U.S. patent application number 14/776525 was filed with the patent office on 2016-02-04 for purification and purity assessment of rna molecules synthesized with modified nucleosides.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Katalin Kariko, Drew Weissman.
Application Number | 20160032316 14/776525 |
Document ID | / |
Family ID | 51625370 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160032316 |
Kind Code |
A1 |
Weissman; Drew ; et
al. |
February 4, 2016 |
Purification and Purity Assessment of RNA Molecules Synthesized
with Modified Nucleosides
Abstract
This invention provides purified preparations of an RNA,
oligoribonucleotide, or polyribonucleotide comprising a modified
nucleoside, and methods of assessing purity of purified
preparations of an RNA, oligoribonucleotide, or polyribonucleotide
comprising a modified nucleoside.
Inventors: |
Weissman; Drew; (Wynnewood,
PA) ; Kariko; Katalin; (Rydal, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
Philadelphia
PA
|
Family ID: |
51625370 |
Appl. No.: |
14/776525 |
Filed: |
March 13, 2014 |
PCT Filed: |
March 13, 2014 |
PCT NO: |
PCT/US14/26140 |
371 Date: |
September 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61783645 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
424/490 ;
435/320.1; 435/69.1; 435/91.3; 514/44R; 536/23.1 |
Current CPC
Class: |
C07K 14/505 20130101;
C12N 2320/30 20130101; C07H 21/02 20130101; C07K 14/475 20130101;
C12N 2310/335 20130101; C07K 14/52 20130101; A61K 48/00 20130101;
C12N 15/85 20130101; C12P 19/34 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C07K 14/505 20060101 C07K014/505; C07K 14/475 20060101
C07K014/475; C12P 19/34 20060101 C12P019/34; C07H 21/02 20060101
C07H021/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
numbers HL087688 and AI050484, awarded by the National Institutes
of Health (NIH). The government has certain rights in the
invention.
Claims
1. A purified preparation of messenger RNA comprising a
1-methyl-pseudouridine residue.
2. The purified preparation of messenger RNA of claim 1, further
comprising a poly-A tail.
3. The purified preparation of messenger RNA of claim 1, further
comprising an m7GpppG cap.
4. The purified preparation of messenger RNA of claim 1, further
comprising a cap-independent translational enhancer.
5. The purified preparation of messenger RNA of claim 1, wherein
the messenger RNA comprises at least about 95% to about 99.9% of
all the nucleic acid present in the purified preparation.
6. The purified preparation of messenger RNA of claim 1, wherein
the messenger RNA is significantly less immunogenic than an
unpurified preparation of messenger RNA with the same sequence.
7. The purified preparation of messenger RNA of claim 1, wherein
the messenger RNA exhibits enhanced ability to be translated by a
target cell than an unpurified preparation of messenger RNA with
the same sequence.
8. The purified preparation of messenger RNA of claim 1, wherein
the messenger RNA exhibits enhanced ability to be translated when
delivered to a mammal than an unpurified preparation of messenger
RNA with the same sequence.
9. The purified preparation of messenger RNA of claim 1, wherein
the messenger RNA is encapsulated in a lipid nanoparticle.
10. The purified preparation of messenger RNA of claim 1, wherein
the modified nucleoside is at least one of 1-methyl-pseudouridine
and m5C.
11. The purified preparation of messenger RNA of claim 1, wherein
the modified nucleoside is at least one of pseudouridine and
m5C.
12. A purified preparation of RNA encoding a protein of interest,
the RNA comprising at least one of a 1-methyl-pseudouridine residue
and a m5C residue.
13. A purified preparation of RNA encoding a protein of interest,
the RNA comprising at least one of a pseudouridine residue and a
m5C residue.
14. The purified preparation of RNA of claim 13, further comprising
a poly-A tail.
15. The purified preparation of RNA of claim 13, further comprising
an m7GpppG cap.
16. The purified preparation of RNA of claim 13, further comprising
a cap-independent translational enhancer.
17. The purified preparation of RNA of claim 13, wherein the
messenger RNA comprises at least about 95% to about 99.9% of all
the nucleic acid present in the purified preparation.
18. The purified preparation of RNA of claim 13, whereby the
messenger RNA is significantly less immunogenic than an unpurified
preparation of messenger RNA with the same sequence.
19. The purified preparation of RNA of claim 13, wherein the RNA
exhibits enhanced ability to be translated by a target cell than an
unpurified preparation of messenger RNA with the same sequence.
20. The purified preparation of RNA of claim 13, wherein the RNA
exhibits enhanced ability to be translated when delivered to a
mammal than an unpurified preparation of messenger RNA with the
same sequence.
21. The purified preparation of RNA of claim 13, wherein the RNA is
encapsulated in a lipid nanoparticle.
22. The purified preparation of RNA of claim 13, wherein the
protein of interest is VEGF-A.
23. The purified preparation of RNA of claim 13, wherein the
protein of interest is erythropoietin (EPO).
24. A method of preparing a purified preparation of messenger RNA
comprising at least one modified nucleoside comprising the steps
of: a. producing a preparation of messenger RNA comprising at least
one modified nucleoside; b. subjecting the preparation of messenger
RNA comprising at least one modified nucleoside to at least one
purification process selected from the group consisting of enzyme
digestion and chromatography; and c. isolating the purified
preparation of messenger RNA comprising at least one modified
nucleoside.
25. The method of claim 24, wherein the at least one modified
nucleoside is 1-methyl-pseudouridine.
26. The method of claim 24, wherein the messenger RNA further
comprises a poly-A tail.
27. The method of claim 24, wherein the messenger RNA further
comprises an m7GpppG cap.
28. The method of claim 24, wherein the messenger RNA further
comprises a cap-independent translational enhancer.
29. The method of claim 24, wherein the messenger RNA comprises at
least about 95% to about 99.9% of all the nucleic acid present in
the purified preparation.
30. The method of claim 24, wherein the messenger RNA is
significantly less immunogenic than an unpurified preparation of
messenger RNA with the same sequence.
31. The method of claim 24, wherein the messenger RNA exhibits
enhanced ability to be translated by a target cell than an
unpurified preparation of messenger RNA with the same sequence.
32. The method of claim 24, wherein the messenger RNA exhibits
enhanced ability to be translated when delivered to a mammal than
an unpurified preparation of messenger RNA with the same
sequence.
33. The method of claim 24, wherein the modified nucleoside
comprises at least one of 1-methyl-pseudouridine and m5C.
34. The method of claim 24, wherein the modified nucleoside
comprises at least one of pseudouridine and m5C.
35. The method of claim 24, wherein the preparation of messenger
RNA is produced by in vitro transcription.
36. The method of claim 24, wherein the at least one purification
process is enzyme digestion.
37. The method of claim 36, wherein the enzyme digestion is
performed using at least one enzyme selected from the group
consisting of RNase III, RNase V1, Dicer, and Chipper.
38. The method of claim 24, wherein the at least one purification
process is chromatography.
39. The method of claim 38, wherein the chromatography is at least
one selected from the group consisting of high performance liquid
chromatography (HPLC) and fast protein liquid chromatography
(FPLC).
40. A method for inducing a mammalian cell to produce a protein of
interest, the method comprising the step of contacting the
mammalian cell with the purified preparation of the RNA of claim 1,
thereby inducing a mammalian cell to produce a protein of
interest.
41. The method of claim 40, wherein the mammalian cell is a
dendritic cell.
42. The method of claim 40, wherein the mammalian cell is an
alveolar cell, an astrocyte, a microglial cell, or a neuron.
43. A purified preparation of in vitro-transcribed RNA, comprising
a at least one modified nucleoside.
44. The purified preparation of in vitro-transcribed RNA of claim
43, wherein the at least one modified nucleoside is
1-methyl-pseudouridine, m5C, m5U, m6A, s2U, .PSI., or
2'-O-methyl-U.
45. The purified preparation of in vitro-transcribed RNA of claim
43, further comprising a poly-A tail.
46. The purified preparation of in vitro-transcribed RNA of claim
43, further comprising an m7GpppG cap.
47. The purified preparation of in vitro-transcribed RNA of claim
43, further comprising a cap-independent translational
enhancer.
48. A purified preparation of an in vitro-synthesized
oligoribonucleotide, comprising at least one modified nucleoside,
wherein the at least one modified nucleoside is
1-methyl-pseudouridine, m5C, m5U, m6A, s2U, .PSI., or
2'-O-methyl-U.
49. The purified preparation of in vitro-synthesized
oligoribonucleotide of claim 48, wherein the in vitro-synthesized
oligoribonucleotide is a therapeutic oligoribonucleotide.
50. A purified preparation of a gene-therapy vector, comprising an
in vitro-synthesized polyribonucleotide encoding a protein of
interest, wherein the polyribonucleotide comprises at least one
modified nucleoside.
51. The purified preparation of gene-therapy vector of claim 50,
wherein the at least one modified nucleoside is
1-methyl-pseudouridine, m5C, m5U, m6A, s2U, .PSI., or
2'-O-methyl-U.
52. The purified preparation of gene-therapy vector of claim 50,
wherein the polyribonucleotide further comprises a poly-A tail.
53. The purified preparation of gene-therapy vector of claim 50,
wherein the polyribonucleotide further comprises an m7GpppG
cap.
54. The purified preparation of gene-therapy vector of claim 50,
wherein the polyribonucleotide further comprises a cap-independent
translational enhancer.
55. A method for delivering a recombinant protein to a subject, the
method comprising the step of contacting a cell of the subject with
the purified preparation of the gene-therapy vector of claim 50,
wherein the cell produces the recombinant protein, thereby
delivering a recombinant protein to a subject.
56. The method of claim 55, wherein the cell is a dendritic
cell.
57. The method of claim 55, wherein the cell is a lung cell, a
brain cell, or a spleen cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/783,645, filed on Mar. 14, 2013, the contents of
which are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0003] The first in vivo delivery of mRNA encoding a therapeutic
protein was reported in 1992 (Jirikowski et al., Science
255:996-998), but only recently has the delivery of mRNA for
scientific and therapeutic purposes gained expanded interest.
Potential uses include: delivery of mRNA encoding transcription
factors to generate induced pluripotent stem (iPS) cells (Angel and
Yanik, 2010, PLoS One 5:e11756; Yakubov et al., 2010, Biochem
Biophys Res Commun 394:189-193; Warren et al., 2010, Cell Stem Cell
7:618-630), in vivo administration to express therapeutic proteins
(Jirikowski et al., Science 255:996-998; Kormann et al., 2011, Nat
Biotechnol 29:154-157; Kariko et al., 2012, Mol Ther 20:948-953),
ex vivo delivery to expanded cells as a cancer therapeutic (Barrett
et al., 2011, Hum Gene Ther 22(12):1575-1586; Almasbak et al.,
2011, Cytotherapy 13:629-640; Zhao et al., 2010, Cancer Res
70:9053-9061; Rabinovich et al., 2009, Hum Gene Ther 20:51-61; Yoon
et al., 2009, Cancer Gene Ther 16:489-497), as the vector for
vaccines (Kariko, et al., 2011, Nucleic Acids Res 39:e142; Kariko
et al., 2008, Mol Ther 16:1833-1840; Weissman et al., 2000, J
Immunol 165:4710-4717), and in vitro delivery to express protein at
a high efficiency (Kariko, et al., 2011, Nucleic Acids Res 39:e142;
Kariko et al., 2008, Mol Ther 16:1833-1840; Weissman et al., 2000,
J Immunol 165:4710-4717).
[0004] All naturally occurring RNA is synthesized from four basic
ribonucleotides ATP, CTP, UTP and GTP, but some of the incorporated
nucleosides are modified post-transcriptionally in almost all types
of RNA. Over one hundred different nucleoside modifications have
been identified in RNA (Rozenski et al., 1999, The RNA Modification
Database: 1999 update. Nucl Acids Res 27:196-197). The extent and
nature of modifications vary and depend on the RNA type as well as
the evolutionary level of the organism from where the RNA is
derived. Ribosomal RNA, the major constituent of cellular RNA,
contains significantly more nucleoside modifications in mammalian
cells than bacteria. Human rRNA, for example, has 10-times more
pseudouridine (.PSI.) and 25-times more 2'-O-methylated nucleosides
than bacterial rRNA, while rRNA from mitochondria has very few
modifications. Transfer RNA (tRNA) is the most heavily modified
subgroup of RNA. In mammalian tRNA, up to 25% of the nucleosides
are modified, while prokaryotic tRNA contains significantly fewer
modifications. Bacterial messenger RNA (mRNA) contains no
nucleoside modifications, while mammalian mRNA contains modified
nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine
(m6A), inosine and 2'-O-methylated nucleosides, in addition to
N7-methylguanosine (m7G), which is part of the 5'-terminal cap.
Nucleoside modifications have a great impact on the
immunostimulatory potential and on the translation efficiency of
RNA.
[0005] The recognition that the immunogenicity of RNA could be
reduced by the incorporation of modified nucleosides with an
associated increase in translation (Kariko et al., 2008, Mol Ther
16:1833-1840; Kariko et al., 2005, Immunity 23:165-175) potentially
allows efficient expression of proteins in vivo and ex vivo without
activation of innate immune receptors. Unfortunately, modified
nucleoside-containing RNA transcribed by phage RNA polymerases
still retains a low level of activation of such pathways (Warren et
al., 2010, Cell Stem Cell 7(5):618-630; Kariko et al., 2008, Mol
Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res
38:5884-5892; Kariko and Weissman, 2007, Curr Opin Drug Discov
Devel 10:523-532). This remaining activation of RNA sensors could
be due to modified nucleosides that do not completely suppress the
RNAs ability to activate sensors (Kormann et al., 2011, Nat
Biotechnol 29:154-157) or dsRNA contaminants that activate even in
the presence of nucleoside modification (Kariko, et al., 2011,
Nucleic Acids Res 39:e142). It is known that RNA transcribed in
vitro by phage polymerase contains multiple aberrant RNAs,
including short RNAs as a result of abortive transcription
initiation events (Milligan et al., 1987, Nucleic Acids Res
15:8783-8798) and double stranded (ds)RNAs generated by RNA
dependent RNA polymerase activity (Arnaud-Barbe et al., 1998,
Nucleic Acids Res 26:3550-3554), RNA-primed transcription from RNA
templates (Nacheva and Berzal-Herranz, 2003, Eur J Biochem
270:1458-1465), and self-complementary 3' extension (Triana-Alonso
et al., 1995, J Biol Chem 270:6298-6307).
[0006] Thus, there is a need in the art for more purified
preparations of RNA containing a reduced amount of contaminants.
The present invention satisfies this need.
SUMMARY OF THE INVENTION
[0007] The invention relates to methods of preparing and assessing
purified preparations of an RNA comprising a modified nucleoside.
Thus, in one embodiment, the invention is a purified preparation of
messenger RNA comprising a 1-methyl-pseudouridine residue. In some
embodiments, the messenger RNA also comprises a poly-A tail. In
some embodiments, the messenger RNA also comprises an m7GpppG cap.
In some embodiments, the messenger RNA also comprises a
cap-independent translational enhancer. In some embodiments, the
messenger RNA comprises at least about 95% to about 99.9% of all
the nucleic acid present in the purified preparation. In some
embodiments, the purified preparation of messenger RNA is
significantly less immunogenic than an unpurified preparation of
messenger RNA with the same sequence. In some embodiments, the
purified preparation of messenger RNA exhibits enhanced ability to
be translated by a target cell than an unpurified preparation of
messenger RNA with the same sequence. In some embodiments, the
purified preparation of messenger RNA exhibits enhanced ability to
be translated when delivered to a mammal than an unpurified
preparation of messenger RNA with the same sequence. In some
embodiments, the messenger RNA is encapsulated in a lipid
nanoparticle. In some embodiments, the modified nucleoside is at
least one of 1-methyl-pseudouridine and m5C. In some embodiments,
the modified nucleoside is at least one of pseudouridine and
m5C.
