U.S. patent application number 17/515859 was filed with the patent office on 2022-05-26 for compositions and methods for delivering messenger rna.
This patent application is currently assigned to ARBUTUS BIOPHARMA CORPORATION. The applicant listed for this patent is ARBUTUS BIOPHARMA CORPORATION. Invention is credited to Michael J. ABRAMS, James HEYES, Adam JUDGE, Kieu Mong LAM, Lorne Ralph PALMER, Stephen P. REID, Edward D. YAWORSKI.
Application Number | 20220160899 17/515859 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220160899 |
Kind Code |
A1 |
ABRAMS; Michael J. ; et
al. |
May 26, 2022 |
COMPOSITIONS AND METHODS FOR DELIVERING MESSENGER RNA
Abstract
The present invention provides compositions comprising nucleic
acid molecules, such as mRNA molecules, encapsulated within lipid
particles. The compositions are useful, for example, to introduce
the mRNA molecules into a human subject where they are translated
to produce a polypeptide that functions to ameliorate one or more
symptoms of a disease.
Inventors: |
ABRAMS; Michael J.; (Custer,
WA) ; HEYES; James; (Vancouver, CA) ; JUDGE;
Adam; (Bainbridge Island, WA) ; LAM; Kieu Mong;
(Richmond, CA) ; PALMER; Lorne Ralph; (Vancouver,
CA) ; REID; Stephen P.; (Vancouver, CA) ;
YAWORSKI; Edward D.; (Maple Ridge, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARBUTUS BIOPHARMA CORPORATION |
Warminster |
PA |
US |
|
|
Assignee: |
ARBUTUS BIOPHARMA
CORPORATION
Warminster
PA
|
Appl. No.: |
17/515859 |
Filed: |
November 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16312162 |
Dec 20, 2018 |
11191849 |
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PCT/US2017/040446 |
Jun 30, 2017 |
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17515859 |
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62375292 |
Aug 15, 2016 |
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62357189 |
Jun 30, 2016 |
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International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/11 20060101 C12N015/11; A61K 9/00 20060101
A61K009/00; A61K 45/06 20060101 A61K045/06; A61K 9/51 20060101
A61K009/51; A61K 31/573 20060101 A61K031/573; A61K 31/58 20060101
A61K031/58; A61K 31/7105 20060101 A61K031/7105; A61K 31/713
20060101 A61K031/713; A61K 47/69 20060101 A61K047/69; A61K 47/54
20060101 A61K047/54; A61K 47/60 20060101 A61K047/60; B82Y 5/00
20060101 B82Y005/00 |
Claims
1. A lipid nanoparticle comprising: (a) a cationic lipid; (b) a
non-cationic lipid; (c) a corticosteroid and; (d) a nucleic acid,
wherein the nucleic acid and the corticosteroid are encapsulated
within the lipid nanoparticle.
2-25. (canceled)
26. A lipid nanoparticle formulation comprising a multiplicity of
lipid nanoparticles, wherein each lipid nanoparticle comprises: (a)
a cationic lipid; (b) a non-cationic lipid; and (c) mRNA
encapsulated within the lipid particle, wherein the lipid
nanoparticle formulation has an IFIT response that is no more than
30 fold greater than a reference IFIT response of phosphate
buffered saline.
27-29. (canceled)
30. A lipid nanoparticle comprising: (a) a cationic lipid; (b) a
PEG-lipid conjugate present in an amount of at least 3 mole
percent; and (c) mRNA encapsulated within the lipid particle;
provided that the lipid particle comprises less than 0.5 mole
percent phospholipid.
31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of priority of
U.S. application Ser. No. 62/357,189, filed Jun. 30, 2016, and of
U.S. application Ser. No. 62/375,292, filed Aug. 15, 2016, which
applications are herein incorporated by reference.
BACKGROUND
[0002] Some diseases in humans are caused by the absence, or
impairment, of a functional protein in a cell type where the
protein is normally present and active. The functional protein can
be completely or partially absent due, for example, to
transcriptional inactivity of the encoding gene, or due to the
presence of a mutation in the encoding gene that renders the
protein completely or partially non-functional.
[0003] Examples of human diseases that are caused by complete or
partial inactivation of a protein include X-linked severe combined
immunodeficiency (X-SCID), and adrenoleukodystrophy (X-ALD). X-SCID
is caused by one or more mutations in the gene encoding the common
gamma chain protein that is a component of the receptors for
several interleukins that are involved in the development and
maturation of B and T cells within the immune system. X-ALD is
caused by one or more mutations in a peroxisomal membrane
transporter protein gene called ABCD1. Individuals afflicted with
X-ALD have very high levels of long chain fatty acids in tissues
throughout the body, which causes a variety of symptoms that may
lead to mental impairment or death.
[0004] Attempts have been made to use gene therapy to treat some
diseases caused by the absence, or impairment, of a functional
protein in a cell type where the protein is normally present and
active. Gene therapy typically involves introduction of a vector
that includes a gene encoding a functional form of the affected
protein, into a diseased person, and expression of the functional
protein to treat the disease. Thus far, gene therapy has met with
limited success.
[0005] As such, there is a continuing need for compositions and
methods for expressing a functional form of a protein within a
human who suffers from a disease caused by the complete or partial
absence of the functional protein, and there is a need for delivery
of nucleic acids (e.g., mRNA) via a methods and compositions that
trigger less of an immune response to the therapy.
BRIEF SUMMARY
[0006] In accordance with the foregoing, the present invention
provides in certain embodiments compositions and methods that can
be used to deliver nucleic acids, e.g., so as to express one or
more mRNA molecules in a living cell (e.g., cells within a human
body). The mRNA molecules can encode one or more polypeptides that
is/are expressed within the living cells. In some embodiments, the
polypeptides are expressed within a diseased organism (e.g.,
mammal, such as a human being), and expression of the polypeptide
ameliorates one or more symptoms of the disease. The compositions
and methods of certain embodiments of the invention are
particularly useful for treating human diseases caused by the
absence, or reduced levels, of a functional polypeptide within the
human body.
[0007] In one aspect, the present invention provides a lipid
nanoparticle (LNP) comprising: (a) a cationic lipid; (b) a
non-cationic lipid; (c) a corticosteroid and; (d) a nucleic acid,
wherein the nucleic acid and the corticosteroid are encapsulated
within the lipid nanoparticle. Certain embodiments of the invention
provide a population of lipid nanoparticles comprising the lipid
nanoparticles. Certain embodiments of the invention provide a
population of lipid particles comprising a multiplicity of lipid
nanoparticles. In certain embodiments, the nucleic acid is
HPLC-purified mRNA. In certain embodiments, the LNP comprises a
PEG-lipid conjugate present in an amount of at least 3 mole
percent. In certain embodiments, the LNP comprises less than 0.5
mole percent phospholipid.
[0008] Certain embodiments of the invention provide a population of
lipid nanoparticles, comprising at least one population of lipid
nanoparticle selected from: (a) a first population of lipid
nanoparticles that each comprise a cationic lipid, a non-cationic
lipid, and a corticosteroid; and (b) a second population of lipid
nanoparticles that each comprise a cationic lipid, a non-cationic
lipid, and a nucleic acid, wherein the first population of lipid
nanoparticles does not comprise a nucleic acid, and wherein the
second population of lipid nanoparticles does not comprise a
corticosteroid. Certain embodiments of the invention provide a
population of lipid nanoparticles comprising the first and second
populations of lipid nanoparticles. In certain embodiments, the
nucleic acid is HPLC-purified mRNA.
[0009] Certain embodiments of the invention provide a lipid
nanoparticle comprising: (a) a cationic lipid; (b) a PEG-lipid
conjugate present in an amount of at least 3 mole percent; and (c)
mRNA encapsulated within the lipid particle; provided that the
lipid particle comprises less than 0.5 mole percent phospholipid.
In certain embodiments, the LNP comprises a corticosteroid. In
certain embodiments, the mRNA is HPLC-purified mRNA.
[0010] Certain embodiments provide a population of lipid
nanoparticles wherein each lipid nanoparticle in the population
comprises: (a) a cationic lipid; (b) a PEG-lipid conjugate present
in an amount of at least 3 mole percent; and (c) mRNA encapsulated
within the lipid nanoparticle; provided that the lipid nanoparticle
comprises less than 0.5 mole percent phospholipid. In certain
embodiments, the population of LNPs comprises LNPs that comprise a
corticosteroid. In certain embodiments, the mRNA is HPLC-purified
mRNA.
[0011] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising a multiplicity of lipid
nanoparticles, wherein each lipid nanoparticle comprises: (a) a
cationic lipid; (b) a non-cationic lipid; and (c) mRNA encapsulated
within the lipid particle, wherein the lipid nanoparticle
formulation has an IFIT response that is no more than 30 fold
greater than a reference IFIT response of phosphate buffered
saline. In certain embodiments, the mRNA is HPLC-purified mRNA. In
certain embodiments, the LNP comprises a PEG-lipid conjugate
present in an amount of at least 3 mole percent. In certain
embodiments, the LNP comprises less than 0.5 mole percent
phospholipid. In certain embodiments, the LNP comprises a
corticosteroid.
[0012] Certain embodiments of the invention provide a method of
making a lipid nanoparticle, comprising combining: (a) a cationic
lipid; (b) a non-cationic lipid; and (c) purified mRNA so as to
form a lipid nanoparticle, wherein the mRNA is encapsulated within
the lipid nanoparticle, and wherein the lipid nanoparticle has an
IFIT response that is no more than 30 fold greater than a reference
IFIT response of phosphate buffered saline. In certain embodiments,
the mRNA is HPLC-purified mRNA. In certain embodiments, the LNP
comprises a PEG-lipid conjugate present in an amount of at least 3
mole percent. In certain embodiments, the LNP comprises less than
0.5 mole percent phospholipid. In certain embodiments, the LNP
comprises a corticosteroid.
[0013] Certain embodiments of the invention provide a method of
making a lipid nanoparticle formulation comprising a multiplicity
of lipid nanoparticles, the method comprising the step of
combining: (a) a cationic lipid; (b) a non-cationic lipid; and (c)
purified mRNA so as to form a lipid nanoparticle formulation
comprising a multiplicity of lipid nanoparticles, wherein the mRNA
is encapsulated within the lipid particles in the lipid
nanoparticle formulation, and wherein the lipid nanoparticle
formulation has an IFIT response that is no more than 30 fold
greater than a reference IFIT response of phosphate buffered
saline. In certain embodiments, the LNP comprises a PEG-lipid
conjugate present in an amount of at least 3 mole percent. In
certain embodiments, the LNP comprises less than 0.5 mole percent
phospholipid. In certain embodiments, the LNP comprises a
corticosteroid.
[0014] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising a multiplicity of lipid
nanoparticles made by a process comprising the steps of combining:
(a) a cationic lipid; (b) a non-cationic lipid; and (c) purified
mRNA so as to form a lipid nanoparticle formulation comprising a
multiplicity of lipid nanoparticles, wherein the mRNA is
encapsulated within the lipid particles in the lipid nanoparticle
formulation, and wherein the lipid nanoparticle formulation has an
IFIT response that is no more than 30 fold greater than a reference
IFIT response of phosphate buffered saline. In certain embodiments,
the LNP comprises a PEG-lipid conjugate present in an amount of at
least 3 mole percent. In certain embodiments, the LNP comprises
less than 0.5 mole percent phospholipid. In certain embodiments,
the LNP comprises a corticosteroid.
[0015] Thus, in one aspect, the present invention provides a lipid
particle comprising a cationic lipid, a non-cationic lipid, and an
mRNA molecule that is encapsulated within the lipid particle.
[0016] The present invention also provides nucleic acid-lipid
particles that each include (a) a lipid particle comprising a
cationic lipid, a PEG-lipid, and a phospholipid; and (b) an mRNA
molecule, wherein the mRNA molecule is encapsulated within the
lipid particle. The lipid particles can optionally include
cholesterol. The mRNA can be completely or partially encapsulated
within the lipid particle. In some embodiments, the nucleic
acid-lipid particle has a lipid:mRNA mass ratio of from about 9:1
to about 20:1. In a specific embodiment, the nucleic acid-lipid
particle has a lipid:mRNA mass ratio of about 12:1. The mRNA can be
chemically modified, such as by the incorporation of pseudouridine
instead of uridine, and/or the incorporation of 5-methylcytidine
instead of cytidine. The present invention also provides
pharmaceutical compositions that include nucleic acid-lipid
particles of the present invention. Typically, the pharmaceutical
compositions include an excipient.
[0017] In another aspect, the present invention provides methods
for introducing an mRNA that encodes a protein into a cell. The
methods each include the step of contacting the cell with a nucleic
acid-lipid particle of the present invention (typically, a
multiplicity of nucleic acid-lipid particles of the present
invention) under conditions whereby the mRNA is introduced into the
cell and expressed therein to produce the protein. The methods can
be practiced in vivo or in vitro. For example, the cell is within a
living body (e.g., a mammalian body, such as a human body), and the
nucleic acid-lipid particle can be introduced into the living body
by injection.
[0018] In a further aspect, the present invention provides methods
for treating and/or ameliorating one or more symptoms associated
with a disease, in a human, caused by impaired expression of a
protein in the human. The methods of this aspect of the invention
include the step of administering to the human a therapeutically
effective amount of a nucleic acid-lipid particle of the present
invention (typically, a multiplicity of nucleic acid-lipid
particles of the present invention), wherein the mRNA encapsulated
within the nucleic acid-lipid particle encodes the protein. The
encoded protein is expressed within the human being, thereby
ameliorating at least one symptom of the disease.
[0019] In one embodiment, the ratio of lipid to nucleic acid (e.g.,
mRNA) in the lipid particles used in the practice of the present
invention is about 13:1.
[0020] The methods and compositions of the invention can be used,
for example, to treat any disease that is caused, at least in part,
by the absence of a polypeptide, or the reduced level of a
polypeptide, or the expression of a non-functional (or partially
functional, or aberrantly functional) form of a polypeptide, in a
cell, tissue, and/or organ of a human body.
[0021] Other objects, features, and advantages of the present
invention will be apparent to one of skill in the art from the
following detailed description and figures.
DETAILED DESCRIPTION
[0022] The nucleic acid-lipid particles, methods, and
pharmaceutical formulations, described herein advantageously
provide significant new compositions and methods for expressing
proteins in a mammalian organism, such as a human being.
Embodiments of the present invention can be administered, for
example, once per day, once per week, or once every several weeks
(e.g., once every two, three, four, five or six weeks), or once per
month, or once per year. Encapsulation of mRNA within lipid
particles confers one or more advantages, such as protecting the
mRNA from nuclease degradation in the bloodstream, allowing
preferential accumulation of the mRNA in target tissue and
providing a means of mRNA entry into the cellular cytoplasm where
the mRNA can express the encoded protein.
[0023] In one aspect, the present invention provides a lipid
nanoparticle comprising: (a) a cationic lipid; (b) a non-cationic
lipid; (c) a corticosteroid and; (d) a nucleic acid, wherein the
nucleic acid and the corticosteroid are encapsulated within the
lipid nanoparticle. Certain embodiments of the invention provide a
population of lipid nanoparticles comprising the lipid
nanoparticles. Certain embodiments of the invention provide a
population of lipid particles comprising a multiplicity of lipid
nanoparticles. In certain embodiments, the nucleic acid is
HPLC-purified mRNA. In certain embodiments, the LNP comprises a
PEG-lipid conjugate present in an amount of at least 3 mole
percent. In certain embodiments, the LNP comprises less than 0.5
mole percent phospholipid.
[0024] Certain embodiments of the invention provide a population of
lipid nanoparticles, comprising at least one population of lipid
nanoparticles selected from: (a) a first population of lipid
nanoparticles that each comprise a cationic lipid, a non-cationic
lipid, and a corticosteroid; and (b) a second population of lipid
nanoparticles that each comprise a cationic lipid, a non-cationic
lipid, and a nucleic acid, wherein the first population of lipid
nanoparticles does not comprise a nucleic acid, and wherein the
second population of lipid nanoparticles does not comprise a
corticosteroid. Certain embodiments of the invention provide a
population of lipid nanoparticles comprising the first and second
populations of lipid nanoparticles. In certain embodiments, the
nucleic acid is HPLC-purified mRNA.
[0025] Certain embodiments of the invention provide a lipid
nanoparticle comprising: (a) a cationic lipid; (b) a PEG-lipid
conjugate present in an amount of at least 3 mole percent; and (c)
mRNA encapsulated within the lipid particle; provided that the
lipid particle comprises less than 0.5 mole percent phospholipid.
In certain embodiments, the LNP comprises a corticosteroid. In
certain embodiments, the mRNA is HPLC-purified mRNA.
[0026] Certain embodiments provide a population of lipid
nanoparticles wherein each lipid nanoparticle in the population
comprises: (a) a cationic lipid; (b) a PEG-lipid conjugate present
in an amount of at least 3 mole percent; and (c) mRNA encapsulated
within the lipid nanoparticle; provided that the lipid nanoparticle
comprises less than 0.5 mole percent phospholipid. In certain
embodiments, the population of LNPs comprises LNPs that comprise a
corticosteroid. In certain embodiments, the mRNA is HPLC-purified
mRNA.
[0027] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising a multiplicity of lipid
nanoparticles, wherein each lipid nanoparticle comprises: (a) a
cationic lipid; (b) a non-cationic lipid; and (c) mRNA encapsulated
within the lipid particle, wherein the lipid nanoparticle
formulation has an IFIT response that is no more than 30 fold
greater than a reference IFIT response of phosphate buffered
saline. In certain embodiments, the mRNA is purified mRNA. In
certain embodiments, the mRNA is HPLC-purified mRNA. In certain
embodiments, the LNP comprises a PEG-lipid conjugate present in an
amount of at least 3 mole percent. In certain embodiments, the LNP
comprises a PEG-lipid conjugate present in an amount of at least
3.5 mole percent. In certain embodiments, the LNP comprises less
than 0.5 mole percent phospholipid. In certain embodiments, the LNP
comprises less than 0.05 mole percent phospholipid. In certain
embodiments, the LNP comprises a corticosteroid. In certain
embodiments, substantially all of the lipid nanoparticles in the
formulation comprise a corticosteroid encapsulated within the lipid
nanoparticle. For example, in certain embodiments, at least about
80% of the lipid nanoparticles in the formulation further comprise
a corticosteroid encapsulated within the lipid nanoparticle. In
certain embodiments, at least about 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% of the lipid nanoparticles in the formulation further comprise
a corticosteroid encapsulated within the lipid nanoparticle.
[0028] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising a multiplicity of lipid
nanoparticles, wherein each lipid nanoparticle comprises: (a) a
cationic lipid; (b) a non-cationic lipid; and (c) mRNA encapsulated
within the lipid particle, wherein the lipid nanoparticle
formulation has an IFIT response that is no more than 30 fold
greater than a reference IFIT response of phosphate buffered
saline, wherein the non-cationic lipid is a PEG-lipid conjugate
present in an amount of at least 3 mole percent, provided that the
lipid nanoparticle comprises less than 0.5 mole percent
phospholipid, and wherein at least 90% of the lipid nanoparticles
in the formulation further comprise a corticosteroid encapsulated
within the lipid nanoparticle.
[0029] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising:
[0030] (a) a first population of lipid nanoparticles that each
comprise a cationic lipid, a non-cationic lipid, and a
corticosteroid encapsulated with the lipid nanoparticle; and
[0031] (b) a second population of lipid nanoparticles that each
comprise a cationic lipid, a non-cationic lipid, and a mRNA
encapsulated within the lipid nanoparticle,
[0032] wherein the first population of lipid nanoparticles does not
comprise a mRNA, wherein the second population of lipid
nanoparticles does not comprise a corticosteroid, and wherein the
lipid nanoparticle formulation has an IFIT response that is no more
than 30 fold greater than a reference IFIT response of phosphate
buffered saline.
[0033] Certain embodiments of the invention provide a method of
making a lipid nanoparticle, comprising combining: (a) a cationic
lipid; (b) a non-cationic lipid; and (c) purified mRNA so as to
form a lipid nanoparticle, wherein the mRNA is encapsulated within
the lipid nanoparticle, and wherein the lipid nanoparticle has an
IFIT response that is no more than 30 fold greater than a reference
IFIT response of phosphate buffered saline. In certain embodiments,
the mRNA is HPLC-purified mRNA. In certain embodiments, the LNP
comprises a PEG-lipid conjugate present in an amount of at least 3
mole percent. In certain embodiments, the LNP comprises less than
0.5 mole percent phospholipid. In certain embodiments, the LNP
comprises a corticosteroid. In certain embodiments, the method
further comprises purifying mRNA to provide the purified mRNA.
[0034] Certain embodiments of the invention provide a method of
making a lipid nanoparticle formulation comprising a multiplicity
of lipid nanoparticles, the method comprising the step of
combining: (a) a cationic lipid; (b) a non-cationic lipid; and (c)
purified mRNA so as to form a lipid nanoparticle formulation
comprising a multiplicity of lipid nanoparticles, wherein the mRNA
is encapsulated within the lipid particles in the lipid
nanoparticle formulation, and wherein the lipid nanoparticle
formulation has an IFIT response that is no more than 30 fold
greater than a reference IFIT response of phosphate buffered
saline. In certain embodiments, the LNP comprises a PEG-lipid
conjugate present in an amount of at least 3 mole percent. In
certain embodiments, the LNP comprises less than 0.5 mole percent
phospholipid. In certain embodiments, the LNP comprises a
corticosteroid. In certain embodiments, the method further
comprises purifying mRNA (e.g., via HPLC) to provide the purified
mRNA.
[0035] Certain embodiments of the invention provide a lipid
nanoparticle formulation comprising a multiplicity of lipid
nanoparticles made by a process comprising the steps of combining:
(a) a cationic lipid; (b) a non-cationic lipid; and (c) purified
mRNA so as to form a lipid nanoparticle formulation comprising a
multiplicity of lipid nanoparticles, wherein the mRNA is
encapsulated within the lipid particles in the lipid nanoparticle
formulation, and wherein the lipid nanoparticle formulation has an
IFIT response that is no more than 30 fold greater than a reference
IFIT response of phosphate buffered saline. In certain embodiments,
the LNP comprises a PEG-lipid conjugate present in an amount of at
least 3 mole percent. In certain embodiments, the LNP comprises
less than 0.5 mole percent phospholipid. In certain embodiments,
the LNP comprises a corticosteroid. In certain embodiments, the
method further comprises purifying mRNA (e.g., via HPLC) to provide
the purified mRNA.
[0036] In certain embodiments, the nucleic acid is mRNA.
[0037] In certain embodiments, the nucleic acid is purified
mRNA.
[0038] In certain embodiments, the mRNA is HPLC-purified mRNA.
[0039] In certain embodiments, the corticosteroid has a log P
greater than 3.0.
[0040] In certain embodiments, substantially all lipid
nanoparticles in a formulation/population comprise a corticosteroid
encapsulated within the lipid nanoparticle. For example, in certain
embodiments, at least about 80% of the lipid nanoparticles in a
formulation/population further comprise a corticosteroid
encapsulated within the lipid nanoparticle. In certain embodiments,
at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the lipid
nanoparticles in the formulation/population further comprise a
corticosteroid encapsulated within the lipid nanoparticle.
[0041] In certain embodiments, the corticosteroid is a
glucocorticoid.
[0042] In certain embodiments, the corticosteroid is a
mineralocorticoid.
[0043] In certain embodiments, the corticosteroid is
clobetasol.
[0044] In certain embodiments, the glucocorticoid is selected from
hydrocortisone, cortisone, corticosterone, deoxycorticosterone,
prednisone, prednisolone, methylprednisolone, dexamethasone,
betamethasone, mometasone, triamcinolone, beclomethasone,
fludrocortisone, aldosterone, fluticasone, clobetasone, clobetasol,
and loteprednol, and pharmaceutically acceptable salts thereof, and
mixtures thereof.
[0045] In certain embodiments, the non-cationic lipid is selected
from a PEG-lipid conjugate and a phospholipid.
[0046] In certain embodiments, the non-cationic lipid is selected
from a PEG-lipid conjugate, a phospholipid, or a mixture of a
PEG-lipid conjugate and a phospholipid.
[0047] In certain embodiments, the non-cationic lipid comprises a
phospholipid.
[0048] In certain embodiments, the non-cationic lipid comprises a
PEG-lipid conjugate.
[0049] In certain embodiments, the non-cationic lipid comprises a
mixture of a PEG-lipid conjugate and a phospholipid.
[0050] In certain embodiments, the non-cationic lipid is a
phospholipid.
[0051] In certain embodiments, the non-cationic lipid is a
PEG-lipid conjugate.
[0052] In certain embodiments, the non-cationic lipid is a mixture
of a PEG-lipid conjugate and a phospholipid.
[0053] In certain embodiments, the lipid nanoparticle further
comprises cholesterol.
[0054] In certain embodiments, the phospholipid comprises
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), or a mixture thereof.
[0055] In certain embodiments, the PEG-lipid conjugate is selected
from the group consisting of a PEG-diacylglycerol (PEG-DAG)
conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a
PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and
a mixture thereof.
[0056] In certain embodiments, the PEG-lipid conjugate is selected
from the group consisting of a PEG-2000-C-DMA, PEG-diacylglycerol
(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a
PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and
a mixture thereof.
[0057] In certain embodiments, the PEG-lipid conjugate is a PEG-DAA
conjugate.
[0058] In certain embodiments, the PEG-DAA conjugate is selected
from the group consisting of a PEG-didecyloxypropyl (C.sub.10)
conjugate, a PEG-dilauryloxypropyl (C.sub.12) conjugate, a
PEG-dimyristyloxypropyl (C.sub.14) conjugate, a
PEG-dipalmityloxypropyl (C.sub.16) conjugate, a
PEG-distearyloxypropyl (C.sub.18) conjugate, and a mixture
thereof.
[0059] In certain embodiments, the lipid nanoparticle has a
lipid:nucleic mass ratio of from about 9:1 to about 20:1.
[0060] In certain embodiments, the multiplicity of lipid
nanoparticles in the lipid nanoparticle formulation has a
lipid:nucleic mass ratio of from about 9:1 to about 20:1.
[0061] In certain embodiments, the mRNA is chemically modified.
[0062] In certain embodiments, the lipid nanoparticle comprises an
electron dense core.
[0063] In certain embodiments, the lipid nanoparticle comprises an
electron dense core and wherein the mRNA is located within the
electron dense core.
[0064] Certain embodiments provide a pharmaceutical composition
comprising a lipid nanoparticle or population thereof as described
herein, and a pharmaceutically acceptable carrier.
[0065] Certain embodiments provide a method for introducing an mRNA
that encodes a protein into a cell, the method comprising
contacting the cell with a lipid nanoparticle or population thereof
as described herein, under conditions whereby the mRNA is
introduced into the cell and expressed therein to produce the
protein.
[0066] Certain embodiments provide a method for treating and/or
ameliorating one or more symptoms associated with a disease in a
human, caused by impaired expression of a protein in the human, the
method comprising administering to the human a therapeutically
effective amount of a lipid nanoparticle or population thereof as
described herein, wherein the mRNA encapsulated within the lipid
nanoparticle encodes the protein.
[0067] In certain embodiments, the phospholipid is
distearoylphosphatidylcholine (DSPC).
[0068] In certain embodiments, the PEG-lipid conjugate is
PEG-2000-C-DMA.
[0069] In certain embodiments, the LNP comprises at least 3.5 mole
percent of the PEG-lipid conjugate (e.g., at least about 3.5, 4,
4.5, 5, 5.5, or 6 mole percent).
[0070] In certain embodiments, the amount of PEG-lipid conjugate is
at least 3 mole percent (e.g., at least 3.1 mole percent, at least
3.2 mole percent, at least 3.3 mole percent, at least 3.4 mole
percent, at least 3.5 mole percent, at least 3.6 mole percent, at
least 3.7 mole percent, at least 3.8 mole percent, at least 3.9
mole percent, at least 4 mole percent). With respect to
phospholipid, in certain embodiments, no phospholipid is used in
the practice of the invention. In certain embodiments, the lipid
particle comprises less than 2 mole percent phospholipid, e.g., 1.9
mol % phospholipid, 1.8 mol % phospholipid, 1.7 mol % phospholipid,
1.6 mol % phospholipid, 1.5 mol % phospholipid, 1.4 mol %
phospholipid, 1.3 mol % phospholipid, 1.2 mol % phospholipid, 1.1
mol % phospholipid, 1.0 mol % phospholipid, 0.9 mol % phospholipid,
0.8 mol % phospholipid, 0.7 mol % phospholipid, 0.6 mol %
phospholipid, 0.5 mol % phospholipid, 0.4 mol % phospholipid, 0.3
mol % phospholipid, 0.2 mol % phospholipid, 0.1 mol % phospholipid,
or 0.0% phospholipid, e.g., less than 1.9 mol % phospholipid, less
than 1.8 mol % phospholipid, less than 1.7 mol % phospholipid, less
than 1.6 mol % phospholipid, less than 1.5 mol % phospholipid, less
than 1.4 mol % phospholipid, less than 1.3 mol % phospholipid, less
than 1.2 mol % phospholipid, less than 1.1 mol % phospholipid, less
than 1.0 mol % phospholipid, less than 0.9 mol % phospholipid, less
than 0.8 mol % phospholipid, less than 0.7 mol % phospholipid, less
than 0.6 mol % phospholipid, less than 0.5 mol % phospholipid, less
than 0.4 mol % phospholipid, less than 0.3 mol % phospholipid, less
than 0.2 mol % phospholipid, less than 0.1 mol % phospholipid.
