U.S. patent application number 17/826946 was filed with the patent office on 2022-09-15 for use of thermostable rna polymerases to produce rnas having reduced immunogenicity.
This patent application is currently assigned to New England Biolabs, Inc.. The applicant listed for this patent is New England Biolabs, Inc.. Invention is credited to G. B. Robb, Bijoyita Roy.
Application Number | 20220288240 17/826946 |
Document ID | / |
Family ID | 1000006366329 |
Filed Date | 2022-09-15 |
United States Patent
Application |
20220288240 |
Kind Code |
A1 |
Roy; Bijoyita ; et
al. |
September 15, 2022 |
Use of Thermostable RNA Polymerases to Produce RNAs Having Reduced
Immunogenicity
Abstract
Provided herein, among other things, is a method for producing
an RNA product that has reduced immunogenicity. In some
embodiments, the method involves transcribing a template DNA with a
thermostable RNA polymerase at a temperature of greater than
44.degree. C.
Inventors: |
Roy; Bijoyita; (Medford,
MA) ; Robb; G. B.; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New England Biolabs, Inc. |
Ipswich |
MA |
US |
|
|
Assignee: |
New England Biolabs, Inc.
Ipswich
MA
|
Family ID: |
1000006366329 |
Appl. No.: |
17/826946 |
Filed: |
May 27, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16616734 |
Nov 25, 2019 |
11376338 |
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PCT/US18/36996 |
Jun 12, 2018 |
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17826946 |
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62522877 |
Jun 21, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/122 20130101;
A61K 48/0091 20130101; C12N 2310/17 20130101; C12N 15/113 20130101;
C12N 9/12 20130101; A61K 31/713 20130101; C12P 19/34 20130101; C12N
2310/141 20130101; A61K 31/7105 20130101; C12N 9/1247 20130101;
C12Y 207/07006 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12P 19/34 20060101 C12P019/34; A61K 31/713 20060101
A61K031/713; C12N 9/12 20060101 C12N009/12; A61K 31/7105 20060101
A61K031/7105; C12N 15/113 20060101 C12N015/113 |
Claims
1. A composition comprising: (a) an RNA polymerase, wherein the RNA
polymerase: (i) has at least 90% sequence identity to SEQ ID NO:1;
and (ii) having (1) at least 90% sequence identity to SEQ ID NO:1,
and (2) at least one amino acid substitution corresponding to a
position selected from the group consisting of: 109, 205, 534, 567,
and 618 of SEQ ID NO:1; and (b) a single-stranded RNA product
having reduced immunogenicity compared with an RNA product produced
by an RNA polymerase at 37.degree. C.
2. A composition according to claim 1, further comprising a
pharmaceutically acceptable excipient.
3. A composition according to claim 1, wherein the RNA polymerase
has a further amino acid substitution at a position selected from
positions 109, 205, 534, and 619.
4. A composition according to claim 1, wherein the RNA polymerase
has an amino acid substitution at a position corresponding to
position 567, wherein the substation is V567P.
5. A composition according to claim 1, wherein the RNA polymerase
has an amino acid substitution at a position corresponding to
position 388.
6. A composition according to claim 1, wherein the RNA product
encodes a therapeutic protein.
7. A composition according to claim 1, wherein the RNA product is a
therapeutic RNA.
8. A composition according to claim 1, wherein the RNA product is
selected from a guide RNA, a short hairpin RNA, a siRNA, a
microRNA, a long noncoding RNA, a mRNA encoding a recombinant
protein or a native protein, an RNA containing modified
nucleotides, and a capped mRNA.
9. A composition according to claim 1, wherein the RNA product
comprises one or more modified nucleotides.
10. A method for producing an RNA product with reduced
immunogenicity, comprising: (a) combining a DNA and a thermostable
RNA polymerase at a temperature of greater than 44.degree. C., the
RNA polymerase variant having an amino acid sequence that has at
least 90% sequence identity with SEQ ID NO:1 and having at least
one amino acid substitution corresponding to a position selected
from the group consisting of: 109, 205, 534, and 618 of SEQ ID
NO:1; and (b) producing the RNA product comprising a single strand
RNA having reduced immunogenicity compared with an RNA product
produced by an RNA polymerase at 37.degree. C.
11. A method according to claim 10, further comprising combining
the single stranded RNA with a pharmaceutically acceptable
excipient.
12. A method according to claim 11, further comprising introducing
the formulation into a mammalian cell.
13. The method of claim 11, further comprising administering the
formulation to a subject.
14. The method of claim 10, wherein the transcribing is done at a
temperature of at least 50.degree. C.
15. The method of claim 10, wherein the RNA polymerase has a
further amino acid substitution at a position selected from
positions 109, 205, 534, and 619.
16. The method of claim 10, wherein the RNA product encodes a
therapeutic protein.
17. The method of claim 10, wherein the RNA product is a
therapeutic RNA.
18. The method of claim 10, wherein the RNA product is selected
from a guide RNA, a short hairpin RNA, a siRNA, a microRNA, a long
noncoding RNA, a mRNA encoding a recombinant protein or a native
protein, an RNA containing modified nucleotides, and a capped mRNA.
Description
BACKGROUND
[0001] Synthetic RNAs are a promising new class of therapeutics for
non-virus-mediated gene therapy, vaccines and protein replacement
therapeutics, as well as in immuno-oncology and personalized cancer
vaccines (Sahin, et al., (2014): Nature Reviews Drug Discovery 13,
759-80; Weissman, (2015), Expert Review of Vaccines, 14(2):265-81).
Synthetic RNAs are commonly manufactured by in vitro transcription
(IVT) of a DNA template that encodes the antigen or protein of
interest (Sahin, et al., (2014); Steinle, et al., (2017), Stem
Cells, 35(1):68-79).
[0002] One limitation associated with the therapeutic use of
synthetic RNA is an immunostimulatory response induced by
double-stranded RNA (dsRNA) contaminants created during IVT
(Devoldere, et al., (2016), Drug Discover Today, 21(1), 11-25;
Loomis et al., (2016), Journal of Materials Chemistry 4:1619-32;
Triana-Alonso et al., (1995), The Journal of Biological Chemistry,
270 (11): 6298-6307). The immunostimulatory response of cells
results from activation of receptors that trigger secretion of
interferons and inflammatory cytokines (Devoldere et al., (2016);
Loomis et al., (2016); Kariko, et al., (2005) Immunity,
23:165-175).