[0008] In another embodiment, the invention is a purified
preparation of RNA encoding a protein of interest, where the RNA
comprises at least one of a 1-methyl-pseudouridine residue and a
m5C residue. In another embodiment, the invention is a purified
preparation of RNA encoding a protein of interest, where the RNA
comprises at least one of a pseudouridine residue and a m5C
residue. In some embodiments, the messenger RNA also comprises a
poly-A tail. In some embodiments, the messenger RNA also comprises
an m7GpppG cap. In some embodiments, the messenger RNA also
comprises a cap-independent translational enhancer. In some
embodiments, the messenger RNA comprises at least about 95% to
about 99.9% of all the nucleic acid present in the purified
preparation. In some embodiments, the purified preparation of
messenger RNA is significantly less immunogenic than an unpurified
preparation of messenger RNA with the same sequence. In some
embodiments, the purified preparation of messenger RNA exhibits
enhanced ability to be translated by a target cell than an
unpurified preparation of messenger RNA with the same sequence. In
some embodiments, the purified preparation of messenger RNA
exhibits enhanced ability to be translated when delivered to a
mammal than an unpurified preparation of messenger RNA with the
same sequence. In some embodiments, the messenger RNA is
encapsulated in a lipid nanoparticle.
[0009] In one embodiment, the invention is a method of preparing a
purified preparation of messenger RNA comprising at least one
modified nucleoside including the steps of producing a preparation
of messenger RNA comprising at least one modified nucleoside,
subjecting the preparation of messenger RNA comprising at least one
modified nucleoside to at least one purification process selected
from the group consisting of enzyme digestion and chromatography;
and isolating the purified preparation of messenger RNA comprising
at least one modified nucleoside. In some embodiments, the at least
one modified nucleoside is 1-methyl-pseudouridine. In some
embodiments, the messenger RNA further comprises a poly-A tail. In
some embodiments, the messenger RNA further comprises an m7GpppG
cap. In some embodiments, the messenger RNA further comprises a
cap-independent translational enhancer. In some embodiments, the
messenger RNA comprises at least about 95% to about 99.9% of all
the nucleic acid present in the purified preparation. In some
embodiments, the messenger RNA is significantly less immunogenic
than an unpurified preparation of messenger RNA with the same
sequence. In some embodiments, the messenger RNA exhibits enhanced
ability to be translated by a target cell than an unpurified
preparation of messenger RNA with the same sequence. In some
embodiments, the messenger RNA exhibits enhanced ability to be
translated when delivered to a mammal than an unpurified
preparation of messenger RNA with the same sequence. In some
embodiments, the modified nucleoside comprises at least one of
1-methyl-pseudouridine and m5C. In some embodiments, the modified
nucleoside comprises at least one of pseudouridine and m5C. In some
embodiments, the preparation of messenger RNA is produced by in
vitro transcription. In some embodiments, the at least one
purification process is enzyme digestion. In various embodiments,
the enzyme digestion is performed using at least one enzyme
selected from the group consisting of RNase III, RNase V1, Dicer,
and Chipper. In some embodiments, the at least one purification
process is chromatography. In various embodiments, the
chromatography is at least one selected from the group consisting
of high performance liquid chromatography (HPLC) and fast protein
liquid chromatography (FPLC).
[0010] In another embodiments, the invention is a method of
inducing a mammalian cell to produce a protein of interest, the
method comprising the step of contacting the mammalian cell with
the purified preparation of the RNA, thereby inducing a mammalian
cell to produce a protein of interest. In some embodiments, the
mammalian cell is a dendritic cell. In some embodiments, the
mammalian cell is an alveolar cell, an astrocyte, a microglial
cell, or a neuron.
[0011] In one embodiment, the invention is a purified preparation
of in vitro-transcribed RNA, comprising a 1-methyl-pseudouridine or
a modified nucleoside. In some embodiments, the modified nucleoside
is m5C, m5U, m6A, s2U, .PSI., or 2'-O-methyl-U. In some
embodiments, the in vitro-transcribed RNA also comprises a poly-A
tail. In some embodiments, the in vitro-transcribed RNA also
comprises an m7GpppG cap. In some embodiments, the in
vitro-transcribed RNA also comprises a cap-independent
translational enhancer.
[0012] In another embodiment, the invention is a purified
preparation of an in vitro-synthesized oligoribonucleotide,
comprising a 1-methyl-pseudouridine or a modified nucleoside, where
the modified nucleoside is m5C, m5U, m6A, s2U, .PSI., or
2'-O-methyl-U. In some embodiments, the in vitro-synthesized
oligoribonucleotide is a therapeutic oligoribonucleotide.
[0013] In one embodiment, the invention is a purified preparation
of a gene-therapy vector, comprising an in vitro-synthesized
polyribonucleotide encoding a protein of interest, where the
polyribonucleotide comprises a 1-methyl-pseudouridine or a modified
nucleoside. In some embodiments, the modified nucleoside is m5C,
m5U, m6A, s2U, .PSI., or 2'-O-methyl-U. In some embodiments, the
polyribonucleotide further comprises a poly-A tail. In some
embodiments, the polyribonucleotide further comprises an m7GpppG
cap. In some embodiments, the polyribonucleotide further the
comprises a cap-independent translational enhancer.
[0014] In another embodiment, the invention is a method for
delivering a recombinant protein to a subject, the method
comprising the step of contacting a cell of the subject with the
purified preparation of the gene-therapy vector, where the cell
produces the recombinant protein, thereby delivering a recombinant
protein to a subject. In some embodiments, the cell is a dendritic
cell. In some embodiments, the cell is a lung cell, a brain cell,
or a spleen cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0016] FIG. 1, comprising FIGS. 1A-1C, depicts the results of
experiments demonstrating that in vitro transcribed RNA is
immunogenic and contains dsRNA contaminants. (FIG. 1A) 200 ng of in
vitro transcripts encoding mEPO and containing the indicated
modified nucleosides were blotted and analyzed with K1 and J2
dsRNA-specific mAbs. The dsRNA positive control contained a 328 bp
long dsRNA (25 ng). (FIG. 1B) DCs were treated with
Lipofectin-complexed Renilla luciferase (T7TSRenA.sub.30), firefly
and Metridia luciferases (T7TSLucA.sub.30, T7TSMetlucA.sub.30), and
mEPO (TEVmEPOA.sub.51) mRNAs. TNF-.alpha. levels were measured in
the supernatants at 24 h. (FIG. 1C) DCs were treated with
TransIT-complexed in vitro transcripts encoding Renilla and firefly
luciferases (T7TSRenA.sub.30, T7TSLucA.sub.30), eGFP
(TEVeGFPA.sub.51) and mEPO (TEVmEPOA.sub.51). IFN-.alpha. levels
were measured in the supernatants at 24 h. Error bars are standard
error of the mean. Data shown is from one experiment that is
representative of greater than 20 experiments using many different
coding sequence mRNAs.
[0017] FIG. 2 depicts a chromatogram of .PSI.-modified
TEVeGFPA.sub.n mRNA. HPLC purification of RNA identifies
contaminants eluting before and after the expected product. RNA was
applied to the HPLC column and eluted using a linear gradient of
Buffer B (0.1 M TEAA, pH 7.0, 25% acetonitrile) in Buffer A (0.1 M
TEAA, pH 7.0). The gradient spanned 38-55% Buffer B over 22 min
(red line). Absorbance at 260 nm was analyzed (black line), which
demonstrated the expected sized RNA as well as smaller and larger
RNA species. Data shown is from one experiment that is
representative of over 200.
[0018] FIG. 3, comprising FIGS. 3A-3D, depicts the results of
analyses demonstrating that HPLC purification of in
vitro-transcribed nucleoside modified mRNA removes dsRNA
contaminants and eliminates immunogenicity. (FIG. 3A) 200 ng of RNA
encoding the indicated protein and containing the indicated
modified nucleosides with or without HPLC-purification were blotted
and analyzed with the J2 dsRNA-specific mAb. (FIG. 3B) 200 ng of
RNA encoding the indicated protein and containing
.PSI.-modifications with or without HPLC-purification were blotted
and analyzed with the J2 dsRNA-specific mAb. Blots were reprobed
with a 32P-labeled probe for the 3' UTR of the RNAs to control for
amount of RNA analyzed. (FIG. 3C) DCs were treated with
TEVRenA.sub.51 RNA containing the indicated nucleoside
modifications with or without HPLC purification and complexed to
Lipofectin. TNF-.alpha. levels were measured in the supernatants at
24 hr. Differences in the effect of nucleoside modification on
immunogenicity of Renilla encoding mRNA compared to FIG. 1B is
likely due to donor variation and differences in UTRs of the RNAs.
(FIG. 3D) DCs were treated with TEVLucA.sub.51 RNA containing the
indicated nucleoside modifications with or without HPLC
purification and complexed to TransIT. IFN-.alpha. levels were
measured in the supernatants at 24 hr. Error bars are standard
error of the mean. Data shown is from one experiment that is
representative of 3 or more.
[0019] FIG. 4, comprising FIGS. 4A and 4B, depicts the results of
analyses demonstrating that HPLC purification of in vitro
transcribed nucleoside-modified mRNA eliminates activation of genes
associated with RNA sensor activation. (FIG. 4A) Heat map
representing changes in expression of genes activated by RNA
sensors were derived from microarray analyses of DCs treated for 6
hr with TransIT alone or transit-complexed TEVRenA.sub.51 RNA with
the indicated modifications with or without HPLC purification. RNA
from medium treated cells was used as the baseline for comparison.
(FIG. 4B) Northern blot of RNA from DCs treated with medium or
TransIT alone or TransIT-complexed TEVRenA.sub.51 RNA with the
indicated modifications with or without HPLC purification and
probed for IFN-.alpha., IFN-.beta., TNF-.alpha., and GAPDH
mRNAs.
[0020] FIG. 5, comprising FIGS. 5A-5D, depicts the results of
analyses demonstrating that HPLC purification of in vitro
transcribed mRNA enhances translation. 293T (FIG. 5A) and human DCs
(FIG. 5B-5C) were transfected with TransIT (FIG. 5A, FIG. 5C) or
Lipofectin (FIG. 5B) complexed TEVRenA.sub.51 or TEVmEPOA.sub.51
mRNA with the indicated modifications with or without HPLC
purification and analyzed for Renilla luciferase activity or levels
of supernatant-associated mEPO protein at 24 hr. (FIG. 5D) Human
DCs were transfected with .PSI.-modified TEVeGFPA.sub.n mRNA with
or without HPLC purification (0.1 .mu.g/well) complexed with
Lipofectin or TransIT and analyzed 24 hr later. Error bars are
standard error of the mean. Data shown is from one experiment that
is representative of 3 or more.
[0021] FIG. 6, comprising FIGS. 6A-6C, depicts the results of
analyses showing that RNA contaminants are removed by HPLC
purification. (FIG. 6A) One hundred .mu.g of .PSI.-modified
T7TSLucA.sub.30 RNA was applied to the HPLC column and 3 fractions
were collected, all RNAs eluting before the main transcription
product (I), the expected RNA (II), and all RNAs eluting after the
main transcription product (III). The gradient began at 38% Buffer
B and increased to 43% Buffer B over 2.5 min and then spanned 43%
to 65% Buffer B over 22 min. Unmodified and m5C/.PSI.-modified
T7TSLucA.sub.30 RNA had similar fractions obtained. (FIG. 6B) The
RNAs from each fraction were complexed to TransIT and added to DCs
and IFN-.alpha. in the supernatant was measured 24 hr later. Error
bars are standard error of the mean. (FIG. 6C) Two hundred ng of
RNA from the 3 fractions and the starting unpurified RNA were
blotted and analyzed with the J2 dsRNA-specific mAb.
[0022] FIG. 7 depicts the results of an exemplary experiment
demonstrating that daily transfection with HPLC-purified
m5C/T-modified mRNA does not reduce cell proliferation. Primary
keratinocytes were transfected daily with TransIT alone or
m5C/.PSI.-modified RNA encoding Renilla luciferase with or without
HPLC-purification complexed with TransIT. Every 2-3 days, cultures
were split and equal numbers of cells for each condition were
plated. Total cell numbers for each condition were divided by the
total cell number in untreated cells to calculate the percent of
control proliferation.
[0023] FIG. 8, comprising FIGS. 8A-8B, depicts the results of
analyses showing that RNA contaminants are removed by treatment
with RNase III. One hundred .mu.g of U, .PSI., m5C/.PSI., or
1-Me-.PSI.-modified TEV-ren-A51 RNA was treated with 0.001 units of
bacterial RNase III for 60 minutes at 37.degree. C. in 66 mM
acetate buffer, pH=7.5. Similar results were obtained with ranges
of RNase III from about 0.001 to about 0.1 units, with treatment
times ranging from about 15 to about 120 minutes, and
concentrations of acetate buffer ranging from about 33 to about 200
mM and pHs ranging from about 7.5 to about 8.0. After
precipitation, washing and resuspension in water, 200 ng of RNA was
analyzed for binding by the dsRNA-specific mAb J2 (FIG. 8A) or 300
ng of RNA was complexed to TransIT and added to primary human
monocyte derived dendritic cells (FIG. 8B). After 24 hrs,
supernatant was analyzed for interferon (IFN)-.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0024] This invention provides methods of preparing and assessing
purified preparations of an RNA, oligoribonucleotide, or
polyribonucleotide comprising pseudouridine or a modified
nucleoside, purified preparations of gene therapy vectors
comprising pseudouridine or a modified nucleoside, as well as
methods of reducing immunogenicity and of increasing translation
through the use of purified preparations of an RNA,
oligoribonucleotide, polyribonucleotide or gene therapy vector
comprising pseudouridine or a modified nucleoside.
[0025] In one embodiment, the present invention provides a purified
preparation of messenger RNA comprising a 1-methyl-pseudouridine
residue. In another embodiment, the messenger RNA encodes a protein
of interest.
[0026] In another embodiment, the present invention provides a
purified preparation of an RNA encoding a protein of interest, the
RNA comprising at least one 1-methyl-pseudouridine residue.
[0027] In another embodiment, the present invention provides a
purified preparation of an in vitro-transcribed RNA molecule,
comprising a 1-methyl-pseudouridine.
[0028] In another embodiment, the present invention provides a
purified preparation of an in vitro-transcribed RNA molecule,
comprising a modified nucleoside.
[0029] As provided herein, the present invention provides methods
for purifying and assessing purity of in vitro-transcribed RNA
molecules, comprising 1-methyl-pseudouridine and/or modified
nucleosides.
[0030] In another embodiment, the present invention provides a
purified preparation of a messenger RNA comprising at least one
1-methyl-pseudouridine residue.
[0031] In another embodiment, the purified preparation of in
vitro-transcribed RNA is synthesized by T7 phage RNA polymerase. In
another embodiment, the purified preparation of in
vitro-transcribed RNA is synthesized by SP6 phage RNA polymerase.
In another embodiment, the purified preparation of in
vitro-transcribed RNA is synthesized by T3 phage RNA polymerase. In
another embodiment, the purified preparation of in
vitro-transcribed RNA is synthesized by any known in vitro chemical
synthesis method, such as those known in the art.
[0032] In another embodiment, the purified preparation of in
vitro-transcribed RNA is an oligoribonucleotide. In another
embodiment, the purified preparation of the in vitro-transcribed
RNA is a polyribonucleotide.
[0033] In another embodiment, the present invention provides a
purified preparation of an in vitro-synthesized
oligoribonucleotide, comprising a 1-methyl-pseudouridine or a
modified nucleoside, wherein the modified nucleoside is
pseudouridine (.PSI.), m5C, m5U, m6A, s2U, 2'-O-methyl-C,
2'-O-methyl-A, 2'-O-methyl-G, or 2'-O-methyl-U.
[0034] In another embodiment, the present invention provides a
purified preparation of an in vitro-synthesized polyribonucleotide,
comprising a 1-methyl-pseudouridine or a modified nucleoside,
wherein the modified nucleoside is pseudouridine (.PSI.), m5C, m5U,
m6A, s2U, 2'-O-methyl-C, 2'-O-methyl-A, 2'-O-methyl-G, or
2'-O-methyl-U.
[0035] In another embodiment, the purified preparation of in
vitro-synthesized oligoribonucleotide or polyribonucleotide is a
short hairpin (sh)RNA. In a particular non-limiting embodiment, the
purified preparation of in vitro-synthesized oligoribonucleotide or
polyribonucleotide is a cas9 guide RNA. In another embodiment, the
purified preparation of in vitro-synthesized oligoribonucleotide is
a small interfering RNA (siRNA). In another embodiment, the
purified preparation of in vitro-synthesized oligoribonucleotide is
any other type of oligoribonucleotide known in the art.
[0036] In another embodiment, the purified preparation of an RNA,
oligoribonucleotide, or polyribonucleotide of the methods and
compositions of the present invention further comprise an open
reading frame that encodes a functional protein. In another
embodiment, the purified preparation of an RNA or a
oligoribonucleotide functions without encoding a functional protein
(e.g., in transcriptional silencing), such as an RNAzyme, etc.
