U.S. patent application number 16/038107 was filed with the patent office on 2019-02-28 for cationic lipid compositions for tissue-specific delivery.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Xavier de MOLLERAT du JEU, Shikha MISHRA.
Application Number | 20190060482 16/038107 |
Document ID | / |
Family ID | 63104088 |
Filed Date | 2019-02-28 |
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United States Patent
Application |
20190060482 |
Kind Code |
A1 |
MISHRA; Shikha ; et
al. |
February 28, 2019 |
CATIONIC LIPID COMPOSITIONS FOR TISSUE-SPECIFIC DELIVERY
Abstract
Provided herein are, inter alia, compositions and methods useful
for the in vivo delivery of bioactive agents (e.g., therapeutic or
diagnostic agents). The compositions provided herein include
cationic lipids, helper lipids and a biostability enhancing agent,
which together form a lipid aggregate with the bioactive agent and
allow for the systemic delivery of the bioactive agent to, for
example, lung tissue without the requirement for biomolecular
targeting.
Inventors: |
MISHRA; Shikha; (San Diego,
CA) ; de MOLLERAT du JEU; Xavier; (Encinitas,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
63104088 |
Appl. No.: |
16/038107 |
Filed: |
July 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62552783 |
Aug 31, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/549 20170801;
A61K 9/1272 20130101; C12N 15/88 20130101; A61K 47/6917 20170801;
A61P 11/00 20180101; A61K 47/541 20170801; A61K 31/7105 20130101;
C12N 9/22 20130101; A61K 9/0019 20130101; A61K 31/713 20130101 |
International
Class: |
A61K 47/69 20060101
A61K047/69; A61K 9/127 20060101 A61K009/127; C12N 9/22 20060101
C12N009/22; A61K 31/7105 20060101 A61K031/7105; A61K 47/54 20060101
A61K047/54; A61P 11/00 20060101 A61P011/00 |
Claims
1. A composition comprising: (i) a first cationic lipid at a
compositional molar ratio from about 0.18 to about 0.32 and of
formula: ##STR00013## wherein R.sup.1 and R.sup.2 are independently
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl; R.sup.3 and
R.sup.4 are independently hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; m is an integer from 1 to 6;
X.sub.a.sup.- is an anion; (ii) a second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula: ##STR00014## wherein R.sup.5 and R.sup.8 are independently
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R.sup.6 and R.sup.7 are independently substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; n is an integer from 1 to 6; and
X.sub.b.sup.- is an anion; (iii) a first helper lipid at a
compositional molar ratio from about 0.20 to about 0.32; (iv) a
second helper lipid at a compositional molar ratio from about 0.01
to about 0.14; and (v) a biostability enhancing agent at a
compositional molar ratio from about 0.01 to about 0.02.
2. The composition of claim 1, wherein m is an integer from about 1
to 5, about 1 to 4, about 1 to 3, or where m is 1, 2, 3, 4, 5, or
6.
3-10. (canceled)
11. The composition of claim 1, wherein n is an integer from about
1 to 5, about 1 to 4, about 1 to 3, or where n is 1, 2, 3, 4, 5, or
6.
12-19. (canceled)
20. The composition of claim 1, wherein said first cationic lipid
is present at a compositional molar ratio of about 0.18 about 0.23,
about 0.24, about 0.25, about 0.27, about 0.28, or about 0.32.
21-26. (canceled)
27. The composition of claim 1, wherein said first cationic lipid
has the formula: ##STR00015## wherein X.sub.a.sup.- is Cl.sup.- or
CH.sub.3COO.sup.-.
28. The composition of claim 27, wherein X.sub.a.sup.- is
CH.sub.3COO.sup.-.
29. The composition of claim 1, wherein said first cationic lipid
is dihydroxy dimyristyl spermidine.
30. The composition of claim 1, wherein said second cationic lipid
is present at a compositional molar ratio of about 0.24, about 0.38
about 0.39 about 0.40 about 0.45 about 0.47 about 0.51.
31-36. (canceled)
37. The composition of claim 1, wherein said second cationic lipid
has the formula: ##STR00016## wherein X.sub.b.sup.- is Cl.sup.- or
CH.sub.3COO.sup.-.
38. The composition of any one of claim 37, wherein X.sub.b.sup.-
is CH.sub.3COO.sup.-.
39. The composition of claim 1, wherein said second cationic lipid
is hydroxy dimyristyl spermidine.
40. The composition of claim 1, wherein said first helper lipid is
present at a compositional molar ratio of about 0.20, about 0.24,
about 0.26, or about 0.32.
41-43. (canceled)
44. The composition of claim 1, wherein said first helper lipid is
dioleoylphosphatidylethanolamine (DOPE).
45. The composition of claim 1, wherein said second helper lipid is
present at a compositional molar ratio of about 0.01, about 0.05,
about 0.08, about 0.10, about 0.14.
46-49. (canceled)
50. The composition of claim 1, wherein said second helper lipid is
cholesterol.
51. The composition of claim 1, wherein said biostability enhancing
agent is present at a compositional molar ratio of about 0.01 or
about 0.02.
52. (canceled)
53. The composition of claim 1, wherein said biostability enhancing
agent is a polyether compound, a PEGylated phospholipid, or
polyethylene glycol.
54. (canceled)
55. (canceled)
56. The composition of claim 1, wherein said biostability enhancing
agent has a molecular weight of about 750 g/mol, about 2000 g/mol,
or about 5000/mol.
57. (canceled)
58. (canceled)
59. The composition of claim 1, wherein said biostability enhancing
agent is C14 polyethylene glycol 750, C14 polyethylene glycol 2000,
or C14 polyethylene glycol 5000.
60-72. (canceled)
73. The composition of claim 1, further comprising a bioactive
agent.
74. The composition of claim 73, wherein said bioactive agent is a
therapeutic agent or a diagnostic agent.
75. The composition of claim 73, wherein said bioactive agent
comprises a nucleic acid, a ribonucleoprotein or a small
molecule.
76. The composition of claim 75, wherein said nucleic acid is an
mRNA, a siRNA, a miRNA or a guide RNA.
77. The composition of claim 76, wherein said bioactive agent
comprises a nucleic acid and a ribonucleoprotein.
78. The composition of claim 77, wherein said ribonucleoprotein is
CRISPR associated protein 9 (Cas9).
79. A pharmaceutical composition comprising a composition of claim
76 and a pharmaceutically acceptable excipient.
80. A cell comprising a composition of claim 76.
81-87. (canceled)
88. The cell of claim 80, wherein said cell is an epithelial cell,
an epithelial lung cell, an endothelial cell, or an endothelial
lung cell.
89-92. (canceled)
93. The cell of claim 80, wherein said cell is in the lung tissue
of a mammal, optionally wherein said mammal is a primate,
optionally wherein said primate is a human patient.
94. A method of delivering a bioactive agent to a cell, said method
comprising: (i) admixing an bioactive agent with a composition of
one of claim 1, thereby forming a bioactive agent-lipid complex;
(ii) contacting a cell with said bioactive agent-lipid complex,
thereby delivering said bioactive agent-lipid complex to said
cell.
95. (canceled)
96. The method of claim 94, wherein said bioactive agent is a
therapeutic agent or a diagnostic agent selected from a nucleic
acid, a ribonucleoprotein a small molecule, or combinations
thereof, wherein said nucleic acid is an mRNA, a siRNA, miRNA or
guide RNA, wherein said bioactive agent comprises a guide RNA and a
ribonucleoprotein, wherein said ribonucleoprotein is CRISPR
associated protein, optionally wherein said CRISPR associated
protein is bound to said guide RNA.
97-101. (canceled)
102. The method of claim 94, wherein said cell is a mammalian cell,
optionally wherein said mammalian cell is a primate cell.
103-108. (canceled)
109. The method of claim 94, wherein said cell is an epithelial
cell, an epithelial lung cell, an endothelial, or cell an
endothelial lung cell.
110-112. (canceled)
113. A method of delivering a bioactive agent to lung tissue in a
subject, said method comprising: (i) admixing an bioactive agent
with a composition of claim 1, thereby forming a bioactive
agent-lipid complex; (ii) systemically administering an effective
amount of said bioactive agent-lipid complex to a subject, thereby
delivering said bioactive agent-lipid complex to a lung tissue in a
subject.
114. (canceled)
115. A method of treating a pulmonary disease in a subject in need
thereof, said method comprising administering to a subject a
therapeutically effective amount of a bioactive agent and a
composition of claim 1, thereby treating said pulmonary disease in
said subject.
116. The method of claim 113, wherein said composition and said
bioactive agent are admixed prior to said administering.
117. The method of claim 113, wherein said bioactive agent
comprises a nucleic acid, a ribonucleoprotein or a small
molecule.
118. The method of claim 117, wherein said nucleic acid is an mRNA,
a siRNA, a miRNA or a guide RNA.
119. The method of one of claim 115, wherein said pulmonary disease
is asthma, chronic obstructive pulmonary disease (COPD), lung
cancer or cystic fibrosis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 62/552,783, filed Aug. 31, 2017, which
disclosure is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Delivery of payloads such as therapeutic nucleic acids to
specific tissues has traditionally been achieved using biomolecular
targeting via ligand or receptor expression on the surface of lipid
nanoparticles which serve as a delivery vehicle. Designing lipid
nanoparticles capable of targeting specific organs, tissues, or
cell types without the use of canonical biomolecular targeting
techniques has been a significant challenge. Manipulation of the
inherent properties of lipid nanoparticles affords a means of
achieving organ, tissue, and cell-type specific targeting. Provided
herein are compositions and methods which cure this and other needs
in the art.
BRIEF SUMMARY OF THE INVENTION
[0003] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio from about 0.18
to about 0.32 and of formula:
##STR00001##
wherein R.sup.1 and R.sup.2 are independently substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.3 and R.sup.4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; m is an integer from 1 to 6;
X.sub.a.sup.- is an anion; (ii) a second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula:
##STR00002##
wherein R.sup.5 and R.sup.8 are independently hydrogen, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.6 and R.sup.7 are
independently substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; n
is an integer from 1 to 6; and X.sub.b.sup.- is an anion; (iii) a
first helper lipid; (iv) a second helper lipid; and (v) a
biostability enhancing agent.
[0004] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio from about 0.18
to about 0.32 and of formula:
##STR00003##
wherein R.sup.1 and R.sup.2 are independently substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.3 and R.sup.4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; m is an integer from 1 to 6;
X.sub.a.sup.- is an anion; (ii) a second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula:
##STR00004##
wherein R.sup.5 and R.sup.8 are independently hydrogen, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.6 and R.sup.7 are
independently substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; n
is an integer from 1 to 6; and X.sub.b.sup.- is an anion; (iii) a
first helper lipid at a compositional molar ratio from about 0.20
to about 0.32; (iv) a second helper lipid; and (v) a biostability
enhancing agent.
[0005] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio from about 0.18
to about 0.32 and of formula:
##STR00005##
wherein R.sup.1 and R.sup.2 are independently substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.3 and R.sup.4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; m is an integer from 1 to 6;
X.sub.a.sup.- is an anion; (ii) a second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula:
##STR00006##
wherein R.sup.5 and R.sup.8 are independently hydrogen, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.6 and R.sup.7 are
independently substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; n
is an integer from 1 to 6; and X.sub.b.sup.- is an anion; (iii) a
first helper lipid at a compositional molar ratio from about 0.20
to about 0.32; (iv) a second helper lipid at a compositional molar
ratio from about 0.01 to about 0.14; and (v) a biostability
enhancing agent.
[0006] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio from about 0.18
to about 0.32 and of formula:
##STR00007##
wherein R.sup.1 and R.sup.2 are independently substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.3 and R.sup.4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl; m is an integer from 1 to 6;
X.sub.a.sup.- is an anion; (ii) a second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula:
##STR00008##
wherein R.sup.5 and R.sup.8 are independently hydrogen, substituted
or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl; R.sup.6 and R.sup.7 are
independently substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl; n
is an integer from 1 to 6; and X.sub.b.sup.- is an anion; (iii) a
first helper lipid at a compositional molar ratio from about 0.20
to about 0.32; (iv) a second helper lipid at a compositional molar
ratio from about 0.01 to about 0.14; and (v) a biostability
enhancing agent at a compositional molar ratio from about 0.01 to
about 0.02.
[0007] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.24,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.05, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0008] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.32,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.39, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.26, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0009] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0010] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.23,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.45, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0011] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.51, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0012] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.27,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0013] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.25,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.26, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0014] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.28,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.24, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.14, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
750.
[0015] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.47, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein said biostability enhancing agent is C14 polyethylene
glycol 5000.
[0016] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.24,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.40, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.24, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
2000.
[0017] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.32,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.08, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0018] In an aspect is provided a pharmaceutical composition
including a composition as provided herein including embodiments
thereof and a pharmaceutically acceptable excipient.
[0019] In an aspect is provided a cell including a composition as
provided herein including embodiments thereof. In embodiments, the
cell is a mammalian cell. In embodiments, the cell is a rodent
cell. In embodiments, the cell is a mouse cell. In embodiments, the
cell is a rat cell. In embodiments, the cell is a porcine cell. In
embodiments, the cell is a canine cell. In embodiments, the cell is
a primate cell. In embodiments, the cell is an epithelial cell. In
embodiments, the cell is an epithelial lung cell. In embodiments,
the cell is an endothelial cell. In embodiments, the cell is an
endothelial lung cell.
[0020] In an aspect is provided, method of delivering a bioactive
agent to a cell, the method including: (i) admixing an bioactive
agent with a composition as provided herein including embodiments
thereof, thereby forming a bioactive agent-lipid complex; (ii)
contacting a cell with the bioactive agent-lipid complex, thereby
delivering the bioactive agent-lipid complex to a cell.
[0021] In another aspect is provided a method of delivering a
bioactive agent to lung tissue in a subject, the method including:
(i) admixing an bioactive agent with a composition as described
herein including embodiments thereof, thereby forming a bioactive
agent-lipid complex; (ii) systemically administering an effective
amount of the bioactive agent-lipid complex to a subject, thereby
delivering the bioactive agent-lipid complex to a lung tissue in a
subject.
[0022] In another aspect is provided a method of expressing a
protein in lung tissue in a subject, the method including: (i)
admixing a mRNA with a composition as described herein including
embodiments thereof, thereby forming a mRNA-lipid complex; (ii)
administering an effective amount of the mRNA-lipid complex to a
subject; and (iii) allowing the mRNA of the mRNA-lipid complex to
express in lung tissue of the subject, thereby expressing a protein
in lung tissue in a subject.
[0023] In an aspect is provided a method of treating a pulmonary
disease in a subject in need thereof, the method including
administering to a subject a therapeutically effective amount of a
bioactive agent and a composition as described herein including
embodiments thereof, thereby treating a pulmonary disease in the
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A-IC. Based on mixture Design of Experiment
optimization, new LNP formulations were made, complexed with mRNA
using the previously developed protocol (FIGS. 1A-1C), and the
subsequent LNPs were screened for delivery in vivo using a
luciferase readout. Top performing formulations were then used to
model and predict second generation formulations for optimized
tissue specific expression. These new formulations were then tested
in vivo to identify the best LNP formulation. FIG. 1A. Cartoon
representation of method for generating new lipid nanoparticle
(LNP) formulations, testing in vivo, and optimizing. FIG. 1B.
Cartoon representation of steps involved in complexing the LNP
formulation with mRNA and testing in vivo. FIG. 1C. Written
procedure of steps involved in complexing the LNP formulation with
mRNA and testing in vivo.
[0025] FIG. 2. In vivo luciferase assay workflow for LNP
testing.