[0003] Several methods have been developed to separate desired IVT
products from contaminating dsRNA. These include various
chromatography techniques such as: ion exchange high-performance
liquid chromatography (HPLC), reverse phase HPLC, hydrophobic
interaction HPLC, low or normal pressure liquid chromatography,
size exclusion chromatography, oligo dT affinity chromatography,
and core bead chromatography (Kariko, et al., (2011) Nucleic Acids
Research, 39 (21), e142; Weissman, et al., (2012) Methods in
Molecular Biology, 969:43-54; Kobuk, et al., (2013) RNA,
10:1449-59; US 2016/0024141; US 2016/0024140 A1; U.S. Pat. No.
8,383,340; WO 2014/144711; US 2016/0032316; US 2014/144767; US
2016/0326575). Enzymatic digestion of dsRNA with RNase III, RNase
V1, Dicer, and Chipper is also implemented to reduce dsRNA (US
2016/0032316).
[0004] The use of a physical separation method to remove the dsRNA
from IVT reactions increases the cost and labor involved in the
production of IVT RNAs that minimally activate innate immune
responses.
SUMMARY
[0005] It has been found that IVT of a DNA template at an elevated
temperature (e.g., at a temperature of greater than 44.degree. C.)
produces an RNA product that is less immunostimulatory than RNA
products that are produced at a lower temperature (at 37.degree.
C.). Without wishing to be bound to any specific theory, it is
believed that RNA products produced at an elevated temperature are
less immunostimulatory than those produced at a lower temperature
because they contain less dsRNA, which is known to have an
immunostimulatory effect. As such, in some embodiments, the RNA
products produced at an elevated temperature can be transfected
into cells without first removing the dsRNA from the RNA product,
i.e., without first purifying the non-dsRNA products from the RNA
product (such as using chromatography or degrading the dsRNA
enzymatically).
[0006] A variety of methods and compositions are described herein.
In some embodiments, the method may comprise: (a) transcribing a
template DNA with a thermostable RNA polymerase at a temperature of
greater than 44.degree. C. to produce an RNA product; and (b)
introducing (e.g. transfecting) the RNA product into mammalian
cells. Because the RNA product produced by this method does not
contain significant amounts of dsRNA (has a reduced dsRNA content
as compared to a control RNA product produced from the same
template using the same RNA polymerase but at a lower temperature
of only 37.degree. C.), the method may be done in the absence of a
step that removes dsRNA from the RNA product prior to introducing
the RNA product into the mammalian cells.
[0007] Embodiments provide a method comprising: (a) transcribing a
template DNA with a thermostable RNA polymerase at a temperature of
greater than 44.degree. C. (such as at a temperature of at least
50.degree. C.) to produce an RNA product; and (b) measuring the
immunogenicity of the RNA product in the absence of a step that
removes any dsRNA from the RNA product.
[0008] The thermostable RNA polymerase may be a variant of a
bacteriophage RNA polymerase, such as a thermostable variant of the
wild type T7 RNA polymerase having the amino acid sequence shown in
SEQ ID NO:1.
[0009] The RNA product may be a protein, such as a therapeutic
protein. The RNA product may be a therapeutic RNA and/or may be a
guide RNA, a short hairpin RNA, a siRNA, a microRNA, a long
noncoding RNA, an mRNA encoding a recombinant protein or a native
protein, an RNA containing modified nucleotides, and a capped
mRNA.
[0010] The immunogenicity of the RNA product may be measured by any
known means. For example, the immunogenicity may be measured by
assaying the RNA product for the presence of dsRNA. As discussed
herein, dsRNA is known to have an immunostimulatory effect. The
presence or amount of dsRNA can therefore be correlated with
immunogenicity of the RNA product. Alternatively, or in addition,
the immunogenicity of the RNA product may be measured by
introducing the RNA product into one or more mammalian cells. The
mammalian cells may be cells of a mammalian subject in vivo (i.e.
the RNA product is administered to a mammal, such as a test mammal;
and an immune response in the mammal is measured). Alternatively,
or in addition, the mammalian cells may be mammalian cells cultured
in vitro or mammalian cells ex vivo (in which case the RNA product
is introduced into the cells, such as by transfection; and an
immune response in the cells is measured, such as using an
enzyme-linked immunosorbent assay (ELISA) to determine the level of
e.g. IFN-.alpha. and/or TNF-.alpha. in the cell supernatant).
[0011] The method may involve comparing the immunogenicity measured
for the RNA product with the immunogenicity measured for a control
RNA product. The control RNA product may be produced by
transcribing the template DNA with the thermostable RNA polymerase
at a temperature of 37.degree. C., in the absence of a step that
removes any dsRNA from the control RNA. Thus, the method may
further comprise: (i) producing a control RNA product by
transcribing the template DNA with the thermostable RNA polymerase
at a temperature of 37.degree. C. and, in the absence of a step
that removes any dsRNA from the control RNA product, measuring the
immunogenicity of the control RNA product; and (ii) comparing the
immunogenicity measured for the RNA product with the immunogenicity
measured for the control RNA product. The immunogenicity of the
control RNA product may be measured by any known means; such as by
assaying the control RNA product for the presence of dsRNA, by
introducing the control RNA product into cells of a mammalian
subject in vivo, or by introducing the control RNA product into
mammalian cells cultured in vitro or into mammalian cells ex
vivo.
[0012] Embodiments also provide a method comprising: (a)
transcribing a template DNA with a thermostable RNA polymerase at a
temperature of greater than 44.degree. C. to produce an RNA
product; and optionally measuring the immunogenicity of the RNA
product (e.g. using any technique described herein); and (b)
combining the RNA product with a pharmaceutically acceptable
excipient; wherein the method is done in the absence of a step that
removes any dsRNA from the RNA product between steps (a) and
(b).
BRIEF DESCRIPTION OF THE FIGURES
[0013] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0014] FIGS. 1A-1B shows the presence of dsRNA contaminants in IVT
RNAs from different DNA templates that were synthesized under
standard conditions (37.degree. C.). The DNA templates used were
pXba SalI (6 kb), pXba HpaI (9 kb), pXba AvrII (2.5 Kb), and Cluc
NotI (3 kb). Crude IVT reactions were subjected to immunoblot
analyses using a monoclonal antibody (mAb-J2) specific for any form
of dsRNA (Schonborn, et al., (1991), Nucleic Acids Research 19(11)
2993-3000; obtained from English and Scientific Consulting,
Budapest, Hungary). The intensity of the blackness on the
immunoblot correlates with antibody binding to dsRNA
contaminants.