[0037] In another embodiment, the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide further comprises a
poly-A tail. In another embodiment, the purified preparation of the
RNA, oligoribonucleotide, or polyribonucleotide does not comprise a
poly-A tail.
[0038] In another embodiment, the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide further comprises an
m7GpppG cap. In another embodiment, the purified preparation of the
RNA, oligoribonucleotide, or polyribonucleotide does not comprise
an m7GpppG cap.
[0039] In another embodiment, the purified preparation of the RNA,
oligoribonucleotide, or polyribonucleotide further comprises a
cap-independent translational enhancer. In another embodiment, the
purified preparation of the RNA, oligoribonucleotide, or
polyribonucleotide does not comprise a cap-independent
translational enhancer. In one embodiment, the cap-independent
translational enhancer is a tobacco etch virus (TEV)
cap-independent translational enhancer.
[0040] In another embodiment, the present invention provides a
purified preparation of a gene-therapy vector, comprising an in
vitro-synthesized polyribonucleotide, wherein the
polyribonucleotide comprises a 1-methyl-pseudouridine or a modified
nucleoside.
[0041] In another embodiment, the purified preparation of an RNA,
oligoribonucleotide, or polyribonucleotide of the methods and
compositions of the present invention comprises a
1-methyl-pseudouridine. In another embodiment, the purified
preparation of the RNA or oligoribonucleotide comprises a modified
nucleoside. In another embodiment, the purified preparation of the
RNA or oligoribonucleotide is an in vitro-synthesized RNA or
oligoribonucleotide.
[0042] In one embodiment, the modified nucleoside is 4'
(pseudouridine). In one embodiment, the modified nucleoside is
mlacp3T (1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In
another embodiment, the modified nucleoside is m1.PSI.
(1-methylpseudouridine). In another embodiment, the modified
nucleoside is .PSI.m (2'-O-methylpseudouridine). In another
embodiment, the modified nucleoside is m5D
(5-methyldihydrouridine). In another embodiment, the modified
nucleoside is m3.PSI. (3-methylpseudouridine). In another
embodiment, the modified nucleoside is a pseudouridine moiety that
is not further modified. In another embodiment, the modified
nucleoside is any other pseudouridine known in the art.
[0043] In another embodiment, the purified preparation of an RNA,
oligoribonucleotide, or polyribonucleotide of methods and
compositions of the present invention is a therapeutic
oligoribonucleotide.
[0044] In another embodiment, the present invention provides a
method for delivering a recombinant protein to a subject, the
method comprising the step of contacting the subject with a
purified preparation of an RNA, oligoribonucleotide,
polyribonucleotide molecule, or a purified preparation of a
gene-therapy vector of the present invention, thereby delivering a
recombinant protein to a subject.
[0045] In another embodiment, the length of an RNA,
oligoribonucleotide, or polyribonucleotide (e.g., a single-stranded
RNA (ssRNA) or dsRNA molecule) of methods and compositions of the
present invention is greater than 30 nucleotides in length. In
another embodiment, the RNA or oligoribonucleotide is greater than
35 nucleotides in length. In another embodiment, the length is at
least 40 nucleotides. In another embodiment, the length is at least
45 nucleotides. In another embodiment, the length is at least 55
nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 80
nucleotides. In another embodiment, the length is at least 90
nucleotides. In another embodiment, the length is at least 100
nucleotides. In another embodiment, the length is at least 120
nucleotides. In another embodiment, the length is at least 140
nucleotides. In another embodiment, the length is at least 160
nucleotides. In another embodiment, the length is at least 180
nucleotides. In another embodiment, the length is at least 200
nucleotides. In another embodiment, the length is at least 250
nucleotides. In another embodiment, the length is at least 300
nucleotides. In another embodiment, the length is at least 350
nucleotides. In another embodiment, the length is at least 400
nucleotides. In another embodiment, the length is at least 450
nucleotides. In another embodiment, the length is at least 500
nucleotides. In another embodiment, the length is at least 600
nucleotides. In another embodiment, the length is at least 700
nucleotides. In another embodiment, the length is at least 800
nucleotides. In another embodiment, the length is at least 900
nucleotides. In another embodiment, the length is at least 1000
nucleotides. In another embodiment, the length is at least 1100
nucleotides. In another embodiment, the length is at least 1200
nucleotides. In another embodiment, the length is at least 1300
nucleotides. In another embodiment, the length is at least 1400
nucleotides. In another embodiment, the length is at least 1500
nucleotides. In another embodiment, the length is at least 1600
nucleotides. In another embodiment, the length is at least 1800
nucleotides. In another embodiment, the length is at least 2000
nucleotides. In another embodiment, the length is at least 2500
nucleotides. In another embodiment, the length is at least 3000
nucleotides. In another embodiment, the length is at least 4000
nucleotides. In another embodiment, the length is at least 5000
nucleotides. In another embodiment, the length is at least 10000
nucleotides. In another embodiment, the length is at least 20000
nucleotides.
[0046] In another embodiment, the purified preparation of mRNA of
methods and compositions of the present invention is manufactured
by in vitro transcription.
[0047] In another embodiment, the step of in vitro transcription
utilizes T7 phage RNA polymerase. In another embodiment, the in
vitro transcription utilizes SP6 phage RNA polymerase. In another
embodiment, the in vitro transcription utilizes T3 phage RNA
polymerase. In another embodiment, the in vitro transcription
utilizes an RNA polymerase selected from the above polymerases. In
another embodiment, the in vitro transcription utilizes any other
RNA polymerase, or modified DNA polymerase, known in the art. In
another embodiment, the in vitro transcription utilizes chemical
synthesis.
[0048] In another embodiment, the nucleoside that is modified in an
RNA, oligoribonucleotide, or polyribonucleotide of the methods and
compositions of the present invention is uridine (U). In another
embodiment, the modified nucleoside is cytidine (C). In another
embodiment, the modified nucleoside is adenine (A). In another
embodiment the modified nucleoside is guanine (G).
[0049] In another embodiment, the modified nucleoside of the
methods and compositions of the present invention is m5C
(5-methylcytidine). In another embodiment, the modified nucleoside
is m5U (5-methyluridine). In another embodiment, the modified
nucleoside is m6A (N6-methyladenosine). In another embodiment, the
modified nucleoside is s2U (2-thiouridine). In another embodiment,
the modified nucleoside is .PSI. (pseudouridine). In another
embodiment, the modified nucleoside is Um (2'-O-methyluridine).
[0050] In other embodiments, the modified nucleoside is m1A
(1-methyladenosine); m2A (2-methyladenosine); Am
(2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine);
i6A (N6-isopentenyladenosine); ms2i6A
(2-methylthio-N6isopentenyladenosine); io6A
(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A
(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A
(N6-glycinylcarbamoyladenosine); t6A
(N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl
carbamoyladenosine); m6t6A
(N6-methyl-N6-threonylcarbamoyladenosine); hn6A
(N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A
(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)
(2'-O-ribosyladenosine (phosphate)); I (inosine); m1I
(1-methylinosine); m1Im (1,2'-O-dimethylinosine); m3C
(3-methylcytidine); Cm (2'-O-methylcytidine); s2C (2-thiocytidine);
ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm
(5,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine);
k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine);
m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G
(N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm
(N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine
(phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW
(hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG
(wyosine); mimG (methylwyosine); Q (queuosine); oQ
(epoxyqueuosine); galQ (galactosyl-queuosine); manQ
(mannosyl-queuosine); preQO (7-cyano-7-deazaguanosine); preQ1
(7-aminomethyl-7-deazaguanosine); G+(archaeosine); D
(dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U
(4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um
(2-thio-2'-O-methyluridine); acp3U
(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U
(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U
(uridine 5-oxyacetic acid methyl ester); chm5U
(5-(carboxyhydroxymethyl)uridine)); mchm5U
(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U
(5-methoxycarbonylmethyluridine); mcm5Um
(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U
(5-methoxycarbonylmethyl-2-thiouridine); nm5s2U
(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);
mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U
(5-methylaminomethyl-2-selenouridine); ncm5U
(5-carbamoylmethyluridine); ncm5Um
(5-carbamoylmethyl-2'-O-methyluridine); cmnm5U
(5-carboxymethylaminomethyluridine); cmnm5Um
(5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U
(5-carboxymethylaminomethyl-2-thiouridine); m62A
(N6,N6-dimethyladenosine); Im (2'-O-methylinosine); m4C
(N4-methylcytidine); m4Cm (N4,2'-O-dimethylcytidine); hm5C
(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U
(5-carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); m62Am
(N6,N6,O-2'-trimethyladenosine); m2,7G (N2,7-dimethylguanosine);
m2,2,7G (N2,N2,7-trimethylguanosine); m3Um
(3,2'-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm
(5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine);
m1Am (1,2'-O-dimethyladenosine); .tau.m5U (5-taurinomethyluridine);
.tau.m5s2U (5-taurinomethyl-2-thiouridine)); imG-14
(4-demethylwyosine); imG2 (isowyosine); or ac6A
(N6-acetyladenosine).
[0051] In another embodiment, the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide of the methods and
compositions of the present invention comprises a combination of
two or more of the above-described modifications. In another
embodiment, the purified preparation of the RNA or
oligoribonucleotide comprises a combination of three or more of the
above-described modifications. In another embodiment, the purified
preparation of the RNA or oligoribonucleotide comprises a
combination of more than three of the above-described
modifications.
[0052] Throughout this disclosure, various aspects of the invention
can be presented in a range format. It should be understood that
the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies
regardless of the breadth of the range.
[0053] In another embodiment, between 0.1% and 100% of the residues
in the RNA, oligoribonucleotide, or polyribonucleotide of the
methods and compositions of the present invention are modified
(e.g., either by the presence of pseudouridine or a modified
nucleoside base). In another embodiment, 0.1% of the residues are
modified. In another embodiment, 0.2% of the residues are modified.
In another embodiment, 0.3% of the residues are modified. In
another embodiment, 0.4% of the residues are modified. In another
embodiment, 0.5% of the residues are modified. In another
embodiment, 0.6% of the residues are modified. In another
embodiment, 0.8% of the residues are modified. In another
embodiment, 1% of the residues are modified. In another embodiment,
1.5% of the residues are modified. In another embodiment, 2% of the
residues are modified. In another embodiment, 2.5% of the residues
are modified. In another embodiment, 3% of the residues are
modified. In another embodiment, 4% of the residues are modified.
In another embodiment, 5% of the residues are modified. In another
embodiment, 6% of the residues are modified. In another embodiment,
8% of the residues are modified. In another embodiment, 10% of the
residues are modified. In another embodiment, 12% of the residues
are modified. In another embodiment, 14% of the residues are
modified. In another embodiment, 16% of the residues are modified.
In another embodiment, 18% of the residues are modified. In another
embodiment, 20% of the residues are modified. In another
embodiment, 25% of the residues are modified. In another
embodiment, 30% of the residues are modified. In another
embodiment, 35% of the residues are modified. In another
embodiment, 40% of the residues are modified. In another
embodiment, 45% of the residues are modified. In another
embodiment, 50% of the residues are modified. In another
embodiment, 60% of the residues are modified. In another
embodiment, 70% of the residues are modified. In another
embodiment, 80% of the residues are modified. In another
embodiment, 90% of the residues are modified. In another
embodiment, 100% of the residues are modified.
[0054] In another embodiment, less than 5% of the residues are
modified. In another embodiment, less than 3% of the residues are
modified. In another embodiment, less than 1% of the residues are
modified. In another embodiment, less than 2% of the residues are
modified. In another embodiment, the fraction is less than 4% of
the residues are modified. In another embodiment, less than 6% of
the residues are modified. In another embodiment, less than 8% of
the residues are modified. In another embodiment, less than 10% of
the residues are modified. In another embodiment, less than 12% of
the residues are modified. In another embodiment, less than 15% of
the residues are modified. In another embodiment, less than 20% of
the residues are modified. In another embodiment, less than 30% of
the residues are modified. In another embodiment, less than 40% of
the residues are modified. In another embodiment, less than 50% of
the residues are modified. In another embodiment, less than 60% of
the residues are modified. In another embodiment, less than 70% of
the residues are modified.
[0055] In another embodiment, 0.1% of the residues of a given
nucleotide (uridine, cytidine, guanosine, or adenine) are modified.
In another embodiment, the fraction of the given nucleotide that is
modified is 0.2%. In another embodiment, the fraction of the given
nucleotide that is modified is 0.3%. In another embodiment, the
fraction of the given nucleotide that is modified is 0.4%. In
another embodiment, the fraction of the given nucleotide that is
modified is 0.5%. In another embodiment, the fraction of the given
nucleotide that is modified is 0.6%. In another embodiment, the
fraction of the given nucleotide that is modified is 0.8%. In
another embodiment, the fraction of the given nucleotide that is
modified is 1%. In another embodiment, the fraction of the given
nucleotide that is modified is 1.5%. In another embodiment, the
fraction of the given nucleotide that is modified is 2%. In another
embodiment, the fraction of the given nucleotide that is modified
is 2.5%. In another embodiment, the fraction of the given
nucleotide that is modified is 3%. In another embodiment, the
fraction of the given nucleotide that is modified is 4%. In another
embodiment, the fraction of the given nucleotide that is modified
is 5%. In another embodiment, the fraction of the given nucleotide
that is modified is 6%. In another embodiment, the fraction of the
given nucleotide that is modified is 8%. In another embodiment, the
fraction of the given nucleotide that is modified is 10%. In
another embodiment, the fraction of the given nucleotide that is
modified is 12%. In another embodiment, the fraction of the given
nucleotide that is modified is 14%. In another embodiment, the
fraction of the given nucleotide that is modified is 16%. In
another embodiment, the fraction of the given nucleotide that is
modified is 18%. In another embodiment, the fraction of the given
nucleotide that is modified is 20%. In another embodiment, the
fraction of the given nucleotide that is modified is 25%. In
another embodiment, the fraction of the given nucleotide that is
modified is 30%. In another embodiment, the fraction of the given
nucleotide that is modified is 35%. In another embodiment, the
fraction of the given nucleotide that is modified is 40%. In
another embodiment, the fraction of the given nucleotide that is
modified is 45%. In another embodiment, the fraction of the given
nucleotide that is modified is 50%. In another embodiment, the
fraction of the given nucleotide that is modified is 60%. In
another embodiment, the fraction of the given nucleotide that is
modified is 70%. In another embodiment, the fraction of the given
nucleotide that is modified is 80%. In another embodiment, the
fraction of the given nucleotide that is modified is 90%. In
another embodiment, the fraction of the given nucleotide that is
modified is 100%.
[0056] In another embodiment, the fraction of the given nucleotide
that is modified is less than 8%. In another embodiment, the
fraction of the given nucleotide that is modified is less than 10%.
In another embodiment, the fraction of the given nucleotide that is
modified is less than 5%. In another embodiment, the fraction of
the given nucleotide that is modified is less than 3%. In another
embodiment, the fraction of the given nucleotide that is modified
is less than 1%. In another embodiment, the fraction of the given
nucleotide that is modified is less than 2%. In another embodiment,
the fraction of the given nucleotide that is modified is less than
4%. In another embodiment, the fraction of the given nucleotide
that is modified is less than 6%. In another embodiment, the
fraction of the given nucleotide that is modified is less than 12%.
In another embodiment, the fraction of the given nucleotide that is
modified is less than 15%. In another embodiment, the fraction of
the given nucleotide that is modified is less than 20%. In another
embodiment, the fraction of the given nucleotide that is modified
is less than 30%. In another embodiment, the fraction of the given
nucleotide that is modified is less than 40%. In another
embodiment, the fraction of the given nucleotide that is modified
is less than 50%. In another embodiment, the fraction of the given
nucleotide that is modified is less than 60%. In another
embodiment, the fraction is less than 70%.
[0057] In another embodiment, the terms "ribonucleotide,"
"oligoribonucleotide," and "polyribonucleotide" refers to a string
of at least 2 base-sugar-phosphate combinations. The term includes,
in another embodiment, compounds comprising nucleotides in which
the sugar moiety is ribose. In another embodiment, the term
includes both RNA and RNA derivatives in which the backbone is
modified. "Nucleotides" refers to the monomeric units of nucleic
acid polymers. RNA may be, in another embodiment, in the form of a
tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal
RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA
(siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and
miRNA has been described (Caudy et al., Genes & Devel 16:
2491-96 and references cited therein). In addition, these forms of
RNA may be single, double, triple, or quadruple stranded. The term
also includes, in another embodiment, artificial nucleic acids that
may contain other types of backbones but the same bases. In another
embodiment, the artificial nucleic acid is a PNA (peptide nucleic
acid). PNA contain peptide backbones and nucleotide bases and are
able to bind, in another embodiment, to both DNA and RNA molecules.
