U.S. patent application number 15/812800 was filed with the patent office on 2018-03-22 for intracellular translation of circular rna.
This patent application is currently assigned to Ribokine LLC. The applicant listed for this patent is Robert KRUSE. Invention is credited to Robert KRUSE.
Application Number | 20180080041 15/812800 |
Document ID | / |
Family ID | 51898809 |
Filed Date | 2018-03-22 |
United States Patent
Application |
20180080041 |
Kind Code |
A1 |
KRUSE; Robert |
March 22, 2018 |
INTRACELLULAR TRANSLATION OF CIRCULAR RNA
Abstract
A circular mRNA molecule possessing features resembling native
mammalian mRNA demonstrates improved translation, while retaining
the properties of an extremely long half-life inside cells. This
circular mRNA is functional inside mammalian cells, being able to
compete against native cellular mRNAs for the eukaryotic
translation initiation machinery. The invention possesses
additional RNA elements compared to a previous invention containing
only an IRES element for successful in vitro or in vivo
translation.
Inventors: |
KRUSE; Robert; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KRUSE; Robert |
Houston |
TX |
US |
|
|
Assignee: |
Ribokine LLC
Houston
TX
|
Family ID: |
51898809 |
Appl. No.: |
15/812800 |
Filed: |
November 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14890799 |
Nov 12, 2015 |
9822378 |
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PCT/US2014/037795 |
May 13, 2014 |
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15812800 |
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61823709 |
May 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/85 20130101;
C12N 2800/50 20130101; C12N 2840/44 20130101; C12N 2999/007
20130101; C12N 2310/16 20130101; C12N 15/115 20130101; C12N 2830/42
20130101; A61K 48/00 20130101; C12N 2840/203 20130101; C12Q 1/6865
20130101; C12Q 1/6865 20130101; C12Q 2521/119 20130101; C12Q
2531/125 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C12N 15/115 20060101 C12N015/115 |
Claims
1) A vector for making circular mRNA, said vector comprising the
following elements operably connected to each other and arranged in
the following sequence: a) an RNA polymerase promoter, b) a self
circularizing intron 5' slice junction, c) an IRES, d) an optional
5' UTR, e) a multiple cloning insertion site for inserting an ORF
into said vector, f) a 3' UTR, g) an optional polyA tract, h) a
self circularizing intron 3' slice junction, and i) an optional RNA
polymerase terminator.
2) The vector of claim 1, wherein the RNA polymerase promoter and
terminator are from the T7 virus, T6 virus, SP6 virus, T3 virus, or
T4 virus.
3) The vector of claim 1, wherein the 3' UTRs are from human beta
globin, human alpha globin, xenopus beta globin, xenopus alpha
globin, human prolactin, human GAP-43, human eEF1a1, human Tau,
human TNF alpha, dengue virus, hantavirus small mRNA, bunyanavirus
small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella
virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH,
human tubulin, hibiscus chlorotic rinsgpot virus, woodchuck
hepatitis virus post-translational regulated element, sindbis
virus, turnip crinkle virus, tobacco etch virus or Venezuelan
equine encephalitis virus.
4) The vector of claim 1, wherein the 5' UTRs are from human beta
globin, xenopus laevis beta globin, human alpha globin, xenopus
laevis alpha globin, rubella virus, tobacco mosaic virus, mouse
Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco
alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or
the adenovirus tripartite leader.
5) The vector of claim 1, wherein the polyA track is at least 30
nucleotides long.
6) The vector of claim 1, wherein the polyA track is at least 60
nucleotides long.
7) The vector of claim 1, wherein the IRES is from Taura syndrome
virus, Triatoma virus, Theiler's encephalomyelitis virus, simian
Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus,
Reticuloendotheliosis virus, fuman poliovirus 1, Plautia stali
intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca
coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca
coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A
virus, Hepatitis GB virus, foot and mouth disease virus, Human
enterovirus 71, Equine rhinitis virus, Ectropis obliqua
picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus,
Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis
virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid
lethal paralysis virus, Avian encephalomyelitis virus, Acute bee
paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine
fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila
antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2,
Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L,
Human LEF1, Mouse HIF1alpha, Human n-myc, Mouse Gtx, Human p27kip1,
Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila
reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA,
Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID,
S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picornavirus,
Turnip crinkle virus, or an aptamer to eIF4G.
8) The vector of claim 1, further including an RNA sequence that
binds eIF4E when transcribed into the circular mRNA.
9) The vector of claim 8, wherein the RNA sequence binding to eIF4E
is from Mouse histone H4, Human cyclin D1, Pea enation mosaic virus
RNA2, Panicum Mosaic Virus, or an RNA aptamer to eIF4E.
10) The vector of claim 1, wherein the IRES is combined with a
second IRES facilitating additional initiation factor recruitment,
ribosome subunit binding, ribosome shunting, ribosome basepairing,
or ribosome translocation.
11) The vector of claim 1, wherein self-circularizing catalytic
intron is a Group I intron or Group II Intron.
12) The vector of claim 1, further comprising a nuclear transport
element selected from Mason Pfizer Monkey Virus Constitutive
Transport Element (CTE), 4E-SE element, woodchuck hepatitis virus
post regulatory element, hepatitis b virus post regulatory element,
or HIV rev response element.
13) The vector of claim 1, said IRES comprising SEQ ID NO. 3.
14) A method of making circular mRNA, said method comprising adding
ribonucleotide triphosphates, inorganic pyrophosphatase, RNase
inhibitor, and an RNA polymerase to the vector of claim 1 in
appropriate reaction buffer, transcribing RNA from said vector, and
allowing self-circularization of said transcribed RNA to produce
circular mRNA.
15) The method of claim 14, said ribonucleotides including modified
ribonucleotides m.sup.5C, m.sup.5U, m.sup.6A, s2U, .PSI., or
2'-O-methyl-U.
16) A method of making circular mRNA, said method comprising
transfecting the vector of claim 1 and a phage polymerase or
nucleic acid encoding a phage polymerase into a eukaryotic cell,
allowing for transcription of said vector inside the cell to
produce transcribed RNA, and allowing self-circularization of said
transcribed RNA to produce circular mRNA.
17) A method of making circular mRNA, said method comprising
transfecting the vector of claim 1 into a eukaryotic cell, wherein
said vector is transcribed by a host cell RNA polymerase.
18) A circular mRNA, wherein said circular mRNA is capable of being
translated intracellularly and has a half-life of at least twice
that of the same mRNA that is linear inside a eukaryotic cell.
19) A method of gene therapy, comprising introducing the vector of
claim 1 into a patient in need thereof.
20) A method of bioproducing a protein, comprising introducing the
vector of claim 1 into a eukaryotic cell or mammal for production
of a protein encoded by said ORF.
Description
PRIOR RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
14/890,799, now U.S. Pat. No. 9,822,378, filed on Nov. 12, 2015,
which is a National Phase under 35 U.S.C. .sctn. 371 of
International Application PCT/US2014/37795, filed May 13, 2014,
which claims priority to U.S. Provisional Application 61/823,709,
filed May 15, 2013. Each application is expressly incorporated in
its entirety by reference herein for all purposes.