[0015] FIG. 1A shows the results from transcription with wild-type
T7 RNA polymerase at 37.degree. C.
[0016] FIG. 1B shows the results from transcription with a
thermostable variant of T7 RNA polymerase at 37.degree. C. At
37.degree. C., both wild-type and the thermostable variant of RNA
polymerase generated the desired RNA product (1.2% agarose gel) and
contaminating dsRNA (immunoblot) regardless of the length of the
DNA template.
[0017] FIGS. 2A-2D shows the reduction in amounts of dsRNA in IVT
reactions that were performed at higher temperatures either because
the wild-type T7 polymerase was inactive or because the
thermostable T7 polymerase did not produce dsRNA. Activity was
determined by the amount of IVT RNA observed on the 1.2% agarose
gel. The observed effect was independent of the length of the DNA
template.
[0018] FIG. 2A shows, using wild-type T7 RNA polymerase, dsRNA
detected by mAb J2 (English and Scientific Consulting, Budapest,
Hungary) in IVT reaction mixtures at temperatures between
37.degree. C. and 55.degree. C. The polymerase activity is lost at
temperatures greater than 43.9.degree. C., as determined by the
absence of IVT Cluc NotI RNA. Moreover, dsRNA was detected in IVT
reaction mixtures using the mAb J2 (English and Scientific
Consulting, Budapest, Hungary) at temperatures between 37.degree.
C. and 43.9.degree. C.
[0019] FIG. 2B shows, using a thermostable T7 polymerase, dsRNA
detected by mAb J2 in IVT reaction mixtures at temperatures between
37.degree. C. and 55.degree. C. The detectable amount of dsRNA is
substantially reduced at temperatures greater than 44.degree. C.
while the amount of IVT Cluc NotI RNA produced at the same time
using thermostable T7 RNA polymerase is significant.
[0020] FIG. 2C shows, using wild-type T7 RNA polymerase, that both
the amount of IVT RNA and dsRNA from DNA templates of different
lengths are substantially reduced at temperatures of 55.degree.
C.
[0021] FIG. 2D shows, using a thermostable T7 RNA polymerase, that
only dsRNA contaminants in IVT mix from DNA templates of different
lengths are substantially reduced at temperatures of 55.degree. C.
while at the same time the yields of IVT were significant and
similar throughout.
[0022] FIG. 3 shows the effect of high temperature on dsRNA
formation in IVT reactions using a commercially available
thermostable T7 RNA polymerase from Toyobo Life Science Department,
Osaka, Japan on Cluc NotI template DNA. IVT was performed at
37.degree. C. or 50.degree. C. Only the amount of dsRNA
contaminants was reduced at 50.degree. C. while significant amounts
of Cluc NotI RNA were detected on a 1.2% agarose gel. This data
demonstrates that the temperature of the reaction rather than the
particular thermostable T7 RNA polymerase is responsible for
reduction of dsRNA.
[0023] FIG. 4 shows the temperature dependent reduction of dsRNA
associated with IVT of Cluc NotI RNA that either lacks a poly A
tail (no tailing) or was polyadenylated with a tail-length of 125
nucleotides (T125). Both RNAs contained a modified
nucleotide-pseudouridine instead of uridine.
[0024] FIGS. 5A-5B shows activation of interferons and cytokines
(represented by IFN-.alpha. and TNF-.alpha. respectively)
indicative of an immune response activation in human dendritic
cells (hDCs) that were transfected with Cluc NotI IVT RNA from
reactions that were performed with wild-type T7 RNA polymerase at
37.degree. C. or with a thermostable variant of T7 RNA polymerase
at 55.degree. C. Poly I:C, a synthetic analog of dsRNA and
Resiquimod (R848), an activator of Toll-like receptors are used as
controls for interferon activation. Negative controls included
TransIT.RTM. (Mirus Bio, Madison, Wis.) transfection reagent alone
and PBS.
[0025] FIG. 5A shows the results of absolute quantification of
IFN-.alpha. (interferon) levels in the cell culture supernatants of
hDCs that were transfected with Cluc NotI IVT RNA using ELISA
(Kariko, et al., (2011)). Cluc NotI IVT RNA (or control RNA--poly
I:C) was introduced into hDCs, and supernatants were collected 24
hours after transfection. The supernatants were then probed for the
secretion of IFN-.alpha.. Higher interferon secretion is observed
with Cluc NotI IVT RNA from 37.degree. C. transcription reactions
without subsequent removal of the dsRNA (IVT 37.degree. C.) as
compared to Cluc NotI IVT RNA from 55.degree. C. transcription
reactions without subsequent removal of the dsRNA (IVT 55.degree.
C.) or HPLC-purified Cluc NotI IVT RNA (IVT 37.degree. C._HPLC)
indicating low immunostimulatory properties of IVT RNA synthesized
at 55.degree. C. Increased secretion of IFN-.alpha. is seen with
polyl:C (positive control). Total rat RNA, that is known to have
reduced immunogenicity, was also used as a control.
[0026] FIG. 5B shows the absolute quantification of Tumor necrosis
factor (TNF)-.alpha. (cytokine) levels in supernatants of hDCs that
were transfected with Cluc NotI IVT RNA using ELISA (Kariko, et
al., (2011)). Cluc NotI IVT RNA (or control RNA--poly I:C) were
introduced into hDCs, and supernatants were collected 24 hours
after transfection. The supernatants were then probed for the
secretion of TNF-.alpha.. Higher cytokine secretion is observed
with Cluc NotI IVT RNA from 37.degree. C. transcription reactions
without subsequent removal of the dsRNA (IVT 37.degree. C.) as
compared to Cluc NotI IVT RNA from 55.degree. C. transcription
reactions without subsequent removal of the dsRNA (IVT 55.degree.
C.) or HPLC-purified Cluc Not I IVT RNA (IVT 37.degree. C._HPLC)
indicating low immunostimulatory properties of IVT RNA synthesized
at 55.degree. C. Resquimod (R848), an imidazoquinoline compound,
used as a positive control showed increased cytokine secretion.
