacies improve the
likelihood that appropriate dosages of the nucleic acid will be
delivered to the site of pathology, while minimizing potential
systemic adverse effects.
[0050] As provided herein, the compositions can include a transfer
vehicle. As used herein, the term "transfer vehicle" includes any
of the standard pharmaceutical carriers, diluents, excipients and
the like which are generally intended for use in connection with
the administration of biologically active agents, including nucleic
acids. The compositions and in particular the transfer vehicles
described herein are capable of delivering nucleic acids of varying
sizes to their target cells or tissues. In one embodiment of the
present invention, the transfer vehicles of the present invention
are capable of delivering large nucleic acid sequences (e.g.,
nucleic acids of at least 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5 kDa, 10
kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or more). The nucleic
acids can be formulated with one or more acceptable reagents, which
provide a vehicle for delivering such nucleic acids 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 nucleic acids (e.g.,
charge), the intended route of administration, the anticipated
biological environment to which such nucleic acids will be exposed
and the specific properties of the intended target cells. In some
embodiments, transfer vehicles, such as liposomes, encapsulate the
nucleic acids without compromising biological activity. In some
embodiments, the transfer vehicle demonstrates preferential and/or
substantial binding to a target cell relative to non-target cells.
In a preferred embodiment, the transfer vehicle delivers its
contents to the target cell such that the nucleic acids are
delivered to the appropriate subcellular compartment, such as the
cytoplasm.
[0051] In some embodiments, the transfer vehicle is a liposomal
vesicle, or other means to facilitate the transfer of a nucleic
acid to target cells and tissues. Suitable transfer vehicles
include, but are not limited to, liposomes, nanoliposomes,
ceramide-containing nanoliposomes, proteoliposomes,
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 transfer
vehicle. (Hum. Gene Ther. 2008 Sep;19(9):887-95). In a preferred
embodiment of the present invention, the transfer vehicle is
formulated as a lipid nanoparticle. As used herein, the phrase
"lipid nanoparticle" refers to a transfer vehicle comprising one or
more lipids (e.g., cationic and/or non-cationic lipids).
Preferably, the lipid nanoparticles are formulated to deliver one
or more nucleic acids (e.g., mRNA) to one or more target cells or
tissues. The use of lipids, either alone or as a component of the
transfer vehicle, 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 transfer vehicles, whether alone or in
combination with other transfer vehicles. Suitable polymers may
include, for example, polyacrylates, polyalkycyanoacrylates,
polylactide, polylactide-polyglycolide copolymers,
polycaprolactones, dextran, albumin, gelatin, alginate, collagen,
chitosan, cyclodextrins and polyethylenimine. In one embodiment,
the transfer vehicle is selected based upon its ability to
facilitate the transfection of a nucleic acid to a target cell.
[0052] In one embodiment of the present invention, the transfer
vehicle may be selected and/or prepared to optimize delivery of the
nucleic acid to the target cell, tissue or organ. For example, if
the target cell is a hepatocyte the properties of the transfer
vehicle (e.g., size, charge and/or pH) may be optimized to
effectively deliver such transfer vehicle 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., mRNA administered for the treatment of
neurodegenerative diseases may specifically target brain or spinal
tissue) selection and preparation of the transfer vehicle must
consider penetration of, and retention within the blood brain
barrier and/or the use of alternate means of directly delivering
such transfer vehicle to such target tissue. In one embodiment, the
compositions of the present invention may be combined with agents
that facilitate the transfer of exogenous nucleic acids (e.g.,
agents which disrupt or improve the permeability of the blood brain
barrier and thereby enhance the transfer of exogenous mRNA to the
target cells).
[0053] The use of liposomal transfer vehicles to facilitate the
delivery of nucleic acids 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 transfer
vehicles 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.).
[0054] In the context of the present invention, a liposomal
transfer vehicle typically serves to transport the nucleic acid to
the target cell. For the purposes of the present invention, the
liposomal transfer vehicles are prepared to contain the desired
nucleic acids. The process of incorporation of a desired entity
(e.g., a nucleic acid) into a liposome is often referred to as
"loading" (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The
liposome-incorporated nucleic acids 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 nucleic acid into
liposomes is also referred to herein as "encapsulation" wherein the
nucleic acid is entirely contained within the interior space of the
Liposome.
[0055] The purpose of incorporating a nucleic acid into a transfer
vehicle, such as a liposome, is often to protect the nucleic acid
from an environment which may contain enzymes or chemicals that
degrade nucleic acids and/or systems or receptors that cause the
rapid excretion of the nucleic acids. Accordingly, in a preferred
embodiment of the present invention, the selected transfer vehicle
is capable of enhancing the stability of the nucleic acid(s) (e.g.,
mRNA encoding a functional protein) contained therein. The liposome
can allow the encapsulated nucleic acid to reach the target cell
and/or may preferentially allow the encapsulated nucleic acid to
reach the target cell, or alternatively limit the delivery of such
nucleic acids to other sites or cells where the presence of the
administered nucleic acid may be useless or undesirable.
Furthermore, incorporating the nucleic acids into a transfer
vehicle, such as for example, a cationic liposome, also facilitates
the delivery of such nucleic acids into a target cell.
[0056] Ideally, liposomal transfer vehicles are prepared to
encapsulate one or more desired nucleic, acids (e.g., mRNA encoding
a urea cycle enzyme) such that the compositions demonstrate a high
transfection efficiency and enhanced stability. While liposomes can
facilitate introduction of nucleic acids into target cells, the
addition of polycations (e.g., poly L-lysine and protamine), as a
copolymer can 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. Caplet, et al., Gene Ther. 1995; 2: 603; S. Li, et al.,
Gene Ther. 1997; 4, 891.)
[0057] The present invention contemplates the use of cationic
lipids and liposomes to encapsulate and/or enhance the delivery of
nucleic acids 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 transfer vehicles 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-dioleyloxy)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 dioleoylphosphatidylethanolamine or
"DOPE" or other cationic or non-cationic lipids into a liposomal
transfer vehicle or a lipid nanoparticle, and such liposomes can be
used to enhance the delivery of nucleic acids into target cells.
Other suitable cationic lipids include, for example,
5-carboxyspermylglycinedioctadecylamide or "DOGS,"
2,3-dioloyloxy-N[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um 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-diolcyl-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-dimethyl-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
WO2005/121348A1).
[0058] 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).
[0059] 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.
[0060] 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-methytheptan-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, as represented by structure (I)
below. In a preferred embodiment, a transfer vehicle (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-methytheptan-2-yl)-2, 3, 4, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16,
17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)pro-
panoate, as represented by structure (I).
