U.S. patent application number 14/521168 was filed with the patent office on 2015-04-23 for cns delivery of mrna and uses thereof.
The applicant listed for this patent is Shire Human Genetic Therapies, Inc.. Invention is credited to Frank DeRosa, Michael Heartlein, Shrirang Karve.
Application Number | 20150110857 14/521168 |
Document ID | / |
Family ID | 51844900 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150110857 |
Kind Code |
A1 |
DeRosa; Frank ; et
al. |
April 23, 2015 |
CNS DELIVERY OF MRNA AND USES THEREOF
Abstract
The present invention provides, among other things, methods and
compositions for effective delivery of messenger RNA (mRNA) to the
central nervous system (CNS). In particular, the present invention
provides methods and compositions for administering intrathecally
to a subject in need of delivery a composition comprising an mRNA
encoding a protein, encapsulated within a liposome, such that the
administering of the composition results in the intracellular
delivery of mRNA in neurons in the brain and/or spinal cord. The
present invention is particularly useful for the treatment of CNS
diseases, disorders or conditions, such as spinal muscular
atrophy.
Inventors: |
DeRosa; Frank; (Lexington,
MA) ; Heartlein; Michael; (Lexington, MA) ;
Karve; Shrirang; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shire Human Genetic Therapies, Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
51844900 |
Appl. No.: |
14/521168 |
Filed: |
October 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61894246 |
Oct 22, 2013 |
|
|
|
62020161 |
Jul 2, 2014 |
|
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Current U.S.
Class: |
424/450 ;
514/44R |
Current CPC
Class: |
A61K 48/005 20130101;
A61P 21/00 20180101; A61K 38/1709 20130101; A61P 25/28 20180101;
A61K 48/0033 20130101; C07H 21/02 20130101; A61P 43/00 20180101;
A61P 25/00 20180101; A61K 9/1272 20130101; C12N 15/88 20130101 |
Class at
Publication: |
424/450 ;
514/44.R |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 38/17 20060101 A61K038/17; A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of delivery of messenger RNA (mRNA) to the central
nervous system (CNS), comprising administering intrathecally to a
subject in need of delivery a composition comprising an mRNA
encoding a protein, encapsulated within a liposome such that the
administering of the composition results in the intracellular
delivery of mRNA in neurons in the brain and/or spinal cord;
wherein the liposome comprises cationic or non-cationic lipid,
cholesterol-based lipid and PEG-modified lipid.
2-4. (canceled)
5. The method of claim 1, wherein the liposome comprises one or
more cationic lipids, one or more neutral lipids, one or more
cholesterol-based lipids and one or more PEG-modified lipids.
6. The method of claim 5, wherein the one or more cationic lipids
are selected from the group consisting of C12-200, MC3, DLinDMA,
DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC,
DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA,
DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP,
DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combination
thereof.
7. The method of claim 6, wherein the one or more cationic lipids
comprise C12-200.
8. The method of claim 6, wherein the one or more cationic lipids
comprise DLinkC2DMA.
9. The method of claim 6, wherein the cationic lipid is cKK-E12:
##STR00008##
10-12. (canceled)
13. The method of claim 5, wherein the one or more PEG-modified
lipids constitute about 1-10% by molar ratio of the total lipid
composition.
14-15. (canceled)
16. The method claim 1, wherein the liposome comprises a
combination selected from C12-200, sphingomyelin, DOPE,
Cholesterol, and DMG PEG; C12-200, DOPE, cholesterol and DMG-PEG2K;
cKK-E12, DOPE, cholesterol and DMG-PEG2K; cKK-E12, sphingomyelin,
DOPE, cholesterol and DMG-PEG2K; HGT5001, DOPE, cholesterol and
DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; DLinKC2DMA,
DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and
DMG-PEG2K; DODMA, DOPE, cholesterol and DMG-PEG2K; or DODMA,
sphingomyelin, DOPE, cholesterol and DMG-PEG2K.
17. The method of claim 1, wherein the liposome has a size ranging
from about 40-100 nm.
18. The method of claim 1, wherein the mRNA has a length of or
greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5
kb, 4 kb, 4.5 kb, or 5 kb.
19. The method of claim 1, wherein the protein encoded by the mRNA
normally functions in the neurons in the brain and/or spinal
cord.
20. (canceled)
21. The method of claim 1, wherein the protein encoded by the mRNA
is the Survival of Motor Neuron (SMN) protein.
22. The method of claim 1, wherein the mRNA is codon optimized.
23. The method of claim 22, wherein the codon-optimized mRNA
comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID
NO:11.
24-29. (canceled)
30. The method of claim 1, wherein the protein encoded by the mRNA
is an enzyme.
31. (canceled)
32. The method of claim 1, wherein the intracellular delivery of
mRNA results in intracellular expression of the protein encoded by
the mRNA within the cytosol of the neurons.
33. The method of claim 1, wherein the intracellular delivery of
mRNA results in expression of the protein encoded by the mRNA and
secretion extracellularly from the neurons after expression.
34. The method of claim 1, wherein the mRNA comprises one or more
modified nucleotides.
35. (canceled)
36. The method of claim 1, wherein the mRNA is unmodified.
37. The method of claim 1, wherein the mRNA is delivered at an
amount ranging from about 0.01 mg/kg to about 10 mg/kg body
weight.
38-39. (canceled)
40. A method of treating a disease, disorder or condition
associated with deficiency of a protein in the central nervous
system (CNS), comprising delivering a messenger RNA (mRNA) encoding
the protein that is deficient to the CNS using a method according
to claim 1.
41. A method of treating spinal muscular atrophy, comprising
administering intrathecally to a subject in need of treatment a
composition comprising an mRNA encoding the Survival of Motor
Neuron (SMN) protein, encapsulated within a liposome such that the
administering of the composition results in the intracellular
delivery of mRNA in neurons in the brain and/or spinal cord;
wherein the liposome comprises cationic or non-cationic lipid,
cholesterol-based lipid and PEG-modified lipid.
42-43. (canceled)
44. The method of claim 41, wherein the codon-optimized mRNA
comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID
NO:11.
45-48. (canceled)
49. The method of claim 41, wherein the liposome comprises a
combination selected from C12-200, sphingomyelin, DOPE,
Cholesterol, and DMG PEG; C12-200, DOPE, cholesterol and DMG-PEG2K;
cKK-E12, DOPE, cholesterol and DMG-PEG2K; cKK-E12, sphingomyelin,
DOPE, cholesterol and DMG-PEG2K; HGT5001, DOPE, cholesterol and
DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; DLinKC2DMA,
DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and
DMG-PEG2K; DODMA, DOPE, cholesterol and DMG-PEG2K; or DODMA,
sphingomyelin, DOPE, cholesterol and DMG-PEG2K.
50. A composition for treating spinal muscular atrophy, comprising
an mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome; wherein the codon optimized mRNA
comprises SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO:11,
and further wherein the liposome comprises cationic or non-cationic
lipid, cholesterol-based lipid and PEG-modified lipid.
51. A composition for treating spinal muscular atrophy, comprising
an mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome, wherein the liposome comprises a
cationic lipid of formula I-c1-a: ##STR00009## or a
pharmaceutically acceptable salt thereof, wherein: each R.sup.2
independently is hydrogen or C.sub.1-3 alkyl; each q independently
is 2 to 6; each R' independently is hydrogen or C.sub.1-3 alkyl;
and each R.sup.1 independently is C.sub.8-12 alkyl.
52. The composition of claim 51, wherein the cationic lipid is
cKK-E12: ##STR00010##
53. A composition for treating spinal muscular atrophy, comprising
an mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome; wherein the liposome comprises a
combination selected from C12-200, sphingomyelin, DOPE,
Cholesterol, and DMG PEG; C12-200, DOPE, cholesterol and DMG-PEG2K;
cKK-E12, DOPE, cholesterol and DMG-PEG2K; cKK-E12, sphingomyelin,
DOPE, cholesterol and DMG-PEG2K; HGT5001, DOPE, cholesterol and
DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; DLinKC2DMA,
DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and
DMG-PEG2K; DODMA, DOPE, cholesterol and DMG-PEG2K; or DODMA,
sphingomyelin, DOPE, cholesterol and DMG-PEG2K.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/020,161, filed Jul. 2, 2014 and U.S. Provisional
Application No. 61/894,246, filed Oct. 22, 2013, the disclosures of
which are hereby incorporated by reference.
SEQUENCE LISTING
[0002] The present specification makes reference to a Sequence
Listing (submitted electronically as a .txt file named
"2006685-0689_SL.txt" on Oct. 22, 2014). The .txt file was
generated on Oct. 22, 2014 and is 19,618 bytes in size. The entire
contents of the Sequence Listing are herein incorporated by
reference.
BACKGROUND
[0003] Effective therapies are still needed for the treatment of
CNS diseases, such as those diseases directly or indirectly
resulting from the loss, aberrant expression or dysregulation of a
neuronal cellular protein. Several hurdles exist in implementing an
effective treatment strategy for CNS diseases, mainly due to the
isolation and sequestration of the CNS tissues by the impermeable
blood brain barrier (BBB).
[0004] For example, spinal muscular atrophy represents a CNS
disease resulting from a protein deficiency. Typically, a healthy
individual has functional copies of each of the survival of motor
neuron (SMN) genes (SMN-1 and SMN-2), which are nearly identical in
sequence. Patients diagnosed with spinal muscular atrophy typically
fail to express a full-length SMN-1 protein, relying solely on low
level expression of full length SMN-2, which is not sufficient to
prevent motor neuron death in the brain.
[0005] In recent years, messenger RNA (mRNA) therapy has become an
increasingly important option for treatment of various diseases, in
particular, for those associated with deficiency of one or more
proteins. While promising for non-neuronal diseases, those skilled
in the art have been dissuaded from implementing such an approach
for treating a CNS disease, due to the inability of liposomes to
permeate the BBB, as well as the unique and complex membrane
composition of neuronal cells which imposes unique challenges for
delivering mRNA inside neuronal cells (Svennerhol et. al.,
Biochimica et Biophysica Acta, 1992, 1128:1-7; and Karthigasan et.
al., Journal of Neurochemistry, 1994, 62:1203-1213).
SUMMARY
[0006] The present invention provides, among other things, improved
methods and compositions for efficient delivery of mRNA, encoding a
therapeutic protein, to neurons and other cell types of the CNS.
The invention is based, in part, on the surprising discovery that
mRNA loaded lipid or polymer based nanoparticles can be
administered directly into the CNS space (e.g., via intrathecal
administration) and effectively penetrate neuronal cell membrane,
resulting in intracellular delivery of mRNA in neurons in the brain
and/or spinal cord. Prior to the present invention, it was reported
that the neuronal cell membranes are characterized with unique and
complex lipid compositions, different than those of the
non-neuronal cells (Svennerhol et. al., Biochimica et Biophysica
Acta, 1992, 1128:1-7; and Karthigasan et. al., Journal of
Neurochemistry, 1994, 62:1203-1213). Therefore, it was thought that
neuronal cell membranes are hard to penetrate. Even those liposomes
effective in delivering nucleic acids to non-neuronal cells were
not expected to be effective in penetrating neuronal cell
membranes. It was indeed surprising that the lipid or polymer based
nanoparticles described herein can effectively deliver mRNA into
neurons, even those located deep within the center of the brain and
the spinal column and those hard to treat motor neurons. Thus, the
present invention provides an improved and effective approach for
the CNS delivery of mRNA and promises an effective mRNA therapy for
treating various CNS diseases.
[0007] Thus, in one aspect, the invention provides methods of
delivering an mRNA to the central nervous system (CNS). In some
embodiments, an inventive method according to the present invention
includes administering intrathecally to a subject in need of
delivery a composition comprising an mRNA encoding a protein,
encapsulated within a liposome such that the administering of the
composition results in the intracellular delivery of mRNA in
neurons in the brain and/or spinal cord; wherein the liposome
comprises cationic or non-cationic lipid, cholesterol-based lipid
and PEG-modified lipid.
[0008] In some embodiments, the mRNA is delivered to neurons
located within the brain. In some embodiments, the mRNA is
delivered to neurons located within the spinal cord. In some
embodiments, the mRNA is delivered to motor neurons. In some
embodiments, the mRNA is delivered to upper motor neurons and/or
lower motor neurons. In some embodiments, the motor neurons are
located within the anterior horn and/or dorsal root ganglia of the
spinal cord.
[0009] In some embodiments, a suitable liposome comprises one or
more cationic lipids, one or more neutral lipids, one or more
cholesterol-based lipids and one or more PEG-modified lipids.
[0010] In some embodiments, suitable cationic lipids are selected
from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA,
cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC, DDAB,
DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE,
CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP,
DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003. In some specific
embodiments, the one or more cationic lipid comprises C12-200. In
some specific embodiments, the cationic lipid comprises
DLinK22DMA.
[0011] In certain embodiments, a cationic lipid suitable for the
present invention has a structure of formula I-c1-a:
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein: each
R.sup.2 independently is hydrogen or C.sub.1-3 alkyl; each q
independently is 2 to 6; each R' independently is hydrogen or
C.sub.1-3 alkyl; and each R.sup.L independently is C.sub.8-12
alkyl.
[0012] In certain embodiments, a suitable cationic lipid is
cKK-E12:
##STR00002##
[0013] In some embodiments, suitable non-cationic lipids are
selected from distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidyl
lipids or a mixture thereof.
[0014] In some embodiments, a suitable non-cationic lipid is a
phosphatidyl lipid. In some embodiments, a suitable phosphatidyl
lipid is a sphingolipid. In some specific embodiments, a suitable
sphingolipid is sphingomylin.
[0015] In some embodiments, one or more cholesterol-based lipids
suitable for the present invention are selected from cholesterol,
PEGylated cholesterol and/or DC-Chol
(N,N-dimethyl-N-ethylcarboxamidocholesterol),
1,4-bis(3-N-oleylamino-propyl)piperazine.
[0016] In some embodiments, one or more PEG-modified lipids
suitable for the present invention comprise a poly(ethylene) glycol
chain of up to 5 kDa in length covalently attached to a lipid with
alkyl chain(s) of C.sub.6-C.sub.20 length. In some embodiments, a
suitable PEG-modified lipid is a derivatized ceramide such as
N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene
Glycol)-2000]. In some embodiments, a suitable PEG-modified or
PEGylated lipid is PEGylated cholesterol or Dimyristoylglycerol
(DMG)-PEG-2K. In some embodiments, the one or more PEG-modified
lipids are selected form the group consisting of DMG-PEG, C8-PEG,
DOG PEG, ceramide PEG, DSPE-PEG and combination thereof. In some
embodiments, the one or more PEG-modified lipids constitute about
1-10% (e.g., about 1-8%, about 1-6%, or about 1-5%) by molar ratio
of the total lipid compositions. In some embodiments, the one or
more PEG-modified lipids constitute about 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10% by molar ratio of the total lipid compositions. In some
specific embodiments, the PEG-modified lipids constitute at least
5% by molar ratio of the total lipid composition.
[0017] In some embodiments, a suitable liposome comprises a
combination selected from C12-200, sphingomyelin, DOPE,
Cholesterol, and DMG PEG; C12-200, DOPE, cholesterol and DMG-PEG2K;
cKK-E12, DOPE, cholesterol and DMG-PEG2K; cKK-E12, sphingomyelin,
DOPE, cholesterol and DMG-PEG2K; HGT5001, DOPE, cholesterol and
DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; DLinKC2DMA,
DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and
DMG-PEG2K; or DODMA, DOPE, cholesterol and DMG-PEG2K; DODMA,
sphingomyelin, DOPE, cholesterol and DMG-PEG2K; and/or combinations
thereof.
[0018] In some embodiments, a suitable liposome comprises a
commercial enhancer. In some embodiments, the liposome comprises a
biodegradable lipid. In some embodiments, the liposome comprises a
ionizable lipid. In some embodiments, the liposome comprises a
cleavable lipid.
[0019] In some embodiments, a suitable liposome has a size of or
less than about 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95
nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50
nm. In some embodiments, a suitable liposome has a size ranging
from about 40-100 nm (e.g., about 40-90 nm, about 40-80 nm, about
40-70 nm, or about 40-60 nm). As used herein, the size of a
liposome is determined by the length of the largest diameter of a
liposome particle.
[0020] In some embodiments, the mRNA has a length of or greater
than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb,
4.5 kb, or 5 kb.
[0021] In some embodiments, the therapeutic protein encoded by the
mRNA is a cytosolic protein. In some embodiments, the therapeutic
protein encoded by the mRNA is a secreted protein. In some
embodiments, the therapeutic protein encoded by the mRNA is an
enzyme. In some embodiments, the enzyme is a lysosomal enzyme. In
some embodiments, the therapeutic protein encoded by the mRNA is a
protein associated with a CNS disease. In some embodiments, the
therapeutic protein encoded by the mRNA normally functions in the
neurons in the brain and/or spinal cord. In some embodiments, the
therapeutic protein encoded by the mRNA normally functions in the
motor neurons in the spinal cord.
[0022] In some embodiments, the therapeutic protein encoded by the
mRNA is a survival of motor neuron protein. In some embodiments,
the therapeutic protein encoded by the mRNA is a survival of motor
neuron-1 protein. In some embodiments, the therapeutic protein
encoded by the mRNA is a splice isoform, fragment or truncated
version of a survival of motor neuron protein-1. In some
embodiments, the SMN-1 protein comprises the amino acid sequence of
SEQ ID NO:2. In some embodiments, the mRNA encoding the SMN-1
protein comprises the nucleic acid sequence of SEQ ID NO:1. In some
embodiments, the mRNA encoding the SMN-1 protein is codon-optimized
and comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID
NO:11.
[0023] In some embodiments, the mRNA suitable for the present
invention comprises a 5' UTR sequence. In some embodiments, the
5'UTR sequence comprises SEQ ID NO:7. In some embodiments, the mRNA
comprises a 3' UTR. In some embodiments, the 3'UTR comprises SEQ ID
NO:8 or SEQ ID NO:9. In some embodiments, the mRNA comprises a cap
structure. In some embodiments, a suitable cap structure is
selected from Cap 0, Cap 1, or Cap 2 structures. In some
embodiments, a suitable cap structure is an Anti-Reverse Cap Analog
(ARCA) or a modified ARCA.
[0024] In some embodiments, the mRNA encoding a therapeutic protein
comprises one or more modified nucleotides. In some embodiments,
the one or more nucleotides are selected from the group consisting
of pseudouridine, 2-aminoadenosine, 2-thiouridine, inosine,
pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5
propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, N-1-methyl
pseudouridine, 2-thiocytidine, and combinations thereof.
[0025] In some embodiments, the mRNA encoding a therapeutic protein
is unmodified.
[0026] In some embodiments, intracellular delivery of the mRNA
results in expression of the protein encoded by the mRNA. In some
embodiments, the encoded protein is expressed within the cytosol of
the neurons. In some embodiments, the encoded protein is expressed
and secreted extracellularly form the neurons after expression.
[0027] In some embodiments, the mRNA is administered at a dose
ranging from about 0.01-10.0 mg/kg body weight, for example, about
0.01-9.0, 0.01-8.0, 0.01-7.0, 0.01-6.0, 0.01-5.0, 0.01-4.0,
0.01-3.0, 0.01-2.5, 0.01-2.0, 0.01-1.5, 0.01-1.0, 0.01-0.5,
0.01-0.25, 0.01-0.1, 0.1-10.0, 0.1-5.0, 0.1-4.0, 0.1-3.0, 0.1-2.0,
0.1-1.0, 0.1-0.5 mg/kg body weight. In some embodiments, the mRNA
is administered at a dose of or less than about 10.0, 9.0, 8.0,
7.0, 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6,
0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.04, 0.03, 0.02, or 0.01 mg/kg body
weight.
[0028] In some embodiments, the mRNA is delivered without inducing
substantial toxicity or immune response.