[0026] FIGS. 3A-3E. Systemic delivery of LNPs results in varied
biodistribution patterns. Whole animal in vivo imaging revealed
organ specific biodistribution patterns. FIG. 3A shows through
whole animal imaging that LNPs distributed in the spleen. FIG. 3B
shows through whole animal imaging that LNPs distributed in the
lung. FIGS. 3C-3E show biodistribution patterns of LNPs determined
from imaging ex vivo isolated organ tissue.
[0027] FIGS. 4A-4B. Performance optimization screening. FIG. 4A.
Lung directed delivery: ex vivo quantification. FIG. 4B. Spleen
directed delivery: ex vivo quantification.
[0028] FIGS. 5A-5C. Dose dependent protein expression. FIG. 5A.
Lung delivery in vivo luciferase expression. FIG. 5B. Lung radiance
as a function of mRNA dose titration (3 mg/kg, 1 mg/kg, 0.5 mg/kg)
quantified 4 hours (4 h) post-injection. FIG. 5C. Lung directed
delivery: in vivo quantification. In vivo radiance over time
(hours) quantified for each dose titration (3 mg/kg, 1 mg/kg, 0.5
mg/kg).
[0029] FIGS. 6A-6C. Time course of protein expression. FIG. 6A. Ex
vivo lung bioluminescence signal. FIG. 6B. In vivo bioluminescence
signal. FIG. 6C. Quantification of ex vivo lung luciferase
signal.
[0030] FIGS. 7A-7J. Toxicity--Cytokine panel. Quantification of
circulating cytokine levels in mouse serum at 2 hrs, 4 hrs, 24 hrs
and 48 hrs post-injection under two mRNA dose conditions: low (1
mg/kg) and high (3 mg/kg). FIG. 7A. Levels of IL5. FIG. 7B. Levels
of IL4. FIG. 7C. Levels of IL2. FIG. 7D. Levels of IL10. FIG. 7E.
Levels of IL1b. FIG. 7F. Levels of IL6. FIG. 7G. Levels of IFNg.
FIG. 7H. Levels of GMCSF. FIG. 7I. Levels of IL12. FIG. 7J. Levels
of TNFa.
[0031] FIGS. 8A-8B. N/P ratio variance allows for tailoring of mRNA
delivery and expression, resulting in exclusive tissue expression
patterns. In vivo whole animal and ex vivo isolated organ imaging
indicates varying N/P ratio can lead to exclusion of protein
expression from certain organs resulting in specific expression in
the lung (FIG. 8A) or spleen (FIG. 8B).
[0032] FIG. 9. Lung delivery quantification assay: Luciferase
assay.
[0033] FIGS. 10A-10B. Low performance formulations. Luciferase
activity of low performance formulations with (FIG. 10B) and
without (FIG. 10A) the best formulation as a benchmark to
compare.
[0034] FIGS. 11A-11F. Jet PEI.RTM. vs Bruce #3.14. In vivo whole
animal imaging shows Bruce #3.14 lipid nanoparticle formulation has
specific biodistribution patterns (FIG. 11A) compared with Jet
PEI.RTM. (FIG. 11B). Ex vivo isolated organ tissue imaging also
demonstrates that Bruce #3.14 lipid nanoparticle formulation has
specific biodistribution patterns (FIG. 11C) compared with Jet
PEI.RTM. (FIG. 11D). FIG. 11E shows Bruce #3.14 lipid nanoparticle
formulation and Jet PEI.RTM. flux. Two animals in the Jet PEI.RTM.
group died. FIG. 11F shows flux in lung after treatment with Bruce
#3.14 lipid nanoparticle formulation or Jet PEI.RTM..
[0035] FIGS. 12A-12C. Formulation #3.10 treatment. FIG. 12A shows
organ flux luciferase activity across organs. FIG. 12B shows in
vivo whole animal luminescence. FIG. 12C shows ex vivo isolated
organ tissue luminescence.
[0036] FIGS. 13A-13C. Formulation #3.14 treatment. FIG. 13A shows
organ flux luciferase activity across organs. FIG. 13B shows in
vivo whole animal luminescence. FIG. 13C shows ex vivo isolated
organ tissue luminescence.
[0037] FIGS. 14A-14C. Formulation #3.19 treatment. FIG. 14A shows
organ flux luciferase activity across organs. FIG. 14B shows in
vivo whole animal luminescence. FIG. 14C shows ex vivo isolated
organ tissue luminescence.
[0038] FIGS. 15A-15C. Formulation #3.20 treatment. FIG. 15A shows
organ flux luciferase activity across organs. FIG. 15B shows in
vivo whole animal luminescence. FIG. 15C shows ex vivo isolated
organ tissue luminescence.
[0039] FIGS. 16A-16C. Formulation #2.2 treatment. FIG. 16A shows
organ flux luciferase activity across organs. FIG. 16B shows in
vivo whole animal luminescence. FIG. 16C shows ex vivo isolated
organ tissue luminescence.
[0040] FIGS. 17A-17K. Bruce #3.14 in vivo/ex vivo time course (3
mg/kg). In vivo whole animal luminescence at 4 hrs (FIG. 17A), 24
hrs (FIG. 17B), 48 hrs (FIG. 17C), 72 hrs (FIG. 17D), and 96 hrs
(FIG. 17E). Ex vivo isolated organ tissue luminescence at 4 hrs
(FIG. 17F), 24 hrs (FIG. 17G), 48 hrs (FIG. 17H), 72 hrs (FIG.
17I), and 96 hrs (FIG. 17J). FIG. 17K shows mean (total flux (p/s))
vs. column 1 over time.
[0041] FIGS. 18A-18C. mRNA dose variation and time course 3.14 (3,
1, 0.5 mg/kg). In vivo whole animal luminescence at 4 hrs (FIG.
18A), 24 hrs (FIG. 18B), and 48 hrs (FIG. 18C) for each dose
variation.
[0042] FIG. 19. Diagram of workflow.
[0043] FIGS. 20A-20B. Reagent comparative analysis. Experimental
design--Reagents: Four different formulations (IVF.RTM. Lung,
DOTMA:DOPE, DOTAP:DOPE, Jet PEI.RTM.), Route of Delivery:
Intravenous (systemic), Payload: Trilink.RTM. Firefly luciferase
mRNA. FIG. 20A shows a bar graph depicting lung radiance for each
formulation. FIG. 20B shows in vivo whole animal radiance for each
formulation.
[0044] FIG. 21. In vivo screening of different mRNA's encoding for
Firefly Luciferase. mRNA expression kinetics can differ based on
optimization of mRNA. Experimental design--Reagent:
Invivofectamine.RTM. Lung, Route of Delivery: Intravenous
(systemic), Payload: 2 different mRNAs encoding for Firefly
Luciferase (Trilink.RTM. and In House). Graph shows lung radiance
for each payload used.
[0045] FIG. 22. siRNA knockdown in the lung. Tyrosine kinase
receptor Tie2, also known as Tek, plays an important role in
embryonic vasculature and persists in adult endothelial cells. It
is expressed almost exclusively in endothelial cells. Knockdown
analysis was performed on isolated lung tissue 48 hours post IV
delivery of LNPs (IVF Rx lung+siRNA) (N=4). Graph shows the
percentage of Tie2 remaining.
[0046] FIG. 23. Cartoon representation of lacZ Cre-reporter strain
and the result of delivering Cre mRNA using IVF.RTM. Lung.
[0047] FIGS. 24A-24D. lacZ mRNA delivery in wild type mice. FIGS.
24A and 24C show lacZ expression via beta-gal staining. FIGS. 24B
and 24D are high magnification images of the regions circled in 24A
and 24C, respectively.
[0048] FIGS. 25A-25B. Cre mRNA delivery in lacZ reporter mice.
Invivofectamine.RTM. Lung delivery in vivo--Immunofluorescent
staining for detection of lacZ expression reveals expression
throughout the lung tissue, and is distinctly visible in bronchiole
structures. FIG. 25A shows 10.times. view of staining. FIG. 25B
shows 25.times. view of staining.
[0049] FIG. 26. Cartoon illustration describing the N/P ratio. N/P
ratio refers to the positive charge contributed by Nitrogen
residues on the cationic lipid versus the negative charge
contributed by the Phosphates on the nucleic acid backbone. High
N/P=Greater amounts of lipid compared to mRNA. Low N/P=Lower
amounts of lipid compared to mRNA. N/P ratio greatly affects the
surface charge of the lipid nanoparticle which strongly governs
transfection efficiency and affects biodistribution.
[0050] FIGS. 27A-27K. N/P ratio affects biodistribution pattern. In
vivo whole animal imaging and ex vivo isolated organ tissue imaging
showing the effect of N/P ratio (10, 8, 6, 4, 2) on
biodistribution. FIGS. 27A and 27B shows N/P ratio 10. FIGS. 27C
and 27D shows N/P ratio 8. FIGS. 27E and 27F shows N/P ratio 6.
FIGS. 27G and 27H shows N/P ratio 4. FIGS. 27I and 27J shows N/P
ratio 2. FIG. 27K shows a side view of N/P ratio 4.
[0051] FIG. 28. Formulation: DHDMS/DOPE with N/P ratio alteration.
No lung delivery seen and varied N/P ratio did not affect lung
distribution. Ex vivo isolated organ tissue images.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0052] The abbreviations used herein have their conventional
meaning within the chemical and biological arts. The chemical
structures and formulae set forth herein are constructed according
to the standard rules of chemical valency known in the chemical
arts.
[0053] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is equivalent to --OCH.sub.2--.
[0054] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight (i.e.,
unbranched) or branched chain, or combination thereof, which may be
fully saturated, mono- or polyunsaturated and can include di- and
multivalent radicals, having the number of carbon atoms designated
(i.e., C.sub.1-C.sub.10 means one to ten carbons). Alkyl is an
uncyclized chain. Examples of saturated hydrocarbon radicals
include, but are not limited to, groups such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl,
(cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl,
n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl
group is one having one or more double bonds or triple bonds.
Examples of unsaturated alkyl groups include, but are not limited
to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),
2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,
3-butynyl, and the higher homologs and isomers. An alkoxy is an
alkyl attached to the remainder of the molecule via an oxygen
linker (--O--).
[0055] The term "alkylene," by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from an alkyl, as exemplified, but not limited by,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred. A "lower
alkyl" or "lower alkylene" is a C.sub.1-C.sub.8 alkyl or alkylene
group.
[0056] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or combinations thereof, consisting of at least one
carbon atom and at least one heteroatom selected from the group
consisting of O, N, P, Si, and S, and wherein the nitrogen and
sulfur atoms may optionally be oxidized, and the nitrogen
heteroatom may optionally be quaternized. The heteroatom(s) 0, N,
P, S, and Si may be placed at any interior position of the
heteroalkyl group or at the position at which the alkyl group is
attached to the remainder of the molecule. Heteroalkyl is an
uncyclized chain. Examples include, but are not limited
to: --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3.
[0057] Similarly, the term "heteroalkylene," by itself or as part
of another substituent, means, unless otherwise stated, a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--. As described above, heteroalkyl groups, as used
herein, include those groups that are attached to the remainder of
the molecule through a heteroatom, such as --C(O)R', --C(O)NR',
--NR'R'', --OR', --SR', and/or --SO.sub.2R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R'' or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like.
[0058] The terms "cycloalkyl" and "heterocycloalkyl," by themselves
or in combination with other terms, mean, unless otherwise stated,
cyclic versions of "alkyl" and "heteroalkyl," respectively.
Cycloalkyl and heteroalkyl are not aromatic. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl,
3-cyclohexenyl, cycloheptyl, and the like. Examples of
heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a
"heterocycloalkylene," alone or as part of another substituent,
means a divalent radical derived from a cycloalkyl and
heterocycloalkyl, respectively.
[0059] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" includes, but is
not limited to, fluoromethyl, difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0060] The term "acyl" means, unless otherwise stated, --C(O)R
where R is a substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0061] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, hydrocarbon substituent, which can be a
single ring or multiple rings (preferably from 1 to 3 rings) that
are fused together (i.e., a fused ring aryl) or linked covalently.
A fused ring aryl refers to multiple rings fused together wherein
at least one of the fused rings is an aryl ring. The term
"heteroaryl" refers to aryl groups (or rings) that contain from one
to four heteroatoms selected from N, O, and S, wherein the nitrogen
and sulfur atoms are optionally oxidized, and the nitrogen atom(s)
are optionally quaternized. Thus, the term "heteroaryl" includes
fused ring heteroaryl groups (i.e., multiple rings fused together
wherein at least one of the fused rings is a heteroaromatic ring).
A 5,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 5 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. Likewise, a
6,6-fused ring heteroarylene refers to two rings fused together,
wherein one ring has 6 members and the other ring has 6 members,
and wherein at least one ring is a heteroaryl ring. And a 6,5-fused
ring heteroarylene refers to two rings fused together, wherein one
ring has 6 members and the other ring has 5 members, and wherein at
least one ring is a heteroaryl ring. A heteroaryl group can be
attached to the remainder of the molecule through a carbon or
heteroatom. Non-limiting examples of aryl and heteroaryl groups
include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,
5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,
purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. An "arylene" and a "heteroarylene," alone or as
part of another substituent, mean a divalent radical derived from
an aryl and heteroaryl, respectively.
[0062] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl, and the like) including those alkyl groups in which
a carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0063] The term "oxo," as used herein, means an oxygen that is
double bonded to a carbon atom.
[0064] The term "alkylsulfonyl," as used herein, means a moiety
having the formula --S(O.sub.2)--R', where R' is an alkyl group as
defined above. R' may have a specified number of carbons (e.g.,
"C.sub.1-C.sub.4 alkylsulfonyl").
[0065] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl," and "heteroaryl") includes both substituted and
unsubstituted forms of the indicated radical.
[0066] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to,
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR'', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN, and --NO.sub.2 in a
number ranging from zero to (2m'+1), where m' is the total number
of carbon atoms in such radical. R', R'', R''', and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),
substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups,
or arylalkyl groups. When a compound disclosed herein includes more
than one R group, for example, each of the R groups is
independently selected as are each R', R'', R''', and R'''' group
when more than one of these groups is present. When R' and R'' are
attached to the same nitrogen atom, they can be combined with the
nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For
example, --NR'R'' includes, but is not limited to, 1-pyrrolidinyl
and 4-morpholinyl. From the above discussion of substituents, one
of skill in the art will understand that the term "alkyl" is meant
to include groups including carbon atoms bound to groups other than
hydrogen groups, such as haloalkyl (e.g., --CF.sub.3 and
--CH.sub.2CF.sub.3) and acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like).
[0067] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R'',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN, --NO.sub.2, --R',
--N.sub.3, --CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''', and R'''' are preferably independently
selected from hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl. When a compound disclosed
herein includes more than one R group, for example, each of the R
groups is independently selected as are each R', R'', R''', and
R'''' groups when more than one of these groups is present.
[0068] Two or more substituents may optionally be joined to form
aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such
so-called ring-forming substituents are typically, though not
necessarily, found attached to a cyclic base structure. In
embodiments, the ring-forming substituents are attached to adjacent
members of the base structure. For example, two ring-forming
substituents attached to adjacent members of a cyclic base
structure create a fused ring structure. In another embodiment, the
ring-forming substituents are attached to a single member of the
base structure. For example, two ring-forming substituents attached
to a single member of a cyclic base structure create a spirocyclic
structure. In yet another embodiment, the ring-forming substituents
are attached to non-adjacent members of the base structure.
[0069] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally form a ring of the formula
-T-C(O)--(CRR').sub.q--U--, wherein T and U are independently
--NR--, --O--, --CRR'--, or a single bond, and q is an integer of
from 0 to 3. Alternatively, two of the substituents on adjacent
atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of the formula -A-(CH.sub.2).sub.r--B--, wherein
A and B are independently --CRR'--, --O--, --NR--, --S--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2NR'--, or a single bond, and r is an
integer of from 1 to 4. One of the single bonds of the new ring so
formed may optionally be replaced with a double bond.