##STR00001##
Without wishing to be bound by a particular theory, it is believed
that the fusogenicity of the imidazole-based cationic lipid ICE is
related to the endosomal disruption which is facilitated by the
imidazole group, which has a lower pKa relative to traditional
cationic lipids. The endosomal disruption in turn promotes osmotic
swelling and the disruption of the liposomal membrane, followed by
the transfection or intracellular release of the nucleic acid(s)
contents loaded therein into the target cell.
[0061] 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 transfer vehicle 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
transfer vehicle, or preferably about 20% to about 70% of the total
lipid present in the transfer vehicle.
[0062] 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 transfer
vehicle (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-nucleic acid 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., C14 or C18). The PEG-modified phospholipid and derivitized
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 transfer vehicle.
[0063] The present invention also contemplates the use of
non-cationic lipids. 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), dipalmitoylphosphatidyicholine
(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 transfer vehicle.
[0064] Preferably, the transfer vehicle (e.g., a lipid
nanoparticle) is prepared by combining multiple lipid and/or
polymer components. For example, a transfer vehicle 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 transfer
vehicle may he 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 transfer vehicle
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 nucleic acids to be
delivered by the liposomal transfer vehicle. 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).
[0065] The liposomal transfer vehicles 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
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. Uni-lamellar 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.
[0066] In certain embodiments of this invention, the compositions
of the present invention comprise a transfer vehicle wherein the
therapeutic RNA (e.g., mRNA encoding OTC) is associated on both the
surface of the transfer vehicle (e.g., a liposome) and encapsulated
within the same transfer vehicle. For example, during preparation
of the compositions of the present invention, cationic liposomal
transfer vehicles may associate with the nucleic acids (e.g., mRNA)
through electrostatic interactions with such therapeutic mRNA.
[0067] In certain embodiments, the compositions of the present
invention may be loaded with diagnostic radionuclide, fluorescent
materials or other materials that are 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.
[0068] During the preparation of liposomal transfer vehicles, 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 nucleic acids), loading of the
nucleic acid 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 nucleic acid, 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 nucleic acid from the
surface of the liposomal transfer vehicle formulation, such
liposomes may be subject to a Diethylaminoethyl SEPHACEL
column.
[0069] In addition to the encapsulated nucleic acid, one or more
therapeutic or diagnostic agents may be included in the transfer
vehicle. 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 U.S. Pat. Nos.
5,194,654 and 5,223,263, which are incorporated by reference
herein).
[0070] There are several methods for reducing the size, or
"sizing", of liposomal transfer vehicles, 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 consists 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.
[0071] A variety of alternative methods known in the art are
available for sizing of a population of liposomal transfer
vehicles. One such sizing method is described in U.S. Pat. No.
4,737,323, 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.
[0072] Selection of the appropriate size of a liposomal transfer
vehicle 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. In some embodiments, it may be desirable to limit
transfection of the nucleic acids to certain cells or tissues. For
example, the liver represents an important 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 one
embodiment of the present invention, the structural characteristics
of the target tissue may be exploited to direct the distribution of
the liposomal transfer vehicle to such target tissues. For example,
to target hepatocytes a liposomal transfer vehicle 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 transfer vehicle can readily penetrate
such endothelial fenestrations to reach the target hepatocytes.
Alternatively, a liposomal transfer vehicle 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. For example, a liposomal transfer vehicle 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 transfer vehicle to hepatocytes. In
such an embodiment, large liposomal transfer vehicles 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 transfer vehicle is within
the range of about 25 to 250 nm, prefereably less than about 250
nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10
nm.
[0073] Similarly, the compositions of the present invention may be
prepared to preferentially distribute to other target tissues,
cells or organs, such as the heart, lungs, kidneys, spleen. For
example, the transfer vehicles 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.
[0074] In some embodiments, the compositions of the present
invention distribute into the cells and tissues of the liver to
facilitate the delivery and the subsequent expression of the
nucleic acids (e.g., mRNA) comprised therein by the cells and
tissues of the liver (e.g., hepatocytes). While such compositions
may preferentially distribute into the cells and tissues of the
liver, the therapeutic effects of the expressed nucleic acids need
not be limited to the target cells and tissues. For example, the
targeted hepatocytes may function as a "reservoir" or "depot"
capable of expressing or producing, and systemically excreting a
functional protein or enzyme. Accordingly, in one embodiment of the
present invention the liposomal transfer vehicle may target
hepatocyes and/or preferentially distribute to the cells and
tissues of the liver and upon delivery. Following transfection of
the target hepatocytes, the mRNA nucleic acids(s) loaded in the
liposomal vehicle are translated and a functional protein product
expressed, excreted and systemically distributed.
[0075] 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 nucleic acids (e.g., mRNA, miRNA, snRNA and
snoRNA) from the transfer vehicle into an intracellular compartment
of the target cell. In one embodiment, the additional molecule
facilitates the delivery of the nucleic acids into, for example,
the cytosol, the lysosome, the mitochondrion, the nucleus, the
nucleolae or the proteasome of a target cell. Also included are
agents that 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.
[0076] In one embodiment, the compositions of the present invention
facilitate a subject's endogenous production of one or more
functional proteins and/or enzymes, and in particular the
production of proteins and/or enzymes which demonstrate less
immunogenicity relative to their recombinantly-prepared
counterparts. In a preferred embodiment of the present invention,
the transfer vehicles comprise nucleic acids which encode mRNA of a
deficient protein or enzyme. Upon distribution of such compositions
to the target tissues and the subsequent transfection of such
target cells, the exogenous mRNA loaded into the liposomal transfer
vehicle (e.g., a lipid nanoparticle) may be translated in vivo to
produce a functional protein or enzyme encoded by the exogenously
administered mRNA (e.g., a protein or enzyme in which the subject
is deficient). Accordingly, the compositions of the present
invention exploit a subject's ability to translate exogenously- or
recombinantly-prepared mRNA to produce an endogenously-translated
protein or enzyme, and thereby produce (and where applicable
excrete) a functional protein or enzyme. The expressed or
translated proteins or enzymes may also be characterized by the in
vivo inclusion of native post-translational modifications which may
often be absent in recombinantly-prepared proteins or enzymes,
thereby further reducing the immunogenicity of the translated
protein or enzyme.
[0077] The administration of mRNA encoding a deficient protein or
enzyme avoids the need to deliver the nucleic acids to specific
organelles within a target cell (e.g., mitochondria). Rather, upon
transfection of a target cell and delivery of the nucleic acids to
the cytoplasm of the target cell, the mRNA contents of a transfer
vehicle may be translated and a functional protein or enzyme
expressed.