[0029] In another aspect, the present invention provides methods of
treating a disease, disorder or condition associated with
deficiency of a protein in the central nervous system (CNS) by
delivering a messenger RNA (mRNA) encoding the protein that is
deficient to the CNS using a method described herein. In some
embodiments, the CNS disease, disorder or condition is the result
of a protein deficiency. In some embodiments, the CNS disease,
disorder or condition is the result of a protein deficiency in the
motor neurons.
[0030] Among other things, the present invention provides methods
and compositions of treating spinal muscular atrophy.
[0031] In one aspect, the present invention provides a method of
treating spinal muscular atrophy by delivering a messenger RNA
(mRNA) encoding a Survival of Motor Neuron (SMN) protein to the CNS
using a method described herein.
[0032] In another aspect, the present invention provides a
composition for treating spinal muscular atrophy, comprising an
mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome; wherein the mRNA comprises SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:11 (corresponding to
codon-optimized human SMN mRNA), and further wherein the liposome
comprises cationic or non-cationic lipid, cholesterol-based lipid
and PEG-modified lipid.
[0033] In a related aspect, the present invention provides a
composition for treating spinal muscular atrophy, comprising an
mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome, wherein the liposome comprises a
cationic lipid of formula I-c1-a:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein: each
R.sup.2 independently is hydrogen or C.sub.1-3 alkyl; each q
independently is 2 to 6; each R' independently is hydrogen or
C.sub.1-3 alkyl; and each R.sup.L independently is C.sub.8-12
alkyl.
[0034] In some embodiments, a suitable cationic lipid is
cKK-E12:
##STR00004##
[0035] In some embodiments, the present invention provides a
composition for treating spinal muscular atrophy, comprising an
mRNA encoding the Survival of Motor Neuron (SMN) protein,
encapsulated within a liposome; wherein the liposome comprises a
combination selected from
[0036] C12-200, sphingomyelin, DOPE, Cholesterol, and DMG PEG;
[0037] C12-200, DOPE, cholesterol and DMG-PEG2K;
[0038] cKK-E12, DOPE, cholesterol and DMG-PEG2K;
[0039] cKK-E12, sphingomyelin, DOPE, cholesterol and DMG-PEG2K;
[0040] HGT5001, DOPE, cholesterol and DMG-PEG2K;
[0041] HGT4003, DOPE, cholesterol and DMG-PEG2K;
[0042] DLinKC2DMA, DOPE, cholesterol and DMG-PEG2K;
[0043] ICE, DOPE, cholesterol and DMG-PEG2K;
[0044] DODMA, DOPE, cholesterol and DMG-PEG2K; or
[0045] DODMA, sphingomyelin, DOPE, cholesterol and DMG-PEG2K.
[0046] Other features, objects, and advantages of the present
invention are apparent in the detailed description, drawings and
claims that follow. It should be understood, however, that the
detailed description, the drawings, and the claims, while
indicating embodiments of the present invention, are given by way
of illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0047] The drawings are for illustration purposes only not for
limitation.
[0048] FIG. 1 illustrates detection via western blot of human SMN-1
protein derived from exogenous hSMN-1 mRNA that was transfected
into BHK-21 cells. Various antibodies specific to human SMN were
employed: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B)
Pierce PA5-27309 a-SMN antibody at 1:10,000 dilution; and (C) LSBio
C138149 a-SMN antibody at 1:10,000 dilution.
[0049] FIG. 2A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 1.
[0050] FIG. 3A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 2.
[0051] FIG. 4A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 3.
[0052] FIG. 5A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 4.
[0053] FIG. 6A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 5.
[0054] FIG. 7A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 6.
[0055] FIG. 8A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 7.
[0056] FIG. 9A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 8.
[0057] FIG. 10A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 9.
[0058] FIG. 11A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 10.
[0059] FIG. 12A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 11.
[0060] FIG. 13A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 12.
[0061] FIG. 14A-C illustrates multiplex nucleic acid in situ
detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A)
Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post
intrathecal delivery using liposome formulation 13.
[0062] FIG. 15 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery of vehicle control. Image is shown at 5.times.
magnification.
[0063] FIG. 16 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery of vehicle control. Image is shown at
10.times. magnification.
[0064] FIG. 17 illustrates in situ detection of UbC mRNA in spinal
tissue, 24 hours post intrathecal delivery of vehicle control.
[0065] FIG. 18 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery of vehicle control. Image is shown at 5.times.
magnification.
[0066] FIG. 19 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery. Image is shown at 5.times. magnification.
[0067] FIG. 20 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery. Image is shown at 10.times.
magnification.
[0068] FIG. 21 illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in spinal tissue, 24 hours post
intrathecal delivery. Image is shown at 10.times. and 20.times.
magnification.
[0069] FIG. 22 illustrates positive detection of human SMN-1
protein produced in the spinal cord of a rat 24 hours
post-intrathecal administration of human SMN-1 mRNA-loaded lipid
nanoparticles. Anti-human SMN 4F11 antibody was employed at 1:2500
dilution. Panel A represents treated rat spinal cord tissue and
panel B represents untreated rat spinal cord tissue.
[0070] FIGS. 23 A-C illustrates in situ detection of human Survival
Motor Neuron (hSMN-1) mRNA in brain tissue, 30 minutes post
intrathecal delivery. In situ detection of the brain (A)
demonstrates a strong signal observed both within the gray and
white matter of the brain. Two regions of the brain (B) Section 1
and (C) Section 2 were further magnified for closer analysis.
DEFINITIONS
[0071] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0072] Alkyl: As used herein, "alkyl" refers to a radical of a
straight-chain or branched saturated hydrocarbon group having from
1 to 15 carbon atoms ("C.sub.1-15 alkyl"). In some embodiments, an
alkyl group has 1 to 3 carbon atoms ("C.sub.1-3 alkyl"). Examples
of C.sub.1-3 alkyl groups include methyl (C.sub.1), ethyl
(C.sub.2), n-propyl (C.sub.3), and isopropyl (C.sub.3). In some
embodiments, an alkyl group has 8 to 12 carbon atoms ("C.sub.8-12
alkyl"). Examples of C.sub.8-12 alkyl groups include, without
limitation, n-octyl (C.sub.8), n-nonyl (C.sub.9), n-decyl
(C.sub.10), n-undecyl (C.sub.11), n-dodecyl (C.sub.12) and the
like. The prefix "n-" (normal) refers to unbranched alkyl groups.
For example, n-C.sub.8 alkyl refers to --(CH.sub.2).sub.7CH.sub.3,
n-C.sub.10 alkyl refers to --(CH.sub.2).sub.9CH.sub.3, etc.
[0073] Approximately or about: As used herein, the term
"approximately" or "about," as applied to one or more values of
interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about"
refers to a range of values that fall within 25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of
the stated reference value unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0074] Amelioration: As used herein, the term "amelioration" is
meant the prevention, reduction or palliation of a state, or
improvement of the state of a subject. Amelioration includes, but
does not require complete recovery or complete prevention of a
disease condition. In some embodiments, amelioration includes
increasing levels of relevant protein or its activity that is
deficient in relevant disease tissues.
[0075] Amino acid: As used herein, term "amino acid," in its
broadest sense, refers to any compound and/or substance that can be
incorporated into a polypeptide chain. In some embodiments, an
amino acid has the general structure H.sub.2N--C(H)(R)--COOH. In
some embodiments, an amino acid is a naturally occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a d-amino acid; in some
embodiments, an amino acid is an 1-amino acid. "Standard amino
acid" refers to any of the twenty standard 1-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. As used herein, "synthetic amino acid"
encompasses chemically modified amino acids, including but not
limited to salts, amino acid derivatives (such as amides), and/or
substitutions. Amino acids, including carboxy- and/or
amino-terminal amino acids in peptides, can be modified by
methylation, amidation, acetylation, protecting groups, and/or
substitution with other chemical groups that can change the
peptide's circulating half-life without adversely affecting their
activity. Amino acids may participate in a disulfide bond. Amino
acids may comprise one or posttranslational modifications, such as
association with one or more chemical entities (e.g., methyl
groups, acetate groups, acetyl groups, phosphate groups, formyl
moieties, isoprenoid groups, sulfate groups, polyethylene glycol
moieties, lipid moieties, carbohydrate moieties, biotin moieties,
etc.). The term "amino acid" is used interchangeably with "amino
acid residue," and may refer to a free amino acid and/or to an
amino acid residue of a peptide. It will be apparent from the
context in which the term is used whether it refers to a free amino
acid or a residue of a peptide.
[0076] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish, insects, and/or worms. In some embodiments, an
animal may be a transgenic animal, genetically-engineered animal,
and/or a clone.
[0077] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any agent that
has activity in a biological system, and particularly in an
organism. For instance, an agent that, when administered to an
organism, has a biological effect on that organism, is considered
to be biologically active. In particular embodiments, where a
protein or polypeptide is biologically active, a portion of that
protein or polypeptide that shares at least one biological activity
of the protein or polypeptide is typically referred to as a
"biologically active" portion.
[0078] Delivery: The term "delivery", when used in connection with
the CNS delivery, encompasses situations in which an mRNA is
delivered intracellularly in neurons and the encoded protein is
expressed and retained within the neurons, and situations in which
an mRNA is delivered intracellularly in neurons and the encoded
protein is expressed and secreted, e.g., to the CSF, and taken up
by other neurons.
[0079] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end formation); (3)
translation of an RNA into a polypeptide or protein; and/or (4)
post-translational modification of a polypeptide or protein. In
this application, the terms "expression" and "production," and
grammatical equivalent, are used inter-changeably.
[0080] Fragment: The term "fragment" as used herein refers to
polypeptides and is defined as any discrete portion of a given
polypeptide that is unique to or characteristic of that
polypeptide. The term as used herein also refers to any discrete
portion of a given polypeptide that retains at least a fraction of
the activity of the full-length polypeptide. Preferably the
fraction of activity retained is at least 10% of the activity of
the full-length polypeptide. More preferably the fraction of
activity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or
90% of the activity of the full-length polypeptide. More preferably
still the fraction of activity retained is at least 95%, 96%, 97%,
98% or 99% of the activity of the full-length polypeptide. Most
preferably, the fraction of activity retained is 100% of the
activity of the full-length polypeptide. The term as used herein
also refers to any portion of a given polypeptide that includes at
least an established sequence element found in the full-length
polypeptide. Preferably, the sequence element spans at least 4-5,
more preferably at least about 10, 15, 20, 25, 30, 35, 40, 45, 50
or more amino acids of the full-length polypeptide.
[0081] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0082] Half-life: As used herein, the term "half-life" is the time
required for a quantity such as nucleic acid or protein
concentration or activity to fall to half of its value as measured
at the beginning of a time period.
[0083] Improve, increase, or reduce: As used herein, the terms
"improve," "increase" or "reduce," or grammatical equivalents,
indicate values that are relative to a baseline measurement, such
as a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control subject
(or multiple control subject) in the absence of the treatment
described herein. A "control subject" is a subject afflicted with
the same form of disease as the subject being treated, who is about
the same age as the subject being treated.
[0084] In Vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
a multi-cellular organism.
[0085] In Vivo: As used herein, the term "in vivo" refers to events
that occur within a multi-cellular organism, such as a human and a
non-human animal. In the context of cell-based systems, the term
may be used to refer to events that occur within a living cell (as
opposed to, for example, in vitro systems).
[0086] Intrathecal administration: As used herein, the term
"intrathecal administration" or "intrathecal injection" refers to
an injection into the spinal canal (intrathecal space surrounding
the spinal cord). Various techniques may be used including, without
limitation, lateral cerebroventricular injection through a burrhole
or cisternal or lumbar puncture or the like. In some embodiments,
"intrathecal administration" or "intrathecal delivery" according to
the present invention refers to IT administration or delivery via
the lumbar area or region, i.e., lumbar IT administration or
delivery. As used herein, the term "lumbar region" or "lumbar area"
refers to the area between the third and fourth lumbar (lower back)
vertebrae and, more inclusively, the L2-S1 region of the spine.
[0087] Lower motor neurons: As used herein, the term "lower motor
neuron" refers to the motor neurons connecting the brainstem and
spinal cord to muscle fibers. In other words, lower motor neurons
bring the nerve impulses from the upper motor neurons out to the
muscles. Typically, a lower motor neuron's axon terminates on an
effector (muscle). Lower motor neurons include "spinal neuron" and
"Anterior horn cells".
[0088] Lysosomal enzyme: As used herein, the term "lysosomal
enzyme" refers to any enzyme that is capable of reducing
accumulated materials in mammalian lysosomes or that can rescue or
ameliorate one or more lysosomal storage disease symptoms.
Lysosomal enzymes suitable for the invention include both wild-type
or modified lysosomal enzymes and can be produced using recombinant
and synthetic methods or purified from nature sources. Exemplary
lysosomal enzymes are listed in Table 2.
[0089] Lysosomal enzyme deficiency: As used herein, "lysosomal
enzyme deficiency" refers to a group of genetic disorders that
result from deficiency in at least one of the enzymes that are
required to break macromolecules (e.g., enzyme substrates) down to
peptides, amino acids, monosaccharides, nucleic acids and fatty
acids in lysosomes. As a result, individuals suffering from
lysosomal enzyme deficiencies have accumulated materials in various
tissues (e.g., CNS, liver, spleen, gut, blood vessel walls and
other organs).
[0090] Lysosomal Storage Disease: As used herein, the term
"lysosomal storage disease" refers to any disease resulting from
the deficiency of one or more lysosomal enzymes necessary for
metabolizing natural macromolecules. These diseases typically
result in the accumulation of un-degraded molecules in the
lysosomes, resulting in increased numbers of storage granules (also
termed storage vesicles). These diseases and various examples are
described in more detail below.
[0091] messenger RNA (mRNA): As used herein, the term "messenger
RNA (mRNA)" refers to a polynucleotide that encodes at least one
polypeptide. mRNA as used herein encompasses both modified and
unmodified RNA. mRNA may contain one or more coding and non-coding
regions. mRNA can be purified from natural sources, produced using
recombinant expression systems and optionally purified, chemically
synthesized, etc. Where appropriate, e.g., in the case of
chemically synthesized molecules, mRNA can comprise nucleoside
analogs such as analogs having chemically modified bases or sugars,
backbone modifications, etc. An mRNA sequence is presented in the
5' to 3' direction unless otherwise indicated. In some embodiments,
an mRNA is or comprises natural nucleosides (e.g., adenosine,
guanosine, cytidine, uridine); nucleoside analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5
propynyl-uridine, 2-aminoadenosine, C5-bromouridine,
C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,
C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;
biologically modified bases (e.g., methylated bases); intercalated
bases; modified sugars (e.g., 2'-fluororibose, ribose,
2'-deoxyribose, arabinose, and hexose); and/or modified phosphate
groups (e.g., phosphorothioates and 5'-N-phosphoramidite
linkages).
[0092] Nucleic acid: As used herein, the term "nucleic acid," in
its broadest sense, refers to any compound and/or substance that is
or can be incorporated into a polynucleotide chain. In some
embodiments, a nucleic acid is a compound and/or substance that is
or can be incorporated into a polynucleotide chain via a
phosphodiester linkage. In some embodiments, "nucleic acid" refers
to individual nucleic acid residues (e.g., nucleotides and/or
nucleosides). In some embodiments, "nucleic acid" refers to a
polynucleotide chain comprising individual nucleic acid residues.
In some embodiments, "nucleic acid" encompasses RNA as well as
single and/or double-stranded DNA and/or cDNA.
[0093] Patient: As used herein, the term "patient" or "subject"
refers to any organism to which a provided composition may be
administered, e.g., for experimental, diagnostic, prophylactic,
cosmetic, and/or therapeutic purposes. Typical patients include
animals (e.g., mammals such as mice, rats, rabbits, non-human
primates, and/or humans). In some embodiments, a patient is a
human. A human includes pre and post natal forms.
[0094] Pharmaceutically acceptable: The term "pharmaceutically
acceptable" as used herein, refers to substances that, within the
scope of sound medical judgment, are suitable for use in contact
with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0095] Pharmaceutically acceptable salt: Pharmaceutically
acceptable salts are well known in the art. For example, S. M.
Berge et al., describes pharmaceutically acceptable salts in detail
in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically
acceptable salts of the compounds of this invention include those
derived from suitable inorganic and organic acids and bases.
Examples of pharmaceutically acceptable, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid
or rnalonic acid or by using other methods used in the art such as
ion exchange. Other pharmaceutically acceptable salts include
adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, sulfonate and aryl sulfonate. Further
pharmaceutically acceptable salts include salts formed from the
quarternization of an amine using an appropriate electrophile,
e.g., an alkyl halide, to form a quarternized alkylated amino
salt.
[0096] Subject: As used herein, the term "subject" refers to a
human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat,
cattle, swine, sheep, horse or primate). A human includes pre- and
post-natal forms. In many embodiments, a subject is a human being.
A subject can be a patient, which refers to a human presenting to a
medical provider for diagnosis or treatment of a disease. The term
"subject" is used herein interchangeably with "individual" or
"patient." A subject can be afflicted with or is susceptible to a
disease or disorder but may or may not display symptoms of the
disease or disorder.
[0097] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" of a therapeutic agent means an
amount that is sufficient, when administered to a subject suffering
from or susceptible to a disease, disorder, and/or condition, to
treat, diagnose, prevent, and/or delay the onset of the symptom(s)
of the disease, disorder, and/or condition. It will be appreciated
by those of ordinary skill in the art that a therapeutically
effective amount is typically administered via a dosing regimen
comprising at least one unit dose.
[0098] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a substance
(e.g., provided compositions) that partially or completely
alleviates, ameliorates, relives, inhibits, delays onset of,
reduces severity of, and/or reduces incidence of one or more
symptoms, features, and/or causes of a particular disease,
disorder, and/or condition (e.g., influenza). Such treatment may be
of a subject who does not exhibit signs of the relevant disease,
disorder and/or condition and/or of a subject who exhibits only
early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition. In some embodiments, treatment may be of
a subject who has been diagnosed as suffering from the relevant
disease, disorder, and/or condition. In some embodiments, treatment
may be of a subject known to have one or more susceptibility
factors that are statistically correlated with increased risk of
development of the relevant disease, disorder, and/or
condition.
[0099] Upper motor neurons: As used herein, the terms "upper motor
neuron" and "corticospinal neuron" are synonymously used to refer
to motor neurons that originate in the motor region of the cerebral
cortex or the brain stem and carry motor information down to the
final common pathway. Typically, upper motor neurons refer to any
motor neurons that are not directly responsible for stimulating the
target muscle.
DETAILED DESCRIPTION
[0100] The present invention provides, among other things, methods
and compositions for effective delivery of messenger RNA (mRNA) to
the central nervous system (CNS). In particular, the present
invention provides methods and compositions for administering
intrathecally to a subject in need of delivery a composition
comprising an mRNA encoding a protein, encapsulated within a
liposome, such that the administering of the composition results in
the intracellular delivery of mRNA in neurons in the brain and/or
spinal cord. The present invention is particularly useful for the
treatment of CNS diseases, disorders or conditions, such as spinal
muscular atrophy. As used herein, the term "liposome" refers to any
lamellar, multilamellar, or solid lipid nanoparticle vesicle.
Typically, a liposome as used herein can be formed by mixing one or
more lipids or by mixing one or more lipids and polymer(s). Thus,
the term "liposome" as used herein encompasses both lipid and
polymer based nanoparticles. In some embodiments, a liposome
suitable for the present invention contains cationic or
non-cationic lipid(s), cholesterol-based lipid(s) and PEG-modified
lipid(s).