Alternatively, two of the substituents on adjacent atoms of the
aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula --(CRR').sub.s--X'--(C''R''').sub.d--,
where s and d are independently integers of from 0 to 3, and X' is
--O--, --NR'--, --S--, --S(O)--, --S(O).sub.2--, or
--S(O).sub.2NR'--. The substituents R, R', R'', and R''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, and substituted or unsubstituted
heteroaryl.
[0070] As used herein, the terms "heteroatom" or "ring heteroatom"
are meant to include oxygen (O), nitrogen (N), sulfur (S),
phosphorus (P), and silicon (Si).
[0071] A "substituent group," as used herein, means a group
selected from the following moieties: [0072] (A) --OH, --NH.sub.2,
--SH, --CN, --CF.sub.3, --NO.sub.2, oxo, halogen, unsubstituted
alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,
unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted
heteroaryl, and [0073] (B) alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl, and heteroaryl, substituted with at least
one substituent selected from: [0074] (i) oxo, --OH, --NH.sub.2,
--SH, --CN, --CF.sub.3, --NO.sub.2, halogen, unsubstituted alkyl,
unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
[0075] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
and heteroaryl, substituted with at least one substituent selected
from: [0076] (a) oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3,
--NO.sub.2, halogen, unsubstituted alkyl, unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
[0077] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
or heteroaryl, substituted with at least one substituent selected
from: oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
halogen, unsubstituted alkyl, unsubstituted heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl, and unsubstituted heteroaryl.
[0078] A "size-limited substituent" or "size-limited substituent
group," as used herein, means a group selected from all of the
substituents described above for a "substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.4-C.sub.8 cycloalkyl, and
each substituted or unsubstituted heterocycloalkyl is a substituted
or unsubstituted 4 to 8 membered heterocycloalkyl.
[0079] A "lower substituent" or "lower substituent group," as used
herein, means a group selected from all of the substituents
described above for a "substituent group," wherein each substituted
or unsubstituted alkyl is a substituted or unsubstituted
C.sub.1-C.sub.8 alkyl, each substituted or unsubstituted
heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or unsubstituted C.sub.5-C.sub.7 cycloalkyl, and each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 5 to 7 membered heterocycloalkyl.
[0080] In some embodiments, each substituted group described in the
compounds herein is substituted with at least one substituent
group. More specifically, in some embodiments, each substituted
alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl,
substituted alkylene, substituted heteroalkylene, substituted
cycloalkylene, substituted heterocycloalkylene, substituted
arylene, and/or substituted heteroarylene described in the
compounds herein are substituted with at least one substituent
group. In other embodiments, at least one or all of these groups
are substituted with at least one size-limited substituent group.
In other embodiments, at least one or all of these groups are
substituted with at least one lower substituent group.
[0081] In other embodiments of the compounds herein, each
substituted or unsubstituted alkyl may be a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.3-C.sub.8 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and/or each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted 5 to 10 membered
heteroaryl. In some embodiments of the compounds herein, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.20 alkylene, each substituted or
unsubstituted heteroalkylene is a substituted or unsubstituted 2 to
20 membered heteroalkylene, each substituted or unsubstituted
cycloalkylene is a substituted or unsubstituted C.sub.3-C.sub.8
cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a substituted or unsubstituted 3 to 8
membered heterocycloalkylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 10 membered heteroarylene.
[0082] In some embodiments, each substituted or unsubstituted alkyl
is a substituted or unsubstituted C.sub.1-C.sub.8 alkyl, each
substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 8 membered heteroalkyl, each substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted
C.sub.3-C.sub.7 cycloalkyl, each substituted or unsubstituted
heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered
heterocycloalkyl, each substituted or unsubstituted aryl is a
substituted or unsubstituted C.sub.6-C.sub.10 aryl, and/or each
substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.8 alkylene, each substituted or
unsubstituted heteroalkylene is a substituted or unsubstituted 2 to
8 membered heteroalkylene, each substituted or unsubstituted
cycloalkylene is a substituted or unsubstituted C.sub.3-C.sub.7
cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a substituted or unsubstituted 3 to 7
membered heterocycloalkylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 9 membered heteroarylene. In some
embodiments, the compound is a chemical species set forth
herein.
[0083] The terms "a" or "an," as used in herein means one or more.
In addition, the phrase "substituted with a[n]," as used herein,
means the specified group may be substituted with one or more of
any or all of the named substituents. For example, where a group,
such as an alkyl or heteroaryl group, is "substituted with an
unsubstituted C.sub.1-C.sub.20 alkyl, or unsubstituted 2 to 20
membered heteroalkyl," the group may contain one or more
unsubstituted C.sub.1-C.sub.20 alkyls, and/or one or more
unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a
moiety is substituted with an R substituent, the group may be
referred to as "R-substituted." Where a moiety is R-substituted,
the moiety is substituted with at least one R substituent and each
R substituent is optionally different.
[0084] Descriptions of compounds of the present invention are
limited by principles of chemical bonding known to those skilled in
the art. Accordingly, where a group may be substituted by one or
more of a number of substituents, such substitutions are selected
so as to comply with principles of chemical bonding and to give
compounds which are not inherently unstable and/or would be known
to one of ordinary skill in the art as likely to be unstable under
ambient conditions, such as aqueous, neutral, and several known
physiological conditions. For example, a heterocycloalkyl or
heteroaryl is attached to the remainder of the molecule via a ring
heteroatom in compliance with principles of chemical bonding known
to those skilled in the art thereby avoiding inherently unstable
compounds.
[0085] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by a
person of ordinary skill in the art. See, e.g., Singleton et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley
& Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR
CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold
Springs Harbor, N Y 1989). Any methods, devices and materials
similar or equivalent to those described herein can be used in the
practice of this invention. The following definitions are provided
to facilitate understanding of certain terms used frequently herein
and are not meant to limit the scope of the present disclosure.
[0086] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single-, double- or
multiple-stranded form, or complements thereof. The term
"polynucleotide" refers to a linear sequence of nucleotides. The
term "nucleotide" typically refers to a single unit of a
polynucleotide, i.e., a monomer. Nucleotides can be
ribonucleotides, deoxyribonucleotides, or modified versions
thereof. Examples of polynucleotides contemplated herein include
single and double stranded DNA, single and double stranded RNA
(including siRNA and mRNA), and hybrid molecules having mixtures of
single and double stranded DNA and RNA. Nucleic acids can be linear
or branched. For example, nucleic acids can be a linear chain of
nucleotides or the nucleic acids can be branched, e.g., such that
the nucleic acids comprise one or more arms or branches of
nucleotides. Optionally, the branched nucleic acids are
repetitively branched to form higher ordered structures such as
dendrimers and the like.
[0087] Nucleic acids, including nucleic acids with a phosphothioate
backbone can include one or more reactive moieties. As used herein,
the term reactive moiety includes any group capable of reacting
with another molecule, e.g., a nucleic acid or polypeptide through
covalent, non-covalent or other interactions. By way of example,
the nucleic acid can include an amino acid reactive moiety that
reacts with an amino acid on a protein or polypeptide through a
covalent, non-covalent or other interaction.
[0088] The terms also encompass nucleic acids containing known
nucleotide analogs or modified backbone residues or linkages, which
are synthetic, naturally occurring, and non-naturally occurring,
which have similar binding properties as the reference nucleic
acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphodiester derivatives including, e.g.,
phosphoramidate, phosphorodiamidate, phosphorothioate (also known
as phosphothioate), phosphorodithioate, phosphonocarboxylic acids,
phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,
methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press); and peptide nucleic acid
backbones and linkages. Other analog nucleic acids include those
with positive backbones; non-ionic backbones, modified sugars, and
non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or
locked nucleic acids (LNA)), including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, Carbohydrate Modifications in Antisense Research,
Sanghui & Cook, eds. Nucleic acids containing one or more
carbocyclic sugars are also included within one definition of
nucleic acids. Modifications of the ribose-phosphate backbone may
be done for a variety of reasons, e.g., to increase the stability
and half-life of such molecules in physiological environments or as
probes on a biochip. Mixtures of naturally occurring nucleic acids
and analogs can be made; alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. In embodiments, the internucleotide
linkages in DNA are phosphodiester, phosphodiester derivatives, or
a combination of both.
[0089] As used herein, the term "about" means a range of values
including the specified value, which a person of ordinary skill in
the art would consider reasonably similar to the specified value.
In embodiments, the term "about" means within a standard deviation
using measurements generally acceptable in the art. In embodiments,
about means a range extending to +/-10% of the specified value. In
embodiments, about means the specified value.
[0090] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues, wherein the polymer may be conjugated to a moiety that
does not consist of amino acids. The terms apply to amino acid
polymers in which one or more amino acid residue is an artificial
chemical mimetic of a corresponding naturally occurring amino acid,
as well as to naturally occurring amino acid polymers and
non-naturally occurring amino acid polymers. The terms apply to
macrocyclic peptides, peptides that have been modified with
non-peptide functionality, peptidomimetics, polyamides, and
macrolactams. A "fusion protein" refers to a chimeric protein
encoding two or more separate protein sequences that are
recombinantly expressed as a single moiety.
[0091] The term "peptidyl" and "peptidyl moiety" means a monovalent
peptide.
[0092] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid. The terms
"non-naturally occurring amino acid" and "unnatural amino acid"
refer to amino acid analogs, synthetic amino acids, and amino acid
mimetics which are not found in nature.
[0093] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0094] An amino acid or nucleotide base "position" is denoted by a
number that sequentially identifies each amino acid (or nucleotide
base) in the reference sequence based on its position relative to
the N-terminus (or 5'-end). Due to deletions, insertions,
truncations, fusions, and the like that must be taken into account
when determining an optimal alignment, in general the amino acid
residue number in a test sequence determined by simply counting
from the N-terminus will not necessarily be the same as the number
of its corresponding position in the reference sequence. For
example, in a case where a variant has a deletion relative to an
aligned reference sequence, there will be no amino acid in the
variant that corresponds to a position in the reference sequence at
the site of deletion. Where there is an insertion in an aligned
reference sequence, that insertion will not correspond to a
numbered amino acid position in the reference sequence. In the case
of truncations or fusions there can be stretches of amino acids in
either the reference or aligned sequence that do not correspond to
any amino acid in the corresponding sequence.
[0095] The terms "numbered with reference to" or "corresponding
to," when used in the context of the numbering of a given amino
acid or polynucleotide sequence, refers to the numbering of the
residues of a specified reference sequence when the given amino
acid or polynucleotide sequence is compared to the reference
sequence.
[0096] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0097] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0098] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M).
[0099] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide or polypeptide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0100] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% identity over a specified region, e.g., of
the entire polypeptide sequences of the invention or individual
domains of the polypeptides of the invention), 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.
Such sequences are then said to be "substantially identical." This
definition also refers to the complement of a test sequence.
Optionally, the identity exists over a region that is at least
about 50 nucleotides in length, or more preferably over a region
that is 100 to 500 or 1000 or more nucleotides in length.
[0101] 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.
[0102] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of, e.g., a full length sequence or from
20 to 600, about 50 to about 200, or about 100 to about 150 amino
acids or nucleotides 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 (1970) Adv.
Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for
similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad.
Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0103] An example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) or 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
[0104] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). 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 or amino
acid 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.
[0105] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross-reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0106] "Contacting" is used in accordance with its plain ordinary
meaning and refers to the process of allowing at least two distinct
species (e.g. chemical compounds including biomolecules or cells)
to become sufficiently proximal to react, interact or physically
touch. It should be appreciated, however, that the resulting
reaction product can be produced directly from a reaction between
the added reagents or from an intermediate from one or more of the
added reagents which can be produced in the reaction mixture. In
embodiments contacting includes, for example, allowing a
ribonucleic acid as described herein to interact with a an
endonuclease and an enhancer element.
[0107] A "control" sample or value refers to a sample that serves
as a reference, usually a known reference, for comparison to a test
sample. For example, a test sample can be taken from a test
condition, e.g., in the presence of a test compound, and compared
to samples from known conditions, e.g., in the absence of the test
compound (negative control), or in the presence of a known compound
(positive control). A control can also represent an average value
gathered from a number of tests or results. One of skill in the art
will recognize that controls can be designed for assessment of any
number of parameters. For example, a control can be devised to
compare therapeutic benefit based on pharmacological data (e.g.,
half-life) or therapeutic measures (e.g., comparison of side
effects). One of skill in the art will understand which standard
controls are most appropriate in a given situation and be able to
analyze data based on comparisons to standard control values.
Standard controls are also valuable for determining the
significance (e.g. statistical significance) of data. For example,
if values for a given parameter are widely variant in standard
controls, variation in test samples will not be considered as
significant.
[0108] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins or other entities which can be
made detectable, e.g., by incorporating a radiolabel into a peptide
or antibody specifically reactive with a target peptide. Any
appropriate method known in the art for conjugating an antibody to
the label may be employed, e.g., using methods described in
Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San
Diego.
[0109] A "labeled protein or polypeptide" is one that is bound,
either covalently, through a linker or a chemical bond, or
noncovalently, through ionic, van der Waals, electrostatic, or
hydrogen bonds to a label such that the presence of the labeled
protein or polypeptide may be detected by detecting the presence of
the label bound to the labeled protein or polypeptide.
Alternatively, methods using high affinity interactions may achieve
the same results where one of a pair of binding partners binds to
the other, e.g., biotin, streptavidin.
[0110] "Biological sample" or "sample" refer to materials obtained
from or derived from a subject or patient. A biological sample
includes sections of tissues such as biopsy and autopsy samples,
and frozen sections taken for histological purposes. Such samples
include bodily fluids such as blood and blood fractions or products
(e.g., serum, plasma, platelets, red blood cells, and the like),
sputum, tissue, cultured cells (e.g., primary cultures, explants,
and transformed cells) stool, urine, synovial fluid, joint tissue,
synovial tissue, synoviocytes, fibroblast-like synoviocytes,
macrophage-like synoviocytes, immune cells, hematopoietic cells,
fibroblasts, macrophages, T cells, etc. A biological sample is
typically obtained from a eukaryotic organism, such as a mammal
such as a primate e.g., chimpanzee or human; cow; dog; cat; a
rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile;
or fish.
[0111] A "cell" as used herein, refers to a cell carrying out
metabolic or other function sufficient to preserve or replicate its
genomic DNA. A cell can be identified by well-known methods in the
art including, for example, presence of an intact membrane,
staining by a particular dye, ability to produce progeny or, in the
case of a gamete, ability to combine with a second gamete to
produce a viable offspring. Cells may include prokaryotic and
eukaryotic cells. Prokaryotic cells include but are not limited to
bacteria. Eukaryotic cells include but are not limited to yeast
cells and cells derived from plants and animals, for example
mammalian, insect (e.g., spodoptera) and human cells.
[0112] The word "expression" or "expressed" as used herein in
reference to a gene means the transcriptional and/or translational
product of that gene. The level of expression of a DNA molecule in
a cell may be determined on the basis of either the amount of
corresponding mRNA that is present within the cell or the amount of
protein encoded by that DNA produced by the cell (Sambrook et al.,
1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).
[0113] Expression of a transfected gene can occur transiently or
stably in a cell. During "transient expression" the transfected
gene is not transferred to the daughter cell during cell division.
Since its expression is restricted to the transfected cell,
expression of the gene is lost over time. In contrast, stable
expression of a transfected gene can occur when the gene is
co-transfected with another gene that confers a selection advantage
to the transfected cell. Such a selection advantage may be a
resistance towards a certain toxin that is presented to the
cell.
[0114] The term "exogenous" refers to a molecule or substance
(e.g., nucleic acid or protein) that originates from outside a
given cell or organism. Conversely, the term "endogenous" refers to
a molecule or substance that is native to, or originates within, a
given cell or organism.