FEDERALLY SPONSORED RESEARCH STATEMENT
[0002] Not applicable.
FIELD OF THE DISCLOSURE
[0003] The disclosure generally relates to a biologic product
comprising a circular RNA that is capable of translation inside a
eukaryotic cell. The invention describes novel combinations of RNA
elements that facilitate the enhanced translation and expression of
encoded polypeptides, and provides vectors for making circular
mRNA, as well as various applications using the circular mRNA
and/or vector.
BACKGROUND OF THE DISCLOSURE
[0004] "Gene therapy" is the use of DNA as an agent to treat
disease. It derives its name from the idea that DNA can be used to
supplement or alter genes within a patient's cells as a therapy to
treat disease. The most common form of gene therapy involves using
DNA that encodes a functional, therapeutic gene to replace a
mutated, non-functional gene.
[0005] Although early clinical failures led many to dismiss gene
therapy as over-hyped, clinical successes have now bolstered new
optimism in the promise of gene therapy. These include successful
treatment of patients with the retinal disease Leber's congenital
amaurosis, X-linked severe combined immunodeficiency (SCID),
adenosine deaminase SCID (ADA-SCID), adrenoleukodystrophy, chronic
lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL),
multiple myeloma and Parkinson's disease. These recent clinical
successes have led to a renewed interest in gene therapy, with
several articles in scientific and popular publications calling for
continued investment in the field.
[0006] RNA is used in antisense and siRNA based therapies, but to
date mRNA has not been used per se for gene therapy, even though
the use of mRNA versus DNA in gene therapy offers potential
advantages. For example, the protein encoded by the mRNA will be
expressed in all cells, so selection of a promoter is not a
problem. No insertional mutagenesis can occur, increasing the
safety of the method, and the transient nature of expression is
advantageous for many applications. The gene of interest can be
easily expressed in dividing or non-dividing cells, as opposed to
the limitations of DNA.
[0007] However, there are considerable technical difficulties to
overcome before mRNA can be successfully used in various
therapeutic methods.
[0008] For example, transfecting mRNA using lipids,
electroporation, and other methods results in an inflammatory
immune response mediated by Toll-like receptors recognizing the
added RNA as foreign. This recognition leads to interferons being
secreted, and if mRNA is attempted for repeated transfection, then
ultimately cell death occurs via apoptosis.
[0009] A recent breakthrough allows the innate immune response to
be avoided, thus providing a way of overcoming this first hurdle.
The strategy incorporates modified nucleotides that cannot bind to
toll-like receptors into the RNA, thus preventing the inflammatory
immune response (e.g., U.S. Pat. No. 8,278,036, US20100047261,
US20120322864). Thus, at least one challenge has been overcome in
the challenges for implementing RNA-based therapeutic
techniques.
[0010] Another difficulty has been the production of a complete and
active mRNA via in vitro transcription. Further, the resulting mRNA
must have all of the features needed for initiation and
translation, and be able to effectively compete against endogenous
mRNAs. Thus, the complete mRNA in the current art needs a 5' cap or
cap analogue, 5' UTR, ORF, 3' UTR, and polyadenylation tail to
mimic the standard mRNA molecule produced by eukaryotic cells. In
some cases, a 5' cap is omitted and an IRES sequence utilized, but
this is much more inefficient and reduces the half-life of the
linear RNA molecule with no protection of the 5' terminus of RNA.
Similarly, a polyadenylation tail can be omitted, but with reduced
translation efficiency and half-life of the linear mRNA
molecule.
[0011] Perhaps the biggest impediment, however, is the difficulty
in handling mRNA. RNA has two adjacent pendant hydroxyls on the
pentose ring of the terminal nucleotide, making it very susceptible
to nucleophilic attack by bases or by ever-present RNAses in water
and on most surfaces. RNAse-free reagents are used for the
production of mRNA and its resultant storage, but even with such
techniques, the extreme sensitivity to degradation presents
considerably difficulty in implementing any RNA based technique.
Yet another impediment is the short half-life of mRNA once inside
the cell. Messenger RNA only affords transient expression inside
cells, generally on the order of 6-12 hours.
[0012] It is well appreciated in the literature that circular RNA
molecules have much longer half-lives than their linear
counterparts, being naturally resistant to any exonuclease activity
or nucleophilic attack. Thus, the use of circular RNA can solve
both of these degradation issues. In fact, the half-life of
circular RNA in vivo was estimated to be greater than 40 hours in
Xenopus embryos. In the same system, linear mRNAs had a half-life
of 6-8 hours. Even in E. coli, a circular RNA being actively
translated was 4-6 times more stable than its linear counterpart
due to resistance to RNase E activity.
[0013] It is also known that a Shine-Dalgarno sequence is necessary
in prokaryotes for ribosome recruitment and can mediate recruit of
ribosomes to any RNA molecule, whether linear or circular. However,
circular RNA was originally thought to be unable to bind to
eukaryotic ribosomes. Fortunately, Chen (1995) demonstrated that
circular mRNA can bind eukaryotic ribosomes with the presence of an
internal ribosome entry site (IRES).
[0014] Chen utilized a picornavirus IRES sequence for this purpose
and demonstrated translation in an in vitro rabbit reticulocyte
system. The primary goal of their strategy focused on the
application of developing polymeric proteins through continuous
translation around the circular RNA molecule. In order for this to
occur, they eliminated the stop codon so that the ribosome would
never be signaled to fall off the RNA molecule. In such constructs,
only the IRES site and the coding sequence was present in the mRNA
molecule, and other signals such as UTRs, polyA tracts,
terminations sites and the like were missing.
[0015] In summary, for eukaryotes, a circular mRNA expression
system has only been demonstrated in vitro in rabbit reticulocytes,
a system that otherwise biases any level of background translation,
even on a linear template without cap or IRES sequences. There was
no data presented for the ability of a circular mRNA to translate
in vivo inside a eukaryotic cell, and results in prokaryotes were
disappointing. For application to an in vivo translation system
inside the cell, more modifications are needed to circular mRNA in
order to allow for its successful competition with native cellular
mRNAs for translation initiation factors.
[0016] The Sarnow and Chen patent (U.S. Pat. No. 5,766,903) claims
the insertion of an IRES into a circular RNA with a gene of
interest. However, this patent fails to describe the necessity of
other regulatory elements in the circular RNA molecule for in vivo
translation. Indeed, there is no data demonstrating successful
intracellular translation of circular mRNA in the patent or
publication literature. There is no discussion of the insertion of
a polyadenylation sequence, or a 3' UTR to function in synergy with
the IRES element. Furthermore, novel IRES elements with improved
translation in circular mRNA were not proposed.
[0017] Furthermore, there were almost no follow-up reports in the
literature demonstrating the utility of circular mRNA, in vitro or
in vivo. In one recent work, it was shown in a rabbit reticulocyte
system in vitro that a circular mRNA template with the SP-A1 IRES
could direct translation (Wang 2009). However, the translation
efficiency of circular RNA in vitro was 15% that of an uncapped
linear RNA with IRES. In the same experiment, a capped linear RNA
had an activity that was 131% that of uncapped linear RNA,
emphasizing how the rabbit reticulocyte system tends to bias
uncapped transcripts toward levels of translation that are
super-physiologic.