[0071] In certain embodiments, the LNP comprises a PEG-lipid
conjugate present in an amount of at least 3 mole percent, provided
that the LNP comprises less than 0.5 mole percent phospholipid. In
certain embodiments, the PEG-lipid conjugate is present in an
amount of at least 3.5 mole percent (e.g., at least about 3.5, 4,
4.5, 5, 5.5, or 6 mole percent). In certain embodiments, the LNP
comprises less than 0.05 mole percent phospholipid.
[0072] In certain embodiments, the lipid nanoparticle has a
lipid:mRNA mass ratio of from about 9:1 to about 20:1.
[0073] Certain embodiments provide a lipid nanoparticle prepared
according to the methods described herein.
[0074] Certain embodiments provide a lipid nanoparticle formulation
comprising a multiplicity of lipid nanoparticles as described
herein.
[0075] Certain embodiments provide a lipid nanoparticle formulation
comprising a multiplicity of lipid nanoparticles as described
herein, wherein the lipid nanoparticle formulation has an IFIT
response that is no more than 30 fold greater than a phosphate
buffered saline control response. In certain embodiments, the lipid
nanoparticle formulation has an IFIT response that is no more than
about 29, 28, 2, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 fold greater than a
phosphate buffered saline control response.
[0076] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0077] An "effective amount" or "therapeutically effective amount"
of a therapeutic nucleic acid such as an mRNA is an amount
sufficient to produce the desired effect, e.g., mRNA-directed
expression of an amount of a protein that causes a desirable
biological effect in the organism within which the protein is
expressed. For example, in some embodiments, the expressed protein
is an active form of a protein that is normally expressed in a cell
type within the body, and the therapeutically effective amount of
the mRNA is an amount that produces an amount of the encoded
protein that is at least 50% (e.g., at least 60%, or at least 70%,
or at least 80%, or at least 90%) of the amount of the protein that
is normally expressed in the cell type of a healthy individual.
Suitable assays for measuring the expression of an mRNA or protein
include, but are not limited to dot blots, Northern blots, in situ
hybridization, ELISA, immunoprecipitation, enzyme function, as well
as phenotypic assays known to those of skill in the art.
[0078] By "decrease," "decreasing," "reduce," or "reducing" of an
immune response by an mRNA is intended to mean a detectable
decrease of an immune response to a given mRNA (e.g., a modified
mRNA). The amount of decrease of an immune response by a modified
mRNA may be determined relative to the level of an immune response
in the presence of an unmodified mRNA. A detectable decrease can be
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the
immune response detected in the presence of the unmodified mRNA. A
decrease in the immune response to mRNA is typically measured by a
decrease in cytokine production (e.g., IFN.gamma., IFN.alpha.,
TNF.alpha., IL-6, or IL-12) by a responder cell in vitro or a
decrease in cytokine production in the sera of a mammalian subject
after administration of the mRNA.
[0079] "Substantial identity" refers to a sequence that hybridizes
to a reference sequence under stringent conditions, or to a
sequence that has a specified percent identity over a specified
region of a reference sequence.
[0080] The phrase "stringent hybridization conditions" refers to
conditions under which a nucleic acid will hybridize to its target
sequence, typically in a complex mixture of nucleic acids, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization.
[0081] Exemplary stringent hybridization conditions can be as
follows: 50% formamide, 5.times.SSC, and 1% SDS, incubating at
42.degree. C., or, 5.times.SSC, 1% SDS, incubating at 65.degree.
C., with wash in 0.2.times.SSC, and 0.1% SDS at 65.degree. C. For
PCR, a temperature of about 36.degree. C. is typical for low
stringency amplification, although annealing temperatures may vary
between about 32.degree. C. and 48.degree. C. depending on primer
length. For high stringency PCR amplification, a temperature of
about 62.degree. C. is typical, although high stringency annealing
temperatures can range from about 50.degree. C. to about 65.degree.
C., depending on the primer length and specificity. Typical cycle
conditions for both high and low stringency amplifications include
a denaturation phase of 90.degree. C.-95.degree. C. for 30 sec. to
2 min., an annealing phase lasting 30 sec. to 2 min., and an
extension phase of about 72.degree. C. for 1 to 2 min. Protocols
and guidelines for low and high stringency amplification reactions
are provided, e.g., in Innis et al., PCR Protocols, A Guide to
Methods and Applications, Academic Press, Inc. N.Y. (1990).
[0082] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
references, e.g., Current Protocols in Molecular Biology, Ausubel
et al., eds.
[0083] The terms "substantially identical" or "substantial
identity," in the context of two or more nucleic acids, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of nucleotides that are the same (i.e., at
least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
This definition, when the context indicates, also refers
analogously to the complement of a sequence. Preferably, the
substantial identity exists over a region that is at least about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in
length.
[0084] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0085] A "comparison window," as used herein, includes reference to
a segment of any one of a number of contiguous positions selected
from the group consisting of from about 5 to about 60, usually
about 10 to about 45, more usually about 15 to about 30, in which a
sequence may be compared to a reference sequence of the same number
of contiguous positions after the two sequences are optimally
aligned. Methods of alignment of sequences for comparison are well
known in the art. Optimal alignment of sequences for comparison can
be conducted, e.g., by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. USA, 85:2444 (1988), by computerized implementations of
these algorithms (GAP, BESALDHIT, FASTA, and ALDHASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by manual alignment and visual
inspection (see, e.g., Current Protocols in Molecular Biology,
Ausubel et al., eds. (1995 supplement)).
[0086] Non-limiting examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al.,
J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST
2.0 are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids of the invention.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). Another example is a global
alignment algorithm for determining percent sequence identity such
as the Needleman-Wunsch algorithm for aligning protein or
nucleotide (e.g., RNA) sequences.
[0087] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0088] The term "nucleic acid" as used herein refers to a polymer
containing at least two deoxyribonucleotides or ribonucleotides in
either single- or double-stranded form and includes DNA and RNA.
DNA may be in the form of, e.g., antisense molecules, plasmid DNA,
pre-condensed DNA, a PCR product, vectors (e.g., P1, PAC, BAC, YAC,
artificial chromosomes), expression cassettes, chimeric sequences,
chromosomal DNA, or derivatives and combinations of these groups.
RNA may be in the form of small interfering RNA (siRNA),
Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical
interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA,
viral RNA (vRNA), and combinations thereof. Nucleic acids include
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, and which have similar
binding properties as the reference nucleic acid. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
Unless specifically limited, the term encompasses nucleic acids
containing known analogues of natural nucleotides that have similar
binding properties as the reference nucleic acid. Nucleic acid
sequence may in certain embodiments include an "unlocked nucleobase
analogue" (abbreviated as "UNA").
[0089] The term "unlocked nucleobase analogue" (abbreviated as
"UNA") refers to an acyclic nucleobase in which the C2' and C3'
atoms of the ribose ring are not covalently linked. The term
"unlocked nucleobase analogue" includes nucleobase analogues having
the following structure identified as Structure A:
##STR00001##
wherein R is hydroxyl, and Base is any natural or unnatural base
such as, for example, adenine (A), cytosine (C), guanine (G) and
thymine (T). UNA useful in the practice of the present invention
include the molecules identified as acyclic 2'-3'-seco-nucleotide
monomers in U.S. patent serial number 8,314,227 which is
incorporated by reference herein in its entirety.
[0090] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions), alleles,
orthologs, SNPs, and complementary sequences as well as the
sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes,
8:91-98 (1994)).
[0091] The term "small-interfering RNA" or "siRNA" as used herein
refers to double stranded RNA (i.e., duplex RNA) that is capable of
reducing or inhibiting the expression of a target gene or sequence
(e.g., by mediating the degradation or inhibiting the translation
of mRNAs which are complementary to the siRNA sequence) when the
siRNA is in the same cell as the target gene or sequence. The siRNA
may have substantial or complete identity to the target gene or
sequence, or may comprise a region of mismatch (i.e., a mismatch
motif). In certain embodiments, the siRNAs may be about 19-25
(duplex) nucleotides in length, and is preferably about 20-24,
21-22, or 21-23 (duplex) nucleotides in length. siRNA duplexes may
comprise 3' overhangs of about 1 to about 4 nucleotides or about 2
to about 3 nucleotides and 5' phosphate termini. Examples of siRNA
include, without limitation, a double-stranded polynucleotide
molecule assembled from two separate stranded molecules, wherein
one strand is the sense strand and the other is the complementary
antisense strand.
[0092] Preferably, siRNA are chemically synthesized. siRNA can also
be generated by cleavage of longer dsRNA (e.g., dsRNA greater than
about 25 nucleotides in length) with the E. coli RNase III or
Dicer. These enzymes process the dsRNA into biologically active
siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA,
99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA,
99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003);
Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et
al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol.
Chem., 243:82 (1968)). Preferably, dsRNA are at least 50
nucleotides to about 100, 200, 300, 400, or 500 nucleotides in
length. A dsRNA may be as long as 1000, 1500, 2000, 5000
nucleotides in length, or longer. The dsRNA can encode for an
entire gene transcript or a partial gene transcript. In certain
instances, siRNA may be encoded by a plasmid (e.g., transcribed as
sequences that automatically fold into duplexes with hairpin
loops).
[0093] "Nucleotides" contain a sugar deoxyribose (DNA) or ribose
(RNA), a base, and a phosphate group. Nucleotides are linked
together through the phosphate groups. "Bases" include purines and
pyrimidines, which further include natural compounds adenine,
thymine, guanine, cytosine, uracil, inosine, and natural analogs,
and synthetic derivatives of purines and pyrimidines, which
include, but are not limited to, modifications which place new
reactive groups such as, but not limited to, amines, alcohols,
thiols, carboxylates, and alkylhalides.
[0094] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises partial length or entire length coding
sequences necessary for the production of a polypeptide or
precursor polypeptide.
[0095] "Gene product," as used herein, refers to a product of a
gene such as an RNA transcript or a polypeptide.
[0096] The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
characterized by being insoluble in water, but soluble in many
organic solvents. They are usually divided into at least three
classes: (1) "simple lipids," which include fats and oils as well
as waxes; (2) "compound lipids," which include phospholipids and
glycolipids; and (3) "derived lipids" such as steroids.
[0097] The term "lipid particle" includes a lipid formulation that
can be used to deliver a therapeutic nucleic acid (e.g., mRNA or
siRNA) to a target site of interest (e.g., cell, tissue, organ, and
the like). In preferred embodiments, the lipid particle of the
invention is a nucleic acid-lipid particle, which is typically
formed from a cationic lipid, a non-cationic lipid (e.g., a
phospholipid), a conjugated lipid that prevents aggregation of the
particle (e.g., a PEG-lipid), and optionally cholesterol.
Typically, the therapeutic nucleic acid (e.g., mRNA) may be
encapsulated in the lipid portion of the particle, thereby
protecting it from enzymatic degradation.
[0098] The term "electron dense core", when used to describe a
lipid particle of the present invention, refers to the dark
appearance of the interior portion of a lipid particle when
visualized using cryo transmission electron microscopy ("cryoTEM").
Some lipid particles of the present invention have an electron
dense core and lack a lipid bilayer structure. Some lipid particles
of the present invention have an electron dense core, lack a lipid
bilayer structure, and have an inverse Hexagonal or Cubic phase
structure. While not wishing to be bound by theory, it is thought
that the non-bilayer lipid packing provides a 3-dimensional network
of lipid cylinders with water and nucleic on the inside, i.e.,
essentially, a lipid droplet interpenetrated with aqueous channels
containing the nucleic acid.
[0099] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP is a particle made from lipids (e.g.,
a cationic lipid, a non-cationic lipid, and a conjugated lipid that
prevents aggregation of the particle), wherein the nucleic acid
(e.g., mRNA) is fully encapsulated within the lipid. In certain
instances, SNALP are extremely useful for systemic applications, as
they can exhibit extended circulation lifetimes following
intravenous (i.v.) injection, they can accumulate at distal sites
(e.g., sites physically separated from the administration site),
and they can mediate mRNA expression at these distal sites. The
nucleic acid may be complexed with a condensing agent and
encapsulated within a SNALP as set forth in PCT Publication No. WO
00/03683, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
[0100] The lipid particles of the invention (e.g., SNALP) typically
have a mean diameter of from about 30 nm to about 150 nm, from
about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from
about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from
about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from
about 90 nm to about 100 nm, from about 70 to about 90 nm, from
about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or
about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70
nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115
nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and
are substantially non-toxic. In addition, nucleic acids, when
present in the lipid particles of the present invention, are
resistant in aqueous solution to degradation with a nuclease.
Nucleic acid-lipid particles and their method of preparation are
disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and
20070042031, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.
[0101] As used herein, "lipid encapsulated" can refer to a lipid
particle that provides a therapeutic nucleic acid such as an mRNA
with full encapsulation, partial encapsulation, or both. In a
preferred embodiment, the nucleic acid (e.g., mRNA) is fully
encapsulated in the lipid particle (e.g., to form a SNALP or other
nucleic acid-lipid particle).
[0102] The term "lipid conjugate" refers to a conjugated lipid that
inhibits aggregation of lipid particles. Such lipid conjugates
include, but are not limited to, PEG-lipid conjugates such as,
e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates),
PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG
coupled to cholesterol, PEG coupled to phosphatidylethanolamines,
and PEG conjugated to ceramides (see, e.g., U.S. Pat. No.
5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid
conjugates, polyamide oligomers (e.g., ATTA-lipid conjugates), and
mixtures thereof. Additional examples of POZ-lipid conjugates are
described in PCT Publication No. WO 2010/006282. PEG or POZ can be
conjugated directly to the lipid or may be linked to the lipid via
a linker moiety. Any linker moiety suitable for coupling the PEG or
the POZ to a lipid can be used including, e.g., non-ester
containing linker moieties and ester-containing linker moieties. In
certain preferred embodiments, non-ester containing linker
moieties, such as amides or carbamates, are used. The disclosures
of each of the above patent documents are herein incorporated by
reference in their entirety for all purposes.
[0103] The term "amphipathic lipid" refers, in part, to any
suitable material wherein the hydrophobic portion of the lipid
material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Hydrophilic
characteristics derive from the presence of polar or charged groups
such as carbohydrates, phosphate, carboxylic, sulfato, amino,
sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity
can be conferred by the inclusion of apolar groups that include,
but are not limited to, long-chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or
more aromatic, cycloaliphatic, or heterocyclic group(s). Examples
of amphipathic compounds include, but are not limited to,
phospholipids, aminolipids, and sphingolipids.
[0104] Representative examples of phospholipids include, but are
not limited to, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, phosphatidic acid,
palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and
dilinoleoylphosphatidylcholine. Other compounds lacking in
phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols, and .beta.-acyloxyacids, are also within the group
designated as amphipathic lipids. Additionally, the amphipathic
lipids described above can be mixed with other lipids including
triglycerides and sterols.
[0105] The term "neutral lipid" refers to any of a number of lipid
species that exist either in an uncharged or neutral zwitterionic
form at a selected pH. At physiological pH, such lipids include,
for example, diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides, and diacylglycerols.
[0106] The term "non-cationic lipid" refers to any amphipathic
lipid as well as any other neutral lipid or anionic lipid.
[0107] The term "anionic lipid" refers to any lipid that is
negatively charged at physiological pH. These lipids include, but
are not limited to, phosphatidylglycerols, cardiolipins,
diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl
phosphatidylethanolamines, N-succinyl phosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
[0108] The term "hydrophobic lipid" refers to compounds having
apolar groups that include, but are not limited to, long-chain
saturated and unsaturated aliphatic hydrocarbon groups and such
groups optionally substituted by one or more aromatic,
cycloaliphatic, or heterocyclic group(s). Suitable examples
include, but are not limited to, diacylglycerol, dialkylglycerol,
N--N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and
1,2-dialkyl-3-aminopropane.
[0109] The terms "cationic lipid" and "amino lipid" are used
interchangeably herein to include those lipids and salts thereof
having one, two, three, or more fatty acid or fatty alkyl chains
and a pH-titratable amino head group (e.g., an alkylamino or
dialkylamino head group). The cationic lipid is typically
protonated (i.e., positively charged) at a pH below the pK.sub.a of
the cationic lipid and is substantially neutral at a pH above the
pK.sub.a. The cationic lipids of the invention may also be termed
titratable cationic lipids. In some embodiments, the cationic
lipids comprise: a protonatable tertiary amine (e.g.,
pH-titratable) head group; C.sub.18 alkyl chains, wherein each
alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double
bonds; and ether, ester, or ketal linkages between the head group
and alkyl chains. Such cationic lipids include, but are not limited
to, DSDMA, DODMA, DLinDMA, DLenDMA, .gamma.-DLenDMA, DLin-K-DMA,
DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K),
DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, .gamma.-DLen-C2K-DMA,
DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as
MC3) and (DLin-MP-DMA)(also known as 1-B11).
[0110] The term "alkylamino" includes a group of formula --N(H)R,
wherein R is an alkyl as defined herein.
[0111] The term "dialkylamino" includes a group of formula
--NR.sub.2, wherein each R is independently an alkyl as defined
herein.
[0112] The term "salts" includes any anionic and cationic complex,
such as the complex formed between a cationic lipid and one or more
anions. Non-limiting examples of anions include inorganic and
organic anions, e.g., hydride, fluoride, chloride, bromide, iodide,
oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen
phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate,
nitrate, nitrite, nitride, bisulfate, sulfide, sulfite, bisulfate,
sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate,
benzoate, citrate, tartrate, lactate, acrylate, polyacrylate,
fumarate, maleate, itaconate, glycolate, gluconate, malate,
mandelate, tiglate, ascorbate, salicylate, polymethacrylate,
perchlorate, chlorate, chlorite, hypochlorite, bromate,
hypobromite, iodate, an alkylsulfonate, an arylsulfonate, arsenate,
arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,
hydroxide, peroxide, permanganate, and mixtures thereof. In
particular embodiments, the salts of the cationic lipids disclosed
herein are crystalline salts.
[0113] The term "alkyl" includes a straight chain or branched,
noncyclic or cyclic, saturated aliphatic hydrocarbon containing
from 1 to 24 carbon atoms. Representative saturated straight chain
alkyls include, but are not limited to, methyl, ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched
alkyls include, without limitation, isopropyl, sec-butyl, isobutyl,
tert-butyl, isopentyl, and the like. Representative saturated
cyclic alkyls include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like, while
unsaturated cyclic alkyls include, without limitation,
cyclopentenyl, cyclohexenyl, and the like.
[0114] The term "alkenyl" includes an alkyl, as defined above,
containing at least one double bond between adjacent carbon atoms.
Alkenyls include both cis and trans isomers. Representative
straight chain and branched alkenyls include, but are not limited
to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, and the like.
[0115] The term "alkynyl" includes any alkyl or alkenyl, as defined
above, which additionally contains at least one triple bond between
adjacent carbons. Representative straight chain and branched
alkynyls include, without limitation, acetylenyl, propynyl,
1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl,
and the like.
[0116] The term "acyl" includes any alkyl, alkenyl, or alkynyl
wherein the carbon at the point of attachment is substituted with
an oxo group, as defined below. The following are non-limiting
examples of acyl groups: --C(.dbd.O)alkyl, --C(.dbd.O)alkenyl, and
--C(.dbd.O)alkynyl.
[0117] The term "heterocycle" includes a 5- to 7-membered
monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which
is either saturated, unsaturated, or aromatic, and which contains
from 1 or 2 heteroatoms independently selected from nitrogen,
oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms
may be optionally oxidized, and the nitrogen heteroatom may be
optionally quaternized, including bicyclic rings in which any of
the above heterocycles are fused to a benzene ring. The heterocycle
may be attached via any heteroatom or carbon atom. Heterocycles
include, but are not limited to, heteroaryls as defined below, as
well as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,
piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
and the like.
[0118] The terms "optionally substituted alkyl", "optionally
substituted alkenyl", "optionally substituted alkynyl", "optionally
substituted acyl", and "optionally substituted heterocycle" mean
that, when substituted, at least one hydrogen atom is replaced with
a substituent. In the case of an oxo substituent (.dbd.O), two
hydrogen atoms are replaced. In this regard, substituents include,
but are not limited to, oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, R.sup.x and
R.sup.y are the same or different and are independently hydrogen,
alkyl, or heterocycle, and each of the alkyl and heterocycle
substituents may be further substituted with one or more of oxo,
halogen, --OH, --CN, alkyl, --OR.sup.x, heterocycle,
--NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, and --SO.sub.nNR.sup.xR.sup.y. The term
"optionally substituted," when used before a list of substituents,
means that each of the substituents in the list may be optionally
substituted as described herein.
[0119] The term "halogen" includes fluoro, chloro, bromo, and
iodo.
[0120] The term "fusogenic" refers to the ability of a lipid
particle, such as a SNALP, to fuse with the membranes of a cell.
The membranes can be either the plasma membrane or membranes
surrounding organelles, e.g., endosome, nucleus, etc.
[0121] As used herein, the term "aqueous solution" refers to a
composition comprising in whole, or in part, water.
[0122] As used herein, the term "organic lipid solution" refers to
a composition comprising in whole, or in part, an organic solvent
having a lipid.
[0123] "Distal site," as used herein, refers to a physically
separated site, which is not limited to an adjacent capillary bed,
but includes sites broadly distributed throughout an organism.
[0124] "Serum-stable" in relation to nucleic acid-lipid particles
such as SNALP means that the particle is not significantly degraded
after exposure to a serum or nuclease assay that would
significantly degrade free DNA or RNA. Suitable assays include, for
example, a standard serum assay, a DNAse assay, or an RNAse
assay.
[0125] "Systemic delivery," as used herein, refers to delivery of
lipid particles that leads to a broad biodistribution of an active
agent such as an mRNA within an organism. Some techniques of
administration can lead to the systemic delivery of certain agents,
but not others. Systemic delivery means that a useful, preferably
therapeutic, amount of an agent is exposed to most parts of the
body. To obtain broad biodistribution generally requires a blood
lifetime such that the agent is not rapidly degraded or cleared
(such as by first pass organs (liver, lung, etc.) or by rapid,
nonspecific cell binding) before reaching a disease site distal to
the site of administration. Systemic delivery of lipid particles
can be by any means known in the art including, for example,
intravenous, subcutaneous, and intraperitoneal. In a preferred
embodiment, systemic delivery of lipid particles is by intravenous
delivery.
[0126] "Local delivery," as used herein, refers to delivery of an
active agent such as an mRNA directly to a target site within an
organism. For example, an agent can be locally delivered by direct
injection into a disease site, other target site, or a target organ
such as the liver, heart, pancreas, kidney, and the like.
[0127] The term "mammal" refers to any mammalian species such as a
human, mouse, rat, dog, cat, hamster, guinea pig, rabbit,
livestock, and the like.
[0128] When used herein to describe the ratio of lipid:mRNA, the
term "lipid" refers to the total lipid in the particle.
[0129] Unless stated otherwise herein, the term "about", when used
in connection with a value or range of values, means plus or minus
5% of the stated value or range of values.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0130] In one aspect, the present invention provides nucleic
acid-lipid particles that each include (a) a lipid particle
comprising a cationic lipid; and (b) an mRNA molecule encapsulated
within the lipid particle. Typically, a population of mRNA
molecules is encapsulated within the lipid particle. The lipid
particles typically include an outer layer defining an interior
portion, wherein the mRNA molecule(s) is located within the
interior portion. The mRNA molecule(s) is typically completely
encapsulated within the lipid particle. The lipid particles can be
spherical or non-spherical. The lipid particles can have an
electron dense core when visualized using cryo TEM. Typically, the
electron dense core is mainly composed of lipid, although aqueous
material may be present in an amount that is less than the amount
of the lipid.
[0131] In one aspect, the present invention provides a lipid
particle comprising a PEG lipid, a non-cationic lipid, a cationic
lipid selected from a trialkyl cationic lipid and a tetra alkyl
cationic lipid, and an mRNA; wherein the lipid particle has an
electron dense core and the mRNA is encapsulated within the
electron dense core.
[0132] In some embodiments of the invention the lipid particles
include (a) a lipid particle comprising a cationic lipid, a
PEG-lipid, and a phospholipid; and (b) an mRNA molecule, wherein
the mRNA molecule is encapsulated within the lipid particle. The
lipid particles can optionally include cholesterol. The mRNA can be
completely or partially encapsulated within the lipid particle.
[0133] The formation of the particle 100 includes, in one or more
embodiments, disposing a lipid into a first fluid, such as ethanol,
disposing mRNA into a second fluid, such as an aqueous buffer, and
mixing the first and second fluids under controlled conditions to
form particle 100. The resulting particle 100 includes an electron
dense core within the lipid particle when viewed by Cryo
Transmission Electron Microscopy.
mRNA
[0134] mRNA useful in the practice of the present invention may
comprise at least one, two, three, four, five, six, seven, eight,
nine, ten, or more modified nucleotides such as 2'OMe nucleotides.
Preferably, uridine and/or guanosine nucleotides in the mRNA are
modified with 2'OMe nucleotides. In some embodiments, the mRNA may
further comprise modified (e.g., 2'OMe-modified) adenosine and/or
modified (e.g., 2'OMe-modified) cytosine nucleotides. In one
aspect, the present invention provides a nucleic acid-lipid
particle (e.g., SNALP) that includes an mRNA. The nucleic
acid-lipid particles (e.g., SNALP) typically comprise one or more
(e.g., a cocktail) mRNA(s), a cationic lipid, and a non-cationic
lipid. In certain instances, the nucleic acid-lipid particles
(e.g., SNALP) further comprise a conjugated lipid that inhibits
aggregation of particles. Preferably, the nucleic acid-lipid
particles (e.g., SNALP) comprise one or more (e.g., a cocktail)
mRNAs, a cationic lipid, a non-cationic lipid, and a conjugated
lipid that inhibits aggregation of particles.
[0135] In some embodiments, the mRNA(s) are fully encapsulated in
the nucleic acid-lipid particle (e.g., SNALP). With respect to
formulations comprising an mRNA cocktail, the different types of
mRNA species present in the cocktail (e.g., mRNA having different
sequences) may be co-encapsulated in the same particle, or each
type of mRNA species present in the cocktail may be encapsulated in
a separate particle. The mRNA cocktail may be formulated in the
particles described herein using a mixture of two or more
individual mRNAs (each having a unique sequence) at identical,
similar, or different concentrations or molar ratios. In one
embodiment, a cocktail of mRNAs (corresponding to a plurality of
mRNAs with different sequences) is formulated using identical,
similar, or different concentrations or molar ratios of each mRNA
species, and the different types of mRNAs are co-encapsulated in
the same particle. In another embodiment, each type of mRNA species
present in the cocktail is encapsulated in different particles at
identical, similar, or different mRNA concentrations or molar
ratios, and the particles thus formed (each containing a different
mRNA payload) are administered separately (e.g., at different times
in accordance with a therapeutic regimen), or are combined and
administered together as a single unit dose (e.g., with a
pharmaceutically acceptable carrier). The particles described
herein are serum-stable, are resistant to nuclease degradation, and
are substantially non-toxic to mammals such as humans.
[0136] The cationic lipid in the nucleic acid-lipid particles of
the invention (e.g., SNALP) may comprise, e.g., one or more
cationic lipids of Formula I-III described herein or any other
cationic lipid species. In one particular embodiment, the cationic
lipid is selected from the group consisting of
1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-di-.gamma.-linolenyloxy-N,N-dimethylaminopropane
(.gamma.-DLenDMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane
(DLin-K-C2-DMA),
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-M-C3-DMA), salts thereof, and
mixtures thereof.
[0137] The non-cationic lipid in the nucleic acid-lipid particles
of the present invention (e.g., SNALP) may comprise, e.g., one or
more anionic lipids and/or neutral lipids. In some embodiments, the
non-cationic lipid comprises one of the following neutral lipid
components: (1) a mixture of a phospholipid and cholesterol or a
derivative thereof; (2) cholesterol or a derivative thereof; or (3)
a phospholipid. In certain preferred embodiments, the phospholipid
comprises dipalmitoylphosphatidylcholine (DPPC), di
stearoylphosphatidylcholine (DSPC), or a mixture thereof. In a
particularly preferred embodiment, the non-cationic lipid is a
mixture of DPPC and cholesterol.
[0138] The lipid conjugate in the nucleic acid-lipid particles of
the invention (e.g., SNALP) inhibits aggregation of particles and
may comprise, e.g., one or more of the lipid conjugates described
herein. In one particular embodiment, the lipid conjugate comprises
a PEG-lipid conjugate. Examples of PEG-lipid conjugates include,
but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and
mixtures thereof. In certain embodiments, the PEG-DAA conjugate in
the lipid particle may comprise a PEG-didecyloxypropyl (C.sub.10)
conjugate, a PEG-dilauryloxypropyl (C.sub.12) conjugate, a
PEG-dimyristyloxypropyl (C.sub.14) conjugate, a
PEG-dipalmityloxypropyl (C.sub.16) conjugate, a
PEG-distearyloxypropyl (C.sub.18) conjugate, or mixtures thereof.