In another embodiment, the nucleotide is oxetane modified. In
another embodiment, the nucleotide is modified by replacement of
one or more phosphodiester bonds with a phosphorothioate bond. In
another embodiment, the artificial nucleic acid contains any other
variant of the phosphate backbone of native nucleic acids known in
the art. The use of phosphothiorate nucleic acids and PNA are known
to those skilled in the art, and are described in, for example,
Neilsen, Curr Opin Struct Biol 9:353-57; and Raz et al., Biochem
Biophys Res Commun. 297:1075-84. The production and use of nucleic
acids is known to those skilled in art and is described, for
example, in Molecular Cloning, (2012), Sambrook and Russell, eds.
and Methods in Enzymology: Methods for molecular cloning in
eukaryotic cells (2003) Purchio and G. C. Fareed. Each nucleic acid
derivative represents a separate embodiment of the present
invention
[0058] In another embodiment, the term "oligoribonucleotide" refers
to a string comprising fewer than 25 nucleotides (nt). In another
embodiment, "oligoribonucleotide" refers to a string of fewer than
24 nucleotides. In another embodiment, "oligoribonucleotide" refers
to a string of fewer than 23 nucleotides. In another embodiment,
"oligoribonucleotide" refers to a string of fewer than 22
nucleotides. In another embodiment, "oligoribonucleotide" refers to
a string of fewer than 21 nucleotides. In another embodiment,
"oligoribonucleotide" refers to a string of fewer than 20
nucleotides. In another embodiment, "oligoribonucleotide" refers to
a string of fewer than 19 nucleotides. In another embodiment,
"oligoribonucleotide" refers to a string of fewer than 18
nucleotides. In another embodiment, "oligoribonucleotide" refers to
a string of fewer than 17 nucleotides. In another embodiment,
"oligoribonucleotide" refers to a string of fewer than 16
nucleotides.
[0059] In another embodiment, the term "polyribonucleotide" refers
to a string comprising more than 25 nucleotides (nt). In another
embodiment, "polyribonucleotide" refers to a string of more than 26
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 28 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 30
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 32 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 35
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 40 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 50
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 60 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 80
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 100 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 120
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 150 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 200
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 300 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 400
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 500 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 600
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 800 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 1000
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 1200 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 1400
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 1600 nucleotides. In another embodiment,
"polyribonucleotide" refers to a string of more than 1800
nucleotides. In another embodiment, "polyribonucleotide" refers to
a string of more than 2000 nucleotides.
[0060] As used herein, "purified preparation" means that the
nucleic acid preparation predominately contains the nucleic acid of
interest and is substantially free of other nucleic acids which are
not the nucleic acid of interest. In one embodiment, at least about
75% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 76% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 77% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 78% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about 79%
the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 80% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 81% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 82% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about 83%
the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 84% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 85% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 86% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about 87%
the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 88% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 89% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 90% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about 91%
the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 92% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 93% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 94% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about 95%
the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 96% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 97% the nucleic acid present in
the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 98.1% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about
98.2% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 98.3% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 98.4% the nucleic acid present
in the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 98.5% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about
98.6% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 98.7% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 98.8% the nucleic acid present
in the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 98.9% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about
99.0% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 99.1% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 99.2% the nucleic acid present
in the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 99.3% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about
99.4% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 99.5% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest. In
another embodiment, at least about 99.6% the nucleic acid present
in the purified preparation of the invention is the nucleic acid of
interest. In another embodiment, at least about 99.7% the nucleic
acid present in the purified preparation of the invention is the
nucleic acid of interest. In another embodiment, at least about
99.8% the nucleic acid present in the purified preparation of the
invention is the nucleic acid of interest. In another embodiment,
at least about 99.9% the nucleic acid present in the purified
preparation of the invention is the nucleic acid of interest.
[0061] The nucleic acid of interest can be purified by any method
known in the art, or any method to be developed, so long as the
method of purification removes contaminants from the nucleic acid
preparation and thereby substantially reduces the immunogenicity
potential of the nucleic acid preparation. In one embodiment, the
nucleic acid of interest is purified using high-performance liquid
chromatography (HPLC). In another embodiment, the nucleic acid of
interest is purified by contacting the nucleic acid of interest
with the bacterial enzyme RNase III. In other various embodiments,
any method of nucleic acid purification that substantially reduces
the immunogenicity of the nucleic acid preparation can be used.
Non-limiting examples of purification methods that can be used with
the compositions and methods of the invention liquid chromatography
separation and enzyme digestion, each used alone or in any
combination, simultaneously or in any order. Non-limiting examples
of liquid chromatography separation include HPLC and fast protein
liquid chromatography (FPLC). Materials useful in the HPLC and FPLC
methods of the invention include, but are not limited to,
cross-linked polystyrene/divinylbenzene (PS/DVB), PS/DVB-C18,
PS/DVB-alkylated, Helix DNA columns (Varian), Eclipse dsDNA
Analysis Columns (Agilent Technologies), Reverse-phase 5 (RPC-5)
exchange material, DNAPac, ProSwift, and bio-inert UltiMate.RTM.
3000 Titanium columns (Dionex). Enzymes useful in the enzyme
digestion methods of the invention include any enzyme able to
digest any contaminant in a nucleic acid preparation of the
invention, such as, for example a dsRNA contaminant, and include
but are not limited to, RNase III, RNase V1, Dicer, and Chipper
(see Fruscoloni et al., 2002, PNAS 100:1639) Non-limiting examples
of assays for assessing the purity of the nucleic acid of interest
include a dot-blot assay, a Northern blot assay, and a dendritic
cell activation assay, as described elsewhere herein.
[0062] In another embodiment, the present invention provides a
method for inducing a mammalian cell to produce a protein of
interest, comprising contacting the mammalian cell with a purified
preparation of an in vitro-synthesized RNA comprising a
1-methyl-pseudouridine or a modified nucleoside and encoding a
recombinant protein, thereby inducing a mammalian cell to produce a
protein of interest. In another embodiment, the protein of interest
is a recombinant protein.
[0063] "Encoding" refers, in another embodiment, to an RNA that
contains a gene that encodes the protein of interest. In another
embodiment, the RNA comprises an open reading frame that encodes
the protein of interest. In another embodiment, one or more other
proteins are also encoded. In another embodiment, the protein of
interest is the only protein encoded.
[0064] In another embodiment, the present invention provides a
method of inducing a mammalian cell to produce a recombinant
protein, comprising contacting the mammalian cell with a purified
preparation of an in vitro-transcribed RNA comprising a
pseudouridine or a modified nucleoside and encoding a recombinant
protein, thereby inducing a mammalian cell to produce a recombinant
protein.
[0065] In another embodiment, the purified preparation of an RNA,
oligoribonucleotide, or polyribonucleotide of methods and
compositions of the present invention is translated in the cell
more efficiently than an unmodified RNA and/or unpurified RNA with
the same sequence. In another embodiment, the purified preparation
of the RNA, oligoribonucleotide, or polyribonucleotide exhibits
enhanced ability to be translated by a target cell. In another
embodiment, translation is enhanced by a factor of 2-fold relative
to its unmodified and/or unpurified counterpart. In another
embodiment, translation is enhanced by a 3-fold factor. In another
embodiment, translation is enhanced by a 5-fold factor. In another
embodiment, translation is enhanced by a 7-fold factor. In another
embodiment, translation is enhanced by a 10-fold factor. In another
embodiment, translation is enhanced by a 15-fold factor. In another
embodiment, translation is enhanced by a 20-fold factor. In another
embodiment, translation is enhanced by a 50-fold factor. In another
embodiment, translation is enhanced by a 100-fold factor. In
another embodiment, translation is enhanced by a 200-fold factor.
In another embodiment, translation is enhanced by a 500-fold
factor. In another embodiment, translation is enhanced by a
1000-fold factor. In another embodiment, translation is enhanced by
a 2000-fold factor. In another embodiment, the factor is
10-1000-fold. In another embodiment, the factor is 10-100-fold. In
another embodiment, the factor is 10-200-fold. In another
embodiment, the factor is 10-300-fold. In another embodiment, the
factor is 10-500-fold. In another embodiment, the factor is
20-1000-fold. In another embodiment, the factor is 30-1000-fold. In
another embodiment, the factor is 50-1000-fold. In another
embodiment, the factor is 100-1000-fold. In another embodiment, the
factor is 200-1000-fold. In another embodiment, translation is
enhanced by any other significant amount or range of amounts.
[0066] Methods of determining translation efficiency are well known
in the art, and include, e.g., measuring the activity of an encoded
reporter protein (e.g., luciferase or Renilla or green fluorescent
protein (Wall et al., J Biol Chem 2005; 280(30): 27670-8), or
measuring radioactive label incorporated into the translated
protein (Ngosuwan et al., 2003, J Biol Chem 278:7034-42).
[0067] In some embodiments, translation is measured from RNA
complexed to Lipofectin.RTM. (Gibco BRL, Gaithersburg, Md., USA)
and injected into the tail vein of mice. In the spleen lysates,
1-methyl-pseudouridine-modified RNA was translated significantly
more efficiently than unmodified RNA (see U.S. Pat. No. 8,278,036).
Under the conditions utilized, efficiency of transfection-based
methods of the present invention correlates with the ability of the
transfection reagent to penetrate into tissues.
[0068] In another embodiment, the enhanced translation is in a cell
(relative to translation in the same cell of an unmodified RNA, or
a modified but unpurified RNA, with the same sequence. In another
embodiment, the enhanced translation is in vitro (e.g. in an in
vitro translation mix or a reticulocyte lysate). In another
embodiment, the enhanced translation is in vivo. In each case, the
enhanced translation is relative to an unmodified RNA, or a
modified but unpurified RNA, with the same sequence, under the same
conditions.
[0069] In another embodiment, the purified preparation of the RNA,
oligoribonucleotide, or polyribonucleotide of methods and
compositions of the present invention is significantly less
immunogenic than an unmodified in vitro-synthesized RNA with the
same sequence, or compared with the unpurified, modified in
vitro-synthesized RNA with the same sequence. In another
embodiment, the purified RNA is 2-fold less immunogenic than its
unpurified counterpart. In another embodiment, immunogenicity is
reduced by a 3-fold factor. In another embodiment, immunogenicity
is reduced by a 5-fold factor. In another embodiment,
immunogenicity is reduced by a 7-fold factor. In another
embodiment, immunogenicity is reduced by a 10-fold factor. In
another embodiment, immunogenicity is reduced by a 15-fold factor.
In another embodiment, immunogenicity is reduced by a 20-fold
factor. In another embodiment, immunogenicity is reduced by a
50-fold factor. In another embodiment, immunogenicity is reduced by
a 100-fold factor. In another embodiment, immunogenicity is reduced
by a 200-fold factor. In another embodiment, immunogenicity is
reduced by a 500-fold factor. In another embodiment, immunogenicity
is reduced by a 1000-fold factor. In another embodiment,
immunogenicity is reduced by a 2000-fold factor. In another
embodiment, immunogenicity is reduced by another fold
difference.
[0070] In another embodiment, "significantly less immunogenic"
refers to a detectable decrease in immunogenicity. In another
embodiment, the term refers to a fold decrease in immunogenicity
(e.g., 1 of the fold decreases enumerated above). In another
embodiment, the term refers to a decrease such that an effective
amount of the purified preparation of RNA, oligoribonucleotide, or
polyribonucleotide can be administered without triggering a
detectable immune response. In another embodiment, the term refers
to a decrease such that the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide can be repeatedly
administered without eliciting an immune response sufficient to
detectably reduce expression of the recombinant protein. In another
embodiment, the decrease is such that the purified preparation of
RNA, oligoribonucleotide, or polyribonucleotide can be repeatedly
administered without eliciting an immune response sufficient to
eliminate detectable expression of the recombinant protein.
[0071] "Effective amount" of the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide refers, in another
embodiment, to an amount sufficient to exert a therapeutic effect.
In another embodiment, the term refers to an amount sufficient to
elicit expression of a detectable amount of the recombinant
protein.
[0072] Methods of determining immunogenicity are well known in the
art, and include, e.g. measuring secretion of cytokines (e.g.
IL-12, IFN-.alpha., TNF-.alpha., RANTES, MIP-1.alpha. or .beta.,
IL-6, IFN-.beta., or IL-8), measuring expression of DC activation
markers (e.g. CD83, HLA-DR, CD80 and CD86), or measuring ability to
act as an adjuvant for an adaptive immune response.
[0073] In another embodiment, the relative immunogenicity of the
modified nucleotide and its unmodified counterpart, as well as the
purified nucleotide and its unpurified counterpart, are determined
by determining the quantity of the nucleotide required to elicit
one of the above responses to the same degree as a given quantity
of the unmodified or unpurified nucleotide. For example, if twice
as much nucleotide (e.g., modified and/or purified) is required to
elicit the same response, than the nucleotide is two-fold less
immunogenic than the unmodified or unpurified nucleotide.
[0074] In another embodiment, the relative immunogenicity of the
purified nucleotide and its unpurified counterpart are determined
by determining the quantity of cytokine (e.g. IL-12, IFN-.alpha.,
TNF-.alpha., RANTES, MIP-1.alpha. or .beta., IL-6, IFN-.beta., or
IL-8) secreted in response to administration of the purified
nucleotide, relative to the same quantity of the unpurified
nucleotide. For example, if one-half as much cytokine is secreted,
than the purified nucleotide is two-fold less immunogenic than the
unpurified nucleotide. In another embodiment, background levels of
stimulation are subtracted before calculating the immunogenicity in
the above methods.
[0075] In another embodiment, a method of present invention further
comprises mixing the purified preparation of RNA,
oligoribonucleotide, or polyribonucleotide with a transfection
reagent prior to the step of contacting a target cell. In another
embodiment, a method of present invention further comprises
administering the purified preparation of RNA, oligoribonucleotide,
or polyribonucleotide together with the transfection reagent. In
another embodiment, the transfection reagent is a cationic lipid
reagent.
[0076] In another embodiment, the transfection reagent is a
lipid-based transfection reagent. In another embodiment, the
transfection reagent is a protein-based transfection reagent. In
another embodiment, the transfection reagent is a
carbohydrate-based transfection reagent. In another embodiment, the
transfection reagent is a polyethyleneimine-based transfection
reagent. In another embodiment, the transfection reagent is calcium
phosphate. In another embodiment, the transfection reagent is
Lipofectin.RTM. or Lipofectamine.RTM.. In another embodiment, the
transfection reagent is a lipid nanoparticle (Semple et al., 2010,
Nat Biotechnol. 28(2):172-176). In another embodiment, the
transfection reagent is any other transfection reagent known in the
art.
[0077] In another embodiment, the transfection reagent forms a
liposome. Liposomes, in another embodiment, increase intracellular
stability, increase uptake efficiency and improve biological
activity. In another embodiment, liposomes are hollow spherical
vesicles composed of lipids arranged in a similar fashion as those
lipids, which make up the cell membrane. They have, in another
embodiment, an internal aqueous space for entrapping water-soluble
compounds and range in size from 0.05 to several microns in
diameter. In another embodiment, liposomes can deliver RNA to cells
in a biologically active form.
[0078] In another embodiment, the target cell of methods of the
present invention is an antigen-presenting cell. In another
embodiment, the cell is an animal cell. In another embodiment, the
cell is a dendritic cell. In another embodiment, the cell is a
neural cell. In another embodiment, the cell is a brain cell. In
another embodiment, the cell is a spleen cell. In another
embodiment, the cell is a lymphoid cell. In another embodiment, the
cell is a lung cell. In another embodiment, the cell is a skin
cell. In another embodiment, the cell is a keratinocyte. In another
embodiment, the cell is an endothelial cell. In another embodiment,
the cell is an astrocyte, a microglial cell, or a neuron. In
another embodiment, the cell is an alveolar cell. In another
embodiment, the cell is a surface alveolar cell. In another
embodiment, the cell is an alveolar macrophage. In another
embodiment, the cell is an alveolar pneumocyte. In another
embodiment, the cell is a vascular endothelial cell. In another
embodiment, the cell is a mesenchymal cell. In another embodiment,
the cell is an epithelial cell. In another embodiment, the cell is
a hematopoietic cell. In another embodiment, the cell is colonic
epithelium cell. In another embodiment, the cell is a lung
epithelium cell. In another embodiment, the cell is a bone marrow
cell.
