U.S. patent application number 13/897362 was filed with the patent office on 2013-09-19 for modified polynucleotides for the production of factor ix.
This patent application is currently assigned to Moderna Therapeutics. The applicant listed for this patent is MODERNA THERAPEUTICS. Invention is credited to Antonin de Fougerolles, Sayda M. Elbashir, Jason P. Schrum.
Application Number | 20130245103 13/897362 |
Document ID | / |
Family ID | 46147671 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130245103 |
Kind Code |
A1 |
de Fougerolles; Antonin ; et
al. |
September 19, 2013 |
MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF FACTOR IX
Abstract
Provided are formulations, compositions and methods for
delivering biological moieties such as modified nucleic acids into
cells to modulate protein expression. Such compositions and methods
include the delivery of biological moieties, and are useful for
production of proteins.
Inventors: |
de Fougerolles; Antonin;
(Brookline, MA) ; Elbashir; Sayda M.; (Cambridge,
MA) ; Schrum; Jason P.; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MODERNA THERAPEUTICS |
Cambridge |
MA |
US |
|
|
Assignee: |
Moderna Therapeutics
Cambridge
MA
|
Family ID: |
46147671 |
Appl. No.: |
13/897362 |
Filed: |
May 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13437034 |
Apr 2, 2012 |
|
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13897362 |
|
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61470451 |
Mar 31, 2011 |
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Current U.S.
Class: |
514/44R ;
536/23.2; 536/23.5 |
Current CPC
Class: |
A61K 47/28 20130101;
A61K 47/18 20130101; A61P 19/00 20180101; A61P 37/02 20180101; C12N
15/87 20130101; A61K 48/00 20130101; A61P 11/00 20180101; A61P 3/00
20180101; A61P 35/00 20180101; C12N 2310/335 20130101; A61K 31/7088
20130101; A61K 38/4846 20130101; C12N 15/67 20130101; A61P 43/00
20180101; C12N 9/644 20130101; A61P 9/00 20180101; A61P 17/00
20180101; A61P 31/12 20180101; A61P 25/00 20180101; A61K 31/7115
20130101; A61K 38/193 20130101; A61P 29/00 20180101; C07K 14/535
20130101; A61P 1/00 20180101; A61P 13/00 20180101; A61K 47/22
20130101; A61P 31/00 20180101; A61P 5/00 20180101; A61P 21/00
20180101; A61P 7/00 20180101 |
Class at
Publication: |
514/44.R ;
536/23.5; 536/23.2 |
International
Class: |
C07K 14/535 20060101
C07K014/535; C12N 9/64 20060101 C12N009/64 |
Claims
1. An isolated polynucleotide comprising a sequence having at least
70% identity to SEQ ID NO: 8.
2. The isolated polynucleotide of claim 1 comprising SEQ ID NO:
8.
3. The isolated polynucleotide of claim 2, wherein the isolated
polynucleotide is an mRNA.
4. The isolated polynucleotide of claim 3 comprising at least one
nucleoside modification.
5. The isolated polynucleotide of claim 3, wherein the mRNA has a
5' cap and wherein the 5'Cap is Cap1.
6. The isolated polynucleotide of claim 5, wherein the mRNA has a
polyA tail and wherein the polyA tail is approximately 160
nucleotides in length.
7. A pharmaceutical composition comprising the isolated
polynucleotide of claim 3.
8. The pharmaceutical composition of claim 7 which is formulated
and wherein the formulation is a lipid formulation.
9. A method of producing a polypeptide of interest in a cell,
tissue or bodily fluid of a mammalian subject in need thereof
comprising administering to said subject a pharmaceutical
composition comprising the isolated polynucleotide of claim 3.
10. The method of claim 9, wherein the isolated polynucleotide
further comprises a 5'Cap1 structure and a polyA tail of
approximately 160 nucleotides in length.
11. The method of claim 10, wherein the pharmaceutical composition
is formulated.
12. The method of claim 11, wherein the formulation is a lipid
formulation.
13. The method of claim 9, where the pharmaceutical composition is
administered at a total dose of about 0.1 mg/kg to about 40
mg/kg.
14. The method of claim 13, wherein administration occurs on a
schedule selected from the group consisting of three time a day,
twice a day, once a day, every other day, every third day, weekly,
biweekly, every three weeks, every four weekly, and monthly.
15. The method of claim 13, wherein the total dose is administered
by multiple administrations.
16. The method of claim 15, wherein the multiple administrations
occur on a schedule selected from the group consisting of three
times a day, twice a day, once a day, every other day, every third
day, weekly, biweekly, every three weeks, every four weekly, and
monthly.
17. The method of claim 9, wherein an increase in the level of the
polypeptide of interest is observed in tissue or bodily fluid
within 8 hours.
18. The method of claim 9, wherein an increase in the level of the
polypeptide of interest is observed in tissue or bodily fluid
within 2 hours.
19. The method of claim 18, wherein the increased level of the
polypeptide of interest is observed in a tissue selected from the
group consisting of liver, spleen, kidney, lung, heart, peri-renal
adipose tissue, thymus and muscle.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/437,034, filed Apr. 2, 2012, entitled
Delivery and Formulation of Engineered Nucleic Acids which claims
priority to U.S. Provisional Patent Application No. 61/470,451,
filed Mar. 31, 2011, entitled Delivery and Formulation of
Engineered Nucleic Acids the contents, the contents of each is
incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled M003USSQLST.txt created on May 17, 2013 which is
17,058 bytes in size. The information in electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to delivery methods. These methods are
specifically useful in therapeutic delivery of modified nucleic
acids such as modified mRNA (mmRNA).
BACKGROUND OF THE INVENTION
[0004] There are multiple problems with prior methodologies of
deliverying pharmaceutical compositions in order to achieve
effective protein expression both for therapeutics and
bioprocessing applications. For example, introduced DNA can
integrate into host cell genomic DNA at some frequency, resulting
in alterations and/or damage to the host cell genomic DNA.
Alternatively, the heterologous deoxyribonucleic acid (DNA)
introduced into a cell can be inherited by daughter cells (whether
or not the heterologous DNA has integrated into the chromosome) or
by offspring.
[0005] In addition, there are multiple steps which must occur after
delivery but before the encoded protein is made which can effect
protein expression. Once inside the cell, DNA must be transported
into the nucleus where it is transcribed into RNA. The RNA
transcribed from DNA must then enter the cytoplasm where it is
translated into protein. Not only do the multiple processing steps
from administered DNA to protein create lag times before the
generation of the functional protein, each step represents an
opportunity for error and damage to the cell. Further, it is known
to be difficult to obtain DNA expression in cells as frequently DNA
enters a cell but is not expressed or not expressed at reasonable
rates or concentrations. This can be a particular problem when DNA
is introduced into primary cells or modified cell lines.
[0006] Assuming the proper management of the foregoing, effective
delivery and achievement of therapeutically relevant levels of
proteins for a time sufficient to product clinical outcomes remains
a significant hurdle.
[0007] Consequently, there is a need in the art for the delivery of
biological modalities to address pitfalls surrounding the
modulation of intracellular translation and processing of nucleic
acids encoding polypeptides and therefore optimizing protein
expression from the delivered modalities.
[0008] The present invention addresses this need by delivering
pharmaceutical compositions which can contain modified nucleic
acids such as modified mRNA (mmRNA) and may further include
formulations to avoid the problems in the art.
SUMMARY OF THE INVENTION
[0009] Described herein are compositions and methods for delivery
of biological moieties, such as modified nucleic acids, engineered
messenger RNA and isolated polynucleotides into cells in order to
modulate protein expression.
[0010] An isolated polynucleotide may comprise a sequence such as,
but not limited to, SEQ ID NO: 4, 7, 8 and 12. The polynucleotide
may further comprise a 5'Cap1 structure and a polyA tail of
approximately 160 nucleotides in length. Further, the isolated
polynucleotide may be formulated in a pharmaceutical composition. A
polypeptide of interest may be produced in a cell, tissue or bodily
fluid in a subject in need thereof by administering to the subject
a pharmaceutical composition comprising a polynucleotide. The
polynucleotide may comprise a sequence selected from the group
consisting of SEQ ID NO: 4, 7, 8 and 12. The polynucleotide may
further comprise a 5'Cap1 structure and a poly-A tail of
approximately 160 nucleotides in length.
[0011] The pharmaceutical composition may be formulated where the
formulation may be selected from, but is not limited to, saline or
a lipid formulation. The pharmaceutical composition may be
administered by any route of administration such as, but not
limited to, intravenous, intramuscular, subcutaneous, and local
administration. The lipid formulation may be selected from, but is
not limited to, such as, but not limited to, liposomes, lipoplexes,
copolymers such as PLGA and lipid nanoparticles
[0012] The pharmaceutical composition may be administered at a
total dose of about 0.1 mg/kg to about 40 mg/kg. The total dose may
be administered by multiple administrations. The administration
and/or the multiple administration may occur on a schedule such as,
but not limited to, three time a day, twice a day, once a day,
every other day, every third day, weekly, biweekly, every three
weeks, every four weekly, and monthly.
[0013] The modified polypeptide may include a polynucleotide
modification such as, but not limited to, a nucleoside
modification. The nucleoside modification may include, but is not
limited to, pyridin-4-one ribonucleoside, 5-aza-uridine,
2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,
5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyluridine, 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine,
5-methyl-uridine, 1-methyl-pseudouridine,
4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,
N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,
2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine,
7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.
[0014] An increase in the level of a polypeptide of interest can be
observed in tissue such as, but not limited to, the liver, spleen,
kidney, lung, heart, peri-renal adipose tissue, thymus and muscle
and/or in a bodily fluid such as, but not limited to, peripheral
blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF),
sputum, saliva, bone marrow, synovial fluid, aqueous humor,
amniotic fluid, cerumen, breast milk, broncheoalveolar lavage
fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory
fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and
peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,
interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,
mucosal secretion, stool water, pancreatic juice, lavage fluids
from sinus cavities, bronchopulmonary aspirates, blastocyl cavity
fluid, and umbilical cord blood. The increased level can be
observed in the tissue and/or bodily fluid of the subject within 2,
8 and/or 24 hours after administration. Further, the increased
level can be determined from the level of a modified polypeptide in
an exosome.
[0015] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 illustrates lipid structures in the prior art useful
in the present invention. Shown are the structures for 98N12-5
(TETA5-LAP), DLin-DMA, DLin-K-DMA
(2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane),
DLin-KC2-DMA, DLin-MC3-DMA and C12-200.
[0017] FIG. 2 is a representative plasmid useful in the IVT
reactions taught herein. The plasmid contains Insert 64818,
designed by the instant inventors.
[0018] FIG. 3 is a histogram showing in vitro screening results for
nanoparticle formulations of DLin-KC2-DMA and 98N12-15 (before and
after purification) that contain mCherry mmRNA. FIG. 3A shows the
screening results in HEK293 cells and FIG. 3B shows the screening
results in HepG2 cells.
[0019] FIG. 4 is a histogram showing in vitro screening results for
mean fluorescence intensity for nanoparticle formulations of
DLin-KC2-DMA and 98N12-15 (before and after purification) that
contain mCherry mmRNA. FIG. 4A shows the screening results in
HEK293 cells and FIG. 4B shows the screening results in HepG2
cells.
[0020] FIG. 5 is a histogram showing in vitro screening results for
nanoparticle formulations of DLin-KC2-DMA and 98N12-15 before and
after purification. FIG. 5A shows the screening results of 98N15-2
in HEK293 cells, and FIGS. 5B and 5C shows the screening results of
DLin-KC2-DMA in HEK293 cells.
[0021] FIG. 6 is a histogram showing in vitro screening results for
nanoparticle formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA,
98N12-5, C12-200 and DLin-MC3-DMA that contain mCherry mmRNA. FIG.
6A shows the mean fluorescence intensity of mCherry in HEK293 cells
containing 60 ng of modified mCherry mRNA per well. FIGS. 6B and 6C
show the mean fluorescence intensity of mCherry in HEK293 cells
which contained nanoparticles formulations having a concentration
of 62.5 ng/well of modified mCherry mRNA. FIGS. 6D and 6E show the
mean fluorescence intensity of mCherry in HepG2 cells which
contained nanoparticle formulations having a concentration of 62.5
ng/well of modified mCherry mRNA.
[0022] FIG. 7 is a histogram showing in vivo screening results of
human erythropoietin in serum after the administration of modified
human erythropoietin mmRNA or luciferase mmRNA in mice. FIG. 7A
shows the concentration in pg/ml of human erythropoietin after
intramuscular administration. FIG. 7B shows the concentration in
pg/ml of human erythropoietin after subcutaneous
administration.
[0023] FIG. 8 shows histograms of in vivo screening results from
biophotoic imaging. FIG. 8A is a histogram of bioluminescence
(photon/sec) from the intramuscular injection of 5 ug in the left
hind leg. FIG. 8B is a histogram of bioluminescence from the
intramuscular injection of 50 ug in the right hind leg. FIG. 8C is
a histogram showing in vivo screening results from biophotoic
imaging after a subcutaneous injection of 50 ug. FIG. 8D is a
histogram showing in vivo screening results from biophotoic imaging
after a administration of 50 ug intravenously.
[0024] FIG. 9 is a histogram showing in vivo screening results for
modified human G-CSF mmRNA administered intramuscularly,
subcutaneously or intravenously in mice.
[0025] FIG. 10 is a histogram showing in vivo screening results for
modified G-CSF administered intramuscularly, subcutaneously or
intravenously.
[0026] FIG. 11 is a histogram showing in vivo screening results of
modified human G-CSF mmRNA administered intramuscularly or
subcutaneously in mice. FIG. 11A shows the concentration in pg/ml
of human G-CSF in serum after the administration of modified G-CSF
intramuscularly. FIG. 11B shows the concentration in pg/ml of human
G-CSF in serum after the administration of modified G-CSF
subcutaneously.
[0027] FIG. 12 is a histogram showing in vivo screening results of
human erythropoietin in serum after the administration of modified
human erythropoietin mmRNA or luciferase mmRNA administered
intramuscularly in mice.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention
pertains.
DETAILED DESCRIPTION
[0029] Described herein are compositions and methods for the
delivery of modified mRNA molecules in order to modulate protein
expression.
[0030] As described herein and as in copending, co-owned
applications International Application PCT/US2011/046861 filed Aug.
5, 2011 and PCT/US2011/054636 filed Oct. 3, 2011, the contents of
which are incorporated by reference herein in their entirety, these
modified nucleic acid molecules are capable of reducing the innate
immune activity of a population of cells into which they are
introduced, thus increasing the efficiency of protein production in
that cell population.
Modified mRNAs (mmRNAs)
[0031] This invention provides nucleic acids, including RNAs,
specifically mRNAs, that encode at least one polypeptide and
contain one or more modified nucleosides (termed "modified nucleic
acids" or "modified nucleic acid molecules" or "engineered nucleic
acids"), which have useful properties including the lack of a
substantial induction of the innate immune response of a cell into
which the mRNA is introduced. Because these mmRNAs enhance the
efficiency of protein production, intracellular retention of
nucleic acids, and viability of contacted cells, as well as possess
reduced immunogenicity, these nucleic acids having these properties
are termed "enhanced" nucleic acids or modified RNAs herein.
[0032] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that comprise a polymer of nucleotides
linked via a phosphohdiester bond. These polymers are often
referred to as oligonucleotides.
[0033] Exemplary nucleic acids include ribonucleic acids (RNAs),
deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol
nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic
acids (LNAs) or hybrids thereof. They may also include
RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs,
antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce
triple helix formation, aptamers, vectors, etc.
[0034] In preferred embodiments, the nucleic acid is one or more
modified messenger RNAs (mmRNAs). As described herein, in some
embodiments the mmRNAs of the invention do not substantially induce
an innate immune response of a cell into which the mRNA is
introduced.
[0035] The mmRNA of the present invention may encode one or more
polypeptides. Generally the polypeptides of interest are those
which are naturally occurring in the mammalian genome.
[0036] According to the present invention, the shortest length of a
modified mRNA, herein "mmRNA," of the present disclosure can be the
length of an mRNA sequence that may be sufficient to encode for a
dipeptide. In another embodiment, the length of the mRNA sequence
may be sufficient to encode for a tripeptide. In another
embodiment, the length of an mRNA sequence may be sufficient to
encode for a tetrapeptide. In another embodiment, the length of an
mRNA sequence may be sufficient to encode for a pentapeptide. In
another embodiment, the length of an mRNA sequence may be
sufficient to encode for a hexapeptide. In another embodiment, the
length of an mRNA sequence may be sufficient to encode for a
heptapeptide. In another embodiment, the length of an mRNA sequence
may be sufficient to encode for an octapeptide. In another
embodiment, the length of an mRNA sequence may be sufficient to
encode for a nonapeptide. In another embodiment, the length of an
mRNA sequence may be sufficient to encode for a decapeptide.
[0037] Generally, the length of a modified mRNA of the present
invention is greater than about 30 nucleotides in length (e.g., at
least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90,
100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,
700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,
1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000,
60,000, 70,000, 80,000, 90,000 or up to and including 100,000
nucleotides).
[0038] In some embodiments, the modified mRNA of the present
invention includes from about 30 to about 100,000 nucleotides
(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to
500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30
to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000,
from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to
500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from
100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to
25,000, from 100 to 50,000, from 100 to 70,000, from 100 to
100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000,
from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500
to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to
70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to
2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to
7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to
50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to
3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to
10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to
70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to
5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to
25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000
to 100,000).