[0078] 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 transfer vehicle in vivo
without relying upon the use of additional excipients or means to
enhance recognition of the transfer vehicle by target cells. For
example, transfer vehicles 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.
[0079] Alternatively, the present invention 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 transfer vehicle to encourage
localization of such transfer vehicle 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 transfer vehicle 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 transfer vehicle and/or its
contents in the target cells and tissues (e.g., the inclusion of an
apolipoprotein-E targeting ligand in or on the transfer vehicle
encourages recognition and binding of the transfer vehicle 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
nucleic acid contents for the target cells or tissues. Suitable
ligands may optionally be bound or linked to the surface of the
transfer vehicle. In some embodiments, the targeting ligand may
span the surface of a transfer vehicle or be encapsulated within
the transfer vehicle, 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 transfer vehicle (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.
[0080] 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.
[0081] As used herein, the term "target cell" refers to a cell or
tissue to which a composition of the invention is to be directed or
targeted. In some embodiments, the target cells are deficient in a
protein or enzyme of interest. For example, where it is desired to
deliver a nucleic acid to a hepatocyte, the hepatocyte represents
the target cell. In some embodiments, the nucleic acids and
compositions of the present invention transfect the target cells on
a discriminatory basis (i.e., do not transfect non-target cells).
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, hepatocytes, epithelial cells,
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.
[0082] Following transfection of one or more target cells by the
compositions and nucleic acids of the present invention, expression
of the protein encoded by such nucleic acid may he preferably
stimulated and the capability of such target cells to express the
protein of interest is enhanced. For example, transfection of a
target cell with an mRNA OTC will allow expression of the protein
product OTC following translation of the nucleic acid.
[0083] The urea cycle metabolic disorders and protein or enzyme
deficiencies generally may be amenable to treatment with the
methods and compositions provided herein. The nucleic acids of the
compositions and/or methods provided herein preferably encode a
product (e.g., a protein, enzyme, polypeptide, peptide, functional
RNA, and/or antisense molecule), and preferably encodes a product
whose in vivo production is desired.
[0084] The urea cycle metabolic disorders represent examples of
protein and enzyme deficiencies which may be treated using the
methods and compositions provided herein. Such urea cycle metabolic
disorders include OTC deficiency, arginosuccinate synthetase
deficiency (ASD) and argininosuccinate lyase deficiency (ALD).
Therefore, in some embodiments, the nucleic acid of the methods and
compositions provided herein encode an enzyme involved in the urea
cycle, including, for example, ornithine transcarbamylase (OTC),
carbamyl phosphate synthetase (CPS), argininosuccinate synthetase 1
(ASS1) argininosuccinate lyase (ASL), and arginase (ARG).
[0085] Five metabolic disorders which result from defects in the
biosynthesis of the enzymes involved in the urea cycle have been
described, and include ornithine transcarbamylase (OTC) deficiency,
carbamyl phosphate synthetase (CPS) deficiency, argininosuccinate
synthetase 1 (ASS1) deficiency (citrullinemia), argininosuccinate
lyase (ASL) deficiency and arginase deficiency (ARG). Of these five
metabolic disorders, OTC deficiency represents the most common,
occurring in an estimated one out of every 80,000 births.
[0086] OTC is a homotrimeric mitochondrial enzyme which is
expressed almost exclusively in the liver and which encodes a
precursor OTC protein that is cleaved in two steps upon
incorporation into the mitochondrial matrix. (Horwich A L., et al.
Cell 1986; 44: 451-459). OTC deficiency is a genetic disorder which
results in a mutated and biologically inactive form of the enzyme
ornithine transcarbamylase. OTC deficiency often becomes evident in
the first few days of life, typically after protein ingestion. In
the classic severe form of OTC deficiency, within the first days of
life patients present with lethargy, convulsions, coma and severe
hyperammonernia, which quickly leads to a deteriorating and fatal
outcome absent appropriate medical intervention. (Monish S., et
al., Genetics for Pediatricians; Remedica, Cold Spring Harbor
Laboratory (2005)). If improperly treated or if left untreated,
complications from OTC deficiency may include developmental delay
and mental retardation. OTC deficient subjects may also present
with progressive liver damage, skin lesions, and brittle hair. In
some affected individuals, signs and symptoms of OTC deficiency may
be less severe, and may not appear until later in life.
[0087] The OTC gene, which is located on the short arm of the X
chromosome within band Xp21.1, spans more than 85 kb and is
comprised of 10 exons encoding a protein of 1062 amino acids.
(Lindgren V., et al. Science 1984; 226: 698-7700; Horwich, A L., et
al. Science 224: 1068-1074, 1984; Horwich, A L, et al., Cell 44:
451-459, 1986; Hata, A., et al., J. Biochem. 100: 717-725, 1986,
which are incorporated herein by reference). The OTC enzyme
catalyzes the conversion or ornithine and carbamoyl phosphate to
citrulline. Since OTC is on the X chromosome, females are primarily
carriers while males with nonconservative mutations rarely survive
past 72 hours of birth.
[0088] In healthy subjects, OTC is expressed almost exclusively
hepatocellular mitochondria. Although not expressed in the brain of
healthy subjects, OTC deficiency can lead to neurological
disorders. For example, one of the usual symptoms of OTC
deficiency, which is heterogeneous in its presentation, is
hyperammonaemic coma (Gordon, N., Eur J Paediatr Neurol
2003;7:115-121,).
[0089] OTC deficiency is very heterogeneous, with over 200 unique
mutations reported and large deletions that account for
approximately 10-15% of all mutations, while the remainder
generally comprises missense point mutations with smaller numbers
of nonsense, splice-site and small deletion mutations, (Monish A.,
et al.) The phenotype of OTC deficiency is extremely heterogeneous,
which can range from acute neonatal hyperammonemic coma to
asymptomatic hemizygous adults. (Gordon N. Eur J Paediatr Neurol
2003; 7; 115-121). Those mutations that result in severe and life
threatening neonatal disease are clustered in important structural
and functional domains in the interior of the protein at sites of
enzyme activity or at the interchain surface, while mutations
associated with late-onset disease are located on the protein
surface (Monish A., et al.) Patients with milder or partial forms
of OTC deficiency may have onset of disease later in life, which
may present as recurrent vomiting, neurobehavioral changes or
seizures associated with hyperammonemia.