[0079] In other embodiments, the target cell is a Claudius' cell,
Hensen cell, Merkel cell, Muller cell, Paneth cell, Purkinje cell,
Schwann cell, Sertoli cell, acidophil cell, acinar cell,
adipoblast, adipocyte, brown or white alpha cell, amacrine cell,
beta cell, capsular cell, cementocyte, chief cell, chondroblast,
chondrocyte, chromaffin cell, chromophobic cell, corticotroph,
delta cell, Langerhans cell, follicular dendritic cell,
enterochromaffin cell, ependymocyte, epithelial cell, basal cell,
squamous cell, endothelial cell, transitional cell, erythroblast,
erythrocyte, fibroblast, fibrocyte, follicular cell, germ cell,
gamete, ovum, spermatozoon, oocyte, primary oocyte, secondary
oocyte, spermatid, spermatocyte, primary spermatocyte, secondary
spermatocyte, germinal epithelium, giant cell, glial cell,
astroblast, astrocyte, oligodendroblast, oligodendrocyte,
glioblast, goblet cell, gonadotroph, granulosa cell,
haemocytoblast, hair cell, hepatoblast, hepatocyte, hyalocyte,
interstitial cell, juxtaglomerular cell, keratinocyte, keratocyte,
lemmal cell, leukocyte, granulocyte, basophil, eosinophil,
neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast, lymphocyte,
B-lymphocyte, T-lymphocyte, helper induced T-lymphocyte, Th1
T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte,
macrophage, Kupffer cell, alveolar macrophage, foam cell,
histiocyte, luteal cell, lymphocytic stem cell, lymphoid cell,
lymphoid stem cell, macroglial cell, mammotroph, mast cell,
medulloblast, megakaryoblast, megakaryocyte, melanoblast,
melanocyte, mesangial cell, mesothelial cell, metamyelocyte,
monoblast, monocyte, mucous neck cell, muscle cell, cardiac muscle
cell, skeletal muscle cell, smooth muscle cell, myelocyte, myeloid
cell, myeloid stem cell, myoblast, myoepithelial cell,
myofibrobast, neuroblast, neuroepithelial cell, neuron,
odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,
parafollicular cell, paraluteal cell, peptic cell, pericyte,
peripheral blood mononuclear cell, phaeochromocyte, phalangeal
cell, pinealocyte, pituicyte, plasma cell, platelet, podocyte,
proerythroblast, promonocyte, promyeloblast, promyelocyte,
pronormoblast, reticulocyte, retinal pigment epithelial cell,
retinoblast, small cell, somatotroph, stem cell, sustentacular
cell, teloglial cell, or zymogenic cell.
[0080] A variety of disorders may be treated by employing the
purified preparations of the methods of the present invention
including, but not limited to, monogenic disorders, infectious
diseases, acquired disorders, cancer, and the like. Exemplary
monogenic disorders include ADA deficiency, cystic fibrosis,
familial-hypercholesterolemia, hemophilia, chronic ganulomatous
disease, Duchenne muscular dystrophy, Fanconi anemia, sickle-cell
anemia, Gaucher's disease, Hunter syndrome, X-linked SCID, and the
like. In another embodiment, the disorder treated involves one of
the proteins listed below.
[0081] In another embodiment, the recombinant protein encoded by
the purified preparation of an RNA, oligoribonucleotide, or
polyribonucleotide of methods and compositions of the present
invention is ecto-nucleoside triphosphate diphosphohydrolase. In
another embodiment, the recombinant protein is erythropoietin
(EPO).
[0082] In another embodiment, the encoded recombinant protein is
mammalian proteins, such as an immunoglobulin, or a fragment
thereof. In other embodiments, the encoded recombinant protein is a
bacterial protein, a viral protein or a phage protein. In one
embodiment, the encoded recombinant protein is streptokinase. In
another embodiment, the encoded recombinant protein is a phage
lysin.
[0083] In other embodiments, the encoded recombinant protein is at
least one of ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4A1; ALPL; AMPD1;
APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; CBA; C8B; CACNA1S; CCV;
CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1;
COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1;
DISCI; DPYD; EKV; ENOL; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A;
FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA; GFND;
GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2;
KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1;
MPZ; MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS;
NTRK1; OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A;
PLOD; PPDX; PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1;
RPE65; SCCD; SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1;
TNFSF6; TNNT2; TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS;
WS2B; ABCB11; ABCG5; ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP;
ALS2; APOB; BDE; BDMR; BJS; BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1;
COL4A3; COL4A4; COL6A3; CPS1; CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI;
DES; DYSF; EDAR; EFEMP1; EIF2AK3; ERCC3; FSHR; GINGF; GLC1B; GPD2;
GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRS 1; ITGA6; KHK;
KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1;
PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB; SLC11A1;
SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24;
WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET;
BBS3; BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP;
CPO; CRV; CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBEl; GLB1; GLC1C;
GNAI2; GNAT1; GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1;
MDS1; MHS4; MITF; MLH1; MYL3; MYMY; OPA1; P2RY12; PBXPl; PCCB;
POU1F1; PPARG; PROS1; PTHR1; RCAl; RHO; SCAT; SCLC1; SCN5A; SI;
SLC25A20; SLC2A2; TF; TGFBR2; THPO; THRB; TKT; TM4SF1; TRH; UMPS;
UQCRC1; USH3A; VHL; WS2A; XPC; ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2;
ALB; ASMD; BFHD; CNGA1; CRBM; DCK; DSPP; DTDP2; ELONG; ENAM; ETFDH;
EVC; F11; FABP2; FGA; FGB; FGFR3; FGG; FSHMD1A; GC; GNPTA; GNRHR;
GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF; JPD; KIT; KLKB1; LQT4;
MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1; PITX2; PKD2;
QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2; WFS1;
WHCR; ADAMTS2; ADRB2; AMCN; AP3B1; APC; ARSB; B4GALT7; BHR1; C6;
C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD;
ERVR; F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS;
LGMD1A; LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1;
PDE6A; PFBI; RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3;
SM1; SMA@; SMN1; SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1;
AS; ASSP2; BCKDHB; BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2;
DYX2; EJM1; ELOVL4; EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1;
GLYS1; GMPR; GSE; HCR; HFE; HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1;
IDDM1; IFNGR1; IGAD1; IGF2R; ISCW; LAMA2; LAP; LCA5; LPA; MCDR1;
MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3; ODDD; OFC1; PARK2; PBCA;
PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7; PLG; POLH; PPAC;
PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCAT; SCZD3;
SIASD; SOD2; ST8; TAP1; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT; TULP1;
WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM;
BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6;
CMT2D; COL1A2; CRS; CYMD; DFNA5; DLD; DYT11; EEC1; ELN; ETV1;
FKBP6; GCK; GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2;
HRX; IAB; IMMP2L; KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW;
PEX1; PGAM2; PMS2; PON1; PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9;
SERPINE1; SGCE; SHFM1; SHH; SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1;
TWIST; ZWS1; ACHM3; ADRB3; ANK1; CA1; CA2; CCAL1; CLN8; CMT4A;
CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2; DECR1; DPYS; DURS1; EBS1;
ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR; GULOP; HR; KCNQ3; KFM;
KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2; NBS1; PLAT; PLEC1;
PRKDC; PXMP3; RP1; SCZD6; SFTPC; SGM1; SPG5A; STAR; TG; TRPS1;
TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD; ALDH1A1;
ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1; CMD1B;
COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD; FRDA;
GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1; LALL;
LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1; PAPPA;
PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH; SPTLC1;
STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2; COL17A1;
CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2;
HK1; HPS1; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT;
OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4; RDPA; RET;
SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA;
ABCC8; ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2;
BWS; CALCA; CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF;
CPT1A; CTSC; DDB1; DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2;
F2; FSHB; FTH1; G6PT1; G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1;
HBG2; HMBS; HND; HOMG2; HRAS; HVBS1; IDDM2; IGER; INS; JBS; KCNJ11;
KCNJ1; KCNQ1; LDHA; LRP5; MEN1; MLL; MYBPC3; MYO7A; NNO1; OPPG;
OPTB1; PAX6; PC; PDX1; PGL2; PGR; PORC; PTH; PTS; PVRL1; PYGM;
RAG1; RAG2; ROM1; RRAS2; SAM; SCA5; SCZD2; SDHD; SERPING1; SMPD1;
TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C; VMD2; VRNI; WT1;
WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2; AMHR2; AOM;
AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1; CYP27B1;
DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2; HMN2;
HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5;
KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH;
PPKB; PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL;
SPPM; SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B;
BRCA2; BRCD1; CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2;
GJB6; IPF1; MBS1; MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG;
SLC10A2; SLC25A15; STARP1; ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1;
DFNB5; EML1; GALC; GCH1; IBGC1; IGH@; IGHC group; IGHG1; IGHM;
IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1; MPS3C; MYH6; MYH7; NP;
NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1; SERPINA3; SERPINA6;
SLC7A7; SPG3A; SPTB; TCL1A; TGM1; TITF1; TMIP; TRA@; TSHR; USH1A;
VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3; CLN6;
CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1;
FES; HCVS; HEXA; IVD; LCS1; LIPC; MYO5A; OCA2; OTSC1; PWCR; RLBP1;
SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2;
CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA;
CYLD; DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1;
HBA2; HBHR; HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV;
MHC2TA; MLYCD; MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1;
SCA4; SCNN1B; SCNN1G; SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA;
ACADVL; ACE; ALDH3A2; APOH; ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1;
CACD; CCA1; CCZS; CHRNB1; CHRNE; CMT1A; COL1A1; CORD5; CTNS; EPX;
ERBB2; G6PC; GAA; GALK1; GCGR; GFAP; GH1; GH2; GP1BA; GPSC; GUCY2D;
ITGA2B; ITGB3; ITGB4; KRT10; KRT12; KRT13; KRT14; KRT14L1; KRT14L2;
KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17; KRT9; MAPT; MDB; MDCR;
MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1; NME1; P4HB;
PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA; PRKWNK4; PRP8;
PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A; SERPINF2; SGCA;
SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9; SSTR2; SYM1;
SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH; ATP8B1;
BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2;
MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2;
AMH; APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO;
CEACAM5; COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2;
EPOR; ERCC2; ETFB; EXT3; EYCL1; FTL; FUT1; FUT2; FUT6; GAMT; GCDH;
GPI; GUSM; HB1; HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR;
LHB; LIG1; LOH19CR1; LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3;
NPHS1; OFC3; OPA3; PEPD; PRPF31; PRTN3; PRX; PSG1; PVR; RYR1;
SLC5A5; SLC7A9; STK11; TBXA2R; TGFB1; TNNI3; TYROBP; ADA; AHCY;
AVP; CDAN2; CDPD1; CHED1; CHED2; CHRNA4; CST3; EDN3; EEGV1; FTLL1;
GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2; MKKS; NBIA1; PCK1; PI3; PPCD;
PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS; COL6A1; COL6A2; CSTB; DCR;
DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO; PRSS7; RUNX1; SOD1;
TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2; CSF2RB; CTHM;
CYP2D6; CYP2D7P1; DGCR; DIA1; EWSR1; GGT1; MGCR; MN1; NAGA; NF2;
OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5A1; SOX10;
TCN2; TIMP3; TST; VCF; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC; AIED;
AIH3; ALAS2; AMCD; AMELX; ANOP1; AR; ARAF1; ARSC2; ARSE; ARTS; ARX;
ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR;
CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39C; CIDX;
CLA2; CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6;
CPX; CVD1; CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD;
DSS; DYT3; EBM; EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5;
FGD1; FGS1; FMR1; FMR2; G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK;
GLA; GPC3; GRPR; GTD; GUST; HMS1; HPRT1; HPT; HTC2; HTR2C; HYR;
IDS; IHG1; IL2RG; INDX; IP1; IP2; JMS; KAL1; KFSD; L1CAM; LAMP2;
MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX; MECP2; MF4; MGC1; MICS;
MIDI; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20; MRX2; MRX3; MRX40;
MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS; NPHL1; NR0B1; NSX; NYS1;
NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1; OPEM; OPN1LW; OPN1MW;
OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX; PHKA1;
PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPS1;
PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3;
RS1; S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2;
SRPX; SRS; STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC;
TIMM8A; TIMP1; TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC;
XIST; XK; XM; XS; ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6;
AZF1; AZF2; DAZ; GCY; RPS4Y; SMCY; SRY; ZFY; ABAT; AEZ; AFA; AFD1;
ASAH1; ASD1; ASMT; CCAT; CECR9; CEPA; CLA3; CLN4; CSF2RA; CTS1; DF;
DIH1; DWS; DYT2; DYT4; EBR3; ECT; EEF1A1L14; EYCL2; FANCB; GCSH;
GCSL; GIP; GTS; HHG; HMI; HOAC; HOKPP2; HRPT1; HSD3B3; HTC1; HV1S;
ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS; KRT18; KSS; LCAT; LHON; LIMM;
MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS; MSS; MTATP6; MTCO1;
MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6; MTRNR1; MTRNR2;
MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD;
OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS;
RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD.
[0084] In another embodiment, the present invention provides a
method for treating anemia in a subject, comprising contacting a
cell of the subject with a purified preparation of an in
vitro-synthesized RNA molecule, the in vitro-synthesized RNA
encoding erythropoietin, thereby treating anemia in a subject. In
another embodiment, the in vitro-synthesized RNA further comprises
a pseudouridine or a modified nucleoside. In another embodiment,
the cell is a subcutaneous tissue cell. In another embodiment, the
cell is a lung cell. In another embodiment, the cell is a
fibroblast. In another embodiment, the cell is a lymphocyte. In
another embodiment, the cell is a smooth muscle cell. In another
embodiment, the cell is any other type of cell known in the
art.
[0085] In another embodiment, the present invention provides a
method for treating a vasospasm in a subject, comprising contacting
a cell of the subject with a purified preparation of an in
vitro-synthesized RNA molecule, the in vitro-synthesized RNA
encoding inducible nitric oxide synthase (iNOS), thereby treating a
vasospasm in a subject.
[0086] In another embodiment, the present invention provides a
method for improving a survival rate of a cell in a subject,
comprising contacting the cell with a purified preparation of an in
vitro-synthesized RNA molecule, the in vitro-synthesized RNA
encoding a heat shock protein, thereby improving a survival rate of
a cell in a subject. In another embodiment, the cell whose survival
rate is improved is an ischemic cell. In another embodiment, the
cell is not ischemic. In another embodiment, the cell has been
exposed to an ischemic environment. In another embodiment, the cell
has been exposed to an environmental stress.
[0087] In another embodiment, the present invention provides a
method for decreasing an incidence of restenosis of a blood vessel
following a procedure that enlarges the blood vessel, comprising
contacting a cell of the blood vessel with a purified preparation
of an in vitro-synthesized RNA molecule, the in vitro-synthesized
RNA encoding a heat shock protein, thereby decreasing an incidence
of restenosis in a subject.
[0088] In another embodiment, the present invention provides a
method for increasing a hair growth from a hair follicle is a scalp
of a subject, comprising contacting a cell of the scalp with a
purified preparation of an in vitro-synthesized RNA molecule, the
in vitro-synthesized RNA encoding a telomerase or an
immunosuppressive protein, thereby increasing a hair growth from a
hair follicle. In another embodiment, the immunosuppressive protein
is .alpha.-melanocyte-stimulating hormone (.alpha.-MSH). In another
embodiment, the immunosuppressive protein is transforming growth
factor-.beta.1 (TGF-.beta.1). In another embodiment, the
immunosuppressive protein is insulin-like growth factor-I (IGF-I).
In another embodiment, the immunosuppressive protein is any other
immunosuppressive protein known in the art.