Polypeptide Variants
[0039] The mmRNA of the present invention may encode variant
polypeptides, which have a certain identity with a reference
polypeptide sequence, for example a wild type mRNA. The term
"identity" as known in the art, refers to a relationship between
the sequences of two or more peptides, as determined by comparing
the sequences. In the art, "identity" also means the degree of
sequence relatedness between peptides, as determined by the number
of matches between strings of two or more amino acid residues.
"Identity" measures the percent of identical matches between the
smaller of two or more sequences with gap alignments (if any)
addressed by a particular mathematical model or computer program
(i.e., "algorithms"). Identity of related peptides can be readily
calculated by known methods. Such methods include, but are not
limited to, those described in Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0040] In some embodiments, the polypeptide variant has the same or
a similar activity as the reference polypeptide. Alternatively, the
variant has an altered activity (e.g., increased or decreased)
relative to a reference polypeptide. Generally, variants of a
particular polynucleotide or polypeptide of the invention will have
at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to that particular reference polynucleotide or polypeptide
as determined by sequence alignment programs and parameters
described herein and known to those skilled in the art.
[0041] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of this invention. For
example, provided herein is any protein fragment of a reference
protein (meaning a polypeptide sequence at least one amino acid
residue shorter than a reference polypeptide sequence but otherwise
identical) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than
100 amino acids in length In another example, any protein that
includes a stretch of about 20, about 30, about 40, about 50, or
about 100 amino acids which are about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95%, or about 100% identical
to any of the sequences described herein can be utilized in
accordance with the invention. In certain embodiments, a protein
sequence to be utilized in accordance with the invention includes
2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of
the sequences provided or referenced herein.
Targeting Moieties
[0042] In embodiments of the invention, mmRNAs are provided to
express a protein-binding partner or a receptor on the surface of
the cell, which functions to target the cell to a specific tissue
space or to interact with a specific moiety, either in vivo or in
vitro. Suitable protein-binding partners include antibodies and
functional fragments thereof, scaffold proteins, or peptides.
Cell Penetrating Peptides
[0043] The mmRNAs disclosed herein may encode a cell-penetrating
polypeptide. As used herein, "cell-penetrating polypeptide" refers
to a polypeptide which may facilitate the cellular uptake of
molecules. It is known in the art that "CPP" refers to
cell-penetration polypeptides and cell-penetrating peptides. When
used herein, it will be clarified as to which of either
cell-penetrating polypeptides or cell-penetrating peptides the
abbreviation CPP refers to.
[0044] A cell-penetrating polypeptide of the present invention may
contain one or more detectable labels. The polypeptides may be
partially labeled or completely labeled throughout. The mmRNA may
encode the detectable label completely, partially or not at all.
The cell-penetrating peptide may also include a signal sequence. As
used herein, a "signal sequence" refers to a sequence of amino acid
residues bound at the amino terminus of a nascent protein during
protein translation. The signal sequence may be used to signal the
secretion of the cell-penetrating polypeptide.
Fusion Proteins
[0045] The modified nucleic acids and mmRNA may encode a fusion
protein. The fusion protein may be created by operably linking a
charged protein to a therapeutic protein. As used herein, "operably
linked" refers to the therapeutic protein and the charged protein
being connected in such a way to permit the expression of the
complex when introduced into the cell. As used herein, "charged
protein" refers to a protein that carries a positive, negative or
overall neutral electrical charge. Preferably, the therapeutic
protein may be covalently linked to the charged protein in the
formation of the fusion protein. The ratio of surface charge to
total or surface amino acids may be approximately 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.
Synthesis of Modified mRNAs
[0046] Nucleic acids for use in accordance with the invention may
be prepared according to any available technique including, but not
limited to chemical synthesis, enzymatic synthesis, which is
generally termed in vitro transcription, enzymatic or chemical
cleavage of a longer precursor, etc. Methods of synthesizing RNAs
are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide
synthesis: a practical approach, Oxford [Oxfordshire], Washington,
D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide
synthesis: methods and applications, Methods in Molecular Biology,
v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of
which are incorporated herein by reference).
[0047] The modified nucleosides and nucleotides used in the
synthesis of modified RNAs disclosed herein can be prepared from
readily available starting materials using the following general
methods and procedures. It is understood that where typical or
preferred process conditions (i.e., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are given;
other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0048] The manufacturing process herein can be monitored according
to any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or
mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
Modification of mRNAs
[0049] Provided are mmRNAs containing a translatable region and
one, two, or more than two different modifications.
[0050] In some embodiments, the chemical modifications can be
located on the nucleobase of the nucleotide.
[0051] In some embodiments, the chemical modifications can be
located on the sugar moiety of the nucleotide.
[0052] In some embodiments, the chemical modifications can be
located on the phosphate backbone of the nucleotide.
[0053] Preparation of modified nucleosides and nucleotides used in
the manufacture or synthesis of modified RNAs of the present
invention can involve the protection and deprotection of various
chemical groups. The need for protection and deprotection, and the
selection of appropriate protecting groups can be readily
determined by one skilled in the art.
[0054] The chemistry of protecting groups can be found, for
example, in Greene, et al., Protective Groups in Organic Synthesis,
2d. Ed., Wiley & Sons, 1991, which is incorporated herein by
reference in its entirety.
[0055] Modified nucleosides and nucleotides can be prepared
according to the synthetic methods described in Ogata et al.
Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al.
Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al.
Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9):
1729-1740, 1992, each of which are incorporated by reference in
their entirety.
[0056] Modified mRNAs need not be uniformly modified along the
entire length of the molecule. Different nucleotide modifications
and/or backbone structures may exist at various positions in the
nucleic acid. One of ordinary skill in the art will appreciate that
the nucleotide analogs or other modification(s) may be located at
any position(s) of a nucleic acid such that the function of the
nucleic acid is not substantially decreased. A modification may
also be a 5' or 3' terminal modification. The nucleic acids may
contain at a minimum one and at maximum 100% modified nucleotides,
or any intervening percentage, such as at least 50% modified
nucleotides, at least 80% modified nucleotides, or at least 90%
modified nucleotides.
[0057] For example, the mmRNAs may contain a modified pyrimidine
such as uracil or cytosine. In some embodiments, at least 5%, at
least 10%, at least 25%, at least 50%, at least 80%, at least 90%
or 100% of the uracil in the nucleic acid may be replaced with a
modified uracil. The modified uracil can be replaced by a compound
having a single unique structure, or can be replaced by a plurality
of compounds having different structures (e.g., 2, 3, 4 or more
unique structures). In some embodiments, at least 5%, at least 10%,
at least 25%, at least 50%, at least 80%, at least 90% or 100% of
the cytosine in the nucleic acid may be replaced with a modified
cytosine. The modified cytosine can be replaced by a compound
having a single unique structure, or can be replaced by a plurality
of compounds having different structures (e.g., 2, 3, 4 or more
unique structures).
[0058] In some embodiments, modified nucleosides include
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,
5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine. In some embodiments, modified
nucleosides include 5-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,
N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0059] In other embodiments, modified nucleosides include
2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0060] In other embodiments, modified nucleosides include inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0061] In some embodiments, the nucleotide can be modified on the
major groove face and can include replacing hydrogen on C-5 of
uracil with a methyl group or a halo group.
[0062] In specific embodiments, a modified nucleoside is
5'-O-(1-Thiophosphate)-Adenosine, 5'-O-(1-Thiophosphate)-Cytidine,
5'-O-(1-Thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or
5'-O-(1-Thiophosphate)-Pseudouridine.
[0063] Further examples of modified nucleotides and modified
nucleotide combinations are provided below in Table 1.
TABLE-US-00001 TABLE 1 Modified Nucleotides Modified Nucleotide
Combinations 6-aza-cytidine .alpha.-thio-cytidine/5-iodo-uridine
2-thio-cytidine .alpha.-thio-cytidine/N1-methyl-pseudo-uridine
.alpha.-thio-cytidine .alpha.-thio-cytidine/.alpha.-thio-uridine
Pseudo-iso-cytidine .alpha.-thio-cytidine/5-methyl-uridine
5-aminoallyl-uridine .alpha.-thio-cytidine/pseudo-uridine
5-iodo-uridine Pseudo-iso-cytidine/5-iodo-uridine N1-methyl-
Pseudo-iso-cytidine/N1-methyl-pseudo-uridine pseudouridine
5,6-dihydrouridine Pseudo-iso-cytidine/.alpha.-thio-uridine
.alpha.-thio-uridine Pseudo-iso-cytidine/5-methyl-uridine
4-thio-uridine Pseudo-iso-cytidine/Pseudo-uridine 6-aza-uridine
Pyrrolo-cytidine 5-hydroxy-uridine Pyrrolo-cytidine/5-iodo-uridine
Deoxy-thymidine Pyrrolo-cytidine/N1-methyl-pseudo-uridine
Pseudo-uridine Pyrrolo-cytidine/.alpha.-thio-uridine Inosine
Pyrrolo-cytidine/5-methyl-uridine .alpha.-thio-guanosine
Pyrrolo-cytidine/Pseudo-uridine 8-oxo-guanosine
5-methyl-cytidine/5-iodo-uridine O6-methyl-
5-methyl-cytidine/N1-methyl-pseudo-uridine guanosine
7-deaza-guanosine 5-methyl-cytidine/.alpha.-thio-uridine No
modification 5-methyl-cytidine/5-methyl-uridine N1-methyl-
5-methyl-cytidine/Pseudo-uridine adenosine 2-amino-6-Chloro-
5-methyl-cytidine purine N6-methyl-2-amino- 25% Pseudo-iso-cytidine
purine 6-Chloro-purine 25% N1-methyl-pseudo-uridine N6-methyl- 25%
N1-Methyl-pseudo-uridine/75%-pseudo-uridine adenosine
.alpha.-thio-adenosine 5-methyl-uridine 8-azido-adenosine
5-iodo-cytidine 7-deaza-adenosine
[0064] In some embodiments, at least 25% of the cytosines are
replaced by a compound of Formula I-a (e.g., at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or about
100%).
[0065] In some embodiments, at least 25% of the uracils are
replaced by a compound of Formula I-a (e.g., at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or about
100%).
[0066] In some embodiments, at least 25% of the cytosines and 25%
of the uracils are replaced by a compound of Formula I-a (e.g., at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100%).
[0067] Other components of nucleic acid are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'UTR are provided, wherein either or both
may independently contain one or more different nucleoside
modifications. In such embodiments, nucleoside modifications may
also be present in the translatable region. Also provided are
nucleic acids containing a Kozak sequence.
Linkers and Payloads
[0068] The nucleobase of the nucleotide, which may be incorporated
into a mmRNA, can be covalently linked at any chemically
appropriate position to a payload, e.g. detectable agent or
therapeutic agent. For example, the nucleobase can be
deaza-adenosine or deaza-guanosine and the linker can be attached
at the C-7 or C-8 positions of the deaza-adenosine or
deaza-guanosine. In other embodiments, the nucleobase can be
cytosine or uracil and the linker can be attached to the N-3 or C-5
positions of cytosine or uracil.
Linker
[0069] The term "linker" as used herein refers to a group of atoms,
e.g., 10-1,000 atoms, and can be comprised of the atoms or groups
such as, but not limited to, carbon, amino, alkylamino, oxygen,
sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be
attached to a modified nucleoside or nucleotide on the nucleobase
or sugar moiety at a first end, and to a payload, e.g., detectable
or therapeutic agent, at a second end. The linker may be of
sufficient length as to not interfere with incorporation into a
nucleic acid sequence.
[0070] Examples of chemical groups that can be incorporated into
the linker include, but are not limited to, an alkyl, an alkene, an
alkyne, an amido, an ether, a thioether or an ester group. The
linker chain can also comprise part of a saturated, unsaturated or
aromatic ring, including polycyclic and heteroaromatic rings
wherein the heteroaromatic ring may be an aryl group containing one
to four heteroatoms, N, O or S. Specific examples of linkers
include, but are not limited to, unsaturated alkanes, polyethylene
glycols, and dextran polymers.
[0071] For example, the linker can include, but is not limited to,
ethylene or propylene glycol monomeric units, e.g., diethylene
glycol, dipropylene glycol, triethylene glycol, tripropylene
glycol, tetraethylene glycol, or tetraethylene glycol. In some
embodiments, the linker can include, but is not limited to, a
divalent alkyl, alkenyl, and/or alkynyl moiety. The linker can
include an ester, amide, or ether moiety.
[0072] Other examples include, but are not limited to, cleavable
moieties within the linker, such as, for example, a disulfide bond
(--S--S--) or an azo bond (--N.dbd.N--), which can be cleaved using
a reducing agent or photolysis. When a cleavable bond which has
been incorporated into the linker and attached to a modified
nucleotide, is cleaved, a short "scar" or chemical modification on
the nucleotide may result. For example, after cleaving, the
resulting scar on a nucleotide base, which formed part of the
modified nucleotide, and is incorporated into a polynucleotide
strand, is unreactive and does not need to be chemically
neutralized. This increases the ease with which a subsequent
nucleotide can be incorporated during sequencing of a nucleic acid
polymer template. For example, conditions include the use of
tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT) and/or
other reducing agents for cleavage of a disulfide bond. A
selectively severable bond that includes an amido bond can be
cleaved for example by the use of TCEP or other reducing agents,
and/or photolysis. A selectively severable bond that includes an
ester bond can be cleaved for example by acidic or basic
hydrolysis.
Detectable Agents
[0073] The mmRNAs of the present invention may also be linked or
conjugated to one or more detectable agents. Examples of detectable
substances include, but are not limited to, various organic small
molecules, inorganic compounds, nanoparticles, enzymes or enzyme
substrates, fluorescent materials, luminescent materials,
bioluminescent materials, chemiluminescent materials, radioactive
materials, and contrast agents.
[0074] Labels, other than those described herein, are contemplated
by the present disclosure, including, but not limited to, other
optically-detectable labels. Labels can be attached to the modified
nucleotide of the present disclosure at any position using standard
chemistries such that the label can be removed from the
incorporated base upon cleavage of the cleavable linker.
Terminal Architecture Modifications: 5'-Capping
[0075] Endogenous eukaryotic cellular messenger RNA (mRNA)
molecules contain a 5'-cap structure on the 5'-end of a mature mRNA
molecule. The 5'-cap contains a 5'-5'-triphosphate linkage between
the 5'-most nucleotide and guanine nucleotide. The conjugated
guanine nucleotide is methylated at the N7 position. Additional
modifications include methylation of the ultimate and penultimate
most 5'-nucleotides on the 2'-hydroxyl group. The 5'-cap structure
is responsible for binding the mRNA Cap Binding Protein (CBP),
which is responsibility for mRNA stability in the cell and
translation competency.
[0076] Multiple distinct 5'-cap structures can be used to generate
the 5'-cap of a synthetic mRNA molecule. Many chemical cap analogs
are used to co-transcriptionally cap a synthetic mRNA molecule. For
example, the Anti-Reverse Cap Analog (ARCA) cap contains a
5'-5'-triphosphate guanine-guanine linkage where one guanine
contains an N7 methyl group as well as a 3'-O-methyl group. While
chemical cap analogs allow for the concomitant capping of an RNA
molecule, up 20% of transcripts remain uncapped and the synthetic
cap analog is not identical to an endogenous 5'-cap structure of an
authentic cellular mRNA. This may lead to reduced
translationally-competency and reduced cellular stability.
[0077] Synthetic mRNA molecules may also be capped
post-transcriptionally using enzymes responsible for generating a
more authentic 5'-cap structure. As used herein the phrase "more
authentic" refers to a feature that closely mirrors or mimics,
either structurally or functionally an endogenous or wild type
feature. More authentic 5' cap structures of the present invention
are those which, among other things, have enhanced binding of cap
binding proteins, increased half life, reduced susceptibility to 5'
endonucleases and/or reduced 5' decapping. For example, recombinant
Vaccinia Virus Capping Enzyme and recombinant
2'-O-methyltransferase enzyme can create a canonical
5'-5'-triphosphate linkage between the 5'-most nucleotide of an
mRNA and a guanine nucleotide where the guanine contains an N7
methylation and the ultimate 5'-nucleotide contains a 2'-O-methyl
generating the Cap1 structure. This results in a cap with higher
translational-competency and cellular stability and reduced
activation of cellular pro-inflammatory cytokines. Because the
synthetic mRNA is capped post-transcriptionally, nearly 100% of the
mRNA molecules are capped in contrast to .about.80% of synthetic
mRNAs containing a chemical cap analog.
Terminal Architecture Modifications: Poly-A Tails
[0078] During RNA processing, a long chain of adenine nucleotides
(poly-A tail) is normally added to a messenger RNA (mRNA) molecules
to increase the stability of the molecule. Immediately after
transcription, the 3' end of the transcript is cleaved to free a 3'
hydroxyl. Then poly-A polymerase adds a chain of adenine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that is between 100 and 250 residues long.
[0079] It has been discovered that unique poly-A tail lengths
provide certain advantages to the modified RNAs of the present
invention.
[0080] Generally, the length of a poly-A tail of the present
invention is greater than 30 nucleotides in length. In another
embodiment, the poly-A tail is greater than 35 nucleotides in
length. In another embodiment, the length is at least 40
nucleotides. In another embodiment, the length is at least 45
nucleotides. In another embodiment, the length is at least 55
nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 80
nucleotides. In another embodiment, the length is at least 90
nucleotides. In another embodiment, the length is at least 100
nucleotides. In another embodiment, the length is at least 120
nucleotides. In another embodiment, the length is at least 140
nucleotides. In another embodiment, the length is at least 160
nucleotides. In another embodiment, the length is at least 180
nucleotides. In another embodiment, the length is at least 200
nucleotides. In another embodiment, the length is at least 250
nucleotides. In another embodiment, the length is at least 300
nucleotides. In another embodiment, the length is at least 350
nucleotides. In another embodiment, the length is at least 400
nucleotides. In another embodiment, the length is at least 450
nucleotides. In another embodiment, the length is at least 500
nucleotides. In another embodiment, the length is at least 600
nucleotides. In another embodiment, the length is at least 700
nucleotides. In another embodiment, the length is at least 800
nucleotides. In another embodiment, the length is at least 900
nucleotides. In another embodiment, the length is at least 1000
nucleotides.