In another embodiment, the lipid conjugate comprises a POZ-lipid
conjugate such as a POZ-DAA conjugate.
[0139] In some embodiments, the present invention provides nucleic
acid-lipid particles (e.g., SNALP) comprising: (a) one or more
(e.g., a cocktail) mRNA molecule(s) that each encode a protein; (b)
one or more cationic lipids or salts thereof comprising from about
50 mol % to about 85 mol % of the total lipid present in the
particle; (c) one or more non-cationic lipids comprising from about
13 mol % to about 49.5 mol % of the total lipid present in the
particle; and (d) one or more conjugated lipids that inhibit
aggregation of particles comprising from about 0.5 mol % to about 2
mol % of the total lipid present in the particle.
[0140] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) one or more (e.g., a cocktail) mRNA
molecule(s) that each encode a protein; (b) a cationic lipid or a
salt thereof comprising from about 52 mol % to about 62 mol % of
the total lipid present in the particle; (c) a mixture of a
phospholipid and cholesterol or a derivative thereof comprising
from about 36 mol % to about 47 mol % of the total lipid present in
the particle; and (d) a PEG-lipid conjugate comprising from about 1
mol % to about 2 mol % of the total lipid present in the particle.
This embodiment of nucleic acid-lipid particle is generally
referred to herein as the "1:57" formulation. In one particular
embodiment, the 1:57 formulation is a four-component system
comprising about 1.4 mol % PEG-lipid conjugate (e.g.,
PEG2000-C-DMA), about 57.1 mol % cationic lipid (e.g.,
DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC),
and about 34.3 mol % cholesterol (or derivative thereof).
[0141] In another aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) one or more (e.g., a cocktail) mRNA
molecule(s) that each encode a protein; (b) a cationic lipid or a
salt thereof comprising from about 56.5 mol % to about 66.5 mol %
of the total lipid present in the particle; (c) cholesterol or a
derivative thereof comprising from about 31.5 mol % to about 42.5
mol % of the total lipid present in the particle; and (d) a
PEG-lipid conjugate comprising from about 1 mol % to about 2 mol %
of the total lipid present in the particle. This embodiment of
nucleic acid-lipid particle is generally referred to herein as the
"1:62" formulation. In one particular embodiment, the 1:62
formulation is a three-component system which is phospholipid-free
and comprises about 1.5 mol % PEG-lipid conjugate (e.g.,
PEG2000-C-DMA), about 61.5 mol % cationic lipid (e.g.,
DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol % cholesterol
(or derivative thereof).
[0142] Additional embodiments related to the 1:57 and 1:62
formulations are described in PCT Publication No. WO 09/127060 and
published US patent application publication number US 2011/0071208
A1, the disclosures of which are herein incorporated by reference
in their entirety for all purposes.
[0143] In other embodiments, the present invention provides nucleic
acid-lipid particles (e.g., SNALP) comprising: (a) one or more
(e.g., a cocktail) mRNA molecule(s) that each encode a protein; (b)
one or more cationic lipids or salts thereof comprising from about
2 mol % to about 50 mol % of the total lipid present in the
particle; (c) one or more non-cationic lipids comprising from about
5 mol % to about 90 mol % of the total lipid present in the
particle; and (d) one or more conjugated lipids that inhibit
aggregation of particles comprising from about 0.5 mol % to about
20 mol % of the total lipid present in the particle.
[0144] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) one or more (e.g., a cocktail) mRNA
molecule(s) that each encode a protein; (b) a cationic lipid or a
salt thereof comprising from about 30 mol % to about 50 mol % of
the total lipid present in the particle; (c) a mixture of a
phospholipid and cholesterol or a derivative thereof comprising
from about 47 mol % to about 69 mol % of the total lipid present in
the particle; and (d) a PEG-lipid conjugate comprising from about 1
mol % to about 3 mol % of the total lipid present in the particle.
This embodiment of nucleic acid-lipid particle is generally
referred to herein as the "2:40" formulation. In one particular
embodiment, the 2:40 formulation is a four-component system which
comprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),
about 40 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt
thereof, about 10 mol % DPPC (or DSPC), and about 48 mol %
cholesterol (or derivative thereof).
[0145] In further embodiments, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) comprising: (a) one or
more (e.g., a cocktail) mRNA molecule(s) that each encode a
protein; (b) one or more cationic lipids or salts thereof
comprising from about 50 mol % to about 65 mol % of the total lipid
present in the particle; (c) one or more non-cationic lipids
comprising from about 25 mol % to about 45 mol % of the total lipid
present in the particle; and (d) one or more conjugated lipids that
inhibit aggregation of particles comprising from about 5 mol % to
about 10 mol % of the total lipid present in the particle.
[0146] In one aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) one or more (e.g., a cocktail) mRNA
molecule(s) that each encode a protein; (b) a cationic lipid or a
salt thereof comprising from about 50 mol % to about 60 mol % of
the total lipid present in the particle; (c) a mixture of a
phospholipid and cholesterol or a derivative thereof comprising
from about 35 mol % to about 45 mol % of the total lipid present in
the particle; and (d) a PEG-lipid conjugate comprising from about 5
mol % to about 10 mol % of the total lipid present in the particle.
This embodiment of nucleic acid-lipid particle is generally
referred to herein as the "7:54" formulation. In certain instances,
the non-cationic lipid mixture in the 7:54 formulation comprises:
(i) a phospholipid of from about 5 mol % to about 10 mol % of the
total lipid present in the particle; and (ii) cholesterol or a
derivative thereof of from about 25 mol % to about 35 mol % of the
total lipid present in the particle. In one particular embodiment,
the 7:54 formulation is a four-component system which comprises
about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54
mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about
7 mol % DPPC (or DSPC), and about 32 mol % cholesterol (or
derivative thereof).
[0147] In another aspect of this embodiment, the nucleic acid-lipid
particle comprises: (a) one or more (e.g., a cocktail) mRNA
molecule(s) that each encode a protein; (b) a cationic lipid or a
salt thereof comprising from about 55 mol % to about 65 mol % of
the total lipid present in the particle; (c) cholesterol or a
derivative thereof comprising from about 30 mol % to about 40 mol %
of the total lipid present in the particle; and (d) a PEG-lipid
conjugate comprising from about 5 mol % to about 10 mol % of the
total lipid present in the particle. This embodiment of nucleic
acid-lipid particle is generally referred to herein as the "7:58"
formulation. In one particular embodiment, the 7:58 formulation is
a three-component system which is phospholipid-free and comprises
about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58
mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and
about 35 mol % cholesterol (or derivative thereof).
[0148] Additional embodiments related to the 7:54 and 7:58
formulations are described in published US patent application
publication number US 2011/0076335 A1, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0149] The present invention also provides pharmaceutical
compositions comprising a nucleic acid-lipid particle such as a
SNALP and a pharmaceutically acceptable carrier.
[0150] The nucleic acid-lipid particles of the present invention
(e.g., SNALP) are useful for the therapeutic delivery of mRNAs that
express one or more proteins (such as full length proteins, or
biologically active fragments of full length proteins). In some
embodiments, a cocktail of mRNAs that express different proteins is
formulated into the same or different nucleic acid-lipid particles,
and the particles are administered to a mammal (e.g., a human)
requiring such treatment. In certain instances, a therapeutically
effective amount of the nucleic acid-lipid particles can be
administered to the mammal.
[0151] In certain embodiments, the present invention provides a
method for introducing one or more mRNA molecules into a cell by
contacting the cell with a nucleic acid-lipid particle described
herein (e.g., a SNALP formulation). In one particular embodiment,
the cell is a reticuloendothelial cell (e.g., monocyte or
macrophage), fibroblast cell, endothelial cell, or platelet
cell.
[0152] In some embodiments, the nucleic acid-lipid particles
described herein (e.g., SNALP) are administered by one of the
following routes of administration: oral, intranasal, intravenous,
intraperitoneal, intramuscular, intra-articular, intralesional,
intratracheal, subcutaneous, and intradermal. In particular
embodiments, the nucleic acid-lipid particles are administered
systemically, e.g., via enteral or parenteral routes of
administration.
[0153] In particular embodiments, the nucleic acid-lipid particles
of the invention (e.g., SNALP) can preferentially deliver a payload
such as an mRNA to the liver as compared to other tissues.
[0154] In certain aspects, the present invention provides methods
for expressing a protein in a mammal (e.g., human) in need thereof,
the method comprising administering to the mammal a therapeutically
effective amount of a nucleic acid-lipid particle (e.g., a SNALP
formulation) comprising one or more mRNAs that encode one or more
proteins under conditions that enable expression of the protein(s)
in the mammal. For example, in embodiments in which the mRNA
encodes a protein that is normally expressed in a healthy mammalian
subject, the level of expression of the protein encoded by the mRNA
encapsulated within the SNALP is at least 10%, or at least 20%, or
at least 30%, or at least 40%, or at least 50%, or at least 60%, or
at least 70%, or at least 80%, or at least 90%, or at least 100%,
or greater than 100%, of the level of the protein that is normally
expressed in a healthy mammalian subject.
[0155] In other aspects, the present invention provides methods for
treating, preventing, reducing the risk or likelihood of developing
(e.g., reducing the susceptibility to), delaying the onset of,
and/or ameliorating one or more symptoms associated with a disease
in a mammal (e.g., human) in need thereof, wherein the disease is
caused (at least in part) by reduced or aberrant expression of a
protein. The methods each include the step of administering to the
mammal a therapeutically effective amount of a nucleic acid-lipid
particle (e.g., a SNALP formulation) comprising one or more mRNA
molecules that encode a protein that is absent, or present at
reduced levels, within the treated subject.
[0156] mRNA molecules useful in the present invention may be
chemically modified or unmodified. Typically mRNA molecules are
chemically modified in order to reduce their ability to induce the
innate immune response of a cell into which the mRNA is
introduced.
[0157] Modifications to mRNA mRNA used in the practice of the
present invention can include one, two, or more than two nucleoside
modifications. In some embodiments, the modified mRNA exhibits
reduced degradation in a cell into which the mRNA is introduced,
relative to a corresponding unmodified mRNA.
[0158] In some embodiments, modified nucleosides include
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methy
1-pseudouridine, 4-thio-1-methy 1-pseudouridine, 2-thio-1-methy
1-pseudouridine, 1-methy 1-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine.
[0159] In some embodiments, modified nucleosides include
5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine,
N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,
5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,
pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0160] In other embodiments, modified nucleosides include
2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyl adenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0161] In specific embodiments, a modified nucleoside is
5'-0-(1-Thiophosphate)-Adenosine, 5'-0-(1-Thiophosphate)-Cytidine,
5'-0-(1-Thiophosphate)-Guanosine, 5'-0-(1-Thiophosphate)-Uridine or
5'-0-(1-Thiophosphate)-Pseudouridine. The .alpha.-thio substituted
phosphate moiety is provided to confer stability to RNA polymers
through the unnatural phosphorothioate backbone linkages.
Phosphorothioate RNA have increased nuclease resistance and
subsequently a longer half-life in a cellular environment.
Phosphorothioate linked nucleic acids are expected to also reduce
the innate immune response through weaker binding/activation of
cellular innate immune molecules.
[0162] In certain embodiments it is desirable to intracellularly
degrade a modified nucleic acid introduced into the cell, for
example if precise timing of protein production is desired. Thus,
the invention provides a modified nucleic acid containing a
degradation domain, which is capable of being acted on in a
directed manner within a cell.
[0163] In other embodiments, modified nucleosides include inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
Optional Components of the Modified Nucleic Acids
[0164] In further embodiments, the modified nucleic acids may
include other optional components, which can be beneficial in some
embodiments. These optional components include, but are not limited
to, untranslated regions, kozak sequences, intronic nucleotide
sequences, internal ribosome entry site (IRES), caps and polyA
tails. For example, a 5' untranslated region (UTR) and/or a 3' UTR
may be provided, wherein either or both may independently contain
one or more different nucleoside modifications. In such
embodiments, nucleoside modifications may also be present in the
translatable region. Also provided are nucleic acids containing a
Kozak sequence.
[0165] Additionally, provided are nucleic acids containing one or
more intronic nucleotide sequences capable of being excised from
the nucleic acid.
Untranslated Regions (UTRs)
[0166] Untranslated regions (UTRs) of a gene are transcribed but
not translated. The 5'UTR starts at the transcription start site
and continues to the start codon but does not include the start
codon; whereas, the 3'UTR starts immediately following the stop
codon and continues until the transcriptional termination signal.
There is growing body of evidence about the regulatory roles played
by the UTRs in terms of stability of the nucleic acid molecule and
translation. The regulatory features of a UTR can be incorporated
into the mRNA used in the present invention to increase the
stability of the molecule. The specific features can also be
incorporated to ensure controlled down-regulation of the transcript
in case they are misdirected to undesired organs sites.
5' Capping
[0167] The 5' cap structure of an mRNA is involved in nuclear
export, increasing mRNA stability and binds the mRNA Cap Binding
Protein (CBP), which is responsible for mRNA stability in the cell
and translation competency through the association of CBP with
poly(A) binding protein to form the mature cyclic mRNA species. The
cap further assists the removal of 5' proximal introns removal
during mRNA splicing.
[0168] Endogenous mRNA molecules may be 5'-end capped generating a
5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and the 5'-terminal transcribed sense nucleotide of the
mRNA molecule. This 5'-guanylate cap may then be methylated to
generate an N7-methyl-guanylate residue. The ribose sugars of the
terminal and/or anteterminal transcribed nucleotides of the 5' end
of the mRNA may optionally also be 2'-0-methylated. 5'-decapping
through hydrolysis and cleavage of the guanylate cap structure may
target a nucleic acid molecule, such as an mRNA molecule, for
degradation.
IRES Sequences
[0169] mRNA containing an internal ribosome entry site (IRES) are
also useful in the practice of the present invention. An IRES may
act as the sole ribosome binding site, or may serve as one of
multiple ribosome binding sites of an mRNA. An mRNA containing more
than one functional ribosome binding site may encode several
peptides or polypeptides that are translated independently by the
ribosomes ("multicistronic mRNA"). When mRNA are provided with an
IRES, further optionally provided is a second translatable region.
Examples of IRES sequences that can be used according to the
invention include without limitation, those from picornaviruses
(e.g. FMDV), pest viruses (CFFV), polio viruses (PV),
encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses
(FMDV), hepatitis C viruses (HCV), classical swine fever viruses
(CSFV), murine leukemia virus (MLV), simian immune deficiency
viruses (S1V) or cricket paralysis viruses (CrPV).
Poly-A Tails
[0170] During RNA processing, a long chain of adenine nucleotides
(poly-A tail) may be added to a polynucleotide such as an mRNA
molecules in order to increase stability. Immediately after
transcription, the 3' end of the transcript may be cleaved to free
a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that can be between 100 and 250 residues long.
[0171] Generally, the length of a poly-A tail is greater than 30
nucleotides in length. In another embodiment, the poly-A tail is
greater than 35 nucleotides in length (e.g., at least or greater
than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500,
and 3,000 nucleotides).
[0172] In this context the poly-A tail may be 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% greater in length than the modified mRNA.
The poly-A tail may also be designed as a fraction of modified
nucleic acids to which it belongs. In this context, the poly-A tail
may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total
length of the modified mRNA or the total length of the modified
mRNA minus the poly-A tail.
Generating mRNA Molecules
[0173] Methods for isolating RNA, synthesizing RNA, hybridizing
nucleic acids, making and screening cDNA libraries, and performing
PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene,
25:263-269 (1983); Sambrook et al., Molecular Cloning, A Laboratory
Manual (2nd ed. 1989)); as are PCR methods (see, U.S. Pat. Nos.
4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and
Applications (Innis et al., eds, 1990)). Expression libraries are
also well known to those of skill in the art. Additional basic
texts disclosing the general methods of use in this invention
include Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Current Protocols in Molecular Biology (Ausubel et al.,
eds., 1994). The disclosures of these references are herein
incorporated by reference in their entirety for all purposes.
Generating siRNA Molecules
[0174] siRNA can be provided in several forms including, e.g., as
one or more isolated small-interfering RNA (siRNA) duplexes, as
longer double-stranded RNA (dsRNA), or as siRNA or dsRNA
transcribed from a transcriptional cassette in a DNA plasmid. In
some embodiments, siRNA may be produced enzymatically or by
partial/total organic synthesis, and modified ribonucleotides can
be introduced by in vitro enzymatic or organic synthesis. In
certain instances, each strand is prepared chemically. Methods of
synthesizing RNA molecules are known in the art, e.g., the chemical
synthesis methods as described in Verma and Eckstein (1998) or as
described herein.
[0175] Methods for isolating RNA, synthesizing RNA, hybridizing
nucleic acids, making and screening cDNA libraries, and performing
PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene,
25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra),
as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202;
PCR Protocols: A Guide to Methods and Applications (Innis et al.,
eds, 1990)). Expression libraries are also well known to those of
skill in the art. Additional basic texts disclosing the general
methods of use in this invention include Sambrook et al., Molecular
Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); and Current
Protocols in Molecular Biology (Ausubel et al., eds., 1994). The
disclosures of these references are herein incorporated by
reference in their entirety for all purposes.
[0176] Preferably, siRNA are chemically synthesized. The
oligonucleotides that comprise the siRNA molecules of the invention
can be synthesized using any of a variety of techniques known in
the art, such as those described in Usman et al., J. Am. Chem.
Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433
(1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and
Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of
oligonucleotides makes use of common nucleic acid protecting and
coupling groups, such as dimethoxytrityl at the 5'-end and
phosphoramidites at the 3'-end. As a non-limiting example, small
scale syntheses can be conducted on an Applied Biosystems
synthesizer using a 0.2 .mu.mol scale protocol. Alternatively,
syntheses at the 0.2 .mu.mol scale can be performed on a 96-well
plate synthesizer from Protogene (Palo Alto, Calif.). However, a
larger or smaller scale of synthesis is also within the scope of
this invention. Suitable reagents for oligonucleotide synthesis,
methods for RNA deprotection, and methods for RNA purification are
known to those of skill in the art.
[0177] siRNA molecules can be assembled from two distinct
oligonucleotides, wherein one oligonucleotide comprises the sense
strand and the other comprises the antisense strand of the siRNA.
For example, each strand can be synthesized separately and joined
together by hybridization or ligation following synthesis and/or
deprotection.
Lipid Particles
[0178] In certain aspects, the present invention provides lipid
particles comprising one or more therapeutic mRNA molecules
encapsulated within the lipid particles.
[0179] In some embodiments, the mRNA is fully encapsulated within
the lipid portion of the lipid particle such that the mRNA in the
lipid particle is resistant in aqueous solution to nuclease
degradation. In other embodiments, the lipid particles described
herein are substantially non-toxic to mammals such as humans. The
lipid particles of the invention typically have a mean diameter of
from about 30 nm to about 150 nm, from about 40 nm to about 150 nm,
from about 50 nm to about 150 nm, from about 60 nm to about 130 nm,
from about 70 nm to about 110 nm, or from about 70 to about 90 nm.
The lipid particles of the invention also typically have a
lipid:mRNA ratio (mass/mass ratio) of from about 1:1 to about
100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1,
from about 3:1 to about 20:1, from about 5:1 to about 15:1, or from
about 5:1 to about 10:1, or from about 10:1 to about 14:1, or from
about 9:1 to about 20:1. In one embodiment, the lipid particles of
the invention have a lipid: mRNA ratio (mass/mass ratio) of about
12:1, such as 12:1. In another embodiment, the lipid particles of
the invention have a lipid: mRNA ratio (mass/mass ratio) of about
13:1, such as 13:1.
[0180] In preferred embodiments, the lipid particles of the
invention are serum-stable nucleic acid-lipid particles (SNALP)
which comprise an mRNA, a cationic lipid (e.g., one or more
cationic lipids of Formula I-III or salts thereof as set forth
herein), a phospholipid, and a conjugated lipid that inhibits
aggregation of the particles (e.g., one or more PEG-lipid
conjugates). The lipid particles can also include cholesterol. The
SNALP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
unmodified and/or modified mRNA that express one or more
polypeptides. Nucleic acid-lipid particles and their method of
preparation are described in, e.g., U.S. Pat. Nos. 5,753,613;
5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and
6,320,017; and PCT Publication No. WO 96/40964, the disclosures of
which are each herein incorporated by reference in their entirety
for all purposes.
[0181] In the nucleic acid-lipid particles of the invention, the
mRNA may be fully encapsulated within the lipid portion of the
particle, thereby protecting the nucleic acid from nuclease
degradation. In preferred embodiments, a SNALP comprising an mRNA
is fully encapsulated within the lipid portion of the particle,
thereby protecting the nucleic acid from nuclease degradation. In
certain instances, the mRNA in the SNALP is not substantially
degraded after exposure of the particle to a nuclease at 37.degree.
C. for at least about 20, 30, 45, or 60 minutes. In certain other
instances, the mRNA in the SNALP is not substantially degraded
after incubation of the particle in serum at 37.degree. C. for at
least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6,
7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36
hours. In other embodiments, the mRNA is complexed with the lipid
portion of the particle. One of the benefits of the formulations of
the present invention is that the nucleic acid-lipid particle
compositions are substantially non-toxic to mammals such as
humans.
[0182] The term "fully encapsulated" indicates that the nucleic
acid (mRNA) in the nucleic acid-lipid particle is not significantly
degraded after exposure to serum or a nuclease assay that would
significantly degrade free RNA. In a fully encapsulated system,
preferably less than about 25% of the nucleic acid in the particle
is degraded in a treatment that would normally degrade 100% of free
nucleic acid, more preferably less than about 10%, and most
preferably less than about 5% of the nucleic acid in the particle
is degraded. "Fully encapsulated" also indicates that the nucleic
acid-lipid particles are serum-stable, that is, that they do not
rapidly decompose into their component parts upon in vivo
administration.
[0183] In the context of nucleic acids, full encapsulation may be
determined by performing a membrane-impermeable fluorescent dye
exclusion assay, which uses a dye that has enhanced fluorescence
when associated with nucleic acid. Specific dyes such as
OliGreen.RTM. and RiboGreen.RTM. (Invitrogen Corp.; Carlsbad,
Calif.) are available for the quantitative determination of plasmid
DNA, single-stranded deoxyribonucleotides, and/or single- or
double-stranded ribonucleotides. Encapsulation is determined by
adding the dye to a liposomal formulation, measuring the resulting
fluorescence, and comparing it to the fluorescence observed upon
addition of a small amount of nonionic detergent.
Detergent-mediated disruption of the liposomal bilayer releases the
encapsulated nucleic acid, allowing it to interact with the
membrane-impermeable dye. Nucleic acid encapsulation may be
calculated as E=(I.sub.o-I)/I.sub.o, where I and I.sub.o refer to
the fluorescence intensities before and after the addition of
detergent (see, Wheeler et al., Gene Ther., 6:271-281 (1999)).
[0184] In other embodiments, the present invention provides a
nucleic acid-lipid particle (e.g., SNALP) composition comprising a
plurality of nucleic acid-lipid particles.
[0185] In some instances, the SNALP composition comprises mRNA that
is fully encapsulated within the lipid portion of the particles,
such that from about 30% to about 100%, from about 40% to about
100%, from about 50% to about 100%, from about 60% to about 100%,
from about 70% to about 100%, from about 80% to about 100%, from
about 90% to about 100%, from about 30% to about 95%, from about
40% to about 95%, from about 50% to about 95%, from about 60% to
about 95%, from about 70% to about 95%, from about 80% to about
95%, from about 85% to about 95%, from about 90% to about 95%, from
about 30% to about 90%, from about 40% to about 90%, from about 50%
to about 90%, from about 60% to about 90%, from about 70% to about
90%, from about 80% to about 90%, or at least about 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range
therein) of the particles have the mRNA encapsulated therein.
[0186] Depending on the intended use of the lipid particles of the
invention, the proportions of the components can be varied and the
delivery efficiency of a particular formulation can be measured
using, e.g., an endosomal release parameter (ERP) assay.
Cationic Lipids
[0187] Any of a variety of cationic lipids or salts thereof may be
used in the lipid particles of the present invention (e.g., SNALP),
either alone or in combination with one or more other cationic
lipid species or non-cationic lipid species. The cationic lipids
include the (R) and/or (S) enantiomers thereof. Typically, the
cationic lipids contain a portion (i.e. a hydrophobic moiety) that
comprises unsaturated and/or saturated hydrocarbon chains.
[0188] In one aspect, cationic lipids of Formula I having the
following structure are useful in the present invention:
##STR00002##
[0189] or salts thereof, wherein:
[0190] R.sup.1 and R.sup.2 are either the same or different and are
independently hydrogen (H) or an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, or R.sup.1 and R.sup.2 may join to form an optionally
substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2
heteroatoms selected from the group consisting of nitrogen (N),
oxygen (O), and mixtures thereof;
[0191] R.sup.3 is either absent or is hydrogen (H) or a
C.sub.1-C.sub.6 alkyl to provide a quaternary amine; R.sup.4 and
R.sup.5 are either the same or different and are independently an
optionally substituted C.sub.10-C.sub.24 alkyl, C.sub.10-C.sub.24
alkenyl, C.sub.10-C.sub.24 alkynyl, or C.sub.10-C.sub.24 acyl,
wherein at least one of R.sup.4 and R.sup.5 comprises at least two
sites of unsaturation; and
[0192] n is 0, 1, 2, 3, or 4.
[0193] In some embodiments, R.sup.1 and R.sup.2 are independently
an optionally substituted C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, or C.sub.2-C.sub.4 alkynyl. In one preferred embodiment,
R.sup.1 and R.sup.2 are both methyl groups. In other preferred
embodiments, n is 1 or 2. In other embodiments, R.sup.3 is absent
when the pH is above the pK.sub.a of the cationic lipid and R.sup.3
is hydrogen when the pH is below the pK.sub.a of the cationic lipid
such that the amino head group is protonated. In an alternative
embodiment, R.sup.3 is an optionally substituted C.sub.1-C.sub.4
alkyl to provide a quaternary amine. In further embodiments,
R.sup.4 and R.sup.5 are independently an optionally substituted
C.sub.12-C.sub.20 or C.sub.14-C.sub.22 alkyl, C.sub.12-C.sub.20 or
C.sub.14-C.sub.22 alkenyl, C.sub.12-C.sub.20 or C.sub.14-C.sub.22
alkynyl, or C.sub.12-C.sub.20 or C.sub.14-C.sub.22 acyl, wherein at
least one of R.sup.4 and R.sup.5 comprises at least two sites of
unsaturation.
[0194] In certain embodiments, R.sup.4 and R.sup.5 are
independently selected from the group consisting of a dodecadienyl
moiety, a tetradecadienyl moiety, a hexadecadienyl moiety, an
octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl
moiety, a tetradectrienyl moiety, a hexadecatrienyl moiety, an
octadecatrienyl moiety, an icosatrienyl moiety, an arachidonyl
moiety, and a docosahexaenoyl moiety, as well as acyl derivatives
thereof (e.g., linoleoyl, linolenoyl, .gamma.-linolenoyl, etc.). In
some instances, one of R.sup.4 and R.sup.5 comprises a branched
alkyl group (e.g., a phytanyl moiety) or an acyl derivative thereof
(e.g., a phytanoyl moiety). In certain instances, the
octadecadienyl moiety is a linoleyl moiety. In certain other
instances, the octadecatrienyl moiety is a linolenyl moiety or a
.gamma.-linolenyl moiety. In certain embodiments, R.sup.4 and
R.sup.5 are both linoleyl moieties, linolenyl moieties, or
.gamma.-linolenyl moieties. In particular embodiments, the cationic
lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),
1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP), or
mixtures thereof.
[0195] In some embodiments, the cationic lipid of Formula I forms a
salt (preferably a crystalline salt) with one or more anions. In
one particular embodiment, the cationic lipid of Formula I is the
oxalate (e.g., hemioxalate) salt thereof, which is preferably a
crystalline salt.
[0196] The synthesis of cationic lipids such as DLinDMA and
DLenDMA, as well as additional cationic lipids, is described in
U.S. Patent Publication No. 20060083780, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.
The synthesis of cationic lipids such as C2-DLinDMA and C2-DLinDAP,
as well as additional cationic lipids, is described in
international patent application number WO2011/000106 the
disclosure of which is herein incorporated by reference in its
entirety for all purposes.
[0197] In another aspect, cationic lipids of Formula II having the
following structure (or salts thereof) are useful in the present
invention:
##STR00003##
wherein R.sup.1 and R.sup.2 are either the same or different and
are independently an optionally substituted C.sub.12-C.sub.24
alkyl, C.sub.12-C.sub.24 alkenyl, C.sub.12-C.sub.24 alkynyl, or
C.sub.12-C.sub.24 acyl; R.sup.3 and R.sup.4 are either the same or
different and are independently an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, or R.sup.3 and R.sup.4 may join to form an optionally
substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2
heteroatoms chosen from nitrogen and oxygen; R.sup.5 is either
absent or is hydrogen (H) or a C.sub.1-C.sub.6 alkyl to provide a
quaternary amine; m, n, and p are either the same or different and
are independently either 0, 1, or 2, with the proviso that m, n,
and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z
are either the same or different and are independently O, S, or NH.