Total rat RNA, that is known to have reduced immunogenicity, was
used as a control.
DETAILED DESCRIPTION
[0027] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are described.
[0028] All patents and publications, including all sequences
disclosed within such patents and publications referred to herein,
as well as U.S. Provisional Application Ser. No. 62/522,877 filed
Jun. 21, 2017, and U.S. patent application Ser. No. 15/820,656
filed Nov. 22, 2017, are expressly incorporated by reference.
[0029] Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
[0030] The headings provided herein are not limitations of the
various aspects or embodiments of the invention. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale
& Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with the general
meaning of many of the terms used herein. Still, certain terms are
defined below for the sake of clarity and ease of reference.
[0032] As used herein, the term "in vitro transcription" (IVT)
refers to a cell-free reaction in which a DNA template (e.g. a
double-stranded DNA template) is copied by a DNA-directed RNA
polymerase to produce a product that contains RNA molecules that
have been copied from the template.
[0033] As used herein, the term "DNA template" or "template DNA"
refers to a double-stranded DNA molecule that is transcribed in an
IVT reaction. DNA templates have a promoter (e.g., a T7, T3 or SP6
promoter) recognized by the RNA polymerase upstream of the region
that is transcribed.
[0034] As used herein, the term "RNA product" refers to the product
of an IVT reaction. The RNA product of IVT contains a mixture of
RNA molecules and, depending on how the transcription is done, may
contain dsRNA molecules. The molecular events that generate dsRNA
molecules in IVT reactions is unknown. dsRNA molecules can be
detected using an antibody that is specific for dsRNA or by liquid
chromatography (e.g., HPLC), for example.
[0035] As used herein, the terms "less immunostimulatory" and "less
immunogenic" (or "reduced immunostimulation" or "reduced
immunogenicity") are used interchangeably to describe a reduction
in an immune response (e.g., a reduction of interferon or cytokine
expression) relative to a reference sample, e.g., a control. A
decrease in immunostimulation or immunogenicity may be a response
that is reduced by at least 20%, at least 40%, at least 60%, at
least 80%, at least a 90%, or at least a 95% relative to the
control.
[0036] As used herein, the terms "reduced dsRNA", "less dsRNA" and
"fewer dsRNA molecules" are used interchangeably to refer to a
sample that has an amount of dsRNA that is at least 20%, at least
40%, at least 60%, at least 80%, at least 90%, or at least 95% less
than the amount of dsRNA in a reference or control sample.
[0037] As used herein, the term "thermostable RNA polymerase"
refers to an RNA polymerase that has a temperature optimum of
greater than 44.degree. C., such as at a temperature optimum of at
least 50.degree. C., at least 55.degree. C., or at least 60.degree.
C. In many embodiments a thermostable RNA polymerase may be a
variant of a wild type mesophilic RNA polymerase, where the wild
type mesophilic RNA polymerase is substantially inactive at the
temperature at which the thermostable variant is optimally active.
The thermostable RNA polymerase may be purified before use. The RNA
polymerase may be stored in a storage buffer before being added to
a reaction mixture or therapeutic formulation.
[0038] As used herein, the term "step that removes dsRNA" refers to
any method that can be used to specifically remove dsRNA, but not
RNA that is not dsRNA, from a sample. For example, dsRNA can be
removed by chromatography (e.g., HPLC). In another example, dsRNA
can be removed using an RNase that is specific for dsRNA, e.g.,
RNase III, RNase V1, Dicer, or Chipper. A step that removes dsRNA
from a sample does not need to remove all of the dsRNA from the
sample. Rather, such a step should remove at least 80%, at least
90% or at least 95% (up to 100%) of the dsRNA from the sample. A
method that is performed "in the absence of a step that removes
dsRNA" does not include any method step that specifically removes
dsRNA, but not RNA that is not dsRNA, from a sample (such as but
not limited to the dsRNA-removing steps discussed above).
[0039] As used herein, the term "variant" refers to a protein that
comprises or consists of an amino acid sequence that is different
from a reference (e.g. naturally occurring) amino acid sequence
(i.e., having less than 100% sequence identity to the amino acid
sequence of a reference (e.g. naturally occurring protein)) but
that is at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 98% or at least 99% identical to the reference
(e.g. naturally occurring) amino acid sequence.
[0040] As used herein, the term "introducing" refers to any means
for introducing a nucleic acid into cell, including, but not
limited to, transfection, microinjection, electroporation and
lipid-mediated methods.
[0041] As used herein, the term "buffering agent", refers to an
agent that allows a solution to resist changes in pH when acid or
alkali is added to the solution. Examples of suitable non-naturally
occurring buffering agents that may be used in the compositions,
kits, and methods of the invention include, for example, Tris,
HEPES, TAPS, MOPS, tricine, or MES.
[0042] The term "non-naturally occurring" refers to a composition
that does not exist in nature.
[0043] Any protein described herein may be non-naturally occurring,
where the term "non-naturally occurring" refers to a protein that
has an amino acid sequence and/or a post-translational modification
pattern that is different from the protein in its natural state.
For example, a non-naturally occurring protein may have one or more
amino acid substitutions, deletions or insertions at the
N-terminus, the C-terminus and/or between the N- and C-termini of
the protein. A "non-naturally occurring" protein may have an amino
acid sequence that is different from a naturally occurring amino
acid sequence (i.e., having less than 100% sequence identity to the
amino acid sequence of a naturally occurring protein) but that is
at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 98% or at least 99% identical to the naturally
occurring amino acid sequence. In certain cases, a non-naturally
occurring protein may contain an N-terminal methionine or may lack
one or more post-translational modifications (e.g., glycosylation,
phosphorylation, etc.) if it is produced by a different (e.g.,
bacterial) cell. A "mutant" protein may have one or more amino acid
substitutions relative to a wild-type protein and may include a
"fusion" protein. The term "fusion protein" refers to a protein
composed of a plurality of polypeptide components that are unjoined
in their native state. Fusion proteins may be a combination of two,
three or even four or more different proteins. The term polypeptide
includes fusion proteins, including, but not limited to, a fusion
of two or more heterologous amino acid sequences, a fusion of a
polypeptide with: a heterologous targeting sequence, a linker, an
epitope tag, a detectable fusion partner, such as a fluorescent
protein, .beta.-galactosidase, luciferase, etc., and the like. A
fusion protein may have one or more heterologous domains added to
the N-terminus, C-terminus, and or the middle portion of the
protein. If two parts of a fusion protein are "heterologous", they
are not part of the same protein in its natural state.