[0090] The compositions and methods of the present invention are
broadly applicable to the delivery of nucleic acids, and in
particular mRNA, to treat a number of disorders. In particular, the
compositions and methods of the present invention are suitable for
the treatment of diseases or disorders relating to the deficiency
of proteins and/or enzymes. In one embodiment, the nucleic acids 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 another embodiment, the
nucleic acids of the present invention encode functional proteins
or enzymes that remain in the cytosol of the target cell (e.g.,
mRNA encoding urea cycle metabolic disorders). Other disorders for
which the present invention are useful include disorders such as
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
(CADASLL); 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 one embodiment, the nucleic acids, 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 crythropoietin, .alpha.1-antitrypsin,
carboxypeptidase N or human growth hormone.
[0091] Alternatively the nucleic acids may encode full length
antibodies or smaller antibodies (e.g., both heavy and light
chains) to confer immunity to a subject. While one embodiment of
the present invention relates to methods and compositions useful
for conferring immunity to a subject (e.g., via the translation of
mRNA nucleic acids 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
effect a functional response in subjects. For example, the mRNA
nucleic acids 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
mRNA nucleic acids 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.
[0092] The compositions of the present invention can be
administered to a subject. In some embodiments, the composition is
formulated in combination with one or more additional nucleic
acids, carriers, targeting ligands or stabilizing reagents, or in
pharmacological compositions where it is mixed with suitable
excipients. For example, in one embodiment, the compositions of the
present invention may be prepared to deliver nucleic acids (e.g.,
mRNA) encoding two or more distinct proteins or enzymes.
Alternatively, the compositions of the present invention may be
prepared to deliver a single nucleic acid and two or more
populations or such compositions may be combined in a single dosage
form or co-administered to a subject. Techniques for formulation
and administration of drugs may be found in "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest
edition.
[0093] A wide range of molecules that can exert pharmaceutical or
therapeutic effects can be delivered into target cells using
compositions and methods of the present invention, The molecules
can be organic or inorganic. Organic molecules can be peptides,
proteins, carbohydrates, lipids, sterols, nucleic acids (including
peptide nucleic acids), or any combination thereof. A formulation
for delivery into target cells can comprise more than one type of
molecule, for example, two different nucleotide sequences, or a
protein, an enzyme or a steroid.
[0094] 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 nucleic acid in the target cell.
[0095] Suitable routes of administration include, for example,
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.
[0096] Alternately, the compositions of the present invention may
be administered in a local rather than systemic manner, for
example, via injection of the pharmaceutical composition directly
into a targeted tissue, preferably in a depot or sustained release
formulation. Local delivery can be affected in various ways,
depending on the tissue to be targeted. For example, aerosols
containing compositions of the present invention can be inhaled
(for nasal, tracheal, or bronchial delivery); compositions of the
present invention can be injected into the site of injury, disease
manifestation, or pain, for example; compositions can be provided
in lozenges for oral, tracheal, or esophageal application; can be
supplied in liquid, tablet or capsule form for administration to
the stomach or intestines, can be supplied in suppository form for
rectal or vaginal application; or can even be delivered to the eye
by use of creams, drops, or even injection. Formulations containing
compositions of the present invention complexed with therapeutic
molecules or ligands can even be surgically administered, for
example in association with a polymer or other structure or
substance that can allow the compositions to diffuse from the site
of implantation to surrounding cells. Alternatively, they can be
applied surgically without the use of polymers or supports.
[0097] In one embodiment, the compositions of the present invention
are formulated such that they are suitable for extended-release of
the nucleic acids contained therein. Such extended-release
compositions may be conveniently administered to a subject at
extended dosing intervals. For example, in one embodiment, the
compositions of the present invention arc 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 vehicles which are formulated for depot
administration (e.g., intramuscularly, subcutaneously,
intravitreally) to either deliver or release a nucleic acids (e.g.,
mRNA) over extended periods of time. Preferably, the
extended-release means employed are combined with modifications
made to the nucleic acid to enhance stability.
[0098] 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.
[0099] 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 he
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
General Preparation of Transfer Vehicles by Solvent Dilution
Technique
[0100] This example generally illustrates a process for the
manufacture of small (<100 nm) liposomal formulations containing
mRNA and the means to evaluate the amount of mRNA encapsulated.
Parameters which may he modified to further optimize transfection
efficiency include, but are not limited to, the selection of lipid,
the ratio of lipids, the molar ratio of the PEG-containing lipid,
the length of the lipid anchor of the PEG-containing lipid and the
sizing of the liposomal transfer vehicles.
[0101] Appropriate quantities of lipids (e.g.,
DSPC/CHOL/DODAP/C8-PEG2000-Cer) to construct a transfer vehicle of
a desired lipid ratio (e.g., a molar ratio of 31:40:25:4) were
weighed and dissolved in absolute ethanol at 70.degree. C. to
obtain the desired lipid ratios and concentrations. In order to
monitor the lipid, a small amount (typically 0.05 mole %) of
rhodamine-dioleoylphosphatidylethanolamine (Rh-PE) was routinely
added to the lipid solution.
[0102] mRNA, for example, encoding for GFP, OTC or Luciferase was
denatured by heating for 10 minutes at 70.degree. C., followed by
cooling on ice. This solution was analyzed to confirm the mRNA
concentration prior to formulation. An aliquot of mRNA was diluted
with water, and then combined with an equal volume of 10 mM citrate
pH 5.0 buffer such that the final citrate content following lipid
addition (from solvent) was 4 mM.
[0103] The mRNA/citrate buffer solutions were then heated to
90.degree. C. for 5 minutes to completely denature the mRNA. While
stirring or vortexing the denatured mRNA, the ethanolic lipid
solution (at 70.degree. C.) was added to the mRNA to generate
multi-lamellar vesicles (MLVs), The MLVs were then cooled to
70.degree. C. prior to extrusion. For samples prepared at high
solvent concentrations (>20%), the MLVs were diluted with 5 mM
pH 5.0 citrate buffer (at 70.degree. C.) to produce a solvent
concentration of 20% before extrusion to generate large unilamellar
vesicles (LUVs).
[0104] MLVs were extruded at 70.degree. C. through 3 stacked 80 nm
polycarbonate filters, using a thermo-jacketed extruder. Five
passes were routinely used to generate large unilamellar vesicles
(LUVs) of the desired size range. Following extrusion, the
formulations were filtered through a 0.2 .mu.m syringe filter to
remove small amounts of particulate material that tended to
interfere with the determination of vesicle size.
[0105] mRNA that was not associated with the liposomes or was
associated with the exterior surface of DODAP-containing liposomes
was removed by anion exchange, such that all remaining associated
mRNA was encapsulated in the liposomes. Two suitable methods
include the use of anion exchange using Acrodisc units with MUSTANG
Q membranes (Pall Life Sciences), or anion exchange using
DEAE-SEPHACEL (Sigma-Aldrich, suspension in 20% ethanol). These
techniques allowed for efficient removal of unencapsulated mRNA
without significant dilution of the formulations.