In a preferred embodiment, q is 2.
[0198] In some embodiments, the cationic lipid of Formula II is
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane,
2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane,
2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane,
2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane,
2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane,
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane,
2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane,
2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane,
2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane,
2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride,
2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane,
2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane, or mixtures
thereof. In preferred embodiments, the cationic lipid of Formula II
is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane.
[0199] In some embodiments, the cationic lipid of Formula II forms
a salt (preferably a crystalline salt) with one or more anions. In
one particular embodiment, the cationic lipid of Formula II is the
oxalate (e.g., hemioxalate) salt thereof, which is preferably a
crystalline salt.
[0200] The synthesis of cationic lipids such as
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane, as well as
additional cationic lipids, is described in PCT Publication No. WO
09/086558, the disclosure of which is herein incorporated by
reference in its entirety for all purposes, and in PCT Application
No. PCT/US2009/060251, entitled "Improved Amino Lipids and Methods
for the Delivery of Nucleic Acids," the disclosure of which is
incorporated herein by reference in its entirety for all
purposes.
[0201] In a further aspect, cationic lipids of Formula III having
the following structure are useful in the present invention:
##STR00004##
or salts thereof, wherein: R.sup.1 and R.sup.2 are either the same
or different and are independently an optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, or R.sup.1 and R.sup.2 may join to form an optionally
substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2
heteroatoms selected from the group consisting of nitrogen (N),
oxygen (O), and mixtures thereof; R.sup.3 is either absent or is
hydrogen (H) or a C.sub.1-C.sub.6 alkyl to provide a quaternary
amine; R.sup.4 and R.sup.5 are either absent or present and when
present are either the same or different and are independently an
optionally substituted C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10
alkenyl; and n is 0, 1, 2, 3, or 4.
[0202] In some embodiments, R.sup.1 and R.sup.2 are independently
an optionally substituted C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, or C.sub.2-C.sub.4 alkynyl. In a preferred embodiment,
R.sup.1 and R.sup.2 are both methyl groups. In another preferred
embodiment, R.sup.4 and R.sup.5 are both butyl groups. In yet
another preferred embodiment, n is 1. In other embodiments, R.sup.3
is absent when the pH is above the pK.sub.a of the cationic lipid
and R.sup.3 is hydrogen when the pH is below the pK.sub.a of the
cationic lipid such that the amino head group is protonated. In an
alternative embodiment, R.sup.3 is an optionally substituted
C.sub.1-C.sub.4 alkyl to provide a quaternary amine. In further
embodiments, R.sup.4 and R.sup.5 are independently an optionally
substituted C.sub.2-C.sub.6 or C.sub.2-C.sub.4 alkyl or
C.sub.2-C.sub.6 or C.sub.2-C.sub.4 alkenyl.
[0203] In an alternative embodiment, the cationic lipid of Formula
III comprises ester linkages between the amino head group and one
or both of the alkyl chains. In some embodiments, the cationic
lipid of Formula III forms a salt (preferably a crystalline salt)
with one or more anions. In one particular embodiment, the cationic
lipid of Formula III is the oxalate (e.g., hemioxalate) salt
thereof, which is preferably a crystalline salt.
[0204] Although each of the alkyl chains in Formula III contains
cis double bonds at positions 6, 9, and 12 (i.e.,
cis,cis,cis-.DELTA..sup.6,.DELTA..sup.9,.DELTA..sup.12) in an
alternative embodiment, one, two, or three of these double bonds in
one or both alkyl chains may be in the trans configuration.
[0205] In one embodiment, the cationic lipid of Formula III has the
structure:
##STR00005##
[0206] The synthesis of cationic lipids such as .gamma.-DLenDMA, as
well as additional cationic lipids, is described in International
Patent Application WO2011/000106, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0207] In particular embodiments, a cationic lipid having the
following structure is useful in the present invention:
##STR00006##
[0208] The synthesis of cationic lipids such as compound 7, as well
as additional cationic lipids, are described in U.S. patent serial
number 8,158,601, and in International Patent Application serial
number PCT/GB2011/000723, the disclosures of which are herein
incorporated by reference in their entirety for all purposes.
[0209] Examples of other cationic lipids or salts thereof which may
be included in the lipid particles of the present invention
include, but are not limited to, cationic lipids such as those
described in WO2011/000106, the disclosure of which is herein
incorporated by reference in its entirety for all purposes, as well
as cationic lipids such as N,N-dioleyl-N,N-dimethylammonium
chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), 3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
(DC-Chol),
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE),
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-
iumtrifluoroacetate (DO SPA), dioctadecylamidoglycyl spermine
(DOGS),
3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA),
2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy-1-(cis,cis-9',1--
2'-octadecadienoxy)propane (CpLinDMA),
N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),
1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),
1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanedio (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 1,2-dioeylcarbamoyloxy-3-dimethylaminopropane
(DO-C-DAP), 1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),
1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),
dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA; also
known as DLin-M-K-DMA or DLin-M-DMA), and mixtures thereof.
Additional cationic lipids or salts thereof which may be included
in the lipid particles of the present invention are described in
U.S. Patent Publication No. 20090023673, the disclosure of which is
herein incorporated by reference in its entirety for all
purposes.
[0210] In another embodiment, a trialkyl cationic lipid can be used
to prepare the lipid particles described herein. Such trialkyl
cationic lipids typically comprise three saturated or unsaturated
hydrocarbon chains having six or more carbons in each chain.
Trialkyl cationic lipids that can be incorporated into the
compositions described herein can be prepared as described in
International Patent Application Publication Number WO
2013/126803.
[0211] For example, a trialkyl cationic lipid of the following
Formula B can be used to make lipid particles of the present
invention:
X-A-Y--Z; (Formula B)
[0212] or salts thereof, wherein:
[0213] X is --N(H)R or --NR.sub.2;
[0214] A is absent, C.sub.1 to C.sub.6alkyl, C.sub.2 to
C.sub.6alkenyl, or C.sub.2 to C.sub.6alkynyl, which C.sub.1 to
C.sub.6alkyl, C.sub.2 to C.sub.6alkenyl, and C.sub.2 to
C.sub.6alkynyl is optionally substituted with one or more groups
independently selected from oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein each alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle;
[0215] Y is selected from the group consisting of absent,
--C(.dbd.O)--, --O--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--N(R.sup.b)C(.dbd.O)--, --C(.dbd.O)N(R.sup.b)--,
--N(R.sup.b)C(.dbd.O)O--, and --OC(.dbd.O)N(R.sup.b)--;
[0216] Z is a hydrophobic moiety comprising three chains wherein
each of the chains is independently selected from C.sub.8 to
C.sub.11alkyl, C.sub.8 to C.sub.11alkenyl, and C.sub.8 to
C.sub.11alkynyl, which C.sub.8 to C.sub.11alkyl, C.sub.8 to
C.sub.11alkenyl, and C.sub.8 to C.sub.11alkynyl is optionally
substituted with one or more groups independently selected from
oxo, halogen, heterocycle, --CN, --OR.sup.x, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle;
[0217] each R is independently alkyl, alkenyl, or alkynyl, that is
optionally substituted with one or more groups independently
selected from oxo, halogen, heterocycle, --CN, --OR.sup.x,
--NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle; and
[0218] each R.sup.b is H or C.sub.1 to C.sub.6alkyl.
[0219] In some embodiments, Z in Formula B has the structure:
##STR00007##
wherein, R.sub.1, R.sub.2, and R.sub.3 are each independently
C.sub.8 to C.sub.11alkyl, C.sub.8 to C.sub.11alkenyl, or C.sub.8 to
C.sub.11alkynyl, which C.sub.8 to C.sub.11alkyl, C.sub.8 to
C.sub.11alkenyl, and C.sub.8 to C.sub.11alkynyl is optionally
substituted with one or more groups independently selected from
oxo, halogen, heterocycle, --CN, --OR.sup.x, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle.
[0220] In another embodiment, cationic lipids of the following
Formula C are used to make lipid particles of the present
invention:
X-A-Y--Z.sup.1; (Formula C)
[0221] or salts thereof, wherein:
[0222] X is --N(H)R or --NR.sub.2;
[0223] A is absent, C.sub.1 to C.sub.6alkyl, C.sub.2 to
C.sub.6alkenyl, or C.sub.2 to C.sub.6alkynyl, which C.sub.1 to
C.sub.6alkyl, C.sub.2 to C.sub.6alkenyl, and C.sub.2 to
C.sub.6alkynyl is optionally substituted with one or more groups
independently selected from oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein each alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle;
[0224] Y is selected from the group consisting of absent,
--C(.dbd.O)--, --O--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--N(R.sup.b)C(.dbd.O)--, --C(.dbd.O)N(R.sup.b)--,
--N(R.sup.b)C(.dbd.O)O--, and --OC(.dbd.O)N(R.sup.b)--;
[0225] Z.sup.1 is a C.sub.1 to C.sub.6alkyl that is substituted
with three or four R.sup.x groups, wherein each R.sup.x is
independently selected from C.sub.6 to C.sub.11alkyl, C.sub.6 to
C.sub.11alkenyl, and C.sub.6 to C.sub.11alkynyl, which C.sub.6 to
C.sub.11alkyl, C.sub.6 to C.sub.11alkenyl, and C.sub.6 to
C.sub.11alkynyl is optionally substituted with one or more groups
independently selected from oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle;
[0226] each R is independently alkyl, alkenyl, or alkynyl, that is
optionally substituted with one or more groups independently
selected from oxo, halogen, heterocycle, --CN, --OR.sup.x,
--NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle; and
[0227] each R.sup.b is H or C.sub.1 to C.sub.6alkyl.
[0228] In some embodiments, Z.sup.1 in Formula C has the
structure:
##STR00008##
wherein, R.sub.1, R.sub.2, and R.sub.3 are each independently
C.sub.8 to C.sub.11alkyl, C.sub.8 to C.sub.11alkenyl, or C.sub.8 to
C.sub.11alkynyl, which C.sub.8 to C.sub.11alkyl, C.sub.8 to
C.sub.11alkenyl, and C.sub.8 to C.sub.11alkynyl is optionally
substituted with one or more groups independently selected from
oxo, halogen, heterocycle, --CN, --OR.sup.x, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle.
[0229] In some embodiments, Z.sup.1 in Formula C has the
structure:
##STR00009##
wherein one of R.sup.1z and R.sup.2z is selected from the group
consisting of:
##STR00010##
and the other of R.sup.1z and R.sup.2z is selected from the group
consisting of:
##STR00011##
wherein each R.sup.3z, R.sup.4z, R.sup.5z, R.sup.6z, and R.sup.7z
is independently selected from C.sub.6 to C.sub.11alkyl, C.sub.6 to
C.sub.11alkenyl, and C.sub.6 to C.sub.11alkynyl, which C.sub.6 to
C.sub.11alkyl, C.sub.6 to C.sub.11alkenyl, and C.sub.6 to
C.sub.11alkynyl is optionally substituted with one or more groups
independently selected from oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x', and --SO.sub.n'NR.sup.x'R.sup.y', wherein n'
is 0, 1, or 2, and R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle.
[0230] In some embodiments, Z' in Formula C has the structure:
##STR00012##
wherein each R.sup.3z, R.sup.4z, R.sup.5z, and R.sup.6z is
independently selected from C.sub.6 to C.sub.11alkyl, C.sub.6 to
C.sub.11alkenyl, and C.sub.6 to C.sub.11alkynyl, which C.sub.6 to
C.sub.11alkyl, C.sub.6 to C.sub.11alkenyl, and C.sub.6 to
C.sub.11alkynyl is optionally substituted with one or more groups
independently selected from oxo, halogen, heterocycle, --CN,
--OR.sup.x, --NR.sup.xR.sup.y, --NR.sup.xC(.dbd.O)R.sup.y,
--NR.sup.xSO.sub.2R.sup.y, --C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x, and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1, or 2, and R.sup.x and
R.sup.y are each independently hydrogen, alkyl, or heterocycle,
wherein any alkyl and heterocycle of R.sup.x and R.sup.y may be
further substituted with one or more groups independently selected
from oxo, halogen, --OH, --CN, alkyl, --OR.sup.x', heterocycle,
--NR.sup.x'R.sup.y', --NR.sup.x'C(.dbd.O)R.sup.y',
--NR.sup.x'SO.sub.2R.sup.y', --C(.dbd.O)R.sup.x',
--C(.dbd.O)OR.sup.x', --C(.dbd.O)NR.sup.x'R.sup.y',
--SO.sub.n'R.sup.x' and --SO.sub.n'NR.sup.x'R.sup.y', wherein n' is
0, 1, or 2, and R.sup.x' and R.sup.y' are each independently
hydrogen, alkyl, or heterocycle.
[0231] In some embodiments the cationic lipid is selected from the
group consisting of:
##STR00013##
and salts thereof.
[0232] The synthesis of cationic lipids such as CLinDMA, as well as
additional cationic lipids, is described in U.S. Patent Publication
No. 20060240554, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. The synthesis of
cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA, DLinDAP,
DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP,
DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is
described in PCT Publication No. WO 09/086558, the disclosure of
which is herein incorporated by reference in its entirety for all
purposes. The synthesis of cationic lipids such as DO-C-DAP, DMDAP,
DOTAP.Cl, DLin-M-C2-DMA, as well as additional cationic lipids, is
described in PCT Application No. PCT/US2009/060251, entitled
"Improved Amino Lipids and Methods for the Delivery of Nucleic
Acids," filed Oct. 9, 2009, the disclosure of which is incorporated
herein by reference in its entirety for all purposes. The synthesis
of a number of other cationic lipids and related analogs has been
described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;
5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO
96/10390, the disclosures of which are each herein incorporated by
reference in their entirety for all purposes. Additionally, a
number of commercial preparations of cationic lipids can be used,
such as, e.g., LIPOFECTIN.RTM. (including DOTMA and DOPE, available
from Invitrogen); LIPOFECTAMINE.RTM. (including DOSPA and DOPE,
available from Invitrogen); and TRANSFECTAM.RTM. (including DOGS,
available from Promega Corp.).
[0233] In some embodiments, the cationic lipid comprises from about
50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %,
from about 50 mol % to about 80 mol %, from about 50 mol % to about
75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol
% to about 65 mol %, from about 50 mol % to about 60 mol %, from
about 55 mol % to about 65 mol %, or from about 55 mol % to about
70 mol % (or any fraction thereof or range therein) of the total
lipid present in the particle. In particular embodiments, the
cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol
%, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60
mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any
fraction thereof) of the total lipid present in the particle.
[0234] In other embodiments, the cationic lipid comprises from
about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol
%, from about 10 mol % to about 50 mol %, from about 20 mol % to
about 50 mol %, from about 20 mol % to about 40 mol %, from about
30 mol % to about 40 mol %, or about 40 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle.
[0235] Additional percentages and ranges of cationic lipids
suitable for use in the lipid particles of the present invention
are described in PCT Publication No. WO 09/127060, U.S. Published
Application No. US 2011/0071208, PCT Publication No. WO2011/000106,
and U.S. Published Application No. US 2011/0076335, the disclosures
of which are herein incorporated by reference in their entirety for
all purposes.
[0236] It should be understood that the percentage of cationic
lipid present in the lipid particles of the invention is a target
amount, and that the actual amount of cationic lipid present in the
formulation may vary, for example, by .+-.5 mol %. For example, in
the 1:57 lipid particle (e.g., SNALP) formulation, the target
amount of cationic lipid is 57.1 mol %, but the actual amount of
cationic lipid may be .+-.5 mol %, .+-.4 mol %, .+-.3 mol %, .+-.2
mol %, 1 mol %, 0.75 mol %, 0.5 mol %, .+-.0.25 mol %, or .+-.0.1
mol % of that target amount, with the balance of the formulation
being made up of other lipid components (adding up to 100 mol % of
total lipids present in the particle).
[0237] By way of non-limiting example, cationic lipids include the
following compounds:
##STR00014## [0238]
N,N-dimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-amine
(5)
[0238] ##STR00015## [0239]
2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)-N,N-dimethyle-
thanamine (6)
[0239] ##STR00016## [0240]
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (7)
[0240] ##STR00017## [0241]
3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethy-
lpropan-1-amine (8)
[0241] ##STR00018## [0242] (Z)-12-((Z)-dec-4-enyl)docos-16-en-11-yl
5-(dimethylamino)pentanoate (53)
[0242] ##STR00019## [0243]
(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl
6-(dimethylamino)hexanoate (11)
[0243] ##STR00020## [0244]
(6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl
5-(dimethylamino)pentanoate (13)
[0244] ##STR00021## [0245] 12-decyldocosan-11-yl
5-(dimethylamino)pentanoate (14)
##STR00022## ##STR00023## ##STR00024## ##STR00025##
[0245] Non-Cationic Lipids
[0246] The non-cationic lipids used in the lipid particles of the
invention (e.g., SNALP) can be any of a variety of neutral
uncharged, zwitterionic, or anionic lipids capable of producing a
stable complex.
[0247] Non-limiting examples of non-cationic lipids include
phospholipids such as lecithin, phosphatidylethanolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM),
cephalin, cardiolipin, phosphatidic acid, cerebrosides,
dicetylphosphate, di stearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoyl-phosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
palmitoyloleyol-phosphatidylglycerol (POPG),
dioleoylphosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl-phosphatidylethanolamine (DPPE),
dimyristoyl-phosphatidylethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE),
monomethyl-phosphatidylethanolamine,
dimethyl-phosphatidylethanolamine,
dielaidoyl-phosphatidylethanolamine (DEPE),
stearoyloleoyl-phosphatidylethanolamine (SOPE),
lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and
mixtures thereof. Other diacylphosphatidylcholine and
diacylphosphatidylethanolamine phospholipids can also be used. The
acyl groups in these lipids are preferably acyl groups derived from
fatty acids having C.sub.10-C.sub.24 carbon chains, e.g., lauroyl,
myristoyl, palmitoyl, stearoyl, or oleoyl.
[0248] Additional examples of non-cationic lipids include sterols
such as cholesterol and derivatives thereof. Non-limiting examples
of cholesterol derivatives include polar analogues such as
5.alpha.-cholestanol, 5.beta.-coprostanol,
cholesteryl-(2'-hydroxy)-ethyl ether,
cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol;
non-polar analogues such as 5.alpha.-cholestane, cholestenone,
5.alpha.-cholestanone, 5.beta.-cholestanone, and cholesteryl
decanoate; and mixtures thereof. In preferred embodiments, the
cholesterol derivative is a polar analogue such as
cholesteryl-(4'-hydroxy)-butyl ether. The synthesis of
cholesteryl-(2'-hydroxy)-ethyl ether is described in PCT
Publication No. WO 09/127060, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0249] In some embodiments, the non-cationic lipid present in the
lipid particles (e.g., SNALP) comprises or consists of a mixture of
one or more phospholipids and cholesterol or a derivative thereof.
In other embodiments, the non-cationic lipid present in the lipid
particles (e.g., SNALP) comprises or consists of one or more
phospholipids, e.g., a cholesterol-free lipid particle formulation.
In yet other embodiments, the non-cationic lipid present in the
lipid particles (e.g., SNALP) comprises or consists of cholesterol
or a derivative thereof, e.g., a phospholipid-free lipid particle
formulation.
[0250] Other examples of non-cationic lipids suitable for use in
the present invention include nonphosphorous containing lipids such
as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl
palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl
myristate, amphoteric acrylic polymers, triethanolamine-lauryl
sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides,
dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and
the like.
[0251] In some embodiments, the non-cationic lipid comprises from
about 10 mol % to about 60 mol %, from about 20 mol % to about 55
mol %, from about 20 mol % to about 45 mol %, from about 20 mol %
to about 40 mol %, from about 25 mol % to about 50 mol %, from
about 25 mol % to about 45 mol %, from about 30 mol % to about 50
mol %, from about 30 mol % to about 45 mol %, from about 30 mol %
to about 40 mol %, from about 35 mol % to about 45 mol %, from
about 37 mol % to about 42 mol %, or about 35 mol %, 36 mol %, 37
mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %,
44 mol %, or 45 mol % (or any fraction thereof or range therein) of
the total lipid present in the particle.
[0252] In embodiments where the lipid particles contain a mixture
of phospholipid and cholesterol or a cholesterol derivative, the
mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55
mol %, or 60 mol % of the total lipid present in the particle.
[0253] In some embodiments, the phospholipid component in the
mixture may comprise from about 2 mol % to about 20 mol %, from
about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol
%, from about 4 mol % to about 15 mol %, or from about 4 mol % to
about 10 mol % (or any fraction thereof or range therein) of the
total lipid present in the particle. In certain preferred
embodiments, the phospholipid component in the mixture comprises
from about 5 mol % to about 10 mol %, from about 5 mol % to about 9
mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to
about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol
%, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle. As a non-limiting example, a 1:57 lipid particle
formulation comprising a mixture of phospholipid and cholesterol
may comprise a phospholipid such as DPPC or DSPC at about 7 mol %
(or any fraction thereof), e.g., in a mixture with cholesterol or a
cholesterol derivative at about 34 mol % (or any fraction thereof)
of the total lipid present in the particle. As another non-limiting
example, a 7:54 lipid particle formulation comprising a mixture of
phospholipid and cholesterol may comprise a phospholipid such as
DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a
mixture with cholesterol or a cholesterol derivative at about 32
mol % (or any fraction thereof) of the total lipid present in the
particle.
[0254] In other embodiments, the cholesterol component in the
mixture may comprise from about 25 mol % to about 45 mol %, from
about 25 mol % to about 40 mol %, from about 30 mol % to about 45
mol %, from about 30 mol % to about 40 mol %, from about 27 mol %
to about 37 mol %, from about 25 mol % to about 30 mol %, or from
about 35 mol % to about 40 mol % (or any fraction thereof or range
therein) of the total lipid present in the particle. In certain
preferred embodiments, the cholesterol component in the mixture
comprises from about 25 mol % to about 35 mol %, from about 27 mol
% to about 35 mol %, from about 29 mol % to about 35 mol %, from
about 30 mol % to about 35 mol %, from about 30 mol % to about 34
mol %, from about 31 mol % to about 33 mol %, or about 30 mol %, 31
mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle. Typically, a 1:57 lipid particle formulation comprising a
mixture of phospholipid and cholesterol may comprise cholesterol or
a cholesterol derivative at about 34 mol % (or any fraction
thereof), e.g., in a mixture with a phospholipid such as DPPC or
DSPC at about 7 mol % (or any fraction thereof) of the total lipid
present in the particle. Typically, a 7:54 lipid particle
formulation comprising a mixture of phospholipid and cholesterol
may comprise cholesterol or a cholesterol derivative at about 32
mol % (or any fraction thereof), e.g., in a mixture with a
phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction
thereof) of the total lipid present in the particle.
[0255] In embodiments where the lipid particles are
phospholipid-free, the cholesterol or derivative thereof may
comprise up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol
%, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in
the particle.
[0256] In some embodiments, the cholesterol or derivative thereof
in the phospholipid-free lipid particle formulation may comprise
from about 25 mol % to about 45 mol %, from about 25 mol % to about
40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol
% to about 40 mol %, from about 31 mol % to about 39 mol %, from
about 32 mol % to about 38 mol %, from about 33 mol % to about 37
mol %, from about 35 mol % to about 45 mol %, from about 30 mol %
to about 35 mol %, from about 35 mol % to about 40 mol %, or about
30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol
%, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle. As a non-limiting example, a 1:62 lipid particle
formulation may comprise cholesterol at about 37 mol % (or any
fraction thereof) of the total lipid present in the particle. As
another non-limiting example, a 7:58 lipid particle formulation may
comprise cholesterol at about 35 mol % (or any fraction thereof) of
the total lipid present in the particle.
[0257] In other embodiments, the non-cationic lipid comprises from
about 5 mol % to about 90 mol %, from about 10 mol % to about 85
mol %, from about 20 mol % to about 80 mol %, about 10 mol % (e.g.,
phospholipid only), or about 60 mol % (e.g., phospholipid and
cholesterol or derivative thereof) (or any fraction thereof or
range therein) of the total lipid present in the particle.
[0258] Additional percentages and ranges of non-cationic lipids
suitable for use in the lipid particles of the present invention
are described in PCT Publication No. WO 09/127060, U.S. Published
Application No. US 2011/0071208, PCT Publication No. WO2011/000106,
and U.S. Published Application No. US 2011/0076335, the disclosures
of which are herein incorporated by reference in their entirety for
all purposes.
[0259] It should be understood that the percentage of non-cationic
lipid present in the lipid particles of the invention is a target
amount, and that the actual amount of non-cationic lipid present in
the formulation may vary, for example, by .+-.5 mol %. For example,
in the 1:57 lipid particle (e.g., SNALP) formulation, the target
amount of phospholipid is 7.1 mol % and the target amount of
cholesterol is 34.3 mol %, but the actual amount of phospholipid
may be .+-.2 mol %, 1.5 mol %, 1 mol %, 0.75 mol %, 0.5 mol %, 0.25
mol %, or .+-.0.1 mol % of that target amount, and the actual
amount of cholesterol may be .+-.3 mol %, .+-.2 mol %, .+-.1 mol %,
.+-.0.75 mol %, .+-.0.5 mol %, .+-.0.25 mol %, or .+-.0.1 mol % of
that target amount, with the balance of the formulation being made
up of other lipid components (adding up to 100 mol % of total
lipids present in the particle). Similarly, in the 7:54 lipid
particle (e.g., SNALP) formulation, the target amount of
phospholipid is 6.75 mol % and the target amount of cholesterol is
32.43 mol %, but the actual amount of phospholipid may be .+-.2 mol
%, .+-.1.5 mol %, .+-.1 mol %, .+-.0.75 mol %, .+-.0.5 mol %,
.+-.0.25 mol %, or .+-.0.1 mol % of that target amount, and the
actual amount of cholesterol may be .+-.3 mol %, .+-.2 mol %, .+-.1
mol %, .+-.0.75 mol %, 0.5 mol %, .+-.0.25 mol %, or .+-.0.1 mol %
of that target amount, with the balance of the formulation being
made up of other lipid components (adding up to 100 mol % of total
lipids present in the particle).
Lipid Conjugates
[0260] In addition to cationic and non-cationic lipids, the lipid
particles of the invention (e.g., SNALP) may further comprise a
lipid conjugate. The conjugated lipid is useful in that it prevents
the aggregation of particles. Suitable conjugated lipids include,
but are not limited to, PEG-lipid conjugates, POZ-lipid conjugates,
ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs),
and mixtures thereof. In certain embodiments, the particles
comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate
together with a CPL.
[0261] In a preferred embodiment, the lipid conjugate is a
PEG-lipid. Examples of PEG-lipids include, but are not limited to,
PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g.,
PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol
(PEG-DAG) as described in, e.g., U.S. Patent Publication Nos.
20030077829 and 2005008689, PEG coupled to phospholipids such as
phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as
described in, e.g., U.S. Pat. No. 5,885,613, PEG conjugated to
cholesterol or a derivative thereof, and mixtures thereof. The
disclosures of these patent documents are herein incorporated by
reference in their entirety for all purposes. Additional PEG-lipids
suitable for use in the invention include, without limitation,
mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The
synthesis of PEG-C-DOMG is described in PCT Publication No. WO
09/086558, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. Yet additional suitable
PEG-lipid conjugates include, without limitation,
1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-.omega.-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis
of 2KPEG-DMG is described in U.S. Pat. No. 7,404,969, the
disclosure of which is herein incorporated by reference in its
entirety for all purposes.
[0262] PEG is a linear, water-soluble polymer of ethylene PEG
repeating units with two terminal hydroxyl groups. PEGs are
classified by their molecular weights; for example, PEG 2000 has an
average molecular weight of about 2,000 daltons, and PEG 5000 has
an average molecular weight of about 5,000 daltons. PEGs are
commercially available from Sigma Chemical Co. and other companies
and include, but are not limited to, the following:
monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene
glycol-succinate (MePEG-S), monomethoxypolyethylene
glycol-succinimidyl succinate (MePEG-S-NHS),
monomethoxypolyethylene glycol-amine (MePEG-NH.sub.2),
monomethoxypolyethylene glycol-tresylate (MePEG-TRES),
monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as
well as such compounds containing a terminal hydroxyl group instead
of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS,
HO-PEG-NH.sub.2, etc.). Other PEGs such as those described in U.S.
Pat. Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are
also useful for preparing the PEG-lipid conjugates of the present
invention. The disclosures of these patents are herein incorporated
by reference in their entirety for all purposes. In addition,
monomethoxypolyethyleneglycol-acetic acid (MePEG-CH.sub.2COOH) is
particularly useful for preparing PEG-lipid conjugates including,
e.g., PEG-DAA conjugates.
[0263] The PEG moiety of the PEG-lipid conjugates described herein
may comprise an average molecular weight ranging from about 550
daltons to about 10,000 daltons. In certain instances, the PEG
moiety has an average molecular weight of from about 750 daltons to
about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000
daltons, from about 1,500 daltons to about 3,000 daltons, from
about 750 daltons to about 3,000 daltons, from about 750 daltons to
about 2,000 daltons, etc.). In preferred embodiments, the PEG
moiety has an average molecular weight of about 2,000 daltons or
about 750 daltons.