[0101] Various aspects of the invention are described in detail in
the following sections. The use of sections is not meant to limit
the invention. Each section can apply to any aspect of the
invention. In this application, the use of "or" means "and/or"
unless stated otherwise.
mRNA Associated with CNS Diseases, Disorders or Conditions
[0102] The present invention can be used to deliver any mRNA to the
central nervous system. In particular, the present invention is
useful to deliver mRNA that encodes a protein associated with or
implicated in a CNS disease, disorder or condition. As used herein,
a "CNS disease, disorder or condition" refers to a disease,
disorder or condition affecting one or more neuronal functions of
the central nervous system (i.e., the brain and/or spinal cord). In
some embodiments, a CNS disease, disorder or condition may be
caused by a protein deficiency or dysfunction in neurons of the CNS
(i.e., the brain and/or spinal cord).
[0103] Exemplary CNS diseases, disorders or conditions include, but
are not limited to, Acid Lipase Disease, Acid Maltase Deficiency,
Acquired Epileptiform Aphasia, Acute Disseminated
Encephalomyelitis, ADHD, Adie's Pupil, Adie's Syndrome,
Adrenoleukodystrophy, Agnosia, Aicardi Syndrome, Aicardi-Goutieres
Syndrome Disorder, Alexander Disease, Alpers' Disease, Alternating
Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis
(ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis,
Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoiditis,
Arnold-Chiari Malformation, Asperger Syndrome, Ataxia, Ataxia
Telangiectasia, Ataxias and Cerebellar or Spinocerebellar
Degeneration, Attention Deficit-Hyperactivity Disorder, Autism,
Autonomic Dysfunction, Barth Syndrome, Batten Disease, Becker's
Myotonia, Behcet's Disease, Bell's Palsy, Bernhardt-Roth Syndrome,
Binswanger's Disease, Bloch-Sulzberger Syndrome, Bradbury-Eggleston
Syndrome, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy,
CADASIL, Canavan Disease, Causalgia, Cavernomas, Cavernous Angioma,
Central Cervical Cord Syndrome, Central Cord Syndrome, Central
Pontine Myelinolysis, Ceramidase Deficiency, Cerebellar
Degeneration, Cerebellar Hypoplasia, Cerebral Beriberi, Cerebral
Gigantism, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome
(COFS), Cholesterol Ester Storage Disease, Chorea,
Choreoacanthocytosis, Chronic Inflammatory Demyelinating
Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Cockayne
Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Congenital
Myasthenia, Corticobasal Degeneration, Cranial Arteritis, Cree
encephalitis, Creutzfeldt-Jakob Disease, Cushing's Syndrome,
Cytomegalic Inclusion Body Disease, Dancing Eyes-Dancing Feet
Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's
Syndrome, Dejerine-Klumpke Palsy, Dentate Cerebellar Ataxia,
Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia,
Devic's Syndrome, Diffuse Sclerosis, Dravet Syndrome, Dysautonomia,
Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia
Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva,
Fabry Disease, Fahr's Syndrome, Familial Dysautonomia, Familial
Hemangioma, Familial Idiopathic Basal Ganglia Calcification,
Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's
Disease, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant
Syndrome, Friedreich's Ataxia, Gaucher Disease, Generalized
Gangliosidoses, Gerstmann's Syndrome,
Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy,
Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell
Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage
Disease, Guillain-Barre Syndrome, Hallervorden-Spatz Disease,
Hemicrania Continua, Hemiplegia Alterans, Hereditary Spastic
Paraplegia, Heredopathia Atactica Polyneuritiformis, Holmes-Adie
syndrome, Holoprosencephaly, Hughes Syndrome, Huntington's Disease,
Hydranencephaly, Hydromyelia, Hypercortisolism, Immune-Mediated,
Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti,
Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Acid Storage
Disease, Iniencephaly, Isaac's Syndrome, Joubert Syndrome,
Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,
Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay
Syndrome (KTS), Kluver-Bucy Syndrome, Korsakoffs Amnesic Syndrome,
Krabbe Disease, Kugelberg-Welander Disease, Lambert-Eaton
Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral, Femoral
Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Leigh's
Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome,
Levine-Critchley Syndrome, Lewy Body Dementia, Lipoid Proteinosis,
Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease,
Lupus--Neurological Sequelae, Lyme Disease, Machado-Joseph Disease,
Macrencephaly, Melkersson-Rosenthal Syndrome, Menkes Disease,
Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly,
Miller Fisher Syndrome, Moebius Syndrome, Multiple Sclerosis,
Muscular Dystrophy, Myasthenia Gravis, Myelinoclastic Diffuse
Sclerosis, Narcolepsy, Neuroacanthocytosis, Neurofibromatosis,
Neuroleptic Malignant Syndrome, Neurosarcoidosis, Niemann-Pick
Disease, Ohtahara Syndrome, Olivopontocerebellar Atrophy,
Opsoclonus Myoclonus, O'Sullivan-McLeod Syndrome, Pantothenate
Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes,
Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis,
Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease,
Pena Shokeir II Syndrome, Periventricular Leukomalacia, Phytanic
Acid Storage Disease, Pick's Disease, Piriformis Syndrome,
Polymyositis, Pompe Disease, Post-Polio Syndrome, Primary Dentatum
Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia,
Prion Diseases, Progressive Hemifacial Atrophy, Progressive
Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy,
Progressive Sclerosing Poliodystrophy, Progressive Supranuclear
Palsy, Prosopagnosia, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome
II, Rasmussen's Encephalitis, Refsum Disease, Rett Syndrome, Reye's
Syndrome, Riley-Day Syndrome, Sandhoff Disease, Schilder's Disease,
Seitelberger Disease, Severe Myoclonic Epilepsy of Infancy (SMEI),
Shy-Drager Syndrome, Sjogren's Syndrome, Spasticity, Spina Bifida,
Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar
Degeneration, Steele-Richardson-Olszewski Syndrome, Striatonigral
Degeneration, Sturge-Weber Syndrome, Tardive Dyskinesia, Tay-Sachs
Disease, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic
Douloureux, Todd's Paralysis, Trigeminal Neuralgia, Tropical
Spastic Paraparesis, Troyer Syndrome, Von Economo's Disease, Von
Hippel-Lindau Disease (VHL), Von Recklinghausen's Disease,
Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff
Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome,
Wilson Disease, Wolman's Disease, X-Linked Spinal and Bulbar
Muscular Atrophy and Zellweger Syndrome.
[0104] Motor Neuron Diseases
[0105] In some embodiments, a CNS disease, disorder or condition is
a disease, disorder or condition that affects one or more functions
of motor neurons, which is also referred to as a motor neuron
disease. In some embodiments, a motor neuron disease may be caused
by a protein deficiency or dysfunction in motor neurons of the CNS
(i.e., the brain and/or spinal cord). As used herein, the term
"motor neurons" refer to those neurons that control voluntary
muscle activity. Typically, motor neurons include upper motor
neurons and lower motor neurons. As used herein, the term "upper
motor neuron" refers to motor neurons that originate in the motor
region of the cerebral cortex or the brain stem and carry motor
information down to the final common pathway. Upper motor neurons
also referred to as "corticospinal neurons". Typically, upper motor
neurons refer to any motor neurons that are not directly
responsible for stimulating the target muscle. As used herein, the
term "lower motor neuron" refers to the motor neurons connecting
the brainstem and spinal cord to muscle fibers. In other words,
lower motor neurons bring the nerve impulses from the upper motor
neurons out to the muscles. Typically, a lower motor neuron's axon
terminates on an effector (muscle). Lower motor neurons include
"spinal neuron" and "Anterior horn cells".
[0106] Exemplary motor neuron diseases, disorders or conditions
include, but are limited to, Amyotrophic Lateral Sclerosis (ALS),
Primary Lateral Sclerosis (PLS), Pseudobulbar Pasly, Hereditary
Spastic Paraplegia, Progressive Muscular Atrophy (PMA), Progressive
Bulbar Palsy (PBP), Distal Hereditary Motor Neuropathies, and
Spinal Muscular Atrophies.
[0107] In some embodiments, a motor neuron disease, disorder or
condition is a form of spinal muscular atrophy. The family of
spinal muscular atrophies are a genetically and clinically
heterogeneous group of rare debilitating disorders characterized by
degeneration of the lower motor neurons. Degeneration of the cells
within the lower motor neurons, which are also known as the
anterior horn cells of the spinal cord, leads to a loss of motor
function resulting in atrophy and excessive wasting of various
muscle groups within the body. Diseases that comprise the family
can be divided into Proximal, Distal, Autosomal Recessive Proximal
and Localized spinal muscular atrophies. However, given that
protein deficiencies are the major cause of the various forms of
spinal muscular atrophy, each disease member is usually classified
according to the gene associated with the condition. Table 1 below
describes six major groups of spinal muscular atrophies.
TABLE-US-00001 TABLE 1 Representative Groups of Spinal Muscular
Atrophies Group Name Gene Inheritance SMA Spinal muscular atrophy
(SMA) SMN-1 Autosomal Recessive XLSMA X-linked spinal muscular
atrophy NR3C4 X-Linked Reccessive type-1 (SMAX1) X-linked spinal
muscular atrophy UBA1 X-Linked Reccessive type-2 (SMAX2) X-linked
spinal muscular atrophy ATP7A X-Linked Reccessive type-3 (SMAX3)
DSMA Distal spinal muscular atrophy type-1 IGHMBP2 Autosomal
Recessive (DSMA1) Distal spinal muscular atrophy type-2 Autosomal
Recessive (DSMA2) Distal spinal muscular atrophy type-3 Autosomal
Recessive (DSMA3) Distal spinal muscular atrophy type-4 PLEKHG5
Autosomal Recessive (DSMA4) Distal spinal muscular atrophy type-5
DNAJB2 Autosomal Recessive (DSMA5) Distal spinal muscular atrophy
VA GARS Autosomal Dominant (DSMA VA) Distal spinal muscular atrophy
VB REEP1 Autosomal Dominant (DSMA VA) Distal spinal muscular
atrophy with SLC5A7 Autosomal Dominant vocal cord paralysis ADSMA
Autosomal dominant distal spinal HSPB8 Autosomal Dominant muscular
atrophy Autosomal dominant juvenile distal Autosomal Dominant
spinal muscular atrophy NTMA Congential distal spinal muscular
TRPV4 Autosomal Dominant atrophy Scapuloperoneal spinal muscular
TRPV4 Autosomal Dominant atrophy (SPSMA) or X-linked Dominant
Juvenile segmental spinal muscular atrophy (JSSMA) Finkel-type
proximal spinal muscular VAPB Autosomal Dominant atrophy (SMA-FK)
Jokela-type spinal muscular atrophy Autosomal Dominant (SMA-J)
Spinal muscular atrophy with lower DYNC1H1 Autosomal Dominant
extremity predominance (SMA-LED) Spinal muscular atrophy with ASAH1
Autosomal Recessive progressive myoclonic epilepsy (SMA-PME) Spinal
muscular atrophy with Autosomal Recessive congenital bone fractures
(SMA-CBF) PCH Spinal muscular atrophy with VRK1 Autosomal Dominant
pontocerebellar hypoplasia (SMA-PCH) MMA Juvenile asymmetric
segmental spinal muscular atrophy (JASSMA)
[0108] Diseases with a CNS Component
[0109] In some embodiments, a CNS disease, disorder or condition is
a disease with a CNS component. Typically, a disease with a CNS
component is caused by a protein deficiency in one or more tissues,
including both CNS and peripheral tissues, of the body, resulting
in one or more CNS etiology and/or symptoms. For example, in some
embodiments, a protein deficiency may result in the excess
accumulation of an intracellular and/or extracellular component
such as: glucosaminoglycans (GAGs), lipids, plaque (i.e.;
Beta-amyloid) or protein. Thus, in some embodiments, a disease with
a CNS component is a lysosomal storage disease caused by a
deficiency in a lysosomal enzyme, which results in the excess
accumulation of glucosaminoglycans (GAGs) in both the CNS and
peripheral tissues.
[0110] In some embodiments, lysosomal storage diseases having CNS
etiology and/or symptoms include, but are not limited to,
aspartylglucosaminuria, cholesterol ester storage disease, Wolman
disease, cystinosis, Danon disease, Fabry disease, Farber
lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis
types I/II, Gaucher disease types I/II/III, globoid cell
leukodystrophy, Krabbe disease, glycogen storage disease II, Pompe
disease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type
I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease,
GM2-gangliosidosis, .alpha.-mannosidosis types I/II,
.beta.-mannosidosis, metachromatic leukodystrophy, mucolipidosis
type I, sialidosis types I/II, mucolipidosis types II/III, I-cell
disease, mucolipidosis type IIIC pseudo-Hurler polydystrophy,
mucopolysaccharidosis type I, mucopolysaccharidosis type II,
mucopolysaccharidosis type IIIA, Sanfilippo syndrome,
mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC,
mucopolysaccharidosis type HID, mucopolysaccharidosis type IVA,
Morquio syndrome, mucopolysaccharidosis type IVB,
mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly
syndrome, mucopolysaccharidosis type IX, multiple sulfatase
deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease,
CLN2 Batten disease, Niemann-Pick disease types A/B, Niemann-Pick
disease type C1, Niemann-Pick disease type C2, pycnodysostosis,
Schindler disease types I/II, Gaucher disease and sialic acid
storage disease.
[0111] A detailed review of the genetic etiology, clinical
manifestations, and molecular biology of the lysosomal storage
diseases are detailed in Scriver et al., eds., The Metabolic and
Molecular Basis of Inherited Disease, 7.sup.th Ed., Vol. II, McGraw
Hill, (1995). Thus, the enzymes deficient in the above diseases are
known to those of skill in the art, some of these are exemplified
in Table 2 below:
TABLE-US-00002 TABLE 2 Lysosomal Diseases and Enzyme Deficiency
Disease Name Enzyme Deficiency Substance Stored Pompe Disease
Acid-a1, 4-Glucosidase Glycogen .alpha.-1-4 linked Oligosaccharides
GM1 Gangliodsidosis .beta.-Galactosidase GM.sub.1 Gangliosides
Tay-Sachs Disease .beta.-Hexosaminidase A GM.sub.2 Ganglioside GM2
Gangliosidosis: AB GM.sub.2 Activator Protein GM.sub.2 Ganglioside
Variant Sandhoff Disease .beta.-Hexosaminidase A&B GM.sub.2
Ganglioside Fabry Disease .alpha.-Galactosidase A Globosides
Gaucher Disease Glucocerebrosidase Glucosylceramide Metachromatic
Arylsulfatase A Sulphatides Leukodystrophy Krabbe Disease
Galactosylceramidase Galactocerebroside Niemann Pick, Types Acid
Sphingomyelinase Sphingomyelin A & B Niemann-Pick, Type C
Cholesterol Esterification Sphingomyelin Defect Niemann-Pick, Type
D Unknown Sphingomyelin Farber Disease Acid Ceramidase Ceramide
Wolman Disease Acid Lipase Cholesteryl Esters Hurler Syndrome (MPS
IH) .alpha.-L-Iduronidase Heparan & Dermatan Scheie Syndrome
(MPS IS) .alpha.-L-Iduronidase Heparan & Dermatan, Sulfates
Hurler-Scheie (MPS IH/S) .alpha.-L-Iduronidase Heparan &
Dermatan Hunter Syndrome (MPS II) Iduronate Sulfatase Heparan &
Dermatan Sanfilippo A (MPS IIIA) Heparan N-Sulfatase Heparan
Sulfate Sanfilippo B (MPS IIIB) .alpha.-N- Heparan Sulfate
Acetylglucosaminidase Sanfilippo C (MPS IIIC) Acetyl-CoA- Heparan
Sulfate Glucosaminide Acetyltransferase Sanfilippo D (MPS IIID)
N-Acetylglucosamine-6- Heparan Sulfate Sulfatase Morquio B (MPS
IVB) .beta.-Galactosidase Keratan Sulfate Maroteaux-Lamy (MPS VI)
Arylsulfatase B Dermatan Sulfate Sly Syndrome (MPS VII)
.beta.-Glucuronidase .alpha.-Mannosidosis .alpha.-Mannosidase
Mannose/Oligosaccharides .beta.-Mannosidosis .beta.-Mannosidase
Mannose/Oligosaccharides Fucosidosis .alpha.-L-Fucosidase
Fucosyl/Oligosaccharides Aspartylglucosaminuria N-Aspartyl-.beta.-
Aspartylglucosamine Glucosaminidase Asparagines Sialidosis
(Mucolipidosis I) .alpha.-Neuraminidase Sialyloligosaccharides
Galactosialidosis Lysosomal Protective Sialyloligosaccharides
(Goldberg Syndrome) Protein Deficiency Schindler Disease
.alpha.-N-Acetyl- Galactosaminidase Mucolipidosis II (I-Cell
N-Acetylglucosamine-1- Heparan Sulfate Disease) Phosphotransferase
Mucolipidosis III (Pseudo- Same as ML II Hurler Polydystrophy)
Cystinosis Cystine Transport Protein Free Cystine Salla Disease
Sialic Acid Transport Free Sialic Acid and Glucuronic Protein Acid
Infantile Sialic Acid Sialic Acid Transport Free Sialic Acid and
Glucuronic Storage Disease Protein Acid Infantile Neuronal Ceroid
Palmitoyl-Protein Lipofuscins Lipofuscinosis Thioesterase
Mucolipidosis IV Unknown Gangliosides & Hyaluronic Acid
Prosaposin Saposins A, B, C or D
[0112] In various embodiments, the present invention may be used to
deliver an mRNA encoding a protein that is deficient in any of the
CNS diseases, disorders or conditions described herein. In some
embodiments, the present invention may be used to deliver an mRNA
encoding a protein that is deficient in a motor neuron disease, for
example, a motor neuron disease shown in Table 1. In particular
embodiments, the present invention may be used to deliver an mRNA
encoding a protein that is deficient in Spinal muscular atrophy
(SMA), e.g., SMN1, which is described in detail below. In some
embodiments, the present invention may be used to deliver an mRNA
encoding a lysosomal enzyme that is deficient in a lysosomal
storage disease with a CNS component. In some embodiments, the
present invention may be used to deliver an mRNA encoding a
lysosomal enzyme selected from Table 2. In some embodiments, an
mRNA suitable for the invention may encoded a wild-type or
naturally occurring amino acid sequence. In some embodiments, an
mRNA suitable for the invention may be a wild-type or naturally
occurring sequence. In some embodiments, an mRNA suitable for the
invention may be a codon-optimized sequence. In some embodiments,
an mRNA suitable for the invention may encode an amino acid
sequence having substantial homology or identify to the wild-type
or naturally-occurring amino acid protein sequence (e.g., having at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
sequence identity to the wild-type or naturally-occurring
sequence).
Survival of Motor Neuron
[0113] In some embodiments, inventive methods and compositions
provided by the present invention are used to deliver an mRNA
encoding a Survival of Motor Neuron protein to the CNS for
treatment of spinal muscular atrophy (SMA).
[0114] A suitable SMN mRNA encodes any full length, fragment or
portion of a SMN protein which can be substituted for
naturally-occurring SMN protein activity or rescue one or more
phenotypes or symptoms associated with spinal muscular atrophy. The
mRNA sequence for human Survival of Motor Neuron-1 (hSMN-1) and
corresponding amino acid sequence of a typical wild-type or
naturally occurring hSMN-1 protein are shown in Table 3.