[0106] Following removal of external mRNA, buffer could be
exchanged by use of PD-10 gel filtration columns (SEPHADEX G-25, GE
Healthcare) using a spin protocol, which permits small molecular
weight constituents (such as solvent and borate) in the liposome
formulation to be retained in the gel and replaced by the
equilibration buffer, without significant dilution of the sample.
Alternatively, in some experiments, solvent may be removed and
buffer exchanged using a Spectrum 500,000 MWCO diafiltration
cartridge. Samples were ultrafiltered to 2-10 mL, then diafiltered
against 10 wash volumes of the desired final buffer to remove
solvent and exchange the buffer. The sample was sometimes further
concentrated by ultrafiltration after the diafiltration
process.
[0107] To quantify mRNA in samples with low lipid:mRNA ratios, a
standard curve of mRNA. was prepared by diluting the stock solution
with water to obtain standards in the range of 0-200 .mu.g/mL.
Samples were diluted (based on expected mRNA concentrations) with
the appropriate buffer to produce mRNA concentrations within the
standard range. 180 .mu.L aliquots of the standards or samples were
combined with 300 .mu.L of 5% SDS and 120 .mu.L of ethanol. The
samples were incubated for 10 min. at 50.degree. C. to dissolve the
lipid. After cooling, the samples were transferred in duplicate
(250 .mu.L aliquots) into the wells of a UV-transparent microplate.
The absorbance at 260 nm was measured and the mRNA concentration in
the samples calculated from the standard curve. In samples where
the lipid:mRNA (weight: weight) ratio was 10:1 (target ratio) or
less, interference from the lipids with the absorbance at 260 nm
was relatively low and could be ignored.
[0108] In samples where the lipid:mRNA (weight: weight) ratio was
greater than 10:1, lipid interference became more significant as
the amount of lipid increased, and therefore the lipid had to be
removed in order to accurately quantify the mRNA content. A
standard curve of mRNA was prepared by diluting the stock solution
with water to obtain standards in the range of 0-250 .mu.g/mL. The
samples to be assessed were diluted (based on expected mRNA
concentrations) with the appropriate buffer to produce mRNA
concentrations within the standard range. 180 .mu.L of the
standards or samples were combined with 20 .mu.L 0.1 M sodium
borate (to increase the pH, thus neutralizing the charge on the
DODAP in the liposome samples, and causing the mRNA to dissociate
from the DODAP). 600 .mu.L of chloroform: methanol (1:2, v:v) was
added to each standard or sample and the samples were vortexed. 200
.mu.L of chloroform was added with vortexing followed by the
addition of 200 .mu.L of water. The samples were vigorously
vortexed and then centrifuged for 2 min. at 1000.times.g to
separate the phases. 250 .mu.L aliquots of the upper (aqueous)
phase were transferred (in duplicate) into the wells of a
UV-transparent microplate and the absorbance at 260 nm was
measured. The mRNA concentration in samples was calculated from the
standard curve. Note that for liposome samples containing DOTAP (or
any other cationic lipid that cannot be neutralized by incubation
at high pH), this assay is unsuitable for determining mRNA
concentration as the mRNA cannot be disassociated from the DOTAP
and a proportion of the mRNA tends to be extracted into the solvent
(CHCl.sub.3) phase in conjunction with the lipid.
[0109] mRNA encapsulation was determined by separation of samples
on DEAE-SEPHACEL (anion exchange gel) columns as follows. Using 2
mL glass Pasteur pipettes plugged with glass wool, columns of
DEAF-SEPHACEL were poured and equilibrated with 5 volumes
(.about.10 mL) of 145 mM sodium chloride-10 mM borate buffer p8.0.
0.5 mL of sample was loaded onto a column and the eluate collected.
The columns were washed with 7.times.0.5 mL aliquots of 145 mM
sodium chloride-10 mM borate buffer pH 8.0, collecting each eluted
fraction separately. The initial sample and each aliquot was
assayed for mRNA and lipid as described above. The % encapsulation
was calculated by 100 .times. (mRNA/lipid) of material eluted from
the column/(mRNA/lipid) of initial sample). Based on the calculated
mRNA concentration from extraction analyses described above
liposomal mRNA samples were diluted to a desired mRNA concentration
(1 .mu.g) in a total volume of 5 .mu.L (i.e. 0.2. mg/mL).
Example 2
[0110] Preparation of DSPC/CHOL/DODAP/C8-PEG-2000 Ceramide (Molar
Ratio of 31:40:25:4)/Renilla Luciferase mRNA (Formulation 1)
[0111] Formulation 1 was prepared by dissolving the appropriate
masses of DSPC, CHOL, DODAP and C8-PEG-2000 ceramide in absolute
ethanol, then adding this to a solution of Renilla Luciferase mRNA
in buffer to produce MLVs at 10.8 mg/mL lipid, 250 .mu.g/mL mRNA,
20% solvent. The MLVs were extruded to produce LUVs, and then
passed through a 0.2 .mu.m filter. The pH was increased by
combining with an equal volume of 100 mM NaCl-50 mM borate pH 8.0
and the external mRNA removed by anion exchange using the
DEAE-Sephacel centrifugation method, as described in Example 1. The
solvent was removed, the external buffer exchanged and the sample
concentrated by diafiltration/ultrafiltration. The concentrated
sample was then passed through a 0.2 .mu.m filter and aliquots were
transferred to vials and stored at 2-8.degree. C.
Example 3
[0112] Preparation of DSPC/CHOL/DOTAP/C8-PEG-2000 Ceramide (Molar
Ratio of 31:40:25:4)/Renilla Luciferase mRNA (Formulation 2)
[0113] Formulation 2 was prepared using a similar methodology as
Formulation 1 with minor changes. In brief, the appropriate masses
of DSPC, CHOL, DOTAP and C8-PEG-2000 ceramide were dissolved in
absolute ethanol and then added to a solution of Renilla Luciferase
mRNA in buffer to produce MLVs at 10.8 mg/mL lipid, 250 .mu.g/mL
mRNA, 20% solvent. The MLVs were extruded to produce LUVs. As DOTAP
was used in this formulation, the external mRNA could not be
effectively removed by anion exchange and therefore this step was
omitted. The solvent was removed, the external buffer exchanged and
the sample concentrated by diafiltration/ultrafiltration. The
concentrated sample was then passed through a 0.2 .mu.m filter and
aliquots were transferred to vials and stored at 2-8.degree. C.