[0081] In one embodiment, the poly-A tail is designed relative to
the length of the overall modified RNA molecule. This design may be
based on the length of the coding region of the modified RNA, the
length of a particular feature or region of the modified RNA (such
as the mRNA), or based on the length of the ultimate product
expressed from the modified RNA. In this context the poly-A tail
may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length
than the modified RNA or feature thereof. The poly-A tail may also
be desiged as a fraction of the modified RNA to which it belongs.
In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70,
80, or 90% or more of the total length of the construct or the
total length of the construct minus the poly-A tail.
Use of Modified mRNAs
[0082] The mmRNAs of the present invention may find uses in many
areas of research, discovery, therapeutics, diagnostics and in kits
and devices.
Therapeutics
[0083] The mmRNAs (modified RNAs) and the proteins translated from
the mmRNAs described herein can be used as therapeutic agents. For
example, an mmRNA described herein can be administered to a
subject, wherein the mmRNA is translated in vivo to produce a
therapeutic polypeptide in the subject. Provided are compositions,
methods, kits, and reagents for treatment or prevention of disease
or conditions in humans and other mammals. The active therapeutic
agents of the invention include mmRNAs, cells containing mmRNAs or
polypeptides translated from the mmRNAs, polypeptides translated
from mmRNAs.
[0084] Provided herein are methods of inducing translation of a
recombinant polypeptide in a cell population using the mmRNAs
described herein. Such translation can be in vivo, ex vivo, in
culture, or in vitro. The cell population is contacted with an
effective amount of a composition containing a mmRNA that has at
least one nucleoside modification, and a translatable region
encoding the recombinant polypeptide. The population is contacted
under conditions such that the mmRNA is localized into one or more
cells of the cell population and the recombinant polypeptide is
translated in the cell from the nucleic acid.
[0085] An effective amount of the composition is provided based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the nucleic acid (e.g.,
size, and extent of modified nucleosides), and other determinants.
In general, an effective amount of the composition provides
efficient protein production in the cell, preferably more efficient
than a composition containing a corresponding unmodified nucleic
acid. Increased efficiency may be demonstrated by increased cell
transfection (i.e., the percentage of cells transfected with the
nucleic acid), increased protein translation from the nucleic acid,
decreased nucleic acid degradation (as demonstrated, e.g., by
increased duration of protein translation from a mmRNA), or reduced
innate immune response of the host cell.
[0086] Aspects of the invention are directed to methods of inducing
in vivo translation of a recombinant polypeptide in a mammalian
subject in need thereof. Therein, an effective amount of a
composition containing a mmRNA that has at least one nucleoside
modification and a translatable region encoding the recombinant
polypeptide is administered to the subject using the delivery
methods and split dosing regimens described herein. The mmRNA is
provided in an amount and under other conditions such that the
nucleic acid is localized into a cell of the subject and the
recombinant polypeptide is translated in the cell from the mmRNA.
The cell in which the mmRNA is localized, or the tissue in which
the cell is present, may be targeted with one or more than one
rounds of mmRNA administration.
[0087] The subject to whom the therapeutic agent is administered
suffers from or is at risk of developing a disease, disorder, or
deleterious condition. Provided are methods of identifying,
diagnosing, and classifying subjects on these bases, which may
include clinical diagnosis, biomarker levels, genome-wide
association studies (GWAS), and other methods known in the art.
[0088] In certain embodiments, the administered mmRNA directs
production of one or more recombinant polypeptides that provide a
functional activity which is substantially absent in the cell in
which the recombinant polypeptide is translated. For example, the
missing functional activity may be enzymatic, structural, or gene
regulatory in nature. In related embodiments, the administered
mmRNA directs production of one or more recombinant polypeptides
that increases (e.g., synergistically) a functional activity which
is present but substantially deficient in the cell in which the
recombinant polypeptide is translated.
[0089] In other embodiments, the administered mmRNA directs
production of one or more recombinant polypeptides that replace a
polypeptide (or multiple polypeptides) that is substantially absent
in the cell in which the recombinant polypeptide is translated.
Such absence may be due to genetic mutation of the encoding gene or
regulatory pathway thereof. In some embodiments, the recombinant
polypeptide increases the level of an endogenous protein in the
cell to a desirable level; such an increase may bring the level of
the endogenous protein from a subnormal level to a normal level or
from a normal level to a super-normal level.
[0090] Alternatively, the recombinant polypeptide functions to
antagonize the activity of an endogenous protein present in, on the
surface of, or secreted from the cell. Usually, the activity of the
endogenous protein is deleterious to the subject; for example, do
to mutation of the endogenous protein resulting in altered activity
or localization. Additionally, the recombinant polypeptide
antagonizes, directly or indirectly, the activity of a biological
moiety present in, on the surface of, or secreted from the cell.
Examples of antagonized biological moieties include lipids (e.g.,
cholesterol), a lipoprotein (e.g., low density lipoprotein), a
nucleic acid, a carbohydrate, a protein toxin such as shiga and
tetanus toxins, or a small molecule toxin such as botulinum,
cholera, and diphtheria toxins. Additionally, the antagonized
biological molecule may be an endogenous protein that exhibits an
undesirable activity, such as a cytotoxic or cytostatic
activity.
[0091] The polypeptides encoded by the mmRNA described herein are
engineered for localization within the cell, potentially within a
specific compartment such as the nucleus, or are engineered for
secretion from the cell or translocation to the plasma membrane of
the cell.
[0092] In one embodiment of the invention are bifunctional mmRNA.
As the name implies, bifunctional mmRNA are those having or capable
of at least two functions.
[0093] The multiple functionalities of bifunctional mmRNAs may be
encoded by the mRNA (the function may not manifest until the
encoded product is translated) or may be a property of the RNA
itself. It may be structural or chemical. Bifunctional modified
RNAs may comprise a function that is covalently associated with the
RNA or electrostatically associated.
[0094] In some embodiments, modified mRNAs and their encoded
polypeptides in accordance with the present invention may be used
for treatment of any of a variety of diseases, disorders, and/or
conditions, including but not limited to one or more of the
following: autoimmune disorders (e.g. diabetes, lupus, multiple
sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders
(e.g. arthritis, pelvic inflammatory disease); infectious diseases
(e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections,
fungal infections, sepsis); neurological disorders (e.g.
Alzheimer's disease, Huntington's disease; autism; Duchenne
muscular dystrophy); cardiovascular disorders (e.g.
atherosclerosis, hypercholesterolemia, thrombosis, clotting
disorders, angiogenic disorders such as macular degeneration);
proliferative disorders (e.g. cancer, benign neoplasms);
respiratory disorders (e.g. chronic obstructive pulmonary disease);
digestive disorders (e.g. inflammatory bowel disease, ulcers);
musculoskeletal disorders (e.g. fibromyalgia, arthritis);
endocrine, metabolic, and nutritional disorders (e.g. diabetes,
osteoporosis); urological disorders (e.g. renal disease);
psychological disorders (e.g. depression, schizophrenia); skin
disorders (e.g. wounds, eczema); blood and lymphatic disorders
(e.g. anemia, hemophilia); etc.
Avoidance of the Innate Immune Response
[0095] The term "innate immune response" includes a cellular
response to exogenous single stranded nucleic acids, generally of
viral or bacterial origin, which involves the induction of cytokine
expression and release, particularly the interferons, and cell
death. Protein synthesis is also reduced during the innate cellular
immune response. While it is advantageous to eliminate the innate
immune response in a cell, the invention provides modified mRNAs
that substantially reduce the immune response, including interferon
signaling, without entirely eliminating such a response. In some
embodiments, the immune response is reduced by 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as
compared to the immune response induced by a corresponding
unmodified nucleic acid. Such a reduction can be measured by
expression or activity level of Type 1 interferons or the
expression of interferon-regulated genes such as the toll-like
receptors (e.g., TLR7 and TLR8). Reduction of innate immune
response can also be measured by decreased cell death following one
or more administrations of modified RNAs to a cell population;
e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95%
less than the cell death frequency observed with a corresponding
unmodified nucleic acid. Moreover, cell death may affect fewer than
50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than 0.01% of
cells contacted with the mmRNAs.
[0096] The invention provides therapeutic methods for the repeated
introduction (e.g., transfection) of mmRNAs into a target cell
population, e.g., in vitro, ex vivo, or in vivo. The step of
contacting the cell population may be repeated one or more times
(such as two, three, four, five or more than five times). In some
embodiments, the step of contacting the cell population with the
mmRNAs is repeated a number of times sufficient such that a
predetermined efficiency of protein translation in the cell
population is achieved. Given the reduced cytotoxicity of the
target cell population provided by the nucleic acid modifications,
such repeated transfections are achievable in a diverse array of
cell types.
Protein Production
[0097] The methods provided herein are useful for enhancing protein
product yield in a cell culture process. In a cell culture
containing a plurality of host cells, introduction of the modified
mRNAs described herein results in increased protein production
efficiency relative to a corresponding unmodified nucleic acid.
Such increased protein production efficiency can be demonstrated,
e.g., by showing increased cell transfection, increased protein
translation from the nucleic acid, decreased nucleic acid
degradation, and/or reduced innate immune response of the host
cell. Protein production can be measured by ELISA, and protein
activity can be measured by various functional assays known in the
art. The protein production may be generated in a continuous or a
fed-batch mammalian process.
[0098] Additionally, it is useful to optimize the expression of a
specific polypeptide in a cell line or collection of cell lines of
potential interest, particularly an engineered protein such as a
protein variant of a reference protein having a known activity. In
one embodiment, provided is a method of optimizing expression of an
engineered protein in a target cell, by providing a plurality of
target cell types, and independently contacting with each of the
plurality of target cell types a modified mRNA encoding an
engineered polypeptide. Additionally, culture conditions may be
altered to increase protein production efficiency. Subsequently,
the presence and/or level of the engineered polypeptide in the
plurality of target cell types is detected and/or quantitated,
allowing for the optimization of an engineered polypeptide's
expression by selection of an efficient target cell and cell
culture conditions relating thereto. Such methods are particularly
useful when the engineered polypeptide contains one or more
post-translational modifications or has substantial tertiary
structure, situations which often complicate efficient protein
production.
Gene Silencing
[0099] The modified mRNAs described herein are useful to silence
(i.e., prevent or substantially reduce) expression of one or more
target genes in a cell population. A modified mRNA encoding a
polypeptide capable of directing sequence-specific histone H3
methylation is introduced into the cells in the population under
conditions such that the polypeptide is translated and reduces gene
transcription of a target gene via histone H3 methylation and
subsequent heterochromatin formation. In some embodiments, the
silencing mechanism is performed on a cell population present in a
mammalian subject. By way of non-limiting example, a useful target
gene is a mutated Janus Kinase-2 family member, wherein the
mammalian subject expresses the mutant target gene suffers from a
myeloproliferative disease resulting from aberrant kinase
activity.
[0100] Co-administration of modified mRNAs and siRNAs are also
provided herein. As demonstrated in yeast, sequence-specific trans
silencing is an effective mechanism for altering cell function.
Fission yeast require two RNAi complexes for siRNA-mediated
heterochromatin assembly: the RNA-induced transcriptional silencing
(RITS) complex and the RNA-directed RNA polymerase complex (RDRC)
(Motamedi et al. Cell 2004, 119, 789-802). In fission yeast, the
RITS complex contains the siRNA binding Argonaute family protein
Ago1, a chromodomain protein Chp1, and Tas3. The fission yeast RDRC
complex is composed of an RNA-dependent RNA Polymerase Rdp1, a
putative RNA helicase Hrr1, and a polyA polymerase family protein
Cid12. These two complexes require the Dicer ribonuclease and Clr4
histone H3 methyltransferase for activity. Together, Ago1 binds
siRNA molecules generated through Dicer-mediated cleavage of Rdp1
co-transcriptionally generated dsRNA transcripts and allows for the
sequence-specific direct association of Chp1, Tas3, Hrr1, and Clr4
to regions of DNA destined for methylation and histone modification
and subsequent compaction into transcriptionally silenced
heterochromatin. While this mechanism functions in cis- with
centromeric regions of DNA, sequence-specific trans silencing is
possible through co-transfection with double-stranded siRNAs for
specific regions of DNA and concomitant RNAi-directed silencing of
the siRNA ribonuclease Eri1 (Buhler et al. Cell 2006, 125,
873-886).
Modulation of Biological Pathways
[0101] The rapid translation of modified mRNAs introduced into
cells provides a desirable mechanism of modulating target
biological pathways. Such modulation includes antagonism or agonism
of a given pathway. In one embodiment, a method is provided for
antagonizing a biological pathway in a cell by contacting the cell
with an effective amount of a composition comprising a modified
nucleic acid encoding a recombinant polypeptide, under conditions
such that the nucleic acid is localized into the cell and the
recombinant polypeptide is capable of being translated in the cell
from the nucleic acid, wherein the recombinant polypeptide inhibits
the activity of a polypeptide functional in the biological pathway.
Exemplary biological pathways are those defective in an autoimmune
or inflammatory disorder such as multiple sclerosis, rheumatoid
arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis
colitis, or Crohn's disease; in particular, antagonism of the IL-12
and IL-23 signaling pathways are of particular utility. (See Kikly
K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6):
670-5).
[0102] Further, provided are modified nucleic acids encoding an
antagonist for chemokine receptors; chemokine receptors CXCR-4 and
CCR-5 are required for, e.g., HIV entry into host cells (et al,
(1996) October 3; 383(6599):400).
[0103] Alternatively, provided are methods of agonizing a
biological pathway in a cell by contacting the cell with an
effective amount of a modified nucleic acid encoding a recombinant
polypeptide under conditions such that the nucleic acid is
localized into the cell and the recombinant polypeptide is capable
of being translated in the cell from the nucleic acid, and the
recombinant polypeptide induces the activity of a polypeptide
functional in the biological pathway. Exemplary agonized biological
pathways include pathways that modulate cell fate determination.
Such agonization is reversible or, alternatively, irreversible.
Cellular Nucleic Acid Delivery
[0104] Methods of the present invention enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside modification and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unmodified nucleic acid.
The retention of the enhanced nucleic acid is greater than the
retention of the unmodified nucleic acid. In some embodiments, it
is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than
200% greater than the retention of the unmodified nucleic acid.
Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained
following repeated rounds of transfection.
[0105] In some embodiments, the enhanced nucleic acid is delivered
to a target cell population with one or more additional nucleic
acids. Such delivery may be at the same time, or the enhanced
nucleic acid is delivered prior to delivery of the one or more
additional nucleic acids. The additional one or more nucleic acids
may be modified nucleic acids or unmodified nucleic acids. It is
understood that the initial presence of the enhanced nucleic acids
does not substantially induce an innate immune response of the cell
population and, moreover, that the innate immune response will not
be activated by the later presence of the unmodified nucleic acids.
In this regard, the enhanced nucleic acid may not itself contain a
translatable region, if the protein desired to be present in the
target cell population is translated from the unmodified nucleic
acids.
Expression of Ligand or Receptor on Cell Surface
[0106] In some aspects and embodiments of the aspects described
herein, the modified RNAs can be used to express a ligand or ligand
receptor on the surface of a cell (e.g., a homing moiety). A ligand
or ligand receptor moiety attached to a cell surface can permit the
cell to have a desired biological interaction with a tissue or an
agent in vivo. A ligand can be an antibody, an antibody fragment,
an aptamer, a peptide, a vitamin, a carbohydrate, a protein or
polypeptide, a receptor, e.g., cell-surface receptor, an adhesion
molecule, a glycoprotein, a sugar residue, a therapeutic agent, a
drug, a glycosaminoglycan, or any combination thereof. For example,
a ligand can be an antibody that recognizes a cancer-cell specific
antigen, rendering the cell capable of preferentially interacting
with tumor cells to permit tumor-specific localization of a
modified cell. A ligand can confer the ability of a cell
composition to accumulate in a tissue to be treated, since a
preferred ligand may be capable of interacting with a target
molecule on the external face of a tissue to be treated. Ligands
having limited cross-reactivity to other tissues are generally
preferred.
[0107] In some cases, a ligand can act as a homing moiety which
permits the cell to target to a specific tissue or interact with a
specific ligand. Such homing moieties can include, but are not
limited to, any member of a specific binding pair, antibodies,
monoclonal antibodies, or derivatives or analogs thereof, including
without limitation: Fv fragments, single chain Fv (scFv) fragments,
Fab' fragments, F(ab')2 fragments, single domain antibodies,
camelized antibodies and antibody fragments, humanized antibodies
and antibody fragments, and multivalent versions of the foregoing;
multivalent binding reagents including without limitation:
monospecific or bispecific antibodies, such as disulfide stabilized
Fv fragments, scFv tandems ((SCFV)2 fragments), diabodies,
tribodies or tetrabodies, which typically are covalently linked or
otherwise stabilized (i.e., leucine zipper or helix stabilized)
scFv fragments; and other homing moieties include for example,
aptamers, receptors, and fusion proteins.