[0264] In certain instances, the PEG can be optionally substituted
by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated
directly to the lipid or may be linked to the lipid via a linker
moiety. Any linker moiety suitable for coupling the PEG to a lipid
can be used including, e.g., non-ester containing linker moieties
and ester-containing linker moieties. In a preferred embodiment,
the linker moiety is a non-ester containing linker moiety. As used
herein, the term "non-ester containing linker moiety" refers to a
linker moiety that does not contain a carboxylic ester bond
(--OC(O)--). Suitable non-ester containing linker moieties include,
but are not limited to, amido (--C(O)NH--), amino (--NR--),
carbonyl (--C(O)--), carbamate (--NHC(O)O--), urea (--NHC(O)NH--),
disulphide (--S--S--), ether (--O--), succinyl
(--(O)CCH.sub.2CH.sub.2C(O)--), succinamidyl
(--NHC(O)CH.sub.2CH.sub.2C(O)NH--), ether, disulphide, as well as
combinations thereof (such as a linker containing both a carbamate
linker moiety and an amido linker moiety). In a preferred
embodiment, a carbamate linker is used to couple the PEG to the
lipid.
[0265] In other embodiments, an ester containing linker moiety is
used to couple the PEG to the lipid. Suitable ester containing
linker moieties include, e.g., carbonate (--OC(O)O--), succinoyl,
phosphate esters (--O--(O)POH--O--), sulfonate esters, and
combinations thereof.
[0266] Phosphatidylethanolamines having a variety of acyl chain
groups of varying chain lengths and degrees of saturation can be
conjugated to PEG to form the lipid conjugate. Such
phosphatidylethanolamines are commercially available, or can be
isolated or synthesized using conventional techniques known to
those of skilled in the art. Phosphatidyl-ethanolamines containing
saturated or unsaturated fatty acids with carbon chain lengths in
the range of C.sub.10 to C.sub.20 are preferred.
Phosphatidylethanolamines with mono- or diunsaturated fatty acids
and mixtures of saturated and unsaturated fatty acids can also be
used. Suitable phosphatidylethanolamines include, but are not
limited to, dimyristoyl-phosphatidylethanolamine (DMPE),
dipalmitoyl-phosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE), and
distearoyl-phosphatidylethanolamine (DSPE).
[0267] The term "ATTA" or "polyamide" includes, without limitation,
compounds described in U.S. Pat. Nos. 6,320,017 and 6,586,559, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. These compounds include a compound
having the formula:
##STR00026##
wherein R is a member selected from the group consisting of
hydrogen, alkyl and acyl; R.sup.1 is a member selected from the
group consisting of hydrogen and alkyl; or optionally, R and
R.sup.1 and the nitrogen to which they are bound form an azido
moiety; R.sup.2 is a member of the group selected from hydrogen,
optionally substituted alkyl, optionally substituted aryl and a
side chain of an amino acid; R.sup.3 is a member selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto,
hydrazino, amino and NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5
are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p
is 1 to 4; and q is 0 or 1. It will be apparent to those of skill
in the art that other polyamides can be used in the compounds of
the present invention.
[0268] The term "diacylglycerol" or "DAG" includes a compound
having 2 fatty acyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons bonded to the 1- and
2-position of glycerol by ester linkages. The acyl groups can be
saturated or have varying degrees of unsaturation. Suitable acyl
groups include, but are not limited to, lauroyl (C.sub.12),
myristoyl (C.sub.14), palmitoyl (C.sub.16), stearoyl (C.sub.18),
and icosoyl (C.sub.20). In preferred embodiments, R.sup.1 and
R.sup.2 are the same, i.e., R.sup.1 and R.sup.2 are both myristoyl
(i.e., dimyristoyl), R.sup.1 and R.sup.2 are both stearoyl (i.e.,
distearoyl), etc. Diacylglycerols have the following general
formula:
##STR00027##
[0269] The term "dialkyloxypropyl" or "DAA" includes a compound
having 2 alkyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons. The alkyl groups can be
saturated or have varying degrees of unsaturation.
Dialkyloxypropyls have the following general formula:
##STR00028##
[0270] In a preferred embodiment, the PEG-lipid is a PEG-DAA
conjugate having the following formula:
##STR00029##
wherein R.sup.1 and R.sup.2 are independently selected and are
long-chain alkyl groups having from about 10 to about 22 carbon
atoms; PEG is a polyethyleneglycol; and L is a non-ester containing
linker moiety or an ester containing linker moiety as described
above. The long-chain alkyl groups can be saturated or unsaturated.
Suitable alkyl groups include, but are not limited to, decyl
(C.sub.10), lauryl (C.sub.12), myristyl (C.sub.14), palmityl
(C.sub.16), stearyl (C.sub.18), and icosyl (C.sub.20). In preferred
embodiments, R.sup.1 and R.sup.2 are the same, i.e., R.sup.1 and
R.sup.2 are both myristyl (i.e., dimyristyl), R.sup.1 and R.sup.2
are both stearyl (i.e., distearyl), etc.
[0271] In Formula VII above, the PEG has an average molecular
weight ranging from about 550 daltons to about 10,000 daltons. In
certain instances, the PEG has an average molecular weight of from
about 750 daltons to about 5,000 daltons (e.g., from about 1,000
daltons to about 5,000 daltons, from about 1,500 daltons to about
3,000 daltons, from about 750 daltons to about 3,000 daltons, from
about 750 daltons to about 2,000 daltons, etc.). In preferred
embodiments, the PEG has an average molecular weight of about 2,000
daltons or about 750 daltons. The PEG can be optionally substituted
with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments,
the terminal hydroxyl group is substituted with a methoxy or methyl
group.
[0272] In a preferred embodiment, "L" is a non-ester containing
linker moiety. Suitable non-ester containing linkers include, but
are not limited to, an amido linker moiety, an amino linker moiety,
a carbonyl linker moiety, a carbamate linker moiety, a urea linker
moiety, an ether linker moiety, a disulphide linker moiety, a
succinamidyl linker moiety, and combinations thereof. In a
preferred embodiment, the non-ester containing linker moiety is a
carbamate linker moiety (i.e., a PEG-C-DAA conjugate). In another
preferred embodiment, the non-ester containing linker moiety is an
amido linker moiety (i.e., a PEG-A-DAA conjugate). In yet another
preferred embodiment, the non-ester containing linker moiety is a
succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).
[0273] In particular embodiments, the PEG-lipid conjugate is
selected from:
##STR00030##
[0274] The PEG-DAA conjugates are synthesized using standard
techniques and reagents known to those of skill in the art. It will
be recognized that the PEG-DAA conjugates will contain various
amide, amine, ether, thio, carbamate, and urea linkages. Those of
skill in the art will recognize that methods and reagents for
forming these bonds are well known and readily available. See,
e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock,
COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss,
VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman
1989). It will also be appreciated that any functional groups
present may require protection and deprotection at different points
in the synthesis of the PEG-DAA conjugates. Those of skill in the
art will recognize that such techniques are well known. See, e.g.,
Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley
1991).
[0275] Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl
(C.sub.10) conjugate, a PEG-dilauryloxypropyl (C.sub.12) conjugate,
a PEG-dimyristyloxypropyl (C.sub.14) conjugate, a
PEG-dipalmityloxypropyl (C.sub.16) conjugate, or a
PEG-distearyloxypropyl (C.sub.18) conjugate. In these embodiments,
the PEG preferably has an average molecular weight of about 750 or
about 2,000 daltons. In one particularly preferred embodiment, the
PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the "2000"
denotes the average molecular weight of the PEG, the "C" denotes a
carbamate linker moiety, and the "DMA" denotes dimyristyloxypropyl.
In another particularly preferred embodiment, the PEG-lipid
conjugate comprises PEG750-C-DMA, wherein the "750" denotes the
average molecular weight of the PEG, the "C" denotes a carbamate
linker moiety, and the "DMA" denotes dimyristyloxypropyl. In
particular embodiments, the terminal hydroxyl group of the PEG is
substituted with a methyl group. Those of skill in the art will
readily appreciate that other dialkyloxypropyls can be used in the
PEG-DAA conjugates of the present invention.
[0276] In addition to the foregoing, it will be readily apparent to
those of skill in the art that other hydrophilic polymers can be
used in place of PEG. Examples of suitable polymers that can be
used in place of PEG include, but are not limited to,
polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyl methacrylamide, polymethacrylamide and
polydimethylacrylamide, polylactic acid, polyglycolic acid, and
derivatized celluloses such as hydroxymethylcellulose or
hydroxyethylcellulose.
[0277] In addition to the foregoing components, the lipid particles
(e.g., SNALP) of the present invention can further comprise
cationic poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g.,
Chen et al., Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No.
6,852,334; PCT Publication No. WO 00/62813, the disclosures of
which are herein incorporated by reference in their entirety for
all purposes).
[0278] Suitable CPLs include compounds of Formula VIII:
A-W--Y (VIII),
wherein A, W, and Y are as described below.
[0279] With reference to Formula VIII, "A" is a lipid moiety such
as an amphipathic lipid, a neutral lipid, or a hydrophobic lipid
that acts as a lipid anchor. Suitable lipid examples include, but
are not limited to, diacylglycerolyls, dialkylglycerolyls,
N--N-dialkylaminos, 1,2-diacyloxy-3-aminopropanes, and
1,2-dialkyl-3-aminopropanes.
[0280] "W" is a polymer or an oligomer such as a hydrophilic
polymer or oligomer. Preferably, the hydrophilic polymer is a
biocompatable polymer that is nonimmunogenic or possesses low
inherent immunogenicity. Alternatively, the hydrophilic polymer can
be weakly antigenic if used with appropriate adjuvants. Suitable
nonimmunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers, and combinations thereof. In a
preferred embodiment, the polymer has a molecular weight of from
about 250 to about 7,000 daltons.
[0281] "Y" is a polycationic moiety. The term polycationic moiety
refers to a compound, derivative, or functional group having a
positive charge, preferably at least 2 positive charges at a
selected pH, preferably physiological pH. Suitable polycationic
moieties include basic amino acids and their derivatives such as
arginine, asparagine, glutamine, lysine, and histidine; spermine;
spermidine; cationic dendrimers; polyamines; polyamine sugars; and
amino polysaccharides. The polycationic moieties can be linear,
such as linear tetralysine, branched or dendrimeric in structure.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to
employ may be determined by the type of particle application which
is desired.
[0282] The charges on the polycationic moieties can be either
distributed around the entire particle moiety, or alternatively,
they can be a discrete concentration of charge density in one
particular area of the particle moiety e.g., a charge spike. If the
charge density is distributed on the particle, the charge density
can be equally distributed or unequally distributed. All variations
of charge distribution of the polycationic moiety are encompassed
by the present invention.
[0283] The lipid "A" and the nonimmunogenic polymer "W" can be
attached by various methods and preferably by covalent attachment.
Methods known to those of skill in the art can be used for the
covalent attachment of "A" and "W." Suitable linkages include, but
are not limited to, amide, amine, carboxyl, carbonate, carbamate,
ester, and hydrazone linkages. It will be apparent to those skilled
in the art that "A" and "W" must have complementary functional
groups to effectuate the linkage. The reaction of these two groups,
one on the lipid and the other on the polymer, will provide the
desired linkage. For example, when the lipid is a diacylglycerol
and the terminal hydroxyl is activated, for instance with NHS and
DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, e.g.,
U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which
are herein incorporated by reference in their entirety for all
purposes), an amide bond will form between the two groups.
[0284] In certain instances, the polycationic moiety can have a
ligand attached, such as a targeting ligand or a chelating moiety
for complexing calcium. Preferably, after the ligand is attached,
the cationic moiety maintains a positive charge. In certain
instances, the ligand that is attached has a positive charge.
Suitable ligands include, but are not limited to, a compound or
device with a reactive functional group and include lipids,
amphipathic lipids, carrier compounds, bioaffinity compounds,
biomaterials, biopolymers, biomedical devices, analytically
detectable compounds, therapeutically active compounds, enzymes,
peptides, proteins, antibodies, immune stimulators, radiolabels,
fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides,
liposomes, virosomes, micelles, immunoglobulins, functional groups,
other targeting moieties, or toxins.
[0285] In some embodiments, the lipid conjugate (e.g., PEG-lipid)
comprises from about 0.1 mol % to about 2 mol %, from about 0.5 mol
% to about 2 mol %, from about 1 mol % to about 2 mol %, from about
0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol
%, from about 0.8 mol % to about 1.7 mol %, from about 0.9 mol % to
about 1.6 mol %, from about 0.9 mol % to about 1.8 mol %, from
about 1 mol % to about 1.8 mol %, from about 1 mol % to about 1.7
mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol
% to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, or
from about 1.4 mol % to about 1.5 mol % (or any fraction thereof or
range therein) of the total lipid present in the particle.
[0286] In other embodiments, the lipid conjugate (e.g., PEG-lipid)
comprises from about 0 mol % to about 20 mol %, from about 0.5 mol
% to about 20 mol %, from about 2 mol % to about 20 mol %, from
about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15
mol %, from about 4 mol % to about 15 mol %, from about 2 mol % to
about 12 mol %, from about 5 mol % to about 12 mol %, or about 2
mol % (or any fraction thereof or range therein) of the total lipid
present in the particle.
[0287] In further embodiments, the lipid conjugate (e.g.,
PEG-lipid) comprises from about 4 mol % to about 10 mol %, from
about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol
%, from about 5 mol % to about 8 mol %, from about 6 mol % to about
9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6
mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction
thereof or range therein) of the total lipid present in the
particle.
[0288] Additional percentages and ranges of lipid conjugates
suitable for use in the lipid particles of the present invention
are described in PCT Publication No. WO 09/127060, U.S. Published
Application No. US 2011/0071208, PCT Publication No. WO2011/000106,
and U.S. Published Application No. US 2011/0076335, the disclosures
of which are herein incorporated by reference in their entirety for
all purposes.
[0289] It should be understood that the percentage of lipid
conjugate (e.g., PEG-lipid) present in the lipid particles of the
invention is a target amount, and that the actual amount of lipid
conjugate present in the formulation may vary, for example, by
.+-.2 mol %. For example, in the 1:57 lipid particle (e.g., SNALP)
formulation, the target amount of lipid conjugate is 1.4 mol %, but
the actual amount of lipid conjugate may be .+-.0.5 mol %, .+-.0.4
mol %, .+-.0.3 mol %, .+-.0.2 mol %, .+-.0.1 mol %, or .+-.0.05 mol
% of that target amount, with the balance of the formulation being
made up of other lipid components (adding up to 100 mol % of total
lipids present in the particle). Similarly, in the 7:54 lipid
particle (e.g., SNALP) formulation, the target amount of lipid
conjugate is 6.76 mol %, but the actual amount of lipid conjugate
may be .+-.2 mol %, .+-.1.5 mol %, 1 mol %, 0.75 mol %, 0.5 mol %,
0.25 mol %, or .+-.0.1 mol % of that target amount, with the
balance of the formulation being made up of other lipid components
(adding up to 100 mol % of total lipids present in the
particle).
[0290] One of ordinary skill in the art will appreciate that the
concentration of the lipid conjugate can be varied depending on the
lipid conjugate employed and the rate at which the lipid particle
is to become fusogenic.
[0291] By controlling the composition and concentration of the
lipid conjugate, one can control the rate at which the lipid
conjugate exchanges out of the lipid particle and, in turn, the
rate at which the lipid particle becomes fusogenic. For instance,
when a PEG-DAA conjugate is used as the lipid conjugate, the rate
at which the lipid particle becomes fusogenic can be varied, for
example, by varying the concentration of the lipid conjugate, by
varying the molecular weight of the PEG, or by varying the chain
length and degree of saturation of the alkyl groups on the PEG-DAA
conjugate. In addition, other variables including, for example, pH,
temperature, ionic strength, etc. can be used to vary and/or
control the rate at which the lipid particle becomes fusogenic.
Other methods which can be used to control the rate at which the
lipid particle becomes fusogenic will become apparent to those of
skill in the art upon reading this disclosure. Also, by controlling
the composition and concentration of the lipid conjugate, one can
control the lipid particle (e.g., SNALP) size.
Preparation of Lipid Particles
[0292] The lipid particles of the present invention, e.g., SNALP,
in which an mRNA is entrapped within the lipid portion of the
particle and is protected from degradation, can be formed by any
method known in the art including, but not limited to, a continuous
mixing method, a direct dilution process, and an in-line dilution
process.
[0293] In certain embodiments, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via a
continuous mixing method, e.g., a process that includes providing
an aqueous solution comprising a nucleic acid (e.g., mRNA) in a
first reservoir, providing an organic lipid solution in a second
reservoir (wherein the lipids present in the organic lipid solution
are solubilized in an organic solvent, e.g., a lower alkanol such
as ethanol), and mixing the aqueous solution with the organic lipid
solution such that the organic lipid solution mixes with the
aqueous solution so as to substantially instantaneously produce a
lipid vesicle (e.g., liposome) encapsulating the nucleic acid
within the lipid vesicle. This process and the apparatus for
carrying out this process are described in detail in U.S. Patent
Publication No. 20040142025, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
[0294] The action of continuously introducing lipid and buffer
solutions into a mixing environment, such as in a mixing chamber,
causes a continuous dilution of the lipid solution with the buffer
solution, thereby producing a lipid vesicle substantially
instantaneously upon mixing. As used herein, the phrase
"continuously diluting a lipid solution with a buffer solution"
(and variations) generally means that the lipid solution is diluted
sufficiently rapidly in a hydration process with sufficient force
to effectuate vesicle generation. By mixing the aqueous solution
comprising a nucleic acid with the organic lipid solution, the
organic lipid solution undergoes a continuous stepwise dilution in
the presence of the buffer solution (i.e., aqueous solution) to
produce a nucleic acid-lipid particle.
[0295] The nucleic acid-lipid particles formed using the continuous
mixing method typically have a size of from about 30 nm to about
150 nm, from about 40 nm to about 150 nm, from about 50 nm to about
150 nm, from about 60 nm to about 130 nm, from about 70 nm to about
110 nm, from about 70 nm to about 100 nm, from about 80 nm to about
100 nm, from about 90 nm to about 100 nm, from about 70 to about 90
nm, from about 80 nm to about 90 nm, from about 70 nm to about 80
nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or
about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70
nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115
nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm (or
any fraction thereof or range therein). The particles thus formed
do not aggregate and are optionally sized to achieve a uniform
particle size.
[0296] In another embodiment, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via a direct
dilution process that includes forming a lipid vesicle (e.g.,
liposome) solution and immediately and directly introducing the
lipid vesicle solution into a collection vessel containing a
controlled amount of dilution buffer. In preferred aspects, the
collection vessel includes one or more elements configured to stir
the contents of the collection vessel to facilitate dilution. In
one aspect, the amount of dilution buffer present in the collection
vessel is substantially equal to the volume of lipid vesicle
solution introduced thereto. As a non-limiting example, a lipid
vesicle solution in 45% ethanol when introduced into the collection
vessel containing an equal volume of dilution buffer will
advantageously yield smaller particles.
[0297] In yet another embodiment, the present invention provides
nucleic acid-lipid particles (e.g., SNALP) produced via an in-line
dilution process in which a third reservoir containing dilution
buffer is fluidly coupled to a second mixing region. In this
embodiment, the lipid vesicle (e.g., liposome) solution formed in a
first mixing region is immediately and directly mixed with dilution
buffer in the second mixing region. In preferred aspects, the
second mixing region includes a T-connector arranged so that the
lipid vesicle solution and the dilution buffer flows meet as
opposing 180.degree. flows; however, connectors providing shallower
angles can be used, e.g., from about 27.degree. to about
180.degree. (e.g., about 90.degree.). A pump mechanism delivers a
controllable flow of buffer to the second mixing region. In one
aspect, the flow rate of dilution buffer provided to the second
mixing region is controlled to be substantially equal to the flow
rate of lipid vesicle solution introduced thereto from the first
mixing region. This embodiment advantageously allows for more
control of the flow of dilution buffer mixing with the lipid
vesicle solution in the second mixing region, and therefore also
the concentration of lipid vesicle solution in buffer throughout
the second mixing process. Such control of the dilution buffer flow
rate advantageously allows for small particle size formation at
reduced concentrations.
[0298] These processes and the apparatuses for carrying out these
direct dilution and in-line dilution processes are described in
detail in U.S. Patent Publication No. 20070042031, the disclosure
of which is herein incorporated by reference in its entirety for
all purposes.
[0299] The nucleic acid-lipid particles formed using the direct
dilution and in-line dilution processes typically have a size of
from about 30 nm to about 150 nm, from about 40 nm to about 150 nm,
from about 50 nm to about 150 nm, from about 60 nm to about 130 nm,
from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,
from about 80 nm to about 100 nm, from about 90 nm to about 100 nm,
from about 70 to about 90 nm, from about 80 nm to about 90 nm, from
about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm,
90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm,
60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105
nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm,
or 150 nm (or any fraction thereof or range therein). The particles
thus formed do not aggregate and are optionally sized to achieve a
uniform particle size.
[0300] If needed, the lipid particles of the invention (e.g.,
SNALP) can be sized by any of the methods available for sizing
liposomes. The sizing may be conducted in order to achieve a
desired size range and relatively narrow distribution of particle
sizes.
[0301] Several techniques are available for sizing the particles to
a desired size. One sizing method, used for liposomes and equally
applicable to the present particles, is described in U.S. Pat. No.
4,737,323, the disclosure of which is herein incorporated by
reference in its entirety for all purposes. Sonicating a particle
suspension either by bath or probe sonication produces a
progressive size reduction down to particles of less than about 50
nm in size. Homogenization is another method which relies on
shearing energy to fragment larger particles into smaller ones. In
a typical homogenization procedure, particles are recirculated
through a standard emulsion homogenizer until selected particle
sizes, typically between about 60 and about 80 nm, are observed. In
both methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination, or QELS.
[0302] Extrusion of the particles through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane is also an
effective method for reducing particle sizes to a relatively
well-defined size distribution. Typically, the suspension is cycled
through the membrane one or more times until the desired particle
size distribution is achieved. The particles may be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in size.
[0303] In other embodiments, the methods may further comprise
adding non-lipid polycations which are useful to effect the
lipofection of cells using the present compositions. Examples of
suitable non-lipid polycations include, hexadimethrine bromide
(sold under the brand name POLYBRENE.RTM., from Aldrich Chemical
Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. Other
suitable polycations include, for example, salts of
poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,
polyallylamine, and polyethyleneimine. Addition of these salts is
preferably after the particles have been formed.
[0304] In some embodiments, the nucleic acid to lipid ratios
(mass/mass ratios) in a formed nucleic acid-lipid particle (e.g.,
SNALP) will range from about 0.01 to about 0.2, from about 0.05 to
about 0.2, from about 0.02 to about 0.1, from about 0.03 to about
0.1, or from about 0.01 to about 0.08. The ratio of the starting
materials (input) also falls within this range. In other
embodiments, the particle preparation uses about 400 .mu.g nucleic
acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of
about 0.01 to about 0.08 and, more preferably, about 0.04, which
corresponds to 1.25 mg of total lipid per 50 .mu.g of nucleic acid.
In other preferred embodiments, the particle has a nucleic
acid:lipid mass ratio of about 0.08.
[0305] In other embodiments, the lipid to nucleic acid ratios
(mass/mass ratios) in a formed nucleic acid-lipid particle (e.g.,
SNALP) will range from about 1 (1:1) to about 100 (100:1), from
about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50
(50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1)
to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from
about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25
(25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1)
to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from
about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20
(20:1), from about 5 (5:1) to about 15 (15:1), from about 5 (5:1)
to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9
(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15
(15:1), 16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21
(21:1), 22 (22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any
fraction thereof or range therein. The ratio of the starting
materials (input) also falls within this range.
[0306] As previously discussed, the conjugated lipid may further
include a CPL. A variety of general methods for making SNALP-CPLs
(CPL-containing SNALP) are discussed herein. Two general techniques
include the "post-insertion" technique, that is, insertion of a CPL
into, for example, a pre-formed SNALP, and the "standard"
technique, wherein the CPL is included in the lipid mixture during,
for example, the SNALP formation steps. The post-insertion
technique results in SNALP having CPLs mainly in the external face
of the SNALP bilayer membrane, whereas standard techniques provide
SNALP having CPLs on both internal and external faces. The method
is especially useful for vesicles made from phospholipids (which
can contain cholesterol) and also for vesicles containing
PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making
SNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;
6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent
Publication No. 20020072121; and PCT Publication No. WO 00/62813,
the disclosures of which are herein incorporated by reference in
their entirety for all purposes.
Administration of Lipid Particles
[0307] Once formed, the lipid particles of the invention (e.g.,
SNALP) are particularly useful for the introduction of nucleic
acids (e.g., mRNA) into cells. Accordingly, the present invention
also provides methods for introducing a nucleic acid (e.g., mRNA)
into a cell. In particular embodiments, the nucleic acid (e.g.,
mRNA) is introduced into an infected cell such as
reticuloendothelial cells (e.g., macrophages, monocytes, etc.) as
well as other cell types, including fibroblasts, endothelial cells
(such as those lining the interior surface of blood vessels),
and/or platelet cells. The methods may be carried out in vitro or
in vivo by first forming the particles as described herein and then
contacting the particles with the cells for a period of time
sufficient for delivery of the mRNA to the cells to occur.
[0308] The lipid particles of the invention (e.g., SNALP) can be
adsorbed to almost any cell type with which they are mixed or
contacted. Once adsorbed, the particles can either be endocytosed
by a portion of the cells, exchange lipids with cell membranes, or
fuse with the cells. Transfer or incorporation of the nucleic acid
(e.g., mRNA) portion of the particle can take place via any one of
these pathways. In particular, when fusion takes place, the
particle membrane is integrated into the cell membrane and the
contents of the particle combine with the intracellular fluid.
[0309] The lipid particles of the invention (e.g., SNALP) can be
administered either alone or in a mixture with a pharmaceutically
acceptable carrier (e.g., physiological saline or phosphate buffer)
selected in accordance with the route of administration and
standard pharmaceutical practice. Generally, normal buffered saline
(e.g., 135-150 mM NaCl) will be employed as the pharmaceutically
acceptable carrier. Other suitable carriers include, e.g., water,
buffered water, 0.4% saline, 0.3% glycine, and the like, including
glycoproteins for enhanced stability, such as albumin, lipoprotein,
globulin, etc. Additional suitable carriers are described in, e.g.,
REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier"
includes any and all solvents, dispersion media, vehicles,
coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption delaying agents, buffers, carrier solutions,
suspensions, colloids, and the like. The phrase "pharmaceutically
acceptable" refers to molecular entities and compositions that do
not produce an allergic or similar untoward reaction when
administered to a human.
[0310] The pharmaceutically acceptable carrier is generally added
following lipid particle formation. Thus, after the lipid particle
(e.g., SNALP) is formed, the particle can be diluted into
pharmaceutically acceptable carriers such as normal buffered
saline.
[0311] The concentration of particles in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2 to 5%, to as much as about 10 to 90%
by weight, and will be selected primarily by fluid volumes,
viscosities, etc., in accordance with the particular mode of
administration selected. For example, the concentration may be
increased to lower the fluid load associated with treatment. This
may be particularly desirable in patients having
atherosclerosis-associated congestive heart failure or severe
hypertension. Alternatively, particles composed of irritating
lipids may be diluted to low concentrations to lessen inflammation
at the site of administration.
[0312] The pharmaceutical compositions of the present invention may
be sterilized by conventional, well-known sterilization techniques.
Aqueous solutions can be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile aqueous solution prior to administration.
The compositions can contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, and calcium chloride.
Additionally, the particle suspension may include lipid-protective
agents which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as alphatocopherol, and water-soluble iron-specific
chelators, such as ferrioxamine, are suitable.
[0313] In some embodiments, the lipid particles of the invention
(e.g., SNALP) are particularly useful in methods for the
therapeutic delivery of one or more mRNA.
In Vivo Administration
[0314] Systemic delivery for in vivo therapy, e.g., delivery of a
therapeutic nucleic acid to a distal target cell via body systems
such as the circulation, has been achieved using nucleic acid-lipid
particles such as those described in PCT Publication Nos. WO
05/007196, WO 05/121348, WO 05/120152, and WO 04/002453, the
disclosures of which are herein incorporated by reference in their
entirety for all purposes. The present invention also provides
fully encapsulated lipid particles that protect the nucleic acid
from nuclease degradation in serum, are non-immunogenic, are small
in size, and are suitable for repeat dosing.
[0315] For in vivo administration, administration can be in any
manner known in the art, e.g., by injection, oral administration,
inhalation (e.g., intransal or intratracheal), transdermal
application, or rectal administration. Administration can be
accomplished via single or divided doses. The pharmaceutical
compositions can be administered parenterally, i.e.,
intraarticularly, intravenously, intraperitoneally, subcutaneously,
or intramuscularly. In some embodiments, the pharmaceutical
compositions are administered intravenously or intraperitoneally by
a bolus injection (see, e.g., U.S. Pat. No. 5,286,634).