[0018] A variety of additional patents concern circular mRNA.
However, these patents fail to provide evidence of actual in vivo
translation of the circular mRNA molecule. Examples of prior art
include U.S. Pat. No. 5,766,903, U.S. Pat. No. 6,210,931, U.S. Pat.
No. 5,773,244 U.S. Pat. No. 5,580,859, US20100137407, U.S. Pat. No.
5,625,047, U.S. Pat. No. 5,712,128 US20110119782. Therefore,
although possibly recognizing the potential of using circular mRNA
for in vivo expression in eukaryotes, such applications were not in
fact enabled.
[0019] Thus, what is needed in the art are methods of making and
using circular mRNA where such molecules have been fully enabled
and shown to work in in vivo or ex vivo eukaryotic systems.
SUMMARY OF THE DISCLOSURE
[0020] The present disclosure relates to a circular mRNA molecule
that can effectively translate inside eukaryotic cells, as well as
to methods of making and using same, and to the vectors used to
produce circular mRNAs.
[0021] A preferred use includes the administration of circular mRNA
molecules into mammalian cells or animals, e.g., for therapy or
bioproduction of useful proteins. The method is advantageous in
providing the production of a desired polypeptide inside eukaryotic
cells with a longer half-life than linear RNA, due to resistance
from ribonucleases and bases.
[0022] The circular mRNA can be transfected as is, or can be
transfected in DNA vector form and transcribed in the cell, as
desired. Cellular transcription can use added polymerases or
nucleic acids encoding same, or preferably can use endogenous
polymerases. We have demonstrated proof of concept herein with
added T7 polymerases, but this is exemplary only, and more
convenient cell based polymerases may be preferred.
[0023] The preferred half-life of a circular mRNA in a eukaryotic
cell is at least 20 hrs, 30 hrs or even at least 40 hrs, as
measured by either a hybridization or quantitative RT-PCR
experiments.
[0024] A preferred embodiment of the invention consists of a
circular mRNA molecule with an IRES, 5' UTR, coding sequence of
interest, 3' UTR and polyadenylation sequence, in that order. It is
well appreciated that many different combinations of these RNA
elements with translation enhancing properties and synergy can be
created. Such combinations include but are not limited to
IRES-ORF-3' UTR-polyA, IRES-ORF-3' UTR, IRES-5' UTR-ORF-3' UTR, and
the like.
[0025] One embodiment of the invention consists of a circular RNA
molecule with modified RNA nucleotides. The possible modified
ribonucleotide bases include 5-methylcytidine and pseudouridine.
These nucleotides provide additional stability and resistance to
immune activation.
[0026] Another embodiment of the invention consists of the in vitro
transcription of a DNA template encoding the circular mRNA molecule
of interest. Inverted intron self-splicing sequences at both ends
of the RNA molecule facilitate the formation of circular RNA
without any additional enzymes being needed.
[0027] An additional embodiment of the invention includes the
production of circular mRNA inside the cell, which can be
transcribed off a DNA template in the cytoplasm by a bacteriophage
RNA polymerase, or in the nucleus by host RNA polymerase II.
[0028] One embodiment of the invention consists of the injection of
circular mRNA into a human or animal, such that a polypeptide
encoded by the circular mRNA molecule is expressed inside the
organism. The polypeptide can either be found intracellularly or
secreted.
[0029] In another embodiment of the invention, circular mRNA can be
transfected inside cells in tissue culture to express desired
polypeptides of interest. In particular, circular mRNA can express
intracellular proteins and membrane proteins in the cells of
interest.
[0030] The invention includes one or more of the following
features, in all possible combinations thereof: [0031] A vector for
making circular mRNA, said vector comprising the following elements
operably connected to each other and arranged in the following
sequence: a) an RNA polymerase promoter, b) a self circularizing
intron 5' slice junction, c) an IRES, d) an optional 5' UTR, e) a
multiple cloning insertion site for inserting an ORF into said
vector, f) a 3' UTR, g) optionally a polyA tract, h) a self
circularizing intron 3' slice junction, and i) an optional RNA
polymerase terminator. [0032] A vector wherein the RNA polymerase
promoter and terminator are from the T7 virus, T6 virus, SP6 virus,
T3 virus, or T4 virus. [0033] A vector wherein the 3' UTRs are from
human beta globin, human alpha globin xenopus beta globin, xenopus
alpha globin, human prolactin, human GAP-43, human eEF1a1, human
Tau, human TNF alpha, dengue virus, hantavirus small mRNA,
bunyanavirus small mRNA, turnip yellow mosaic virus, hepatitis C
virus, rubella virus, tobacco mosaic virus, human IL-8, human
actin, human GAPDH, human tubulin, hibiscus chlorotic rinsgpot
virus, woodchuck hepatitis virus post translationally regulated
element, sindbis virus, turnip crinkle virus, tobacco etch virus,
or Venezuelan equine encephalitis virus. [0034] A vector wherein
the 5' UTRs are from human beta globin, Xenopus laevis beta globin,
human alpha globin, Xenopus laevis alpha globin, rubella virus,
tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein
70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch
virus, turnip crinkle virus, or the adenovirus tripartite leader.