TABLE-US-00003 TABLE 3 Human SMN-1 Human SMN-1
GGGGACCCGCGGGUUUGCUAUGGCGAUGAGCAGCGGCGGCAGUGGUGGCGGCGU (mRNA)
CCCGGAGCAGGAGGAUUCCGUGCUGUUCCGGCGCGGCACAGGCCAGAGCGAUGA
UUCUGACAUUUGGGAUGAUACAGCACUGAUAAAAGCAUAUGAUAAAGCUGUGGC
UUCAUUUAAGCAUGCUCUAAAGAAUGGUGACAUUUGUGAAACUUCGGGUAAACC
AAAAACCACACCUAAAAGAAAACCUGCUAAGAAGAAUAAAAGCCAAAAGAAGAA
UACUGCAGCUUCCUUACAACAGUGGAAAGUUGGGGACAAAUGUUCUGCCAUUUG
GUCAGAAGACGGUUGCAUUUACCCAGCUACCAUUGCUUCAAUUGAUUUUAAGAG
AGAAACCUGUGUUGUGGUUUACACUGGAUAUGGAAAUAGAGAGGAGCAAAAUCU
GUCCGAUCUACUUUCCCCAAUCUGUGAAGUAGCUAAUAAUAUAGAACAAAAUGC
UCAAGAGAAUGAAAAUGAAAGCCAAGUUUCAACAGAUGAAAGUGAGAACUCCAG
GUCUCCUGGAAAUAAAUCAGAUAACAUCAAGCCCAAAUCUGCUCCAUGGAACUC
UUUUCUCCCUCCACCACCCCCCAUGCCAGGGCCAAGACUGGGACCAGGAAAGCC
AGGUCUAAAAUUCAAUGGCCCACCACCGCCACCGCCACCACCACCACCCCACUU
ACUAUCAUGCUGGCUGCCUCCAUUUCCUUCUGGACCACCAAUAAUUCCCCCACC
ACCUCCCAUAUGUCCAGAUUCUCUUGAUGAUGCUGAUGCUUUGGGAAGUAUGUU
AAUUUCAUGGUACAUGAGUGGCUAUCAUACUGGCUAUUAUAUGGGUUUCAGACA
AAAUCAAAAAGAAGGAAGGUGCUCACAUUCCUUAAAUUAAGGAGAAAUGCUGGC
AUAGAGCAGCACUAAAUGACACCACUAAAGAAACGAUCAGACAGAUCUGGAAUG
UGAAGCGUUAUAGAAGAUAACUGGCCUCAUUUCUUCAAAAUAUCAAGUGUUGGG
AAAGAAAAAAGGAAGUGGAAUGGGUAACUCUUCUUGAUUAAAAGUUAUGUAAUA
ACCAAAUGCAAUGUGAAAUAUUUUACUGGACUCUAUUUUGAAAAACCAUCUGUA
AAAGACUGGGGUGGGGGUGGGAGGCCAGCACGGUGGUGAGGCAGUUGAGAAAAU
UUGAAUGUGGAUUAGAUUUUGAAUGAUAUUGGAUAAUUAUUGGUAAUUUUUAUG
AGCUGUGAGAAGGGUGUUGUAGUUUAUAAAAGACUGUCUUAAUUUGCAUACUUA
AGCAUUUAGGAAUGAAGUGUUAGAGUGUCUUAAAAUGUUUCAAAUGGUUUAACA
AAAUGUAUGUGAGGCGUAUGUGGCAAAAUGUUACAGAAUCUAACUGGUGGACAU
GGCUGUUCAUUGUACUGUUUUUUUCUAUCUUCUAUAUGUUUAAAAGUAUAUAAU
AAAAAUAUUUAAUUUUUUUUUAAAAAAAAAAAAAAAAAAACAAAAAAAAAAAA (SEQ ID NO:
1) Human SMN-1
MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHAL (Amino Acid
KNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCI Seq.)
YPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENE
SQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNG
PPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMS
GYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO: 2)
[0115] Thus, in some embodiments, a suitable mRNA for the present
invention is a wild-type hSMN-1 mRNA sequence (SEQ ID NO:1). In
some embodiments, a suitable mRNA may be a codon optimized hSMN-1
mRNA sequence represented by (SEQ ID NO:3):
TABLE-US-00004 AUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAG
GACAGCGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAU
AUCUGGGACGAUACCGCUCUGAUCAAGGCCUACGACAAGGCCGUGGCC
AGCUUCAAGCACGCCCUGAAAAACGGCGACAUCUGCGAGACCAGCGGC
AAGCCCAAGACAACCCCCAAGAGAAAGCCCGCCAAGAAGAAUAAGAGC
CAGAAAAAGAACACCGCCGCCAGCCUGCAGCAGUGGAAGGUGGGCGAC
AAGUGCAGCGCCAUCUGGAGCGAGGACGGCUGCAUCUACCCCGCCACC
AUCGCCAGCAUCGACUUCAAGAGAGAGACCUGCGUGGUCGUGUACACC
GGCUACGGCAACAGAGAGGAGCAGAACCUGAGCGACCUGCUGAGCCCC
AUUUGUGAGGUGGCCAAUAACAUCGAACAGAACGCCCAGGAGAACGAG
AAUGAAAGCCAGGUGAGCACCGACGAGAGCGAGAACAGCAGAUCUCCU
GGCAACAAGAGCGACAACAUCAAGCCUAAGUCUGCCCCUUGGAACAGC
UUCCUGCCCCCUCCUCCACCCAUGCCCGGACCCAGACUGGGACCCGGA
AAACCUGGCCUGAAGUUCAACGGACCACCUCCCCCUCCACCUCCUCCC
CCACCUCAUCUCCUGAGCUGCUGGCUGCCACCCUUCCCCAGCGGACCC
CCUAUCAUCCCACCACCCCCUCCCAUCUGCCCCGACAGCCUGGACGAC
GCCGAUGCCCUGGGCAGCAUGCUGAUCAGCUGGUACAUGAGCGGCUAC
CACACAGGAUACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGA
UGCUCCCACUCCCUGAACUGA
[0116] Alternatively, in some embodiments, a suitable mRNA may be a
codon optimized hSMN-1 mRNA sequence represented by (SEQ ID
NO:4):
TABLE-US-00005 AUGGCCAUGAGCAGCGGAGGAAGCGGAGGAGGAGUGCCAGAACAGGAA
GAUAGCGUGCUGUUUCGCCGGGGCACCGGACAAUCGGACGACAGCGAU
AUUUGGGACGACACUGCGCUCAUCAAGGCCUACGACAAGGCGGUGGCU
UCGUUCAAGCACGCUCUGAAGAACGGGGAUAUCUGUGAAACCAGCGGU
AAACCAAAAACUACGCCGAAAAGGAAACCCGCCAAAAAGAACAAGUCA
CAGAAGAAGAAUACCGCUGCGAGCUUGCAGCAGUGGAAGGUGGGCGAC
AAGUGCUCCGCGAUUUGGUCGGAAGAUGGUUGCAUCUACCCGGCAACC
AUCGCCUCCAUCGACUUUAAGCGGGAGACUUGCGUCGUGGUCUACACC
GGAUACGGCAAUAGAGAGGAACAGAAUCUGUCAGACCUUCUGUCGCCA
AUCUGCGAGGUCGCCAACAAUAUCGAACAAAACGCCCAAGAGAACGAG
AAUGAGUCCCAAGUGUCCACGGACGAAUCGGAAAACUCACGGUCCCCU
GGGAACAAGUCAGAUAACAUCAAGCCUAAAUCGGCACCAUGGAACUCC
UUCCUGCCGCCUCCGCCUCCGAUGCCGGGCCCGCGCCUGGGACCGGGU
AAACCCGGGCUCAAGUUCAAUGGACCGCCACCCCCACCCCCGCCACCG
CCGCCCCACCUCCUCUCGUGCUGGCUGCCGCCGUUCCCUUCCGGACCG
CCUAUCAUUCCGCCACCUCCACCUAUCUGCCCAGACAGCCUGGAUGAU
GCCGACGCAUUGGGCUCCAUGCUCAUCUCAUGGUACAUGUCGGGAUAC
CAUACUGGGUAUUACAUGGGCUUCAGACAGAACCAGAAGGAAGGACGC
UGUUCCCAUAGCCUGAACUAG
[0117] In some embodiments, a suitable mRNA encodes a full length,
fragment or portion of human Survival of Motor Neuron-2 (hSMN-2)
protein. The mRNA sequence for hSMN-2 and corresponding amino acid
sequence of a typical wild-type or naturally occurring hSMN-2
protein are shown in Table 4.
TABLE-US-00006 TABLE 4 Human SMN-2 Human SMN-2
GGGGCCCCACGCUGCGCACCCGCGGGUUUGCUAUGGCGAUGAGCAGCGGCGGCA (mRNA)
GUGGUGGCGGCGUCCCGGAGCAGGAGGAUUCCGUGCUGUUCCGGCGCGGCACAG
GCCAGAGCGAUGAUUCUGACAUUUGGGAUGAUACAGCACUGAUAAAAGCAUAUG
AUAAAGCUGUGGCUUCAUUUAAGCAUGCUCUAAAGAAUGGUGACAUUUGUGAAA
CUUCGGGUAAACCAAAAACCACACCUAAAAGAAAACCUGCUAAGAAGAAUAAAA
GCCAAAAGAAGAAUACUGCAGCUUCCUUACAACAGUGGAAAGUUGGGGACAAAU
GUUCUGCCAUUUGGUCAGAAGACGGUUGCAUUUACCCAGCUACCAUUGCUUCAA
UUGAUUUUAAGAGAGAAACCUGUGUUGUGGUUUACACUGGAUAUGGAAAUAGAG
AGGAGCAAAAUCUGUCCGAUCUACUUUCCCCAAUCUGUGAAGUAGCUAAUAAUA
UAGAACAGAAUGCUCAAGAGAAUGAAAAUGAAAGCCAAGUUUCAACAGAUGAAA
GUGAGAACUCCAGGUCUCCUGGAAAUAAAUCAGAUAACAUCAAGCCCAAAUCUG
CUCCAUGGAACUCUUUUCUCCCUCCACCACCCCCCAUGCCAGGGCCAAGACUGG
GACCAGGAAAGCCAGGUCUAAAAUUCAAUGGCCCACCACCGCCACCGCCACCAC
CACCACCCCACUUACUAUCAUGCUGGCUGCCUCCAUUUCCUUCUGGACCACCAA
UAAUUCCCCCACCACCUCCCAUAUGUCCAGAUUCUCUUGAUGAUGCUGAUGCUU
UGGGAAGUAUGUUAAUUUCAUGGUACAUGAGUGGCUAUCAUACUGGCUAUUAUA
UGGAAAUGCUGGCAUAGAGCAGCACUAAAUGACACCACUAAAGAAACGAUCAGA
CAGAUCUGGAAUGUGAAGCGUUAUAGAAGAUAACUGGCCUCAUUUCUUCAAAAU
AUCAAGUGUUGGGAAAGAAAAAAGGAAGUGGAAUGGGUAACUCUUCUUGAUUAA
AAGUUAUGUAAUAACCAAAUGCAAUGUGAAAUAUUUUACUGGACUCUAUUUUGA
AAAACCAUCUGUAAAAGACUGAGGUGGGGGUGGGAGGCCAGCACGGUGGUGAGG
CAGUUGAGAAAAUUUGAAUGUGGAUUAGAUUUUGAAUGAUAUUGGAUAAUUAUU
GGUAAUUUUAUGAGCUGUGAGAAGGGUGUUGUAGUUUAUAAAAGACUGUCUUAA
UUUGCAUACUUAAGCAUUUAGGAAUGAAGUGUUAGAGUGUCUUAAAAUGUUUCA
AAUGGUUUAACAAAAUGUAUGUGAGGCGUAUGUGGCAAAAUGUUACAGAAUCUA
ACUGGUGGACAUGGCUGUUCAUUGUACUGUUUUUUUCUAUCUUCUAUAUGUUUA
AAAGUAUAUAAUAAAAAUAUUUAAUUUUUUUUUAAAAA (SEQ ID NO: 5) Human SMN-2
MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHAL (Amino Acid
KNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCI Seq.)
YPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENE
SQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNG
PPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMS GYHTGYYMEMLA
(SEQ ID NO: 6)
[0118] Thus, in some embodiments, a suitable mRNA for the present
invention is a wild-type hSMN-2 mRNA sequence (SEQ ID NO:5). In
some embodiments, a suitable mRNA may be a codon optimized hSMN-1
mRNA sequence.
[0119] In some embodiments, a suitable mRNA sequence may be an mRNA
sequence encoding a homologue or an analogue of human SMN-1(SEQ ID
NO. 2) or human SMN-2 (SEQ ID NO. 6) proteins. For example, a
homologue or an analogue of human SMN-1 or SMN-2 protein may be a
modified human SMN-1 or SMN-2 protein containing one or more amino
acid substitutions, deletions, and/or insertions as compared to a
wild-type or naturally-occurring human SMN-1 protein (e.g., SEQ ID
NO:2) or human SMN-2 protein (e.g., SEQ ID NO:6), while retaining
substantial SMN-1 or SMN-2 protein activity. In some embodiments,
an mRNA suitable for the present invention encodes an amino acid
sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID
NO:2 or SEQ ID NO:6. In some embodiments, an mRNA suitable for the
present invention encodes a protein substantially identical to
human SMN-1 protein (SEQ ID NO:2) or human SMN-2 protein (SEQ ID
NO:6). In some embodiments, an mRNA suitable for the present
invention encodes an amino acid sequence at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to SEQ ID NO:2 or SEQ ID NO:6. In some
embodiments, an mRNA suitable for the present invention encodes a
fragment or a portion of human SMN-1 or human SMN-2 protein,
wherein the fragment or portion of the protein still maintains
SMN-1 or SMN-2 activity similar to that of their respective
wild-type proteins. In some embodiments, an mRNA suitable for the
present invention comprises a nucleotide sequence at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:4 or SEQ ID NO:5.
[0120] Human SMN-1 gene may undergo alternative processing and
transcriptional modification to produce alternative splice
isoforms. For example, there are five known hSMN-1 splice isoforms:
hSMN-1 isoform b, c, e, f and g. Human SMN-2 gene can also undergo
alternative processing and transcriptional modification to produce
alternative splice isoforms. There are four known hSMN-2 splice
isoforms: hSMN-2 isoform a, b, c and d. In some embodiments, the
present invention is used to deliver an mRNA encoding an hSMN-1
isoform (e.g., isoform b, c, e, f, or g). In some embodiments, the
present invention is used to deliver an mRNA encoding an hSMN-2
isoform (e.g., isoform a, b, c or d). The nucleotide and amino acid
sequence of the hSMN-1 and hSMN-2 isoforms are known in the art.
Thus, in some embodiments, the present invention can be used to
deliver an mRNA encoding an hSMN-1 isoform or an hSMN-2 protein or
an isoform thereof. In some embodiments, an mRNA suitable for the
invention may be a wild-type or naturally occurring hSMN-1 or
hSMN-2 isoform sequence. In some embodiments, an mRNA suitable for
the invention may be a codon-optimized hSMN-1 or hSMN-2 isoform
sequence. In some embodiments, an mRNA suitable for the invention
may encode an amino acid sequence having substantial homology or
identify to the wild-type or naturally-occurring hSMN-1 or hSMN-2
isoform sequence (e.g., having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type or
naturally-occurring hSMN-1 or hSMN-2 isoform sequence).
mRNA Synthesis
[0121] mRNAs according to the present invention may be synthesized
according to any of a variety of known methods. For example, mRNAs
according to the present invention may be synthesized via in vitro
transcription (IVT). Briefly, IVT is typically performed with a
linear or circular DNA template containing a promoter, a pool of
ribonucleotide triphosphates, a buffer system that may include DTT
and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7
or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse
inhibitor. The exact conditions will vary according to the specific
application.
[0122] In some embodiments, for the preparation of mRNA according
to the invention, a DNA template is transcribed in vitro. A
suitable DNA template typically has a promoter, for example a T3,
T7 or SP6 promoter, for in vitro transcription, followed by desired
nucleotide sequence for desired mRNA and a termination signal.
[0123] Desired mRNA sequence(s) according to the invention may be
determined and incorporated into a DNA template using standard
methods. For example, starting from a desired amino acid sequence
(e.g., an enzyme sequence), a virtual reverse translation is
carried out based on the degenerated genetic code. Optimization
algorithms may then be used for selection of suitable codons.
Typically, the G/C content can be optimized to achieve the highest
possible G/C content on one hand, taking into the best possible
account the frequency of the tRNAs according to codon usage on the
other hand. The optimized RNA sequence can be established and
displayed, for example, with the aid of an appropriate display
device and compared with the original (wild-type) sequence. A
secondary structure can also be analyzed to calculate stabilizing
and destabilizing properties or, respectively, regions of the
RNA.
[0124] Modified mRNA
[0125] In some embodiments, mRNA according to the present invention
may be synthesized as unmodified or modified mRNA. Typically, mRNAs
are modified to enhance stability. Modifications of mRNA can
include, for example, modifications of the nucleotides of the RNA.
An modified mRNA according to the invention can thus include, for
example, backbone modifications, sugar modifications or base
modifications. In some embodiments, mRNAs may be synthesized from
naturally occurring nucleotides and/or nucleotide analogues
(modified nucleotides) including, but not limited to, purines
(adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine
(C), uracil (U)), and as modified nucleotides analogues or
derivatives of purines and pyrimidines, such as e.g.
1-methyl-adenine, 2-methyl-adenine,
2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,
N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,
4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,
1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,
7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil
(5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil,
5-carboxymethylaminomethyl-2-thio-uracil,
5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,
5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,
5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,
5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,
5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil,
uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),
1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine,
wybutoxosine, and phosphoramidates, phosphorothioates, peptide
nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine
and inosine. The preparation of such analogues is known to a person
skilled in the art e.g. from the U.S. Pat. No. 4,373,071, U.S. Pat.
No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066,
U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No.
4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S.
Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the
disclosures of which are incorporated by reference in their
entirety.
[0126] In some embodiments, mRNAs may contain RNA backbone
modifications. Typically, a backbone modification is a modification
in which the phosphates of the backbone of the nucleotides
contained in the RNA are modified chemically. Exemplary backbone
modifications typically include, but are not limited to,
modifications from the group consisting of methylphosphonates,
methylphosphoramidates, phosphoramidates, phosphorothioates (e.g.
cytidine 5'-O-(1-thiophosphate)), boranophosphates, positively
charged guanidinium groups etc., which means by replacing the
phosphodiester linkage by other anionic, cationic or neutral
groups.
[0127] In some embodiments, mRNAs may contain sugar modifications.
A typical sugar modification is a chemical modification of the
sugar of the nucleotides it contains including, but not limited to,
sugar modifications chosen from the group consisting of
2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine
5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate),
2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine
5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate),
2'-O-alkyloligoribonucleotide,
2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine
5'-triphosphate, 2'-methyluridine 5'-triphosphate),
2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine
5'-triphosphate, 2'-arauridine 5'-triphosphate), or
azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate,
2'-azido-2'-deoxyuridine 5'-triphosphate).
[0128] In some embodiments, mRNAs may contain modifications of the
bases of the nucleotides (base modifications). A modified
nucleotide which contains a base modification is also called a
base-modified nucleotide. Examples of such base-modified
nucleotides include, but are not limited to, 2-amino-6-chloropurine
riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate,
2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate,
4-thiouridine 5'-triphosphate, 5-aminoallylcytidine
5'-triphosphate, 5-aminoallyluridine 5'-triphosphate,
5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate,
5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate,
5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate,
6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate,
6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine
5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine
5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole
riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate,
N1-methylguanosine 5'-triphosphate, N6-methyladenosine
5'-triphosphate, 06-methylguanosine 5'-triphosphate, pseudouridine
5'-triphosphate, puromycin 5'-triphosphate or xanthosine
5'-triphosphate.
[0129] Typically, mRNA synthesis includes the addition of a "cap"
on the N-terminal (5') end, and a "tail" on the C-terminal (3')
end. The presence of the cap is important in providing resistance
to nucleases found in most eukaryotic cells. The presence of a
"tail" serves to protect the mRNA from exonuclease degradation.