Intracellular nucleic acid delivery has also been discussed in
Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et
al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther.
Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274
(1993). Still other methods of administering lipid-based
therapeutics are described in, for example, U.S. Pat. Nos.
3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and
4,588,578. The lipid particles can be administered by direct
injection at the site of disease or by injection at a site distal
from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY,
MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71(1994)). The
disclosures of the above-described references are herein
incorporated by reference in their entirety for all purposes.
[0316] In embodiments where the lipid particles of the present
invention (e.g., SNALP) are administered intravenously, at least
about 5%, 10%, 15%, 20%, or 25% of the total injected dose of the
particles is present in plasma about 8, 12, 24, 36, or 48 hours
after injection. In other embodiments, more than about 20%, 30%,
40% and as much as about 60%, 70% or 80% of the total injected dose
of the lipid particles is present in plasma about 8, 12, 24, 36, or
48 hours after injection. In certain instances, more than about 10%
of a plurality of the particles is present in the plasma of a
mammal about 1 hour after administration. In certain other
instances, the presence of the lipid particles is detectable at
least about 1 hour after administration of the particle. In some
embodiments, the presence of a therapeutic nucleic acid such as an
mRNA molecule is detectable in cells at about 8, 12, 24, 36, 48,
60, 72 or 96 hours after administration. In other embodiments,
expression of a polypeptide encoded by an mRNA introduced into a
living body in accordance with the present invention is detectable
at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after
administration. In further embodiments, the presence or effect of
an mRNA in cells at a site proximal or distal to the site of
administration is detectable at about 12, 24, 48, 72, or 96 hours,
or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28
days after administration. In additional embodiments, the lipid
particles (e.g., SNALP) of the invention are administered
parenterally or intraperitoneally.
[0317] The compositions of the present invention, either alone or
in combination with other suitable components, can be made into
aerosol formulations (i.e., they can be "nebulized") to be
administered via inhalation (e.g., intranasally or intratracheally)
(see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0318] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering nucleic acid compositions
directly to the lungs via nasal aerosol sprays have been described,
e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212. Likewise, the
delivery of drugs using intranasal microparticle resins and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are
also known in the pharmaceutical arts. Similarly, transmucosal drug
delivery in the form of a polytetrafluoroetheylene support matrix
is described in U.S. Pat. No. 5,780,045. The disclosures of the
above-described patents are herein incorporated by reference in
their entirety for all purposes.
[0319] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions are preferably administered, for
example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically, or intrathecally.
[0320] Generally, when administered intravenously, the lipid
particle formulations are formulated with a suitable pharmaceutical
carrier. Many pharmaceutically acceptable carriers may be employed
in the compositions and methods of the present invention. Suitable
formulations for use in the present invention are found, for
example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing
Company, Philadelphia, Pa., 17th ed. (1985). A variety of aqueous
carriers may be used, for example, water, buffered water, 0.4%
saline, 0.3% glycine, and the like, and may include glycoproteins
for enhanced stability, such as albumin, lipoprotein, globulin,
etc. Generally, normal buffered saline (135-150 mM NaCl) will be
employed as the pharmaceutically acceptable carrier, but other
suitable carriers will suffice. These compositions can be
sterilized by conventional liposomal sterilization techniques, such
as filtration. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc. These compositions can be sterilized using the
techniques referred to above or, alternatively, they can be
produced under sterile conditions. The resulting aqueous solutions
may be packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration.
[0321] In certain applications, the lipid particles disclosed
herein may be delivered via oral administration to the individual.
The particles may be incorporated with excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
pills, lozenges, elixirs, mouthwash, suspensions, oral sprays,
syrups, wafers, and the like (see, e.g., U.S. Pat. Nos. 5,641,515,
5,580,579, and 5,792,451, the disclosures of which are herein
incorporated by reference in their entirety for all purposes).
These oral dosage forms may also contain the following: binders,
gelatin; excipients, lubricants, and/or flavoring agents. When the
unit dosage form is a capsule, it may contain, in addition to the
materials described above, a liquid carrier. Various other
materials may be present as coatings or to otherwise modify the
physical form of the dosage unit. Of course, any material used in
preparing any unit dosage form should be pharmaceutically pure and
substantially non-toxic in the amounts employed.
[0322] Typically, these oral formulations may contain at least
about 0.1% of the lipid particles or more, although the percentage
of the particles may, of course, be varied and may conveniently be
between about 1% or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of
particles in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0323] Formulations suitable for oral administration can consist
of: (a) liquid solutions, such as an effective amount of a packaged
therapeutic nucleic acid (e.g., mRNA) suspended in diluents such as
water, saline, or PEG 400; (b) capsules, sachets, or tablets, each
containing a predetermined amount of a therapeutic nucleic acid
(e.g., mRNA), as liquids, solids, granules, or gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions.
Tablet forms can include one or more of lactose, sucrose, mannitol,
sorbitol, calcium phosphates, corn starch, potato starch,
microcrystalline cellulose, gelatin, colloidal silicon dioxide,
talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers, binders, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers. Lozenge forms can
comprise a therapeutic nucleic acid (e.g., mRNA) in a flavor, e.g.,
sucrose, as well as pastilles comprising the therapeutic nucleic
acid in an inert base, such as gelatin and glycerin or sucrose and
acacia emulsions, gels, and the like containing, in addition to the
therapeutic nucleic acid, carriers known in the art.
[0324] In another example of their use, lipid particles can be
incorporated into a broad range of topical dosage forms. For
instance, a suspension containing nucleic acid-lipid particles such
as SNALP can be formulated and administered as gels, oils,
emulsions, topical creams, pastes, ointments, lotions, foams,
mousses, and the like.
[0325] When preparing pharmaceutical preparations of the lipid
particles of the invention, it is preferable to use quantities of
the particles which have been purified to reduce or eliminate empty
particles or particles with therapeutic agents such as nucleic acid
associated with the external surface.
[0326] The methods of the present invention may be practiced in a
variety of hosts. Preferred hosts include mammalian species, such
as primates (e.g., humans and chimpanzees as well as other nonhuman
primates), canines, felines, equines, bovines, ovines, caprines,
rodents (e.g., rats and mice), lagomorphs, and swine.
[0327] The amount of particles administered will depend upon the
ratio of therapeutic nucleic acid (e.g., mRNA) to lipid, the
particular therapeutic nucleic acid used, the disease or disorder
being treated, the age, weight, and condition of the patient, and
the judgment of the clinician, but will generally be between about
0.01 and about 50 mg per kilogram of body weight, preferably
between about 0.1 and about 5 mg/kg of body weight, or about
10.sup.8-10.sup.10 particles per administration (e.g.,
injection).
In Vitro Administration
[0328] For in vitro applications, the delivery of therapeutic
nucleic acids (e.g., mRNA) can be to any cell grown in culture,
whether of plant or animal origin, vertebrate or invertebrate, and
of any tissue or type. In preferred embodiments, the cells are
animal cells, more preferably mammalian cells, and most preferably
human cells.
[0329] Contact between the cells and the lipid particles, when
carried out in vitro, takes place in a biologically compatible
medium. The concentration of particles varies widely depending on
the particular application, but is generally between about 1
.mu.mol and about 10 mmol. Treatment of the cells with the lipid
particles is generally carried out at physiological temperatures
(about 37.degree. C.) for periods of time of from about 1 to 48
hours, preferably of from about 2 to 4 hours.
[0330] In one group of preferred embodiments, a lipid particle
suspension is added to 60-80% confluent plated cells having a cell
density of from about 10.sup.3 to about 10.sup.5 cells/ml, more
preferably about 2.times.10.sup.4 cells/ml. The concentration of
the suspension added to the cells is preferably of from about 0.01
to 0.2 .mu.g/ml, more preferably about 0.1 .mu.g/ml.
[0331] To the extent that tissue culture of cells may be required,
it is well-known in the art. For example, Freshney, Culture of
Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New
York (1994), Kuchler et al., Biochemical Methods in Cell Culture
and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and the
references cited therein provide a general guide to the culture of
cells. Cultured cell systems often will be in the form of
monolayers of cells, although cell suspensions are also used.
[0332] Using an Endosomal Release Parameter (ERP) assay, the
delivery efficiency of the SNALP or other lipid particle of the
invention can be optimized. An ERP assay is described in detail in
U.S. Patent Publication No. 20030077829, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.
More particularly, the purpose of an ERP assay is to distinguish
the effect of various cationic lipids and helper lipid components
of SNALP or other lipid particle based on their relative effect on
binding/uptake or fusion with/destabilization of the endosomal
membrane. This assay allows one to determine quantitatively how
each component of the SNALP or other lipid particle affects
delivery efficiency, thereby optimizing the SNALP or other lipid
particle. Usually, an ERP assay measures expression of a reporter
protein (e.g., luciferase, .beta.-galactosidase, green fluorescent
protein (GFP), etc.), and in some instances, a SNALP formulation
optimized for an expression plasmid will also be appropriate for
encapsulating an mRNA. By comparing the ERPs for each of the
various SNALP or other lipid particles, one can readily determine
the optimized system, e.g., the SNALP or other lipid particle that
has the greatest uptake in the cell.
Cells for Delivery of Lipid Particles
[0333] The present invention can be practiced on a wide variety of
cell types from any vertebrate species, including mammals, such as,
e.g, canines, felines, equines, bovines, ovines, caprines, rodents
(e.g., mice, rats, and guinea pigs), lagomorphs, swine, and
primates (e.g. monkeys, chimpanzees, and humans).
Detection of Lipid Particles
[0334] In some embodiments, the lipid particles of the present
invention (e.g., SNALP) are detectable in the subject at about 1,
2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, the lipid
particles of the present invention (e.g., SNALP) are detectable in
the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or about
6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after
administration of the particles. The presence of the particles can
be detected in the cells, tissues, or other biological samples from
the subject. The particles may be detected, e.g., by direct
detection of the particles, and/or detection of an mRNA sequence
encapsulated within the lipid particles, and/or detection of a
polypeptide expressed from an mRNA.
Detection of Particles
[0335] Lipid particles of the invention such as SNALP can be
detected using any method known in the art. For example, a label
can be coupled directly or indirectly to a component of the lipid
particle using methods well-known in the art. A wide variety of
labels can be used, with the choice of label depending on
sensitivity required, ease of conjugation with the lipid particle
component, stability requirements, and available instrumentation
and disposal provisions. Suitable labels include, but are not
limited to, spectral labels such as fluorescent dyes (e.g.,
fluorescein and derivatives, such as fluorescein isothiocyanate
(FITC) and Oregon Green.TM.; rhodamine and derivatives such Texas
red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin,
biotin, phycoerythrin, AMCA, CyDyes.TM., and the like; radiolabels
such as .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, .sup.33P,
etc.; enzymes such as horse radish peroxidase, alkaline
phosphatase, etc.; spectral colorimetric labels such as colloidal
gold or colored glass or plastic beads such as polystyrene,
polypropylene, latex, etc. The label can be detected using any
means known in the art.
Detection of Nucleic Acids
[0336] Nucleic acids (e.g., mRNA) are detected and quantified
herein by any of a number of means well-known to those of skill in
the art. The detection of nucleic acids may proceed by well-known
methods such as Southern analysis, Northern analysis, gel
electrophoresis, PCR, radiolabeling, scintillation counting, and
affinity chromatography. Additional analytic biochemical methods
such as spectrophotometry, radiography, electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), and hyperdiffusion chromatography
may also be employed.
[0337] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in, e.g., "Nucleic
Acid Hybridization, A Practical Approach," Eds. Hames and Higgins,
IRL Press (1985).
[0338] The sensitivity of the hybridization assays may be enhanced
through the use of a nucleic acid amplification system which
multiplies the target nucleic acid being detected. In vitro
amplification techniques suitable for amplifying sequences for use
as molecular probes or for generating nucleic acid fragments for
subsequent subcloning are known. Examples of techniques sufficient
to direct persons of skill through such in vitro amplification
methods, including the polymerase chain reaction (PCR), the ligase
chain reaction (LCR), Q.beta.-replicase amplification, and other
RNA polymerase mediated techniques (e.g., NASBA.TM.) are found in
Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (2000); and Ausubel et al., SHORT
PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002);
as well as U.S. Pat. No. 4,683,202; PCR Protocols, A Guide to
Methods and Applications (Innis et al. eds.) Academic Press Inc.
San Diego, Calif. (1990); Arnheim & Levinson (Oct. 1, 1990),
C&EN 36; The Journal Of NIH Research, 3:81 (1991); Kwoh et al.,
Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc.
Natl. Acad. Sci. USA, 87:1874 (1990); Lomell et al., J. Clin.
Chem., 35:1826 (1989); Landegren et al., Science, 241:1077 (1988);
Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560
(1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan and
Malek, Biotechnology, 13:563 (1995). Improved methods of cloning in
vitro amplified nucleic acids are described in U.S. Pat. No.
5,426,039. Other methods described in the art are the nucleic acid
sequence based amplification (NASBA.TM., Cangene, Mississauga,
Ontario) and Q.beta.-replicase systems. These systems can be used
to directly identify mutants where the PCR or LCR primers are
designed to be extended or ligated only when a select sequence is
present. Alternatively, the select sequences can be generally
amplified using, for example, nonspecific PCR primers and the
amplified target region later probed for a specific sequence
indicative of a mutation. The disclosures of the above-described
references are herein incorporated by reference in their entirety
for all purposes.
[0339] Nucleic acids for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as inhibitor
components are typically synthesized chemically according to the
solid phase phosphoramidite triester method described by Beaucage
et al., Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an
automated synthesizer, as described in Needham VanDevanter et al.,
Nucleic Acids Res., 12:6159 (1984). Purification of
polynucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion exchange HPLC as
described in Pearson et al., J. Chrom., 255:137 149 (1983). The
sequence of the synthetic polynucleotides can be verified using the
chemical degradation method of Maxam and Gilbert (1980) in Grossman
and Moldave (eds.) Academic Press, New York, Methods in Enzymology,
65:499.
[0340] An alternative means for determining the level of
transcription is in situ hybridization. In situ hybridization
assays are well-known and are generally described in Angerer et
al., Methods Enzymol., 152:649 (1987). In an in situ hybridization
assay, cells are fixed to a solid support, typically a glass slide.
If DNA is to be probed, the cells are denatured with heat or
alkali. The cells are then contacted with a hybridization solution
at a moderate temperature to permit annealing of specific probes
that are labeled. The probes are preferably labeled with
radioisotopes or fluorescent reporters.
EXAMPLES
[0341] The present invention will be described in greater detail by
way of specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results. The art worker will also
understand the while a "Table 1" may be included in more than one
Example, references to a `Table 1` in Example 1 refer to the Table
1 present in Example 1.
Examples 1-13 Co-Delivery of a Nucleic Acid Payload with a Steroid
in Lipid Nanoparticles
[0342] Corticosteroids are a class of steroid hormones that are
produced in the adrenal cortex of vertebrates, as well as the
synthetic analogues of these hormones. Corticosteroids are involved
in a wide range of physiological processes, including stress
response, immune response, and regulation of inflammation,
carbohydrate metabolism, protein catabolism, blood electrolyte
levels, and behavior.
[0343] There are two classes of corticosteroids. Glucocorticoids
such as cortisol control carbohydrate, fat and protein metabolism,
and are anti-inflammatory by preventing phospholipid release,
decreasing eosinophil action and a number of other mechanisms.
Mineralocorticoids such as aldosterone control electrolyte and
water levels, mainly by promoting sodium retention in the
kidney.
[0344] The term "glucocorticoid" refers to any of a group of
natural or synthetic steroid hormones that control carbohydrate,
protein, and fat metabolism and have anti-inflammatory and/or
immunosuppressive properties. Suitable glucocorticoids for use in
certain embodiments of the present invention include, but are not
limited to, hydrocortisone, cortisone, corticosterone,
deoxycorticosterone, prednisone, prednisolone, methylprednisolone,
dexamethasone, betamethasone, mometasone, triamcinolone,
beclomethasone, fludrocortisone, aldosterone, fluticasone,
clobetasone, clobetasol, and loteprednol, and pharmaceutically
acceptable salts thereof, and mixtures thereof."
[0345] Steroids are often used in the treatment of various diseases
as they can help with treatment in a number of ways. In cancer
treatment, for example, steroids can reduce nausea associated with
chemotherapy and radiation, decrease inflammation, reduce allergic
reactions (before transfusions, for example), or simply to help
improve quality of life by enabling the patient to sleep, eat, and
feel better.
[0346] While the use of lipid nanoparticles is a proven delivery
platform, it is necessary to further expand on the therapeutic
utility and patient convenience of LNP by overcoming a number of
safety and tolerability concerns. In the clinic, and the absence of
premedication, most LNP treatment related adverse events are
consistent with infusion-type reactions associated with increases
in certain inflammatory biomarkers that may represent dose limiting
toxicities. (see, e.g., Judge et al., Review: Overcoming the Innate
Immune Response to Small Interfering RNA, Human Gene Therapy, 19,
2008)
[0347] The effects of co-formulating glucocorticoids into LNP have
been investigated, to see if inflammatory response following
intravenous administration could be dampened, and the therapeutic
index of the platform broadened. Preclinical data generated in
murine models have indicated that steroid co-formulation with LNP
is a viable strategy to achieve reduced immune stimulation, while
maintaining the same level of gene silencing ability. Furthermore,
early preclinical data in NHP and porcine models correlated well
with murine data, providing additional assurance to this novel
strategy.
General Procedures
Lipid Nanoparticle Formulating
[0348] Lipid nanoparticles were made by either direct dilution or
in-line dilution methods described by Jeffs et al. (see U.S. Pat.
No. 9,005,654). Lipid composition typically contained the following
lipids in the respective molar ratios, except where otherwise
noted: PEG-lipid (PEG2000-C-DMA, 1.1 mol %); Cationic lipid
(Compound 13, 54.9 mol %); cholesterol (33.0 mol %); and
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, 11.0 mol %).
Nucleic acid was solubilized in 20 mM EDTA, pH 4.5. The solutions
were combined in a T-connector at a flow rate of 400 mL/min,
diluting (in-line or directly into) with PBS at pH 7.4. Ethanol was
then removed and carrier buffer replaced with PBS, pH 7.4 by
tangential flow ultrafilitration using Midgee hoop cartridges (MW
cut off of 500K, GE Healthcare). The LNP were sterile filtered (0.2
.mu.m syringe filter) and sample concentration determined by either
DENAX-HPLC or RiboGreen Assay. Particle size and polydispersity
were determined using a Malvern Nano Series Zetasizer. Final lipid
and steroid concentrations were determined by UPLC.
Animal Models
LPS-Primed Cytokine Mouse Model
[0349] Female ICR mice (n=5 per group, 5-6 weeks old) are
pre-treated with 0.05 mg/kg of lipopolysaccharide (LPS) at time=0
to prime the immune system. At t=2 h they receive LNP at a 1 mg/kg
dose by intravenous administration. Blood samples are collected
into sodium EDTA microtainer tubes at 4 h post-LNP treatment
(terminal bleed) and are processed to plasma by centrifugation at
16000.times.g for 5 min at 16.degree. C. Plasma samples are
analyzed for Interleukin-1 beta (IL-1.beta.), Interleukin-6 (IL-6),
and Monocyte Chemotactic Protein 1 (MCP-1) cytokine levels by
ELISA.
Acute Cytokine Mouse Model
[0350] Female ICR mice (n=5 per group, 5-6 weeks old) are treated
with 10 mg/kg of LNP by intravenous administration. At 2 h
post-treatment, blood is collected into tubes containing 50 mM EDTA
via tail nicks and processed to plasma by centrifugation at
16000.times.g for 5 min at 16.degree. C. At 6 h post-treatment,
terminal blood samples are collected into sodium EDTA microtainer
tubes and are processed to plasma using the same procedure as
above. Plasma samples are analyzed by ELISA for Interleukin-6
(IL-6) and Monocyte Chemotactic Protein 1 (MCP-1) cytokine
levels.
Activity Mouse Model
[0351] LNP formulated with a siRNA targeting Apolipoprotein B
(ApoB) were administered IV to generate dose response curve
(typical doses: 0.01 mg/kg to 0.05 mg/kg total siRNA) in female
Balb/C mice (n=3 per group, 5-6 weeks old). Terminal time point is
at 48 hours post-intravenous administration of LNP. The left liver
lobe is collected into RNAlater and assayed for ApoB levels by
QuantiGene 2.0 analysis. Results are normalized to the house
keeping gene GAPDH.
Example 1: LNP Containing Dexamethasone 21-Palmitate in LPS-Primed
Cytokine Mouse Model
[0352] Dexamethasone 21-palmitate (Dex-P) is effectively a pro-drug
of dexamethasone, requiring enzymatic action to release the steroid
(Dexamethasone). This is less favorable compared to the steroid in
its free form (since it requires conversion to the active form in
vivo); however, dexamethasone 21-palmitate has a higher log P than
free dexamethasone, facilitating better incorporation into LNP.
[0353] Using the base composition described in General Procedures,
a titration series of compositions was prepared with a steadily
increasing Dex-P content (0.5 mol %, 2 mol %, or 5 mol % Dex-P).
Dex-P was simply included as an additional lipid component in the
90% ethanol lipid stock. Particle characteristics were essentially
identical to the base composition (Table 1).
TABLE-US-00001 TABLE 1 Formulation characteristics of Base
Formulation vs Dex-P LNP Formulation Z avg (nm) PDI % Encap Base
Formulation 77 0.04 98 Base + 5% DexP 77 0.03 98 Base + 2% DexP 77
0.02 99 Base + 0.5% DexP 74 0.03 98
[0354] The above formulations were tested in the LPS-primed mouse
cytokine model described in General Procedures. As an additional
control, the Base Formulation was co-administered with free
Dexamethasone. The free steroid was administered intravenously at a
dose of 0.3 mg/kg (a clinically relevant dose).
[0355] ICR mice pre-treated with lipopolysaccharide (LPS) at 0.05
mg/kg induced cytokine response compared to animals pre-treated
with PBS. Following intravenous administration of LNP, there were
further increases in all 3 cytokines. Representative cytokine data
(MCP-1) is shown in Table 2. The cytokine levels were highest for
the group treated with the Base Formulation in the absence of
steroid. Co-formulating Dex-P with the LNP reduced cytokine levels
significantly. Furthermore, the cytokine levels were similar
between the low (0.5 mol %) and high (5 mol %) steroid doses when
co-formulated in LNP, indicating a potentially saturating effect.
With 5 mol % Dex-P incorporated into the LNP, the equivalent dose
of free dexamethasone administered in a single intravenous
injection was 0.3 mg/kg. The cytokine levels achieved with Dex-P
LNP at this dose was similar to co-administering the control LNP
with 0.3 mg/kg of free dexamethasone in two separate, consecutive
injections.
TABLE-US-00002 TABLE 2 MCP-1 ELISA Results of Base Composition vs
Dex-P LNP in LPS-primed Cytokine Mouse Model Average MCP-1 Stdev
Pre-Treatment Treatment (pg/mL) (pg/mL) PBS PBS 95 35 LPS PBS 4170
2345 LPS 1.1:55 (`Base`) 39403 12135 LPS Base + 5% DexP 8978 5817
LPS Base + 2% DexP 13039 4824 LPS Base + 0.5% DexP 12899 4705 LPS
1.1:55 (`Base`) 0.3 mg/kg Free Dex 8801 4327
Example 2: LNP Containing Dexamethasone 21-Palmitate in Acute
Cytokine Mouse Model
[0356] The concept of corticosteroid co-formulation was further
supported with data from the acute cytokine model. The same
formulation panel was tested (dosed at 10 mg/kg in this model) and
data again indicated that co-formulated Dex-P LNP is an effective
means of reducing immune-stimulation, achieving similar cytokine
levels to co-administering LNP with free dexamethasone, at the same
or lower doses (Table 3).
TABLE-US-00003 TABLE 3 MCP-1 ELISA Results of Base Composition vs
Dex-P LNP in Acute Cytokine Mouse Model 2h 6h Average MCP-1 Stdev
Average MCP-1 Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS
88 14 104 18 1.1:55 (`Base`) 3804 1145 1377 553 Base + 5% DexP 104
24 118 43 Base + 2% DexP 148 66 139 84 Base + 0.5% DexP 193 62 203
61 1.1:55 (`Base`) 0.3 mg/kg Free Dex 190 132 252 155
Example 3: LNP Containing Reduced Dexamethasone 21-Palmitate in LPS
Primed Model
[0357] The concentrations of Dex-P were titrated down further (to
0.5 mol %, 0.1 mol % and 0.01 mol %). Post-formulation analysis by
UPLC indicated that Dex-P was readily incorporated into the
particles. Particle characteristics were comparable between LNP
formulations (Table 4).
TABLE-US-00004 TABLE 4 Formulation characteristics of Base
Formulation vs Dex-P LNP Formulation Z-avg (nm) PDI % Encap Base
Formulation 75 0.10 99 Base + 0.5% DexP 74 0.03 99 Base + 0.1% DexP
75 0.07 98 Base + 0.01% DexP 74 0.01 98
[0358] Results for a representative cytokine readout (MCP-1) are
shown in Table 5. A correlation between Dex-P concentration and
cytokine levels was observed (more Dex-P gave a better reduction in
cytokines). The 0.5% Dex-P LNP is equivalent to approximately a
0.03 mg/kg dose of free Dexamethasone. Data comparing these two
groups showed that the reduction in cytokines is significantly
better when the corticosteroid was incorporated in the LNP. In
fact, this LNP (0.5% Dex-P) actually performed as well as a 0.3
mg/kg dose of free Dexamethasone--a 10-fold greater dose of
corticosteroid. It was hypothesized that this surprising result may
be due to much more effective, `targeted delivery` of the
corticosteroid. Immune cells which take up LNP, and may otherwise
have triggered an immune response, are simultaneously receiving the
immune suppressive corticosteroid.
TABLE-US-00005 TABLE 5 MCP-1 ELISA Results of Base Composition vs
Dex-P LNP in LPS-primed Cytokine Mouse Model Average MCP-1 Stdev
Pre-Treatment Treatment (pg/mL) (pg/mL) PBS PBS 135 21 LPS PBS 3821
432 LPS 1.1:55 (`Base`) 28020 6224 LPS Base + 0.5% DexP 12304 7171
LPS Base + 0.1% DexP 24119 9935 LPS Base + 0.01% DexP 29276 5392
LPS 1.1:55 (`Base`) 0.3 mg/kg Free Dex 13982 4350 LPS 1.1:55
(`Base`) 0.03 mg/kg Free Dex 27893 7566 LPS 1.1:55 (`Base`) 0.006
mg/kg Free Dex 23901 4254
Example 4: Activity of LNP Co-Formulated with Dex-P
[0359] To verify that incorporation of Dex-P into LNP did not
impact potency, formulations were assessed in the Activity Mouse
Model described in General Procedures. Samples containing various
amounts of Dex-P were tested at 2 doses; 0.025 mg/kg and 0.05
mg/kg. Similar silencing activity was observed for all formulations
at each dose. The Base Formulation containing 0.5% Dex-P and a
non-targeting control siRNA (siLuc-2) was included as a negative
control. No silencing is expected or observed with this payload.
Results are shown in Table 6.
TABLE-US-00006 TABLE 6 ApoB Silencing Activity of 1.1:55 Base vs
LNP co-formulated with Dex-P in Mouse Model Treatment Dose (mg/kg)
% PBS % Error PBS n/a.sup.1 100 21 1.1:55 (`Base`) 0.025 55 6 0.05
47 7 Base + 5% Dex-P 0.025 81 12 0.05 46 5 Base + 2.5% Dex-P 0.025
57 19 0.05 34 6 Base + 0.5% Dex-P 0.025 54 8 0.05 35 10 Base + 0.1%
Dex-P 0.025 67 8 0.05 36 14 Base + Free Dex 0.025 64 15 0.05 29 6
Base + 0.5% Dex-P (siLuc-2) 0.025 86 8 0.05 92 20
.sup.1 PBS administered intravenously at 10 mL/kg.
Example 5: LNP Containing Clobetasol in LPS Primed Mouse Model
[0360] It was then attempted to co-formulate the corticosteroid
clobetasol-17-propionate (clobetasol) into LNP, again to study its
ability to suppress inflammatory responses to the particle. A 40 mM
acetate, pH 4.5 buffer was used to solubilize the nucleic acid.
Final lipid composition is shown in Table 7. Clobetasol, having a
lower log P (.about.3.5), didn't incorporate as readily into the
LNP as Dex-P (log P.about.9). Therefore an 8-fold greater
concentration of clobetasol than was actually desired in the end
product was input into the process. Analysis by UPLC confirmed
incorporation of clobetasol.
TABLE-US-00007 TABLE 7 Lipid composition of 1.1:55 Base vs LNP
containing Clobetasol Lipid Composition (mol %) PEG2000-C- DMA
Compound 13 Cholesterol Phospholipid.sup.1 Clobetasol 1.1:55
(`Base`) 1.1 55 33 11 -- Base + 1% 1.1 55 33 11 1 Clobetasol
.sup.11,2-di-O-octadecyl-sn-glycero-3-phosphocholine used instead
of DSPC in LNP containing Clobetasol
[0361] As outlined in Table 8, formulation characteristics were
comparable between the two compositions. Particle size,
polydispersity and payload encapsulation indicated uniform particle
populations.