[0044] In the context of a nucleic acid, the term "non-naturally
occurring" refers to a nucleic acid that contains: a) a sequence of
nucleotides that is different from a nucleic acid in its natural
state (i.e., having less than 100% sequence identity to a naturally
occurring nucleic acid sequence), b) one or more non-naturally
occurring nucleotide monomers (which may result in a non-natural
backbone or sugar that is not G, A, T or C) and/or c) may contain
one or more other modifications (e.g., an added label or other
moiety) to the 5'-end, the 3' end, and/or between the 5'- and
3'-ends of the nucleic acid.
[0045] In the context of a preparation, the term "non-naturally
occurring" refers to: a) a combination of components that are not
combined by nature, e.g., because they are at different locations,
in different cells or different cell compartments; b) a combination
of components that have relative concentrations that are not found
in nature; c) a combination that lacks something that is usually
associated with one of the components in nature; d) a combination
that is in a form that is not found in nature, e.g., dried, freeze
dried, crystalline, aqueous; and/or e) a combination that contains
a component that is not found in nature. For example, a preparation
may contain a "non-naturally occurring" buffering agent (e.g.,
Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a
reaction enhancer or inhibitor, an oxidizing agent, a reducing
agent, a solvent or a preservative that is not found in nature.
[0046] In some embodiments, a method for introducing an IVT RNA
product into mammalian cells is provided. In embodiments, the IVT
RNA being tested does not contain pseudouridine. The method may be
performed in vitro or in vivo. For example, the mammalian cells may
be in vitro or may be ex vivo. Alternatively, the IVT RNA product
may be introduced into mammalian cells by administering the IVT RNA
product to a mammalian subject. In any of these embodiments, the
method may comprise: (a) transcribing a template DNA with a
thermostable RNA polymerase at a temperature of greater than
44.degree. C. (e.g., a temperature of at least 45.degree. C., at
least 50.degree. C., at least 55.degree. C. or at least 60.degree.
C., up to about 70.degree. C. or 75.degree. C.) to produce an RNA
product; and (b) introducing the RNA product into mammalian cells.
The RNA product is generally introduced into mammalian cells at
temperatures of about 37.degree. C. Because the RNA product
contains significantly reduced amounts of dsRNA as compared to an
RNA product (made in the presence of U, A, G and C nucleotides)
produced by transcription from the template DNA with the polymerase
at a lower temperature of e.g. about 37.degree. C., the method may
be done in the absence of a step that removes any dsRNA from the
RNA product (i.e., a purification step or enzyme treatment step)
prior to introducing the RNA product into the cells, i.e., between
steps (a) and (b) of the method. In this method, the RNA product
obtained in step (a) and introduced into the cells in step (b) is
believed to be less immunostimulatory than a control RNA product
produced by transcribing the template DNA with the polymerase at a
temperature of 37.degree. C., when the immunostimulatory effect of
the control RNA (containing nucleotides U, A, G and C but not
modified nucleotides of U) is evaluated by introducing the control
RNA into the cells in the absence of a step that would remove dsRNA
from control RNA product. As illustrated below, the
immunostimulatory effect of an RNA product can be measured by
introducing the RNA product to mammalian cells and measuring the
expression of markers for innate immunity (e.g., interferons and
cytokines, among many others) by the cells. Immunogenicity may be
measured by ELISAs (e.g. as described previously (Kariko, et al.,
(2011); Weissman, et al., (2012)). For example, mammalian cells are
transfected with the IVT RNA product, the cells are harvested, and
the cell supernatant is assayed for levels of IFN-.alpha. and/or
TNF-.alpha..
[0047] Because the results shown below indicate that this effect
appears to be based on the temperature of the IVT reaction rather
than the polymerase used, the method may be done using any suitable
thermostable variants of a bacteriophage RNA polymerase. In some
embodiments, the polymerase may be a thermostable variant of the
T7, T3 and SP6 RNA polymerases, which have been well characterized.
Guidance for making thermostable variants of those RNA polymerases
can be found in PCT/US2017/013179 and U.S. application Ser. No.
15/594,090. In some embodiments, the thermostable RNA polymerase
used in the method may be a variant of the wild type T7 RNA
polymerase of SEQ ID NO:1. In particular embodiments, the
thermostable RNA polymerase may have an amino acid sequence that is
at least 80% or at least 90% identical to SEQ ID NO:1 (but less
than 100% identical to SEQ ID NO:1) and may comprise an amino acid
substitution at one or more positions corresponding to positions
selected from 109, 205, 388, 534, 567 and 618 of SEQ ID NO:1, as
described in PCT/US2017/013179. For example, the variant may
include a mutation at a position corresponding to 567 in SEQ ID
NO:1 for example V567P.
[0048] The amino acid sequence of the full length T7 RNA polymerase
is shown below (SEQ ID NO: 1)
TABLE-US-00001 MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA
[0049] In some embodiments, the RNA product may encode a protein,
e.g., a therapeutic protein or a protein expected to alter the
cells into which it is introduced and, as such, the RNA molecules
in the RNA product may have a 5' untranslated region (5' UTR), one
or more coding sequences, and a 3' translated region (3' UTR),
where the 3' and 5' UTRs facilitate translation of the one or more
coding sequence to produce a protein within the cells. In other
embodiments, the RNA product may be a therapeutic RNA. In some
embodiments the RNA product may be a guide RNA, a short hairpin
RNA, a siRNA, a microRNA, a long noncoding RNA, or a protein-coding
RNA (which may encode a recombinant protein or a protein that is
native to the cells). In some embodiments, the RNA product may
contain modified nucleotides (triphosphates for which can be added
to the IVT reaction). In these embodiments, modified nucleotides
may be incorporated into the IVT RNA. Incorporation of modified
nucleotides can increase in translation efficiency of the RNA and
increased stability of the RNA. Modifications can be present either
in the sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose,
arabinose, and hexose); and/or in the phosphate groups (e.g.,
phosphorothioates and 5'-N-phosphoramidite linkages); and/or in the
nucleotide base (for example, see: U.S. Pat. No. 8,383,340; WO
2013/151666; U.S. Pat. No. 9,428,535 B2; US 2016/0032316). In some
embodiments, the RNA product may be altered during or after the
transcription reaction, e.g., to decrease the rate at which the RNA
products are degraded in the cells. In some embodiment, the RNA
product may contain capped RNAs (see, for example: WO 2016/090262;
WO 2014/152673; WO 2009/149253; WO 2009/149253; Strenkowska, et
al., (2016), Nucleic Acids Research, 44(20):9578-90). RNAs with
poly A tails of varying length and labeled RNAs can also be
produced.