[0115] A "cell culture" is an in vitro population of cells residing
outside of an organism. The cell culture can be established from
primary cells isolated from a cell bank or animal, or secondary
cells that are derived from one of these sources and immortalized
for long-term in vitro cultures.
[0116] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent,
i.e., and a variety of other pharmaceutically acceptable
components. See Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa., 1980). The preferred form depends
on the intended mode of administration and therapeutic application.
The compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0117] The compositions can be administered for therapeutic or
prophylactic treatments. In therapeutic applications, compositions
are administered to a patient suffering from a disease (e.g.,
pulmonary disease) in a "therapeutically effective dose." Amounts
effective for this use will depend upon the severity of the disease
and the general state of the patient's health. Single or multiple
administrations of the compositions may be administered depending
on the dosage and frequency as required and tolerated by the
patient. A "patient" or "subject" for the purposes of the present
invention includes both humans and other animals, particularly
mammals. Thus the methods are applicable to both human therapy,
veterinary applications, and in research use settings, for example
in experimental animal models including rodent, canine, and primate
animal models. In certain embodiments the subject or patient is a
mammal, preferably a primate, and in the most preferred embodiment
the patient is human. In other embodiments the subject or patient
is a mammal, preferably a rodent, and in the most preferred
embodiments a mouse or rat.
[0118] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0119] The compositions provided herein, 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. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane,
nitrogen, and the like.
[0120] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intratumoral, 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 can be administered,
for example, by intravenous infusion, intraperitoneally,
intravesically or intrathecally. Parenteral administration, and
intravenous administration are the preferred methods of
administration. The formulations of compounds can be presented in
unit-dose or multi-dose sealed containers, such as ampules and
vials.
[0121] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described.
[0122] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as powders in vials or
ampoules. The composition can, if desired, also contain other
compatible therapeutic agents.
[0123] The combined administrations contemplates co-administration,
using separate formulations or a single pharmaceutical formulation,
and consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0124] Effective doses of the compositions provided herein vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
However, a person of ordinary skill in the art would immediately
recognize appropriate and/or equivalent doses looking at dosages of
approved compositions for treating and preventing lung/pulmonary
disorders for guidance.
[0125] The terms "disease" or "condition" refer to a state of being
or health status of a patient or subject capable of being treated
with a compound, pharmaceutical composition, or method provided
herein. In embodiments, the disease is a pulmonary disease (e.g.
lung cancer, asthma, chronic obstructive pulmonary disease (COPD),
cystic fibrosis).
[0126] The term "associated" or "associated with" in the context of
a substance or substance activity or function associated with a
disease (e.g., lung disease, lung cancer, asthma, chronic
obstructive pulmonary disease (COPD), cystic fibrosis) is caused by
(in whole or in part), or a symptom of the disease is caused by (in
whole or in part) the substance or substance activity or
function.
[0127] The terms "transfection", "transduction", "transfecting" or
"transducing" can be used interchangeably and are defined as a
process of introducing a nucleic acid molecule and/or a protein
and/or a ribonucleoprotein to a cell in culture or in a tissue in
vivo. The nucleic acid molecule can be a sequence encoding complete
proteins, ribonucleoproteins or functional portions thereof.
Typically, a nucleic acid encoding proteins, ribonucleoproteins or
functional portions thereof comprises the elements necessary for
expression of the protein or functional portion thereof (e.g., a
promoter, transcription start site, etc.). Non-viral methods of
transfection include any appropriate method that does not use viral
DNA or viral particles as a delivery system to introduce the
nucleic acid molecule into the cell. Exemplary non-viral
transfection methods include liposomal transfection. The terms
"transfection" or "transduction" also refer to introducing nucleic
acids and/or proteins into a cell from the external environment.
Through transfection, the nucleic acid molecule and/or a protein
and/or a ribonucleoprotein is delivered into the interior of the
cell or the cells constituting the tissue. Transfection of a
nucleic acid molecule and/or a protein and/or a ribonucleoprotein
into a cell or tissue may be performed with the purpose of
modifying the biological function of the cell. Alternatively,
transfection of a nucleic acid molecule and/or a protein and/or a
ribonucleoprotein may be performed with the purpose of delivering a
detectable label to a cell or tissue to facilitate identification
of a cell or tissue. For example, nucleic acids (e.g., DNA, RNA,
mRNA, siRNA, miRNA, guide RNA) and/or ribonucleoproteins (e.g., CAS
9) can be introduced into a cell or tissue via lipid-mediated
delivery (e.g., liposomal transfection). The nucleic acid molecule
may alternatively be an mRNA, a siRNA, a miRNA or a guide RNA. In
some instances, the nucleic acid molecule may be bound to a
ribonucleoprotein (e.g., Cas9 bound to a guide RNA).
[0128] For specific proteins described herein, the named protein
includes any of the protein's naturally occurring forms, or
variants or homologs that maintain the protein transcription factor
activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%,
99% or 100% activity compared to the native protein). In some
embodiments, variants or homologs have at least 90%, 95%, 96%, 97%,
98%, 99% or 100% amino acid sequence identity across the whole
sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200
continuous amino acid portion) compared to a naturally occurring
form. In other embodiments, the protein is the protein as
identified by its NCBI sequence reference. In other embodiments,
the protein is the protein as identified by its NCBI sequence
reference or functional fragment or homolog thereof.
[0129] Thus, a "CRISPR associated protein 9," "Cas9" or "Cas9
protein" as referred to herein includes any of the recombinant or
naturally-occurring forms of the Cas9 endonuclease or variants or
homologs thereof that maintain Cas9 endonuclease enzyme activity
(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
100% activity compared to Cas9). In some aspects, the variants or
homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino
acid sequence identity across the whole sequence or a portion of
the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid
portion) compared to a naturally occurring Cas9 protein. In
embodiments, the Cas9 protein is substantially identical to the
protein identified by the UniProt reference number Q99ZW2 or a
variant or homolog having substantial identity thereto. Cas9 refers
to the protein also known in the art as "nickase". In embodiments,
Cas9 binds a CRISPR (clustered regularly interspaced short
palindromic repeats) nucleic acid sequence. In embodiments, the
CRISPR nucleic acid sequence is a prokaryotic nucleic acid
sequence.
[0130] As used herein, the term "lipid" refers to lipid molecules
that can include fats, waxes, steroids, cholesterol, fat-soluble
vitamins, monoglycerides, diglycerides, phospholipids,
sphingolipids, glycolipids, cationic or anionic lipids, derivatized
lipids, and the like, as described in detail below.
[0131] Suitable phospholipids include but are not limited to
phosphatidylcholine (PC), phosphatidic acid (PA),
phosphatidylethanolamine (PE), phosphatidylglycerol (PG),
phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl
phosphatidyl choline (DMPC), distearoyl phosphatidyl choline
(DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl
phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol
(DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl
phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol
(DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl
phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS),
dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl
ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), and
cardiolipin. In some embodiments, the phospholipid is DOPE. In
other embodiments, the phospholipid is DSPC. Lipid extracts, such
as egg PC, heart extract, brain extract, liver extract, and soy PC,
are also useful in the present invention. In some embodiments, soy
PC can include Hydro Soy PC (HSPC). In certain embodiments, the
lipids can include derivatized lipids, such as PEGylated lipids.
Derivatized lipids can include, for example, DSPE-PEG2000,
cholesterol-PEG2000, DSPE-polyglycerol, or other derivatives
generally known in the art.
[0132] A "cationic lipid" as provided herein refers, in the usual
and customary sense, to a net positively charged lipid which can
facilitate the formation of lipid aggregates. Cationic lipids
contain positively charged functional groups under physiological
conditions. Cationic lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA),
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE),
N-[1-(2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE), 3.beta.-[N--(N',N'-dimethylaminoethane)
carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB)
and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA). In
embodiments, the cationic lipid (e.g., first cationic lipid, second
cationic lipid) is a compound of formula (I) or (II). In
embodiments, the first cationic lipid is dihydroxy dimyristyl
spermidine (DHDMS). In embodiments, the second cationic lipid is
hydroxy dimyristyl spermidine (HDMS).
[0133] Lipids can form micelles, monolayers, and bilayer membranes.
The lipids can self-assemble into liposomes or lipid
aggregates.
[0134] The term "lipid aggregate" refers to a lipid structure
including a plurality of lipids or type of lipids, forming a higher
order structure (e.g., secondary, tertiary or quaternary
structure). Non-limiting examples of lipid aggregates include
liposomes, unilamellar vesicles, multilamellar vesicles, micelles,
amorphous aggregates, and the like. The lipid aggregates of the
present invention can contain any suitable lipid, including
cationic lipids, zwitterionic lipids, neutral lipids, or anionic
lipids. In embodiments, the lipid aggregate includes a cationic
lipid or a cationic lipid type. In embodiments, the lipid aggregate
includes a cationic lipid or a cationic lipid type in combination
with a non-cationic (e.g., neutral) lipid or a non-cationic lipid
type. In embodiments, the lipid aggregate has a net positive
charge. In embodiments, the lipid aggregate includes a cationic
lipid and a neutral lipid. In embodiments, the cationic lipid is a
cationic lipid as described in U.S. Pat. No. 8,785,200 which is
hereby incorporated by reference and for all purposes.
[0135] In embodiments, the lipid aggregate includes a single lipid.
In embodiments, the lipid aggregate includes a plurality of
different lipids (e.g., first cationic lipid, second cationic
lipid, helper lipid). Where the lipid aggregate includes a
plurality of different lipids the lipid aggregate may include a
lipid blend. A "lipid blend" as provided herein is a mixture of a
plurality of lipid types. In embodiments, the lipid blend includes
a first lipid type, a second lipid type or a third lipid type. The
first, second and third lipid type may be independently different
(e.g., cationic lipid and non-cationic lipid). Therefore, a person
having ordinary skill in the art will immediately recognize that
the terms "lipid" and "lipid type(s)" have the same meaning and can
be used interchangeably.
[0136] In embodiments, the lipid aggregate provided herein is a
liposome. As used herein, the term "liposome" encompasses any
compartment enclosed by a lipid bilayer. The term liposome includes
unilamellar vesicles which are comprised of a single lipid bilayer
and generally have a diameter in the range of about 20 to about 400
nm. Liposomes can also be multilamellar having a diameter in the
range of approximately 1 .mu.m to approximately 10 .mu.m.
Multilamellar liposomes may consist of several (anywhere from two
to hundreds) unilamellar vesicles forming one inside the other in
diminishing size, creating a multilamellar structure of concentric
phospholipid spheres separated by layers of water. Alternatively,
multilamellar liposomes may consist of many smaller non concentric
spheres of lipid inside a large liposome. In embodiments, liposomes
include multilamellar vesicles (MLV), large unilamellar vesicles
(LUV), and small unilamellar vesicles (SUV). The liposomes of the
present invention can contain any suitable lipid, including
cationic lipids, zwitterionic lipids, neutral lipids, or anionic
lipids.
Compositions
[0137] Provided herein are, inter alia, compositions and methods
useful for the in vivo delivery of bioactive agents (e.g.,
therapeutic, biologically active, or diagnostic agents). The
compositions and methods provided herein including embodiments
thereof may be, inter alia, used for the delivery of bioactive
agents (e.g., nucleic acid molecules, ribonucleoproteins, small
molecules or combinations thereof) to the lung (e.g., including but
not limited to endothelial lung cells, epithelial lung cells) of a
subject. The compositions provided herein include cationic lipids,
helper lipids and a biostability enhancing agent, which together
form a lipid aggregate with the bioactive agent and allow for the
systemic delivery of the bioactive agent to, for example, lung
tissue without the requirement for biomolecular targeting.
[0138] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio from about 0.18
to about 0.32 and of formula:
##STR00009##
In formula (I) R.sup.1 and R.sup.2 are independently substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. R.sup.3 and R.sup.4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl. m is an integer from 1 to 6.
X.sub.a.sup.- is an anion. (ii) A second cationic lipid at a
compositional molar ratio from about 0.24 to about 0.51 and of
formula:
##STR00010##
In formula (II) R.sup.5 and R.sup.8 are independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl. R.sup.6 and
R.sup.7 are independently substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl. n is an integer from 1 to 6.
X.sub.b.sup.- is an anion. (iii) A first helper lipid at a
compositional molar ratio from about 0.20 to about 0.32. (iv) A
second helper lipid at a compositional molar ratio from about 0.01
to about 0.14; and (v) a biostability enhancing agent at a
compositional molar ratio from about 0.01 to about 0.02.
[0139] In embodiments, R.sup.1 and R.sup.2 are independently
substituted or unsubstituted alkyl. In embodiments, R.sup.1 and
R.sup.2 are independently unsubstituted alkyl. R.sup.1 and R.sup.2
are independently unsubstituted C.sub.1-C.sub.20 alkyl. In
embodiments, R.sup.1 and R.sup.2 are independently unsubstituted
C.sub.5-C.sub.20 alkyl. In embodiments, R.sup.1 and R.sup.2 are
independently unsubstituted C.sub.10-C.sub.20 alkyl. In
embodiments, R.sup.1 and R.sup.2 are independently unsubstituted
C.sub.12-C.sub.18 alkyl. In embodiments, R.sup.1 and R.sup.2 are
independently unsubstituted C.sub.14-C.sub.16 alkyl. In
embodiments, R.sup.1 is unsubstituted C.sub.14 alkyl. In
embodiments, R.sup.2 is unsubstituted C.sub.14 alkyl. In
embodiments, R.sup.1 is unsubstituted C.sub.15 alkyl. In
embodiments, R.sup.2 is unsubstituted C.sub.15 alkyl. In
embodiments, R.sup.1 is unsubstituted C.sub.16 alkyl. In
embodiments, R.sup.2 is unsubstituted C.sub.16 alkyl. In
embodiments, R.sup.1 is --(CH.sub.2).sub.13CH.sub.3. In
embodiments, R.sup.2 is --(CH.sub.2).sub.13CH.sub.3.
[0140] In embodiments, R.sup.3 and R.sup.4 are independently
hydrogen or substituted or unsubstituted alkyl. In embodiments,
R.sup.3 and R.sup.4 are independently hydrogen.
[0141] In embodiments, R.sup.5, R.sup.6 and R.sup.7 are
independently hydrogen, substituted or unsubstituted alkyl. In
embodiments, R.sup.5, R.sup.6 and R.sup.7 are independently
hydrogen or unsubstituted alkyl. In embodiments, R.sup.5, R.sup.6
and R.sup.7 are independently hydrogen or unsubstituted
C.sub.1-C.sub.20 alkyl. In embodiments, R.sup.5, R.sup.6 and
R.sup.7 are independently hydrogen or unsubstituted
C.sub.5-C.sub.20 alkyl. In embodiments, R.sup.5, R.sup.6 and
R.sup.7 are independently hydrogen or unsubstituted
C.sub.10-C.sub.20 alkyl. In embodiments, R.sup.5, R.sup.6 and
R.sup.7 are independently hydrogen or unsubstituted
C.sub.12-C.sub.18 alkyl. In embodiments, R.sup.5, R.sup.6 and
R.sup.7 are independently hydrogen or unsubstituted
C.sub.14-C.sub.16 alkyl. In embodiments, R.sup.5, R.sup.6 and
R.sup.7 are independently hydrogen or unsubstituted C.sub.14 alkyl.
In embodiments, R.sup.5, R.sup.6 and R.sup.7 are independently
hydrogen or unsubstituted C.sub.15 alkyl. In embodiments, R.sup.5,
R.sup.6 and R.sup.7 are independently hydrogen or unsubstituted
C.sub.16 alkyl. In embodiments, R.sup.5 is unsubstituted C.sub.14
alkyl. In embodiments, R.sup.7 is unsubstituted C.sub.14 alkyl. In
embodiments, R.sup.5 is --(CH.sub.2).sub.13CH.sub.3. In
embodiments, R.sup.6 is hydrogen. In embodiments, R.sup.7 is
--(CH.sub.2).sub.13CH.sub.3.