Example 4
[0114] Preparation of DSPC/CHOL/DODAP/C8-PEG-2000 Ceramide (Molar
Ratio of 31:40:25:4) Firefly Luciferase mRNA (Formulation 3)
[0115] To prepare Formulation 3 the appropriate masses of DSPC,
CHOL, DODAP and C8-PEG-2000 ceramide were dissolved in absolute
ethanol, then added to a solution of Firefly Luciferase mRNA in
buffer to produce MLVs at 10.8 mg/mL lipid, 250 .mu.g/mL mRNA, 20%
solvent. The MLVs were extruded to produce LUVs, and then passed
through a 0.2 .mu.m filter. The pH was increased by combining with
0.1 volumes of 0.1 M sodium borate and the external mRNA removed by
anion exchange using the DEAF-Sephacel column method described in
Example 1. The solvent was removed, the external buffer exchanged
and the sample concentrated by diafiltration/ultrafiltration. The
concentrated sample was then passed through a 0.2 .mu.m filter and
aliquots were transferred to vials and stored at 2-8.degree. C.
Example 5
[0116] Preparation of DSPC/CHOL/DODAP/C8-PEG-2000 Ceramide (Molar
Ratio of 31:40:2:4)/Murine OTC mRNA (Formulation 4)
[0117] Formulation 4 was prepared by dissolving the appropriate
mass of DSPC, CHOL, DODAP and C8-PEG-2000 ceramide in absolute
ethanol, then adding this to a solution of murine OTC mRNA in
buffer to produce MLVs at 10.8 mg/mL lipid, 250 .mu.g/mL mRNA, 20%
solvent. The MLVs were extruded to produce LUVs, and then passed
through a 0.2 .mu.m filter. The pH was increased by combining with
0.1 volumes of 0.1 M sodium borate and the external mRNA removed by
anion exchange using the DEAE-Sephacel column method as described
in Example 1. The solvent was removed, the external buffer
exchanged and the sample concentrated by
diafiltration/ultrafiltration. The concentrated sample was then
passed through a 0.2 .mu.m filter and aliquots were transferred to
vials and stored at 2-8.degree. C.
Example 6
[0118] Preparation and Characterization of the Imidiazole
Cholesterol Ester Lipid (3S, 10R, 13R, 17R)-10,
13-dimethyl-17-((R)-6-methytheptan-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; Imidazole Cholesterol Ester
(ICE)
[0119] FIG. 1 depicts the reaction scheme for the synthesis of ICE.
A mixture of trityl-deamino-histidine (1), (1.97 g, 5.15 mmol),
cholesterol (2), (1.97 g, 5.1 mmol), dicyclohexylcarbodiimide
(DCC), (2.12 g, 5.2 mmol) and dimethylaminopyridine (DMAP), (0.13
g, 1.0 mmol) in anhydrous benzene (100 ml) was stirred at ambient
temperature for two days. The resulting suspension was filtered
through Celite and the filtrate was removed under reduced pressure.
The resulting foam was dried under high vacuum overnight to provide
crude ester (3) which was used on the following step without
purification.
[0120] The crude ester (3) was dissolved in anhydrous
dichloromethane (DCM), (200 ml) and trifluoroacetic acid (TFA), (50
ml) was added at room temperature. The resulting solution was
stirred at ambient temperature for 4 hours. Aqueous saturated
NaHCO.sub.3 (250 ml) was added carefully, followed by solid
Na.sub.2CO.sub.3 until slightly basic.
[0121] The phases were separated and the aqueous layer was
extracted with DCM (200 ml). The organic phases were washed with
brine (200 ml), dried (Na.sub.2SO.sub.4) and filtered. The
resulting filtrate was evaporated and the residue was dried under
high vacuum overnight. Flash chromatography purification (silica
gel, 0-10% methanol in chloroform) afforded the desired pure
product (4) (1.07 g, 37% yield for two steps) as a white solid (mp:
192-194.degree. C.).
[0122] .sup.1H NMR (CDCl.sub.3): .delta.0.66 (s, 3H), 0.84-1.64 (m,
33H), 1.76-2.05 (m, 5H), 2.29 (d, 2H), 2.63 (t, 2H), 2.90 (t, 2H),
4.61 (m, 1H), 5.36 (d, 1H), 6.80 (s, 1H), 7.53 (s, 1H). .sup.13C
NMR (CDCl.sub.3): .delta.11.9, 18.8, 19.4, 21.1, 21.6, 22.6, 22.9,
23.9, 24.4, 27.8, 28.1, 28.3, 31.9, 34.5, 35.9, 36.3, 36.7, 37.0,
38.2, 39.6, 39.8, 42.4, 50.1, 56.2, 56.8, 74.1, 122.8, 134.7,
139.6, 173,4. APCI(+)-MS (m/z): Calcd. 509, Found 509. Elem. Anal.
(C,H,N): Calcd. 77.90, 10.30, 5.51; Found 77.65, 10.37, 5.55.
Example 7
Formulation Protocol
[0123] A codon-optimized firefly luciferase messenger RNA
represented by SEQ ID NO: 1 (FFL mRNA) was synthesized by in vitro
transcription from a plasmid DNA template encoding the gene, which
was followed by the addition of a 5' cap structure (Cap1) and a 3'
poly(A) tail of approximately 200 nucleotides in length as
determined by gel electrophoresis, (See, e.g., Fechter, P. et al.,
J. Gen. Virology, 86, 1239-1249 (2005), the contents of which are
incorporated herein by reference in its entirety.) The 5' and 3'
untranslated regions present in the FFL mRNA product are underlined
(SEQ ID NO: 1).
[0124] Nanoparticulate transfer vehicles were formed via standard
ethanol injection methods. (See, e.g., Ponsa, M., et al., Int.
Pharm. 95, 51-56 (1993), the contents of which are incorporated
herein by reference.) Ethanolic stock solutions of the lipids were
prepared ahead of time at a concentration of 50 mg/mL and stored at
-20.degree. C., FFL mRNA was stored in water at a final
concentration of 1 mg/mL at -80.degree. C. until the time of
use.
[0125] All mRNA concentrations were determined via the Ribogreen
assay (invitrogen). Encapsulation of mRNA was calculated by
performing the Ribogreen assay both with and without the presence
of 0.1% Triton-X 100. Particle sizes (dynamic light scattering
(DLS)) and zeta potentials were determined using a Malvern
Zetasizer instrument in 1x PBS and 1 mM KCl solutions,
respectively.