[0050] In some embodiments, the method may further comprise testing
the RNA product for an immune-stimulatory effect, without
performing a step that removes the dsRNA from the RNA product. As
noted above, this may be done in a variety of different ways. The
method may comprise measuring the immunogenicity of the RNA product
obtained in step (a) before or after step (b). In some embodiments,
the method comprises the steps of comparing the immunogenicity of
the RNA product with the immunogenicity of a control RNA product.
For example, the method may comprise the steps of: (i) producing a
control RNA product by transcribing the template DNA with the
thermostable RNA polymerase at a temperature of 37.degree. C. and,
in the absence of a step that removes any dsRNA from the control
RNA product, measuring the measuring the immunogenicity of the
control RNA product; and (ii) comparing the immunogenicity measured
for the RNA product with the immunogenicity measured for the
control RNA product. For example, in some embodiments, the amount
of dsRNA in the RNA product may be measured using a dsRNA-specific
antibody or by liquid chromatography, for example.
[0051] In any embodiment, the in vitro transcription may be done
using natural NTPs, i.e., GTP, CTP, UTP and ATP to produce a
product that does not contain not contain modified nucleosides.
[0052] In any embodiment, the in vitro transcription may be done
using NTPs corresponding to G, C, U and A in the absence of
pseudo-uridine triphosphate to produce a product that does not
contain not contain pseudo-uridine. The cells into which the RNA
product is introduced may be in vitro (i.e., cells that have been
cultured in vitro on a synthetic medium). In these embodiments, the
RNA product may be transfected into the cells. In other
embodiments, the cells into which the RNA product is introduced may
be in vivo (cells that are part of a mammal). In these embodiments,
the introducing may be done by administering the RNA product to a
subject in vivo. In some embodiments, the cells into which the RNA
product is introduced may present ex vivo (cells that are part of a
tissue, e.g., a soft tissue that has been removed from a mammal or
isolated from the blood of a mammal).
[0053] Methods for making a formulation are also provided. In some
embodiments, the method may comprise combining an RNA product made
by transcribing a template DNA with a thermostable RNA polymerase
at a temperature of greater than 44.degree. C. with a
pharmaceutically acceptable excipient to produce a formulation. In
some embodiments, the RNA product may be combined with the
pharmaceutically acceptable excipient in the absence of a step that
removes any dsRNA from the RNA product. In some embodiments, the
method comprises (a) transcribing a template DNA with a
thermostable RNA polymerase at a temperature of greater than
44.degree. C. to produce an RNA product; and (b) combining the RNA
product with a pharmaceutically acceptable excipient; wherein the
method is done in the absence of a step that removes any dsRNA from
the RNA product between steps (a) and (b).
[0054] In some embodiments, the RNA can be formulated in a suitable
excipient and effective therapeutic dose for introducing into a
host for achieving a desired therapeutic effect. The formulation
should lack adverse immune-stimulatory effects caused by dsRNA.
[0055] The product RNA is not packaged into a virus particle prior
to administration.
[0056] In some embodiments, this method may further comprise
administering the formulation to a subject, where the subject may
be a human or any non-human animal (e.g., mouse, rat, rabbit, dog,
cat, cattle, swine, sheep, horse or primate). Depending on the
subject, the RNA (modified or unmodified) can be introduced into
the cell directly by injecting the RNA or indirectly via the
surrounding medium. Administration can be performed by standardized
methods. The RNA can either be naked or formulated in a suitable
form for administration to a subject, e.g., a human. Formulations
can include liquid formulations (solutions, suspensions,
dispersions), topical formulations (gels, ointments, drops,
creams), liposomal formulations (such as those described in: U.S.
Pat. No. 9,629,804 B2; US 2012/0251618 A1; WO 2014/152211; US
2016/0038432 A1).
[0057] In some embodiments, the in vitro synthesized RNA product
can be delivered into the cells by packaging them into
nanoparticles such as cationic lipids and polymers, non-viral
carriers like protamine. Direct introduction of the RNA into the
cell using microinjection, electroporation, sonoporation can also
be implemented. The delivery (localized or systemic) and the
packaging of the RNA (with or without modifications) can be
performed at temperatures optimal for the delivery approach or the
formulation used (such as those described in: U.S. Pat. No.
9,629,804 B2; US 2012/0251618 A1; WO 2014/152211; US 2016/0038432
A1; US 2016/0032316 A1; U.S. Pat. No. 9,597,413 B2; US
2012/0258176).
[0058] A therapeutic formulation is also provided. In some
embodiments, the formulation may comprise: (a) an RNA product
produced by transcribing a template DNA using a thermostable RNA
polymerase at a temperature of greater than 44.degree. C.; and (b)
pharmaceutically acceptable excipient. Consistent with the above,
the formulation may be made in the absence of a step that removes
dsRNA from the RNA product.
[0059] Also provided is an RNA product produced by transcribing a
template DNA using a thermostable RNA polymerase at a temperature
of greater than 44.degree. C., for use as a medicament. Also
provided is a therapeutic formulation of the invention, for use as
a medicament.
[0060] Also provided is a method comprising: (a) transcribing a
template DNA with a thermostable RNA polymerase at a temperature of
greater than 44.degree. C. to produce an RNA product; and (b)
measuring an immunostimulatory effect of the RNA product in the
absence of a step that removes dsRNA from the RNA product. As
discussed above, the immunostimulatory effect of an RNA product can
be measured by introducing the RNA product to mammalian cells and
measuring the expression of markers for innate immunity (e.g.,
interferons and cytokines, among many others) by the cells. These
cells may be in vitro, in vivo or ex vivo.
[0061] In some embodiments, the methods and compositions described
herein may be used to make polyribonucleotides which when
transfected into eukaryotic cells or prokaryotic cells in vivo or
in vitro can change the cell phenotype by production of proteins or
by affecting expression of targets in the cell. This is best
achieved if one can avoid generating an immunostimulatory response
(triggered by dsRNA) that would undermine the viability of the
target cells.