[0142] In embodiments, R.sup.8 is hydrogen or substituted or
unsubstituted alkyl. In embodiments, R.sup.8 is hydrogen.
[0143] In embodiments, m is an integer from about 1 to 6. In
embodiments, m is an integer from about 1 to 5. In embodiments, m
is an integer from about 1 to 4. In embodiments, m is an integer
from about 1 to 3. In embodiments, m is an integer from 1 to 6. In
embodiments, m is an integer from 1 to 5. In embodiments, m is an
integer from 1 to 4. In embodiments, m is an integer from 1 to 3.
In embodiments, m is 1. In embodiments, m is 2. In embodiments, m
is 3. In embodiments, m is 4. In embodiments, m is 5. In
embodiments, m is 6.
[0144] In embodiments, n is an integer from about 1 to 6. In
embodiments, n is an integer from about 1 to 5. In embodiments, n
is an integer from about 1 to 4. In embodiments, n is an integer
from about 1 to 3. In embodiments, n is an integer from 1 to 6. In
embodiments, n is an integer from 1 to 5. In embodiments, n is an
integer from 1 to 4. In embodiments, n is an integer from 1 to 3.
In embodiments, n is 1. In embodiments, n is 2. In embodiments, n
is 3. In embodiments, n is 4. In embodiments, n is 5. In
embodiments, n is 6.
[0145] In one embodiment, R.sup.1 is --(CH.sub.2).sub.13CH.sub.3,
R.sup.2 is --(CH.sub.2).sub.13CH.sub.3, R.sup.3 is hydrogen,
R.sup.4 is hydrogen, m is 4 and X.sub.a.sup.- is
CH.sub.3COO.sup.-.
[0146] In one embodiment, R.sup.5 is --(CH.sub.2).sub.13CH.sub.3,
R.sup.6 is hydrogen, R.sup.7 is --(CH.sub.2).sub.13CH.sub.3,
R.sup.8 is hydrogen, n is 4 and X.sub.b.sup.- is
CH.sub.3COO.sup.-.
[0147] The term "compositional molar ratio" of a compound (e.g.,
cationic lipid, helper lipid, biostability enhancing agent) refers
to the ratio of the number of solute moles of an individual
compound to the total number of solute moles of all compounds in a
solution. For example, the total number of solute moles may be 33.8
and the number of solute moles of an individual compound (e.g.,
first cationic lipid) may be 8.1 resulting in a compositional ratio
for the individual compound of 0.24. In embodiments, the total
number of solute moles is 33.8 and the number of solute moles of a
single compound is 10.8 moles resulting in a compositional ratio of
the single compound of 0.32.
[0148] The compositions provided herein include two or more helper
lipids (e.g., a first helper lipid, a second helper lipid). A
"helper lipid" as provided herein refers to a lipid capable of
increasing delivery of the bioactive agent to a cell relative to
the absence of the helper lipid. Thus, the delivery efficiency of a
bioactive agent to a cell is higher in the presence of a helper
lipid relative to the delivery efficiency in the absence of said
helper lipid. Delivery of a bioactive agent into a cell includes,
for example, uptake of the bioactive agent into a cell (penetration
through the cell membrane), endosomal release of the bioactive
agent in a cell, enhancing stability of the bioactive agent and/or
the compounds forming the lipid aggregate during the process of
delivery. Helper lipids useful in this invention include, without
limitation: lecithins; phosphotidylethanolamine;
phosphatidylethanolamines, such as DOPE
(dioleoylphosphatidylethanolamine), DPhPE
(diphytanoylphosphatidylethanolamine), DPPE
(dipalmitoylphosphatidylethanolamine),
dipalmiteoylphosphatidylethanolamine, POPE
(palmitoyloleoylphosphatidylethanolamine) and
distearoylphosphatidylethanolamine; phosphotidylcholine;
phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine),
DPPC (dipalmitoylphosphatidylcholine) POPC
(palmitoyloleoylphosphatidylcholine) and
distearoylphosphatidylcholine; phosphatidylglycerol;
phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol),
DPPG (dipalmitoylphosphatidyl-glycerol), and
distearoylphosphatidylglycerol; phosphatidylserine;
phosphatidylserines, such as dioleoyl- or
dipalmitoylphosphatidylserine; diphosphatidylglycerols; fatty acid
esters; glycerol esters; sphingolipids; cardolipin; cerebrosides;
and ceramides; and mixtures thereof. Helper lipids also include
cholesterol and other 3.beta.OH-sterols.
[0149] A "biostability enhancing agent" as provided herein is a
compound capable of increasing the physical and chemical stability
of the compositions (e.g., lipid aggregates including a bioactive
agent) provided herein relative to the absence of the compound.
Upon administration to a subject a biostability enhancing agent may
increase the biodistribution of the compounds provided herein. In
embodiments, the biostability enhancing agent is a polyether
compound. In embodiments, the biostability enhancing agent is a
PEGylated phospholipid. In embodiments, the biostability enhancing
agent is polyethylene glycol.
[0150] In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.18. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.18.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.23. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.23.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.24. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.24.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.25. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.25.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.27. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.27.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.28. In embodiments, the first
cationic lipid is present at a compositional molar ratio of 0.28.
In embodiments, the first cationic lipid is present at a
compositional molar ratio of about 0.32. In embodiments, the first
cationic lipid is present at a compositional molar ratio of
0.32.
[0151] In embodiments, the first cationic lipid has the
formula:
##STR00011##
wherein X.sub.a.sup.- is Cl.sup.- or CH.sub.3COO.sup.-. In
embodiments, X.sub.a.sup.- is CH.sub.3COO.sup.-. In embodiments,
the first cationic lipid is dihydroxy dimyristyl spermidine.
[0152] In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.24. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.24.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.38. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.38.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.39. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.39.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.40. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.40.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.45. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.45.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.47. In embodiments, the second
cationic lipid is present at a compositional molar ratio of 0.47.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.51. In embodiments, the second
cationic lipid is present at a compositional molar ratio of
0.51.
[0153] In embodiments, the second cationic lipid has the
formula:
##STR00012##
wherein X.sub.b.sup.- is Cl.sup.- or CH.sub.3COO.sup.-. In
embodiments, X.sub.b.sup.- is CH.sub.3COO.sup.-. In embodiments,
the second cationic lipid is hydroxy dimyristyl spermidine.
[0154] In embodiments, the first helper lipid is present at a
compositional molar ratio of about 0.20. In embodiments, the first
helper lipid is present at a compositional molar ratio of 0.20. In
embodiments, the first helper lipid is present at a compositional
molar ratio of about 0.24. In embodiments, the first helper lipid
is present at a compositional molar ratio of 0.24. In embodiments,
the first helper lipid is present at a compositional molar ratio of
about 0.26. In embodiments, the first helper lipid is present at a
compositional molar ratio of 0.26. In embodiments, the first helper
lipid is present at a compositional molar ratio of about 0.32. In
embodiments, the first helper lipid is present at a compositional
molar ratio of 0.32. In embodiments, the first helper lipid is
dioleoylphosphatidylethanolamine (DOPE).
[0155] In embodiments, the second helper lipid is present at a
compositional molar ratio of about 0.01. In embodiments, the second
helper lipid is present at a compositional molar ratio of 0.01. In
embodiments, the second helper lipid is present at a compositional
molar ratio of about 0.05. In embodiments, the second helper lipid
is present at a compositional molar ratio of 0.05. In embodiments,
the second helper lipid is present at a compositional molar ratio
of about 0.08. In embodiments, the second helper lipid is present
at a compositional molar ratio of 0.08. In embodiments, the second
helper lipid is present at a compositional molar ratio of about
0.10. In embodiments, the second helper lipid is present at a
compositional molar ratio of 0.10. In embodiments, the second
helper lipid is present at a compositional molar ratio of about
0.14. In embodiments, the second helper lipid is present at a
compositional molar ratio of 0.14. In embodiments, the second
helper lipid is cholesterol.
[0156] In embodiments, the biostability enhancing agent is present
at a compositional molar ratio of about 0.01. In embodiments, the
biostability enhancing agent is present at a compositional molar
ratio of 0.01. In embodiments, the biostability enhancing agent is
present at a compositional molar ratio of about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio of 0.02. In embodiments, the biostability
enhancing agent is a polyether compound. In embodiments, the
biostability enhancing agent is a PEGylated phospholipid. In
embodiments, the biostability enhancing agent is polyethylene
glycol.
[0157] In embodiments, the biostability enhancing agent has a
molecular weight from about 750 g/mol to about 5000 g/mol. In
embodiments, the biostability enhancing agent has a molecular
weight from about 800/mol to about 5000 g/mol. In embodiments, the
biostability enhancing agent has a molecular weight from about 850
g/mol to about 5000 g/mol. In embodiments, the biostability
enhancing agent has a molecular weight from about 900 g/mol to
about 5000 g/mol. In embodiments, the biostability enhancing agent
has a molecular weight from about 950 g/mol to about 5000 g/mol. In
embodiments, the biostability enhancing agent has a molecular
weight from about 1000 g/mol to about 5000 g/mol. In embodiments,
the biostability enhancing agent has a molecular weight from about
1500 g/mol to about 5000 g/mol. In embodiments, the biostability
enhancing agent has a molecular weight from about 2000 g/mol to
about 5000 g/mol. In embodiments, the biostability enhancing agent
has a molecular weight from about 2500 g/mol to about 5000 g/mol.
In embodiments, the biostability enhancing agent has a molecular
weight from about 3000 g/mol to about 5000 g/mol. In embodiments,
the biostability enhancing agent has a molecular weight from about
3500 g/mol to about 5000 g/mol. In embodiments, the biostability
enhancing agent has a molecular weight from about 4000 g/mol to
about 5000 g/mol. In embodiments, the biostability enhancing agent
has a molecular weight from about 4500 g/mol to about 5000
g/mol.
[0158] In embodiments, the biostability enhancing agent has a
molecular weight of about 750 g/mol. In embodiments, the
biostability enhancing agent has a molecular weight of 750 g/mol.
In embodiments, the biostability enhancing agent has a molecular
weight of about 2000 g/mol. In embodiments, the biostability
enhancing agent has a molecular weight of 2000 g/mol. In
embodiments, the biostability enhancing agent has a molecular
weight of about 5000 g/mol. In embodiments, the biostability
enhancing agent has a molecular weight of 5000 g/mol.
[0159] In embodiments, the biostability enhancing agent is C14
polyethylene glycol 750. In embodiments, the biostability enhancing
agent is C14 polyethylene glycol 2000. In embodiments, biostability
enhancing agent is C14 polyethylene glycol 5000.
[0160] In embodiments, the first cationic lipid is present at a
compositional molar ratio from about 0.18 to about 0.32, from about
0.19 to about 0.32, from about 0.20 to about 0.32, from about 0.21
to about 0.32, from about 0.22 to about 0.32, from about 0.23 to
about 0.32, from about 0.24 to about 0.32, from about 0.25 to about
0.32, from about 0.26 to about 0.32, from about 0.27 to about 0.32,
from about 0.28 to about 0.32, from about 0.29 to about 0.32, or
from about 0.30 to about 0.32. In embodiments, the first cationic
lipid is present at a compositional molar ratio from 0.18 to 0.32,
from 0.19 to 0.32, from 0.20 to 0.32, from 0.21 to 0.32, from 0.22
to 0.32, from 0.23 to 0.32, from 0.24 to 0.32, from 0.25 to 0.32,
from 0.26 to 0.32, from 0.27 to 0.32, from 0.28 to 0.32, from 0.29
to 0.32, or from 0.30 to 0.32.
[0161] In embodiments, the first cationic lipid is present at a
compositional molar ratio from 0.18 to 0.32. In embodiments, the
first cationic lipid is present at a compositional molar ratio from
0.19 to 0.32. In embodiments, the first cationic lipid is present
at a compositional molar ratio from 0.20 to 0.32. In embodiments,
the first cationic lipid is present at a compositional molar ratio
from 0.21 to 0.32. In embodiments, the first cationic lipid is
present at a compositional molar ratio from 0.22 to 0.32. In
embodiments, the first cationic lipid is present at a compositional
molar ratio from 0.23 to 0.32. In embodiments, the first cationic
lipid is present at a compositional molar ratio from 0.24 to 0.32.
In embodiments, the first cationic lipid is present at a
compositional molar ratio from 0.25 to 0.32. In embodiments, the
first cationic lipid is present at a compositional molar ratio from
0.26 to 0.32. In embodiments, the first cationic lipid is present
at a compositional molar ratio from 0.27 to 0.32. In embodiments,
the first cationic lipid is present at a compositional molar ratio
from 0.28 to 0.32. In embodiments, the first cationic lipid is
present at a compositional molar ratio from 0.29 to 0.32. In
embodiments, the first cationic lipid is present at a compositional
molar ratio from 0.30 to 0.32.
[0162] In embodiments, the first cationic lipid is present at a
compositional molar ratio from 0.18 to 0.31. In embodiments, the
first cationic lipid is present at a compositional molar ratio from
0.18 to 0.30. In embodiments, the first cationic lipid is present
at a compositional molar ratio from 0.18 to 0.29. In embodiments,
the first cationic lipid is present at a compositional molar ratio
from 0.18 to 0.28. In embodiments, the first cationic lipid is
present at a compositional molar ratio from 0.18 to 0.27. In
embodiments, the first cationic lipid is present at a compositional
molar ratio from 0.18 to 0.26. In embodiments, the first cationic
lipid is present at a compositional molar ratio from 0.18 to 0.25.
In embodiments, the first cationic lipid is present at a
compositional molar ratio from 0.18 to 0.24. In embodiments, the
first cationic lipid is present at a compositional molar ratio from
0.18 to 0.23. In embodiments, the first cationic lipid is present
at a compositional molar ratio from 0.18 to 0.22. In embodiments,
the first cationic lipid is present at a compositional molar ratio
from 0.18 to 0.21. In embodiments, the first cationic lipid is
present at a compositional molar ratio from 0.18 to 0.20.
[0163] In embodiments, the first cationic lipid is present at a
compositional molar ratio of 0.18, 0.19, 0.20, 0.21, 0.22, 0.23,
0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 or 0.32. In
embodiments, the first cationic lipid is present at a compositional
molar ratio of about 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24,
0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 or 0.32. In further
embodiments, the second cationic lipid is present at a
compositional molar ratio from about 0.24 to about 0.51, the first
helper lipid is present at a compositional molar ratio from about
0.20 to about 0.32, the second helper lipid is present at a
compositional molar ratio from about 0.01 to about 0.14 and the
biostability enhancing agent is present at a compositional molar
ratio from about 0.01 to about 0.02.
[0164] In embodiments, the second cationic lipid is present at a
compositional molar ratio from about 0.24 to about 0.51, from about
0.25 to about 0.51, from about 0.26 to about 0.51, from about 0.27
to about 0.51, from about 0.28 to about 0.51, from about 0.29 to
about 0.51, from about 0.30 to about 0.51, from about 0.31 to about
0.51, from about 0.32 to about 0.51, from about 0.33 to about 0.51,
from about 0.34 to about 0.51, from about 0.35 to about 0.51, from
about 0.36 to about 0.51, from about 0.37 to about 0.51, from about
0.38 to about 0.51, from about 0.39 to about 0.51, from about 0.40
to about 0.51, from about 0.41 to about 0.51, from about 0.42 to
about 0.51, from about 0.43 to about 0.51, from about 0.44 to about
0.51, from about 0.45 to about 0.51, from about 0.46 to about 0.51,
from about 0.47 to about 0.51, from about 0.48 to about 0.51, or
from about 0.49 to about 0.51.