[0035] A vector wherein the polyA track is at least 30 nucleotides
long or at least 60 nucleotides long. [0036] A vector wherein the
IRES is from Taura syndrome virus, Triatoma virus, Theiler's
encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus
1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human
poliovirus 1, Plautia stali intestine virus, Kashmir bee virus,
Human rhinovirus 2, Homalodisca coagulata virus-1, Human
Immunodeficiency Virus type 1, Homalodisca coagulata virus-1,
Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis
GB virus, Foot and mouth disease virus, Human enterovirus 71,
Equine rhinitis virus, Ectropis obliqua picorna-like virus,
Encephalomyocarditis virus, Drosophila C Virus, Human
coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus,
Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal
paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis
virus, Hibiscus chlorotic ringspot virus, Classical swine fever
virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila
antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2,
Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L,
Human LEF1, Mouse HIF1alpha, Human n.myc, Mouse Gtx, Human p27kip1,
Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila
reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA,
Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID,
S. cerevisiae YAP1, WO0155369, tobacco etch virus, turnip crinkle
virus, or an aptamer to eIF4G. [0037] A vector including an RNA
sequence that binds eIF4E when transcribed into the circular mRNA,
functioning as an IRES element. [0038] A vector wherein the RNA
sequence binding to eIF4E is from Mouse histone H4, Human cyclin
D1, Pea enation mosaic virus RNA2, Panicum Mosaic Virus, or an RNA
aptamer to eIF4E. [0039] A vector wherein the IRES is combined with
a second IRES facilitating additional initiation factor
recruitment, ribosome subunit binding, ribosome shunting, ribosome
basepairing, or ribosome translocation. [0040] A vector wherein in
self-circularizing catalytic intron is a Group I intron or Group II
Intron. [0041] A vector comprising a nuclear transport element
selected from Mason Pfizer Monkey Virus Constitutive Transport
Element (CTE), 4E-SE element, woodchuck hepatitis virus post
regulatory element, hepatitis b virus post regulatory element, or
HIV rev response element. [0042] A vector wherein said IRES
comprises SEQ ID NO. 3. [0043] A method of making circular mRNA,
said method comprising adding ribonucleotide triphosphates,
inorganic pyrophosphatase, RNase inhibitor, and an RNA polymerase
to a vector herein described in appropriate reaction buffer,
transcribing RNA from said vector, and allowing
self-circularization of said transcribed RNA to produce circular
mRNA. [0044] A method as herein described, wherein said
ribonucleotides including modified ribonucleotides m5C, m5U, m6A,
s2U, .PSI., or 2'-O-methyl-U. [0045] A method of making circular
mRNA, said method comprising transfecting the vector herein
described and a phage polymerase or nucleic acid encoding a phage
polymerase into a eukaryotic cell, allowing for transcription of
said vector inside the cell to produce transcribed RNA, and
allowing self-circularization of said transcribed RNA to produce
circular mRNA. [0046] A method of making circular mRNA, said method
comprising transfecting a vector herein described into a eukaryotic
cell, wherein said vector is transcribed by a host cell RNA
polymerase. [0047] A circular mRNA made by any method or vector
herein. [0048] A circular mRNA with a half-life of at least 20 hrs
in a eukaryotic cell or with a half-life of at least twice that of
the same mRNA that is linear inside a eukaryotic cell. [0049] A
method of gene therapy, comprising introducing a circular mRNA into
a patient in need thereof. [0050] A method of gene therapy,
comprising introducing a vector as described herein into a patient
in need thereof. [0051] A method of bioproducing a protein,
comprising introducing a vector herein described into a eukaryotic
cell or a mammal for production of a protein encoded by said ORF.
[0052] A method of bioproducing a protein, comprising introducing a
circular mRNA into a eukaryotic cell or a mammal for production of
a protein encoded by said ORF.
[0053] By "gene" herein what is a meant is a DNA molecule that
includes at least promoter, ORF, and termination sequence and any
other desired expression control sequences.
[0054] By "ORF" what is meant is an open reading frame, typically
encoding a protein of interest.
[0055] By "in vivo" what is meant is translation of mRNA inside a
cell, versus translation "in vitro" where a mixture of purified
components included eukaryotic translation initiation factors,
ribosomes, tRNAs charged with amino acids, and mRNA are mixed
together without intact cells. "Ex vivo" means inside living cells
that originated from a multicellular organism, but are now grown as
cell cultures.
[0056] By "vector" or "cloning vector" what is meant is a small
piece of DNA, taken from a virus, plasmid, or cell of a higher
organism, that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted for cloning and/or
expression purposes. A vector typically has an origin of
replication, a selectable marker or reporter gene, such as
antibiotic resistance or GFP, and usually contains a multiple
cloning site. The term includes plasmid vectors, viral vectors,
cosmids, bacterial artificial chromosomes (BACs), yeast artificial
chromosomes (YACs), and the like.
[0057] In some embodiments the vector may also contain integration
sequences, allowing for integration into a host genome, and such
may be particularly preferred for cell based bioreactors because of
increased stability.
[0058] An "expression vector" is a vector that also contains all of
the sequences needed for transcription and translation of an ORF.
These include a strong promoter, the correct translation initiation
sequence such as a ribosomal binding site and start codon, a strong
termination codon, and a transcription termination sequence. There
are differences in the machinery for protein synthesis between
prokaryotes and eukaryotes, therefore the expression vectors must
have the elements for expression that is appropriate for the chosen
host. For example, prokaryotes expression vectors would have a
Shine-Dalgarno sequence at its translation initiation site for the
binding of ribosomes, while eukaryotes expression vectors contains
the Kozak consensus sequence.
[0059] A "multiple cloning site" or "MCS", also called a
"polylinker," is a short segment of DNA which contains many (up to
.about.20) restriction sites and is a standard feature of
engineered plasmids and other vectors. Restriction sites within an
MCS are typically unique, occurring only once within a given
plasmid, and can therefore be used to insert an ORF of interest
into a vector. Furthermore, expression vectors are often designed
so that the MCS can insert the ORF in the correct reading frame by
choosing the correct insertion site, and/or the user can select the
reading frame by choice of vectors, which are often available in
all three frames.
[0060] "Aptamers" are oligonucleic acid or peptide molecules that
bind to a specific target molecule. Aptamers are usually created by
selecting them from a large random sequence pool, but natural
aptamers also exist in riboswitches. Aptamers can be used for both
basic research and clinical purposes as macromolecular drugs.
Aptamers can be combined with ribozymes to self-cleave in the
presence of their target molecule. These compound molecules have
additional research, industrial and clinical applications.
[0061] More specifically, nucleic acid aptamers can be classified
as DNA or RNA or XNA aptamers. They consist of (usually short)
strands of oligonucleotides. Peptide aptamers consist of a short
variable peptide domain, attached at both ends to a protein
scaffold.
[0062] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims or the specification means
one or more than one, unless the context dictates otherwise.
[0063] The term "about" means the stated value plus or minus the
margin of error of measurement or plus or minus 10% if no method of
measurement is indicated.
[0064] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or if the alternatives are mutually exclusive.
[0065] The terms "comprise", "have", "include" and "contain" (and
their variants) are open-ended linking verbs and allow the addition
of other elements when used in a claim.
[0066] The phrase "consisting of" is closed, and excludes all
additional elements.
[0067] The phrase "consisting essentially of" excludes additional
material elements, but allows the inclusions of non-material
elements that do not substantially change the nature of the
invention, such as instructions for use, buffers, and the like.
TABLE-US-00001 ABBREVIATION TERM GFP Green fluorescent protein ORF
Open reading frame IRES Internal ribosome entry site UTR
Untranslated region DAPI 4',6-diamidino-2-phenylindole is a
fluorescent stain that binds strongly to A-T rich regions in DNA.
HEK Human embryonic kidney IRES Interal Ribosome Entry Site CITE
Cap independent Translation Element PEMV Pea enation mosaic virus
4E-SE 4E sensitive element EMCV Encephalomyocarditis virus, a
picornavirus PFA Paraformaldehyde
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1. Shows a vector designed to produce a circular mRNA
molecule. The vector is shown with an ORF inserted thereinto, but
before insertion a MCS would be shown instead.
[0069] FIG. 2. Shows the GFP reporter protein imaging results from
circular mRNA translation in HEK cells after T7 RNA polymerase
driven expression.
[0070] FIG. 3. Shows the GFP and DAPI (nuclear stain) imaging
results from circular mRNA translation in HEK cells after T7 RNA
polymerase driven expression.