[0108] In some embodiments, the homing moiety may be a
surface-bound antibody, which can permit tuning of cell targeting
specificity. This is especially useful since highly specific
antibodies can be raised against an epitope of interest for the
desired targeting site. In one embodiment, multiple antibodies are
expressed on the surface of a cell, and each antibody can have a
different specificity for a desired target. Such approaches can
increase the avidity and specificity of homing interactions.
[0109] A skilled artisan can select any homing moiety based on the
desired localization or function of the cell, for example an
estrogen receptor ligand, such as tamoxifen, can target cells to
estrogen-dependent breast cancer cells that have an increased
number of estrogen receptors on the cell surface. Other
non-limiting examples of ligand/receptor interactions include CCR1
(e.g., for treatment of inflamed joint tissues or brain in
rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g.,
targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g., to target
to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),
CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., for
treatment of inflammation and inflammatory disorders, bone marrow),
Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4NCAM-1
(e.g., targeting to endothelium). In general, any receptor involved
in targeting (e.g., cancer metastasis) can be harnessed for use in
the methods and compositions described herein.
Mediators of Cell Death
[0110] In one embodiment, a modified nucleic acid molecule
composition can be used to induce apoptosis in a cell (e.g., a
cancer cell) by increasing the expression of a death receptor, a
death receptor ligand or a combination thereof. This method can be
used to induce cell death in any desired cell and has particular
usefulness in the treatment of cancer where cells escape natural
apoptotic signals.
[0111] Apoptosis can be induced by multiple independent signaling
pathways that converge upon a final effector mechanism consisting
of multiple interactions between several "death receptors" and
their ligands, which belong to the tumor necrosis factor (TNF)
receptor/ligand superfamily. The best-characterized death receptors
are CD95 ("Fas"), TNFRI (p55), death receptor 3 (DR3 or
Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). The final effector
mechanism of apoptosis may be the activation of a series of
proteinases designated as caspases. The activation of these
caspases results in the cleavage of a series of vital cellular
proteins and cell death. The molecular mechanism of death
receptors/ligands-induced apoptosis is well known in the art. For
example, Fas/FasL-mediated apoptosis is induced by binding of three
FasL molecules which induces trimerization of Fas receptor via
C-terminus death domains (DDs), which in turn recruits an adapter
protein FADD (Fas-associated protein with death domain) and
Caspase-8. The oligomerization of this trimolecular complex,
Fas/FAIDD/caspase-8, results in proteolytic cleavage of proenzyme
caspase-8 into active caspase-8 that, in turn, initiates the
apoptosis process by activating other downstream caspases through
proteolysis, including caspase-3. Death ligands in general are
apoptotic when formed into trimers or higher order of structures.
As monomers, they may serve as antiapoptotic agents by competing
with the trimers for binding to the death receptors.
[0112] In one embodiment, the modified nucleic acid molecule
composition encodes for a death receptor (e.g., Fas, TRAIL, TRAMO,
TNFR, TLR etc). Cells made to express a death receptor by
transfection of modified RNA become susceptible to death induced by
the ligand that activates that receptor. Similarly, cells made to
express a death ligand, e.g., on their surface, will induce death
of cells with the receptor when the transfected cell contacts the
target cell. In another embodiment, the modified RNA composition
encodes for a death receptor ligand (e.g., FasL, TNF, etc). In
another embodiment, the modified RNA composition encodes a caspase
(e.g., caspase 3, caspase 8, caspase 9 etc). Where cancer cells
often exhibit a failure to properly differentiate to a
non-proliferative or controlled proliferative form, in another
embodiment, the synthetic, modified RNA composition encodes for
both a death receptor and its appropriate activating ligand. In
another embodiment, the synthetic, modified RNA composition encodes
for a differentiation factor that when expressed in the cancer
cell, such as a cancer stem cell, will induce the cell to
differentiate to a non-pathogenic or nonself-renewing phenotype
(e.g., reduced cell growth rate, reduced cell division etc) or to
induce the cell to enter a dormant cell phase (e.g., G.sub.0
resting phase).
[0113] One of skill in the art will appreciate that the use of
apoptosis-inducing techniques may require that the modified nucleic
acid molecules are appropriately targeted to e.g., tumor cells to
prevent unwanted wide-spread cell death. Thus, one can use a
delivery mechanism (e.g., attached ligand or antibody, targeted
liposome etc) that recognizes a cancer antigen such that the
modified nucleic acid molecules are expressed only in cancer
cells.
Formulations of Modified mRNAs
[0114] Provided herein are formulations containing an effective
amount of an mmRNA.
[0115] In certain embodiments, the formulations include one or more
cell penetration agents, e.g., transfection agents. In one specific
embodiment, an mmRNA is mixed or admixed with a transfection agent
(or mixture thereof) and the resulting mixture is employed to
transfect cells. Preferred transfection agents are cationic lipid
compositions, particularly monovalent and polyvalent cationic lipid
compositions, more particularly LIPOFECTIN.RTM., LIPOFECTACE.RTM.,
LIPOFECTAMINE.TM., CELLFECTIN.RTM., DMRIE-C, DMRIE, DOTAP, DOSPA,
and DOSPER, and dendrimer compositions, particularly G5-G10
dendrimers, including dense star dendrimers, PAMAM dendrimers,
grafted dendrimers, and dendrimers known as dendrigrafts and
SUPERFECT.RTM..
[0116] In a second specific transfection method, a ribonucleic acid
is conjugated to a nucleic acid-binding group, for example a
polyamine and more particularly a spermine, which is then
introduced into the cell or admixed with a transfection agent (or
mixture thereof) and the resulting mixture is employed to transfect
cells. In a third specific embodiment, a mixture of one or more
transfection-enhancing peptides, proteins, or protein fragments,
including fusagenic peptides or proteins, transport or trafficking
peptides or proteins, receptor-ligand peptides or proteins, or
nuclear localization peptides or proteins and/or their modified
analogs (e.g., spermine modified peptides or proteins) or
combinations thereof are mixed with and complexed with a
ribonucleic acid to be introduced into a cell, optionally being
admixed with transfection agent and the resulting mixture is
employed to transfect cells. Further, a component of a transfection
agent (e.g., lipids, cationic lipids or dendrimers) is covalently
conjugated to selected peptides, proteins, or protein fragments
directly or via a linking or spacer group. Of particular interest
in this embodiment are peptides or proteins that are fusagenic,
membrane-permeabilizing, transport or trafficking, or which
function for cell-targeting. The peptide- or protein-transfection
agent complex is combined with a ribonucleic acid and employed for
transfection.
[0117] In certain embodiments, the formulations include a
pharmaceutically acceptable carrier that causes the effective
amount of mmRNA to be substantially retained in a target tissue
containing the cell.
[0118] In certain embodiments, the formulation may include at least
an mmRNA and a delivery agent. In some embodiments, the delivery
agent may comprise lipidoid-based formulations allowed for
localized and systemic delivery of mmRNA.
[0119] Also provided are compositions for generation of an in vivo
depot containing an engineered ribonucleotide. For example, the
composition contains a bioerodible, biocompatible polymer, a
solvent present in an amount effective to plasticize the polymer
and form a gel therewith, and an engineered ribonucleic acid. In
certain embodiments the composition also includes a cell
penetration agent as described herein. In other embodiments, the
composition also contains a thixotropic amount of a thixotropic
agent mixable with the polymer so as to be effective to form a
thixotropic composition. Further compositions include a stabilizing
agent, a bulking agent, a chelating agent, or a buffering
agent.
[0120] In other embodiments, provided are sustained-release
delivery depots, such as for administration of a mmRNA to an
environment (meaning an organ or tissue site) in a patient. Such
depots generally contain a mmRNA and a flexible chain polymer where
both the mmRNA and the flexible chain polymer are entrapped within
a porous matrix of a crosslinked matrix protein. Usually, the pore
size is less than 1 mm, such as 900 nm, 800 nm, 700 nm, 600 nm, 500
nm, 400 nm, 300 nm, 200 nm, 100 nm, or less than 100 nm. Usually
the flexible chain polymer is hydrophilic. Usually the flexible
chain polymer has a molecular weight of at least 50 kDa, such as 75
kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa,
or greater than 500 kDa. Usually the flexible chain polymer has a
persistence length of less than 10%, such as 9, 8, 7, 6, 5, 4, 3,
2, 1 or less than 1% of the persistence length of the matrix
protein. Usually the flexible chain polymer has a charge similar to
that of the matrix protein. In some embodiments, the flexible chain
polymer alters the effective pore size of a matrix of crosslinked
matrix protein to a size capable of sustaining the diffusion of the
mmRNA from the matrix into a surrounding tissue comprising a cell
into which the mmRNA is capable of entering.
Formulation Using Lipidoids
[0121] The pharmaceutical compositions described herein include
lipidoid-based formulations allowing for localized and systemic
delivery of mmRNA. The synthesis of lipidoids has been extensively
described and formulations containing these compounds are
particularly suited for delivery of polynucleotides (see Mahon et
al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern
Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569;
Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart
et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which
are incorporated herein by reference in their entireties).
[0122] According to the present invention, complexes, micelles,
liposomes or particles can be prepared containing these lipidoids
and therefore, result in an effective delivery of mmRNA, as judged
by the production of an encoded protein, following the injection of
an mmRNA-formulated lipidoids via localized and systemic routes of
administration. Modified mRNA-lipidoid complexes can be
administered by various means disclosed herein.
[0123] The characteristics of optimized lipidoid formulations for
intramuscular or subcutaneous routes may vary significantly
depending on the target cell type and the ability of formulations
to diffuse through the extracellular matrix into the blood stream.
While a particle size of less than 150 nm may be desired for
effective hepatocyte delivery due to the size of the endothelial
fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein
incorporated by reference), use of lipidoid oligonucleotides to
deliver the formulation to other cells types including, but not
limited to, endothelial cells, myeloid cells, and muscle cells may
not be similarly size-limited.
[0124] In one aspect, effective delivery to myeloid cells, such as
monocytes, lipidoid formulations may have a similar component molar
ratio. Different ratios of lipidoids and other components
including, but not limited to, disteroylphosphatidyl choline,
cholesterol and PEG-DMG, may be used to optimize the formulation of
the mmRNA molecule for delivery to different cell types including,
but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
For example, the component molar ratio may include, but is not
limited to, 50% lipid, 10% disteroylphosphatidyl choline, 38.5%
cholesterol, and %1.5 PEG. The lipid may be selected from, but is
not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,
C12-200 (including variants and derivatives), DLin-MC3-DMA and
analogs thereof. The use of lipidoid formulations for the localized
delivery of nucleic acids to cells (such as, but not limited to,
adipose cells and muscle cells) via either subcutaneous or
intramuscular delivery, may also not require all of the formulation
components which may be required for systemic delivery, and as such
may comprise the lipidoid and the mmRNA.
[0125] In a further embodiment, combinations of different lipidoids
may be used to improve the efficacy of mmRNA-directed protein.
[0126] According to the present invention, modified mRNA may be
formulated by mixing the mmRNA with the lipidoid at a set ratio
prior to addition to cells. In vivo formulations may require the
addition of extra ingredients to facilitate circulation throughout
the body. To test the ability of these lipidoids to form particles
suitable for in vivo work, a standard formulation process used for
siRNA-lipidoid formulations may be used as a starting point.
Initial mmRNA-lipidoid formulations consist of particles composed
of 42% lipidoid, 48% cholesterol and 10% PEG, with further
optimization of ratios possible. After formation of the particle,
mmRNA is added and allowed to integrate with the complex. The
encapsulation efficiency is determined using a standard dye
exclusion assays.
[0127] In vivo delivery of nucleic acids may be affected by many
parameters, including, but not limited to, the formulation
composition, nature of particle PEGylation, degree of loading,
oligonucleotide to lipid ratio, and biophysical parameters such as
particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein
incorporated by reference in its entirety). As an example, small
changes in the anchor chain length of poly(ethylene glycol) (PEG)
lipids may result in significant effects on in vivo efficacy.
Formulations with the different lipidoids, including, but not
limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine
hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al.,
Analytical Biochemistry, 401:61 (2010)), C12-200 (including
derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA
and DLin-MC3-DMA (see FIG. 1), can be tested for in vivo
activity.
[0128] The lipidoid referred to herein as "98N12-5" is disclosed by
Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by
reference in its entirety. (See FIG. 1)
[0129] The lipidoid referred to herein as "C12-200" is disclosed by
Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG.
1) and Liu and Huang, Molecular Therapy. 2010 669-670 (see FIG. 1);
both of which are herein incorporated by reference in their
entirety. The lipidoid formulations can include particles
comprising either 3 or 4 or more components in addition to
polynucleotide, primary construct, or mmRNA. As an example,
formulations with certain lipidoids, include, but are not limited
to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10%
PEG (C14 alkyl chain length). As another example, formulations with
certain lipidoids, include, but are not limited to, C12-200 and may
contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5%
cholesterol, and 1.5% PEG-DMG.
[0130] The ratio of mmRNA to lipidoid used to test for in vitro
transfection is tested empirically at different lipidoid:mmRNA
ratios. Previous work using siRNA and lipidoids have utilized
2.5:1, 5:1, 10:1, and 15:1 lipidoid:siRNA wt:wt ratios. Given the
longer length of mmRNA relative to siRNA, a lower wt:wt ratio of
lipidoid to mmRNA is likely to be effective. In addition, for
comparison mmRNA are also formulated using RNAiMax (Invitrogen,
Carlsbad, Calif.) or TRANSIT-mRNA (Minis Bio, Madison Wis.)
cationic lipid delivery vehicles.
[0131] The ability of lipidoid-formulated mmRNA to express the
desired protein product can be confirmed by luminescence for
luciferase expression, flow cytometry for expression, and by ELISA
for secretion.
[0132] The expression of mmRNA-encoded proteins can be assessed
both within the muscle or subcutaneous tissue and systemically in
blood and other organs and fluids such as the liver and spleen,
urine, saliva, etc.
[0133] For example, single dose studies allow an assessment of the
magnitude, dose responsiveness, and longevity of expression of the
desired product. After formulation of mmRNA with the lipidoid
formulations, as described previously, animals are divided into
groups receiving either a saline formulation, or a
lipidoid-formulation containing one of several different mmRNA.
Prior to injection, mmRNA-containing lipidoid formulations are
diluted in PBS and animals administered a single intramuscular dose
of formulated mmRNA ranging from 50 mg/kg to doses as low as 1
ng/kg with a preferred range to be 10 mg/kg to 100 ng/kg. If the
animal tested is a mouse the maximum dose can be roughly 1 mg mmRNA
or as low as 0.02 ng mmRNA if administered once into the hind limb.
Likewise for subcutaneous administration, mmRNA-containing lipidoid
formulations are diluted in PBS before the animals are administered
a single subcutaneous dose of formulated mmRNA ranging from 400
mg/kg- to doses as low as 1 ng/kg. A preferred dosage range
comprises 80 mg/kg to 100 ng/kg. If the animal tested is a mouse,
the maximum dose administered can be roughly 8 mg mmRNA or as low
as 0.02 ng mmRNA if the dose is administered once
subcutaneously.
[0134] It is preferred that the volume of a single intramuscular
injection is maximally 0.025 ml and of a single subcutaneous
injection is maximally 0.2 ml for a 20 gram mouse. The dose of the
mmRNA administered to the animal is calculated depending on the
body weight of the animal. At various points in time points
following the administration of the mmRNA-lipidoid, serum, tissues,
and tissue lysates can be obtained and the level of the
mmRNA-encoded product determined. The ability of
lipidoid-formulated mmRNA to express the desired protein product
can be confirmed by luminescence for luciferase expression, flow
cytometry, and by ELISA.
[0135] Additional studies for a multi-dose regimen can also be
performed to determine the maximal expression using mmRNA, to
evaluate the saturability of the mmRNA-driven expression (achieved
by giving a control and active mmRNA formulation in parallel or in
sequence), and to determine the feasibility of repeat drug
administration (by giving mmRNA in doses separated by weeks or
months and then determining whether expression level is affected by
factors such as immunogenicity).
Administration
[0136] The present invention provides methods comprising
administering modified mRNAs and or complexes in accordance with
the invention to a subject in need thereof. mmRNA or complexes, or
pharmaceutical, imaging, diagnostic, or prophylactic compositions
thereof, may be administered to a subject using any amount and any
route of administration which may be effective for preventing,
treating, diagnosing, or imaging a disease, disorder, and/or
condition (e.g., a disease, disorder, and/or condition relating to
working memory deficits). The exact amount required will vary from
subject to subject, depending on factors such as, but not limited
to, the species, age, and general condition of the subject, the
severity of the disease, the particular composition, its mode of
administration, its mode of activity, and the like.
[0137] mmRNA to be delivered and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof may be administered to
animals, such as mammals (e.g., humans, domesticated animals, cats,
dogs, mice, rats, etc.). In some embodiments, pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof are
administered to humans.
[0138] mmRNA may be administered by any route. In some embodiments,
mmRNA are administered by one or more of a variety of routes,
including, but not limited to, local, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
subcutaneous, intraventricular, transdermal, interdermal, rectal,
intravaginal, intraperitoneal, topical (e.g. by powders, ointments,
creams, gels, lotions, and/or drops), mucosal, nasal, buccal,
enteral, vitreal, intratumoral, sublingual; by intratracheal
instillation, bronchial instillation, and/or inhalation; as an oral
spray, nasal spray, and/or aerosol, and/or through a portal vein
catheter.
[0139] In some embodiments, mmRNA are administered by systemic
intravenous injection. In specific embodiments, mmRNA may be
administered intravenously and/or orally. In specific embodiments,
mmRNA may be administered in a way which allows the mmRNA to cross
the blood-brain barrier, vascular barrier, or other epithelial
barrier.