[0165] In embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.24 to 0.51, from 0.25 to 0.51,
from 0.26 to 0.51, from 0.27 to 0.51, from 0.28 to 0.51, from 0.29
to 0.51, from 0.30 to 0.51, from 0.31 to 0.51, from 0.32 to 0.51,
from 0.33 to 0.51, from 0.34 to 0.51, from 0.35 to 0.51, from 0.36
to 0.51, from 0.37 to 0.51, from 0.38 to 0.51, from 0.39 to 0.51,
from 0.40 to 0.51, from 0.41 to 0.51, from 0.42 to 0.51, from 0.43
to 0.51, from 0.44 to 0.51, from 0.45 to 0.51, from 0.46 to 0.51,
from 0.47 to 0.51, from 0.48 to 0.51, or from 0.49 to 0.51.
[0166] In embodiments, the second cationic lipid is present at a
compositional molar ratio from about 0.24 to about 0.51, from about
0.24 to about 0.50, from about 0.24 to about 0.49, from about 0.24
to about 0.48, from about 0.24 to about 0.47, from about 0.24 to
about 0.46, from about 0.24 to about 0.45, from about 0.24 to about
0.44, from about 0.24 to about 0.43, from about 0.24 to about 0.42,
from about 0.24 to about 0.41, from about 0.24 to about 0.40, from
about 0.24 to about 0.39, from about 0.24 to about 0.38, from about
0.24 to about 0.37, from about 0.24 to about 0.36, from about 0.24
to about 0.35, from about 0.24 to about 0.34, from about 0.24 to
about 0.33, from about 0.24 to about 0.32, from about 0.24 to about
0.31, from about 0.24 to about 0.30, from about 0.24 to about 0.29,
from about 0.24 to about 0.28, from about 0.24 to about 0.27, or
from about 0.24 to about 0.26.
[0167] In embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.24 to 0.51, from 0.24 to 0.50,
from 0.24 to 0.49, from 0.24 to 0.48, from 0.24 to 0.47, from 0.24
to 0.46, from 0.24 to 0.45, from 0.24 to 0.44, from 0.24 to 0.43,
from 0.24 to 0.42, from 0.24 to 0.41, from 0.24 to 0.40, from 0.24
to 0.39, from 0.24 to 0.38, from 0.24 to 0.37, from 0.24 to 0.36,
from 0.24 to 0.35, from 0.24 to 0.34, from 0.24 to 0.33, from 0.24
to 0.32, from 0.24 to 0.31, from 0.24 to 0.30, from 0.24 to 0.29,
from 0.24 to 0.28, from 0.24 to 0.27, or from 0.24 to 0.26.
[0168] In embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.24 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.25 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.26 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.27 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.28 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.29 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.30 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.31 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.32 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.33 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.34 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.35 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.36 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.37 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.38 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.39 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.40 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.41 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.42 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.43 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.44 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.45 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio
from 0.46 to 0.51. In embodiments, the second cationic lipid is
present at a compositional molar ratio from 0.47 to 0.51. In
embodiments, the second cationic lipid is present at a
compositional molar ratio from 0.48 to 0.51. In embodiments, the
second cationic lipid is present at a compositional molar ratio or
from 0.49 to 0.51.
[0169] In embodiments, the second cationic lipid is present at a
compositional molar ratio of 0.24, 0.25, 0.26, 0.27, 0.28, 0.29,
0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40,
0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50 or 0.51.
In embodiments, the second cationic lipid is present at a
compositional molar ratio of about 0.24, 0.25, 0.26, 0.27, 0.28,
0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39,
0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50 or
0.51. In further embodiments, the first cationic lipid is present
at a compositional molar ratio from about 0.18 to about 0.32, the
first helper lipid is present at a compositional molar ratio from
about 0.20 to about 0.32, the second helper lipid is present at a
compositional molar ratio from about 0.01 to about 0.14 and the
biostability enhancing agent is present at a compositional molar
ratio from about 0.01 to about 0.02.
[0170] In embodiments, the first helper lipid is present at a
compositional molar ratio from 0.20 to 0.32, from 0.21 to 0.32,
from 0.22 to 0.32, from 0.23 to 0.32, from 0.24 to 0.32, from 0.25
to 0.32, from 0.26 to 0.32, from 0.27 to 0.32, from 0.28 to 0.32,
from 0.29 to 0.32, or from 0.30 to 0.32. In embodiments, the first
helper lipid is present at a compositional molar ratio from about
0.20 to about 0.32, from about 0.21 to about 0.32, from about 0.22
to about 0.32, from about 0.23 to about 0.32, from about 0.24 to
about 0.32, from about 0.25 to about 0.32, from about 0.26 to about
0.32, from about 0.27 to about 0.32, from about 0.28 to about 0.32,
from about 0.29 to about 0.32, or from about 0.30 to about
0.32.
[0171] In embodiments, the first helper lipid is present at a
compositional molar ratio from 0.20 to 0.31, from 0.20 to 0.30,
from 0.20 to 0.29, from 0.20 to 0.28, from 0.20 to 0.27, from 0.20
to 0.26, from 0.20 to 0.25, from 0.20 to 0.24, from 0.20 to 0.23,
or from 0.20 to 0.22. In embodiments, the first helper lipid is
present at a compositional molar ratio from about 0.20 to about
0.31, from about 0.20 to about 0.30, from about 0.20 to about 0.29,
from about 0.20 to about 0.28, from about 0.20 to about 0.27, from
about 0.20 to about 0.26, from about 0.20 to about 0.25, from about
0.20 to about 0.24, from about 0.20 to about 0.23, or from about
0.20 to about 0.22.
[0172] In embodiments, the first helper lipid is present at a
compositional molar ratio from 0.20 to 0.31. In embodiments, the
first helper lipid is present at a compositional molar ratio from
0.20 to 0.30. In embodiments, the first helper lipid is present at
a compositional molar ratio from 0.20 to 0.29. In embodiments, the
first helper lipid is present at a compositional molar ratio from
0.20 to 0.28. In embodiments, the first helper lipid is present at
a compositional molar ratio from 0.20 to 0.27. In embodiments, the
first helper lipid is present at a compositional molar ratio from
0.20 to 0.26. In embodiments, the first helper lipid is present at
a compositional molar ratio from 0.20 to 0.25. In embodiments, the
first helper lipid is present at a compositional molar ratio from
0.20 to 0.24. In embodiments, the first helper lipid is present at
a compositional molar ratio from 0.20 to 0.23. In embodiments, the
first helper lipid is present at a compositional molar ratio or
from 0.20 to 0.22.
[0173] In embodiments, the first helper lipid is present at a
compositional molar ratio from 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,
0.26, 0.27, 0.28, 0.29, 0.30, 0.31, or 0.32. In embodiments, the
first helper lipid is present at a compositional molar ratio from
about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29,
0.30, 0.31, or 0.32. In further embodiments, the first cationic
lipid is present at a compositional molar ratio from about 0.18 to
about 0.32, the second cationic lipid is present at a compositional
molar ratio from about 0.24 to about 0.51, the second helper lipid
is present at a compositional molar ratio from about 0.01 to about
0.14 and the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.02.
[0174] In embodiments, the second helper lipid is present at a
compositional molar ratio from 0.01 to 0.14, from 0.01 to 0.13,
from 0.01 to 0.12, from 0.01 to 0.11, from 0.01 to 0.10, from 0.01
to 0.9, from 0.01 to 0.8, from 0.01 to 0.7, from 0.01 to 0.6, from
0.01 to 0.5, from 0.01 to 0.4, from 0.01 to 0.3, from 0.01 to 0.2,
from 0.01 to 0.1, from 0.01 to 0.09, from 0.01 to 0.08, from 0.01
to 0.07, from 0.01 to 0.06, from 0.01 to 0.05, from 0.01 to 0.04,
or from 0.01 to 0.03. In embodiments, the second helper lipid is
present at a compositional molar ratio from about 0.01 to about
0.14, from about 0.01 to about 0.13, from about 0.01 to about 0.12,
from about 0.01 to about 0.11, from about 0.01 to about 0.10, from
about 0.01 to about 0.9, from about 0.01 to about 0.8, from about
0.01 to about 0.7, from about 0.01 to about 0.6, from about 0.01 to
about 0.5, from about 0.01 to about 0.4, from about 0.01 to about
0.3, from about 0.01 to about 0.2, from about 0.01 to about 0.1,
from about 0.01 to about 0.09, from about 0.01 to about 0.08, from
about 0.01 to about 0.07, from about 0.01 to about 0.06, from about
0.01 to about 0.05, from about 0.01 to about 0.04, or from about
0.01 to about 0.03.
[0175] In embodiments, the second helper lipid is present at a
compositional molar ratio from 0.02 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.03 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.04 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.05 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.06 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.07 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.08 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.09 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.1 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.2 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.3 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.4 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.5 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.6 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.7 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.8 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.9 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.1 to 0.14. In embodiments, the second helper lipid is present at
a compositional molar ratio from 0.11 to 0.14. In embodiments, the
second helper lipid is present at a compositional molar ratio from
0.12 to 0.14.
[0176] In embodiments, the second helper lipid is present at a
compositional molar ratio from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,
0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, or 0.14. In embodiments,
the second helper lipid is present at a compositional molar ratio
from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.10, 0.11, 0.12, 0.13, or 0.14. In further embodiments, the first
cationic lipid is present at a compositional molar ratio from about
0.18 to about 0.32, the second cationic lipid is present at a
compositional molar ratio from about 0.24 to about 0.51, the first
helper lipid is present at a compositional molar ratio from about
0.20 to about 0.32 and the biostability enhancing agent is present
at a compositional molar ratio from about 0.01 to about 0.02.
[0177] In embodiments, the biostability enhancing agent is present
at a compositional molar ratio from 0.01 to 0.02. In embodiments,
the biostability enhancing agent is present at a compositional
molar ratio from 0.011 to 0.02. In embodiments, the biostability
enhancing agent is present at a compositional molar ratio from
0.012 to 0.02. In embodiments, the biostability enhancing agent is
present at a compositional molar ratio from 0.013 to 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from 0.014 to 0.02. In embodiments, the
biostability enhancing agent is present at a compositional molar
ratio from 0.015 to 0.02. In embodiments, the biostability
enhancing agent is present at a compositional molar ratio from
0.016 to 0.02. In embodiments, the biostability enhancing agent is
present at a compositional molar ratio from 0.017 to 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from 0.018 to 0.02.
[0178] In embodiments, the biostability enhancing agent is present
at a compositional molar ratio from about 0.01 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.011 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.012 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.013 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.014 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.015 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.016 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.017 to about 0.02. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.018 to about 0.02.
[0179] In embodiments, the biostability enhancing agent is present
at a compositional molar ratio from about 0.01 to about 0.019. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.018. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.017. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.016. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.015. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.014. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.013. In
embodiments, the biostability enhancing agent is present at a
compositional molar ratio from about 0.01 to about 0.012.
[0180] In embodiments, the biostability enhancing agent is present
at a compositional molar ratio from 0.01, 0.011, 0.012, 0.013,
0.014, 0.015, 0.016, 0.017, 0.018, 0.019 or 0.020. In embodiments,
the biostability enhancing agent is present at a compositional
molar ratio from about 0.01, 0.011, 0.012, 0.013, 0.014, 0.015,
0.016, 0.017, 0.018, 0.019 or 0.020. In further embodiments, the
first cationic lipid is present at a compositional molar ratio from
about 0.18 to about 0.32, the second cationic lipid is present at a
compositional molar ratio from about 0.24 to about 0.51, the first
helper lipid is present at a compositional molar ratio from about
0.20 to about 0.32 and the second helper lipid is present at a
compositional molar ratio from about 0.01 to about 0.14.
[0181] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.24,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.05, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0182] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.32,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.39, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.26, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0183] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0184] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.23,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.45, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0185] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.51, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0186] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.27,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0187] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.25,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.26, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.01,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0188] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.28,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.24, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.14, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
750.
[0189] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.18,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.47, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.32, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.01, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein said biostability enhancing agent is C14 polyethylene
glycol 5000.
[0190] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.24,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.40, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.24, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.10, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
2000.
[0191] In an aspect is provided a composition including: (i) a
first cationic lipid at a compositional molar ratio of about 0.32,
wherein the first cationic lipid is dihydroxy dimyristyl
spermidine; (ii) a second cationic lipid at a compositional molar
ratio of about 0.38, wherein the second cationic lipid is hydroxy
dimyristyl spermidine; (iii) a first helper lipid at a
compositional molar ratio of about 0.20, wherein the first helper
lipid is dioleoylphosphatidylethanolamine; (iv) a second helper
lipid at a compositional molar ratio of about 0.08, wherein the
second helper lipid is cholesterol; and (v) a biostability
enhancing agent at a compositional molar ratio of about 0.02,
wherein the biostability enhancing agent is C14 polyethylene glycol
5000.
[0192] In embodiments, the composition as provided herein including
embodiments thereof, further includes a bioactive agent. A
"bioactive agent" as provided herein refers to a compound that upon
administration to a cell, tissue or organism has a detectable
effect on the biological function of said cell, tissue or organism.
In embodiments, the detectable effect is a biological effect. In
embodiments, the detectable effect is a therapeutic effect. In
embodiments, the detectable effect is a diagnostic effect. The
bioactive agent is capable of forming a lipid aggregate with the
compositions provided herein including embodiments thereof. In
embodiments, the bioactive agent is a test compound. A "test
compound" as provided herein is a compound whose effect on a
biological function is determined relative to a control compound. A
"control compound" as provided herein refers to a compound having a
known effect on a biological function. In embodiments, the
bioactive agent is a control compound. In embodiments, the
bioactive agent is a therapeutic agent or a diagnostic agent. In
embodiments, the bioactive agent is a therapeutic agent or a
diagnostic agent. In embodiments, the bioactive agent is a
therapeutic agent. In embodiments, the bioactive agent is a
diagnostic agent. In embodiments, the bioactive agent includes a
nucleic acid, a ribonucleoprotein or a small molecule. In
embodiments, the bioactive agent includes a nucleic acid. In
embodiments, the bioactive agent includes a ribonucleoprotein. In
embodiments, the bioactive agent includes a small molecule. In
embodiments, the nucleic acid is an mRNA, a siRNA, a miRNA or a
guide RNA. In embodiments, the nucleic acid is an mRNA. In
embodiments, the nucleic acid is a siRNA. In embodiments, the
nucleic acid is a miRNA. In embodiments, the nucleic acid is a
guide RNA. In embodiments, the bioactive agent includes a nucleic
acid and a ribonucleoprotein. In embodiments, the ribonucleoprotein
is CRISPR associated protein 9 (Cas9). An "mRNA" as provided herein
refers to a ribonucleic acid molecule, including one or more than
one expressible nucleic acid sequences encoding one or more
proteins or polypeptides, or other DNA molecules.
Pharmaceutical Composition
[0193] In an aspect is provided a pharmaceutical composition
including a composition as provided herein including embodiments
thereof and a pharmaceutically acceptable excipient.
[0194] A therapeutically effective amount as provided herein refers
to an amount effective to achieve its intended purpose. The actual
amount effective for a particular application will depend, inter
alia, on the condition being treated. When administered in methods
to treat a disease, the pharmaceutical compositions described
herein will contain an amount of active bioactive agent effective
to achieve the desired result, e.g., modulating the activity of a
target molecule and/or reducing, eliminating, or slowing the
progression of disease symptoms (e.g., pulmonary disease).
Determination of a therapeutically effective amount of a bioactive
agent forming a lipid aggregate with the compositions provided
herein is well within the capabilities of those skilled in the art,
especially in light of the detailed disclosure herein.