[0130] Cap Structure
[0131] In some embodiments, mRNAs include a 5' cap structure. A 5'
cap is typically added as follows: first, an RNA terminal
phosphatase removes one of the terminal phosphate groups from the
5' nucleotide, leaving two terminal phosphates; guanosine
triphosphate (GTP) is then added to the terminal phosphates via a
guanylyl transferase, producing a 5'5'5 triphosphate linkage; and
the 7-nitrogen of guanine is then methylated by a
methyltransferase. Examples of cap structures include, but are not
limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
[0132] Naturally occurring cap structures comprise a 7-methyl
guanosine that is linked via a triphosphate bridge to the 5'-end of
the first transcribed nucleotide, resulting in a dinucleotide cap
of m.sup.7G(5')ppp(5')N, where N is any nucleoside. In vivo, the
cap is added enzymatically. The cap is added in the nucleus and is
catalyzed by the enzyme guanylyl transferase. The addition of the
cap to the 5' terminal end of RNA occurs immediately after
initiation of transcription. The terminal nucleoside is typically a
guanosine, and is in the reverse orientation to all the other
nucleotides, i.e., G(5')ppp(5')GpNpNp.
[0133] A common cap for mRNA produced by in vitro transcription is
m.sup.7G(5')ppp(5')G, which has been used as the dinucleotide cap
in transcription with T7 or SP6 RNA polymerase in vitro to obtain
RNAs having a cap structure in their 5'-termini. The prevailing
method for the in vitro synthesis of capped mRNA employs a
pre-formed dinucleotide of the form m.sup.7G(5')ppp(5')G
("m.sup.7GpppG") as an initiator of transcription.
[0134] To date, a usual form of a synthetic dinucleotide cap used
in in vitro translation experiments is the Anti-Reverse Cap Analog
("ARCA") or modified ARCA, which is generally a modified cap analog
in which the 2' or 3' OH group is replaced with --OCH.sub.3.
[0135] Additional cap analogs include, but are not limited to, a
chemical structures selected from the group consisting of
m.sup.7GpppG, m.sup.7GpppA, m.sup.7GpppC; unmethylated cap analogs
(e.g., GpppG); dimethylated cap analog (e.g., m.sup.2,7GpppG),
trimethylated cap analog (e.g., m.sup.2,2,7GpppG), dimethylated
symmetrical cap analogs (e.g., m.sup.7Gpppm.sup.7G), or anti
reverse cap analogs (e.g., ARCA; m.sup.7,2'OmeGpppG,
m.sup.72'dGpppG, m.sup.7,3'OmeGpppG, m.sup.7,3'dGpppG and their
tetraphosphate derivatives) (see, e.g., Jemielity, J. et al.,
"Novel `anti-reverse` cap analogs with superior translational
properties", RNA, 9: 1108-1122 (2003)).
[0136] In some embodiments, a suitable cap is a 7-methyl guanylate
("m.sup.7G") linked via a triphosphate bridge to the 5'-end of the
first transcribed nucleotide, resulting in m.sup.7G(5')ppp(5')N,
where N is any nucleoside. A preferred embodiment of a m.sup.7G cap
utilized in embodiments of the invention is
m.sup.7G(5')ppp(5')G.
[0137] In some embodiments, the cap is a Cap0 structure. Cap0
structures lack a 2'-O-methyl residue of the ribose attached to
bases 1 and 2. In some embodiments, the cap is a Cap1 structure.
Cap1 structures have a 2'-O-methyl residue at base 2. In some
embodiments, the cap is a Cap2 structure. Cap2 structures have a
2'-O-methyl residue attached to both bases 2 and 3.
[0138] A variety of m.sup.7G cap analogs are known in the art, many
of which are commercially available. These include the m.sup.7GpppG
described above, as well as the ARCA 3'-OCH.sub.3 and 2'-OCH.sub.3
cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)).
Additional cap analogs for use in embodiments of the invention
include N7-benzylated dinucleoside tetraphosphate analogs
(described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),
phosphorothioate cap analogs (described in Grudzien-Nogalska, E.,
et al., RNA, 13: 1745-1755 (2007)), and cap analogs (including
biotinylated cap analogs) described in U.S. Pat. Nos. 8,093,367 and
8,304,529, incorporated by reference herein.
[0139] Tail Structure
[0140] Typically, the presence of a "tail" serves to protect the
mRNA from exonuclease degradation. The poly A tail is thought to
stabilize natural messengers and synthetic sense RNA. Therefore, in
certain embodiments a long poly A tail can be added to an mRNA
molecule thus rendering the RNA more stable. Poly A tails can be
added using a variety of art-recognized techniques. For example,
long poly A tails can be added to synthetic or in vitro transcribed
RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology.
1996; 14: 1252-1256). A transcription vector can also encode long
poly A tails. In addition, poly A tails can be added by
transcription directly from PCR products. Poly A may also be
ligated to the 3' end of a sense RNA with RNA ligase (see, e.g.,
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991
edition)).
[0141] In some embodiments, mRNAs include a 3' poly(A) tail
structure. Typically, the length of the poly A tail can be at least
about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some
embodiments, a poly-A tail on the 3' terminus of mRNA typically
includes about 10 to 300 adenosine nucleotides (e.g., about 10 to
200 adenosine nucleotides, about 10 to 150 adenosine nucleotides,
about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine
nucleotides, or about 20 to 60 adenosine nucleotides). In some
embodiments, mRNAs include a 3' poly(C) tail structure. A suitable
poly-C tail on the 3' terminus of mRNA typically include about 10
to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine
nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70
cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about
10 to 40 cytosine nucleotides). The poly-C tail may be added to the
poly-A tail or may substitute the poly-A tail.
[0142] In some embodiments, the length of the poly A or poly C tail
is adjusted to control the stability of a modified sense mRNA
molecule of the invention and, thus, the transcription of protein.
For example, since the length of the poly A tail can influence the
half-life of a sense mRNA molecule, the length of the poly A tail
can be adjusted to modify the level of resistance of the mRNA to
nucleases and thereby control the time course of polynucleotide
expression and/or polypeptide production in a target cell.
[0143] 5' and 3' Untranslated Region
[0144] In some embodiments, mRNAs include a 5' and/or 3'
untranslated region. In some embodiments, a 5' untranslated region
includes one or more elements that affect an mRNA's stability or
translation, for example, an iron responsive element. In some
embodiments, a 5' untranslated region may be between about 50 and
500 nucleotides in length.
[0145] In some embodiments, a 3' untranslated region includes one
or more of a polyadenylation signal, a binding site for proteins
that affect an mRNA's stability of location in a cell, or one or
more binding sites for miRNAs. In some embodiments, a 3'
untranslated region may be between 50 and 500 nucleotides in length
or longer.
[0146] Exemplary 3' and/or 5' UTR sequences can be derived from
mRNA molecules which are stable (e.g., globin, actin, GAPDH,
tubulin, histone, or citric acid cycle enzymes) to increase the
stability of the sense mRNA molecule. For example, a 5' UTR
sequence may include a partial sequence of a CMV immediate-early 1
(IE1) gene, or a fragment thereof to improve the nuclease
resistance and/or improve the half-life of the polynucleotide. Also
contemplated is the inclusion of a sequence encoding human growth
hormone (hGH), or a fragment thereof to the 3' end or untranslated
region of the polynucleotide (e.g., mRNA) to further stabilize the
polynucleotide. Generally, these modifications improve the
stability and/or pharmacokinetic properties (e.g., half-life) of
the polynucleotide relative to their unmodified counterparts, and
include, for example modifications made to improve such
polynucleotides' resistance to in vivo nuclease digestion.
Delivery Vehicles
[0147] According to the present invention, mRNA may be delivered to
the CNS as naked RNA (unpackaged) or via delivery vehicles. As used
herein, the terms "delivery vehicle," "transfer vehicle,"
"Nanoparticle" or grammatical equivalent, are used
interchangeably.
[0148] In some embodiments, mRNAs may be delivered via a single
delivery vehicle. In some embodiments, mRNAs may be delivered via
one or more delivery vehicles each of a different composition.
According to various embodiments, suitable delivery vehicles
include, but are not limited to, polymer based carriers, such as
polyethyleneimine (PEI), lipid nanoparticles and liposomes,
nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes,
both natural and synthetically-derived exosomes, natural, synthetic
and semi-synthetic lamellar bodies, nanoparticulates, calcium
phosphor-silicate nanoparticulates, calcium phosphate
nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline
particulates, semiconductor nanoparticulates, poly(D-arginine),
sol-gels, nanodendrimers, starch-based delivery systems, micelles,
emulsions, niosomes, multi-domain-block polymers (vinyl polymers,
polypropyl acrylic acid polymers, dynamic polyconjugates), dry
powder formulations, plasmids, viruses, calcium phosphate
nucleotides, aptamers, peptides and other vectorial tags.
[0149] Liposomal Delivery Vehicles
[0150] In some embodiments, a suitable delivery vehicle is a
liposomal delivery vehicle, e.g. a lipid nanoparticle. As used
herein, liposomal delivery vehicles, e.g. lipid nanoparticles, are
usually characterized as microscopic vesicles having an interior
aqua space sequestered from an outer medium by a membrane of one or
more bilayers. Bilayer membranes of liposomes are typically formed
by amphiphilic molecules, such as lipids of synthetic or natural
origin that comprise spatially separated hydrophilic and
hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).
Typically, a liposomal delivery vehicle (e.g., a lipid nanoparticle
or liposome) suitable for the present invention is formed by
combining one or more different lipids and/or polymers. In some
embodiments, a liposomal delivery vehicle (e.g., a lipid
nanoparticle or liposome) contains one or more cationic lipids, one
or more non-cationic/helper lipids, one or more cholesterol based
lipids, and/or one or more PEGylated lipids.
[0151] Cationic Lipids
[0152] In some embodiments, a suitable delivery vehicle contains a
cationic lipid. As used herein, the phrase "cationic lipid" refers
to any of a number of lipid species that have a net positive charge
at a selected pH, such as physiological pH. Some cationic lipids,
in particular, those known as titratable or pH-titratable cationic
lipids are particularly effective in delivering mRNA. Several
cationic (e.g., titratable) lipids have been described in the
literature, many of which are commercially available. Particularly
suitable cationic lipids for use in the compositions and methods of
the invention include those described in international patent
publications WO 2010/053572 (and particularly, CI 2-200 described
at paragraph [00225]) and WO 2012/170930, both of which are
incorporated herein by reference. In some embodiments, the cationic
lipid N-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium
chloride or "DOTMA" is used. (Feigner et al. (Proc. Nat'l Acad.
Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be
formulated alone or can be combined with the neutral lipid,
dioleoylphosphatidyl-ethanolamine or "DOPE" or other cationic or
non-cationic lipids into a liposomal transfer vehicle or a lipid
nanoparticle, and such liposomes can be used to enhance the
delivery of nucleic acids into target cells. Other suitable
cationic lipids include, for example,
5-carboxyspermylglycinedioctadecylamide or "DOGS,"
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium or "DOSPA" (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982
(1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761),
1,2-Dioleoyl-3-Dimethylammonium-Propane or "DODAP",
1,2-Dioleoyl-3-Trimethylammonium-Propane or "DOTAP". Contemplated
cationic lipids also include
1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA",
1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA",
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA",
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA",
N-dioleyl-N,N-dimethylammonium chloride or "DODAC",
N,N-distearyl-N,N-dimethylarnrnonium bromide or "DDAB",
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide or "DMRIE",
3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane or "CLinDMA",
2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy
1-1-(cis,cis-9',1-2'-octadecadienoxy)propane or "CpLinDMA",
N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA",
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP",
2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP",
1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane or
"DLincarbDAP", 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or
"DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or
"Dlin- -DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
or "DLin-K-XTC2-DMA", and
2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-di-
methylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et
al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof.
(Heyes, J., et al., J Controlled Release 107: 276-287 (2005);
Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005);
PCT Publication WO2005/121348A1).
[0153] In some embodiments, one or more of the cationic lipids
present in such a composition are chosen from XTC
(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3
(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate), ALNY-100
((3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahyd-
ro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5
(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tet-
raazahexadecane-1,16-diamide), DODAP
(1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,
the teachings of which are incorporated herein by reference in
their entirety), ICE (WO 2011/068810, the teachings of which are
incorporated herein by reference in their entirety).
[0154] In certain embodiments, the compositions and methods of the
invention employ a lipid nanoparticles comprising an ionizable
cationic lipid described in U.S. provisional patent application
61/617,468, filed Mar. 29, 2012 (incorporated herein by reference),
such as, e.g,
(15Z,18Z)--N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,1-
8-dien-1-amine (HGT5000),
(15Z,18Z)--N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,1-
5,18-trien-1-amine (HGT5001), and
(15Z,18Z)--N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,1-
5,18-trien-1-amine (HGT5002).
[0155] In some embodiments, provided liposomes include a cationic
lipid described in WO 2013063468 and in U.S. provisional
application entitled "Lipid Formulations for Delivery of Messernger
RNA" filed concurrently with the present application on even date,
both of which are incorporated by reference herein. In some
embodiments, a cationic lipid comprises a compound of formula
I-c1-a:
##STR00005##
[0156] or a pharmaceutically acceptable salt thereof, wherein:
[0157] each R.sup.2 independently is hydrogen or C.sub.1-3 alkyl;
[0158] each q independently is 2 to 6; [0159] each R' independently
is hydrogen or C.sub.1-3 alkyl; [0160] and each R.sup.1
independently is C.sub.8-12 alkyl.
[0161] In some embodiments, each R.sup.2 independently is hydrogen,
methyl or ethyl. In some embodiments, each R.sup.2 independently is
hydrogen or methyl. In some embodiments, each R.sup.2 is
hydrogen.
[0162] In some embodiments, each q independently is 3 to 6. In some
embodiments, each q independently is 3 to 5. In some embodiments,
each q is 4.
[0163] In some embodiments, each R' independently is hydrogen,
methyl or ethyl. In some embodiments, each R' independently is
hydrogen or methyl. In some embodiments, each R' independently is
hydrogen.
[0164] In some embodiments, each R.sup.L independently is
C.sub.8-12 alkyl. In some embodiments, each R.sup.L independently
is n-C.sub.8-12 alkyl. In some embodiments, each R.sup.L
independently is C.sub.9-11 alkyl. In some embodiments, each
R.sup.L independently is n-C.sub.9-11 alkyl. In some embodiments,
each R.sup.L independently is C.sub.10 alkyl. In some embodiments,
each R.sup.L independently is n-C.sub.10 alkyl.
[0165] In some embodiments, each R.sup.2 independently is hydrogen
or methyl; each q independently is 3 to 5; each R' independently is
hydrogen or methyl; and each R.sup.L independently is C.sub.8-12
alkyl.
[0166] In some embodiments, each R.sup.2 is hydrogen; each q
independently is 3 to 5; each R' is hydrogen; and each R.sup.L
independently is C.sub.8-12 alkyl.
[0167] In some embodiments, each R.sup.2 is hydrogen; each q is 4;
each R' is hydrogen; and each R.sup.L independently is C.sub.8-12
alkyl.
[0168] In some embodiments, a cationic lipid comprises a compound
of formula I-g:
##STR00006##
or a pharmaceutically acceptable salt thereof, wherein each R.sup.L
independently is C.sub.8-12 alkyl. In some embodiments, each
R.sup.L independently is n-C.sub.8-12 alkyl. In some embodiments,
each R.sup.L independently is C.sub.9-11 alkyl. In some
embodiments, each R.sup.L independently is n-C.sub.9-11 alkyl. In
some embodiments, each R.sup.L independently is C.sub.10 alkyl. In
some embodiments, each R.sup.L is n-C.sub.10 alkyl.
[0169] In particular embodiments, provided liposomes include a
cationic lipid cKK-E12, or
(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).
Structure of cKK-E12 is shown below:
##STR00007##
[0170] In some embodiments, suitable lipid nanoparticles of the
invention comprise at least one of the following cationic lipids:
C12-200, DLin-KC2-DMA, cKK-E12, Re-1, DODMA, DODAP, HGT4003, ICE,
XTC, DSPC, MC3, HGT5000, or HGT5001.
[0171] In some embodiments, the percentage of cationic lipid in a
liposome may be greater than about 10%, greater than about 20%,
greater than about 30%, greater than about 40%, greater than about
50%, greater than about 60%, or greater than about 70% by molar
ratio. In some embodiments, cationic lipid(s) constitute(s) about
30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about
35-45%, or about 35-40%) of the liposome by molar ratio. In some
embodiments, the cationic lipid constitutes about 30%, about 35%,
about 40%, about 45%, or about 50% of the liposome by molar
ratio.
[0172] Non-Cationic/Helper Lipids
[0173] In some embodiments, provided liposomes contain one or more
non-cationic ("helper") lipids. As used herein, the phrase
"non-cationic lipid" refers to any neutral, zwitterionic or anionic
lipid. As used herein, the phrase "anionic lipid" refers to any of
a number of lipid species that carry a net negative charge at a
selected H, such as physiological pH. In some embodiments, a
non-cationic lipid is a neutral lipid, i.e., a lipid that does not
carry a net charge in the conditions under which the composition is
formulated and/or administered. Non-cationic lipids include, but
are not limited to, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture
thereof.
[0174] In some embodiments, suitable non-cationic ("helper") lipids
include one or more phosphatidyl lipids, for example, the
phosphatidyl compounds (e.g., phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine and
phosphatidylethanolamine).
[0175] In some embodiments, suitable non-cationic ("helper") lipids
include one or more Sphingolipids, for example, sphigosine,
ceramide, sphingomyelin, cerebroside and ganglioside.
[0176] In some embodiments, non-cationic ("helper") lipids may
constitute about 5% to about 90% (e.g., about 10-80%, 10-70%,
10-60%, 10-50%, 10-40%, 10-30%, or 10-20%) of the total lipid
present in a liposome by molar ratio. In some embodiments, the
percentage of non-cationic ("helper") lipids in a liposome may be
greater than about 5%, greater than about 10%, greater than about
15%, greater than 20%, greater than about 25%, greater than 30%,
greater than about 35%, or greater than 40% by molar ratio.
[0177] Cholesterol-Based Lipids
[0178] In some embodiments, provided liposomes comprise one or more
cholesterol-based lipids. For example, suitable cholesterol-based
cationic lipids include, for example, cholesterol, PEGylated
cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol),
1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem.
Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23,
139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments,
cholesterol-based lipids may constitute about 2% to about 30%, or
about 5% to about 20% of the total lipid present in a liposome by
molar ratio. In some embodiments, The percentage of
cholesterol-based lipid in the lipid nanoparticle may be greater
than 5, %, 10%, greater than 20%, greater than 30%, or greater than
40% by molar ratio.
[0179] PEGylated Lipids
[0180] In some embodiments, provided lipid nanoparticles comprise
one or more PEGylated lipids. For example, the use of polyethylene
glycol (PEG)-modified phospholipids and derivatized lipids such as
derivatized ceramides (PEG-CER), including
N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene
Glycol)-2000] (C8 PEG-2000 ceramide) is contemplated by the present
invention in combination with one or more of the cationic and, in
some embodiments, other lipids. In some embodiments, suitable
PEGylated lipids comprise PEG-ceramides having shorter acyl chains
(e.g., C.sub.14 or C.sub.18). In some embodiments, the PEGylated
lipid DSPE-PEG-Maleimide-Lectin may be used. Other contemplated
PEG-modified lipids include, but are not limited to, a polyethylene
glycol chain of up to 5 kDa in length covalently attached to a
lipid with alkyl chain(s) of C.sub.6-C.sub.20 length. Without
wishing to be bound by a particular theory, it is contemplated that
the addition of PEGylated lipids may prevent complex aggregation
and increase circulation lifetime to facilitate the delivery of the
lipsome encapsulated mRNA to the target cell.