[0089] In another embodiment, the present invention provides a
method of inducing expression of an enzyme with antioxidant
activity in a cell, comprising contacting the cell with a purified
preparation of an in vitro-synthesized RNA molecule, the in
vitro-synthesized RNA encoding the enzyme, thereby inducing
expression of an enzyme with antioxidant activity in a cell. In
another embodiment, the enzyme is catalase. In another embodiment,
the enzyme is glutathione peroxidase. In another embodiment, the
enzyme is phospholipid hydroperoxide glutathione peroxidase. In
another embodiment, the enzyme is superoxide dismutase-1. In
another embodiment, the enzyme is superoxide dismutase-2. In
another embodiment, the enzyme is any other enzyme with antioxidant
activity that is known in the art. In another embodiment, the
present invention provides a method for treating cystic fibrosis in
a subject, comprising contacting a cell of the subject with a
purified preparation of an in vitro-synthesized RNA molecule, the
in vitro-synthesized RNA encoding Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR), thereby treating cystic fibrosis in a
subject.
[0090] In another embodiment, the present invention provides a
method for treating damage to heart muscle with a purified,
nucleoside modified mRNA encoding VEGF-A. In another embodiment,
the present invention provides a method for treating an X-linked
agammaglobulinemia in a subject, comprising contacting a cell of
the subject with a purified preparation of an in vitro-synthesized
RNA molecule, the in vitro-synthesized RNA encoding a Bruton's
tyrosine kinase, thereby treating an X-linked
agammaglobulinemia.
[0091] In another embodiment, the present invention provides a
method for treating an adenosine deaminase severe combined
immunodeficiency (ADA SCID) in a subject, comprising contacting a
cell of the subject with a purified preparation of a purified
preparation of an in vitro-synthesized RNA molecule, the in
vitro-synthesized RNA encoding an ADA, thereby treating an ADA
SCID.
[0092] In another embodiment, the present invention provides a
method for reducing immune responsiveness of the skin and improve
skin pathology, comprising contacting a cell of the subject with a
purified preparation of an in vitro-synthesized RNA molecule, the
in vitro-synthesized RNA encoding an ecto-nucleoside triphosphate
diphosphohydrolase, thereby reducing immune responsiveness of the
skin and improve skin pathology.
[0093] In another embodiment, the purified preparation of an RNA or
ribonucleotide of the present invention is encapsulated in a
nanoparticle. Methods for nanoparticle packaging are well known in
the art, and are described, for example, in Bose et al., (2004, J.
Virol. 78:8146); Dong et al., (2005, Biomaterials 26:6068);
Lobenberg et al., (1998, J Drug Target 5:171); Sakuma et al.,
(1999, Int J Pharm 177:161. 1999); Virovic et al., (2005, Expert
Opin Drug Deliv 2:707); and Zimmermann et al., (2001, Eur J Pharm
Biopharm 52:203).
[0094] Various embodiments of dosage ranges of compounds of the
present invention can be used in methods of the present invention.
In one embodiment, the dosage is in the range of 1-10 .mu.g/day. In
another embodiment, the dosage is 2-10 .mu.g/day. In another
embodiment, the dosage is 3-10 .mu.g/day. In another embodiment,
the dosage is 5-10 .mu.g/day. In another embodiment, the dosage is
2-20 .mu.g/day. In another embodiment, the dosage is 3-20
.mu.g/day. In another embodiment, the dosage is 5-20 .mu.g/day. In
another embodiment, the dosage is 10-20 .mu.g/day. In another
embodiment, the dosage is 3-40 .mu.g/day. In another embodiment,
the dosage is 5-40 .mu.g/day. In another embodiment, the dosage is
10-40 .mu.g/day. In another embodiment, the dosage is 20-40
.mu.g/day. In another embodiment, the dosage is 5-50 .mu.g/day. In
another embodiment, the dosage is 10-50 .mu.g/day. In another
embodiment, the dosage is 20-50 .mu.g/day. In one embodiment, the
dosage is 1-100 .mu.g/day. In another embodiment, the dosage is
2-100 .mu.g/day. In another embodiment, the dosage is 3-100
.mu.g/day. In another embodiment, the dosage is 5-100 .mu.g/day. In
another embodiment the dosage is 10-100 .mu.g/day. In another
embodiment the dosage is 20-100 .mu.g/day. In another embodiment
the dosage is 40-100 .mu.g/day. In another embodiment the dosage is
60-100 .mu.g/day.
[0095] In another embodiment, the dosage is 0.1 .mu.g/day. In
another embodiment, the dosage is 0.2 .mu.g/day. In another
embodiment, the dosage is 0.3 .mu.g/day. In another embodiment, the
dosage is 0.5 .mu.g/day. In another embodiment, the dosage is 1
.mu.g/day. In another embodiment, the dosage is 2 mg/day. In
another embodiment, the dosage is 3 .mu.g/day. In another
embodiment, the dosage is 5 .mu.g/day. In another embodiment, the
dosage is 10 .mu.g/day. In another embodiment, the dosage is 15
.mu.g/day. In another embodiment, the dosage is 20 .mu.g/day. In
another embodiment, the dosage is 30 .mu.g/day. In another
embodiment, the dosage is 40 .mu.g/day. In another embodiment, the
dosage is 60 .mu.g/day. In another embodiment, the dosage is 80
.mu.g/day. In another embodiment, the dosage is 100 .mu.g/day.
[0096] In another embodiment, the dosage is 10 .mu.g/dose. In
another embodiment, the dosage is 20 .mu.g/dose. In another
embodiment, the dosage is 30 .mu.g/dose. In another embodiment, the
dosage is 40 .mu.g/dose. In another embodiment, the dosage is 60
.mu.g/dose. In another embodiment, the dosage is 80 .mu.g/dose. In
another embodiment, the dosage is 100 .mu.g/dose. In another
embodiment, the dosage is 150 .mu.g/dose. In another embodiment,
the dosage is 200 .mu.g/dose. In another embodiment, the dosage is
300 .mu.g/dose. In another embodiment, the dosage is 400
.mu.g/dose. In another embodiment, the dosage is 600 .mu.g/dose. In
another embodiment, the dosage is 800 .mu.g/dose. In another
embodiment, the dosage is 1000 .mu.g/dose. In another embodiment,
the dosage is 1.5 mg/dose. In another embodiment, the dosage is 2
mg/dose. In another embodiment, the dosage is 3 mg/dose. In another
embodiment, the dosage is 5 mg/dose. In another embodiment, the
dosage is 10 mg/dose. In another embodiment, the dosage is 15
mg/dose. In another embodiment, the dosage is 20 mg/dose. In
another embodiment, the dosage is 30 mg/dose. In another
embodiment, the dosage is 50 mg/dose. In another embodiment, the
dosage is 80 mg/dose. In another embodiment, the dosage is 100
mg/dose.
[0097] In another embodiment, the dosage is 10-20 .mu.g/dose. In
another embodiment, the dosage is 20-30 .mu.g/dose. In another
embodiment, the dosage is 20-40 .mu.g/dose. In another embodiment,
the dosage is 30-60 .mu.g/dose. In another embodiment, the dosage
is 40-80 .mu.g/dose. In another embodiment, the dosage is 50-100
.mu.g/dose. In another embodiment, the dosage is 50-150 .mu.g/dose.
In another embodiment, the dosage is 100-200 .mu.g/dose. In another
embodiment, the dosage is 200-300 .mu.g/dose. In another
embodiment, the dosage is 300-400 .mu.g/dose. In another
embodiment, the dosage is 400-600 .mu.g/dose. In another
embodiment, the dosage is 500-800 .mu.g/dose. In another
embodiment, the dosage is 800-1000 .mu.g/dose. In another
embodiment, the dosage is 1000-1500 .mu.g/dose. In another
embodiment, the dosage is 1500-2000 .mu.g/dose. In another
embodiment, the dosage is 2-3 mg/dose. In another embodiment, the
dosage is 2-5 mg/dose. In another embodiment, the dosage is 2-10
mg/dose. In another embodiment, the dosage is 2-20 mg/dose. In
another embodiment, the dosage is 2-30 mg/dose. In another
embodiment, the dosage is 2-50 mg/dose. In another embodiment, the
dosage is 2-80 mg/dose. In another embodiment, the dosage is 2-100
mg/dose. In another embodiment, the dosage is 3-10 mg/dose. In
another embodiment, the dosage is 3-20 mg/dose. In another
embodiment, the dosage is 3-30 mg/dose. In another embodiment, the
dosage is 3-50 mg/dose. In another embodiment, the dosage is 3-80
mg/dose. In another embodiment, the dosage is 3-100 mg/dose. In
another embodiment, the dosage is 5-10 mg/dose. In another
embodiment, the dosage is 5-20 mg/dose. In another embodiment, the
dosage is 5-30 mg/dose. In another embodiment, the dosage is 5-50
mg/dose. In another embodiment, the dosage is 5-80 mg/dose. In
another embodiment, the dosage is 5-100 mg/dose. In another
embodiment, the dosage is 10-20 mg/dose. In another embodiment, the
dosage is 10-30 mg/dose. In another embodiment, the dosage is 10-50
mg/dose. In another embodiment, the dosage is 10-80 mg/dose. In
another embodiment, the dosage is 10-100 mg/dose.
[0098] In another embodiment, the dosage is a daily dose. In
another embodiment, the dosage is a weekly dose. In another
embodiment, the dosage is a monthly dose. In another embodiment,
the dosage is an annual dose. In another embodiment, the dose is
one is a series of a defined number of doses. In another
embodiment, the dose is a one-time dose. As described below, in
another embodiment, an advantage of RNA, oligoribonucleotide, or
polyribonucleotide molecules of the present invention is their
greater potency, enabling the use of smaller doses.
[0099] In another embodiment, the present invention provides a
method for producing a recombinant protein, comprising contacting
an in vitro translation apparatus with an in vitro-synthesized
oligoribonucleotide, the in vitro-synthesized oligoribonucleotide
comprising a 1-methyl-pseudouridine or a modified nucleoside,
thereby producing a recombinant protein.
[0100] In another embodiment, the present invention provides a
method for producing a recombinant protein, comprising contacting
an in vitro translation apparatus with an in vitro-transcribed RNA
of the present invention, the in vitro-transcribed RNA comprising a
pseudouridine or a modified nucleoside, thereby producing a
recombinant protein.
[0101] In another embodiment, the present invention provides an in
vitro transcription apparatus, comprising: an unmodified
nucleotide, a nucleotide containing a pseudouridine or a modified
nucleoside, and a polymerase. In another embodiment, the present
invention provides an in vitro transcription kit, comprising: an
unmodified nucleotide, a nucleotide containing a pseudouridine or a
modified nucleoside, a polymerase and instructional material. As
used herein, an "instructional material" includes a publication, a
recording, a diagram, or any other medium of expression, which can
be used to communicate the usefulness of a compound, composition,
or method of the invention in a kit. The instructional material of
the kit of the invention can, for example, be affixed to a
container which contains the identified compound, composition, or
method of the invention or be shipped together with a container,
which contains the identified compound, composition, or method of
the invention. Alternatively, the instructional material can be
shipped separately from the container with the intention that the
instructional material and the compound, composition, or method of
the invention be used cooperatively by the recipient.
[0102] In another embodiment, the present invention provides a
method of reducing the immunogenicity of an oligoribonucleotide or
RNA molecule, the method comprising the step of replacing a
nucleotide of the oligoribonucleotide or RNA with a modified
nucleotide that contains a modified nucleoside or a
1-methyl-pseudouridine, and purifying the preparation of modified
oligoribonucleotide or RNA molecule, thereby reducing the
immunogenicity of an oligoribonucleotide or RNA molecule.
[0103] In another embodiment, the present invention provides a
method of reducing the immunogenicity of a gene-therapy vector
comprising a polyribonucleotide or RNA molecule, the method
comprising the step of replacing a nucleotide of the
polyribonucleotide or RNA with a modified nucleotide that contains
a modified nucleoside or a 1-methyl-pseudouridine, and purifying
the preparation of modified oligoribonucleotide or RNA molecule,
thereby reducing the immunogenicity of a gene-therapy vector.
[0104] In another embodiment, the present invention provides a
method of enhancing in vitro translation from an
oligoribonucleotide or RNA molecule, the method comprising the step
of replacing a nucleotide of the oligoribonucleotide or RNA with a
modified nucleotide that contains a modified nucleoside or a
1-methyl-pseudouridine, and purifying the preparation of modified
oligoribonucleotide or RNA molecule, thereby enhancing in vitro
translation from an oligoribonucleotide or RNA molecule.
[0105] In another embodiment, the present invention provides a
method of enhancing in vivo translation from a gene-therapy vector
comprising a polyribonucleotide or RNA molecule, the method
comprising the step of replacing a nucleotide of the
polyribonucleotide or RNA with a modified nucleotide that contains
a modified nucleoside or a 1-methyl-pseudouridine, and purifying
the preparation of modified oligoribonucleotide or RNA molecule,
thereby enhancing in vivo translation from a gene-therapy
vector.
[0106] In another embodiment, the present invention provides a
method of increasing efficiency of delivery of a recombinant
protein by a gene therapy vector comprising a polyribonucleotide or
RNA molecule, the method comprising the step of replacing a
nucleotide of the polyribonucleotide or RNA with a modified
nucleotide that contains a modified nucleoside or a
1-methyl-pseudouridine, and purifying the preparation of modified
oligoribonucleotide or RNA molecule, thereby increasing efficiency
of delivery of a recombinant protein by a gene therapy vector.
[0107] In another embodiment, the present invention provides a
method of increasing in vivo stability of gene therapy vector
comprising a polyribonucleotide or RNA molecule, the method
comprising the step of replacing a nucleotide of the
polyribonucleotide or RNA with a modified nucleotide that contains
a modified nucleoside or a 1-methyl-pseudouridine, and purifying
the preparation of modified oligoribonucleotide or RNA molecule,
thereby increasing in vivo stability of gene therapy vector.
[0108] In another embodiment, the present invention provides a
method of reducing the ability of an RNA to stimulate signaling by
TLR3, comprising modifying a nucleoside of the RNA by a method of
the present invention, and purifying the preparation of modified
RNA. In another embodiment, the present invention provides a method
of reducing the ability of an RNA to stimulate signaling by TLR7,
comprising modifying a nucleoside of the RNA by a method of the
present invention, and purifying the preparation of modified RNA.
In another embodiment, the present invention provides a method of
reducing the ability of an RNA to stimulate signaling by TLR8,
comprising modifying a nucleoside of the RNA by a method of the
present invention, and purifying the preparation of modified
RNA.
[0109] In another embodiment, the present invention provides a
method of reducing the ability of an RNA to stimulate signaling by
PKR, comprising modifying a nucleoside of the RNA by a method of
the present invention, and purifying the preparation of modified
RNA. In another embodiment, the present invention provides a method
of reducing the ability of an RNA to stimulate signaling by
oligoadenylates synthase, comprising modifying a nucleoside of the
RNA by a method of the present invention, and purifying the
preparation of modified RNA. In another embodiment, the present
invention provides a method of reducing the ability of an RNA to
stimulate signaling by RIG-I, comprising modifying a nucleoside of
the RNA by a method of the present invention, and purifying the
preparation of modified RNA. In another embodiment, the present
invention provides a method of reducing the ability of an RNA to
stimulate signaling by MDA5, comprising modifying a nucleoside of
the RNA by a method of the present invention, and purifying the
preparation of modified RNA. In another embodiment, the present
invention provides a method of reducing the ability of an RNA to
stimulate signaling by NOD2, comprising modifying a nucleoside of
the RNA by a method of the present invention, and purifying the
preparation of modified RNA. In another embodiment, the present
invention provides a method of reducing the ability of an RNA to
stimulate signaling by DDX41, comprising modifying a nucleoside of
the RNA by a method of the present invention, and purifying the
preparation of modified RNA. In another embodiment, the present
invention provides a method of reducing the ability of an RNA to
stimulate signaling by NALP3, comprising modifying a nucleoside of
the RNA by a method of the present invention, and purifying the
preparation of modified RNA.
[0110] In another embodiment, all the inter-nucleotide linkages in
the RNA, oligoribonucleotide, or polyribonucleotide are
phosphodiester. In another embodiment, the inter-nucleotide
linkages are predominantly phosphodiester. In another embodiment,
most of the inter-nucleotide linkages are phosphorothioate. In
another embodiment, most the inter-nucleotide linkages are
phosphodiester.
[0111] In another embodiment, the percentage of the
inter-nucleotide linkages that are phosphodiester is above 50%. In
another embodiment, the percentage is above 10%. In another
embodiment, the percentage is above 15%. In another embodiment, the
percentage is above 20%. In another embodiment, the percentage is
above 25%. In another embodiment, the percentage is above 30%. In
another embodiment, the percentage is above 35%. In another
embodiment, the percentage is above 40%. In another embodiment, the
percentage is above 45%. In another embodiment, the percentage is
above 55%. In another embodiment, the percentage is above 60%. In
another embodiment, the percentage is above 65%. In another
embodiment, the percentage is above 70%. In another embodiment, the
percentage is above 75%. In another embodiment, the percentage is
above 80%. In another embodiment, the percentage is above 85%. In
another embodiment, the percentage is above 90%. In another
embodiment, the percentage is above 95%.