[0062] RNA products of IVT can be used for encoding proteins such
as antigens for vaccines, for cancer immunotherapies (such as those
described in: U.S. Pat. No. 8,217,016 B2; US 2012/0009221 A1; US
2013/0202645A1; U.S. Pat. No. 9,587,003 B2; Sahin, et al., (2014);
allergy tolerance (such as those described in Sahin, et al.,
(2014), for producing recombinant or naturally occurring protein
for protein replacement therapeutics (such as those described in:
US 2016/0032316 A1; US 2016/0032316; U.S. Pat. No. 8,680,069; WO
2013/151736; WO 2014/152940; U.S. Pat. Nos. 9,181,321; 9,220,792
B2; 9,233,141 B2; Sahin, et al., (2014)), supplementation
therapeutics (such as those described in Sahin, et al., (2014)),
cell reprogramming (such as those described in: US 2011/0143436 A1;
U.S. Pat. Nos. 8,802,438; 9,371,544; WO/2009077134 A2; Sahin, et
al., (2014)), genome editing/engineering (such as those described
in Sahin, et al., (2014)).
Embodiments
[0063] Embodiment 1. A method, comprising: (a) transcribing a
template DNA with a thermostable RNA polymerase at a temperature of
greater than 44.degree. C. to produce an RNA product; and (b)
introducing the RNA product into mammalian cells, wherein the
method is done in the absence of a step that removes any dsRNA from
the RNA product between steps (a) and (b).
[0064] Embodiment 2. The method of embodiment 1, wherein the RNA
product administered in (b) is less immunostimulatory than a
control RNA product produced by transcribing the template DNA with
the polymerase at a temperature of 37.degree. C., wherein the
control RNA is introduced into the cells in the absence of a step
that removes any dsRNA from the control RNA product.
[0065] Embodiment 3. The method of any prior embodiment, wherein
the transcribing is done at a temperature of at least 50.degree.
C.
[0066] Embodiment 4. The method of any prior embodiment, wherein
the thermostable RNA polymerase is a variant of a bacteriophage RNA
polymerase.
[0067] Embodiment 5. The method of any prior embodiment, wherein
the thermostable RNA polymerase is a variant of the wild type T7
RNA polymerase of SEQ ID NO:1.
[0068] Embodiment 6. The method of any prior embodiment, including
one or more of the following: (i) the RNA product encodes a
therapeutic protein, (ii) the RNA product does not include
pseudouridine and/or (iii) the RNA product is preferably not
delivered in a virus particle.
[0069] Embodiment 7. The method of any prior embodiment, wherein
the RNA product is a therapeutic RNA.
[0070] Embodiment 8. The method of any prior embodiment, wherein
the RNA product is selected from a guide RNA, a short hairpin RNA,
a siRNA, a microRNA, a long noncoding RNA, a mRNA encoding a
recombinant protein or a native protein, an RNA containing modified
nucleotides, and a capped mRNA.
[0071] Embodiment 9. The method of any prior embodiment, further
comprising assaying the RNA product of step (a) for the presence of
dsRNA, without performing a step that removes any dsRNA from the
RNA product.
[0072] Embodiment 10. The method of any prior embodiment, wherein
the cells are cells cultured in vitro.
[0073] Embodiment 11. The method of any of embodiments 1-9, wherein
the introducing is done by administering the RNA product to a
subject in vivo.
[0074] Embodiment 12. The method of any of embodiments 1-9, wherein
the cells are ex vivo.
[0075] Embodiment 13. A method, comprising: combining an RNA
product made by transcribing a template DNA with a thermostable RNA
polymerase at a temperature of greater than 44.degree. C. with a
pharmaceutically acceptable excipient to produce a formulation.
[0076] Embodiment 14. The method of embodiment 13, further
comprising administering the formulation to a subject.
[0077] Embodiment 15. The method of any of embodiments 13 and 14,
wherein the RNA product is combined with the pharmaceutically
acceptable excipient in the absence of a step that removes any
dsRNA from the RNA product.
[0078] Embodiment 16. A therapeutic formulation comprising: (a) an
RNA product produced by transcribing a template DNA using a
thermostable RNA polymerase at a temperature of greater than
44.degree. C.; and (b) pharmaceutically acceptable excipient.
[0079] Embodiment 17. A method, comprising: (a) transcribing a
template DNA with a thermostable RNA polymerase at a temperature of
greater than 44.degree. C. to produce an RNA product; (b) measuring
the immunogenicity of the RNA product in the absence of a step that
removes any dsRNA from the RNA product.
Examples
[0080] Aspects of the present teachings can be further understood
in light of the following examples, which should not be construed
as limiting the scope of the present teachings in any way.
[0081] Synthesis of IVT mRNA--IVT reactions were performed in 41 mM
Tris-HCl pH 8.0, 50 mM NaCl, 19 mM MgCl.sub.2, 5.5 mM DTT, 1 mM
spermidine, 4 mM of each ribonucleotide, 4.15 units/5 mL yeast
inorganic pyrophosphatase, 1000 units/mL murine ribonuclease
inhibitor, 30 nM DNA template and 30 nM T7 RNA polymerase. The DNA
template was plasmid DNA that was linearized using restriction
endonucleases at specific sites downstream of the T7 promoter.
Reactions were performed at various temperatures ranging from
37.degree. C. to 55.degree. C. For the synthesis of modified mRNA,
UTP was replaced with triphosphate derivatives of pseudouridine
(Trilink Biotechnologies, San Diego, Calif.) in the IVT reaction. A
125-nt poly(A) tail was template-encoded in mRNAs that were used
for transfection experiments. IVT mRNAs were processed through a
spin column (MEGAClear.TM., Thermo Fisher Scientific, Waltham,
Mass.) to remove unincorporated nucleotides before performing
capping reactions or HPLC purification. Capped mRNAs were generated
for the transfection experiments using vaccinia capping enzyme (New
England Biolabs, Ipswich, Mass.), and 2' O-methyltransferase (New
England Biolabs, Ipswich, Mass.) was added to the reaction to
attain the Cap1 structure required for efficient translation.
[0082] dsRNA immunoblot--IVT RNAs were blotted onto Nytran.TM.