[0181] In some embodiments, PEG-modified phospholipids and/or
derivitized lipids may constitute from about 0% to about 20%, about
0% to about 15%, about 0% to about 10%, about 1% to about 10%,
about 1% to about 8%, 1% to about 6%, 1% to about 5%, about 2% to
about 10%, about 4% to about 10%, of the total lipids present in
the liposome by molar ratio. In some embodiments, the percentage of
PEG-modified phospholipids and/or derivitized lipids may be of or
less than about 20%, about 15%, about 10%, about 9%, about 8%,
about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or
about 1% of the total lipids present in the liposome by molar
ratio. In some embodiments, the percentage of PEG-modified
phospholipids and/or derivitized lipids may be of or greater than
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 15%, or about 20% of the
total lipids present in the liposome by molar ratio.
[0182] Polymers
[0183] In some embodiments, a suitable delivery vehicle is
formulated using a polymer as a carrier, alone or in combination
with other carriers including various lipids described herein.
Thus, in some embodiments, liposomal delivery vehicles, as used
herein, also encompass polymer containing nanoparticles. Suitable
polymers may include, for example, polyacrylates,
polyalkycyanoacrylates, polylactide, polylactide-polyglycolide
copolymers, polycaprolactones, dextran, albumin, gelatin, alginate,
collagen, chitosan, cyclodextrins, protamine, PEGylated protamine,
PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present,
it may be linear or branched PEI of a molecular weight ranging from
10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).
[0184] In various embodiments, a suitable delivery vehicle (e.g., a
lipid nanoparticle) is prepared by combining one or more lipids
and/or polymer components described herein. For example, a lipid
nanoparticle may be prepared by combining C12-200, sphingomyelin,
DOPE, Cholesterol, and DMG PEG; or C12-200, DOPE, cholesterol and
DMG-PEG2K; or cKK-E12, DOPE, cholesterol and DMG-PEG2K; or cKK-E12,
sphingomyelin, DOPE, cholesterol and DMG-PEG2K; or HGT5001, DOPE,
cholesterol and DMG-PEG2K; or HGT4003, DOPE, cholesterol and
DMG-PEG2K; or DLinKC2DMA, DOPE, cholesterol and DMG-PEG2K; or ICE,
DOPE, cholesterol and DMG-PEG2K; or DODMA, DOPE, cholesterol and
DMG-PEG2K; or DODMA, sphingomyelin, DOPE, cholesterol and
DMG-PEG2K; or Re-1, DOPE, cholesterol, DMG-PEG2K; or cKK-EE12,
DOPE, cholesterol, DMG-PEG2K and/or DSPE-PEG-Maleimide-Lectin.
[0185] In various embodiments, the cationic lipids, non-cationic
lipids, cholesterol and/or PEG-modified lipids can be combined at
various relative molar ratios. For example, the ratio of cationic
lipid (e.g., cKK-E12, C12-200, etc.) to non-cationic lipid (e.g.,
DOPE, sphingomyelin, etc.) to cholesterol-based lipid (e.g.,
cholesterol) to PEGylated lipid (e.g., DMG-PEG2K) may be between
about 30-60:25-35:20-30:1-15, respectively. In some embodiments,
the ratio of cationic lipid (e.g., cKK-E12, C12-200, etc.) to
non-cationic lipid (e.g., DOPE, sphingomyelin, etc.) to
cholesterol-based lipid (e.g., cholesterol) to PEGylated lipid
(e.g., DMG-PEG2K) is approximately 40:30:20:10, respectively. In
some embodiments, the ratio of cationic lipid (e.g., cKK-E12,
C12-200, etc.) to non-cationic lipid (e.g., DOPE, sphingomyelin,
etc.) to cholesterol-based lipid (e.g., cholesterol) to PEGylated
lipid (e.g., DMG-PEG2K) is approximately 40:30:25:5, respectively.
In some embodiments, the ratio of cationic lipid (e.g., cKK-E12,
C12-200, etc.) to non-cationic lipid (e.g., DOPE, sphingomyelin,
etc.) to cholesterol-based lipid (e.g., cholesterol) to PEGylated
lipid (e.g., DMG-PEG2K) is approximately 40:32:25:3, respectively.
In some embodiments, the ratio of cationic lipid (e.g., cKK-E12,
C12-200, etc.) to non-cationic lipid (e.g., DOPE, sphingomyelin,
etc.) to cholesterol-based lipid (e.g., cholesterol) to PEGylated
lipid (e.g., DMG-PEG2K) is approximately 50:25:20:5.
Lipid Nanoparticle Preparation
[0186] Delivery vehicles, such as lipid nanoparticles, for use in
the present invention can be prepared by various techniques which
are presently known in the art. Multilamellar vesicles (MLV) may be
prepared conventional techniques, for example, by depositing a
selected lipid on the inside wall of a suitable container or vessel
by dissolving the lipid in an appropriate solvent, and then
evaporating the solvent to leave a thin film on the inside of the
vessel or by spray drying. An aqueous phase may then added to the
vessel with a vortexing motion which results in the formation of
MLVs. Uni-lamellar vesicles (ULV) can then be formed by
homogenization, sonication or extrusion of the multi-lamellar
vesicles. In addition, unilamellar vesicles can be formed by
detergent removal techniques.
[0187] In certain embodiments of this invention, the compositions
of the present invention comprise a transfer vehicle wherein the
mRNA is associated on both the surface of the transfer vehicle and
encapsulated within the same transfer vehicle. For example, during
preparation of the compositions of the present invention, cationic
liposomal transfer vehicles may associate with the mRNA through
electrostatic interactions.
[0188] Bilayer membranes of the liposomes can also be formed by
amphophilic polymers and surfactants (e.g., polymerosomes,
niosomes, etc.). The process of incorporation of a desired mRNA
into a liposome is often referred to as "loading". Exemplary
methods are described in Lasic, et al., FEBS Lett., 312: 255-258,
1992, which is incorporated herein by reference. The
liposome-incorporated nucleic acids may be completely or partially
located in the interior space of the liposome, within the bilayer
membrane of the liposome, or associated with the exterior surface
of the liposome membrane. The incorporation of a nucleic acid into
liposomes is also referred to herein as "encapsulation" wherein the
nucleic acid is entirely contained within the interior space of the
liposome. The purpose of incorporating a mRNA into a transfer
vehicle, such as a liposome, is often to protect the nucleic acid
from an environment which may contain enzymes or chemicals that
degrade nucleic acids and/or systems or receptors that cause the
rapid excretion of the nucleic acids. Accordingly, in some
embodiments, a suitable delivery vehicle is capable of enhancing
the stability of the mRNA contained therein and/or facilitate the
delivery of mRNA to the target CNS cell or tissue.
[0189] Suitable liposomal delivery vehicles according to the
present invention may be made in various sizes. In some
embodiments, the size of a liposome is determined by the length of
the largest diameter of the lipososme particle. In some
embodiments, a suitable liposomal delivery vehicle has a size no
greater than about 250 nm (e.g., no greater than about 225 nm, 200
nm, 175 nm, 150 nm, 125 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50
nm, or 40 nm). In some embodiments, a suitable liposomal delivery
vehicle has a size ranging from about 40-100 nm (e.g., ranging from
about 40-90 nm, about 40-80 nm, about 40-70 nm, about 40-60 nm,
about 40-50 nm, about 50-100 nm, about 50-90 nm, about 50-80 nm,
about 50-70 nm, about 50-60 nm, about 60-100 nm, about 60-90 nm,
about 60-80 nm, about 60-70 nm, about 70-100 nm, about 70-90 nm,
about 70-80 nm, about 80-100 nm, about 80-90 nm, or about 90-100
nm).
[0190] A variety of methods known in the art are available for
sizing of a population of liposomal transfer vehicles. One such
sizing method is described in U.S. Pat. No. 4,737,323, incorporated
herein by reference. Sonicating a liposome suspension either by
bath or probe sonication can produces a progressive size reduction
down to desired small ULV. Homogenization is another method that
relies on shearing energy to fragment large liposomes into smaller
ones. In a typical homogenization procedure, MLV are recirculated
through a standard emulsion homogenizer until selected liposome
sizes. The size of the liposomal vesicles may be determined by
quasi-electric light scattering (QELS) as described in Bloomfield,
Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein
by reference. Average liposome diameter may be reduced by
sonication of formed liposomes. Intermittent sonication cycles may
be alternated with QELS assessment to guide efficient liposome
synthesis.
CNS Delivery
[0191] mRNAs or mRNA containing delivery vehicles (e.g., mRNA
loaded lipid nanoparticles) as described herein, are suitable for
CNS delivery. In some embodiments, mRNA loaded lipid nanoparticles
can be delivered to the CNS via various techniques and routes
including, but not limited to, intraparenchymal, intracerebral,
intravetricular cerebral (ICV), intrathecal (e.g., IT-Lumbar,
IT-cisterna magna) administrations and any other techniques and
routes for injection directly or indirectly to the CNS and/or
CSF.
[0192] Intrathecal Delivery
[0193] In some embodiments, mRNA loaded lipid nanoparticles are
delivered to the CNS by injecting into the cerebrospinal fluid
(CSF) of a subject in need of treatment. In some embodiments,
intrathecal administration is used for injecting mRNA or mRNA
loaded nanoparticles to the CSF. As used herein, intrathecal
administration (also referred to as intrathecal injection) refers
to an injection into the spinal canal (intrathecal space
surrounding the spinal cord). Various techniques may be used
including, without limitation, lateral cerebroventricular injection
through a burrhole or cistemal or lumbar puncture or the like.
Exemplary methods are described in Lazorthes et al. Advances in
Drug Delivery Systems and Applications in Neurosurgery, 143-192 and
Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of
which are incorporated herein by reference.
[0194] According to the present invention, mRNA or mRNA loaded
nanoparticles may be injected at any region surrounding the spinal
canal. In some embodiments, mRNA or mRNA loaded nanoparticles are
injected into the lumbar area or the cisterna magna or
intraventricularly into a cerebral ventricle space. As used herein,
the term "lumbar region" or "lumbar area" refers to the area
between the third and fourth lumbar (lower back) vertebrae and,
more inclusively, the L2-S1 region of the spine. Typically,
intrathecal injection via the lumbar region or lumber area is also
referred to as "lumbar IT delivery" or "lumbar IT administration."
The term "cisterna magna" refers to the space around and below the
cerebellum via the opening between the skull and the top of the
spine. Typically, intrathecal injection via cisterna magna is also
referred to as "cisterna magna delivery." The term "cerebral
ventricle" refers to the cavities in the brain that are continuous
with the central canal of the spinal cord. Typically, injections
via the cerebral ventricle cavities are referred to as
intravetricular Cerebral (ICV) delivery.
[0195] In some embodiments, "intrathecal administration" or
"intrathecal delivery" according to the present invention refers to
lumbar IT administration or delivery, for example, delivered
between the third and fourth lumbar (lower back) vertebrae and,
more inclusively, the L2-S1 region of the spine.
[0196] In some embodiments, intrathecal administration may be
performed by either lumbar puncture (i.e., slow bolus) or via a
port-catheter delivery system (i.e., infusion or bolus). In some
embodiments, the catheter is inserted between the laminae of the
lumbar vertebrae and the tip is threaded up the thecal space to the
desired level (generally L3-L4).
[0197] Administration
[0198] The present invention contemplate single as well as multiple
administrations of a therapeutically effective amount of mRNA or
mRNA loaded nanoparticles described herein. mRNA or mRNA loaded
nanoparticles can be administered at regular intervals, depending
on the nature, severity and extent of the subject's CNS disease or
condition. In some embodiments, a therapeutically effective amount
of mRNA or mRNA loaded nanoparticles may be administered
intrathecally periodically at regular intervals (e.g., once every
year, once every six months, once every five months, once every
three months, bimonthly (once every two months), monthly (once
every month), biweekly (once every two weeks), weekly, daily or
continuously).
[0199] In some embodiments, the CNS disease is associated with
peripheral symptoms. Thus, in some embodiments, intrathecal
administration may be used in conjunction with other routes of
administration (e.g., intravenous, subcutaneously, intramuscularly,
parenterally, transdermally, or transmucosally (e.g., orally or
nasally)).
[0200] As used herein, the term "therapeutically effective amount"
is largely determined base on the total amount of mRNA contained in
the pharmaceutical compositions of the present invention.
Generally, a therapeutically effective amount is sufficient to
achieve a meaningful benefit to the subject (e.g., treating,
modulating, curing, preventing and/or ameliorating the underlying
disease or condition). For example, a therapeutically effective
amount may be an amount sufficient to achieve a desired therapeutic
and/or prophylactic effect. Generally, the amount of mRNA
administered to a subject in need thereof will depend upon the
characteristics of the subject. Such characteristics include the
condition, disease severity, general health, age, sex and body
weight of the subject. One of ordinary skill in the art will be
readily able to determine appropriate dosages depending on these
and other related factors. In addition, both objective and
subjective assays may optionally be employed to identify optimal
dosage ranges.
[0201] In some embodiments, a therapeutically effective dose ranges
from about 0.001 mg/kg body weight to 10 mg/kg body weight, from
about 0.005 mg/kg body weight to 10 mg/kg body weight, from about
0.01 mg/kg body weight to 10 mg/kg body weight, from about 0.01
mg/kg body weight to 9 mg/kg body weight, from about 0.01 mg/kg
body weight to 8 mg/kg body weight, from about 0.01 mg/kg body
weight to 7 mg/kg body weight, from about 0.01 mg/kg body weight to
6 mg/kg body weight, from about 0.01 mg/kg body weight to 5 mg/kg
body weight, from about 0.01 mg/kg body weight to 4 mg/kg body
weight, from about 0.01 mg/kg body weight to 3 mg/kg body weight,
from about 0.01 mg/kg body weight to 2 mg/kg body weight, from
about 0.01 mg/kg body weight to 1 mg/kg body weight, from about
0.01 mg/kg body weight to 0.5 mg/kg body weight, from about 0.1
mg/kg body weight to 10 mg/kg body weight, from about 0.1 mg/kg
body weight to 5 mg/kg body weight, from about 0.5 mg/kg body
weight to 10 mg/kg body weight, or from about 0.5 mg/kg body weight
to 5 mg/kg body weight.
[0202] In some embodiments, a therapeutically effective dose ranges
from about 0.001 mg/kg brain weight to 100 mg/kg brain weight, from
about 0.001 mg/kg brain weight to 90 mg/kg brain weight, from about
0.001 mg/kg brain weight to 80 mg/kg brain weight, from about 0.001
mg/kg brain weight to 70 mg/kg brain weight, from about 0.001 mg/kg
brain weight to 60 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 50 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 40 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 30 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 20 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 10 mg/kg brain weight, from about 0.001 mg/kg brain
weight to 5 mg/kg brain weight, from about 0.001 mg/kg brain weight
to 1 mg/kg brain weight, from about 0.01 mg/kg brain weight to 100
mg/kg brain weight, from about 0.05 mg/kg brain weight to 100 mg/kg
brain weight, from about 0.1 mg/kg brain weight to 100 mg/kg brain
weight, or from about 0.5 mg/kg brain weight to 100 mg/kg brain
weight.
[0203] As one skilled in the art would appreciate, the brain
weights and body weights can be correlated. Dekaban A S. "Changes
in brain weights during the span of human life: relation of brain
weights to body heights and body weights," Ann Neurol 1978;
4:345-56. Thus, in some embodiments, the dosages can be converted
as shown in Table 5.
TABLE-US-00007 TABLE 5 Correlation between Brain Weights, body
weights and ages of males Age (year) Brain weight (kg) Body weight
(kg) 3 (31-43 months) 1.27 15.55 4-5 1.30 19.46
[0204] Delivery to Neurons and Other Cell Types in the Brain and/or
Spinal Cord
[0205] Inventive methods according to the present invention result
in delivery of mRNA in various neurons and other cell types in the
brain and/or spinal cord. In some embodiments, mRNA encoding a
therapeutic protein is delivered to various cells in the brain
including, but not limited to, neurons, glial cells, perivascular
cells and/or meningeal cells. In particular, inventive methods
according to the present invention result in delivery of mRNA in
various neurons and other cell types affected by a CNS disease
and/or deficiency, or various neurons and other cell types in which
the deficient protein associated with the CNS disease is normally
expressed. In some embodiments, inventive methods according to the
present invention result in delivery of mRNA in various neurons and
other cell types in the CNS in which there is a detectable or
abnormally high amount of enzyme substrate, for example stored in
the cellular lysosomes of the tissue, in patients suffering from or
susceptible to the lysosomal storage disease. In some embodiments,
inventive methods according to the present invention result in
delivery of mRNA in various neurons and other cell types that
display disease-associated pathology, symptom, or feature. For
example, mRNA may be delivered to neurons or other cell types that
are deteriorating, degenerating or undergoing apoptosis such as
those neurons or non-neuronal cells associated with
neurodenegrative diseases (e.g., Alzheimer's disease, Parkinson's
disease, and Huntington's disease) or motor neurons associated with
motor neuron diseases (e.g., Amyotrophic Lateral Sclerosis (ALS),
Primary Lateral Sclerosis (PLS), Pseudobulbar Pasly, Hereditary
Spastic Paraplegia, Progressive Muscular Atrophy (PMA), Progressive
Bulbar Palsy (PBP), Distal Hereditary Motor Neuropathies, and
Spinal Muscular Atrophies).
[0206] In some embodiments, mRNA is delivered to neurons and/or
non-neuronal cells located within the brain. In some embodiments,
mRNA is delivered to neurons and/or non-neuronal cells located
within the spinal cord. In some embodiments, mRNA is delivered to
motor neurons. In some embodiments, the mRNA is delivered to upper
motor neurons and/or lower motor neurons. In some embodiments, the
motor neurons are located within the anterior horn and/or dorsal
root ganglia of the spinal cord.
[0207] In some embodiments, mRNA is delivered intracellularly in
various neurons and other cell types in the brain and/or spinal
cord. In some embodiments, mRNA is delivered to the axons of
neurons. In some embodiments, mRNA delivery according to the
present invention results in intracellular expression of the
protein encoded by the mRNA within cytosol of the neurons. In some
embodiments, mRNA delivery according to the present invention
results in expression of the protein encoded by the mRNA in
subcellular compartment of the neurons, e.g., lysosomes,
mitochondria, transmembrane, and the like. In some embodiments,
mRNA delivery according to the present invention results in
expression of the protein encoded by the mRNA and secretion
extracellularly from the neurons.
[0208] Additional exemplary neurons and other cell types in the
brain and/or spinal cord are described below.
[0209] Brain
[0210] In general, inventive methods according to the present
invention can be used to deliver mRNA and encoded protein to
neurons and other cell types in various regions of the brain.
Typically, brain can be divided into different regions, layers and
tissues. For example, meningeal tissue is a system of membranes
which envelops the central nervous system, including the brain. The
meninges contain three layers, including dura mater, arachnoid
mater, and pia mater. In general, the primary function of the
meninges and of the cerebrospinal fluid is to protect the central
nervous system. In some embodiments, mRNA and the encoded protein
is delivered to neurons or non-neuronal cells in one or more layers
of the meninges.
[0211] The brain has three primary subdivisions, including the
cerebrum, cerebellum, and brain stem. The cerebral hemispheres,
which are situated above most other brain structures, are covered
with a cortical layer. Underneath the cerebrum lies the brainstem,
which resembles a stalk on which the cerebrum is attached. At the
rear of the brain, beneath the cerebrum and behind the brainstem,
is the cerebellum.
[0212] The diencephalon, which is located near the midline of the
brain and above the mesencephalon, contains the thalamus,
metathalamus, hypothalamus, epithalamus, prethalamus, and
pretectum. The mesencephalon, also called the midbrain, contains
the tectum, tegumentum, ventricular mesocoelia, and cerebral
peduncels, the red nucleus, and the cranial nerve III nucleus. The
mesencephalon is associated with vision, hearing, motor control,
sleep/wake, alertness, and temperature regulation.