[0112] In another embodiment, a method of the present invention
comprises increasing the number, percentage, or frequency of
modified nucleosides in the RNA to decrease immunogenicity or
increase efficiency of translation. As provided herein, the number
of modified residues in an RNA, oligoribonucleotide, or
polyribonucleotide determines, in another embodiment, the magnitude
of the effects observed in the present invention.
[0113] In another embodiment, the present invention provides a
method for introducing a recombinant protein into a cell of a
subject, comprising contacting the subject with a purified
preparation of an in vitro-transcribed RNA encoding the recombinant
protein, the in vitro-transcribed RNA further comprising a modified
nucleoside, thereby introducing a recombinant protein into a cell
of a subject.
[0114] In another embodiment, the present invention provides a
method for decreasing TNF-.alpha. production in response to a gene
therapy vector in a subject, comprising the step of engineering the
vector to contain a pseudouridine or a modified nucleoside base,
and purifying the preparation of the gene therapy vector, thereby
decreasing TNF-.alpha. production in response to a gene therapy
vector in a subject.
[0115] In another embodiment, the present invention provides a
method for decreasing IFN-.alpha. production in response to a gene
therapy vector in a subject, comprising the step of engineering the
vector to contain a pseudouridine or a modified nucleoside base,
and purifying the preparation of the gene therapy vector, thereby
decreasing IFN-.alpha. production in response to a gene therapy
vector in a subject.
[0116] In another embodiment, the present invention provides a
method for decreasing IFN-.beta. production in response to a gene
therapy vector in a subject, comprising the step of engineering the
vector to contain a pseudouridine or a modified nucleoside base,
and purifying the preparation of the gene therapy vector, thereby
decreasing IFN-.beta. production in response to a gene therapy
vector in a subject.
[0117] In another embodiment, the present invention provides a
method for decreasing IL-12 production in response to a gene
therapy vector in a subject, comprising the step of engineering the
vector to contain a pseudouridine or a modified nucleoside base,
and purifying the preparation of the gene therapy vector, thereby
decreasing IL-12 production in response to a gene therapy vector in
a subject.
[0118] In another embodiment, the present invention provides a
method of reducing an immunogenicity of a gene therapy vector,
comprising introducing a modified nucleoside into the gene therapy
vector, and purifying the preparation of the gene therapy vector,
thereby reducing an immunogenicity of a gene therapy vector.
[0119] In another embodiment, an advantage of the purified
preparation of an RNA, oligoribonucleotide, and polyribonucleotide
of the present invention is that RNA does not incorporate to the
genome (as opposed to DNA-based vectors). In another embodiment, an
advantage is that translation of RNA, and therefore appearance of
the encoded product, is instant. In another embodiment, an
advantage is that the amount of protein generated from the mRNA can
be regulated by delivering more or less RNA. In another embodiment,
an advantage is that repeated delivery of unmodified RNA could
induce autoimmune reactions. In another embodiment, an advantage is
that reducing or removing contaminants from the RNA preparation
reduces or eliminates immunogenicity of the purified RNA
preparation as compared with the unpurified RNA preparation. In
another embodiment, an advantage is that reducing or removing dsRNA
from the RNA preparation reduces or eliminates immunogenicity of
the purified RNA preparation as compared with the unpurified RNA
preparation.
[0120] In another embodiment, an advantage is lack of
immunogenicity, enabling repeated delivery without generation of
inflammatory cytokines.
[0121] In another embodiment, the present invention provides a kit
comprising a reagent utilized in performing a method of the present
invention. In another embodiment, the present invention provides a
kit comprising a composition, tool, instructional material, or
instrument of the present invention.
[0122] In another embodiment, a treatment protocol of the present
invention is therapeutic. In another embodiment, the protocol is
prophylactic.
[0123] In one embodiment, the phrase "contacting a cell" or
"contacting a population" refers to a method of exposure, which can
be direct or indirect. In one method such contact comprises direct
injection of the cell through any means well known in the art, such
as microinjection. In another embodiment, supply to the cell is
indirect, such as via provision in a culture medium that surrounds
the cell, or administration to a subject, or via any route known in
the art. In another embodiment, the term "contacting" means that
the purified preparation of the present invention is introduced
into a subject receiving treatment, and the purified preparation is
allowed to come in contact with the cell in vivo.
[0124] In various embodiments, the purified preparations of the
compositions of the present invention can be administered to a
subject by any method known to a person skilled in the art, such as
parenterally, paracancerally, transmucosally, transdermally,
intramuscularly, intravenously, intra-dermally, subcutaneously,
intra-peritonealy, intra-ventricularly, intra-cranially,
intra-vaginally or intra-tumorally.
[0125] In another embodiment, the purified preparations of the
methods and compositions of the present invention, the compositions
are administered orally, and are thus formulated in a form suitable
for oral administration, i.e., as a solid or a liquid preparation.
Suitable solid oral formulations include tablets, capsules, pills,
granules, pellets and the like. Suitable liquid oral formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In another embodiment of the present invention, the
active ingredient is formulated in a capsule. In accordance with
this embodiment, the compositions of the present invention
comprise, in addition to the active compound and the inert carrier
or diluent, a hard gelating capsule.
[0126] In other embodiments, the pharmaceutical compositions are
administered by intravenous, intra-arterial, or intra-muscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In another embodiment, the pharmaceutical compositions
are administered intravenously and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical compositions are administered intra-arterially and
are thus formulated in a form suitable for intra-arterial
administration. In another embodiment, the pharmaceutical
compositions are administered intra-muscularly and are thus
formulated in a form suitable for intra-muscular
administration.
[0127] In another embodiment, the pharmaceutical compositions are
administered topically to body surfaces and are thus formulated in
a form suitable for topical administration. Suitable topical
formulations include gels, ointments, creams, lotions, drops and
the like. For topical administration, the compositions or their
physiologically tolerated derivatives are prepared and applied as
solutions, suspensions, or emulsions in a physiologically
acceptable diluent with or without a pharmaceutical carrier.
[0128] In another embodiment, the composition is administered as a
suppository, for example a rectal suppository or a urethral
suppository. In another embodiment, the pharmaceutical composition
is administered by subcutaneous implantation of a pellet. In
another embodiment, the pellet provides for controlled release of
agent over a period of time.
[0129] In another embodiment, the active compound is delivered in a
vesicle, e.g. a liposome (see Langer, 1990, Science 249:1527-1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.
353-365 (1989)).
[0130] As used herein "pharmaceutically acceptable carriers or
diluents" are well known to those skilled in the art. The carrier
or diluent may be may be, in various embodiments, a solid carrier
or diluent for solid formulations, a liquid carrier or diluent for
liquid formulations, or mixtures thereof.
[0131] In another embodiment, solid carriers/diluents include, but
are not limited to, a gum, a starch (e.g. corn starch,
pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose,
dextrose), a cellulosic material (e.g. microcrystalline cellulose),
an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium
oxide, talc, or mixtures thereof.
[0132] In other embodiments, pharmaceutically acceptable carriers
for liquid formulations may be aqueous or non-aqueous solutions,
suspensions, emulsions or oils. Examples of non-aqueous solvents
are propylene glycol, polyethylene glycol, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Examples of oils are those of petroleum,
animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver
oil.
[0133] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, and glycols such as propylene glycols or polyethylene
glycol are preferred liquid carriers, particularly for injectable
solutions. Examples of oils are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
[0134] In another embodiment, the compositions further comprise
binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl
cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl
cellulose, povidone), disintegrating agents (e.g. cornstarch,
potato starch, alginic acid, silicon dioxide, croscarmelose sodium,
crospovidone, guar gum, sodium starch glycolate), buffers (e.g.,
Tris-HCI, acetate, phosphate) of various pH and ionic strength,
additives such as albumin or gelatin to prevent absorption to
surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile
acid salts), protease inhibitors, surfactants (e.g. sodium lauryl
sulfate), permeation enhancers, solubilizing agents (e.g.,
glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers
(e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose),
viscosity increasing agents (e.g. carbomer, colloidal silicon
dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame,
citric acid), preservatives (e.g., Thimerosal, benzyl alcohol,
parabens), lubricants (e.g. stearic acid, magnesium stearate,
polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g.
colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate,
triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl
cellulose, sodium lauryl sulfate), polymer coatings (e.g.,
poloxamers or poloxamines), coating and film forming agents (e.g.
ethyl cellulose, acrylates, polymethacrylates) and/or
adjuvants.
[0135] In another embodiment, the pharmaceutical compositions
provided herein are controlled-release compositions, i.e.
compositions in which the compound is released over a period of
time after administration. Controlled- or sustained-release
compositions include formulation in lipophilic depots (e.g. fatty
acids, waxes, oils). In another embodiment, the composition is an
immediate-release composition, i.e., a composition in which the
entire compound is released immediately after administration.
[0136] In another embodiment, molecules of the present invention
are modified by the covalent attachment of water-soluble polymers
such as polyethylene glycol, copolymers of polyethylene glycol and
polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl
alcohol, polyvinylpyrrolidone or polyproline. The modified
compounds are known to exhibit substantially longer half-lives in
blood following intravenous injection than do the corresponding
unmodified compounds. Such modifications also increase, in another
embodiment, the compound's solubility in aqueous solution,
eliminate aggregation, enhance the physical and chemical stability
of the compound, and greatly reduce the immunogenicity and
reactivity of the compound. As a result, the desired in vivo
biological activity may be achieved by the administration of such
polymer-compound abducts less frequently or in lower doses than
with the unmodified compound.
[0137] An active component is, in another embodiment, formulated
into the composition as neutralized pharmaceutically acceptable
salt forms. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the
polypeptide or antibody molecule), which are formed with inorganic
acids such as, for example, hydrochloric or phosphoric acids, or
such organic acids as acetic, oxalic, tartaric, mandelic, and the
like. Salts formed from the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine, and the like.
EXPERIMENTAL EXAMPLE
[0138] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0139] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure.
Example 1
Purification and Purity Assessment of RNA Molecules Synthesized
with Modified Nucleosides
[0140] As described herein, the purification of mRNA results in up
to a 5,000-fold increase in translation and protein production, in
primary cells and in vivo compared to unpurified in vitro
transcribed RNA through the removal of contaminants, such as dsRNA
and other RNAs that do not encode the sequence of interest that
activate RNA sensors that inhibit protein translation directly or
indirectly. In addition, when such purified RNA contains certain
modified nucleosides, it can also ablate activation of innate
immune RNA sensors resulting in a highly translatable,
low-immunogenic RNA. Described herein are three methods, both
quantitative and qualitative, to measure the purification of mRNA
preparations.
[0141] The materials and methods are now described.
In Vitro-Transcribed RNA
[0142] RNA containing modified nucleosides was synthesized
essentially as described in U.S. Pat. No. 8,278,036. RNA containing
modified nucleosides was purified using high-performance liquid
chromatography (HPLC) and/or RNase III treatment.
[0143] RNA was purified by HPLC (Akta Purifier, GE Healthcare)
using a column matrix of alkylated nonporous
polystyrene-divinylbenzene copolymer microspheres (2.1 .mu.m) (21
mm.times.100 mm column). Buffer A contained 0.1 M triethylammonium
acetate (TEAA), pH=7.0 and Buffer B contained 0.1 M TEAA, pH=7.0
and 25% acetonitrile (Transgenomics). Columns were equilibrated
with 38% Buffer B, loaded with RNA, and run with a single or 2
linear gradients to 55 or 65% Buffer B over 20-30 minutes at 5
ml/minute. RNA analyses were performed with the same column matrix
and buffer system using a 7.8 mm.times.50 mm column at 1.0
ml/min.
[0144] RNA content from desired fractions was concentrated and
desalted using Amicon Ultra-15 centrifugal filter units (30K
membrane) (Millipore) by successive centrifugation at 4,000.times.g
for 10 min (4.degree. C.) in a Sorvall ST16R centrifuge (Thermo
Scientific) and dilution with nuclease free water. The RNA was
recovered by overnight precipitation at -20.degree. C. in NaOAc
(0.3 M, pH 5.5), isopropanol (1 volume) (Fisher) and glycogen (3
.mu.l) (Roche).
[0145] RNase III purification was performed by making the RNA
buffer 66 mM Na acetate, pH=7.5 to a volume of 100 .mu.l and adding
0.001 units of RNase III and incubating at 37.degree. C. for 60
minutes. RNA was purified away from enzyme and salt using LiCl
precipitation.
Dot Blot
[0146] A major contaminant found in modified nucleoside RNA
preparations is dsRNA. Binding of dsRNA-specific mAb J2 occurs even
when the dsRNA contains modified nucleosides, e.g.,
1-methyl-pseudouridine, pseudouridine and/or 5-methylcytidine,
while binding of the other dsRNA-specific mAb K1 is reduced when
dsRNA contains such modifications (Kariko, et al., 2011, Nucleic
Acids Res 39:e142).
[0147] RNA (200 ng) was blotted onto super charged Nytran, dried,
blocked with 5% non-fat dried milk in TBS-T buffer (50 mM Tris-HCl,
150 mM NaCl, 0.05% Tween-20, pH 7.4), and incubated with
dsRNA-specific mAb J2 or K1 (English & Scientific Consulting)
for 60 min. Membranes were washed six times with TBS-T and reacted
with HRP-conjugated donkey anti-mouse Ig (Jackson Immunology),
washed six times and detected with ECL Plus Western blot detection
reagent (Amersham).
[0148] Images were captured on a Fujifilm LAS1000 digital imaging
system. dsRNA (25 ng) used as a positive control was derived from
sense and antisense strands of T7TS UTR sequence (328 bp). Blots
were reprobed with .sup.32P-labeled DNA complementary to the 3'-UTR
of the RNA to document the presence of RNA. The assay can be
further quantitated by varying the amount of RNA blotted onto the
membrane and creating a standard curve of known double-stranded RNA
using increasing amounts blotted onto membranes. This allows the
quantitation of the amount of dsRNA in a known quantity of
mRNA.
Northern Blot
[0149] The mRNA of interest is derived from a specific region of
the plasmid bounded by a T7 promoter and a sequence corresponding
to a poly(A) tail. RNA contaminants can be derived from any region
of the plasmid containing the RNA sequence of interest. To identify
contaminants, the in vitro transcribed mRNA is analyzed by Northern
blot using probes derived from all of the regions of the plasmid
excluding the coding sequence of interest. The mRNA of interest and
signal from contaminating RNA can be quantitated and represented as
the percent impurity.
[0150] Northern blot analyses were performed with in vitro
synthesized RNA. The RNA pellet was reconstituted in 25 .mu.l of
nuclease-free water (Promega) and stored at -20.degree. C. RNA
samples (5 .mu.l) containing 1 .mu.g of RNA were denatured and then
separated in a 1.4% denaturing agarose, 0.22 M formaldehyde gel
that was submerged into morpholinepropanesulfonic acid
(MOPS)-EDTA-sodium acetate buffer (Sigma) supplemented with
formaldehyde (0.22 M). RNA was transferred to NYTRAN SuperCharge
filters (Schleicher and Schuell, Keene, N.H.) and UV cross-linked.
The filters were prehybridized at 68.degree. C. for 1 h in
MiracleHyb (Stratagene, La Jolla, Calif.). To probe the Northern
blots, 50 ng of DNA probe corresponding to the coding sequence,
UTRs, or regions of the plasmid used to make the RNA was labeled
with Redivue [.alpha.-32P]dCTP (Amersham, Arlington Heights, Ill.)
with a random prime-labeling kit (Boehringer Mannheim). The filters
were hybridized at 68.degree. C. for 20 h with MiracleHyb
containing the labeled and denatured probe. The filters were washed
and exposed to Kodak film with an intensifier screen at -70.degree.
C. for 2 to 72 h.
HPLC Analysis
[0151] Although the Northern blot method accurately quantitates
contaminating RNA and allows the quantitation of the increase in
purity by HPLC, it does not measure RNA contaminants that are
derived from the coding sequence of interest on the plasmid,
derived by aberrant start or stop sites or transcription from the
opposite strand. These contaminants, as well as all other
contaminants, can be accurately quantitated by HPLC analysis using
the same parameters as used for purification. HPLC purification
results in mRNA that ranges between 98% and 99.9% pure starting
with in vitro transcribed RNA that is typically 70-93% pure. Thus,
the HPLC method is the very accurate and predictive of RNA
purity.