[0195] Acceptable carriers, excipients or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and
include buffers such as phosphate, citrate, or acetate at a pH
typically of 5.0 to 8.0, most often 6.0 to 7.0; salts such as
sodium chloride, potassium chloride, etc. to make isotonic;
antioxidants, preservatives, low molecular weight polypeptides,
proteins, hydrophilic polymers such as polysorbate 80, amino acids
such as glycine, carbohydrates, chelating agents, sugars, and other
standard ingredients known to those skilled in the art (Remington's
Pharmaceutical Science 16.sup.th edition, Osol, A. Ed. 1980).
[0196] A pharmaceutical composition including a composition
provided herein including embodiments thereof (e.g., a lipid
aggregate complexed with a bioactive agent) can be administered by
a variety of methods known in the art. The route and/or mode of
administration vary depending upon the desired results. In
embodiments, administration is intravenous, intramuscular,
intraperitoneal, or subcutaneous, or administered proximal to the
site of the target. Pharmaceutically acceptable excipients can be
suitable for intravenous, intramuscular, subcutaneous, parenteral,
spinal or epidermal administration (e.g., by injection or
infusion).
[0197] Pharmaceutical compositions of the composition provided
herein including embodiments thereof (e.g., a lipid aggregate
complexed with a bioactive agent) can be prepared in accordance
with methods well known and routinely practiced in the art. See,
e.g., Remington: The Science and Practice of Pharmacy, Mack
Publishing Co., 20.sup.th ed., 2000; and Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker,
Inc., New York, 1978. Pharmaceutical compositions are preferably
manufactured under GMP conditions. Typically, a therapeutically
effective dose or efficacious dose of the composition provided
herein including embodiments thereof (e.g., a lipid aggregate
complexed with a bioactive agent) is employed in the pharmaceutical
compositions of the invention. The composition provided herein
including embodiments thereof (e.g., a lipid aggregate complexed
with a bioactive agent) can be formulated into pharmaceutically
acceptable dosage forms by conventional methods known to those of
skill in the art. Dosage regimens are adjusted to provide the
optimum desired response (e.g., a therapeutic response). For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation. It may be advantageous to formulate the
composition provided herein including embodiments thereof (e.g., a
lipid aggregate complexed with a bioactive agent) in combination
with other therapies or agents. It can be advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit contains a
predetermined quantity of a composition provided herein including
embodiments thereof (e.g., a lipid aggregate complexed with a
bioactive agent) calculated to produce the desired therapeutic
effect in association with the required pharmaceutical
excipient.
[0198] Actual dosage levels of the bioactive agent in the
pharmaceutical compositions of the present invention can be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level
depends upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular antibody being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors.
[0199] A physician or veterinarian can start doses of the
composition provided herein including embodiments thereof (e.g., a
lipid aggregate complexed with a bioactive agent) employed in the
pharmaceutical composition at levels lower than that required to
achieve the desired therapeutic effect and gradually increase the
dosage until the desired effect is achieved. In general, effective
doses of the compositions of the present invention vary depending
upon many different factors, including the specific disease or
condition to be treated, means of administration, target site,
physiological state of the patient, whether the patient is human or
an animal, other medications administered, and whether treatment is
prophylactic or therapeutic. Treatment dosages need to be titrated
to optimize safety and efficacy.
[0200] Composition provided herein including embodiments thereof
(e.g., a lipid aggregate complexed with a bioactive agent) can be
administered on multiple occasions. Intervals between single
dosages can be weekly, monthly or yearly. Intervals can also be
irregular as indicated by measuring blood levels of the composition
in the patient. Dosage and frequency vary depending on the
half-life of the composition in the patient. The dosage and
frequency of administration can vary depending on whether the
treatment is prophylactic or therapeutic. In prophylactic
applications, a relatively low dosage is administered at relatively
infrequent intervals over a long period of time. Some patients
continue to receive treatment for the rest of their lives. In
therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
Cellular Compositions
[0201] In an aspect is provided a cell including a composition as
provided herein including embodiments thereof. In embodiments, the
cell is a mammalian cell. In embodiments, the cell is a rodent
cell. In embodiments, the cell is a mouse cell. In embodiments, the
cell is a rat cell. In embodiments, the cell is a porcine cell. In
embodiments, the cell is a canine cell. In embodiments, the cell is
a primate cell. In embodiments, the cell is a human cell. In
embodiments, the cell is an epithelial cell. In embodiments, the
cell is an epithelial lung cell. In embodiments, the cell is an
endothelial cell. In embodiments, the cell is an endothelial lung
cell. In embodiments, the cell forms part of an organism. In
embodiments, the organism is human. In embodiments, the organism is
rat. In embodiments, the organism is mouse.
Methods of Delivery
[0202] In an aspect is provided, method of delivering a bioactive
agent to a cell, the method including: (i) admixing an bioactive
agent with a composition as provided herein including embodiments
thereof, thereby forming a bioactive agent-lipid complex; (ii)
contacting a cell with the bioactive agent-lipid complex, thereby
delivering the bioactive agent-lipid complex to a cell. The
bioactive agent may be any bioactive agent as described herein
(e.g. a nucleic acid). The bioactive agent-lipid complex as
provided herein is a lipid aggregate as described herein.
[0203] In embodiments, the method as described herein including
embodiments thereof, further includes allowing the bioactive
agent-lipid complex to enter the cell. In embodiments, the
bioactive agent is a therapeutic agent or a diagnostic agent. In
embodiments, the bioactive agent includes a nucleic acid, a
ribonucleoprotein or a small molecule. In embodiments, the nucleic
acid is an mRNA, a siRNA, miRNA or guide RNA. In embodiments, the
bioactive agent includes a guide RNA and a ribonucleoprotein. In
embodiments, the ribonucleoprotein is CRISPR associated protein 9
(Cas9). In embodiments, the ribonucleoprotein is bound to the guide
RNA.
[0204] In embodiments, the cell is a mammalian cell. In
embodiments, the cell is a rodent cell. In embodiments, the cell is
a mouse cell. In embodiments, the cell is a rat cell. In
embodiments, the cell is a porcine cell. In embodiments, the cell
is a canine cell. In embodiments, the cell is a primate cell. In
embodiments, the cell is an epithelial cell. In embodiments, the
cell is an epithelial lung cell. In embodiments, the cell is an
endothelial cell. In embodiments, the cell is an endothelial lung
cell.
[0205] In another aspect is provided a method of delivering a
bioactive agent to lung tissue in a subject, the method including:
(i) admixing an bioactive agent with a composition as described
herein including embodiments thereof, thereby forming a bioactive
agent-lipid complex; (ii) systemically administering an effective
amount of the bioactive agent-lipid complex to a subject, thereby
delivering the bioactive agent-lipid complex to a lung tissue in a
subject.
[0206] In another aspect is provided a method of expressing a
protein in lung tissue in a subject, the method including: (i)
admixing a mRNA with a composition as described herein including
embodiments thereof, thereby forming a mRNA-lipid complex; (ii)
administering an effective amount of the mRNA-lipid complex to a
subject; and (iii) allowing the mRNA of the mRNA-lipid complex to
express in lung tissue of the subject, thereby expressing a protein
in lung tissue in a subject.
Methods of Treatment
[0207] In an aspect is provided a method of treating a pulmonary
disease in a subject in need thereof, the method including
administering to a subject a therapeutically effective amount of a
bioactive agent and a composition as described herein including
embodiments thereof, thereby treating a pulmonary disease in the
subject.
[0208] In embodiments, the composition and the bioactive agent are
admixed prior to the administering. In embodiments, bioactive agent
includes a nucleic acid, a ribonucleoprotein or a small molecule.
In embodiments, the nucleic acid is an mRNA, a siRNA, a miRNA or a
guide RNA.
[0209] In embodiments, the pulmonary disease is asthma, chronic
obstructive pulmonary disease (COPD), lung cancer or cystic
fibrosis. In embodiments, the pulmonary disease is asthma. In
embodiments, the pulmonary disease is chronic obstructive pulmonary
disease (COPD). In embodiments, the pulmonary disease is lung
cancer. In embodiments, the pulmonary disease is or cystic
fibrosis.
[0210] The terms "pulmonary disease," "pulmonary disorder," "lung
disease," etc. are used interchangeably herein. The term is used to
broadly refer to lung disorders characterized by difficulty
breathing, coughing, airway discomfort and inflammation, increased
mucus, and/or pulmonary fibrosis.
[0211] The terms "dose" and "dosage" are used interchangeably
herein. A dose refers to the amount of active ingredient given to
an individual at each administration. For the present invention,
the dose will generally refer to the amount of pulmonary disease
treatment. The dose will vary depending on a number of factors,
including the range of normal doses for a given therapy, frequency
of administration; size and tolerance of the individual; severity
of the condition; risk of side effects; and the route of
administration. One of skill will recognize that the dose can be
modified depending on the above factors or based on therapeutic
progress. The term "dosage form" refers to the particular format of
the pharmaceutical, and depends on the route of administration. For
example, a dosage form can be in a liquid form for nebulization,
e.g., for inhalants, or a saline solution, e.g., for injection.
[0212] As used herein, the terms "treat" and "prevent" are not
intended to be absolute terms. Treatment can refer to any delay in
onset, reduction in the frequency or severity of symptoms,
amelioration of symptoms, improvement in patient comfort and/or
respiratory function, etc. The effect of treatment can be compared
to an individual or pool of individuals not receiving a given
treatment, or to the same patient prior to, or after cessation of,
treatment.
[0213] "Treating" or "treatment" as used herein (and as
well-understood in the art) also broadly includes any approach for
obtaining beneficial or desired results in a subject's condition,
including clinical results. Beneficial or desired clinical results
can include, but are not limited to, alleviation or amelioration of
one or more symptoms or conditions, diminishment of the extent of a
disease, stabilizing (i.e., not worsening) the state of disease,
prevention of a disease's transmission or spread, delay or slowing
of disease progression, amelioration or palliation of the disease
state, diminishment of the reoccurrence of disease, and remission,
whether partial or total and whether detectable or undetectable. In
other words, "treatment" as used herein includes any cure,
amelioration, or prevention of a disease. Treatment may prevent the
disease from occurring; inhibit the disease's spread; relieve the
disease's symptoms (e.g., ocular pain, seeing halos around lights,
red eye, very high intraocular pressure), fully or partially remove
the disease's underlying cause, shorten a disease's duration, or do
a combination of these things.
[0214] "Treating" and "treatment" as used herein include
prophylactic treatment. Treatment methods include administering to
a subject a therapeutically effective amount of an active agent.
The administering step may consist of a single administration or
may include a series of administrations. The length of the
treatment period depends on a variety of factors, such as the
severity of the condition, the age of the patient, the
concentration of active agent, the activity of the compositions
used in the treatment, or a combination thereof. It will also be
appreciated that the effective dosage of an agent used for the
treatment or prophylaxis may increase or decrease over the course
of a particular treatment or prophylaxis regime. Changes in dosage
may result and become apparent by standard diagnostic assays known
in the art. In some instances, chronic administration may be
required. For example, the compositions are administered to the
subject in an amount and for a duration sufficient to treat the
patient.
[0215] The term "prevent" refers to a decrease in the occurrence of
pulmonary disease symptoms in a patient. As indicated above, the
prevention may be complete (no detectable symptoms) or partial,
such that fewer symptoms are observed than would likely occur
absent treatment.
[0216] The term "therapeutically effective amount," as used herein,
refers to that amount of the therapeutic agent sufficient to
ameliorate the disorder, as described above. For example, for the
given parameter, a therapeutically effective amount will show an
increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%,
60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also
be expressed as "-fold" increase or decrease. For example, a
therapeutically effective amount can have at least a 1.2-fold,
1.5-fold, 2-fold, 5-fold, or more effect over a control.
[0217] The term "diagnosis" refers to a relative probability that a
pulmonary disease is present in the subject. Similarly, the term
"prognosis" refers to a relative probability that a certain future
outcome may occur in the subject. For example, in the context of
the present invention, prognosis can refer to the likelihood that
an individual will develop a pulmonary disease, or the likely
severity of the disease (e.g., severity of symptoms, rate of
functional decline, survival, etc.). The terms are not intended to
be absolute, as will be appreciated by any one of skill in the
field of medical diagnostics.
EXAMPLES
Example 1: Novel Lipid Nanoparticles for In Vivo Lung Targeted mRNA
and siRNA Delivery
[0218] The rapidly expanding utilization of nucleic acids as a
therapeutic tool has presented the field with the task of
optimizing and innovating delivery methods. Lipid nanoparticles are
a common delivery vehicle due to their ability to facilitate
cellular uptake while protecting the payload from extracellular
enzyme degradation. Organ and tissue specific delivery of sensitive
payloads such as mRNA is of great importance in tailoring
therapeutic functionality. This is commonly achieved using
biomolecular targeting via ligand or receptor expression on the
surface of the nanoparticle. However, manipulation of the inherent
properties of nanoparticles affords the opportunity to tailor the
location of delivery to a specific organ of interest.
[0219] Applicants have developed novel lipid nanoparticles that are
specifically optimized for use in vivo, and engineered to
inherently target and deliver nucleic acid payloads (mRNA and
siRNA) to murine lungs and spleen without the use of biomolecular
targeting. Development of the lipid nanoparticle was performed
using multivariable Design of Experiment modeling, which allowed
Applicants to understand and optimize the lipid formulation
components and composition. Characterization of the lipid
nanoparticles showed uniform size and high encapsulation
efficiency. Following multiple design iterations, in vivo
functional testing to assess biodistribution identified a novel
formulation capable of exclusively targeting lung tissue and
achieving highly efficient mRNA transfection. Transfection
efficiency was measured following in vivo systemic delivery of a
chemically modified luciferase encoding mRNA. Quantification was
performed using the IVIS imaging system to measure in vivo and ex
vivo bioluminescence measurements.
Example 2: Development of Novel Lipid Nanoparticles for In Vivo
Lung Targeted mRNA Delivery
[0220] Abstract.
[0221] The rapidly expanding utilization of mRNA as a therapeutic
tool has presented the field with the task of optimizing and
innovating delivery methods. As the applications for RNA based
therapeutics continues to rise, a parallel emerging need to improve
upon and develop novel technology has come to the forefront. Lipid
nanoparticles area common delivery vehicle for mRNA due to their
ability to facilitate cellular uptake while protecting the mRNA
from extracellular enzyme degradation. Organ and tissue specific
delivery of mRNA is of great importance in tailoring therapeutic
functionality. This is commonly achieved using biomolecular
targeting via ligand or receptor expression on the surface of the
nanoparticle.
[0222] Applicants have developed novel lipid nanoparticles that are
specifically optimized for use in vivo, and engineered to
inherently target and deliver mRNA to murine lungs and spleen
without the use of biomolecular targeting. Development of the lipid
nanoparticle was performed using multivariable Design of Experiment
modeling, which allowed Applicants to understand and optimize the
lipid formulation components and composition. Characterization of
the lipid nanoparticles showed uniform size and high encapsulation
efficiency. Following multiple design iterations, in vivo
functional testing to assess biodistribution identified a novel
formulation capable of exclusively targeting the lung tissue and
achieving highly efficient mRNA transfection. Transfection
efficiency was measured following in vivo systemic delivery of a
chemically modified luciferase encoding mRNA. Quantification was
performed using the IVIS imaging system to measure in vivo and ex
vivo bioluminescence measurements. Lung specific expression levels
could be modulated by varying the dose of mRNA, and significant
protein expression was sustained over the course of 48 hours
following a single administration. This novel lipid nanoparticle is
well tolerated in vivo, with no qualitative gross toxicity, and
quantitatively analyzed by a comprehensive cytokine profiling
performed on murine serum samples. Further optimization of
biodistribution to achieve exclusive targeting to the lung is
currently underway, and preliminary data indicates that variance in
charge ratio enhances delivery and expression exclusively to the
lung, and depletes expression in other organs.