[0071] FIG. 4 shows SEQ ID NOs. 1-9. SEQ ID NO. 1: T7 RNA
Polymerase Promoter (21 bp); SEQ ID NO. 2: 5' Group I Intron
sequence (167 bp); SEQ ID NO. 3: eIF4E aptamer 1 sequence (86 bp);
SEQ ID NO. 4: Human Beta Globin 5' UTR (50 bp); SEQ ID NO. 5: Human
Beta Globin 3' UTR (133 bp); SEQ ID NO. 6: 3' Group I Intron
sequence (107 bp); SEQ ID NO. 7: T7 RNA Polymerase Terminator (47
bp); SEQ ID NO. 8: Polyadenylation Sequence (33 bp); SEQ ID NO. 9:
EMCV IRES (593 bp).
[0072] FIG. 5 shows SEQ ID NO. 10: pBSK-CR sequence=synthesized DNA
sequence based on the outline provided in FIG. 5. Genes of Interest
(GOI) can be cloned between NcoI and SalI and expressed as a
circular mRNA with eIF4E aptamer-beta globin 5'UTR-GOI-beta globin
3'UTR-polyA.
[0073] FIG. 6 shows SEQ ID NO. 11: Gene 1LG--Expresses uncapped
Linear mRNA containing IRES GFP.
[0074] FIG. 7 shows SEQ ID NO. 12: Gene 2CI--Expresses Circular
mRNA containing IRES GFP.
[0075] FIG. 8 shows SEQ ID NO. 13: Gene 3CIA--Expresses Circular
mRNA containing IRES GFP, beta globin 3'UTR and polyadenylation
sequence.
[0076] FIG. 9 shows SEQ ID NO. 14: Gene 4EA--Expresses Circular
mRNA containing eIF4E aptamer, beta globin 5'UTR, GFP, beta globin
3'UTR, and polyadenylation sequence.
[0077] FIG. 10. shows one exemplary vector sequence as SEQ ID NO.
15: Circular RNA IRES-MCS-UTR-polyA--Vector contains a multiple
cloning site after an EMCV IRES and before beta globin 3'UTR and
polyA sequence to allow for insertion of genes in multiple reading
frames. Expression yields circular mRNA.
DETAILED DESCRIPTION
[0078] The current disclosure describes circular mRNA molecules
that can successfully translate inside mammalian cells, as well as
methods of making same, vectors for making same, and methods of
using either the vector or the circular mRNA.
[0079] The circular mRNA features additional regions beyond the
IRES and ORF in order to help recruit ribosomes to the circular
mRNA. The circular mRNA has an IRES site, an ORF for protein of
interest, a 3'UTR, and an optional polyA track. In some embodiments
of the invention, there can be both an IRES and a 5' UTR, depending
upon how the IRES functions. Note that given the wide diversity of
IRES sequences in nature, there will be a wide range of
translational efficiencies when these IRES sequences are
substituted in the proposed vector. In general, however, the
invention will increase the efficiency of circular mRNA product
regardless of the nature of the IRES in question because of the use
of the polyA tail and 3' UTR elements, both of which help recruit
ribosomes for translation.
[0080] In order for circular mRNAs to translate efficiently inside
cells, they must compete in vivo against cellular mRNAs also
recruiting the translation machinery. While IRES sequences have
fewer translation initiation requirements, the canonical
recruitment process via eIF4E still recruits ribosomes much more
efficiently in a head to head comparison. The present invention
describes in addition to viral IRES sequences, an IRES sequence for
circular mRNA that can recruit eIF4E itself.
[0081] In one embodiment of the invention, the IRES is an aptamer
to eIF4E (SEQ ID NO. 3) with specificity for the face of the
protein that binds the guanosine cap. This aptamer has never been
used as an IRES before, and represents a novel aspect of the
disclosure. The aptamer is able to bind to eIF4E and thereby the
rest of the translation initiation complex, similar to cellular
mRNA molecules. Helping translation initiation is the presence of
the 3' UTR and polyA tail, meaning the circular mRNA in this
embodiment of the invention can recruit ribosomes in almost the
exact same way as cellular RNA. Cellular RNA circularizes by the
way of a physical connection of PABP to eIF4G to eIF4E proteins,
whereas the circular RNA in the invention is held together by a
physical link.
[0082] Recruitment of eIF4E for cap-independent translation can be
achieved by tethering eIF4E via a peptide tag to an RNA structure
that specifically binds the tag. This suggests that if an RNA
structure or aptamer or stem-loop could bind eIF4E directly, than
cap-independent translation could be achieved. Indeed, this has
been observed already for a plant virus, Pea enation mosaic virus
(PEMV). Its RNA2 molecule contains a pseudoknot RNA structure that
directly binds to plant eIF4E protein.
[0083] Certain eukaryotic transcripts are also able to recruit the
eIF4E protein via RNA motifs in their 3' UTRs or coding sequences,
acting independently of the cap binding mechanisms. This motif is
called a 4E sensitive element, or 4E-SE. Examples of 4E-SEs are
found in Mouse histone H4 mRNA and Human cyclin D1 mRNA. Their role
in these mRNAs is to regulate nuclear localization and export, as
well as modulating translation. For the purposes of a circular
mRNA, it can easily be imagined that the 4E-SE could be used to
similarly recruit eIF4E to the mRNA independent of cap and
stimulate translation.
[0084] In another embodiment of the invention, the IRES is a 4E-SE
element, taken from sequences in cellular mRNAs, which mediates
direct binding to eIF4E. In this embodiment, a 5'UTR or IRES
downstream of the 4E-SE may be added that promotes ribosome
shunting as a way to stimulate non-canonical translation. An
example would be the mouse Gtx 5'UTR or any viral or cellular IRES
sequence.
[0085] As an alternative to using a 4E-SE sequence to bind eIF4E,
an aptamer directed against the cap-binding pocket of eIF4E is
proposed to be able to replicate the effects the normal guanosine
cap in promoting translation. An example eIF4E aptamer sequence is
given in SEQ ID NO. 3. As is known in the art, aptamers of many
degenerate sequences can be generated against a given protein, and
this is only one exemplary sequence.
[0086] In a similar fashion, novel IRESes could be developed that
bind directly to eIF4G, skipping the necessary recruitment via
eIF4E. An example of this strategy would be to develop aptamers
against eIF4G that do not inhibit translation, but mediate strong
binding to eIF4G.
[0087] Beyond utilizing novel IRES sequences, adding other RNA
elements to the circular mRNA molecule allow for translation inside
cells. It is readily recognized for example, that while the cap is
an important structure for eukaryotic linear mRNA translation, the
5' UTR, 3' UTR and polyA tails also play important roles in
translation.
[0088] The preferred embodiment of the invention contains a
polyadenylation sequence within the circular RNA molecule of about
30-ribonucleotides of adenosine, which is able to bind to a single
complex of human poly(A)-binding protein. This polyadenylation
sequence would be located after the ORF, 3'UTR and before the
splice site and termination signal.
[0089] Polyadenylation of mRNAs have been shown to increase the
expression of viral IRES driven expression. The added polyA
sequence in the circular mRNA might also function as a type of
additional IRES site, as suggested by a report that a polyA 5' RNA
leader could allow the bypass of initiation factors in mediating
translation.
[0090] In addition to viral IRESes, cellular mRNAs can also have
the translation efficiency of their IRES sequences increased with
polyadenylation tails. The c-myc and BiP mRNA IRES activity could
be enhanced though the addition of a polyA tail, even without
intact eIF4G or PABP, factors which would normally mediate such an
interaction.