[0140] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing agents, wetting agents,
and/or suspending agents. Sterile injectable preparations may be
sterile injectable solutions, suspensions, and/or emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P., and isotonic sodium chloride solution. Sterile,
fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. Fatty acids such as
oleic acid can be used in the preparation of injectables.
[0141] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0142] Dosage forms for local, topical and/or transdermal
administration of a composition may include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants and/or
patches. Additionally, the present invention contemplates the use
of transdermal patches, which often have the added advantage of
providing controlled delivery of a compound to the body. Such
dosage forms may be prepared, for example, by dissolving and/or
dispensing the compound in the proper medium. Alternatively or
additionally, rate may be controlled by either providing a rate
controlling membrane and/or by dispersing the compound in a polymer
matrix and/or gel.
[0143] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi liquid preparations such
as liniments, lotions, oil in water and/or water in oil emulsions
such as creams, ointments and/or pastes, and/or solutions and/or
suspensions. Topically-administrable formulations may, for example,
comprise from about 1% to about 10% (w/w) active ingredient,
although the concentration of active ingredient may be as high as
the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0144] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form
of eye drops including, for example, a 0.1/1.0% (w/w) solution
and/or suspension of the active ingredient in an aqueous or oily
liquid excipient. Such drops may further comprise buffering agents,
salts, and/or one or more other of any additional ingredients
described herein. Other opthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form and/or in a liposomal preparation. Ear
drops and/or eye drops are contemplated as being within the scope
of this invention.
[0145] In general the most appropriate route of administration will
depend upon a variety of factors including the nature of the mmRNA
to be delivered (e.g., its stability in the environment of the
gastrointestinal tract, bloodstream, etc.), the condition of the
patient (e.g., whether the patient is able to tolerate particular
routes of administration), etc. The invention encompasses the
delivery of the mmRNA by any appropriate route taking into
consideration likely advances in the sciences of drug delivery.
[0146] In certain embodiments, compositions in accordance with the
present invention may be administered at dosage levels sufficient
to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40
mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01
mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or
from about 1 mg/kg to about 25 mg/kg, of subject body weight per
day, one or more times a day, to obtain the desired therapeutic,
diagnostic or prophylactic effect. The desired dosage may be
delivered three times a day, two times a day, once a day, every
other day, every third day, every week, every two weeks, every
three weeks, or every four weeks. In certain embodiments, the
desired dosage may be delivered using multiple administrations
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, or more administrations). When
multiple administration is employed, split dosing regimens such as
those described herein may be used.
[0147] According to the present invention, it has been discovered
that administration of mmRNA in split-dose regimens produce higher
levels of proteins in mammalian subjects. As used herein, a "split
dose" is the division of single unit dose or total daily dose into
two or more doses. As used herein, a "single unit dose" is a dose
of any therapeutic administed in one dose/at one time/single
route/single point of contact, i.e., single administration event.
As used herein, a "total daily dose" is an amount given or
prescribed in 24 hr period. It may be administered as a single unit
dose. In one embodiment, the mmRNA of the present invention are
administed to a subject in split doses. The mmRNA may be formulated
in buffer only or in a formulation described herein.
[0148] Modified nucleic acid molecules or complexes may be used or
administered in combination with one or more other therapeutic,
prophylactic, diagnostic, or imaging agents. By "in combination
with," it is not intended to imply that the agents must be
administered at the same time and/or formulated for delivery
together, although these methods of delivery are within the scope
of the present disclosure. Compositions can be administered
concurrently with, prior to, or subsequent to, one or more other
desired therapeutics or medical procedures. In general, each agent
will be administered at a dose and/or on a time schedule determined
for that agent. In some embodiments, the present disclosure
encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or imaging compositions in combination with agents that
may improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body.
[0149] It will further be appreciated that therapeutically,
prophylactically, diagnostically, or imaging active agents utilized
in combination may be administered together in a single composition
or administered separately in different compositions. In general,
it is expected that agents utilized in combination with be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually. In one
embodiment, the combinations, each or together may be administered
according to the split dosing regimens described herein.
[0150] The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will take into
account compatibility of the desired therapeutics and/or procedures
and the desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, a composition useful for
treating cancer in accordance with the invention may be
administered concurrently with a chemotherapeutic agent), or they
may achieve different effects (e.g., control of any adverse
effects).
[0151] Compositions containing mmRNAs are formulated for
administration intramuscularly, transarterially, intraocularly,
vaginally, rectally, intraperitoneally, intravenously,
intranasally, subcutaneously, endoscopically, transdermally,
intramuscularly, intraventricularly, intradermally, intrathecally,
topically (e.g. by powders, ointments, creams, gels, lotions,
and/or drops), mucosally, nasal, enterally, intratumorally, by
intratracheal instillation, bronchial instillation, and/or
inhalation; nasal spray and/or aerosol, and/or through a portal
vein catheter.
[0152] The compositions may also be formulated for direct delivery
to an organ or tissue in any of several ways in the art including,
but not limited to, direct soaking or bathing, via a catheter, by
gels, powder, ointments, creams, gels, lotions, and/or drops, by
using substrates such as fabric or biodegradable materials coated
or impregnated with the compositions, and the like. In some
embodiments, the composition is formulated for extended release. In
specific embodiments, mmRNA molecules or complexes, and/or
pharmaceutical, prophylactic, diagnostic, or imaging compositions
thereof, may be administered in a way which allows the mmRNA
molecules or complex to cross the blood-brain barrier, vascular
barrier, or other epithelial barrier.
[0153] In some aspects of the invention, the nucleic acids
(particularly ribonucleic acids encoding polypeptides) are
spatially retained within or proximal to a target tissue. Provided
are method of providing a composition to a target tissue of a
mammalian subject by contacting the target tissue (which contains
one or more target cells) with the composition under conditions
such that the composition, in particular the nucleic acid
component(s) of the composition, is substantially retained in the
target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70,
80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99%
of the composition is retained in the target tissue.
Advantageously, retention is determined by measuring the amount of
the nucleic acid present in the composition that enters one or more
target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60,
70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than
99.99% of the nucleic acids administered to the subject are present
intracellularly at a period of time following administration. For
example, intramuscular injection to a mammalian subject is
performed using an aqueous composition containing a ribonucleic
acid and a transfection reagent, and retention of the composition
is determined by measuring the amount of the ribonucleic acid
present in the muscle cells.
[0154] Aspects of the invention are directed to methods of
providing a composition to a target tissue of a mammalian subject,
by contacting the target tissue (containing one or more target
cells) with the composition under conditions such that the
composition is substantially retained in the target tissue. The
composition contains an effective amount of a ribonucleic acid
engineered to avoid an innate immune response of a cell into which
the ribonucleic acid enters, where the ribonucleic acid contains a
nucleotide sequence encoding a polypeptide of interest, under
conditions such that the polypeptide of interest is produced in at
least one target cell. The compositions generally contain a cell
penetration agent, although "naked" nucleic acid (such as nucleic
acids without a cell penetration agent or other agent) is also
contemplated, and a pharmaceutically acceptable carrier.
[0155] In some circumstances, the amount of a protein produced by
cells in a tissue is desirably increased. Preferably, this increase
in protein production is spatially restricted to cells within the
target tissue. Thus, provided are methods of increasing production
of a protein of interest in a tissue of a mammalian subject. A
composition is provided that contains a ribonucleic acid that is
engineered to avoid an innate immune response of a cell into which
the ribonucleic acid enters and encodes the polypeptide of interest
and the composition is characterized in that a unit quantity of
composition has been determined to produce the polypeptide of
interest in a substantial percentage of cells contained within a
predetermined volume of the target tissue. In some embodiments, the
composition includes a plurality of different ribonucleic acids,
where one or more than one of the ribonucleic acids is engineered
to avoid an innate immune response of a cell into which the
ribonucleic acid enters, and where one or more than one of the
ribonucleic acids encodes a polypeptide of interest. Optionally,
the composition also contains a cell penetration agent to assist in
the intracellular delivery of the ribonucleic acid. A determination
is made of the dose of the composition required to produce the
polypeptide of interest in a substantial percentage of cells
contained within the predetermined volume of the target tissue
(generally, without inducing significant production of the
polypeptide of interest in tissue adjacent to the predetermined
volume, or distally to the target tissue). Subsequent to this
determination, the determined dose is introduced directly into the
tissue of the mammalian subject.
[0156] Formulations which may be administered intramuscularly
and/or subcutaneously may include, but are not limited to,
polymers, copolymers, and gels. The polymers, copolymers and/or
gels may further be adjusted to modify release kinetics by
adjusting factors such as, but not limited to, molecular weight,
particle size, payload and/or ratio of the monomers. As a
non-limiting example, formulations administered intramuscularly
and/or subcutaneously may include a copolymer such as
poly(lactic-co-glycolic acid).
[0157] Localized delivery of the compositions described herein may
be administered by methods such as, but not limited to, topical
delivery, ocular delivery, transdermal delivery, and the like. The
composition may also be administered locally to a part of the body
not normally available for localized delivery such as, but not
limited to, when a subject's body is open to the environment during
treatment. The composition may further be delivered by bathing,
soaking and/or surrounding the body part with the composition.
[0158] However, the present disclosure encompasses the delivery of
mmRNA molecules or complexes, and/or pharmaceutical, prophylactic,
diagnostic, or imaging compositions thereof, by any appropriate
route taking into consideration likely advances in the sciences of
drug delivery.
[0159] The level or concentration of a mmRNA may be characterized
using exosomes. A level or concentration of the mmRNA in exosomes
can represent an expression level, presence, absence, truncation or
alteration of the mmRNA. The level or concentration may be
determined by a method such as, but not limited to, an assay using
construct specific probes, cytometry, qRT-PCR, realtime PCR, PCR,
flow cytometry, electrophoresis, mass spectrometry, or combinations
thereof. Further, the level or concentration may be associated with
a clinical phenotype. For analysis, the exosome may be isolated by
a method such as, but not limited to, immunohistochemcial methods
such as enzyme linked immonsorbent assay (ELISA) methods, size
exclusion chromatography, density gradient centrifugation,
differential centrifugation, nanomembrane ultrafiltration,
immunoabsorbent capture, affinity purification, microfluidic
separation, or combinations thereof.
Pharmaceutical Compositions
[0160] When administered to a subject the pharmaceutical
compositions described herein may provide proteins which have been
generated from modified mRNAs. Pharmaceutical compositions may
optionally comprise one or more additional therapeutically active
substances. In accordance with some embodiments, a method of
administering pharmaceutical compositions comprising one or more
proteins to be delivered to a subject in need thereof is provided.
In some embodiments, compositions are administered to human
subjects. In a further embodiment, the compositions are
administered to a subject who is a patient.
[0161] Pharmaceutical compositions may optionally comprise one or
more additional therapeutically active substances.
[0162] In some embodiments, compositions are administered to
humans. For the purposes of the present disclosure, the phrase
"active ingredient" generally refers to a mmRNA to be delivered as
described herein.
[0163] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions is contemplated include, but are not
limited to, humans and/or other primates; mammals, including
commercially relevant mammals such as cattle, pigs, horses, sheep,
cats, dogs, mice, and/or rats; and/or birds, including commercially
relevant birds such as chickens, ducks, geese, and/or turkeys.
[0164] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, shaping and/or packaging
the product into a desired single- or multi-dose unit.
[0165] A pharmaceutical composition in accordance with the
invention may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient which would be administered
to a subject and/or a convenient fraction of such a dosage such as,
for example, one-half or one-third of such a dosage.
[0166] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
invention will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100% (w/w)
active ingredient.
[0167] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes any and all solvents, dispersion media, diluents, or other
liquid vehicles, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional excipient
medium is incompatible with a substance or its derivatives, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical composition, its use is contemplated to be
within the scope of this invention.
[0168] In some embodiments, a pharmaceutically acceptable excipient
is at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% pure. In some embodiments, an excipient is approved
for use in humans and for veterinary use.
[0169] In some embodiments, an excipient is approved by United
States Food and Drug Administration. In some embodiments, an
excipient is pharmaceutical grade. In some embodiments, an
excipient meets the standards of the United States Pharmacopoeia
(USP), the European Pharmacopoeia (EP), the British Pharmacopoeia,
and/or the International Pharmacopoeia.
[0170] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, dispersing and/or granulating agents,
surface active agents and/or emulsifiers, disintegrating agents,
binding agents, preservatives, buffering agents, lubricating
agents, and/or oils. Such excipients may optionally be included in
pharmaceutical formulations. Excipients such as cocoa butter and
suppository waxes, coloring agents, coating agents, sweetening,
flavoring, and/or perfuming agents can be present in the
composition, according to the judgment of the formulator.
[0171] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium carbonate, calcium phosphate, dicalcium
phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose, cellulose, microcrystalline cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch,
cornstarch, powdered sugar, etc., and/or combinations thereof.
[0172] Exemplary granulating and/or dispersing agents include, but
are not limited to, potato starch, corn starch, tapioca starch,
sodium starch glycolate, clays, alginic acid, guar gum, citrus
pulp, agar, bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds,
etc., and/or combinations thereof.
[0173] Exemplary surface active agents and/or emulsifiers include,
but are not limited to, natural emulsifiers (e.g. acacia, agar,
alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,
xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol,
wax, and lecithin), colloidal clays (e.g. bentonite [aluminum
silicate] and Veegum.RTM. [magnesium aluminum silicate]), long
chain amino acid derivatives, high molecular weight alcohols (e.g.
stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate, ethylene glycol distearate, glyceryl monostearate,
and propylene glycol monostearate, polyvinyl alcohol), carbomers
(e.g. carboxy polymethylene, polyacrylic acid, acrylic acid
polymer, and carboxyvinyl polymer), carrageenan, cellulosic
derivatives (e.g. carboxymethylcellulose sodium, powdered
cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate
[TWEEN.RTM.20], polyoxyethylene sorbitan [TWEEN.RTM.60],
polyoxyethylene sorbitan monooleate [TWEEN.RTM.80], sorbitan
monopalmitate [SPAN.RTM.40], sorbitan monostearate [SPAN.RTM.60],
sorbitan tristearate [SPAN.RTM.65], glyceryl monooleate, sorbitan
monooleate [SPAN.RTM.80]), polyoxyethylene esters (e.g.
polyoxyethylene monostearate [MYRJ.RTM.45], polyoxyethylene
hydrogenated castor oil, polyethoxylated castor oil,
polyoxymethylene stearate, and SOLUTOL.RTM.), sucrose fatty acid
esters, polyethylene glycol fatty acid esters (e.g.
CREMOPHOR.RTM.), polyoxyethylene ethers, (e.g. polyoxyethylene
lauryl ether [BRIJ.RTM.30]), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, PLURONIC.RTM.F 68, POLOXAMER.RTM.188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof.
[0174] Exemplary binding agents include, but are not limited to,
starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol,
mannitol,); natural and synthetic gums (e.g. acacia, sodium
alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage
of isapol husks, carboxymethylcellulose, methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, microcrystalline cellulose,
cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum
silicate (VEEGUM.RTM.), and larch arabogalactan); alginates;
polyethylene oxide; polyethylene glycol; inorganic calcium salts;
silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and
combinations thereof.
[0175] Exemplary preservatives may include, but are not limited to,
antioxidants, chelating agents, antimicrobial preservatives,
antifungal preservatives, alcohol preservatives, acidic
preservatives, and/or other preservatives. Exemplary antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid,
acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and/or sodium sulfite. Exemplary chelating
agents include ethylenediaminetetraacetic acid (EDTA), citric acid
monohydrate, disodium edetate, dipotassium edetate, edetic acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric
acid, and/or trisodium edetate. Exemplary antimicrobial
preservatives include, but are not limited to, benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and/or thimerosal.
Exemplary antifungal preservatives include, but are not limited to,
butyl paraben, methyl paraben, ethyl paraben, propyl paraben,
benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium
sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
Exemplary alcohol preservatives include, but are not limited to,
ethanol, polyethylene glycol, phenol, phenolic compounds,
bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl
alcohol. Exemplary acidic preservatives include, but are not
limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric
acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid,
and/or phytic acid. Other preservatives include, but are not
limited to, tocopherol, tocopherol acetate, deteroxime mesylate,
cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened
(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl
ether sulfate (SLES), sodium bisulfite, sodium metabisulfite,
potassium sulfite, potassium metabisulfite, GLYDANT PLUS.RTM.,
PHENONIP.RTM., methylparaben, GERMALL.RTM.115, GERMABEN.RTM.II,
NEOLONE.TM., KATHON.TM., and/or EUXYL.RTM..
[0176] Exemplary buffering agents include, but are not limited to,
citrate buffer solutions, acetate buffer solutions, phosphate
buffer solutions, ammonium chloride, calcium carbonate, calcium
chloride, calcium citrate, calcium glubionate, calcium gluceptate,
calcium gluconate, D-gluconic acid, calcium glycerophosphate,
calcium lactate, propanoic acid, calcium levulinate, pentanoic
acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium
phosphate, calcium hydroxide phosphate, potassium acetate,
potassium chloride, potassium gluconate, potassium mixtures,
dibasic potassium phosphate, monobasic potassium phosphate,
potassium phosphate mixtures, sodium acetate, sodium bicarbonate,
sodium chloride, sodium citrate, sodium lactate, dibasic sodium
phosphate, monobasic sodium phosphate, sodium phosphate mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic
acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl
alcohol, etc., and/or combinations thereof.
[0177] Exemplary lubricating agents include, but are not limited
to, magnesium stearate, calcium stearate, stearic acid, silica,
talc, malt, glyceryl behanate, hydrogenated vegetable oils,
polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate,
etc., and combinations thereof.