TABLE-US-00008 TABLE 8 Characterization of 1.1:55 Base vs LNP
containing Clobetasol Formulation Z avg (nm) PDI % Encap Base
Formulation 79 0.04 96 Base + 1% clobetasol 65 0.04 98
[0362] The two compositions were assessed in the LPS-primed mouse
model. A representative cytokine readout (MCP-1) is shown in Table
9. The inflammatory response to the LNP is significantly reduced
with the clobetasol LNP. The MCP-1 levels are similar to LNP
containing Dex-P; however, clobetasol is not a pro-drug, and does
not rely on enzymatic action for activation. This is a significant
advantage over Dex-P as this corticosteroid would work
spontaneously upon delivery of the LNP to its targeted site of
action.
TABLE-US-00009 TABLE 9 MCP-1 ELISA Results of Base Composition vs
LNP containing Clobetasol in LPS-primed Cytokine Mouse Model
Average MCP-1 Stdev Pre-Treatment Treatment (pg/mL) (pg/mL) PBS PBS
201 13 LPS PBS 3877 2138 LPS 1.1:55 (`Base`) 49069 9784 LPS Base +
1% Clobetasol 11550 7598
Example 6: LNP Containing Clobetasol in Acute Cytokine Mouse
Model
[0363] The same LNP compositions (Base and Base+Clobetasol) were
tested in the acute cytokine model. MCP-1 and IL-6 cytokine levels
were measured at both 2 h and 6 h time points. Both cytokines
reported significantly lower levels for the co-formulated
clobetasol LNP. The latter formulation reported only baseline
levels of IL-6 at both time points (Table 10).
TABLE-US-00010 TABLE 10 IL-6 ELISA Results of Base Composition vs
LNP containing Clobetasol in Acute Cytokine Mouse Model 2 h 6 h
Average IL-6 Stdev Average IL-6 Stdev Treatment (pg/mL) (pg/mL)
(pg/mL) (pg/mL) PBS 17 4 10 7 1.1:55 (`Base`) 2232 493 152 137 Base
+ 1% Clobetasol 27 13 5 2
[0364] An expanded acute cytokine study was performed with more
time points, to ensure that the peak cytokine response had not
simply shifted to an earlier or later time point with the
clobetasol LNP. Peak cytokine response is usually observed at 2 h
post-LNP treatment in this model. Results confirmed that this
steroid was effective at suppressing the immune response throughout
the time course of the study (Table 11).
TABLE-US-00011 TABLE 11 MCP-1 ELISA Results of Base Composition vs
LNP containing Clobetasol in an Expanded Time Course Study, Acute
Cytokine Mouse Model Base + 1% PBS 1.1:55 (`Base`) Clobetasol Time
Avg Avg Avg Point MCP-1 Stdev MCP-1 Stdev MCP-1 Stdev (h) (pg/mL)
(pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) 0.25 74 10 83 13 68 5 0.5
150 52 314 196 75 9 1 91 7 356 202 99 7 2 100 12 1447 602 248 198 3
98 21 946 815 186 156 4 113 22 646 411 191 99 6 84 16 735 561 106
24
Example 7: Activity of LNP Containing Clobetasol in a Mouse Dose
Response Study
[0365] To verify that incorporation of clobetasol into LNP and the
slight modifications to the process did not impact potency,
formulations were assessed in the Activity Model described in
General Procedures. Similar silencing activity was observed for
both formulations at each dose (Table 12). This highlights that the
clobetasol LNP have a dramatically improved Therapeutic Index; they
have equal potency, but far superior tolerability.
TABLE-US-00012 TABLE 12 ApoB Silencing Activity of 1.1:55 Base
Composition vs LNP containing Clobetasol Treatment Dose (mg/kg) %
PBS % Error PBS n/a.sup.1 100 20 1.1:55 (`Base`) 0.01 45 7 0.025 21
1 0.05 12 1 Base + 1% 0.01 44 11 Clobetasol 0.025 18 5 0.05 9 1
.sup.1PBS administered intravenously at 10 mL/kg.
Example 8: Immune Suppression with LNP Containing Ciclesonide
[0366] Next the corticosteroid Ciclesonide was tested. Final lipid
composition is shown in Table 13. Analysis by UPLC confirmed
incorporation of Ciclesonide. Ciclesonide has a log P of .about.5.3
and incorporated readily into the particle.
TABLE-US-00013 TABLE 13 Lipid composition of 1.1:55 Base vs LNP
containing Clobetasol Lipid Composition (mol %) PEG2000-C- DMA
Compound 13 Cholesterol DSPC Ciclesonide Base Formulation 1.1 55 33
11 -- Base + 2% Ciclesonide 1.1 55 33 11 2
[0367] As outlined in Table 14, formulation characteristics were
comparable between the two compositions. Particle size,
polydispersity and payload encapsulation indicated uniform particle
populations.
TABLE-US-00014 TABLE 14 Characterization of 1.1:55 Base vs LNP
containing Clobetasol Formulation Z-avg (nm) PDI % Encap Base
Formulation 78 0.06 96 Base + 2% Ciclesonide 77 0.04 97
[0368] The Ciclesonide LNP were tested in the LPS-primed mouse
model, and compared to the Base formulation as well as Dex-P LNP.
The Ciclesonide LNP also exhibited a significantly reduced
inflammatory cytokine response, compared to the Base formulation.
Data for a representative cytokine (MCP-1) is shown in Table
15.
TABLE-US-00015 TABLE 15 MCP-1 ELISA Results of Base Composition vs
LNP containing Steroid in LPS-primed Cytokine Mouse Model Average
MCP-1 Stdev Pre-Treatment Treatment (pg/mL) (pg/mL) PBS PBS 170 9
LPS PBS 6012 2774 LPS 1.1:55 (`Base`) 54909 18542 LPS Base + 2%
Dex-P 26468 4561 LPS Base + 2% Ciclesonide 17913 4352
Example 9: Activity of LNP Containing Ciclesonide
[0369] Ciclesonide LNP were also assessed in the Activity Model
described in General Procedures. Like Dex-P LNP, the Ciclesonide
LNP exhibited similar potency to the Base Formulation (Table
16).
TABLE-US-00016 TABLE 16 ApoB Silencing Activity of 1.1:55 Base
Composition vs LNP containing Ciclesonide and Dex-P Treatment Dose
(mg/kg) % PBS % Error PBS n/a.sup.1 100 17 1.1:55 (`Base`) 0.01 31
3 0.025 16 2 0.05 12 2 Base + 2% Dex-P 0.01 43 11 0.025 17 9 0.05
17 3 Base + 2% 0.01 48 16 Ciclesonide 0.025 27 2 0.05 13 4
.sup.1PBS administered intravenously at 10 mL/kg.
Example 10: LNP Containing Clobetasol Reduced Immune-Stimulation in
Non-Human Primates
[0370] An intravenous pharmacology study was conducted in
cynomolgus monkeys to evaluate a clobetasol LNP formulation, and
compared to a control formulation (the `Base Formulation` used
previously). Similar to Example 5, an 8-fold greater concentration
of clobetasol was input into the process to get the desired 1 mol %
in the final composition (Table 17). A 40 mM acetate, pH 4.5 buffer
was used to solubilize the nucleic acid.
TABLE-US-00017 TABLE 17 Lipid composition of 1.1:55 Base vs LNP
containing Clobetasol Lipid Composition (mol %) PEG-2000- Compound
C-DMA 13 Cholesterol DSPC Clobetasol 1.1:55 (`Base`) 1.1 55 33 11
-- Base + 1% 1.0 61 18.5 18.5 1 Clobetasol
[0371] LNP formulation was administered to a group of four
cynomolgus monkeys (Cambodian origin; 2 males, 2 females, 2-5 years
of age) via a 60 minute intravenous infusion at a dose of 2.0 mg/kg
total siRNA. Blood draws at pre-dose and 2, 6, and 24 h
post-infusion were tested for a panel of inflammatory markers. The
clobetasol LNP demonstrated a significant reduction in a number of
inflammatory markers, further confirming the effectiveness of this
strategy. Table 18 compares MCP-1, IL-6, and IL-1ra (Interleukin-1
receptor antagonist) levels between the two compositions at 6 hours
post-infusion.
TABLE-US-00018 TABLE 18 Comparison of Inflammatory Response (at 6
hours post- infusion) to Base Composition vs LNP containing
Clobetasol in Cynomolgus Monkeys Avg Avg Avg MCP-1 Stdev IL-6 Stdev
IL-1ra Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL)
(pg/mL) Saline 660 334 26 31 420 344 1.1:55 5548 7280 703 1164 2571
3114 (`Base`) Base + 1% 214 28 7 3 204 160 Clobetasol
Example 11: An Examination of the Cardiovascular Effects of a
Single Infusion of LNP Formulations in Anesthetized Female
Gottingen Mini-Pigs
[0372] Test article-related effects of a single 1-hour infusion of
LNP formulations on hemodynamic parameters and inflammatory
biomarkers were evaluated in anesthetized female Gottingen
mini-pigs. LNP were formulated with the compositions in Table 19.
Similar to Example 5, an 8-fold greater concentration of clobetasol
was input into the process to get the desired 1 mol % in the final
composition. A 40 mM acetate, pH 4.5 buffer was used to solubilize
the nucleic acid.
TABLE-US-00019 TABLE 19 Lipid composition of 1.1:55 Base vs LNP
containing Clobetasol Lipid Composition (mol %) PEG-2000- Compound
C-DMA 13 Cholesterol DSPC Clobetasol 1.1:55 (`Base`) 1.1 55 33 11
-- Base + 1% 1.0 61 18.5 18.5 1 Clobetasol
[0373] Three (3) naive mini-pigs were surgically instrumented and
baseline data was collected prior to a single administration of
vehicle (saline), base LNP, or clobetasol LNP via a 60 minute
intravenous infusion. LNP formulations were administered at a dose
of 0.3 mg/kg total nucleic acid. Just prior to the first infusion,
and at approximately 5, 60, 90, 120, 180, and 240 minutes after the
start of infusion, a blood sample was taken, processed to plasma
and serum, and analyzed for cytokines and thromboxane
(11-dehydrothromboxane B2). Hemodynamic data was collected
continuously throughout the experiment, for a total time period of
4 hours. At 4 hours post-infusion, pigs were euthanized under
anesthesia via barbiturate overdose.
[0374] Treatment with the Base Formulation resulted in
considerable, measureable increases in thromboxane (Table 20) and
cytokines (Table 21) with the Base Formulation. Furthermore,
hemodynamic changes such as increases in pulmonary artery pressure
were observed (Table 22). These parameters are all indicative of an
inflammatory response to the Base LNP that is suppressed by
incorporation of clobetasol into the LNP formulation. This is
likely a glucocorticoid-mediated inhibition of phospholipase A2 and
transrepression of inflammatory cytokine transcription when the
animals are treated with co-formulated steroid LNP.
TABLE-US-00020 TABLE 20 Comparison of Thromboxane Levels between
1.1:55 Base and LNP containing Clobetasol in Anesthetized Mini-Pigs
TXB2 Change from Baseline (pg/mL) Time Relative to Start of
Infusion (minutes) -15 5 60 90 120 180 240 Saline Mean 0.0 -9.2 1.5
-13.0 -13.0 -7.0 -9.0 sem 0.0 3.5 3.6 2.3 2.3 1.5 2.2 1.1:55 Mean
0.0 4.8 21.5 24.4 19.4 14.0 4.8 (`Base`) sem 0.0 2.0 1.4 8.1 7.2
6.6 2.7 Base + 1% Mean 0.0 -7.3 9.9 -0.9 -0.1 -7.3 -7.3 Clobetasol
sem 0.0 7.3 8.5 0.9 7.3 7.3 7.3
TABLE-US-00021 TABLE 21 Comparison of IL-6 Cytokine Levels between
1.1:55 Base and LNP containing Clobetasol in Anesthetized Mini-Pigs
Fold Change in IL-6 from Baseline Time Relative to Start of
Infusion (minutes) -15 5 60 90 120 180 240 Saline Mean 1.0 0.9 3.3
5.4 8.0 16.6 40.3 sem 0.0 0.1 1.5 2.3 1.6 1.4 20.0 1.1:55 Mean 1.0
1.0 4.2 9.0 21.4 76.5 210.5 (`Base`) sem 0.0 0.0 3.2 8.0 15.9 39.9
58.9 Base + 1% Mean 1.0 0.9 3.9 10.0 15.8 1.0 0.4 Clobetasol sem
0.0 0.5 2.9 9.0 14.7 0.6 0.3
TABLE-US-00022 TABLE 22 Comparison of Mean Pulmonary Artery
Pressure (PAP) between 1.1:55 Base and LNP containing Clobetasol in
Anesthetized Mini-Pigs Mean PAP--Change from Baseline (mmHg) Time
Relative to Start of Infusion (minutes) -15 0 15 30 60 90 120 150
180 240 Saline Mean 0 0 0 0 0 0 0 0 0 0 sem 0 0 0 1 1 1 1 1 1 1
1.1:55 Mean 0 0 4 1 4 6 4 3 2 1 (`Base`) sem 0 0 2 0 2 3 2 2 1 1
Base + 1% Mean 0 0 4 1 1 1 1 1 0 1 Clobetasol sem 0 0 0 0 1 1 1 0 0
1
Example 12 Co-Formulated Clobetasol is Effective at Reducing Immune
Response to LNP Bearing mRNA Payloads
[0375] The concept of steroid co-formulation with LNP was further
tested bearing an mRNA payload. This example demonstrates how a
reduction in mRNA-LNP immune stimulation can be achieved by
incorporation of the corticosteroid clobetasol.
[0376] LNP (PL-containing & PL-free) were prepared by the
direct dilution method described by Jeffs et al. In brief, lipid
stocks were prepared in 100% ethanol at a total lipid concentration
of 6-7 mg/mL. An mRNA transcript encoding Luciferase (TriLink
BioTechnologies), a reporter gene, was solubilized in 40 mM EDTA,
pH 4.5 at 0.366 mg/mL. Equal volumes of these solutions were
combined in a T-connector at a flow rate of 250 mL/min, immediately
diluting into PBS (4.times. volume of lipid stock) at pH 7.4.
Ethanol was then removed and carrier buffer was replaced with PBS
by dialysis (Slide-A-Lyzer unit, MWCO 10k), dialyzing overnight
against 100 volumes of PBS. Following dialysis the formulations
were concentrated to .about.0.3 mg/mL using VivaSpin concentrator
units (MWCO 100,000). As with siRNA formulations, the low log P of
clobetasol necessitated it being input at .about.8.times. the
amount desired in the final composition, as only .about.15%
incorporates in the LNP particles. The remainder is lost during
dialysis. The LNP samples were sterile filtered (0.2 .mu.m syringe
filter) and sample concentration determined by RiboGreen Assay.
Particle size and polydispersity were determined using a Malvern
Nano Series Zetasizer. The amount of clobetasol and other lipids in
the final composition was determined by UPLC and is displayed in
Table 23.
TABLE-US-00023 TABLE 23 Formulation characteristics of Base
Formulation vs LNP containing Clobetasol Lipid Composition (mol %)
Characterization Phos- % PEG2000- Cmpd Chol- pho Clo- Size En-
C-DMA 13 esterol lipid .sup.1 betasol (nm) PDI cap 1.6:55 1.6 55 33
11 0 73 0.07 97 Base Base + 1.6 55 33 11 1 67 0.04 96 1.0% Clo-
betasol .sup.1 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine
[0377] Prior to injection formulations were diluted to 0.05 mg/mL.
Balb/c mice (n=5) were injected at 0.5 mg/kg (mRNA) via the
intravenous route through the lateral tail. Four hours following
injection the animals were euthanized with a lethal dose of
ketamine/xylazine. A small amount (20 .mu.L) of the terminal blood
was collected into a tube containing 5 .mu.L of 50 mg/L heparin,
while the rest of the blood was collected into sodium EDTA
microtainer tubes. All of these tubes were centrifuged for 5 min at
16000.times.g & 16.degree. C. to isolate plasma. A small
portion (.about.200 mg) of the left lateral lobe of the liver was
collected and stored overnight in RNALater @ 4.degree. C.
[0378] The heparin plasma (diluted 1:4000 with ELISA Diluent) was
used in a standard Murine EPO ELISA analysis (kit from R&D
Systems). As shown in Table 24, the incorporation of clobetasol
within the mRNA-LNP possibly resulted in a slight reduction in
potency, though likely within the boundary of experimental
variability. The regular level of EPO seen within the plasma of
these mice is 0.1-0.2 ng/mL. Therefore, the incorporation of
clobetasol within the mRNA-LNP does not really affect the efficacy
of the formulation.
TABLE-US-00024 TABLE 24 Efficacy of Murine mEPO-LNP in Liver 4 h
Post-IV Administration of Base Composition vs LNP containing
Clobetasol Treatment mEPO (ng/mL) Stdev (ng/mL) 1.6:55 (`Base`)
6041 1305 Base + Clobetasol 4438 781
[0379] To assess the effect on immune stimulation, EDTA plasma
samples were diluted (1:8) and analyzed for cytokines (MCP-1 and
IL-6) by ELISA (ELISA assays and capture & detection antibodies
from BD Biosciences). Table 25 demonstrates that cytokine
production is significantly reduced when clobetasol is incorporated
in the formulation.
TABLE-US-00025 TABLE 25 MCP-1 and IL-6 ELISA Results of Base
Compositions vs LNP containing Clobetasol MCP-1 IL-6 Average Stdev
Average Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 57 4 15
3 1.6:55 (`Base`) 5911 3592 353 194 Base + Clobetasol 479 146 78
24
[0380] The liver IFIT (Interferon Induced proteins with
Tetratricopeptide repeats) response to the two formulations was
also measured. The IFIT biomarker indicates a type I interferon
response to the payload. Liver samples (20-25 mg) were homogenized
and the QuantiGene 2.0 assay (Affymetrix) used to assess IFIT
levels in the liver (normalized to the housekeeping gene GAPDH).
Results are plotted as a fold increase over the PBS control group,
and demonstrate that clobetasol co-formulation is also effective in
suppressing the IFIT response to the mRNA payload (Table 26).
TABLE-US-00026 TABLE 26 Hepatic IFIT Induction of Base Composition
vs LNP containing Clobetasol Fold Increase Over Treatment PBS Stdev
1.6:55 (`Base`) 591 116 Base + Clobetasol 334 31
[0381] Taken together these results demonstrate that the
incorporation of clobetasol into the LNP particle significantly
abrogates the immune stimulation to mRNA-LNP, and an increase in
the Therapeutic Index is observed.
Example 13 Co-Formulated Dexamethasone Palmitate is Effective at
Reducing Immune Response to LNP Bearing mRNA Payloads
[0382] This example demonstrates how a steroid pro-drug,
dexamethasone 21-palmitate (Dex-P), can also be used to reduce
inflammatory responses to LNP bearing and mRNA payload. An mRNA-LNP
containing Dex-P was formulated as described in Example 12, with
the compositions described in Table 27. A murine EPO mRNA
transcript (TriLink BioTechnologies) was used for the payload.
TABLE-US-00027 TABLE 27 Formulation characteristics of Base
Formulation vs LNP containing Dex-P Lipid Composition (mol %)
Characterization PEG2000- Cmpd Chol- Dex- Size % C-DMA 13 esterol
DSPC P (nm) PDI Encap 1.1:55 1.1 55 33 11 0 85 0.06 97 Base Base +
2% 1.1 55 33 11 2 81 0.05 97 Dex-P
[0383] The formulations were injected intravenously at a dose of
0.5 mg/kg (total mRNA) into Balb/C mice (n=5). Six hours following
injection, the animals were euthanized with a lethal dose of
ketamine/xylazine. A small amount (75 .mu.L) of the terminal blood
was collected into a tube containing 5 .mu.L of 50 mg/L heparin,
while the rest of the blood was collected into sodium EDTA
microtainer tubes. Blood samples were centrifuged for 5 min at
16000.times.g & 16.degree. C. to isolate plasma.
[0384] The heparin plasma (diluted 1:4000 with ELISA Diluent) was
analyzed by ELISA for EPO levels (kit from R&D Systems). As
shown in Table 28, the incorporation of Dex-P within the mRNA-LNP
possibly resulted in an increase in potency, though possibly within
experimental variability.
TABLE-US-00028 TABLE 28 Efficacy of Murine mEPO-LNP in Liver 4 h
Post-IV Administration of Base Composition vs LNP containing Dex-P
Treatment mEPO (ng/mL) Stdev (ng/mL) 1.1:55 (`Base`) 2009 1179 Base
+ 2% Dex-P 3659 1313
[0385] EDTA plasma was analyzed by ELISA (BD Biosciences) for
cytokine levels (MCP-1 & IL-6). Table 29 demonstrates that
incorporation of Dex-P into the mRNA-LNP yields a marked reduction
in cytokine levels.
TABLE-US-00029 TABLE 29 MCP-1 and IL-6 ELISA Results of Base
Composition vs LNP containing Dex-P MCP-1 IL-6 Average Stdev
Average Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 36 15
27 13 1.1:55 (`Base`) 4471 1279 515 194 Base + 2% Dex-P 1281 737 83
16
This data demonstrates that incorporation of
dexamethasone-palmitate reduced the inflammatory response to
mRNA-LNP, and results significantly improved therapeutic index for
mRNA-LNP.
Examples 14-20. Reduced or Absent Phospholipid and High PEG in
LNP
[0386] The presence of phospholipid can reduce the shelf-life of
lipid nanoparticles (LNPs) because the phosphate ester bond is
thought to be quite labile. Most or all the phospholipid was
removed from LNPs to test the effect on shelf-life of LNP
comprising mRNA. During these experiments, it was discovered that
the immunogenicity of the formulations was greatly reduced without
affecting potency. As described herein, certain embodiments of the
present invention are directed to LNPs possessing the combination
of low or absent phospholipid and higher than usual PEG.
Interestingly, the combination of low phospholipid and high mol
percent PEG reduces immunostimulation for mRNA more effectively
than for siRNA.
[0387] In certain embodiments, the amount of PEG is at least 3 mole
percent (e.g., at least 3.1 mole percent, at least 3.2 mole
percent, at least 3.3 mole percent, at least 3.4 mole percent, at
least 3.5 mole percent, at least 3.6 mole percent, at least 3.7
mole percent, at least 3.8 mole percent, at least 3.9 mole percent,
at least 4 mole percent). With respect to phospholipid, in certain
embodiments, no phospholipid is used in the practice of the
invention. In certain embodiments, the lipid particle comprises
less than 2 mole percent phospholipid, e.g., 1.9 mol %
phospholipid, 1.8 mol % phospholipid, 1.7 mol % phospholipid, 1.6
mol % phospholipid, 1.5 mol % phospholipid, 1.4 mol % phospholipid,
1.3 mol % phospholipid, 1.2 mol % phospholipid, 1.1 mol %
phospholipid, 1.0 mol % phospholipid, 0.9 mol % phospholipid, 0.8
mol % phospholipid, 0.7 mol % phospholipid, 0.6 mol % phospholipid,
0.5 mol % phospholipid, 0.4 mol % phospholipid, 0.3 mol %
phospholipid, 0.2 mol % phospholipid, 0.1 mol % phospholipid, or
0.0% phospholipid, e.g., less than 1.9 mol % phospholipid, less
than 1.8 mol % phospholipid, less than 1.7 mol % phospholipid, less
than 1.6 mol % phospholipid, less than 1.5 mol % phospholipid, less
than 1.4 mol % phospholipid, less than 1.3 mol % phospholipid, less
than 1.2 mol % phospholipid, less than 1.1 mol % phospholipid, less
than 1.0 mol % phospholipid, less than 0.9 mol % phospholipid, less
than 0.8 mol % phospholipid, less than 0.7 mol % phospholipid, less
than 0.6 mol % phospholipid, less than 0.5 mol % phospholipid, less
than 0.4 mol % phospholipid, less than 0.3 mol % phospholipid, less
than 0.2 mol % phospholipid, less than 0.1 mol % phospholipid.
General Procedures
[0388] Lipid Nanoparticle Formulating
[0389] LNP formulations were prepared by the LipoMixer method
described by Jeffs et al, using either direct dilution or in-line
dilution. Lipid compositions were as described, typically
comprising 3 or 4 lipids in the molar ratios described. Lipids were
solubilized in 100% ethanol at a total lipid concentration of
approx. 12 mg/mL. Nucleic acid was solubilized in 20 mM EDTA, pH
4.5 when phospholipid is present in the LNP and in 40 mM EDTA, pH
4.5 in the absence of phospholipid. Equal volumes of these
solutions were combined in a T-connector at a flow rate of 400
mL/min, immediately diluting (in-line or directly into) with PBS
(4.times. volume of lipid stock) at pH 7.4. Ethanol was then
removed and carrier buffer was replaced with PBS by either dialysis
(Slide-A-Lyzer unit, MWCO 10k) or tangential flow ultrafilitration
using Midgee hoop cartridges (MWCO 500k, GE Healthcare). The LNP
samples were sterile filtered (0.2 .mu.m syringe filter) and sample
concentration determined by either DENAX-HPLC or RiboGreen Assay.
Particle size and polydispersity were determined using a Malvern
Nano Series Zetasizer. Final lipid were determined by UPLC.
Animal Models
[0390] 3 animal models were used to assess formulations; two models
of immune stimulation, and one of potency. Descriptions as
follows:
[0391] LPS-Primed Cytokine Mouse Model
[0392] Female ICR mice (n=5, 5-6 weeks old) are pre-treated with
0.05 mg/kg of lipopolysaccharide (LPS) at time=0 to prime the
immune system. At t=2 h they receive LNP at a 1 mg/kg dose by
intravenous administration. Blood samples are collected into sodium
EDTA microtainer tubes at 4 h post-LNP treatment (terminal bleed)
and are processed to plasma by centrifugation at 16000.times.g for
5 min at 16.degree. C. Plasma samples are analyzed for IL-10, IL-6,
and MCP-1 cytokine levels by ELISA.
[0393] Acute Cytokine Mouse Model
[0394] Female ICR mice (n=5, 5-6 weeks old) are treated with 10
mg/kg of LNP by intravenous administration. At 2 h post-treatment,
blood is collected into tubes containing 50 mM EDTA via tail nicks
and processed to plasma by centrifugation at 16000.times.g for 5
min at 16.degree. C. At 6 h post-treatment, terminal blood samples
are collected into sodium EDTA microtainer tubes and are processed
to plasma using the same procedure as above. Plasma samples are
analyzed by ELISA for IL-6 and MCP-1 cytokine levels.
[0395] Activity Mouse Model
[0396] LNP formulated with a siRNA targeting Apolipoprotein B.
Administered IV to generate dose response curve (typical doses:
0.01 mg/kg to 0.05 mg/kg total siRNA encapsulated in LNP) in female
Balb/C mice (n=3, 5-6 weeks old). Terminal time point is at 48
hours post-intravenous administration of LNP. The left liver lobe
is collected into RNAlater and assayed for ApoB levels by
QuantiGene 2.0 analysis. Results are normalized to the house
keeping gene GAPDH, and expressed as a % of the PBS control. A
group with an ApoB readout that is `20% of PBS` has experienced
more profound gene silencing activity than one with `80% of
PBS`.
Example 14: Removing Phospholipid from LNP Formulation Reduces
Immune Stimulation without Impairing Potency
[0397] Two LNP formulations were made with an oligonucleotide siRNA
payload targeting Apolipoprotein B (ApoB). A `base` composition,
and another with the phospholipid omitted (`phospholipid free`, or
`PL-free`). Composition details are outlined in Table 30.
TABLE-US-00030 TABLE 30 Lipid composition of a 1.6:55 (`Base`) and
1.8:61 (`PL-free`) Formulation Lipid Composition (mol %)
PEG-2000-C- Cationic DMA Compound 13 Cholesterol DSPC 1.6:55
(`Base`) 1.6 55 33 11 1.8:61 (`PL- 1.8 61 37 0 free`)
[0398] In the LPS-primed mouse cytokine model, results clearly
indicate that the PL-free LNP is less stimulatory than its parent
composition that contains phospholipid (Table 31).
TABLE-US-00031 TABLE 31 MCP-1 ELISA Results of Base Composition vs
PL-free Composition in LPS-Primed Cytokine Mouse Model Average
MCP-1 Stdev Pre-Treatment Treatment (pg/mL) (pg/mL) PBS PBS 160 11
LPS PBS 3237 782 LPS 1.6:55 (`Base`) 35691 17620 LPS 1.8:61
(`PL-free`) 10485 3545
[0399] While reducing inflammatory responses is an important
objective, it is also important that potency is not simultaneously
impaired. The same panel of LNP were therefore assessed in the ApoB
Silencing Activity Model (described under General Procedures). Gene
silencing data (Table 32) reveals that removing the phospholipid
does not impair potency.
TABLE-US-00032 TABLE 32 ApoB Silencing Activity of Base Composition
vs PL-free Composition in Activity Mouse Model Treatment Dose
(mg/kg) % PBS % Error PBS n/a.sup.1 100 9.2 0.01 45 8.6 1.6:55
(`Base`) 0.025 17 2.1 0.05 11 1.3 0.01 43 8.6 1.8:61 (`PL-free`)
0.025 29 1.5 0.05 20 5.2
.sup.1 PBS administered intravenously at 10 mL/kg.