[0091] In one embodiment of the invention, a pair of viral 5' and
3' UTRs may be utilized that naturally communicate with each other
to mediate translation. The 5' and 3' UTRs of many viruses
communicate through RNA-RNA or RNA-protein interactions to
facilitate increased translation or regulation of translation. This
suggests that optimizing the use of the said UTRs or by bringing
the ends together permanently through circularization might lead to
enhanced translation. One example of synergistic UTRs useful in the
circular RNA invention is the pair of 5' and 3' UTRs from the
dengue virus, which together possess IRES activity.
[0092] In the invention, the ability of 3' certain viral UTR
sequences to augment or replace some of the canonical components of
mRNA is also proposed. As an example, the 3'UTR of the Andes
Hantavirus Small mRNA can functionally replace the polyA tail and
can act in synergism with cap-dependent translation.
[0093] In another embodiment of the invention, a 5' UTR will be
utilized that will facilitate the delivery of the ribosome to the
first codon of the polypeptide to be translated. The mechanism of
ribosomal tethering and delivery to downstream AUG codons would
also be useful in circular mRNA molecules. This process is also
referred to "ribosomal shunting." An example of a sequence that
mediates shunting is an mRNA element from the 5' UTR of the Gtx
homodomain mRNA, which basepairs to 18S rRNA, and the adenovirus
tripartite leader.
[0094] While modified RNA nucleotides have received much attention
for their resistance to nucleases in the setting of siRNA among
other applications, modified RNA nucleotides produce only moderate
improvements in translation efficiency and transcript half-life.
Thus, circular RNA represents an improvement in the ability to
achieve the longest transcript half-lives compared to all other
methods today, while at the same time providing a much more robust
and cheaper method of mRNA production requiring only the single RNA
polymerase enzyme. This is compared to other mRNA in vitro product
protocols in the prior art that require up to 3 enzyme reactions
total (e.g., RNA polymerase, polyadenylase, and capping enzyme).
Furthermore, RNA yields from transcription reactions mixed with cap
analogue are generally 2-6 times lower than without, representing
another production advantage for circular mRNA.
[0095] The circular mRNA described herein can also be produced in
vivo inside the cell. There are two different embodiments for in
vivo production of circular mRNA. In the first embodiment, DNA is
delivered or integrated into nucleus. Transcription will be driven
by a promoter recruiting a RNA polymerase II that is endogenous to
that cell. Self-splicing would occur within the nucleus. Given that
the 5' cap has been shown to be important for mRNA export, an
alternative means may need to be added in order to increase
circular mRNA export. An example is the Mason Pfizer Monkey Virus
constitutive transport element (CTE), an RNA sequence which helps
mediate non-canonical mRNA export.
[0096] The other means of in vivo circular mRNA generation would
consist of transfecting linear or circular DNA containing an e.g.,
T7 promoter inside the cell, and adding e.g., T7. T7 polymerase
protein could be transfected along with the plasmid DNA, whereafter
in the cytoplasm it would bind the T7 promoter on the vector DNA
and begin transcribing circular mRNA. In one embodiment of this
method, the transcription cassette lacks a T7 terminator leading to
continuous rolling circle transcription of RNA where T7 never
dissociates from the DNA template.
[0097] In other embodiments, DNA encoding T7 DNA or even T7 mRNA
could be added to the cell, allowing transcription and translation
to produce the T7 inside the cell. Of course, T7 is exemplary only
and any similar RNA polymerase could be used, such as T6, T4, T3,
SP6, or RNA Polymerase I and the like.
[0098] The technologies required to produce circular RNA have been
described in the literature previously. Commonly, group I
self-splicing by a permuted intron-exon sequences from the T4
bacteriophage is used. This reaction can occur in prokaryotic
cells, eukaryotic cells, or in vitro since it is catalyzed by RNA
alone. However, a variety of different methods exist in that prior
art concerning ways to synthesize circular RNA. It is understood
that the proposed enhanced circular mRNA molecule could use any of
these methods in its production (e.g., U.S. Pat. No. 6,210,931,
U.S. Pat. No. 5,773,244).
[0099] Examples of group I intron self-splicing sequences include
self-splicing permuted intron-exon sequences derived from T4
bacteriophage gene td. The intervening sequence (IVS) rRNA of
Tetrahymena also contains an example of a Group I intron self
splicing sequences. Given the widespread existence of group I and
group II catalytic introns across nature, many possible sequences
could be used for creating circular RNA.
[0100] Self-splicing occurs for rare introns that form a ribozyme,
performing the functions of the spliceosome by RNA alone. There are
three kinds of self-splicing introns, Group I, Group II and Group
III. Group I and II introns perform splicing similar to the
spliceosome without requiring any protein. This similarity suggests
that Group I and II introns may be evolutionarily related to the
spliceosome. Self-splicing may also be very ancient, and may have
existed in an RNA world present before protein.
[0101] Cytoplasmic expression systems have been used before as an
alternative to nuclear dependent transcription, or the transfection
of mRNA itself. These systems rely on the co-transfection of a
phage RNA polymerase (usually T7 DNA polymerase) with a DNA
template. Sometimes, the T7 is expressed as a gene from a nuclear
promoter, or the mRNA encoding T7 polymerase is transfected inside
the cell. These provide alternatives to protein transfection of T7
polymerase. Furthermore, T7 polymerase could direct the synthesis
of more T7 polymerase in certain systems, creating a
self-sustaining autogene effect. Such autogene systems achieve
unparalleled expression levels, and are only limited by the amount
of triphosphate-ribonucleotides in the cytoplasm among other
factors.
[0102] In another application of the invention, circular mRNAs
could be generated continuously off a circular template, due to the
highly processive nature of T7 RNA polymerase, which rarely falls
off a DNA template during the elongation phase. T7 RNA polymerase
can circle around plasmids many times if no proper termination
sequence is provided. This has been shown in an shRNA system to
produced a greatly increased yield of RNA product.
[0103] In an effort to mediate translation of RNA based
technologies to clinical use, advances have been made in purifying
mRNA on a large scale, eliminating double-stranded RNA impurities
that can activate the innate immune system (e.g., EP2510099,
EP2092064).
[0104] A related application distinct from circular mRNA molecules
describes a circular RNA interference effector molecules (e.g.,
WO2010084371). Also, it has been recently published in the
literature that human cells possess natural circular RNA molecules
that appear to function as micro-RNA sponges. These circular RNA
molecules were tested, however, and showed no translation activity,
despite possessing exon sequences from proteins. In a slightly
different application, the circular RNA molecules serve as
substrates for Dicer and further processing to produce siRNA (e.g.,
EP2143792).
[0105] The following experiments are exemplary only and serve to
provide proof of concept experiments for the invention generally.
However, the invention and the claims should not be limited by the
specific exemplars provided.