[0178] Exemplary oils include, but are not limited to, almond,
apricot kernel, avocado, babassu, bergamot, black current seed,
borage, cade, camomile, canola, caraway, carnauba, castor,
cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton
seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba,
kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary oils include, but are not limited
to, butyl stearate, caprylic triglyceride, capric triglyceride,
cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl
myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone
oil, and/or combinations thereof.
[0179] Liquid dosage forms for oral and parenteral administration
include, but are not limited to, pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups, and/or
elixirs. In addition to active ingredients, liquid dosage forms may
comprise inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, oral compositions can include adjuvants
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, and/or perfuming agents. In certain
embodiments for parenteral administration, compositions are mixed
with solubilizing agents such as Cremophor.RTM., alcohols, oils,
modified oils, glycols, polysorbates, cyclodextrins, polymers,
and/or combinations thereof.
[0180] General considerations in the formulation and/or manufacture
of pharmaceutical agents may be found, for example, in Remington:
The Science and Practice of Pharmacy 21.sup.st ed., Lippincott
Williams & Wilkins, 2005 (incorporated herein by
reference).
[0181] In order to prolong the effect of an active ingredient, it
is often desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
[0182] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing
compositions with suitable non-irritating excipients such as cocoa
butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
ingredient. Solid dosage forms for oral administration include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, an active ingredient is mixed with at least one
inert, pharmaceutically acceptable excipient such as sodium citrate
or dicalcium phosphate and/or fillers or extenders (e.g. starches,
lactose, sucrose, glucose, mannitol, and silicic acid), binders
(e.g. carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.
glycerol), disintegrating agents (e.g. agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate), solution retarding agents (e.g. paraffin),
absorption accelerators (e.g. quaternary ammonium compounds),
wetting agents (e.g. cetyl alcohol and glycerol monostearate),
absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g.
talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate), and mixtures thereof. In the case
of capsules, tablets and pills, the dosage form may comprise
buffering agents.
[0183] Solid compositions of a similar type may be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. Solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally comprise opacifying agents and can be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type may be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polyethylene glycols and the
like.
[0184] Dosage forms for topical and/or transdermal administration
of a composition may include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants and/or patches.
Generally, an active ingredient is admixed under sterile conditions
with a pharmaceutically acceptable excipient and/or any needed
preservatives and/or buffers as may be required.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of active ingredient may be as high as the solubility
limit of the active ingredient in the solvent. Formulations for
topical administration may further comprise one or more of the
additional ingredients described herein.
[0185] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for pulmonary administration
via the buccal cavity. Such a formulation may comprise dry
particles which comprise the active ingredient and which have a
diameter in the range from about 0.5 nm to about 7 nm or from about
1 nm to about 6 nm. Such compositions are suitably in the form of
dry powders for administration using a device comprising a dry
powder reservoir to which a stream of propellant may be directed to
disperse the powder and/or using a self propelling solvent/powder
dispensing container such as a device comprising the active
ingredient dissolved and/or suspended in a low-boiling propellant
in a sealed container. Such powders comprise particles wherein at
least 98% of the particles by weight have a diameter greater than
0.5 nm and at least 95% of the particles by number have a diameter
less than 7 nm. Alternatively, at least 95% of the particles by
weight have a diameter greater than 1 nm and at least 90% of the
particles by number have a diameter less than 6 nm. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0186] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50% to 99.9%
(w/w) of the composition, and active ingredient may constitute 0.1%
to 20% (w/w) of the composition. A propellant may further comprise
additional ingredients such as a liquid non-ionic and/or solid
anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0187] Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of droplets
of a solution and/or suspension. Such formulations may be prepared,
packaged, and/or sold as aqueous and/or dilute alcoholic solutions
and/or suspensions, optionally sterile, comprising active
ingredient, and may conveniently be administered using any
nebulization and/or atomization device. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, a flavoring agent such as saccharin sodium, a
volatile oil, a buffering agent, a surface active agent, and/or a
preservative such as methylhydroxybenzoate. Droplets provided by
this route of administration may have an average diameter in the
range from about 0.1 nm to about 200 nm.
[0188] Formulations described herein as being useful for pulmonary
delivery are useful for intranasal delivery of a pharmaceutical
composition. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient
and having an average particle from about 0.2 .mu.m to 500 .mu.m.
Such a formulation is administered in the manner in which snuff is
taken, i.e. by rapid inhalation through the nasal passage from a
container of the powder held close to the nose.
[0189] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of active ingredient, and may comprise one or more of
the additional ingredients described herein. A pharmaceutical
composition may be prepared, packaged, and/or sold in a formulation
suitable for buccal administration. Such formulations may, for
example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, 0.1% to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable
and/or degradable composition and, optionally, one or more of the
additional ingredients described herein. Alternately, formulations
suitable for buccal administration may comprise a powder and/or an
aerosolized and/or atomized solution and/or suspension comprising
active ingredient. Such powdered, aerosolized, and/or aerosolized
formulations, when dispersed, may have an average particle and/or
droplet size in the range from about 0.1 nm to about 200 nm, and
may further comprise one or more of any additional ingredients
described herein
Properties of the Pharmaceutical Compositions
[0190] The pharmaceutical compositions described herein can be
characterized by one or more of the following properties:
[0191] Bioavailability
[0192] The mmRNA molecules, when formulated into a composition with
a delivery agent as described herein, can exhibit an increase in
bioavailability as compared to a composition lacking a delivery
agent as described herein. As used herein, the term
"bioavailability" refers to the systemic availability of a given
amount of a mmRNA molecule administered to a mammal.
Bioavailability can be assessed by measuring the area under the
curve (AUC) or the maximum serum or plasma concentration
(C.sub.max) of the unchanged form of a compound following
administration of the compound to a mammal. AUC is a determination
of the area under the curve plotting the serum or plasma
concentration of a compound along the ordinate (Y-axis) against
time along the abscissa (X-axis). Generally, the AUC for a
particular compound can be calculated using methods known to those
of ordinary skill in the art and as described in G. S. Banker,
Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,
Marcel Dekker, New York, Inc., 1996, herein incorporated by
reference.
[0193] The C.sub.max value is the maximum concentration of the
compound achieved in the serum or plasma of a mammal following
administration of the compound to the mammal. The C.sub.max value
of a particular compound can be measured using methods known to
those of ordinary skill in the art. The phrases "increasing
bioavailability" or "improving the pharmacokinetics," as used
herein mean that the systemic availability of a first mmRNA
molecule, measured as AUC, C.sub.max, or C.sub.min in a mammal is
greater, when co-administered with a delivery agent as described
herein, than when such co-administration does not take place. In
some embodiments, the bioavailability of the mmRNA molecule can
increase by at least about 2%, at least about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100%.
[0194] Therapeutic Window
[0195] The mmRNA molecules, when formulated into a composition as
described herein, can exhibit an increase in the therapeutic window
of the administered mmRNA molecule composition as compared to the
therapeutic window of the administered mmRNA molecule composition
lacking a delivery agent as described herein. As used herein
"therapeutic window" refers to the range of plasma concentrations,
or the range of levels of therapeutically active substance at the
site of action, with a high probability of eliciting a therapeutic
effect. In some embodiments, the therapeutic window of the mmRNA
molecule when co-administered with a delivery agent as described
herein can increase by at least about 2%, at least about 5%, at
least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or about 100%.
[0196] Volume of Distribution
[0197] The mmRNA molecules, when formulated into a composition as
described herein, can exhibit an improved volume of distribution
(V.sub.dist). The volume of distribution (V.sub.dist) relates the
amount of the drug in the body to the concentration of the drug in
the blood or plasma. As used herein, the term "volume of
distribution" refers to the fluid volume that would be required to
contain the total amount of the drug in the body at the same
concentration as in the blood or plasma: V.sub.dist equals the
amount of drug in the body/concentration of drug in blood or
plasma. For example, for a 10 mg dose and a plasma concentration of
10 mg/L, the volume of distribution would be 1 liter. The volume of
distribution reflects the extent to which the drug is present in
the extravascular tissue. A large volume of distribution reflects
the tendency of a compound to bind to the tissue components
compared with plasma protein binding. In a clinical setting,
V.sub.dist can be used to determine a loading dose to achieve a
steady state concentration. In some embodiments, the volume of
distribution of the mmRNA molecule when co-administered with a
delivery agent as described herein can decrease at least about 2%,
at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%.
Devices and Methods for Multi-Administration
[0198] Methods and devices for multi-administration may be employed
to deliver the mmRNA of the present invention according to the
split dosing regimens taught herein. Such methods and devices are
described below.
[0199] Method and devices known in the art for multi-administration
to cells, organs and tissues are contemplated for use in
conjunction with the methods and compositions disclosed herein as
embodiments of the present invention. These include, for example,
those methods and devices having multiple needles, hybrid devices
employing for example lumens or catheters as well as devices
utilizing heat, electric current or radiation driven
mechanisms.
[0200] According to the present invention, these
multi-administration devices may be utilized to deliver the split
doses contemplated herein.
[0201] Suitable devices for use in delivering intradermal
pharmaceutical compositions described herein include short needle
devices such as those described in U.S. Pat. Nos. 4,886,499;
5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496;
and 5,417,662. Intradermal compositions may be administered by
devices which limit the effective penetration length of a needle
into the skin, such as those described in PCT publication WO
99/34850 and functional equivalents thereof. Jet injection devices
which deliver liquid compositions to the dermis via a liquid jet
injector and/or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis are suitable. Jet injection
devices are described, for example, in U.S. Pat. Nos. 5,480,381;
5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;
5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;
5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460;
and PCT publications WO 97/37705 and WO 97/13537. Ballistic
powder/particle delivery devices which use compressed gas to
accelerate vaccine in powder form through the outer layers of the
skin to the dermis are suitable. Alternatively or additionally,
conventional syringes may be used in the classical mantoux method
of intradermal administration.
[0202] A method for delivering therapeutic agents to a solid tissue
has been described by Bahrami et al and is taught for example in US
Patent Publication 20110230839, the contents of which are
incorporated herein by reference in their entirety. According to
Bahrami, an array of needles is incorporated into a device which
delivers a substantially equal amount of fluid at any location in
said solid tissue along each needle's length.
[0203] A device for delivery of biological material across the
biological tissue has been described by Kodgule et al and is taught
for example in US Patent Publication 20110172610, the contents of
which are incorporated herein by reference in their entirety.
According to Kodgule, multiple hollow micro-needles made of one or
more metals and having outer diameters from about 200 microns to
about 350 microns and lengths of at least 100 microns are
incorporated into the device which delivers peptides, proteins,
carbohydrates, nucleic acid molecules, lipids and other
pharmaceutically active ingredients or combinations thereof.
[0204] A delivery probe for delivering a therapeutic agent to a
tissue has been described by Gunday et al and is taught for example
in US Patent Publication 20110270184, the contents of which are
incorporated herein by reference in their entirety. According to
Gunday, multiple needles are incorporated into the device which
moves the attached capsules between an activated position and an
inactivated position to force the agent out of the capsules through
the needles.
[0205] A multiple-injection medical apparatus has been described by
Assaf and is taught for example in US Patent Publication
20110218497, the contents of which are incorporated herein by
reference in their entirety. According to Assaf, multiple needles
are incorporated into the device which has a chamber connected to
one or more of said needles and a means for continuously refilling
the chamber with the medical fluid after each injection.
[0206] An at least partially implantable system for injecting a
substance into a patient's body, in particular a penis erection
stimulation system has been described by Forsell and is taught for
example in US Patent Publication 20110196198, the contents of which
are incorporated herein by reference in their entirety. According
to Forsell, multiple needles are incorporated into the device which
is implanted along with one or more housings adjacent the patient's
left and right corpora cavernosa. A reservoir and a pump are also
implanted to supply drugs through the needles.
[0207] A method for the transdermal delivery of a therapeutic
effective amount of iron has been described by Berenson and is
taught for example in US Patent Publication 20100130910, the
contents of which are incorporated herein by reference in their
entirety. According to Berenson, multiple needles may be used to
create multiple micro channels in stratum corneum to enhance
transdermal delivery of the ionic iron on an iontophoretic
patch.
[0208] A method for delivery of biological material across the
biological tissue has been described by Kodgule et al and is taught
for example in US Patent Publication 20110196308, the contents of
which are incorporated herein by reference in their entirety.
According to Kodgule, multiple biodegradable microneedles
containing a therapeutic active ingredient are incorporated in a
device which delivers proteins, carbohydrates, nucleic acid
molecules, lipids and other pharmaceutically active ingredients or
combinations thereof.
[0209] A transdermal patch comprising a botulinum toxin composition
has been described by Donovan and is taught for example in US
Patent Publication 20080220020, the contents of which are
incorporated herein by reference in their entirety. According to
Donovan, multiple needles are incorporated into the patch which
delivers botulinum toxin under stratum corneum through said needles
which project through the stratum corneum of the skin without
rupturing a blood vessel.
[0210] A cryoprobe for administration of an active agent to a
location of cryogenic treatment has been described by Toubia and is
taught for example in US Patent Publication 20080140061, the
contents of which are incorporated herein by reference in their
entirety. According to Toubia, multiple needles are incorporated
into the probe which receives the active agent into a chamber and
administers the agent to the tissue.
[0211] A method for treating or preventing inflammation or
promoting healthy joints has been described by Stock et al and is
taught for example in US Patent Publication 20090155186, the
contents of which are incorporated herein by reference in their
entirety. According to Stock, multiple needles are incorporated in
a device which administers compositions containing signal
transduction modulator compounds.
[0212] A multi-site injection system has been described by Kimmell
et al and is taught for example in US Patent Publication
20100256594, the contents of which are incorporated herein by
reference in their entirety. According to Kimmell, multiple needles
are incorporated into a device which delivers a medication into a
stratum corneum through the needles.
[0213] A method for delivering interferons to the intradermal
compartment has been described by Dekker et al and is taught for
example in US Patent Publication 20050181033, the contents of which
are incorporated herein by reference in their entirety. According
to Dekker, multiple needles having an outlet with an exposed height
between 0 and 1 mm are incorporated into a device which improves
pharmacokinetics and bioavailability by delivering the substance at
a depth between 0.3 mm and 2 mm.
[0214] A method for delivering genes, enzymes and biological agents
to tissue cells has described by Desai and is taught for example in
US Patent Publication 20030073908, the contents of which are
incorporated herein by reference in their entirety. According to
Desai, multiple needles are incorporated into a device which is
inserted into a body and delivers a medication fluid through said
needles.
[0215] A method for treating cardiac arrhythmias with fibroblast
cells has been described by Lee et al and is taught for example in
US Patent Publication 20040005295, the contents of which are
incorporated herein by reference in their entirety. According to
Lee, multiple needles are incorporated into the device which
delivers fibroblast cells into the local region of the tissue.
[0216] A method using a magnetically controlled pump for treating a
brain tumor has been described by Shachar et al and is taught for
example in U.S. Pat. No. 7,799,012 (method) and 7,799,016 (device),
the contents of which are incorporated herein by reference in their
entirety. According Shachar, multiple needles were incorporated
into the pump which pushes a medicating agent through the needles
at a controlled rate.
[0217] Methods of treating functional disorders of the bladder in
mammalian females have been described by Versi et al and are taught
for example in U.S. Pat. No. 8,029,496, the contents of which are
incorporated herein by reference in their entirety. According to
Versi, an array of micro-needles is incorporated into a device
which delivers a therapeutic agent through the needles directly
into the trigone of the bladder.
[0218] A micro-needle transdermal transport device has been
described by Angel et al and is taught for example in U.S. Pat. No.
7,364,568, the contents of which are incorporated herein by
reference in their entirety. According to Angel, multiple needles
are incorporated into the device which transports a substance into
a body surface through the needles which are inserted into the
surface from different directions.
[0219] A device for subcutaneous infusion has been described by
Dalton et al and is taught for example in U.S. Pat. No. 7,150,726,
the contents of which are incorporated herein by reference in their
entirety. According to Dalton, multiple needles are incorporated
into the device which delivers fluid through the needles into a
subcutaneous tissue.
[0220] A device and a method for intradermal delivery of vaccines
and gene therapeutic agents through microcannula have been
described by Mikszta et al and are taught for example in U.S. Pat.
No. 7,473,247, the contents of which are incorporated herein by
reference in their entirety. According to Mitszta, at least one
hollow micro-needle is incorporated into the device which delivers
the vaccines to the subject's skin to a depth of between 0.025 mm
and 2 mm.
[0221] A method of delivering insulin has been described by Pettis
et al and is taught for example in U.S. Pat. No. 7,722,595, the
contents of which are incorporated herein by reference in their
entirety. According to Pettis, two needles are incorporated into a
device wherein both needles insert essentially simultaneously into
the skin with the first at a depth of less than 2.5 mm to deliver
insulin to intradermal compartment and the second at a depth of
greater than 2.5 mm and less than 5.0 mm to deliver insulin to
subcutaneous compartment.
[0222] Cutaneous injection delivery under suction has been
described by Kochamba et al and is taught for example in U.S. Pat.
No. 6,896,666, the contents of which are incorporated herein by
reference in their entirety. According to Kochamba, multiple
needles in relative adjacency with each other are incorporated into
a device which injects a fluid below the cutaneous layer.
[0223] A device for withdrawing or delivering a substance through
the skin has been described by Down et al and is taught for example
in U.S. Pat. No. 6,607,513, the contents of which are incorporated
herein by reference in their entirety. According to Down, multiple
skin penetrating members which are incorporated into the device
have lengths of about 100 microns to about 2000 microns and are
about 30 to 50 gauge.