[0213] In some embodiments, mRNA and the encoded protein is
delivered to neurons and/or non-neuronal cells of one or more
tissues of the cerebellum. In certain embodiments, the targeted one
or more tissues of the cerebellum are selected from the group
consisting of tissues of the molecular layer, tissues of the
Purkinje cell layer, tissues of the Granular cell layer, cerebellar
peduncles, and combination thereof. In some embodiments, mRNA and
the encoded protein is delivered to one or more deep tissues of the
cerebellum including, but not limited to, tissues of the Purkinje
cell layer, tissues of the Granular cell layer, deep cerebellar
white matter tissue (e.g., deep relative to the Granular cell
layer), and deep cerebellar nuclei tissue.
[0214] In some embodiments, mRNA and the encoded protein is
delivered to one or more tissues of the brainstem.
[0215] In some embodiments, mRNA and encoded protein is delivered
to various brain tissues including, but not limited to, gray
matter, white matter, periventricular areas, pia-arachnoid,
meninges, neocortex, cerebellum, deep tissues in cerebral cortex,
molecular layer, caudate/putamen region, midbrain, deep regions of
the pons or medulla, and combinations thereof. In some embodiments,
mRNA and encoded protein is delivered to oligodendrocytes of deep
white matter.
[0216] Spinal Cord
[0217] In some embodiments, inventive methods according to the
present invention can be used to deliver mRNA and encoded protein
to neurons and other cell types in various regions of the spinal
cord. In general, regions or tissues of the spinal cord can be
characterized based on the depth of the tissues. For example,
spinal cord tissues can be characterized as surface or shallow
tissues, mid-depth tissues, and/or deep tissues.
[0218] In some embodiments, mRNA and the encoded protein is
delivered to one or more surface or shallow tissues of the spinal
cord. In some embodiments, a targeted surface or shallow tissue of
the spinal cord contains pia mater and/or the tracts of white
matter.
[0219] In some embodiments, mRNA and the encoded protein is
delivered to one or more deep tissues of the spinal cord. In some
embodiments, a targeted deep tissue of the spinal cord contains
spinal cord grey matter and/or ependymal cells.
[0220] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All literature citations are
incorporated by reference.
Examples
Example 1
Formulations and Messenger RNA Material
[0221] This example provides exemplary liposome formulations for
effective delivery and expression of mRNA in the CNS. In general,
the formulations described herein include a multi-component lipid
mixture of varying ratios employing one or more cationic lipids,
neutral lipids, cholesterol and/or PEGylated lipids designed to
encapsulate various nucleic acid-based materials.
Messenger RNA Material
[0222] Codon-optimized human Survival of Motor Neuron-1(hSMN-1)
messenger RNA was synthesized by in vitro transcription from a
plasmid DNA template encoding the gene, which was followed by the
addition of a 5' cap structure (Cap 1) (Fechter, P.; Brownlee, G.
G. "Recognition of mRNA cap structures by viral and cellular
proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A)
tail of approximately 250 nucleotides in length as determined by
gel electrophoresis. The 5' and 3' untranslated regions present in
each mRNA product are represented as X and Y, respectively and
defined as stated.
TABLE-US-00008 Survival of Motor Neuron (hSMN-1) mRNA: X-SEQ ID NO:
3-Y. 5' and 3' UTR Sequences X (5' UTR Sequence)= (SEQ ID NO: 7)
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGA
AGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGA
ACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG Y (3' UTR Sequence)=
(SEQ ID NO: 8) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGA
AGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUG CAUCAAGCU OR (SEQ
ID NO: 9) GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAA
GUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGC AUCAAAGCU
[0223] For example, the codon-optimized human Survival of Motor
Neuron-1(hSMN-1) messenger RNA comprised:
TABLE-US-00009 (SEQ ID NO: 10)
AGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGA
ACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGG
CCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAGGACA
GCGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAUAUCU
GGGACGAUACCGCUCUGAUCAAGGCCUACGACAAGGCCGUGGCCAGCU
UCAAGCACGCCCUGAAAAACGGCGACAUCUGCGAGACCAGCGGCAAGC
CCAAGACAACCCCCAAGAGAAAGCCCGCCAAGAAGAAUAAGAGCCAGA
AAAAGAACACCGCCGCCAGCCUGCAGCAGUGGAAGGUGGGCGACAAGU
GCAGCGCCAUCUGGAGCGAGGACGGCUGCAUCUACCCCGCCACCAUCG
CCAGCAUCGACUUCAAGAGAGAGACCUGCGUGGUCGUGUACACCGGCU
ACGGCAACAGAGAGGAGCAGAACCUGAGCGACCUGCUGAGCCCCAUUU
GUGAGGUGGCCAAUAACAUCGAACAGAACGCCCAGGAGAACGAGAAUG
AAAGCCAGGUGAGCACCGACGAGAGCGAGAACAGCAGAUCUCCUGGCA
ACAAGAGCGACAACAUCAAGCCUAAGUCUGCCCCUUGGAACAGCUUCC
UGCCCCCUCCUCCACCCAUGCCCGGACCCAGACUGGGACCCGGAAAAC
CUGGCCUGAAGUUCAACGGACCACCUCCCCCUCCACCUCCUCCCCCAC
CUCAUCUCCUGAGCUGCUGGCUGCCACCCUUCCCCAGCGGACCCCCUA
UCAUCCCACCACCCCCUCCCAUCUGCCCCGACAGCCUGGACGACGCCG
AUGCCCUGGGCAGCAUGCUGAUCAGCUGGUACAUGAGCGGCUACCACA
CAGGAUACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGAUGCU
CCCACUCCCUGAACUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUG
CCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUC
CUAAUAAAAUUAAGUUGCAUCAAGCU
or the codon-optimized human Survival of Motor Neuron-1(hSMN-1)
messenger RNA comprised:
TABLE-US-00010 (SEQ ID NO: 11)
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUA
GAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAU
UGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACA
CGAUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCA
GGAGGACAGCGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGAC
AGCGAUAUCUGGGACGAUACCGCUCUGAUCAAGGCCUACGACAAGG
CCGUGGCCAGCUUCAAGCACGCCCUGAAAAACGGCGACAUCUGCGA
GACCAGCGGCAAGCCCAAGACAACCCCCAAGAGAAAGCCCGCCAAG
AAGAAUAAGAGCCAGAAAAAGAACACCGCCGCCAGCCUGCAGCAGU
GGAAGGUGGGCGACAAGUGCAGCGCCAUCUGGAGCGAGGACGGCUG
CAUCUACCCCGCCACCAUCGCCAGCAUCGACUUCAAGAGAGAGACC
UGCGUGGUCGUGUACACCGGCUACGGCAACAGAGAGGAGCAGAACC
UGAGCGACCUGCUGAGCCCCAUUUGUGAGGUGGCCAAUAACAUCGA
ACAGAACGCCCAGGAGAACGAGAAUGAAAGCCAGGUGAGCACCGAC
GAGAGCGAGAACAGCAGAUCUCCUGGCAACAAGAGCGACAACAUCA
AGCCUAAGUCUGCCCCUUGGAACAGCUUCCUGCCCCCUCCUCCACC
CAUGCCCGGACCCAGACUGGGACCCGGAAAACCUGGCCUGAAGUUC
AACGGACCACCUCCCCCUCCACCUCCUCCCCCACCUCAUCUCCUGA
GCUGCUGGCUGCCACCCUUCCCCAGCGGACCCCCUAUCAUCCCACC
ACCCCCUCCCAUCUGCCCCGACAGCCUGGACGACGCCGAUGCCCUG
GGCAGCAUGCUGAUCAGCUGGUACAUGAGCGGCUACCACACAGGAU
ACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGAUGCUCCCA
CUCCCUGAACUGAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCU
CUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCC
UAAUAAAAUUAAGUUGCAUCAAAGCU.
Exemplary Formulation Protocol
[0224] Lipid nanoparticles (LNP) were formed via standard ethanol
injection methods (Ponsa, M.; Foradada, M.; Estelrich, J.
"Liposomes obtained by the ethanol injection method" Int. J. Pharm.
1993, 95, 51-56). For the various lipid components, a 50 mg/ml
ethanolic stock solutions was prepared and stored at -20.degree. C.
In preparation of each exemplary formulation listed in Table 5
below, the indicated lipid components were added to an ethanol
solution to achieve a predetermined final concentration and molar
ratio, and scaled to a 3 ml final volume of ethanol. Separately, an
aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of
hSMN-1 mRNA was prepared from a 1 mg/ml stock. The lipid solution
was injected rapidly into the aqueous mRNA solution, either
manually or via syringe pump, and shaken to yield a final
suspension in 20% ethanol. The resulting nanoparticle suspension
was filtered and dialysed against 1.times.PBS (pH 7.4),
concentrated and stored between 2-8.degree. C. SMN-1 mRNA
concentration was determined via the Ribogreen assay (Invitrogen).
Encapsulation of mRNA was calculated by performing the Ribogreen
assay with and without the presence of 0.1% Triton-X 100. Particle
sizes (dynamic light scattering (DLS)) and zeta potentials were
determined using a Malvern Zetasizer instrument in 1.times.PBS and
1 mM KCl solutions, respectively.
TABLE-US-00011 TABLE 6 Exemplary Lipid Nanoparticle Formulations
Molar Ratio Final mRNA Formulations Components of lipids
Concentration Zeta Parameters 1 C12-200 40:30:25:5 2.5 mg/ml
Z.sub.ave = 82 nm; DOPE Dv.sub.(50) = 53 nm; Cholesterol
Dv.sub.(90) = 97 nm DMG-PEG2K hSMN-1 mRNA 2 C12-200 40:15:20:20:5
1.28 mg/ml Z.sub.ave = 90 nm; Sphingomyelin Dv.sub.(50) = 75 nm;
DOPE Dv.sub.(90) = 104 nm Cholesterol DMG-PEG-2K hSMN-1 mRNA 3
DLin-KC2-DMA 40:30:25:5 2.05 mg/ml Z.sub.ave = 72 nm; DOPE
Dv.sub.(50) = 48 nm; Cholesterol Dv.sub.(90) = 85 nm DMG-PEG-2K
hSMN-1 mRNA 4 cKK-E12 40:30:25:5 1.85 mg/ml Z.sub.ave = 71 nm; DOPE
Dv.sub.(50) = 44 nm; Cholesterol Dv.sub.(90) = 93 nm DMG-PEG-2K
hSMN-1 mRNA 5 cKK-E12 40:30:25:5 1.8 mg/ml Z.sub.ave = 72 nm; DOPE
Dv.sub.(50) = 49 nm; Cholesterol Dv.sub.(90) = 90 nm DMG-PEG-5K
hSMN-1 mRNA 6 Re-1 40:30:25:5 1.8 mg/ml Z.sub.ave = 81 nm; DOPE
Dv.sub.(50) = 66 nm; Cholesterol Dv.sub.(90) = 97 nm DMG-PEG-5K
hSMN-1 mRNA 7 HGT5001 40:30:25:5 1.5 mg/ml Z.sub.ave = 82 nm; DOPE
Dv.sub.(50) = 53 nm; Cholesterol Dv.sub.(90) = 99 nm DMG-PEG-5K
hSMN-1 mRNA 8 ICE 40:30:25:5 1.96 mg/ml Z.sub.ave = 63 nm; DOPE
Dv.sub.(50) = 41 nm; Cholesterol Dv.sub.(90) = 83 nm DMG-PEG-5K
hSMN-1 mRNA 9 HGT4003 40:30:25:5 1.5 mg/ml Z.sub.ave = 82 nm; DOPE
Dv.sub.(50) = 53 nm; Cholesterol Dv.sub.(90) = 99 nm DMG-PEG-5K
hSMN-1 mRNA 10 DODMA 40:30:25:5 1.6 mg/ml Z.sub.ave = 78 nm; DOPE
Dv.sub.(50) = 49 nm; Cholesterol Dv.sub.(90) = 96 nm DMG-PEG-5K
hSMN-1 mRNA 11 cKK-EE12 40:30:25:2:3 1.4 mg/ml Z.sub.ave = 95 nm;
DOPE Dv.sub.(50) = 72 nm; Cholesterol Dv.sub.(90) = 103 nm
DMG-PEG-2K DSPE-PEG-Maleimide-Lectin hSMN-1 mRNA 12 C12-200
40:30:25:5 1.2 mg/ml Z.sub.ave = 74 nm; DOPE Dv.sub.(50) = 50 nm;
Cholesterol Dv.sub.(90) = 93 nm DOG-PEG-2K hSMN-1 mRNA 13 cKK-EE12
40:15:20:20:5 1.6 mg/ml Z.sub.ave = 74 nm; Sphingomyelin
Dv.sub.(50) = 41 nm; DOPE Dv.sub.(90) = 90 nm Cholesterol
DMG-PEG-2K hSMN-1 mRNA
Example 2
Intrathecal Administration of mRNA Loaded Liposome
Nanoparticles
[0225] This example illustrates exemplary methods of administering
intrathecally mRNA-loaded liposome nanoparticles and methods for
analyzing delivered mRNA in neurons.
[0226] All studies were performed with either rats or mice of
approximately 6-8 weeks of age at the beginning of each experiment.
At the start of the experiment, each animal was anesthetized with
isoflurane (1-3%, to effect) by inhalation. Once anesthetized, each
animal was shaved at the exact injection site (L4-L5 or L5-L6).
Following insertion of the needle, reflexive flick of the tail was
used to indicate puncture of the dura and confirm intrathecal
placement. Each animal received a single bolus intrathecal
injection of one of the test formulations listed in Table 6. All
animals were sacrificed 24 hours post injection and perfused with
saline.
Isolation of Organ Tissues for Analysis
[0227] All animals had the whole brain and spinal cord harvested.
The brain was cut longitudinally and placed in one histology
cassette per animal. The whole spinal cord was stored ambient in a
15 ml tube containing 10% neutral buffered formalin (NBF) for at
least 24 hours and no more than 72 hours before transfer into 70%
histology grade alcohol solution. Each spinal cord sample was cut
into cervical, thoracic and lumbar sections. Each spinal cord
section cut in half and both halves were placed in individual
cassettes per section (cervical, thoracic and lumbar) for
processing. All three cassettes were embedded into one paraffin
block per animal. When applicable, portions of brain and spinal
cord were snap frozen and stored at -80.degree. C.
hSMN-1 Western Blot Analysis
[0228] Standard western blot procedures were followed employing
various antibodies that recognizes hSMN protein, such as: (A)
anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309
a-SMN antibody at 1:1,000 dilution; and (C) LSBio C138149 a-SMN
antibody at 1:1,000 dilution. For each experiment one microgram of
hSMN mRNA was transfected into 1.times.10.sup.6 BHK-21 cells using
Lipofectamine 2000. Cells were treated with OptiMem and harvested
16-18 hours post-transfection. Cell lysates were harvested,
processed and loaded on to an 8-16% Tris Glycine gel. The gel was
transferred using a PVDF membrane and treated with the respective
primary antibody. Goat anti-mouse HRP antibody was used as the
secondary antibody at 1:10,000 dilution for 45 minutes at room
temperature followed by washing and development. The data
demonstrates that each antibody tested showed a strong signal for
hSMN-1 and was specific for human SMN, as indicated by an absence
in a cross-reactive signal for untreated BHK cells (FIG. 1).
In Situ Hybridzation (ISH) Analysis
[0229] Tissue from each representative sample, was assayed for
hSMN-1 mRNA using two different in situ hybridization methods. For
the first approach, manual in situ hybridization analysis was
performed using RNAscope.RTM. (Advanced Cell Diagnostic) "ZZ" probe
technology. Probes were generated based on the codon-optimized
sequence of human SMN messenger RNA (SEQ ID NO:3). Briefly, the
RNAscope.RTM. assay is an in situ hybridisation assay designed to
visualize single RNA molecules per cell in formalin-fixed,
paraffin-embedded (FFPE) tissue mounted on slides. Each embedded
tissue sample was pretreated according to the manufacturers
protocol and incubated with a target specific hSMN-1 RNA probe. The
hSMN-1 probe was shown to be specific for human SMN-1 and had
little to no cross reactivity with mouse or rat SMN-1. Once bound,
the hSMN-1 probe is hybridized to a cascade of signal amplification
molecules, through a series of 6 consecutive rounds of
amplification. The sample was then treated with an HRP-labeled
probe specific to the signal amplification cassette and assayed by
chromatic visualization using 3,3'-diaminobenzidine (DAB). A probe
specific for Ubiquitin C was used as the positive control. Positive
SMN signal was compared to that of untreated and vehicle control
treated rat or mouse tissue. Stained samples were visualized under
a standard bright field microscope.
[0230] For the second approach, a fully automated in situ
hybridization analysis was performed using the Leica Bond Rx
detection system. Probes were generated based on the
codon-optimized sequence of human SMN messenger RNA (SEQ ID NO:3).
Briefly, eash embedded tissue sample was pretreated according to
the manufacturers protocol and incubated with a target specific
HRP-labeled hSMN-1 RNA probe. A Ubiquitin C probe was used as the
positive control (FIG. 18) and a DapB probe was used as the
negative probe control (FIG. 17). Hybridized was assayed using
Fast-Red, a chromatic substrate for alkaline phosphatase.
Immunohistochemical Analysis
[0231] Human SMN-1 mRNA-loaded lipid nanoparticles were
administered to rats via intrathecal injection, and tissue samples
collected and processed 24 hours post administration in accordance
with the methods described above. Rat spinal tissue samples were
then assayed for hSMN-1 protein expression. Briefly, fixed tissue
embedded in paraffin was processed and placed on slides. The slides
were dewaxed, rehydrated and antigen retrieval was performed using
a pressure cooker with citrate buffer. Several blocking buffers
were employed followed by primary antibody incubation overnight at
4.degree. C., using the 4F11 antibody at a 1:2500 dilution. The
resulting slides were washed and incubated at ambient temperature
with the secondary antibody polymer followed by washing and
subsequent chromagen development. The data demonstrates that in as
little as 24 hours post intrathecal administration of hSMN-1 mRNA,
staining is observed for human SMN-1 protein when compared to
no-treatment control (FIG. 22). This supports the previous findings
which demonstrate delivery of hSMN-1 mRNA to the spinal tissue.
Furthermore, the data demonstrates that once delivered to the cell
hSMN-1 mRNA is effectively expressed to generate hSMN-1
protein.
Example 3
Effective Intracellular Delivery of mRNA in Neurons
[0232] The data presented in this example demonstrates that
intrathecal administration of hSMN-1 mRNA loaded liposomes (e.g.,
lipid or polymer-based nanoparticles) results in successful
intracellular delivery of mRNA in neurons in the brain and spinal
cord, including those difficult to treat cells, such as anterior
horn cells and dorsal root ganglia.
[0233] The results have shown that mRNA encapsulated within a lipid
nanoparticle can be effectively delivered to various tissues of the
CNS following interthecal administrations. Using the thirteen
different formulations disclosed in Table 6, mRNA was effectively
delievered and internalized within various neurons of the spinal
cord (FIGS. 2A-14C), as verified by two independent in situ
hybridization assays. Surprisingly, intracellular mRNA delivery was
demonstrated in the difficult to reach neuronal cells of the
anterior horn, located deep within the tissues of the spinal
column, were it was expressed as protein (FIGS. 19-22). Little to
no background was observed with mouse or rat SMN-1, indicating
specificity for the human SMN-1 probe (FIGS. 15-17). Positive SMN
signal was compared to that of untreated and vehicle control
treated rat or mouse tissue. Stained samples were visualized under
a standard bright field microscope.
[0234] These data demonstrates that the lipid or polymer
nanoparticle based mRNA delivery approach described herein were
able to successfully permeate the complex and dense cell membrane
of the spinal cord neurons and deliver the mRNA payload for the
production of encoded proteins inside neurons. It was particularly
surprising that the mRNA delivery approach described herein was
equally successful in permeate those difficult to treat neurons
such as anterior horn cell and dorsal root ganglia. Thus, the data
presented herein demonstrates that lipid or polymer nanoparticles
based mRNA delivery approach is a promising option for treating a
CNS disease. In particular, the present invention demonstrates that
hSMN mRNA loaded nanoparticles can be effectively delivered to
neurons including those difficult to treat motor neurons in the
spinal cord for the production of SMN protein and treatment of
spinal muscular atrophy.