Example 15: Increasing PEG Lipid Content in Phospholipid-Free LNP
Resulted in Further Reductions in Cytokine Levels, without
Significantly Impacting Potency
[0400] The following LNP were formulated using an ApoB siRNA
payload. Starting from a `base` composition, the phospholipid was
first removed, and then PEG content either doubled or tripled
(Table 33):
TABLE-US-00033 TABLE 33 Lipid composition of a 1.1:55 Base and
PL-free Formulations with Increasing PEG Content Lipid Composition
(mol %) PEG-2000-C- Cationic DMA Compound 13 Cholesterol DSPC
1.1:55 ("Base") 1.1 55 33 11 1.1:57 (PL-free) 1.1 57 42 0 2.2:56
(PL-free) 2.2 56 42 0 3.3:55 (PL-free) 3.3 55 41 0
This panel of LNP was evaluated in the acute mouse cytokine model,
with time points at 2 h and 6 h. Again a reduction in cytokines was
observed when phospholipid was removed from the composition.
Representative cytokine data (MCP-1) is displayed in Table 34.
Subsequently, as the PEG content was increased, an even more
pronounced reduction in cytokines was observed.
TABLE-US-00034 TABLE 34 MCP-1 ELISA Results of 1.1:55 Base
Composition vs PL-free Compositions with Increasing PEG Lipid
Content in Acute Cytokine Mouse Model 2 h 6 h Average Average MCP-1
Stdev MCP-1 Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 29
4 36 6 1.1:55 ("Base") 1967 916 973 801 1.1:57 (PL-free) 1072 317
745 342 2.2:56 (PL-free) 704 147 183 73 3.3:55 (PL-free) 157 72 45
7
Interestingly, the potency of these high PEG, PL-free compositions
was not significantly impacted (Table 35). In previous experience,
formulations with higher PEG content were often found to have
compromised activity. For example three (3) related LNP in a
similar PEG titration series (Table 36), but with a standard
phospholipid content, were evaluated and representative activity
data is shown in Table 37. A significant attenuation of activity if
observed as PEG content increases. It is therefore surprising to
see the significant reduction in cytokines without a more profound,
commensurate loss of potency with the high PEG, PL-free systems
above.
TABLE-US-00035 TABLE 35 ApoB Silencing Activity of 1.1:55 Base
Composition vs PL-free Compositions with Increasing PEG Lipid
Content in Activity Mouse Model Treatment Dose (mg/kg) % PBS %
Error PBS n/a.sup.1 100 13 0.01 37 2 1.1:55 (`Base`) 0.025 15 2
0.05 9 3 0.01 37 7 1.1:57 (`PL-free`) 0.025 21 5 0.05 12 2 0.01 45
7 2.2:56 (`PL-free) 0.025 20 5 0.05 11 2 0.01 48 7 3.3:55
(`PL-free`) 0.025 31 8 0.05 22 6 .sup.1PBS administered
intravenously at 10 mL/kg.
TABLE-US-00036 TABLE 36 Lipid composition of a 1.1:55 Base and
PL-containing LNP with Increasing PEG Content Lipid Composition
(mol %) PEG-2000-C- Cationic DMA Compound 13 Cholesterol DSPC
1.1:55 ("Base") 1.1 55 33 11 2.2:54 2.2 54 33 11 4.3:53 4.3 53 32
11
TABLE-US-00037 TABLE 37 ApoB Silencing Activity of 1.1:55 Base and
PL-containing LNP with Increasing PEG content in Activity Mouse
Model Dose % % Treatment (mg/kg) PBS Error PBS n/a.sup.1 100 30
1.1:55 (`Base`) 0.05 16 5 2.2:55 0.05 33 3 4.4:55 0.05 100 38
Example 16: Reduction in Phospholipid Content Also Effective at
Reducing Inflammatory Response
[0401] It was hypothesized that it may not be necessary to
completely remove phospholipid from the LNP formulation to abrogate
immune stimulation, and that this effect can be achieved by simply
reducing the PL content. A panel of compositions was prepared
(Table 38), using reduced PL content (3 mol % or 8 mol %) vs the
parent composition (11 mol %):
TABLE-US-00038 TABLE 38 Lipid composition of a 1.1:55 Base and
PL-containing LNP with Reduced PL Content Lipid Composition (mol %)
Cationic PEG-2000- Compound Choles- C-DMA 13 terol DSPC 1.1:55
("Base") 1.1 55 33 11 1.1:57 1.1 57 34 8 1.1:55 1.1 55 41 3 1.6:52
1.6 52 39 8 1.6:55 1.6 55 41 3
These compositions were assessed for immune stimulation in the
acute cytokine model. As seen in Table 39, the compositions with
reduced phospholipid had a reduced tendency to stimulate the
production of cytokines. Further, reduction of phospholipid content
had no significant impact on potency (Table 40).
TABLE-US-00039 TABLE 39 MCP-1 ELISA Results of 1.1:55 Base vs
PL-containing LNP with Reduced PL Content in Acute Cytokine Mouse
Model 2 h 6 h Average Average MCP-1 Stdev MCP-1 Stdev Treatment
(pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 721 487 85 23 1.1:55 ("Base")
3644 3250 894 759 1.1:57 831 786 388 295 1.1:55 335 213 117 39
1.6:52 447 174 76 14 1.6:55 221 159 91 35
TABLE-US-00040 TABLE 40 ApoB Silencing Activity of 1.1:55 Base
Composition vs PL-containing LNP with Reduced PL Content in Mouse
Model Treatment Dose (mg/kg) % PBS % Error PBS n/a.sup.1 100 6
1.1:55 (`Base`) 0.01 47 4 0.025 18 2 1.1:57 0.01 37 4 0.025 15 2
1.1:55 0.01 60 13 0.025 20 5 1.6:52 0.01 66 14 0.025 27 3 1.6:55
0.01 46 10 0.025 28 2 .sup.1PBS administered intravenously at 10
mL/kg.
Example 17 Removing Phospholipid Reduces Immune Stimulation of LNP
Containing mRNA without Impairing Potency
[0402] This example demonstrates how a reduction in mRNA-LNP immune
stimulation can be achieved by removing the phospholipid from the
LNP composition (i.e. production of a PL-free mRNA-LNP). The
mRNA-LNP formulations were made with the lipid compositions shown
in Table 41. Two different base formulations (1.1:55 and 1.6:55)
had their phospholipid content removed (`PL-free` compositions),
whilst either maintaining or slightly increasing the cholesterol
content.
TABLE-US-00041 TABLE 41 Formulation characteristics of Base
Formulations vs PL-free mLuc-LNP Lipid Composition (mol %)
Characterization PEG-2000- Cmpd Chol- Size % C-DMA 13 esterol DSPC
(nm) PDI Encap 1.1:55 1.1 55 33 11 104 0.01 96 (`Base`) 1.6:55 1.6
55 33 11 80 0.11 99 (`Base`) 1.1:57 1.1 57 42 0 106 0.08 96
(`PL-free`) 1.7:57 1.7 57 42 0 94 0.08 98 (`PL-free`) 1.8:61 1.8 61
37 0 99 0.09 95 (`PL-free`)
LNP (PL-containing & PL-free) were prepared by the direct
dilution method described by Jeffs et al. In brief, lipid stocks
were prepared in 100% ethanol at a total lipid concentration of 6-7
mg/mL. An mRNA transcript encoding Luciferase, a reporter gene, was
solubilized in 40 mM EDTA, pH 4.5 at 0.366 mg/mL. Equal volumes of
these solutions were combined in a T-connector at a flow rate of
250 mL/min, immediately diluting into PBS (4.times. volume of lipid
stock) at pH 7.4. Ethanol was then removed and carrier buffer was
replaced with PBS by dialysis (Slide-A-Lyzer unit, MWCO 10k),
dialyzing overnight against 100 volumes of PBS. Following dialysis
the formulations were concentrated to .about.0.6 mg/mL using
VivaSpin concentrator units (MWCO 100,000). The LNP samples were
sterile filtered (0.2 .mu.m syringe filter) and sample
concentration determined by RiboGreen Assay. Particle size and
polydispersity were determined using a Malvern Nano Series
Zetasizer.
[0403] Prior to injection, LNP were diluted to 0.05 mg/mL. Balb/c
mice (n=4) were administered doses (using a volume dose of 10
mL/kg) of 0.5 mg/kg (total mRNA) via lateral tail vein. Animals
were euthanized at 4 h post-treatment with ketamine/xylazine.
Terminal blood was collected into sodium EDTA microtainer tubes and
centrifuged for 5 min at 16000.times.g & 16.degree. C. to
isolate plasma. Liver sections were collected into FastPrep tubes
and stored at -80.degree. C. until analysis.
[0404] To assess potency, livers were homogenized in 1.times. Cell
Culture Lysis Reagent (CCLR) buffer, then analyzed for luciferase
activity using the Luciferase Assay (Promega). As seen in Table 42,
there is no detrimental effect on potency when phospholipid was
removed from the compositions.
TABLE-US-00042 TABLE 42 Luciferase Gene Expression in Liver 6 h
Post-IV Administration of Base Compositions vs PL-free mLuc-LNP
Luciferase Activity Stdev Treatment (ng/g liver) (ng/g) 1.1:55
(`Base`) 849 33 1.6:55 (`Base`) 1092 42 1.1:57 (`PL-free`) 991 27
1.7:57 (`PL-free`) 846 22 1.8:61 (`PL-free`) 1156 65
[0405] To assess the effect on immune stimulation, plasma samples
were diluted (1:8) and analyzed for cytokines (MCP-1 and IL-6) by
ELISA (ELISA assays and capture & detection antibodies from BD
Biosciences). Table 43 demonstrates that cytokine production is
significantly reduced for both of the `Base` formulations when the
phospholipid is removed. The effect on the 1.1:55 Base is
particularly profound.
TABLE-US-00043 TABLE 43 MCP-1 and IL-6 ELISA Results of Base
Compositions vs PL-free mLuc-LNP MCP-1 IL-6 Average Stdev Average
Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 28 7 14 5
1.1:55 (`Base`) 8022 3166 566 177 1.6:55 (`Base`) 2293 293 136 23
1.1:57 (`PL-free`) 1434 657 41 29 1.7:57 (`PL-free`) 705 144 28 6
1.8:61 (`PL-free`) 540 82 23 6
[0406] Taken together these results demonstrate that the use of
PL-free formulations results in a reduction in the immune
stimulation of the formulation compared to PL-containing
formulations. Given that the efficacy does not seem to be affected
this should result in an increase in the therapeutic index of the
mRNA-LNP.
Example 18 Removing Phospholipid Reduces Immune Stimulation of LNP
Containing mRNA without Impairing Potency
[0407] The result in Example 17 was further corroborated with a
subset of the compositions formulated with two different mRNA
transcripts, mLuc (Table 44a) & mEPO (Table 44b). Transcripts
used encoded either luciferase (reporter gene) or erythropoietin (a
hormone that controls erythropoiesis, or red blood cell
production).
TABLE-US-00044 TABLE 44a Formulation characteristics of 1.6:55 Base
Formulation vs PL-free mLuc-LNP Lipid Composition (mol %)
Characterization PEG- Com- % 2000-C- pound Chol- Size En- DMA 13
esterol DSPC (nm) PDI cap 1.6:55 1.6 55 33 11 77 0.10 95 (`Base`)
1.7:57 1.7 57 42 0 80 0.06 93 (`PL-free`) 1.8:61 1.8 61 37 0 90
0.06 91 (`PL-free`)
TABLE-US-00045 TABLE 44b Formulation characteristics of 1.6:55 Base
Formulation vs PL-free mEPO-LNP Lipid Composition (mol %)
Characterization PEG- Com- % 2000-C- pound Chol- Size En- DMA 13
esterol DSPC (nm) PDI cap 1.6:55 1.6 55 33 11 76 0.10 95 (`Base`)
1.7:57 1.7 57 42 0 78 0.09 95 (`PL-free`) 1.8:61 1.8 61 37 0 82
0.02 93 (`PL-free`)
[0408] The mRNA-LNP were prepared as described in Example 17.
Female Balb/C (n=4) received a 0.5 mg/kg (mRNA) intravenous dose of
LNP. Six hours later, animals were euthanized with
ketamine/xylazine. A small amount (75 .mu.L) of the terminal blood
was collected into a tube containing 18.8 .mu.L of 50 mg/L heparin,
while the rest of the blood was collected into sodium EDTA
microtainer tubes. All of these tubes were centrifuged for 5 min at
16000.times.g & 16.degree. C. and plasma was isolated. Also,
part of the liver was collected into FastPrep tubes which were
placed at -80.degree. C. until analysis.
[0409] The livers were homogenized in 1.times.CCLR buffer and were
analyzed for luciferase activity using the Luciferase Assay
(Promega). As can be seen in Table 45 the efficacy of the PL-free
mRNA-LNP formulations are similar to those of the Base
(PL-containing) formulation.
TABLE-US-00046 TABLE 45 Luciferase Gene Expression in Liver 6 h
Post-IV Administration of Base Compositions vs PL-free mLuc-LNP
Luciferase Activity Stdev Treatment (ng/g liver) (ng/g liver)
1.6:55 (`Base`) 1708 73 1.7:57 (`PL-free`) 1651 70 1.8:61
(`PL-free`) 1867 62
[0410] The heparin plasma (diluted 1:4000 with ELISA Diluent) was
used in a standard Murine EPO ELISA analysis (R&D Systems). As
seen in Table 46, the efficacy trend for the mEPO-LNP was similar
to that seen with the mLuc-LNP where the efficacies of the PL-free
Luc mRNA-LNP are similar to those of the Base (PL-containing)
formulation.
TABLE-US-00047 TABLE 46 Efficacy of Murine mEPO-LNP in Liver 6 h
Post-IV Administration of Base Compositions vs PL-free mEPO-LNP
Treatment mEPO (ng/mL) Stdev (ng/mL) PBS 0.124 0.009 1.6:55
(`Base`) 2839 313 1.7:57 (`PL-free`) 2460 611 1.8:61 (`PL-free`)
3986 426
[0411] To assess immune stimulation, plasma samples were analyzed
by ELISA for cytokines MCP-1 and IL-6 (ELISA assays and
capture/detection antibodies from BD Biosciences). Results in Table
47a (mEPO) and Table 47b (mLuc) show again how the inflammatory
response to the PL-free LNP compositions markedly reduced compared
to the Base composition. This is true for both sets of LNP
(luciferase and EPO). Given that the efficacy of these PL-free
compositions has not been impaired, their reduced cytokine
induction furnishes a significantly improved therapeutic index.
TABLE-US-00048 TABLE 47a MCP-1 and IL-6 ELISA Results of Base
Compositions vs PL-free mEPO-LNP MCP-1 IL-6 Average Stdev Average
Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 47 3 15 3
1.6:55 (`Base`) 3947 440 236 29 1.7:57 (`PL-free`) 873 431 39 14
1.8:61 (`PL-free`) 851 110 43 17
TABLE-US-00049 TABLE 47b MCP-1 and IL-6 ELISA Results of Base
Compositions vs PL-free mLuc-LNP MCP-1 IL-6 Average Stdev Average
Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 47 5 14 3
1.6:55 (`Base`) 2474 446 208 31 1.7:57 (`PL-free`) 1231 687 27 6
1.8:61 (`PL-free`) 856 745 30 8
Example 19 PL-Free LNP with Increased PEG Content are Less
Stimulatory
[0412] This example demonstrates how a further reduction in
mRNA-LNP immune stimulation can be achieved by increasing the PEG
component of the PL-free mRNA-LNP, again without impairing
activity. The following mRNA-LNP compositions (Table 48) were
prepared as described in Example 17, with an EPO mRNA payload:
TABLE-US-00050 TABLE 48 Formulation characteristics of 1.6:55 Base
Formulation vs PL-free mRNA-LNP Lipid Composition (mol %)
Characterization PEG- Com- % 2000-C- pound Chol- Size En DMA 13
esterol DSPC (nm) PDI cap 1.6:55 1.6 55 33 11 73 0.07 97 (`Base`)
1.8:61 1.8 61 37 0 86 0.06 96 (`PL-free`) 3.3:55 3.3 55 41 0 59
0.06 96 (`PL-free`)
[0413] Female Balb/C (n=4) then received a 0.5 mg/kg (mRNA)
intravenous administration of LNP. Six hours later, animals were
euthanized with ketamine/xylazine. Blood was collected into sodium
EDTA microtainer tubes. All of these tubes were centrifuged for 5
min at 16000.times.g & 16.degree. C. and plasma was isolated.
Plasma samples were analyzed by ELISA for cytokines MCP-1 and IL-6
(ELISA assays and capture/detection antibodies from BD
Biosciences). Results in Table 49 shows again how inflammatory
response to the PL-free LNP compositions markedly reduced compared
to the Base composition. Given that the efficacy of these PL-free
compositions has not been impaired, their reduced cytokine
induction furnishes a significantly improved therapeutic index.
TABLE-US-00051 TABLE 49 MCP-1 and IL-6 ELISA Results of Base
Compositions vs High PEG, PL-free mEPO-LNP MCP-1 IL-6 Average Stdev
Average Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 57 4 15
3 1.6:55 (`Base`) 2875 2317 244 112 1.8:61 (`PL-free`) 1204 460 77
22 3.3:55 (`PL-free`) 712 186 37 11
[0414] These results demonstrate that by increasing the amount of
PEG lipid to 3.3 mol % within the PL-free formulation (3.3:55
Compound 13 PL-free mRNA-LNP), a further reduction in the immune
stimulation of the mRNA-LNP can be achieved. The potency of the
3.3:55 composition was next examined.
Example 20 High PEG, PL-Free mRNA-LNP Remain Surprisingly Potent,
and Exhibit a Larger Therapeutic Index
[0415] This example demonstrates that the high PEG, PL-free 3.3:55
mRNA-LNP composition is just as efficacious as the Base,
PL-containing composition. It again shows the reduced immune
stimulation of the 3.3:55 composition and therefore the improved
Therapeutic Index. The following LNP were prepared using the
process described in Example 17 (Table 50), with an EPO
payload:
TABLE-US-00052 TABLE 50 Formulation characteristics of 1.6:55 Base
Formulation vs High PEG, PL-free mEPO-LNP Lipid Composition (mol %)
Characterization PEG- Com- % 2000-C- pound Chol- Size En- DMA 13
esterol DSPC (nm) PDI cap 1.6:55 1.6 55 33 11 74 0.07 97 (`Base`)
3.3:55 3.3 55 41 0 63 0.06 96 (`PL-free`)
[0416] Female Balb/C (n=4) then received a 0.5 mg/kg (mRNA)
intravenous administration of LNP. Six hours later, animals were
euthanized with ketamine/xylazine. At time points of 2 h, 3 h, 4 h
& 5 h blood draws were performed and 20 .mu.L of the blood was
collected into a tube containing 5 .mu.L of 50 mg/L heparin. Then
at 6 hour time point, the animals were euthanized with a lethal
dose of ketamine/xylazine. A small amount (20 .mu.L) of the
terminal blood was collected into a tube containing 5 .mu.L of 50
mg/L heparin, while the rest of the blood was collected into sodium
EDTA microtainer tubes. Tubes were centrifuged for 5 min at
16000.times.g & 16.degree. C. and to isolate plasma.
[0417] Heparin plasma analyzed by ELISA (kit from R&D Systems)
for EPO concentrations. As seen in Table 51, at all the time points
the efficacy of the PL-containing mRNA-LNP and the 3.3:55 PL-free
formulation are equivalent.
TABLE-US-00053 TABLE 51 Efficacy of Murine mEPO-LNP in Liver 6 h
Post-IV Administration of Base Compositions vs High PEG, PL-free
mEPO-LNP Time 1.6:55 (`Base`) 3.3:55 (`PL-free`) Point EPO Stdev
EPO Stdev (h) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 2 5065 773 4832 843 3
6174 514 6412 1188 4 5194 485 5902 1093 5 3961 643 4313 768 6 2936
528 3055 521
[0418] Plasma samples were analyzed by ELISA for cytokines MCP-1
and IL-6 (ELISA assays and capture/detection antibodies from BD
Biosciences). Results in Table 52 corroborate the data in Example
19, and show again how the inflammatory response to the high PEG,
PL-free LNP compositions is markedly reduced compared to the Base
composition.
TABLE-US-00054 TABLE 52 MCP-1 and IL-6 ELISA Results of Base
Compositions vs High PEG, PL-free mEPO-LNP MCP-1 IL-6 Average Stdev
Average Stdev Treatment (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 67 3 22
4 1.6:55 (`Base`) 2402 1317 163 74 3.3:55 (`PL-free`) 485 210 36
12
[0419] Taken together these results demonstrate that by using a
PL-free formulation the therapeutic index of an mRNA-LNP
formulation can be increased and further by changing the
formulation to include higher amounts of PEG (e.g., 3.3 mol %) the
therapeutic index can be further advanced.
Example 21 Use of HPLC Purified mRNA for Immune Stimulation
Abrogation of mRNA-LNP
[0420] This example demonstrates how a reduction in mRNA-LNP immune
stimulation can be achieved by replacing regular silica membrane
purified mRNA with Reverse Phase (RP) HPLC-purified mRNA in LNP.
Murine erythropoietin (EPO) mRNA purified by either method was
formulated in LNP and injected via the iv route into Balb/C mice.
Four hours following injection the animals were sacrificed and
plasma & liver tissue were analyzed for efficacy and immune
stimulation.
[0421] LNP were prepared by the regular LipoMixer technology. In
brief, a 7.36 mg/mL lipid solution in 100% ethanol was prepared
containing the lipids DSPC:Chol:PEG.sub.2000-C-DMA:Compound 13 in
the following molar ratios: 10.9:32.8:1.64:54.6, mol %. The mRNA
payload was solubilized in 40 mM EDTA (pH 4.5) at a concentration
of 0.366 mg/mL. Equal volumes of each solution (1.6 mL) were
blended at 250 mL/min through a T-connector using the Direct
Dilution method described by Jeffs et al. The resulting mixture was
subsequently collected directly into a tube containing .about.4
volumes (5.9 mL) of PBS, pH 7.4. These formulations were placed in
Slide-A-Lyzer dialysis units (MWCO 10,000) and were dialyzed
overnight against 100 volumes of PBS, pH 7.4. Following dialysis
the formulations were concentrated to .about.0.6 mg/mL using
VivaSpin concentrator units (MWCO 100,000).
[0422] Female Balb/C mice (n=5) received an intravenous (tail vein)
administration of either PBS, or LNP bearing either a regularly
purified (silica membrane) or HPLC-purified mRNA payload. The mRNA
transcript encoded mouse erythropoietin (EPO). Each animal received
a dose corresponding to 0.5 mg/kg mRNA. Four hours following
injection the animals were euthanized with a lethal dose of
ketamine/xylazine. A small amount (20 .mu.L) of the terminal blood
was collected into a tube containing 5 .mu.L of 50 mg/L heparin,
while the rest of the blood was collected into Na EDTA microtainer
tubes. All tubes were centrifuged for 5 min at 16000.times.g &
16.degree. C. to isolate plasma. Half of the left lateral lobe was
collected into 1.5 mL of RNALater and stored at 4.degree. C. for at
least 16 h.
[0423] The heparin plasma (diluted 1:4000 with ELISA Diluent) was
used in a standard Murine EPO ELISA analysis (kit from R&D
Systems). As can be seen in Table 53 the efficacy of the LNP
containing the highly (HPLC) purified EPO mRNA is very similar to
that of the regular (Silica Membrane) purified EPO mRNA. The
regular level of EPO seen within the plasma of mice treated with
PBS is 100-200 pg/mL. Therefore, regardless of the purity of the
EPO mRNA incorporated the level of liver gene expression of EPO
from the encapsulated mEPO is extremely high.
[0424] To determine the level of immune stimulation caused by
mRNA-LNP, the fold increase of IFIT mRNA within the livers for the
LNP treated animals (over the PBS treated animals) was determined.
This is one example of an assay the art worker can use to assess
the level of immune stimulation caused by mRNA-LNP. In certain
embodiments, the HPLC-purified mRNA-LNP induces significantly less
immune stimulation than a control, which in certain embodiments is
characterized by the IFIT response. The IFN-inducible IFIT1 mRNA,
the most strongly induced mRNA in response to type I IFN, was used
as a more sensitive measure of immune stimulation. Strong IFIT1
mRNA induction in both liver and spleen can be observed in mice
treated with LNP bearing nucleic acids, even in the absence of
detectable plasma IFN protein. This likely reflects local IFN
induction that does not manifest as a systemic cytokine response.
In the IFIT1 assay, the amount of IFIT1 mRNA in hepatocytes was
quantified and normalized to mRNA levels of a housekeeping gene
(usually GAPD), which typically remains constant. A QuantiGene 2.0
kit (a branched DNA based assay) from Affymetrix was used to
determine mRNA levels of both genes.
[0425] Approximately 20-25 mg of the left lateral lobe of the
livers (stored in RNALater) were homogenized. Relative mRNA levels
of both IFIT & and the house keeping gene GAPD were determined
using the QuantiGene 2.0 Assay. IFIT readouts were normalized to
GAPD for each group of animals, and expressed as a fold increase
over the PBS control group. The results in Table 54 demonstrate
that LNP bearing an mRNA payload purified by silica membrane
yielded a 627-fold increase in IFIT, vs the PBS group. Mice treated
with LNP bearing an HPLC-purified payload exhibited only a 21-fold
increase vs PBS. Thus they are approx. 30-fold less stimulatory
than particles bearing silica-membrane purified mRNA. In certain
embodiments, the lipid nanoparticle formulation having
HPLC-purified mRNA has an IFIT response that is no more than 30
fold greater than a reference IFIT response of phosphate buffered
saline. In certain embodiments, the lipid nanoparticle formulation
having HPLC-purified mRNA has an IFIT response that is no more than
10 or 20 or 30 or 40 or 50 or 60 or 70 or 80 or 90 or 100 fold
greater than a reference IFIT response of phosphate buffered
saline.
[0426] Further comparison of immune stimulatory capacity was
afforded by analysis of the EDTA plasma in cytokine (MCP-1 and
IL-6) ELISA assays. Plasma was diluted (1:8 or 1:80) in ELISA
diluent and was analyzed for levels of MCP-1 & IL-6 presence
using ELISA assays with capture & detection antibodies from BD
Biosciences. The level of MCP-1 is dramatically reduced (37106
pg/mL down to 105 pg/mL) with the incorporation of the HPLC
purified mRNA. Similarly, another cytokine (IL-6) is also
dramatically reduced (4805 pg/mL reduced to 22 pg/mL) when using
the HPLC purified mRNA. Results are shown in Table 55.
[0427] Taken together these results demonstrate that LNP particles
bearing an HPLC-purified mRNA payload are significantly less
stimulatory than those with a less refined (e.g. regular silica
membrane purified) mRNA payload. Given that the efficacy is not
adversely affected, this represents a dramatic increase in the
therapeutic index of the mRNA-LNP.
TABLE-US-00055 TABLE 53 EPO Efficacy of 0.5 mg/kg 1.6:55 Compound
13 Containing Regular Silica Membrane Purified or HPLC Purified
Murine mEPO 4 h Following IV Dosing in Balb/C Mice (n = 5) EPO
Stdev Treatment (ng/mL) (ng/mL) 1.6:55 (`Base`) with Regular
Purified Murine 4050 450 mEPO 1.6:55 (`Base`) with HPLC Purified
Murine mEPO 4525 385
TABLE-US-00056 TABLE 54 IFIT Induction of 0.5 mg/kg 1.6:55 Compound
13 Containing Regular Silica Membrane Purified or HPLC Purified
Murine mEPO 4 h Following IV Dosing in Balb/C Mice (n = 5) IFIT
(Fold Increase Over PBS) Treatment Average Stdev PBS 1.0 0.3 1.6:55
(`Base`) with Regular Purified 627 52 Murine mEPO 1.6:55 (`Base`)
with HPLC Purified 21 12 Murine mEPO
TABLE-US-00057 TABLE 55 MCP-1 and IL-6 ELISA Results in Balb/C Mice
4 h Following IV Dosing of 0.5 mg/kg of 1.6:55 Compound 13
Containing Regular Silica Membrane Purified or HPLC Purified Murine
mEPO (n = 5) MCP-1 IL-6 Average Stdev Average Stdev Treatment
(pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS 56 3 17 2 1.6:55 (`Base`) with
Regular 37106 12621 4805 1075 Purified Murine mEPO 1.6:55 (`Base`)
with HPLC 105 18 22 6 Purified Murine mEPO
[0428] All documents cited herein are incorporated by reference.
While certain embodiments of invention are described, and many
details have been set forth for purposes of illustration, certain
of the details can be varied without departing from the basic
principles of the invention.
[0429] The use of the terms "a" and "an" and "the" and similar
terms in the context of describing embodiments of invention are to
be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. In addition to the order detailed herein, the
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate embodiments of invention and does not necessarily impose
a limitation on the scope of the invention unless otherwise
specifically recited in the claims. No language in the
specification should be construed as indicating that any
non-claimed element is essential to the practice of the
invention.
* * * * *
References