[0223] Introduction.
[0224] Due to the unstable nature of mRNA, protection against
degradation during the delivery phase is a serious challenge to
overcome in the field of mRNA therapeutics. Applicants have
developed efficient lipid nanoparticle (LNP) systems that
encapsulate and protect the mRNA from degradation and clearance in
the blood; thus translating to improved efficacy and reduced
toxicity in vivo. Optimization of Applicant's LNP systems has
maximized delivery efficiency with organ specific uptake patterns
such as the lung and spleen via intravenous delivery.
[0225] Materials and Methods.
[0226] Based on mixture Design of Experiment optimization, new LNP
formulations were made, complexed with mRNA using the previously
developed protocol shown in FIGS. 1A-1C, and the subsequent LNPs
were screened for delivery in vivo using a luciferase readout. Top
performing formulations were then used to model and predict second
generation formulations for optimized tissue specific expression.
These new formulations were then tested in vivo to identify the
best LNP formulation.
[0227] Firefly Luciferase mRNA was complexed with each LNP
formulation and injected intravenously (FIG. 2). Following 4 hours
incubation, Luciferin substrate was injected IP, and luciferase
expression was measured in vivo and ex vivo via the IVIS Lumina LT.
Bioluminescence (p/sec/cm 2/sr) was quantified with Living
Image.RTM. Software (FIG. 2).
[0228] Results.
[0229] First pass screening of LNPs revealed organ specific
delivery patterns based on formulation composition or N/P ratio
variance. Whole animal in vivo imaging indicated organ specific
patterns (FIGS. 3A-3B), and ex vivo analysis allowed for extensive
evaluation of biodistribution patterns (FIG. 3C-3E). Using the
performance data from the first generation of LNPs, expression was
optimized using DoE design to maximize tissue specific performance,
focusing mainly on enhancement of expression in the spleen and
lung.
[0230] Over 100 new LNP formulations, spanning 3 generations of
optimization were screened for performance. For screening, mRNA
encoding for firefly luciferase was complexed with each newly
designed formulation and injected intravenously. Ex vivo
quantification of bioluminescence signal in isolated lung tissue
(FIG. 4A) or isolated spleen tissue (FIG. 4B) from BALB/c mice,
4-hrs post IV injection is summarized in the graphs. The
bioluminescence (p/sec/cm 2/sr) was quantified using Living
Image.RTM. Software.
[0231] Dose titration of mRNA was performed, and luciferase
expression was used as a readout to measure protein expression.
Mice were injected at Time 0 h, and imaged over the course of 120
hours (5 days). In vivo images, and corresponding quantification at
4 hours is shown in FIGS. 5A-5B. Sustained luciferase activity
measurement were repeated over the duration of the experiment on
the same mice; measurements were taken using the whole body in vivo
imaging technique which is reflected by the lower starting signal
values compared to ex vivo measurements. The bioluminescence
(p/sec/cm 2/sr) was quantified using Living Image.RTM. Software and
is summarized in the graph in FIG. 5C.
[0232] Following optimization of mRNA dose, a time course of
luciferase expression levels was performed and performance was
measured following removal of the lung tissue and performing ex
vivo measurements over the course of 96 hours (FIGS. 6A-6C). Mice
were injected intravenously with the LNPs, whole body imaging was
performed at each specified time point, and then ex vivo
measurements were obtained on isolated lung tissue.
[0233] Quantification of circulating cytokine levels in mouse serum
at 2 hrs, 4 hrs, 24 hrs and 48 hrs post-injection under two mRNA
dose conditions: low (1 mg/kg) and high (3 mg/kg) (FIGS.
7A-7J).
[0234] Preliminary results indicate that varying N/P ratio of the
LNPs can lead to exclusion of protein expression from certain
organs, resulting in a tissue specific biodistribution of mRNA
(FIG. 8A-8B).
CONCLUSION
[0235] LNP formulations were extensively optimized for targeted
lung delivery using Design of Experiment based testing. Through in
vivo systemic delivery, high fidelity luciferase expression was
achieved in lung tissue. Transient expression was sustained for 48
hours post transfection. Modulation of expression was achieved by
varying dose of mRNA.
[0236] Expression in spleen tissue was also achieved, and
specificity of delivery is currently being optimized through
variance of N/P ratio. Ongoing and future studies involve:
Determination of transfection efficiency of specific cell type
populations within the targeted organ of interest, Increase single
organ specificity following systemic delivery, Stability of LNP
formulation before and after mRNA complexation.
Example 3: Preparation and Complexation
[0237] Formulation Preparation.
[0238] Prepare stock solutions of the individual lipid components
in 100% Ethanol (EtOH) at appropriate concentrations based on
individual lipid solubility. Heat to 50.degree. C. to dissolve
lipids completely in the EtOH. In a glass vial, combine all 5 lipid
components using a pipette in volumes calculated according to Molar
ratios listed in Table 1. Dilute the combined lipids using 200 mM
Sodium Acetate at a dilution ratio of 1:4 by volume. Seal lid with
paraffin. Store at 4.degree. C.
[0239] Complexation Preparation.
[0240] Concentration of lipid is delivered at a 3 mg/kg
concentration. Concentration of mRNA is a 10:1 dilution, so 0.3
mg/mL total mRNA are administered. Label 2 screw-cap plastic sample
tubes as `lipid` and `mRNA`. Transfer 100 .mu.L of prepared lipid
formulation to tube labeled `lipid`. Calculate amount of mRNA
needed for 200 .mu.L total volume of complex. (0.6 mg/mL) Since
lipid formulation is at a 25% EtOH solution following the 1:4
dilution with Sodium Acetate, the mRNA needs to be diluted in a 25%
EtOH solution. Add 25 .mu.L of 100% EtOH to tube labeled `mRNA`.
Add appropriate volume of mRNA to `mRNA` tube according to
calculations. Bring up the volume of sample to 100 uL using sterile
filtered water. Add contents of mRNA tube to lipid tube mixing
well. Vortex briefly. Heat complex at 50.degree. C. for 30 minutes.
Transfer complex to dialysis columns with an 8-10 kD molecular
weight cut off, and allow to dialyze for 2 hours in 100% PBS.
Transfer sample to air-tight screw capped sample tube and either
use immediately or store at -20.degree. C. until use.
[0241] Complex Administration.
[0242] If using frozen complex, thaw to room temperature before
use. According to the institutional guidelines for the care and use
of laboratory animals, complex is administered to mouse via tail
vein injection.
[0243] Preparation of formulations was performed as follows.
Individual powder lipid components were resuspended in 100% Ethanol
(EtOH). Stock solutions were prepared in the following
concentrations: DHDMS at 50 mg/mL, HDMS at 15 mg/mL, Cholesterol at
25 mg/mL, DOPE at 50 mg/mL and C14 PEG 5000 (Avanti Polar Lipids,
#880210) at 100 mg/mL. Lipid solutions were heated to 50.degree. C.
using a heat block to achieve complete solubility during
formulation preparation.
[0244] The 5 formulation components were combined in a glass screw
top vial using a pipette in the volumes listed in Table 1,
calculated based on the desired molar ratio and the stock
concentration of the lipid. Once all five of the components were
added at the appropriate volumes to the glass vial, the volume was
brought up to 100 .mu.L using 100% EtOH. This volume was then
diluted at a ratio of 1:4 using a 200 mM Sodium Acetate solution.
The resulting solution was used as the transfection reagent, and
stored at 4.degree. C.
[0245] Preparation of the lipid/mRNA complexes was performed as
follows. The final target concentration of 0.3 mg/mL of mRNA was
chosen as the desired concentration for delivery, and the basis of
the calculations for sample preparation. 2 screw-cap plastic sample
tubes were labeled as `lipid` and `mRNA`. 100 uL of the prepared
lipid formulation was transferred to tube labeled `lipid`.
[0246] For the mRNA solution preparation: the amount of mRNA needed
for a total complex volume of 200 .mu.L, based on the desired 0.3
mg/mL final concentration is 0.6 mg/mL. Based on the concentration
of mRNA solution, calculate the necessary volume using the formula
(C1)(V1)=(C2)(V2), where C1=stock concentration mRNA, V1=variable,
C2=0.6 mg/mL final mRNA concentration and V2=100 .mu.L of solution
volume. Stock mRNA was thawed on ice, and mRNA solution was
prepared on ice. For experiments, Firefly luciferase mRNA
(Trilink.RTM. #L-6107) was purchased at a concentration of 2 mg/mL.
To achieve the desired concentration, Applicants used 30 .mu.L of
the stock mRNA solution. To maintain a concentration of 25% EtOH
(lipid formulation is at a 25% EtOH) the mRNA is prepared in a
solution of 25% EtOH. To the tube labeled mRNA, 30 .mu.L of
undiluted mRNA, 25 uL of 100% EtOH and 45 .mu.L of Ultrapure
DNase/RNase-Free distilled water (Thermofisher #10977-015) were
combined.
[0247] Once both tubes were prepared, mRNA tube was added to the
lipid tube and mixed well. Tube was briefly vortexed and then
heated at 50.degree. C. for 30 minutes. Following complexation,
complex was transferred to a dialysis column with an 8-10 kD
molecular weight cut off (Spectrum labs, Float-a-Lyzer dialysis
device #G235031) and dialyzed in 100% PBS (Gibco PBS #10010023) for
2 hours. Following dialysis, sample was transferred to screw-capped
plastic sample tube, and either used immediately, or stored at
-20.degree. C. until use.
[0248] Complex administration in vivo was performed as follows: If
using frozen complex, thaw to room temperature before use, if using
freshly dialyzed samples, proceed immediately. The 200 .mu.L
injection volume is according to use of a 20 gram mouse. Adjust
volume for mice weighing more or less accordingly. To perform tail
vein injection, restrain animal using a restraining device. A warm
water bath or heat lamp can be used to help dilate and visualize
the vein. Disinfect the tail using alcohol. Insert needle into vein
and slowly inject solution into vein, (20-40 .mu.L/second).
Successful needle insertion and injection will cause blood to clear
from vein. If this does not occur, and resistance is felt upon
injection, remove needle and repeat proximal to primary injection
site. Following complete injection of the complex solution,
withdraw the needle and apply gentle compression to the site of
injection to stop bleeding.
[0249] 3 hours following injection of luciferase mRNA, 50 .mu.L of
luciferin substrate was injected IP into the mouse and allowed to
incubate for 15 minutes. Immediately, tissues were collected for
luciferase activity assay. For experiments, Applicant's utilized an
IVIS Lumina LT pre-clinical in vivo imaging system (Perkin-Elmer
#IVISLMLT) to measure the luciferase activity in the individual
organs. Using Living Image.RTM. software, the flux of signal
representing bioluminescence from a specified region of interest
was measured and quantified.
TABLE-US-00001 TABLE 1 Volume of Stock solutions (.mu.L) DHDMS HDMS
DOPE Cholesterol PEG EtOH 3.10 62.7 568 160.9 26.1 19.2 163 3.14
83.6 582.9 130.8 5.2 38.5 159 3.19 60.1 672.6 100.6 52.3 38.5 76
38.53.20 47 568 160.9 52.3 38.5 133.3 2.2 146.3 358.7 160.9 73.2
9.6 251.3
TABLE-US-00002 TABLE 2 Top producing lipid nanoparticle
formulations. PEG PEG CHO- chain Mol DH- LES- Formulation length
Weight DMS HDMS DOPE TEROL PEG Bruce #3.10 14 5000 0.24 0.38 0.32
0.05 0.01 Bruce #3.14 14 5000 0.32 0 39 0.26 0.01 0.02 Bruce #3.20
14 5000 0.18 0.38 0.32 0.10 0.02 Bruce #3.19 14 5000 0.23 0.45 0.20
0.10 0.02 Bruce #3.11 14 5000 0.18 0.51 0.20 0.10 0.01 Bruce #3.15
14 5000 0.27 0.38 0.32 0.01 0.02 Bruce #3.12 14 5000 0.25 0.38 0.26
0.10 0.01 Bruce #2.2 18 750 0.28 0.24 0.32 0.14 0.02 Bruce #3.16 14
5000 0.18 0.47 0.32 0.01 0.02 Bruce #4 14 2000 0.24 0.40 0.24 0.10
0.02 Bruce #3.18 14 5000 0.32 0.38 0.20 0.08 0.02 MOLAR 0.18- 0.24-
0.20- 0.01- 0.01- RANGES 0.32 0.51 0.32 0.14 0.02
TABLE-US-00003 TABLE 3 Non-targeting, low performance lipid
nanoparticle formulation. PEG PEG CHO- chain Mol DH- LES-
Formulation length Weight DMS HDMS DOPE TEROL PEG Bruce #23 14 5000
0.10 0.10 0.10 0.38 0.02 Bruce #64 14 5000 0.10 0.10 0.40 0.30 0.10
Bruce #24 14 5000 0.10 0.10 0.40 0.38 0.02 Bruce #52 18 750 0.22
0.10 0.22 0.40 0.06 Bruce #61 14 2000 0.22 0.22 0.22 0.24 0.10
Bruce #27 14 5000 0.10 0.38 0.10 0.40 0.02 WORKING 0.18- 0.24-
0.20- 0.01- 0.01- MOLAR 0.32 0.51 0.32 0.14 0.02 RANGES
Example 4: Novel In Vivo Platform for Lung Delivery
[0250] FIG. 19 shows Invivofectamine.RTM. Lung (IVF.RTM. Lung)
research and development workflow.
[0251] Reagent comparative analysis was performed using four
different formulations (IVF.RTM. Lung, DOTMA:DOPE, DOTAP:DOPE, and
Jet PEI.RTM.). Animals received systemic delivery of formulations
through intravenous injection. All formulations delivered
Trilink.RTM. Firefly luciferase mRNA as a payload. FIGS. 20A-20B
show experimental results.
[0252] Multiple mRNA sequences were tested to determine delivery of
mRNA to organ (i.e., lung) using IVF.RTM. Lung. To determine the
delivery of Firefly Luciferase, obtained from Trilink.RTM. or
developed in house, bioluminescence imaging of organ and mouse was
used. To determine delivery of lacZ (obtained from Trilink.RTM.),
histology on lung tissue was completed by staining with beta-gal.
To determine delivery of Cre (obtained from Trilink.RTM.) in a lacZ
Cre-reporter strain, immunofluorescence staining to detect protein
expression was used.
[0253] mRNA expression kinetics can differ based on optimization of
mRNA. FIG. 21 shows lung radiance following systemic (intravenous)
delivery of Invivofectamine.RTM. Lung including either Trilink.RTM.
Firefly Luciferase or in house optimized Firefly Luciferase.
[0254] To assess IVF.RTM. Lung delivery of lacZ mRNA and IVF.RTM.
Lung delivery of Cre mRNA animals were injected intravenously with
LNP complexed. Tissues was harvested 4 hours post-delivery and
immediately cryopreserved. Cryosectioning of isolated lung tissue
was subsequently stained for beta-gal and tissue was counterstained
for anatomical features (FIGS. 24A-24D) or used for
immunofluorescence detection of lacZ protein expression (FIGS.
25A-25B), depending on the experiment. Delivery of mRNA was seen
demonstrated by positive 3-gal staining and immunofluorescent
staining. Using the reporter strain, specific delivery to the
airways is seen with the fluorescent staining.
[0255] FIG. 22 demonstrates the ability of IVF.RTM. Lung to not
only delivery siRNA to the lung but to endothelial cells as
demonstrated by knockdown of Tie2, and endothelial cell specific
marker.
[0256] FIGS. 27A-27K show that the N/P ratio (see FIG. 26) affects
biodistribution patterns. However, using the core lipid (DHDMS)
formulated with DOPE resulted in no lung delivery and varied N/P
ratio did not affect lung distribution (FIG. 28).
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* * * * *
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