SuPerCharge (SPC) Blotting Membranes (GE Healthcare, Marlborough,
Mass.). The dried membranes were blocked for at least 1 hour in
blocking buffer (TBS Blotto, Santa Cruz Biotechnologies, Dallas,
Tex.) and then incubated with mAb-J2 (1:500 dilution; English and
Scientific Consulting, Budapest, Hungary) for at least 4 hours.
IRDye-800-conjugated donkey anti-mouse secondary antibody (1:5000;
LiCor, Lincoln, Nebr.) was used for detection using an Odyssey
imaging system (LiCor, Lincoln, Nebr.).
[0083] HPLC purification of IVT RNA--HPLC purification of IVT RNA
was performed as described previously (Kariko, et al., (2011);
Weissman, et al., (2012)). RNA from IVT reactions was loaded onto
an analytical column with a matrix composed of alkylated non-porous
polystyrene-divinylbenzene copolymer microspheres that was obtained
from Transgenomic, Omaha, Nebr. The column was equilibrated with
38% buffer B (0.1 M triethylammonium acetate pH 7.0, 25%
acetonitrile) and loaded with the IVT RNA (10 .mu.g per run)
followed by a linear gradient of buffer B (38% to 65%). Fractions
were collected for the major IVT RNA peak (II), and the fractions
were desalted and concentrated using Amicon.RTM. Ultra-15
centrifugation units (EMD Millipore, Billerica, Mass.). RNA was
diluted with nuclease-free water and subjected to capping.
[0084] Cell culture and transfection--hDCs were cultured in
lymphocyte growth medium with 50 ng/mL GM-CSF and 50 ng/mL IL-4
(Lonza, Portsmouth, N.H.) for four days prior to transfection.
HEK293 cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 2 mM L-glutamine and 10% fetal calf serum
(Thermo Fisher Scientific, Waltham, Mass.). Capped poly-adenylated
IVT mRNA (500 ng) was complexed with either Lipofectamine (Thermo
Fisher Scientific, Waltham, Mass.) or TransIT.RTM.. Transfection
was performed as recommended by the manufacturer.
[0085] Translation efficiency--The translation efficiency was
measured as relative luciferase activity from the transfected IVT
Cluc NotI mRNA, as measured from the supernatant of transfected
cells using the BioLux.RTM. Cypridina Luciferase Assay Kit (New
England Biolabs, Ipswich, Mass.). Luminescence was measured using
the Centro LB 960 Microplate Luminometer from Berthold Technologies
(Wildbad, Germany). For each reaction, 20 .mu.L of the supernatant
was assayed 12 hours after transfection. The results in triplicate
showed that the translation efficiency for the IVT RNA synthesized
by thermostable T7 RNA polymerase at 55.degree. C. was similar to
the translation efficiency of IVT RNA synthesized by wild type T7
RNA polymerase at 37.degree. C. and by IVT RNA synthesized by wild
type T7 RNA polymerase at 37.degree. C. after an HPLC column
treatment at this incubation time (where each sample provided
300,000-400,000 relative luciferase units). The negative controls
(TransIT), PBS, Polyinosinic-polycytidylic acid (Polyl:C) synthetic
dsRNA (Invivogen, San Diego, Calif.), and Resiquimod (R848) small
molecule immune activator (Invivogen, San Diego, Calif.)) all were
consistently negative with zero detectable relative luciferase
units.
[0086] Immunogenicity assays--Immunogenicity was measured by ELISAs
as described previously (Kariko et al., (2011); Weissman, et al.,
(2012)). Supernatant from cells that were transfected with IVT mRNA
was harvested 24 hours after transfection and assayed for levels of
IFN-.alpha. (PBL Interferon Source, Piscataway, N.J.) (for
TransIT-complexed RNA) and TNF-.alpha. (Thermo Fisher Scientific,
Waltham, Mass.) (for Lipofectamine-complexed RNA). A standard curve
using purified IFN-.alpha. or TNF-.alpha. was used to quantify the
cytokines.
[0087] Results of experiments performed using the protocols
described above are shown in FIGS. 1A-1B, 2A-2B, 3, 4 and 5A-5B.
These results show that IVT at a temperature greater than
44.degree. C. (e.g., at a temperature of greater than 44.degree.
C.) results in a product that has less dsRNA and is less
immunogenic than a product transcribed at a lower temperature
(e.g., a temperature of 37.degree. C. or below).
Sequence CWU 1
1
11883PRTArtificial SequenceSynthetic construct 1Met Asn Thr Ile Asn
Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro
Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu
Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg
Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala
Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75
80Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro
Glu 100 105 110Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys
Leu Thr Ser 115 120 125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser
Ala Ile Gly Arg Ala 130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg
Ile Arg Asp Leu Glu Ala Lys145 150 155 160His Phe Lys Lys Asn Val
Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys
Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly
Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200
205Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly
Gln Asp225 230 235 240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala
Glu Ala Ile Ala Thr 245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser
Pro Met Phe Gln Pro Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr
Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro
Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg
Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile305 310 315
320Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu
Asp Ile 340 345 350Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro
Glu Asp Ile Asp 355 360 365Met Asn Pro Glu Ala Leu Thr Ala Trp Lys
Arg Ala Ala Ala Ala Val 370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys
Ser Arg Arg Ile Ser Leu Glu Phe385 390 395 400Met Leu Glu Gln Ala
Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn
Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn
Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440
445Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg
Ile Lys465 470 475 480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala
Cys Ala Lys Ser Pro 485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln
Asp Ser Pro Phe Cys Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala
Gly Val Gln His His Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro
Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser
Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn545 550 555
560Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr
Asp Asn 580 585 590Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu
Ile Ser Glu Lys 595 600 605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly
Gln Trp Leu Ala Tyr Gly 610 615 620Val Thr Arg Ser Val Thr Lys Arg
Ser Val Met Thr Leu Ala Tyr Gly625 630 635 640Ser Lys Glu Phe Gly
Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile
Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala
Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680
685Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile
Leu Arg705 710 715 720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp
Gly Phe Pro Val Trp 725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr
Arg Leu Asn Leu Met Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro
Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys
Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775 780Ser Gln Asp
Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu785 790 795
800Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu
Thr Met 820 825 830Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp
Phe Tyr Asp Gln 835 840 845Phe Ala Asp Gln Leu His Glu Ser Gln Leu
Asp Lys Met Pro Ala Leu 850 855 860Pro Ala Lys Gly Asn Leu Asn Leu
Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880Ala Phe Ala
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