Dendritic Cell Activation
[0152] The addition of RNA to human primary DCs and the measurement
of the resultant inflammatory cytokine protein or mRNA is a very
sensitive measure that the mRNA has been purified to its optimum.
This assay cannot quantitate the purity of the RNA, but it is the
most sensitive and accurate at determining whether the mRNA is
purified to its optimal amount. The optimal level of purity needed
varies for each mRNA coding sequence and cannot be predicted. The
DC activation assay is used to discern whether an RNA preparation
is optimally purified.
[0153] Leukophoresis samples were obtained from HIV-uninfected
volunteers through an IRB-approved protocol. Peripheral blood
mononuclear cells were purified by Ficoll-Hypaque density gradient
purification. DCs were produced by adhering monocytes to plastic
6-well plates and removing unbound cells. GM-CSF (50 .mu.g/ml) and
IL-4 (100 .mu.g/ml, R & D Systems) were added. The resulting
immature DCs were used between 6 and 9 days after the initial
culture of monocytes.
[0154] Lipofectin (Invitrogen, Carlsbad, Calif.) and mRNA were
complexed in phosphate buffer that has been shown to enhance
transfection in vitro and in vivo. To assemble a 50-pi complex of
RNA-lipofectin, first 0.4 .mu.l potassium phosphate buffer (0.4
mol/l, pH 6.2) containing 10 .mu.g/.mu.l bovine serum albumin
(Sigma, St. Louis, Mo.) was added to 6.7 .mu.l DMEM, then 0.8 .mu.l
lipofectin was mixed in and the sample was incubated for 10
minutes. In a separate tube, RNA was added to DMEM to a final
volume of 3.3 .mu.l. Diluted RNA was added to the lipofectin mix
and incubated for 10 minutes. Finally, the RNA-lipofectin complex
was further diluted by adding 38.8 .mu.l DMEM. Fifty microliter of
such a complex was used to transfect cells present in 1 well of a
96-well plate. Complexing of RNA to TransIT mRNA (Mirus Bio) was
performed according to the manufacturer combining RNA (0.1 mg) with
TransIT mRNA (0.3 ml) and boost (0.2 ml) reagents in 17 .mu.l of
serum free medium, which was then added to DCs. Culture
supernatants were collected at 24 h and analyzed for inflammatory
cytokines (TNF-.alpha., IFN-.alpha., IL-6, and others) by ELISA
assay. Cellular RNA was obtained 6 hrs after stimulation and
analyzed by Northern blot, as described above with the following
changes. Total RNA was isolated from cells with guanidinium
thiocyanate (Master Blaster; Bio-Rad, Hercules, Calif.). To enhance
the RNA yield, 70 .mu.g of glycogen (Boehringer Mannheim,
Indianapolis, Ind.) was added as carrier, and the precipitation was
performed in siliconized tubes at -20.degree. C. overnight. Probes
were derived from plasmids and were specific for the coding regions
of human IFN-.alpha.13, IFN-.beta. (Open Biosystems), TNF-.alpha.,
or GAPDH (ATCC).
[0155] The results of this example are now described.
[0156] Experiments were performed that demonstrate that in vitro
transcribed RNA is immunogenic and contains dsRNA contaminants. 200
ng of in vitro transcripts encoding mEPO and containing the
indicated modified nucleosides were blotted and analyzed with K1
and J2 dsRNA-specific mAbs. The dsRNA positive control contained a
328 bp long dsRNA (25 ng) (FIG. 1A). DCs were treated with
Lipofectin-complexed Renilla luciferase (T7TSRenA.sub.30), firefly
and Metridia luciferases (T7TSLucA.sub.30, T7TSMetlucA.sub.30), and
mEPO (TEVmEPOA.sub.51) mRNAs. TNF-.alpha. levels were measured in
the supernatants at 24 h (FIG. 1B). DCs were treated with
TransIT-complexed in vitro transcripts encoding Renilla and firefly
luciferases (T7TSRenA.sub.30, T7TSLucA.sub.30), eGFP
(TEVeGFPA.sub.51) and mEPO (TEVmEPOA.sub.51). IFN-.alpha. levels
were measured in the supernatants at 24 h. Error bars are standard
error of the mean. Data shown is from one experiment that is
representative of greater than 20 experiments using many different
coding sequence mRNAs (FIG. 1C).
[0157] HPLC purification of .PSI.-modified TEVeGFPA.sub.n mRNA
identified contaminants eluting before and after the expected
product (FIG. 2). RNA was applied to the HPLC column and eluted
using a linear gradient of Buffer B (0.1 M TEAA, pH 7.0, 25%
acetonitrile) in Buffer A (0.1 M TEAA, pH 7.0). The gradient
spanned 38-55% Buffer B over 22 min (red line). Absorbance at 260
nm was analyzed (black line), which demonstrated the expected sized
RNA as well as smaller and larger RNA species. Data shown is from
one experiment that is representative of over 200.
[0158] HPLC purification of in vitro-transcribed nucleoside
modified mRNA was found to remove dsRNA contaminants and eliminate
immunogenicity. 200 ng of RNA encoding the indicated protein and
containing the indicated modified nucleosides with or without
HPLC-purification were blotted and analyzed with the J2
dsRNA-specific mAb (FIG. 3A). 200 ng of RNA encoding the indicated
protein and containing .PSI.-modifications with or without
HPLC-purification were blotted and analyzed with the J2
dsRNA-specific mAb. Blots were reprobed with a 32P-labeled probe
for the 3' UTR of the RNAs to control for amount of RNA analyzed
(FIG. 3B). DCs were treated with TEVRenA.sub.51 RNA containing the
indicated nucleoside modifications with or without HPLC
purification and complexed to Lipofectin. TNF-.alpha. levels were
measured in the supernatants at 24 hr. Differences in the effect of
nucleoside modification on immunogenicity of Renilla encoding mRNA
compared to FIG. 1B is likely due to donor variation and
differences in UTRs of the RNAs (FIG. 3C). DCs were treated with
TEVLucA.sub.51 RNA containing the indicated nucleoside
modifications with or without HPLC purification and complexed to
TransIT. IFN-.alpha. levels were measured in the supernatants at 24
hr. Error bars are standard error of the mean. Data shown is from
one experiment that is representative of 3 or more (FIG. 3D).
[0159] HPLC purification of in vitro transcribed
nucleoside-modified mRNA was found to eliminate activation of genes
associated with RNA sensor activation. Heat map representing
changes in expression of genes activated by RNA sensors were
derived from microarray analyses of DCs treated for 6 hr with
TransIT alone or transit-complexed TEVRenA.sub.51 RNA with the
indicated modifications with or without HPLC purification. RNA from
medium treated cells was used as the baseline for comparison (FIG.
4A). Northern blot of RNA from DCs treated with medium or TransIT
alone or TransIT-complexed TEVRenA.sub.51 RNA with the indicated
modifications with or without HPLC purification and probed for
IFN-.alpha., IFN-.beta., TNF-.alpha., and GAPDH mRNAs (FIG.
4B).
[0160] HPLC purification of in vitro transcribed mRNA was found to
enhance translation. 293T (FIG. 5A) and human DCs (FIG. 5B-5C) were
transfected with TransIT (FIG. 5A, 5C) or Lipofectin (FIG. 5B)
complexed TEVRenA.sub.51 or TEVmEPOA.sub.51 mRNA with the indicated
modifications with or without HPLC purification and analyzed for
Renilla luciferase activity or levels of supernatant-associated
mEPO protein at 24 hr. (FIG. 5D) Human DCs were transfected with
.PSI.-modified TEVeGFPA.sub.n mRNA with or without HPLC
purification (0.1 .mu.g/well) complexed with Lipofectin or TransIT
and analyzed 24 hr later. Error bars are standard error of the
mean. Data shown is from one experiment that is representative of 3
or more.
[0161] HPLC purification was found to remove RNA contaminants.
(FIG. 6A) One hundred .mu.g of .PSI.-modified T7TSLucA.sub.30 RNA
was applied to the HPLC column and 3 fractions were collected, all
RNAs eluting before the main transcription product (I), the
expected RNA (II), and all RNAs eluting after the main
transcription product (III). The gradient began at 38% Buffer B and
increased to 43% Buffer B over 2.5 min and then spanned 43% to 65%
Buffer B over 22 min. Unmodified and m5C/.PSI.-modified
T7TSLucA.sub.30 RNA had similar fractions obtained. (FIG. 6B) The
RNAs from each fraction were complexed to TransIT and added to DCs
and IFN-.alpha. in the supernatant was measured 24 hr later. Error
bars are standard error of the mean. (FIG. 6C) 200 ng of RNA from
the 3 fractions and the starting unpurified RNA were blotted and
analyzed with the J2 dsRNA-specific mAb.
[0162] Daily transfection with HPLC-purified m5C/T-modified mRNA
does not reduce cell proliferation (FIG. 7). Primary keratinocytes
were transfected daily with TransIT alone or m5C/.PSI.-modified RNA
encoding Renilla luciferase with or without HPLC-purification
complexed with TransIT. Every 2-3 days, cultures were split and
equal numbers of cells for each condition were plated. Total cell
numbers for each condition were divided by the total cell number in
untreated cells to calculate the percent of control
proliferation.
[0163] RNA contaminants were found to be removed by treatment with
RNase III (FIG. 8). One hundred .mu.g of U, .PSI., m5C/.PSI., or
1-Me-.PSI.-modified TEV-ren-A51 RNA was treated with 0.001 units of
bacterial RNase III for 60 minutes at 37.degree. C. in 66 mM
acetate buffer, pH=7.5. Similar results were obtained with ranges
of RNase III from about 0.001 to about 0.1 units, with treatment
times ranging from about 15 to about 120 minutes, and
concentrations of acetate buffer ranging from about 33 to about 200
mM and pHs ranging from about 7.5 to about 8.0. After
precipitation, washing and resuspension in water, 200 ng of RNA was
analyzed for binding by the dsRNA-specific mAb J2 (FIG. 8A) or 300
ng of RNA was complexed to TransIT and added to primary human
monocyte derived dendritic cells (FIG. 8B). After 24 hrs,
supernatant was analyzed for interferon (IFN)-.alpha..
[0164] Multiple exemplary RNA preparations have been produced and
have been found to contain contaminants, including dsRNA
contaminants, and they have been purified using the methods
described elsewhere herein to remove immunogenicity. Exemplary RNA
preparations that have been produced and purified using the methods
described elsewhere herein include bacterial Cas9, Firefly
luciferase, Metridia luciferase, Renilla luciferase, green
fluorescent protein, enhanced green fluorescent protein, mouse
CD40L, mouse 4-1BBL, mouse CD70, HIV envelope, HIV gag, HIV
protease, mouse OX40L, mouse CD80, mouse Wnt10b, 6RHU3-SL,
D2-1BiMAB-S, 6RHU3-SL, SIINFEKL, gp70AH5mini, murine tyrosinase,
influenza HA, TLR3, TLR4, mouse GM-CSF, human, macaque, and mouse
EPO, beta-gal, NGF-beta-endorphin, VRC01 monoclonal antibody, human
iNos, dsRed, Semliki forest virus, Zinc finger nucleases, mouse
SFTPD-Myc/DDK, mouse and human CD39, mouse and human CD73, mouse
and human DNaseI, mouse and human RNaseI, mouse and human
telomerase, Klf4, Lin28, cMyc, Nanog, Sox2, Oct4, IL18, CCL5,
ILiBeta, TNFa, FCAR, 7SL, Let7a2, premir16, and b-lactamase.
TABLE-US-00001 TABLE 1 Binding by double stranded Immuno- RNA-
genicity specific Purity Purity Enhanced Enhanced Enhanced DCs mAb
RNA Before After Translation Translation Translation after after
Number Backbone Coding Sequence HPLC HPLC in 293T (log) in DC (log)
in Animal (log) HPLC HPLC 6181 + 6190 pppTEV VRC01-light-A101-Y
76.39% ~96-99.5% 0.95 >3 0 0 6182 + 6191 pppTEV VRC01-heavy-2PA-
92.66% ~96-99.5% 1.1 0 0 light-A101-Y 6198 pppTEV VRC01-heavy-2PA-
93.70% ~96-99.5% 1.1 0 0 light-A101-Y 6210 pppTEV dsRED-A101-Y
93.18% ~96-99.5% 0 0 6192 NrcapT7TS bGAL-Y 91.99% ~96-99.5% 0 0
6166 pppTEV IR3A HIV envelope- 83.19% ~96-99.5% >3 0 0 A101-Y
6183 pppTEV NGFs-betaendorphine- 87.69% ~96-99.5% 0 0 A101-Y 6183
pppTEV NGFs-betaendorphine- 88.02% ~96-99.5% 0 0 A101-Y 5649 pppTEV
oPL-A51-Y 80.88% ~96-99.5% 0 0 6088 pppTEV eGFP-A101-Y 87.01%
~96-99.5% 0.65 1.8 >3 0 0 6096 pppTEV gag-A101-Y 92.33%
~96-99.5% 0.72 2.2 >3 0 0 6091 pppTEV CD40L-A101-Y 73.22%
~96-99.5% 1.9 >3 0 0 6092 pppTEV caTLR4-A101-Y 80.80% ~96-99.5%
1.7 >3 0 0 6093 pppTEV caTLR3-A101-Y 78.50% ~96-99.5% 1.75 >3
0 0 6063 pppTEV huTert RT--A101-Y 82.37% ~96-99.5% 0 0 6049 pppTEV
CD39-Flagtag-A101-Y 89.23% ~96-99.5% >3 0 0 6050 pppTEV
CD73-Flagtag-A101-Y 80.43% ~96-99.5% >3 0 0 6051 pppTEV
DNasel-Flagtag-A101-Y 88.74% ~96-99.5% >3 0 0 6052 pppTEV
mRNasel-Flagtag- 90.47% ~96-99.5% >3 0 0 A101-Y 6022 Nrcap-T7TS
huiNOS-A30-Y 85.68% ~96-99.5% 0 0 5807 pppTEV Niv-M-A101-Y 87.00%
~96-99.5% 0 0 5893 cap1-Oct4-Y 83.66% ~96-99.5% 0 0 5892
cap1-Sox2-Y 81.63% ~96-99.5% 0 0 5891 cap1-Nanog-Y 87.20% ~96-99.5%
0 0 5890 cap1-cMyc-T58A-Y 80.25% ~96-99.5% 0 0 5889 cap1-Lin28-Y
86.82% ~96-99.5% 0 0 5888 cap1-Klf4-Y 88.84% ~96-99.5% 0 0 5867
pppTEV luc-A101-Y 64.59% ~96-99.5% 0.75 1.9 3.5 0 0 5841 pppTEV
gag-eGFP-A101-Y 87.31% ~96-99.5% 0.87 2.4 >3 0 0 5705 cap1-TEV
HIV envelope-A51-Y 82.85% ~96-99.5% 1.4 >3 0 0 5706 cap1-TEV HIV
envelope 82.34% ~96-99.5% 1.45 >3 0 0 D/R-A51-Y 5717 ppp-TEV
omEPO-A51-Y 87.50% ~96-99.5% 0.98 2.1 3.15 0 0 5647 cap1-TEV
pontellaFP-A101-Y 84.17% ~96-99.5% 2.2 0 0 5648 cap1-TEV
luc-tmtomato-A101-Y 84.38% ~96-99.5% 0 0 5649 pppTEV oPL-A51-Y
88.43% ~96-99.5% 0 0 5560 cap1-TEV NivM-GFP-A101-Y 85.85% ~96-99.5%
0 0 5519 cap1-TEV beta-lactamasestop- 75.47% ~96-99.5% 0 0 A101-Y
5515 cap1-TEV ren-A101-Y 83.47% ~96-99.5% 0.52 2.3 0 0 5424
cap1-TEV eIF4E-A101-Y 79.73% ~96-99.5% 0 0 5340 cap1-TEV
huAntiTripsinA101- 89.18% ~96-99.5% 0 0 m5C/Y 5286 with cap1-TEV2
eRRhEPO-A101- 76.23% ~96-99.5% 0.88 2.3 >3 0 0 Cap1 m5C/Y
* * * * *