[0224] A device for delivering a substance to the skin has been
described by Palmer et al and is taught for example in U.S. Pat.
No. 6,537,242, the contents of which are incorporated herein by
reference in their entirety. According to Palmer, an array of
micro-needles is incorporated into the device which uses a
stretching assembly to enhance the contact of the needles with the
skin and provides a more uniform delivery of the substance.
[0225] A perfusion device for localized drug delivery has been
described by Zamoyski and is taught for example in U.S. Pat. No.
6,468,247, the contents of which are incorporated herein by
reference in their entirety. According to Zamoyski, multiple
hypodermic needles are incorporated into the device which injects
the contents of the hypodermics into a tissue as said hypodermics
are being retracted.
[0226] A method for enhanced transport of drugs and biological
molecules across tissue by improving the interaction between
micro-needles and human skin has been described by Prausnitz et al
and is taught for example in U.S. Pat. No. 6,743,211, the contents
of which are incorporated herein by reference in their entirety.
According to Prausnitz, multiple micro-needles are incorporated
into a device which is able to present a more rigid and less
deformable surface to which the micro-needles are applied.
[0227] A device for intraorgan administration of medicinal agents
has been described by Ting et al and is taught for example in U.S.
Pat. No. 6,077,251, the contents of which are incorporated herein
by reference in their entirety. According to Ting, multiple needles
having side openings for enhanced administration are incorporated
into a device which by extending and retracting said needles from
and into the needle chamber forces a medicinal agent from a
reservoir into said needles and injects said medicinal agent into a
target organ.
[0228] A multiple needle holder and a subcutaneous multiple channel
infusion port has been described by Brown and is taught for example
in U.S. Pat. No. 4,695,273, the contents of which are incorporated
herein by reference in their entirety. According to Brown, multiple
needles on the needle holder are inserted through the septum of the
infusion port and communicate with isolated chambers in said
infusion port.
[0229] A dual hypodermic syringe has been described by Horn and is
taught for example in U.S. Pat. No. 3,552,394, the contents of
which are incorporated herein by reference in their entirety.
According to Horn, two needles incorporated into the device are
spaced apart less than 68 mm and may be of different styles and
lengths, thus enabling injections to be made to different
depths.
[0230] A syringe with multiple needles and multiple fluid
compartments has been described by Hershberg and is taught for
example in U.S. Pat. No. 3,572,336, the contents of which are
incorporated herein by reference in their entirety. According to
Hershberg, multiple needles are incorporated into the syringe which
has multiple fluid compartments and is capable of simultaneously
administering incompatible drugs which are not able to be mixed for
one injection.
[0231] A surgical instrument for intradermal injection of fluids
has been described by Eliscu et al and is taught for example in
U.S. Pat. No. 2,588,623, the contents of which are incorporated
herein by reference in their entirety. According to Eliscu,
multiple needles are incorporated into the instrument which injects
fluids intradermally with a wider disperse.
[0232] An apparatus for simultaneous delivery of a substance to
multiple breast milk ducts has been described by Hung and is taught
for example in EP 1818017, the contents of which are incorporated
herein by reference in their entirety. According to Hung, multiple
lumens are incorporated into the device which inserts though the
orifices of the ductal networks and delivers a fluid to the ductal
networks.
[0233] A catheter for introduction of medications to the tissue of
a heart or other organs has been described by Tkebuchava and is
taught for example in WO2006138109, the contents of which are
incorporated herein by reference in their entirety. According to
Tkebuchava, two curved needles are incorporated which enter the
organ wall in a flattened trajectory.
[0234] Devices for delivering medical agents have been described by
Mckay et al and are taught for example in WO2006118804, the content
of which are incorporated herein by reference in their entirety.
According to Mckay, multiple needles with multiple orifices on each
needle are incorporated into the devices to facilitate regional
delivery to a tissue, such as the interior disc space of a spinal
disc.
[0235] A method for directly delivering an immunomodulatory
substance into an intradermal space within a mammalian skin has
been described by Pettis and is taught for example in WO2004020014,
the contents of which are incorporated herein by reference in their
entirety. According to Pettis, multiple needles are incorporated
into a device which delivers the substance through the needles to a
depth between 0.3 mm and 2 mm.
[0236] Methods and devices for administration of substances into at
least two compartments in skin for systemic absorption and improved
pharmacokinetics have been described by Pettis et al and are taught
for example in WO2003094995, the contents of which are incorporated
herein by reference in their entirety. According to Pettis,
multiple needles having lengths between about 300 um and about 5 mm
are incorporated into a device which delivers to intradermal and
subcutaneous tissue compartments simultaneously.
[0237] A drug delivery device with needles and a roller has been
described by Zimmerman et al and is taught for example in
WO2012006259, the contents of which are incorporated herein by
reference in their entirety. According to Zimmerman, multiple
hollow needles positioned in a roller are incorporated into the
device which delivers the content in a reservoir through the
needles as the roller rotates.
Methods and Devices Utilizing Catheters and/or Lumens
[0238] Methods and devices using catheters and lumens may be
employed to administer the mmRNA of the present invention on a
split dosing schedule. Such methods and devices are described
below.
[0239] A catheter-based delivery of skeletal myoblasts to the
myocardium of damaged hearts has been described by Jacoby et al and
is taught for example in US Patent Publication 20060263338, the
contents of which are incorporated herein by reference in their
entirety. According to Jacoby, multiple needles are incorporated
into the device at least part of which is inserted into a blood
vessel and delivers the cell composition through the needles into
the localized region of the subject's heart.
[0240] An apparatus for treating asthma using neurotoxin has been
described by Deem et al and is taught for example in US Patent
Publication 20060225742, the contents of which are incorporated
herein by reference in their entirety. According to Deem, multiple
needles are incorporated into the device which delivers neurotoxin
through the needles into the bronchial tissue.
[0241] A method for administering multiple-component therapies has
been described by Nayak and is taught for example in U.S. Pat. No.
7,699,803, the contents of which are incorporated herein by
reference in their entirety. According to Nayak, multiple injection
cannulas may be incorporated into a device wherein depth slots may
be included for controlling the depth at which the therapeutic
substance is delivered within the tissue.
[0242] A surgical device for ablating a channel and delivering at
least one therapeutic agent into a desired region of the tissue has
been described by McIntyre et al and is taught for example in U.S.
Pat. No. 8,012,096, the contents of which are incorporated herein
by reference in their entirety. According to McIntyre, multiple
needles are incorporated into the device which dispenses a
therapeutic agent into a region of tissue surrounding the channel
and is particularly well suited for transmyocardial
revascularization operations.
[0243] Methods of treating functional disorders of the bladder in
mammalian females have been described by Versi et al and are taught
for example in U.S. Pat. No. 8,029,496, the contents of which are
incorporated herein by reference in their entirety. According to
Versi, an array of micro-needles is incorporated into a device
which delivers a therapeutic agent through the needles directly
into the trigone of the bladder.
[0244] A device and a method for delivering fluid into a flexible
biological barrier have been described by Yeshurun et al and are
taught for example in U.S. Pat. No. 7,998,119 (device) and
8,007,466 (method), the contents of which are incorporated herein
by reference in their entirety. According to Yeshurun, the
micro-needles on the device penetrate and extend into the flexible
biological barrier and fluid is injected through the bore of the
hollow micro-needles.
[0245] A method for epicardially injecting a substance into an area
of tissue of a heart having an epicardial surface and disposed
within a torso has been described by Bonner et al and is taught for
example in U.S. Pat. No. 7,628,780, the contents of which are
incorporated herein by reference in their entirety. According to
Bonner, the devices have elongate shafts and distal injection heads
for driving needles into tissue and injecting medical agents into
the tissue through the needles.
[0246] A device for sealing a puncture has been described by
Nielsen et al and is taught for example in U.S. Pat. No. 7,972,358,
the contents of which are incorporated herein by reference in their
entirety. According to Nielsen, multiple needles are incorporated
into the device which delivers a closure agent into the tissue
surrounding the puncture tract.
[0247] A method for myogenesis and angiogenesis has been described
by Chiu et al and is taught for example in U.S. Pat. No. 6,551,338,
the contents of which are incorporated herein by reference in their
entirety. According to Chiu, 5 to 15 needles having a maximum
diameter of at least 1.25 mm and a length effective to provide a
puncture depth of 6 to 20 mm are incorporated into a device which
inserts into proximity with a myocardium and supplies an exogeneous
angiogenic or myogenic factor to said myocardium through the
conduits which are in at least some of said needles.
[0248] A method for the treatment of prostate tissue has been
described by Bolmsj et al and is taught for example in U.S. Pat.
No. 6,524,270, the contents of which are incorporated herein by
reference in their entirety. According to Bolmsj, a device
comprising a catheter which is inserted through the urethra has at
least one hollow tip extendible into the surrounding prostate
tissue. An astringent and analgesic medicine is administered
through said tip into said prostate tissue.
[0249] A method for infusing fluids to an intraosseous site has
been described by Findlay et al and is taught for example in U.S.
Pat. No. 6,761,726, the contents of which are incorporated herein
by reference in their entirety. According to Findlay, multiple
needles are incorporated into a device which is capable of
penetrating a hard shell of material covered by a layer of soft
material and delivers a fluid at a predetermined distance below
said hard shell of material.
[0250] A device for injecting medications into a vessel wall has
been described by Vigil et al and is taught for example in U.S.
Pat. No. 5,713,863, the contents of which are incorporated herein
by reference in their entirety. According to Vigil, multiple
injectors are mounted on each of the flexible tubes in the device
which introduces a medication fluid through a multi-lumen catheter,
into said flexible tubes and out of said injectors for infusion
into the vessel wall.
[0251] A catheter for delivering therapeutic and/or diagnostic
agents to the tissue surrounding a bodily passageway has been
described by Faxon et al and is taught for example in U.S. Pat. No.
5,464,395, the contents of which are incorporated herein by
reference in their entirety. According to Faxon, at least one
needle cannula is incorporated into the catheter which delivers the
desired agents to the tissue through said needles which project
outboard of the catheter.
[0252] Balloon catheters for delivering therapeutic agents have
been described by Orr and are taught for example in WO2010024871,
the contents of which are incorporated herein by reference in their
entirety. According to Orr, multiple needles are incorporated into
the devices which deliver the therapeutic agents to different
depths within the tissue.
Methods and Devices Utilizing Electrical Current
[0253] Methods and devices utilizing electric current may be
employed to deliver the mmRNA of the present invention according to
the split dosing regimens taught herein. Such methods and devices
are described below.
[0254] An electro collagen induction therapy device has been
described by Marquez and is taught for example in US Patent
Publication 20090137945, the contents of which are incorporated
herein by reference in their entirety. According to Marquez,
multiple needles are incorporated into the device which repeatedly
pierce the skin and draw in the skin a portion of the substance
which is applied to the skin first.
[0255] An electrokinetic system has been described by Etheredge et
al and is taught for example in US Patent Publication 20070185432,
the contents of which are incorporated herein by reference in their
entirety. According to Etheredge, micro-needles are incorporated
into a device which drives by an electrical current the medication
through the needles into the targeted treatment site.
[0256] An iontophoresis device has been described by Matsumura et
al and is taught for example in U.S. Pat. No. 7,437,189, the
contents of which are incorporated herein by reference in their
entirety. According to Matsumura, multiple needles are incorporated
into the device which is capable of delivering ionizable drug into
a living body at higher speed or with higher efficiency.
[0257] Intradermal delivery of biologically active agents by
needle-free injection and electroporation has been described by
Hoffmann et al and is taught for example in U.S. Pat. No.
7,171,264, the contents of which are incorporated herein by
reference in their entirety. According to Hoffmann, one or more
needle-free injectors are incorporated into an electroporation
device and the combination of needle-free injection and
electroporation is sufficient to introduce the agent into cells in
skin, muscle or mucosa.
[0258] A method for electropermeabilization-mediated intracellular
delivery has been described by Lundkvist et al and is taught for
example in U.S. Pat. No. 6,625,486, the contents of which are
incorporated herein by reference in their entirety. According to
Lundkvist, a pair of needle electrodes is incorporated into a
catheter. Said catheter is positioned into a body lumen followed by
extending said needle electrodes to penetrate into the tissue
surrounding said lumen. Then the device introduces an agent through
at least one of said needle electrodes and applies electric field
by said pair of needle electrodes to allow said agent pass through
the cell membranes into the cells at the treatment site.
[0259] A delivery system for transdermal immunization has been
described by Levin et al and is taught for example in WO2006003659,
the contents of which are incorporated herein by reference in their
entirety. According to Levin, multiple electrodes are incorporated
into the device which applies electrical energy between the
electrodes to generate micro channels in the skin to facilitate
transdermal delivery.
[0260] A method for delivering RF energy into skin has been
described by Schomacker and is taught for example in WO2011163264,
the contents of which are incorporated herein by reference in their
entirety. According to Schomacker, multiple needles are
incorporated into a device which applies vacuum to draw skin into
contact with a plate so that needles insert into skin through the
holes on the plate and deliver RF energy.
Devices and Kits
[0261] Devices may also be used in conjunction with the present
invention. In one embodiment, a device is used to assess levels of
a protein which has been administered in the form of a modified
mRNA. The device may comprise a blood, urine or other biofluidic
test. It may be as large as to include an automated central lab
platform or a small decentralized bench top device. It may be point
of care or a handheld device. The device may be useful in drug
discovery efforts as a companion diagnostic.
[0262] In some embodiments the device is self-contained, and is
optionally capable of wireless remote access to obtain instructions
for synthesis and/or analysis of the generated nucleic acid. The
device is capable of mobile synthesis of at least one nucleic acid,
and preferably an unlimited number of different nucleic acid
sequences. In certain embodiments, the device is capable of being
transported by one or a small number of individuals. In other
embodiments, the device is scaled to fit on a benchtop or desk. In
other embodiments, the device is scaled to fit into a suitcase,
backpack or similarly sized object. In further embodiments, the
device is scaled to fit into a vehicle, such as a car, truck or
ambulance, or a military vehicle such as a tank or personnel
carrier. The information necessary to generate a modified mRNA
encoding protein of interest is present within a computer readable
medium present in the device.
[0263] In some embodiments, the device is capable of communication
(e.g., wireless communication) with a database of nucleic acid and
polypeptide sequences. The device contains at least one sample
block for insertion of one or more sample vessels. Such sample
vessels are capable of accepting in liquid or other form any number
of materials such as template DNA, nucleotides, enzymes, buffers,
and other reagents. The sample vessels are also capable of being
heated and cooled by contact with the sample block. The sample
block is generally in communication with a device base with one or
more electronic control units for the at least one sample block.
The sample block preferably contains a heating module, such heating
molecule capable of heating and/or cooling the sample vessels and
contents thereof to temperatures between about -20 C and above +100
C. The device base is in communication with a voltage supply such
as a battery or external voltage supply. The device also contains
means for storing and distributing the materials for RNA
synthesis.
[0264] Optionally, the sample block contains a module for
separating the synthesized nucleic acids. Alternatively, the device
contains a separation module operably linked to the sample block.
Preferably the device contains a means for analysis of the
synthesized nucleic acid. Such analysis includes sequence identity
(demonstrated such as by hybridization), absence of non-desired
sequences, measurement of integrity of synthesized mRNA (such has
by microfluidic viscometry combined with spectrophotometry), and
concentration and/orpotency of modified RNA (such as by
spectrophotometry).
[0265] In certain embodiments, the device is combined with a means
for detection of pathogens present in a biological material
obtained from a subject, e.g., the IBIS PLEX-ID system (Abbott) for
microbial identification.
[0266] The present invention provides for devices which incorporate
mmRNA that encode proteins of interest. These devices may be
implantable in an animal subject or may supply mmRNA formulations
via a catheter or lumen. The device may be connected to or
incorporate a pump. Such devices include those which can deliver
therapeutics to areas of the body not readily accessible such as
the CNS or across the blood brain barrier. In this embodiment the
split dosing regimen can be implemented using a regulated pump.
Kits
[0267] The invention provides a variety of kits for conveniently
and/or effectively carrying out methods of the present invention.
Typically kits will comprise sufficient amounts and/or numbers of
components to allow a user to perform multiple treatments of a
subject(s) and/or to perform multiple experiments.
[0268] In one aspect, the present invention provides kits for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and a nucleic acid modification,
wherein the nucleic acid may be capable of evading an innate immune
response of a cell into which the first isolated nucleic acid may
be introduced, and packaging and instructions. The kit may further
comprise a delivery agent to form a formulation composition. The
delivery composition may comprise a lipidoid. The lipoid may be
selected from, but is not limited to, C12-200, 98N12-5, MD1,
DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA and analogs
thereof.
[0269] In one aspect, the present invention provides kits for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and a nucleoside modification,
wherein the nucleic acid exhibits reduced degradation by a cellular
nuclease, and packaging and instructions.
[0270] In one aspect, the present invention provides kits for
protein production, comprising a first isolated nucleic acid
comprising a translatable region and at least two different
nucleoside modifications, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease, and packaging and
instructions.
[0271] In some embodiments, kits would provide split doses or
instructions for the administration of split dosages of the mmRNA
of the kit.
DEFINITIONS
[0272] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges. For example, the term "C.sub.1-6 alkyl" is
specifically intended to individually disclose methyl, ethyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0273] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans at any stage of development. In some embodiments,
"animal" refers to non-human animals at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[0274] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0275] Associated with: As used herein, the terms "associated
with," "conjugated," "linked," "attached," and "tethered," when
used with respect to two or more moieties, means that the moieties
are physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. An
"association" need not be strictly through direct covalent chemical
bonding. It may also suggest ionic or hydrogen bonding or a
hybridization based connectivity sufficiently stable such that the
"associated" entities remain physically associated.