Vector Construction
[0106] A series of vectors were prepared to make circular mRNA
matching the scheme outlined in FIG. 1. This template was then used
to construct a series of different GFP encoding genes, which yield
different types of mRNA molecules. The genes 1LG and 2CI produce
linear mRNA molecules that exist in the prior art, while the genes
3CIA and 4CEA produce circular mRNA molecules that are novel to the
current invention. As outlined below standard cloning procedures
were utilized to produce the final vector DNA sequences.
[0107] The plasmid, pBSK-CR was prepared with a synthesized DNA
sequence matching FIG. 5 (Seq. ID No. 10). Another plasmid,
pIRES-GFP, containing the EMCV IRES followed by a GFP sequence was
also obtained. The following cloning steps were undertaken to
produce the vectors used to generate circular mRNAs herein:
[0108] Vector 1LG, which produces a linear, uncapped RNA molecule
with IRES GFP-Beta Globin 3' UTR-polyA, was constructed by
digestion with BamHI and SalI in both pIRES-GFP and pBSK-CR,
followed by ligation of the IRES-GFP insert into the pBSK-CR
sequence.
[0109] Vector 2CI, which produces a circular RNA molecule with
IRES-GFP alone, was constructed by digestion of pIRES-GFP and
pBSK-CR with XhoI and XbaI, followed by ligation of the IRES-GFP
insert into the pBSK-CR sequence.
[0110] Vector 3CIA, which produces a circular RNA molecule with
IRES-GFP-Beta Globin 3' UTR-polyA, was constructed by digestion of
pIRES-GFP and pBSK-CR with XhoI and SalI, followed by ligation of
the IRES-GFP insert into the pBSK-CR sequence.
[0111] Vector 4CEA, which produces a circular RNA molecule with
eIF4E aptamer-beta globin 5' UTR-GFP-beta globin 3' UTR-polyA, was
constructed by digestion of pBSK-CR and pIRES-GFP with NcoI and
SalI, followed by ligation of the GFP insert into the pBSK-CR
sequence.
[0112] Prior art plasmid pIRES-GFP produces a canonical linear
capped mRNA with polyA tail that is produced inside the nucleus of
a cell driven by a CMV promoter. This plasmid allows us to compare
our novel circular mRNA with the expression of linear capped mRNAs
and provides a direct comparison with the prior art. The relative
mRNA levels produced will be different between the two systems
given their different promoters, however.
Intracellular T7 Driven mRNA Expression
[0113] The purpose of this experiment was to generate mRNA inside
the cell with T7 polymerase, eliminating variables of toxic effects
of RNA during transfection, or the possible degradation of the mRNA
by abundant RNases in the environment during experimental handling.
The goal was to co-transfect plasmid DNA (combinations shown below)
into HEK 293 cells together with active T7 RNA polymerase protein
in a 24-well format, with four wells per condition. All amounts and
volumes are given on a per well basis.
TABLE-US-00002 Conditions GFP expression Expressed mRNA Sequences
pIRES-GFP + Lipofectamine Positive Control Linear capped
IRES-GFP-polyA 1LG + T7 + Lipofectamine Positive Control Linear
uncapped IRES-GFP-3' UTR-polyA 1LG + Lipofectamine Negative Control
None (because no T7 added) 2CI + T7 + Lipofectamine Test condition
Circular IRES-GFP 3CIA + T7 + Lipofectamine Test condition Circular
IRES-GFP-3' UTR-polyA 4CEA + T7 + Lipofectamine Test condition
Circular eIF4E aptamer-5' UTR-GFP-3' UTR-polyA
[0114] 1. The day before transfection, HEK cells were trypsinized
and counted. Cells were plated at 1.0.times.10.sup.5 cells per well
in 0.5 ml of complete growth medium.
[0115] 2. 2 .mu.g of DNA and 50 U of T7 RNA polymerase (NEB.RTM.)
in 50 .mu.l of serum-free OPTIMEM medium were combined, and
incubated for 10 minutes at room temperature.
[0116] 3. Pure lipofectamine (5 .mu.l) was added to the plasmid/T7
RNA polymerase complex, the mixture incubated for 45 min, and then
diluted to 200 .mu.l with OPTIMEM medium.
[0117] 4. After a further 30-minute incubation, 200 .mu.l of the
DNA-T7 polymerase-Lipofectamine reagent complexes were added
directly to each well containing cells and mixed gently by rocking
the plate back and forth. The DNA/protein/lipofectamine complexes
do not have to be removed following transfection.
[0118] 5. The cells are incubated at 37.degree. C. in 5% CO.sub.2
for 24 hours.
[0119] 6. Pictures of the HEK cells were then taken using
fluorescent microscope at 24 hours to detect GFP expression. In
some experiments, the cells were fixed at 48 hours using 4% PFA,
and then stained with DAPI to detect the outline of the nucleus of
cells and improve GFP visualization. Localization of GFP could then
be observed in reference to the position of the nucleus.
[0120] The results of the experiment showed that the linear
uncapped IRES-GFP-3' UTR-polyA expressed GFP (1LG), as has been
observed in several systems. The 2CI circular mRNA, which matches
the prior art of circular RNA with IRES and ORF elements only,
failed to show GFP expression in repeated experiments when imaged
during live cells (FIG. 2) or after cell fixation (FIG. 3). Thus,
merely circularizing an RNA is not sufficient for eukaryotic
expression in eukaryotic cells, even when the same mRNA is
transcribable in linear form (not shown herein, but demonstrated in
the prior art).
[0121] The 3CIA and 4CEA circular mRNAs of the invention exhibited
distinct GFP expression, which was similar in GFP intensity to the
linear uncapped 1LG mRNA.
TABLE-US-00003 GFP Conditions expression Sequences Results
pIRES-GFP + Positive Control Linear capped IRES-GFP-polyA +++
Lipofectamine 1LG + T7 + Positive Control Linear uncapped
IRES-GFP-3' UTR-polyA ++ Lipofectamine 1LG + Negative none -
Lipofectamine Control 2CI + T7 + Test condition Circular IRES-GFP -
Lipofectamine 3CIA + T7 + Test condition Circular IRES-GFP-3'
UTR-polyA ++ Lipofectamine 4CEA + T7 + Test condition Circular
eIF4E aptamer-5' UTR-GFP-3' UTR- ++ Lipofectamine polyA
mRNA TRANSCRIPTION AND TRANSFECTION
[0122] The same vectors 1LG, 2CI, 3CIA, and 4EA from the previous
experiment were used as templates for in vitro mRNA transcription.
The process of in vitro mRNA transcription is well known in the
field and consists of obtaining a DNA template with a phage
promoter of short length followed by the gene of interest on the
same sense strand. This DNA template is oftentimes linearized due
to the high processivity of RNA polymerases, but can remain
circular if a polymerase terminator sequence follows after the
gene.
[0123] For the experiments herein, an in vitro mRNA transcription
reaction was set up using the MEGAscript kit from Ambion.RTM.. A
mixture of ribonucleotides, T7 polymerase and DNA template was
added in a 20 .mu.L reaction mixture. The reaction was allowed to
proceed for 2 hours at 37.degree. C. The mRNA transcripts were then
purified using a standard lithium chloride protocol to remove
excess ribonucleotides, DNA and protein.