Example 4
Effective Intracellular Delivery of mRNA in Brain White and Grey
Matter
[0235] The data presented in this example demonstrate that
intrathecal administration of hSMN-1 mRNA loaded liposomes (e.g.,
lipid or polymer-based nanoparticles) results in successful
intracellular delivery of mRNA in neurons in the brain, including
difficult to treat tissues located deep within the brain, such a
white matter.
[0236] The study was performed with rats of approximately 6-8 weeks
of age at the beginning of each experiment, using the methods and
techniques described above. Briefly, at the start of the
experiment, each animal was anesthetized with isoflurane (1-3%, to
effect) by inhalation. Once anesthetized, each animal was shaved at
the exact injection site (L4-L5 or L5-L6). Following insertion of
the needle, reflexive flick of the tail was used to indicate
puncture of the dura and confirm intrathecal placement. Each animal
received a single bolus intrathecal injection of one of the test
formulations listed in Table 6. All animals were sacrificed 30
minutes of 24 hours post injection and perfused with saline. The
data presented in Example 4, demonstrate the results of mRNA
delivery using formulation 13 of Table 6 above.
In Situ Hybridzation (ISH) Analysis
[0237] Human SMN-1 mRNA-loaded lipid nanoparticles were
administered to rats via intrathecal injection, and tissue samples
collected 30 min. and 24 hours post administration, processed and
assayed for hSMN-1 mRNA using RNAscope.RTM. (Advanced Cell
Diagnostic) "ZZ" probe technology, as described above. Each
embedded tissue sample was pretreated according to the
manufacturers protocol and incubated with a target specific hSMN-1
RNA probe. The data demonstrates that in as little as 30 minutes
post intrathecal administration of hSMN-1 mRNA, staining is
observed for human SMN-1 mRNA throughout the tissue of the brain,
compared to no-treatment control (FIG. 23A). This supports the
previous findings and highlights the speed and effectiveness of the
mRNA delivery method, which results in mRNA delivery in as little
as 30 minutes post IT delivery. Furthermore, the data clearly
demonstrates the surprising and unexpected discovery that mRNA
delivery in accordance with the invention, resusts in effective
mRNA delivery to both grey matter tissue (located at the external
periphery of the brain) and white matter tissue (located deep
within the brain). Thus suggesting that the current approach can
serve as an viable therapy in treating neurological or
neuromuscular diseases, which manifest as a result of dysregution
of cells located deep within the hard to reach white matter tissue
of the brain.
EQUIVALENTS
[0238] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims:
Sequence CWU 1
1
1511511RNAHomo sapiens 1ggggacccgc ggguuugcua uggcgaugag cagcggcggc
agugguggcg gcgucccgga 60gcaggaggau uccgugcugu uccggcgcgg cacaggccag
agcgaugauu cugacauuug 120ggaugauaca gcacugauaa aagcauauga
uaaagcugug gcuucauuua agcaugcucu 180aaagaauggu gacauuugug
aaacuucggg uaaaccaaaa accacaccua aaagaaaacc 240ugcuaagaag
aauaaaagcc aaaagaagaa uacugcagcu uccuuacaac aguggaaagu
300uggggacaaa uguucugcca uuuggucaga agacgguugc auuuacccag
cuaccauugc 360uucaauugau uuuaagagag aaaccugugu ugugguuuac
acuggauaug gaaauagaga 420ggagcaaaau cuguccgauc uacuuucccc
aaucugugaa guagcuaaua auauagaaca 480aaaugcucaa gagaaugaaa
augaaagcca aguuucaaca gaugaaagug agaacuccag 540gucuccugga
aauaaaucag auaacaucaa gcccaaaucu gcuccaugga acucuuuucu
600cccuccacca ccccccaugc cagggccaag acugggacca ggaaagccag
gucuaaaauu 660caauggccca ccaccgccac cgccaccacc accaccccac
uuacuaucau gcuggcugcc 720uccauuuccu ucuggaccac caauaauucc
cccaccaccu cccauauguc cagauucucu 780ugaugaugcu gaugcuuugg
gaaguauguu aauuucaugg uacaugagug gcuaucauac 840uggcuauuau
auggguuuca gacaaaauca aaaagaagga aggugcucac auuccuuaaa
900uuaaggagaa augcuggcau agagcagcac uaaaugacac cacuaaagaa
acgaucagac 960agaucuggaa ugugaagcgu uauagaagau aacuggccuc
auuucuucaa aauaucaagu 1020guugggaaag aaaaaaggaa guggaauggg
uaacucuucu ugauuaaaag uuauguaaua 1080accaaaugca augugaaaua
uuuuacugga cucuauuuug aaaaaccauc uguaaaagac 1140uggggugggg
gugggaggcc agcacggugg ugaggcaguu gagaaaauuu gaauguggau
1200uagauuuuga augauauugg auaauuauug guaauuuuua ugagcuguga
gaaggguguu 1260guaguuuaua aaagacuguc uuaauuugca uacuuaagca
uuuaggaaug aaguguuaga 1320gugucuuaaa auguuucaaa ugguuuaaca
aaauguaugu gaggcguaug uggcaaaaug 1380uuacagaauc uaacuggugg
acauggcugu ucauuguacu guuuuuuucu aucuucuaua 1440uguuuaaaag
uauauaauaa aaauauuuaa uuuuuuuuua aaaaaaaaaa aaaaaaaaca
1500aaaaaaaaaa a 15112294PRTHomo sapiens 2Met Ala Met Ser Ser Gly
Gly Ser Gly Gly Gly Val Pro Glu Gln Glu 1 5 10 15 Asp Ser Val Leu
Phe Arg Arg Gly Thr Gly Gln Ser Asp Asp Ser Asp 20 25 30 Ile Trp
Asp Asp Thr Ala Leu Ile Lys Ala Tyr Asp Lys Ala Val Ala 35 40 45
Ser Phe Lys His Ala Leu Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly 50
55 60 Lys Pro Lys Thr Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys
Ser 65 70 75 80 Gln Lys Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys
Val Gly Asp 85 90 95 Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys
Ile Tyr Pro Ala Thr 100 105 110 Ile Ala Ser Ile Asp Phe Lys Arg Glu
Thr Cys Val Val Val Tyr Thr 115 120 125 Gly Tyr Gly Asn Arg Glu Glu
Gln Asn Leu Ser Asp Leu Leu Ser Pro 130 135 140 Ile Cys Glu Val Ala
Asn Asn Ile Glu Gln Asn Ala Gln Glu Asn Glu 145 150 155 160 Asn Glu
Ser Gln Val Ser Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro 165 170 175
Gly Asn Lys Ser Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser 180
185 190 Phe Leu Pro Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro
Gly 195 200 205 Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro
Pro Pro Pro 210 215 220 Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro
Phe Pro Ser Gly Pro 225 230 235 240 Pro Ile Ile Pro Pro Pro Pro Pro
Ile Cys Pro Asp Ser Leu Asp Asp 245 250 255 Ala Asp Ala Leu Gly Ser
Met Leu Ile Ser Trp Tyr Met Ser Gly Tyr 260 265 270 His Thr Gly Tyr
Tyr Met Gly Phe Arg Gln Asn Gln Lys Glu Gly Arg 275 280 285 Cys Ser
His Ser Leu Asn 290 3885RNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 3auggccauga gcagcggagg
cagcggcgga ggagugcccg agcaggagga cagcgugcug 60uucaggagag gcaccggcca
gagcgaugac agcgauaucu gggacgauac cgcucugauc 120aaggccuacg
acaaggccgu ggccagcuuc aagcacgccc ugaaaaacgg cgacaucugc
180gagaccagcg gcaagcccaa gacaaccccc aagagaaagc ccgccaagaa
gaauaagagc 240cagaaaaaga acaccgccgc cagccugcag caguggaagg
ugggcgacaa gugcagcgcc 300aucuggagcg aggacggcug caucuacccc
gccaccaucg ccagcaucga cuucaagaga 360gagaccugcg uggucgugua
caccggcuac ggcaacagag aggagcagaa ccugagcgac 420cugcugagcc
ccauuuguga gguggccaau aacaucgaac agaacgccca ggagaacgag
480aaugaaagcc aggugagcac cgacgagagc gagaacagca gaucuccugg
caacaagagc 540gacaacauca agccuaaguc ugccccuugg aacagcuucc
ugcccccucc uccacccaug 600cccggaccca gacugggacc cggaaaaccu
ggccugaagu ucaacggacc accucccccu 660ccaccuccuc ccccaccuca
ucuccugagc ugcuggcugc cacccuuccc cagcggaccc 720ccuaucaucc
caccaccccc ucccaucugc cccgacagcc uggacgacgc cgaugcccug
780ggcagcaugc ugaucagcug guacaugagc ggcuaccaca caggauacua
caugggcuuc 840agacagaacc agaaggaggg cagaugcucc cacucccuga acuga
8854885RNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 4auggccauga gcagcggagg aagcggagga
ggagugccag aacaggaaga uagcgugcug 60uuucgccggg gcaccggaca aucggacgac
agcgauauuu gggacgacac ugcgcucauc 120aaggccuacg acaaggcggu
ggcuucguuc aagcacgcuc ugaagaacgg ggauaucugu 180gaaaccagcg
guaaaccaaa aacuacgccg aaaaggaaac ccgccaaaaa gaacaaguca
240cagaagaaga auaccgcugc gagcuugcag caguggaagg ugggcgacaa
gugcuccgcg 300auuuggucgg aagaugguug caucuacccg gcaaccaucg
ccuccaucga cuuuaagcgg 360gagacuugcg ucguggucua caccggauac
ggcaauagag aggaacagaa ucugucagac 420cuucugucgc caaucugcga
ggucgccaac aauaucgaac aaaacgccca agagaacgag 480aaugaguccc
aaguguccac ggacgaaucg gaaaacucac gguccccugg gaacaaguca
540gauaacauca agccuaaauc ggcaccaugg aacuccuucc ugccgccucc
gccuccgaug 600ccgggcccgc gccugggacc ggguaaaccc gggcucaagu
ucaauggacc gccaccccca 660cccccgccac cgccgcccca ccuccucucg
ugcuggcugc cgccguuccc uuccggaccg 720ccuaucauuc cgccaccucc
accuaucugc ccagacagcc uggaugaugc cgacgcauug 780ggcuccaugc
ucaucucaug guacaugucg ggauaccaua cuggguauua caugggcuuc
840agacagaacc agaaggaagg acgcuguucc cauagccuga acuag
88551442RNAHomo sapiens 5ggggccccac gcugcgcacc cgcggguuug
cuauggcgau gagcagcggc ggcaguggug 60gcggcguccc ggagcaggag gauuccgugc
uguuccggcg cggcacaggc cagagcgaug 120auucugacau uugggaugau
acagcacuga uaaaagcaua ugauaaagcu guggcuucau 180uuaagcaugc
ucuaaagaau ggugacauuu gugaaacuuc ggguaaacca aaaaccacac
240cuaaaagaaa accugcuaag aagaauaaaa gccaaaagaa gaauacugca
gcuuccuuac 300aacaguggaa aguuggggac aaauguucug ccauuugguc
agaagacggu ugcauuuacc 360cagcuaccau ugcuucaauu gauuuuaaga
gagaaaccug uguugugguu uacacuggau 420auggaaauag agaggagcaa
aaucuguccg aucuacuuuc cccaaucugu gaaguagcua 480auaauauaga
acagaaugcu caagagaaug aaaaugaaag ccaaguuuca acagaugaaa
540gugagaacuc caggucuccu ggaaauaaau cagauaacau caagcccaaa
ucugcuccau 600ggaacucuuu ucucccucca ccacccccca ugccagggcc
aagacuggga ccaggaaagc 660caggucuaaa auucaauggc ccaccaccgc
caccgccacc accaccaccc cacuuacuau 720caugcuggcu gccuccauuu
ccuucuggac caccaauaau ucccccacca ccucccauau 780guccagauuc
ucuugaugau gcugaugcuu ugggaaguau guuaauuuca ugguacauga
840guggcuauca uacuggcuau uauauggaaa ugcuggcaua gagcagcacu
aaaugacacc 900acuaaagaaa cgaucagaca gaucuggaau gugaagcguu
auagaagaua acuggccuca 960uuucuucaaa auaucaagug uugggaaaga
aaaaaggaag uggaaugggu aacucuucuu 1020gauuaaaagu uauguaauaa
ccaaaugcaa ugugaaauau uuuacuggac ucuauuuuga 1080aaaaccaucu
guaaaagacu gagguggggg ugggaggcca gcacgguggu gaggcaguug
1140agaaaauuug aauguggauu agauuuugaa ugauauugga uaauuauugg
uaauuuuaug 1200agcugugaga aggguguugu aguuuauaaa agacugucuu
aauuugcaua cuuaagcauu 1260uaggaaugaa guguuagagu gucuuaaaau
guuucaaaug guuuaacaaa auguauguga 1320ggcguaugug gcaaaauguu
acagaaucua acugguggac auggcuguuc auuguacugu 1380uuuuuucuau
cuucuauaug uuuaaaagua uauaauaaaa auauuuaauu uuuuuuuaaa 1440aa
14426282PRTHomo sapiens 6Met Ala Met Ser Ser Gly Gly Ser Gly Gly
Gly Val Pro Glu Gln Glu 1 5 10 15 Asp Ser Val Leu Phe Arg Arg Gly
Thr Gly Gln Ser Asp Asp Ser Asp 20 25 30 Ile Trp Asp Asp Thr Ala
Leu Ile Lys Ala Tyr Asp Lys Ala Val Ala 35 40 45 Ser Phe Lys His
Ala Leu Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly 50 55 60 Lys Pro
Lys Thr Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys Ser 65 70 75 80
Gln Lys Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys Val Gly Asp 85
90 95 Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys Ile Tyr Pro Ala
Thr 100 105 110 Ile Ala Ser Ile Asp Phe Lys Arg Glu Thr Cys Val Val
Val Tyr Thr 115 120 125 Gly Tyr Gly Asn Arg Glu Glu Gln Asn Leu Ser
Asp Leu Leu Ser Pro 130 135 140 Ile Cys Glu Val Ala Asn Asn Ile Glu
Gln Asn Ala Gln Glu Asn Glu 145 150 155 160 Asn Glu Ser Gln Val Ser
Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro 165 170 175 Gly Asn Lys Ser
Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser 180 185 190 Phe Leu
Pro Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly 195 200 205
Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro 210
215 220 Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly
Pro 225 230 235 240 Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp
Ser Leu Asp Asp 245 250 255 Ala Asp Ala Leu Gly Ser Met Leu Ile Ser
Trp Tyr Met Ser Gly Tyr 260 265 270 His Thr Gly Tyr Tyr Met Glu Met
Leu Ala 275 280 7140RNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 7ggacagaucg ccuggagacg ccauccacgc
uguuuugacc uccauagaag acaccgggac 60cgauccagcc uccgcggccg ggaacggugc
auuggaacgc ggauuccccg ugccaagagu 120gacucaccgu ccuugacacg
1408105RNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 8cggguggcau cccugugacc ccuccccagu
gccucuccug gcccuggaag uugccacucc 60agugcccacc agccuugucc uaauaaaauu
aaguugcauc aagcu 1059105RNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 9ggguggcauc ccugugaccc
cuccccagug ccucuccugg cccuggaagu ugccacucca 60gugcccacca gccuuguccu
aauaaaauua aguugcauca aagcu 105101130RNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
10ggacagaucg ccuggagacg ccauccacgc uguuuugacc uccauagaag acaccgggac
60cgauccagcc uccgcggccg ggaacggugc auuggaacgc ggauuccccg ugccaagagu
120gacucaccgu ccuugacacg auggccauga gcagcggagg cagcggcgga
ggagugcccg 180agcaggagga cagcgugcug uucaggagag gcaccggcca
gagcgaugac agcgauaucu 240gggacgauac cgcucugauc aaggccuacg
acaaggccgu ggccagcuuc aagcacgccc 300ugaaaaacgg cgacaucugc
gagaccagcg gcaagcccaa gacaaccccc aagagaaagc 360ccgccaagaa
gaauaagagc cagaaaaaga acaccgccgc cagccugcag caguggaagg
420ugggcgacaa gugcagcgcc aucuggagcg aggacggcug caucuacccc
gccaccaucg 480ccagcaucga cuucaagaga gagaccugcg uggucgugua
caccggcuac ggcaacagag 540aggagcagaa ccugagcgac cugcugagcc
ccauuuguga gguggccaau aacaucgaac 600agaacgccca ggagaacgag
aaugaaagcc aggugagcac cgacgagagc gagaacagca 660gaucuccugg
caacaagagc gacaacauca agccuaaguc ugccccuugg aacagcuucc
720ugcccccucc uccacccaug cccggaccca gacugggacc cggaaaaccu
ggccugaagu 780ucaacggacc accucccccu ccaccuccuc ccccaccuca
ucuccugagc ugcuggcugc 840cacccuuccc cagcggaccc ccuaucaucc
caccaccccc ucccaucugc cccgacagcc 900uggacgacgc cgaugcccug
ggcagcaugc ugaucagcug guacaugagc ggcuaccaca 960caggauacua
caugggcuuc agacagaacc agaaggaggg cagaugcucc cacucccuga
1020acugacgggu ggcaucccug ugaccccucc ccagugccuc uccuggcccu
ggaaguugcc 1080acuccagugc ccaccagccu uguccuaaua aaauuaaguu
gcaucaagcu 1130111130RNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 11ggacagaucg
ccuggagacg ccauccacgc uguuuugacc uccauagaag acaccgggac 60cgauccagcc
uccgcggccg ggaacggugc auuggaacgc ggauuccccg ugccaagagu
120gacucaccgu ccuugacacg auggccauga gcagcggagg cagcggcgga
ggagugcccg 180agcaggagga cagcgugcug uucaggagag gcaccggcca
gagcgaugac agcgauaucu 240gggacgauac cgcucugauc aaggccuacg
acaaggccgu ggccagcuuc aagcacgccc 300ugaaaaacgg cgacaucugc
gagaccagcg gcaagcccaa gacaaccccc aagagaaagc 360ccgccaagaa
gaauaagagc cagaaaaaga acaccgccgc cagccugcag caguggaagg
420ugggcgacaa gugcagcgcc aucuggagcg aggacggcug caucuacccc
gccaccaucg 480ccagcaucga cuucaagaga gagaccugcg uggucgugua
caccggcuac ggcaacagag 540aggagcagaa ccugagcgac cugcugagcc
ccauuuguga gguggccaau aacaucgaac 600agaacgccca ggagaacgag
aaugaaagcc aggugagcac cgacgagagc gagaacagca 660gaucuccugg
caacaagagc gacaacauca agccuaaguc ugccccuugg aacagcuucc
720ugcccccucc uccacccaug cccggaccca gacugggacc cggaaaaccu
ggccugaagu 780ucaacggacc accucccccu ccaccuccuc ccccaccuca
ucuccugagc ugcuggcugc 840cacccuuccc cagcggaccc ccuaucaucc
caccaccccc ucccaucugc cccgacagcc 900uggacgacgc cgaugcccug
ggcagcaugc ugaucagcug guacaugagc ggcuaccaca 960caggauacua
caugggcuuc agacagaacc agaaggaggg cagaugcucc cacucccuga
1020acugagggug gcaucccugu gaccccuccc cagugccucu ccuggcccug
gaaguugcca 1080cuccagugcc caccagccuu guccuaauaa aauuaaguug
caucaaagcu 113012500DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 12aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
480aaaaaaaaaa aaaaaaaaaa 50013300DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 13aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 30014200DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
14cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc
60cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc
120cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc
cccccccccc 180cccccccccc cccccccccc 20015250DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
15aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 240aaaaaaaaaa 250
* * * * *