[0126] Aliquots of 50 mg/mL ethanolic solutions of an imidazole
cholesterol ester lipid (ICE), DOPE and DMG-PEG-2000 were mixed and
diluted with ethanol to a final volume of 3 mL. The molar ratio of
the prepared ICE:DOPE:DMG-PEG-2000 transfer vehicle was 70:25:5.
Separately, an aqueous buffered solution (10 mM citrate/150 mM
NaCl, pH 4.5) of FFL mRNA was prepared from a 1 mg/mL stock. The
lipid solution was injected rapidly into the aqueous mRNA solution
and shaken to yield a final suspension in 20% ethanol. The
resulting nanoparticulate suspension was filtered, diafiltrated
with 1x PBS (pH 7.4), concentrated and stored at 2-8.degree. C.,
The final concentration was equal to 1.73 mg/mL CO-FE mRNA
(encapsulated), the Z.sub.ave was equal to 68.0 nm (with a
Dv.sub.(50) of 41.8 nm, and a Dv.sub.(90) of 78.0 nm) and the Zeta
potential was equal to +25.7 mV.
Biodistribution Analysis
[0127] All studies were performed using female CD-1 mice of
approximately 3-weeks age at the beginning of each experiment.
Samples were introduced by a single bolus tail-vein injection of an
equivalent total dose of 200 .mu.g of encapsulated FEL mRNA. Four
hours post-injection the mice were sacrificed and perfused with
saline.
[0128] The liver and spleen of each mouse was harvested,
apportioned into three parts, and stored in either, (i) 10% neutral
buffered formalin, (ii) snap-frozen and stored at -80.degree. C.
for bioluminescence analysis (see below), or for in situ
hybridization studies, or (iii) liver sections were isolated in
isopentane (2-methylbutane) bath, maintained at -35.degree. C.,
rinsed with 1x PBS, lightly patted with a kimwipe to remove any
excess fluid, placed in the bath for approximately 5-7 minutes,
after which the liver was removed, wrapped in foil and stored in a
small sterile plastic bag at -80.degree. C. until ready for
assay.
[0129] The bioluminescence assay was conducted using a Promega
Luciferase Assay System (item # E1500 Promega). Tissue preparation
was performed as follows: Portions of the desired tissue sample
(snap-frozen) were thawed, washed with RODI water and placed in a
ceramic bead homogenization tube. The tissue was treated with lysis
buffer and homogenized. Upon subjection to five freeze/thaw cycles
followed by centrifugation at 4.degree. C., the supernatant was
transferred to new microcentrifuge tubes. Repeat and store tissue
extracts at -80.degree. C.
[0130] The Luciferase Assay Reagent was prepared by adding 10 mL of
Luciferase Assay Buffer to Luciferase Assay Substrate and mix via
vortex. 20 .mu.L of homogenate samples was loaded onto a 96-well
plate followed by 20 .mu.L of plate control to each sample.
Separately, 120 .mu.L of Luciferase Assay Reagent (prepared as
described above) was loaded onto each well of a 96-well flat
bottomed plate. Each plate was inserted into the appropriate
chambers using a Molecular Device Flex Station instrument and
measure the luminescence (measured in relative light units
(RLU)).
In Situ Hybridization
Tissue Slide Preparation
[0131] Slide preparation and analysis was performed as follows:
Each liver was frozen at -35.degree. C. according to the procedure
described above. The frozen livers were cut into 6 micrometer
sections and mounted onto glass microscope slides. Prior to in situ
hybridization, the sections were fixed in 4% formaldehyde in
phosphate buffered saline (PBS), treated with
trienthanolamine/acetic anhydride and washed and dehydrated through
a series of ethanol solutions.
cRNA Probe Preparation
[0132] DNA templates were designed consisting of pBSKII+ vector
containing EcoRI and XbaI restriction sites for generation of the
antisense and sense strands, respectively. cRNA transcripts were
synthesized from these DNA templates (antisense and sense strands,
each 700 bp) with T3 and T7 RNA polymerase, respectively, Templates
were validated by cold RNA probe synthesis prior to making
riboprobes with .sup.35S-UTP. Both antisense and sense radiolabeled
riboprobes were synthesized in vitro according to the
manufacturer's protocol (Ambion) and labeled with 35S-UTP
(>1,000 Ci/mmol).
Hybridization and Washing Procedures
[0133] Sections were hybridized overnight at 55.degree. C. in
deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM
EDTA, 10 mM Na.sub.2HPO.sub.4, 10% dextran sulfate, 1.times.
Denhardt's reagent, 50 .mu.g/mL total yeast RNA and 50-80,000
cpm/.mu.L 35S labeled cRNA probe. The tissues were subjected to
stringent washing at 65.degree. C. in 50% formamide, 2.times. SSC,
10 mM DTT and washed in PBS before treatment with 20 .mu.g/ml RNAse
A at 37.degree. C. for 30 minutes. Following washes in 2.times. SSC
and 0.1.times. SSC for 10 minutes at 37.degree. C., the slides were
dehydrated and exposed to Kodak BioMaxMR x-ray film for 90 minutes
then submitted to emulsion autoradiography for 11 and 24 hours
exposure times.
Imaging of Liver Sections
[0134] Photographic development was carried out in Kodak D-19,
Sections were counterstained lightly with cresyl violet and
analyzed using brightfield and darkfield microscopy. Sense
(control) riboprobes established the level of background
signal.
In Vivo Bioluminescence Results
[0135] Animals were injected intravenously with a single 200 .mu.g
dose of encapsulated mRNA and sacrificed after four hours. Activity
of expressed firefly luciferase protein in livers and spleens was
determined in a bioluminescence assay. As demonstrated in FIG. 2,
detectable signal over baseline was observed in every animal
tested. The presence of a luminescent signal over background infers
the expression of firefly luciferase protein from the exogenous
mRNA. Luminescence observed in the liver was enhanced over similar
signals observed in the spleen.
In Situ Hybridization Results
[0136] In situ hybridization studies were performed on liver taken
from two different animals from the group of mice treated using an
ICE:DOPE:DMG-PEG-2000 transfer vehicle (prepared as previously
described) and one control liver from the untreated group of mice.
X-Ray film autoradiography was employed for the detection of
codon-optimized firefly luciferase mRNA via .sup.35S-U labeled
riboprobes. (See, Wilcox, J. N. J. Histochem. Cytochem. 41,
1725-1733 (1993)). FIG. 3 demonstrates both brightfield
illumination (cresyl violet counterstain) and darkfield
illumination of control and treated livers under low (2.times.)
magnification, CO-FF luciferase mRNA was detected in both treated
livers (B1 and B2, thin arrows) but not the control liver (Ct) when
using the antisense riboprobe (FIG. 3B). High-level mRNA labeling
was observed in the liver marginal tissue band (large arrow). No
signal was detected in any liver when applying the control (sense)
riboprobe (FIG. 3C).