[0124] The purified mRNA was then transfected into HEK 293 cells
using Lipofectamine, as follows:
[0125] 1. The day before transfection, HEK cells were trypsinized
and counted. Cells were plated at 1.0.times.10.sup.5 cells per well
in 0.5 ml of complete growth medium.
[0126] 2. 0.5-1 .mu.g of RNA was added to 2.5 .mu.L Lipofectamine
2000, the mixture was incubated for 45 min, and then diluted to 200
.mu.l with OPTIMEM medium.
[0127] 4. After a further 30-minute incubation, 200 .mu.l of the
mRNA-Lipofectamine Reagent complexes were added directly to each
well containing cells and mixed by gently by rocking the plate back
and forth. Complexes were not removed following transfection.
[0128] 5. Cells were further incubated at 37.degree. C. in 5%
CO.sub.2 for 24 hours.
[0129] 6. Pictures of the HEK cells were taken using fluorescent
microscope at 24 hours to detect GFP expression. In some
experiments, the cells were fixed at 48 hours using 4% PFA, and
then stain with DAPI to detect the outline of the nucleus of cells.
Localization of GFP could then be observed in reference to the
position of the nucleus.
[0130] The results of the mRNA transfection experiment matched the
results of intracellular T7 driven mRNA expression, as expected.
Linear uncapped IRES-GFP-3' UTR-polyA mRNA expressed GFP (1LG), as
has been observed in several prior art systems. The 2CI circular
mRNA, which matches the prior art of EMCV IRES and ORF alone (see
U.S. Pat. No. 5,766,903), failed to show GFP expression in repeated
experiments when imaged during live cells or after cell fixation.
Thus, consistent with the above experiments, and IRES and ORF alone
are insufficient for intracellular transcription of a circular
mRNA. Furthermore, it is predicted to not be sufficient for live
animal (in vivo) expression either.
[0131] The 3CIA and 4CEA circular mRNAs exhibited GFP expression,
which was similar in GFP intensity to the linear uncapped 1LG
mRNA.
TABLE-US-00004 GFP Conditions expression Sequences Results
pIRES-GFP + Positive Linear capped +++ Lipofectamine Control
IRES-GFP-polyA 1LG + T7 + Positive Linear IRES-GFP-3' ++
Lipofectamine Control UTR-polyA 1LG + Negative none - Lipofectamine
Control 2CI + T7 + Test condition Circular IRES-GFP - Lipofectamine
3CIA + T7 + Test condition Circular IRES-GFP-3' ++ Lipofectamine
UTR-polyA 4CEA + T7 + Test condition Circular eIF4E aptamer-5' ++
Lipofectamine UTR-GFP-3' UTR-polyA
CONCLUSION
[0132] Using two different methods of circular mRNA production, it
was observed for the first time that circular mRNA can be
translated intracellularly in a eukaryotic cell in direct
competition with host capped mRNAs. This is a significant finding
that previous researchers were unable to accomplish. Furthermore,
circular mRNAs that translate inside eukaryotic cells have not been
found to exist in nature so far, and thus these results are
unexpected. Indeed, while circular exons and introns are now
appreciated to exist inside eukaryotic cells, evolution appears not
to have selected for a circular mRNA capable translation by
ribosomes. A summary of the experimental results is listed in the
table below.
TABLE-US-00005 Vector Expressed RNA Molecule Intracellular
expression pIRES- Linear capped IRES-GFP-polyA Demonstrated in GFP
Previous Studies 1LG Linear IRES-GFP-3' UTR-polyA Demonstrated in
Current and Previous Studies 2CI Circular IRES-GFP No Expression in
Current Study 3CIA Circular IRES-GFP-3' UTR-polyA Demonstrated in
Current Study 4CEA Circular 4E aptamer-GFP-3'UTR- Demonstrated in
Current polyA Study
[0133] The gene 2CI was constructed to produce a circular mRNA
molecule that matches the prior art containing the same EMCV IRES
and ORF construction (see U.S. Pat. No. 5,766,903). The 2CI gene
thus serves as a comparison with the current invention, which
contains multiple RNA translation enhancing elements. One observes
that the 2CI circular mRNA encoding EMCV IRES-GFP alone fails to
produce any discernable GFP expression both in live cell imaging
and after fixation inside cells. This contrasts with its reported
positive expression in an in vitro rabbit reticulocyte system (Chen
& Sarnow, Science, 1995).
[0134] On the other hand, the circular mRNAs 3CIA and 4CEA produce
expression patterns similar to the expression of linear uncapped
mRNA 1LG. The linear mRNA 1LG containing uncapped EMCV
IRES-GFP-beta globin 3'UTR-polyA is known in the literature to
produce GFP after transfection, but we have demonstrated the first
confirmed showing of expression of a circular version of the same
mRNA.
[0135] The difference in GFP expression between circular mRNA in
2CI (no expression) and circular mRNA in 3CIA (expression) is
remarkable, considering that the only additional sequences were the
beta globin 3'UTR and polyadenylation sequence. This indicates that
these added sequences were able to allow the EMCV IRES to
effectively recruit ribosomes inside the cell, likely through
helping recruit additional initiation factors to the IRES to
increase its efficiency. For example, PABP binds to polyadenylation
sequence and to eIF4G, which is a targeted protein by the EMCV
IRES.
[0136] The present invention also describes for the first time the
use of an eIF4E binding RNA sequence as an IRES-like element in
recruiting ribosomes to circular RNA. So far, no mammalian viruses
or cellular genes have been described that utilize eIF4E
recruitment as an exclusive mechanism of ribosome recruitment. The
demonstration of an eIF4E aptamer facilitating translation thus
represents a novel finding for eukaryotic mRNA translation
initiation.
[0137] Future experiments will explore optimization of circular
mRNA genes using different combinations of IRES, 5' and 3' UTR, and
length of polyadenylation sequences. The firefly luciferase gene
will be utilized as the transfected ORF to allow for quantitative
measurements of protein amounts produced after mRNA
translation.
[0138] We also plan a future experiment to measure the half-life of
our circular mRNA in eukaryotic cells, using quantitative RT-PCR
and/or RNA purification and hybridization experiments. Based on the
prior art teachings, we expect the half-life to be at least
2.times., 3.times., 4.times. or 5.times. higher than a control
capped mRNA having a half-life of 10 hours Thus, we expect
half-lives of at least 20 hrs, 30 hrs, 40 hrs or more.
Materials
TABLE-US-00006 [0139] Reduced Serum Media Appropriate tissue
culture plates and supplies T7 Polymerase (New England Biosciences
.RTM.) Lipofectamine 2000 (Invitrogen .RTM.) HEK 293 cells
maintained in Dulbecco's Modified Eagle Medium (DMEM) medium
(Invitrogen .RTM.) supplemented with 4 mM L-Glutamine (Invitrogen
.RTM.), 10% fetal bovine serum (Invitrogen .RTM.). HEK 293 cells at
37.degree. C. with 5% CO.sub.2. Plasmid DNA of interest
Lipofectamine 2000 Reagent (store at +4.degree. C. until ready to
use) Opti-MEM .RTM. MEGAscript kit (Ambion .RTM.).
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