[0137] Under a dark field illumination labeled FFL mRNA was
detected as bright spots (100.times. magnification) in the livers
of injected animals by hybridization of an antisense probe of FFL
mRNA (FIG. 4A), while the same liver showed few bright spots when a
sense strand probe of FFL mRNA was used for hybridization (FIG.
4C). A control liver taken from an animal that did not receive any
nanoparticles by injection did not produce any significant signal
under dark field illumination when either the antisense (FIG. 4E)
or sense probes (FIG. 4G) were used for hybridization.
Example 8
Immunohistochemical Analysis Results
[0138] The FFL mRNA was packaged and delivered via a lipid transfer
vehicle formulation consisting of cholesterol, DOPE, DLinDMA, and
DMG-PEG2000 in a manner similar to that described supra.
[0139] The translation of the FFL mRNA into its respective protein
has been successfully identified via immunohistochemical analysis
(FIG. 5). Using an anti firefly antibody, the detection of
expressed firefly protein can be observed in the hepatocytes of
treated mice (FIGS. 5B and 5C). The analysis of control mice
treated with 1x PBS demonstrated no detectable firefly protein
(FIG. 5A).
Discussion
[0140] A synthetic messenger RNA encapsulted in lipid-based
materials can be used for the delivery and expression of genes in
vivo in liver including leptocytes. Mixtures of cationic,
non-cationic and PEG-modified lipids were used to express a
reporter protein molecule. The imidazole-based cationic lipid ICE
resulted in enriched delivery of mRNA to liver versus spleen in
vivo. The observation of a bioluminescent signal demonstrates that
a protein reporter molecule was translated from the exogenous mRNA
that was delivered in a lipid nanoparticle in vivo. In situ
hybridization studies demonstrated the direct detection of the
exogenous mRNA through .sup.35S-U riboprobe labeling. Emulsion
autoradiography produced a signal that can be used to localize the
mRNA to liver tissue and more specifically to hepatocytes present
in the livers of treated animals (See, FIGS. 3 and 4). FFL mRNA was
not detected in the livers of untreated control mice.
[0141] The successful delivery of such mRNA to the liver and in
particular to hepatocytes supports the conclusion that the methods,
formulations and compositions of the present invention can be used
for the treatment and the correction of in-born errors of
metabolism that are localized to the liver. For example, diseases
such as ASD, ARG, CPS, ASS1 and OTC deficiencies, as well as other
disorders may be treated through mRNA replacement therapy of a
missing or malfunctioning gene. Metabolic zonation of the urea
cycle to hepatocytes means that replacement of the missing enzyme
activity in these cells should greatly improve normal biochemical
processing in subjects afflicted by an enzyme deficiency, and in
particular subjects afflicted with a urea cycle disorder.
Sequence CWU 1
1
311672RNAArtificialCO-FF Luciferase mRNA 1gggauccuac cauggaagau
gccaaaaaca uuaagaaggg cccagcgcca uucuacccac 60ucgaagacgg gaccgccggc
gagcagcugc acaaagccau gaagcgcuac gcccuggugc 120ccggcaccau
cgccuuuacc gacgcacaua ucgaggugga cauuaccuac gccgaguacu
180ucgagaugag cguucggcug gcagaagcua ugaagcgcua ugggcugaau
acaaaccauc 240ggaucguggu gugcagcgag aauagcuugc aguucuucau
gcccguguug ggugcccugu 300ucaucggugu ggcuguggcc ccagcuaacg
acaucuacaa cgagcgcgag cugcugaaca 360gcaugggcau cagccagccc
accgucguau ucgugagcaa gaaagggcug caaaagaucc 420ucaacgugca
aaagaagcua ccgaucauac aaaagaucau caucauggau agcaagaccg
480acuaccaggg cuuccaaagc auguacaccu ucgugacuuc ccauuugcca
cccggcuuca 540acgaguacga cuucgugccc gagagcuucg accgggacaa
aaccaucgcc cugaucauga 600acaguagugg caguaccgga uugcccaagg
gcguagcccu accgcaccgc accgcuugug 660uccgauucag ucaugcccgc
gaccccaucu ucggcaacca gaucaucccc gacaccgcua 720uccucagcgu
ggugccauuu caccacggcu ucggcauguu caccacgcug ggcuacuuga
780ucugcggcuu ucgggucgug cucauguacc gcuucgagga ggagcuauuc
uugcgcagcu 840ugcaagacua uaagauucaa ucugcccugc uggugcccac
acuauuuagc uucuucgcua 900agagcacucu caucgacaag uacgaccuaa
gcaacuugca cgagaucgcc agcggcgggg 960cgccgcucag caaggaggua
ggugaggccg uggccaaacg cuuccaccua ccaggcaucc 1020gccagggcua
cggccugaca gaaacaacca gcgccauucu gaucaccccc gaaggggacg
1080acaagccugg cgcaguaggc aagguggugc ccuucuucga ggcuaaggug
guggacuugg 1140acaccgguaa gacacugggu gugaaccagc gcggcgagcu
gugcguccgu ggccccauga 1200ucaugagcgg cuacguuaac aaccccgagg
cuacaaacgc ucucaucgac aaggacggcu 1260ggcugcacag cggcgacauc
gccuacuggg acgaggacga gcacuucuuc aucguggacc 1320ggcugaagag
ccugaucaaa uacaagggcu accagguagc cccagccgaa cuggagagca
1380uccugcugca acaccccaac aucuucgacg ccggggucgc cggccugccc
gacgacgaug 1440ccggcgagcu gcccgccgca gucgucgugc uggaacacgg
uaaaaccaug accgagaagg 1500agaucgugga cuauguggcc agccagguua
caaccgccaa gaagcugcgc ggugguguug 1560uguucgugga cgaggugccu
aaaggacuga ccggcaaguu ggacgcccgc aagauccgcg 1620agauucucau
uaaggccaag aagggcggca agaucgccgu guaauuugaa uu
16722157RNAArtificial5' CMV Sequence 2uaauacgacu cacuauagga
cagaucgccu ggagacgcca uccacgcugu uuugaccucc 60auagaagaca ccgggaccga
uccagccucc gcggccggga acggugcauu ggaacgcgga 120uuccccgugc
caagagugac ucaccguccu ugacacg 1573100RNAArtificial3' hGH Sequence
3cggguggcau cccugugacc ccuccccagu gccucuccug gcccuggaag uugccacucc
60agugcccacc agccuugucc uaauaaaauu aaguugcauc 100
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