[0276] Bifunctional: As used herein, the term "bifunctional" refers
to any substance, molecule or moiety which is capable of or
maintains at least two functions. The functions may effect the same
outcome or a different outcome. The structure that produces the
function may be the same or different. For example, bifunctional
modified RNAs of the present invention may encode a cytotoxic
peptide (a first function) while those nucleosides which comprise
the encoding RNA are, in and of themselves, cytotoxic (second
function). In this example, delivery of the bifunctional modified
RNA to a cancer cell would produce not only a peptide or protein
molecule which may ameliorate or treat the cancer but would also
deliver a cytotoxic payload of nucleosides to the cell should
degredation, instead of translation of the modified RNA, occur.
[0277] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
that has activity in a biological system and/or organism. For
instance, a substance that, when administered to an organism, has a
biological affect on that organism, is considered to be
biologically active. In particular embodiments, a nucleic acid
molecule of the present invention may be considered biologically
active if even a portion of the nucleic acid molecule is
biologically active or mimics an activity considered biologically
relevant.
[0278] Chemical terms: As used herein, the term "alkyl" is meant to
refer to a saturated hydrocarbon group which is straight-chained or
branched. Example alkyl groups include methyl (Me), ethyl (Et),
propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl,
isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl),
and the like. An alkyl group can contain from 1 to about 20, from 2
to about 20, from 1 to about 12, from 1 to about 8, from 1 to about
6, from 1 to about 4, or from 1 to about 3 carbon atoms.
[0279] As used herein, "alkenyl" refers to an alkyl group having
one or more double carbon-carbon bonds. Example alkenyl groups
include ethenyl, propenyl, and the like.
[0280] As used herein, "alkoxy" refers to an --O-alkyl group.
Example alkoxy groups include methoxy, ethoxy, propoxy (e.g.,
n-propoxy and isopropoxy), t-butoxy, and the like.
[0281] As used herein, "alkenyl" refers to an alkyl, as defined
above, containing at least one double bond between adjacent carbon
atoms. Alkenyls include both cis and trans isomers. Representative
straight chain and branched alkenyls include ethylenyl, propylenyl,
1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like.
[0282] As used herein, "alkynyl" refers to an alkyl group having
one or more triple carbon-carbon bonds. Example alkynyl groups
include ethynyl, propynyl, and the like.
[0283] As used herein, "aryl" refers to monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as,
for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl,
indenyl, and the like. In some embodiments, aryl groups have from 6
to about 20 carbon atoms.
[0284] As used herein, "halo" or "halogen" includes fluoro, chloro,
bromo, and iodo.
[0285] Compound: As used herein, the term "compound," is meant to
include all stereoisomers, geometric isomers, tautomers, and
isotopes of the structures depicted.
[0286] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present disclosure that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds of the present disclosure are described
and may be isolated as a mixture of isomers or as separated
isomeric forms.
[0287] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond and the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Examples prototropic tautomers include ketone-enol
pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic
acid pairs, enamine-imine pairs, and annular forms where a proton
can occupy two or more positions of a heterocyclic system, such as,
1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and
2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in
equilibrium or sterically locked into one form by appropriate
substitution.
[0288] Compounds of the present disclosure also include all of the
isotopes of the atoms occurring in the intermediate or final
compounds. "Isotopes" refers to atoms having the same atomic number
but different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium.
[0289] The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to form
solvates and hydrates by routine methods.
[0290] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[0291] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence may apply to the entire length of an oligonucleotide or
polypeptide or may apply to a portion, region or feature
thereof.
[0292] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound, substance, entity, moiety, cargo
or payload.
[0293] Delivery Agent: As used herein, "delivery agent" refers to
any substance which facilitates, at least in part, the in vivo
delivery of a nucleic acid molecule to targeted cells.
[0294] Detectable label: As used herein, "detectable label" refers
to one or more markers, signals, or moieties which are attached,
incorporated or associated with another entity that is readily
detected by methods known in the art including radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance and
the like. Detectable labels include radioisotopes, fluorophores,
chromophores, enzymes, dyes, metal ions, ligands such as biotin,
avidin, strepavidin and haptens, quantum dots, and the like.
Detectable labels may be located at any position in the peptides or
proteins disclosed herein. They may be within the amino acids, the
peptides, or proteins, or located at the N- or C-termini.
[0295] Distal: As used herein "distal" means farther from center
mass or line of symmetry of subject or reference point. For limbs,
it is farther from body.
[0296] Dosing regimen: As used herein, a "dosing regimen" is a
schedule of administration or physician determined regimen of
treatment, prophylaxis, or palliative care.
[0297] Dose splitting factor (DSF)--ratio of PUD of dose split
treatment divided by PUD of total daily dose or single unit dose.
The value is derived from comparison of dosing regimens groups.
[0298] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an RNA into a polypeptide or protein; and (4)
post-translational modification of a polypeptide or protein.
[0299] Formulation: As used herein, a "formulation" includes at
least a modified nucleic acid molecule and a delivery agent.
[0300] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0301] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or at least 99%
identical. In some embodiments, polymeric molecules are considered
to be "homologous" to one another if their sequences are at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 99% similar. The term "homologous" necessarily
refers to a comparison between at least two sequences
(polynucleotide or polypeptide sequences).
[0302] In accordance with the invention, two polynucleotide
sequences are considered to be homologous if the polypeptides they
encode are at least about 50% identical, at least about 60%
identical, at least about 70% identical, at least about 80%
identical, or at least about 90% identical for at least one stretch
of at least about 20 amino acids.
[0303] In some embodiments, homologous polynucleotide sequences are
characterized by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. For polynucleotide sequences less
than 60 nucleotides in length, homology is determined by the
ability to encode a stretch of at least 4-5 uniquely specified
amino acids. In accordance with the invention, two protein
sequences are considered to be homologous if the proteins are at
least about 50% identical, at least about 60% identical, at least
about 70% identical, at least about 80% identical, or at least
about 90% identical for at least one stretch of at least about 20
amino acids.
[0304] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g., between
oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two polynucleotide sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using methods such as those described
in Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleotide sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Atschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0305] Inhibit expression of a gene: As used herein, the phrase
"inhibit expression of a gene" means to cause a reduction in the
amount of an expression product of the gene. The expression product
can be an RNA transcribed from the gene (e.g., an mRNA) or a
polypeptide translated from an mRNA transcribed from the gene.
Typically a reduction in the level of an mRNA results in a
reduction in the level of a polypeptide translated therefrom. The
level of expression may be determined using standard techniques for
measuring mRNA or protein.
[0306] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[0307] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[0308] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been separated from at least some of
the components with which it was associated (whether in nature or
in an experimental setting). Isolated substances may have varying
levels of purity in reference to the substances from which they
have been associated. Isolated substances and/or entities may be
separated from at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or more of
the other components with which they were initially associated. In
some embodiments, isolated agents are more than about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, or more than about
99% pure. As used herein, a substance is "pure" if it is
substantially free of other components. Substantially isolated: By
"substantially isolated" is meant that the compound is
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in the compound of the present disclosure.
Substantial separation can include compositions containing at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
at least about 99% by weight of the compound of the present
disclosure, or salt thereof. Methods for isolating compounds and
their salts are routine in the art.
[0309] Modified: As used herein "modified" refers to a changed
state or structure of a molecule of the invention. Molecules may be
modified in many ways including chemically, structurally, and
functionally. In one embodiment, the mRNA molecules of the present
invention are modified by the introduction of non-natural
nucleosides and/or nucleotides. Modified, as it pertains to a
modified mRNA may also mean any alteration which is different from
the wild type.
[0310] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[0311] Patient: As used herein, "patient" refers to a subject who
may seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition.
[0312] Peptide: As used herein, "peptide" is less than or equal to
50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids long.
[0313] Prodrug: The present disclosure also includes prodrugs of
the compounds described herein. As used herein, "prodrugs" refer to
any substance, molecule or entity which is in a form predicate for
that substance, molecule or entity to act as a therapeutic upon
chemical or physical alteration. Prodrugs may by covalently bonded
or sequestested in some way and which release or are converted into
the active drug moiety prior to, upon or after administered to a
mammalian subject. Prodrugs can be prepared by modifying functional
groups present in the compounds in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compounds. Prodrugs include compounds wherein
hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any
group that, when administered to a mammalian subject, cleaves to
form a free hydroxyl, amino, sulfhydryl, or carboxyl group
respectively. Preparation and use of prodrugs is discussed in T.
Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol.
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, ed. Edward B. Roche, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
incorporated by reference in their entirety.
[0314] Proliferate: As used herein, the term "proliferate" means to
grow, expand or increase or cause to grow, expand or increase
rapidly. "Proliferative" means having the ability to proliferate.
"Anti-proliferative" means having properties counter to or
inapposite to proliferative properties.
[0315] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0316] Pharmaceutically acceptable salts: The present disclosure
also includes pharmaceutically acceptable salts of the compounds
described herein. As used herein, "pharmaceutically acceptable
salts" refers to derivatives of the disclosed compounds wherein the
parent compound is modified by converting an existing acid or base
moiety to its salt form. Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or organic acid
salts of basic residues such as amines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like. The
pharmaceutically acceptable salts of the present disclosure include
the conventional non-toxic salts of the parent compound formed, for
example, from non-toxic inorganic or organic acids. The
pharmaceutically acceptable salts of the present disclosure can be
synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, nonaqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17.sup.th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418
and Journal of Pharmaceutical Science, 66, 2 (1977), each of which
is incorporated herein by reference in its entirety.
[0317] Polypeptide: As used herein, "polypeptide" means a polymer
of amino acid residues linked together by peptide bonds. The term,
as used herein, refers to proteins, polypeptides, and peptides of
any size, structure, or function. Typically, however, a polypeptide
will be at least 50 amino acids long. In some instances the
polypeptide encoded is smaller than about 50 amino acids and the
polypeptide is termed a peptide. If the polypeptide is a peptide,
it will be at least about 5 amino acid residues long. Thus,
polypeptides include gene products, naturally occurring
polypeptides, synthetic polypeptides, homologs, orthologs,
paralogs, fragments and other equivalents, variants, and analogs of
the foregoing. A polypeptide may be a single molecule or may be a
multi-molecular complex such as a dimer, trimer or tetramer. The
term polypeptide may also apply to amino acid polymers in which one
or more amino acid residues are an artificial chemical analogue of
a corresponding naturally occurring amino acid.
[0318] Polypeptide per unit drug (PUD): As used herein, a PUD or
product per unit drug, is defined as a subdivided portion of total
daily dose, usually 1 mg, pg, kg, etc., of a product (such as a
polypeptide) as measured in body fluid or tissue, usually defined
in concentration such as pmol/mL, mmol/mL, etc divided by the
measure in the body fluid.
[0319] Proximal: As used herein, "proximal" means closer to center
mass or line of symmetry of subject or reference point. For limbs,
it is closer to body.
[0320] Sample: As used herein, the term "sample" refers to a subset
of its tissues, cells or component parts (e.g. body fluids,
including but not limited to peripheral blood, serum, plasma,
ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone
marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen,
breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid,
cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair,
tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid,
lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum,
vomit, vaginal secretions, mucosal secretion, stool water,
pancreatic juice, lavage fluids from sinus cavities,
bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical
cord blood). A sample further may include a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, organs. A sample further refers to a medium, such as
a nutrient broth or gel, which may contain cellular components,
such as proteins or nucleic acid molecule.
[0321] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[0322] Stable: As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0323] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition in accordance with
the invention may be administered, e.g., for experimental,
diagnostic, prophylactic, and/or therapeutic purposes. Typical
subjects include animals (e.g., mammals such as mice, rats,
rabbits, non-human primates, and humans) and/or plants.
[0324] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0325] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0326] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term means within 2 seconds.
[0327] Simultaneously: As used herein, "simultaneously" means
within scientific reproducibility, at same time.
[0328] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of a disease, disorder, and/or
condition.
[0329] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or may not exhibit symptoms of the disease, disorder, and/or
condition but harbors a propensity to develop a disease or its
symptoms. In some embodiments, an individual who is susceptible to
a disease, disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0330] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or polypeptides or other molecules of the present
invention may be chemical or enzymatic.
[0331] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administed in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[0332] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr period. It may be administered
as a single unit dose.
[0333] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0334] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells may be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism may be an animal, preferably a mammal, more preferably a
human and most preferably a patient.
[0335] Therapeutic Agent: The term "therapeutic agent" refers to
any agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect.
[0336] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to a
disease, disorder, and/or condition, to treat, improve symptoms of,
diagnose, prevent, and/or delay the onset of the disease, disorder,
and/or condition.
[0337] Transcription factor: As used herein, the term
"transcription factor" refers to a DNA-binding protein that
regulates transcription of DNA into RNA, for example, by activation
or repression of transcription. Some transcription factors effect
regulation of transcription alone, while others act in concert with
other proteins. Some transcription factor can both activate and
repress transcription under certain conditions. In general,
transcription factors bind a specific target sequence or sequences
highly similar to a specific consensus sequence in a regulatory
region of a target gene. Transcription factors may regulate
transcription of a target gene alone or in a complex with other
molecules.
[0338] Treating: As used herein, the term "treating" refers to
partially or completely alleviating, ameliorating, improving,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of a particular disease, disorder, and/or condition. For
example, "treating" cancer may refer to inhibiting survival,
growth, and/or spread of a tumor. Treatment may be administered to
a subject who does not exhibit signs of a disease, disorder, and/or
condition and/or to a subject who exhibits only early signs of a
disease, disorder, and/or condition for the purpose of decreasing
the risk of developing pathology associated with the disease,
disorder, and/or condition.
[0339] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in any way.
Unmodified may, but does not always, refer to the wild type or
native form of a biomolecule. Molecules may undergo a series of
modifications whereby each modified molecule may serve as the
"unmodified" starting molecule for a subsequent modification.
Equivalents and Scope
[0340] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0341] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context.
[0342] The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0343] It is also noted that the term "comprising" is intended to
be open and permits the inclusion of additional elements or
steps.
[0344] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0345] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0346] As used herein and in the claims, the singular forms include
the plural reference and vice versa unless the context clearly
indicates otherwise. Other than in the operating examples, or where
otherwise indicated, all numbers expressing quantities of
ingredients or reaction conditions used herein should be understood
as modified in all instances by the term "about."
[0347] All patents, oligonucleotide sequences identified by gene
identification numbers, and other publications identified herein
are expressly incorporated by reference for the purpose of
describing and disclosing, for example, the methodologies described
in such publications that might be used in connection with the
present invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0348] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
EXAMPLES
Example 1
Modified mRNA Production
[0349] Modified mRNAs (mmRNA) according to the invention may be
made using standard laboratory methods and materials. The open
reading frame (ORF) of the gene of interest may be flanked by a 5'
untranslated region (UTR) which may contain a strong Kozak
translational initiation signal and/or an alpha-globin 3' UTR which
may include an oligo(dT) sequence for templated addition of a
poly-A tail. The modified mRNAs may be modified to reduce the
cellular innate immune response. The modifications to reduce the
cellular response may include pseudouridine (.psi.) and
5-methyl-cytidine (5meC or m.sup.5C). (see, Kariko K et al.
Immunity 23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40
(2008), Anderson B R et al. NAR (2010); herein incorporated by
reference).
[0350] The ORF may also include various upstream or downstream
additions (such as, but not limited to, .beta.-globin, tags, etc.)
may be ordered from an optimization service such as, but limited
to, DNA2.0 (Menlo Park, Calif.) and may contain multiple cloning
sites which may have XbaI recognition. Upon receipt of the
construct, it may be reconstituted and transformed into chemically
competent E. coli.
[0351] For the present invention, NEB DH5-alpha Competent E. coli
are used. Transformations are performed according to NEB
instructions using 100 ng of plasmid. The protocol is as follows:
[0352] 1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice
for 10 minutes. [0353] 2. Add 1-5 .mu.l containing 1 pg-100 ng of
plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times
to mix cells and DNA. Do not vortex. [0354] 3. Place the mixture on
ice for 30 minutes. Do not mix. [0355] 4. Heat shock at 42.degree.
C. for exactly 30 seconds. Do not mix. [0356] 5. Place on ice for 5
minutes. Do not mix. [0357] 6. Pipette 950 .mu.l of room
temperature SOC into the mixture. [0358] 7. Place at 37.degree. C.
for 60 minutes. Shake vigorously (250 rpm) or rotate. [0359] 8.
Warm selection plates to 37.degree. C. [0360] 9. Mix the cells
thoroughly by flicking the tube and inverting. [0361] 10. Spread
50-100 .mu.l of each dilution onto a selection plate and incubate
overnight at 37.degree. C. Alternatively, incubate at 30.degree. C.
for 24-36 hours or 25.degree. C. for 48 hours.
[0362] A single colony is then used to inoculate 5 ml of LB growth
media using the appropriate antibiotic and then allowed to grow
(250 RPM, 37.degree. C.) for 5 hours. This is then used to
inoculate a 200 ml culture medium and allowed to grow overnight
under the same conditions.
[0363] To isolate the plasmid (up to 850 .mu.g), a maxi prep is
performed using the Invitrogen PURELINK.TM. HiPure Maxiprep Kit
(Carlsbad, Calif.), following the manufacturer's instructions.
[0364] In order to generate cDNA for In Vitro Transcription (IVT),
the plasmid (an Example of which is shown in FIG. 2) is first
linearized using a restriction enzyme such as XbaI. A typical
restriction digest with XbaI will comprise the following: Plasmid
1.0 .mu.g; 10.times. Buffer 1.0 IA; XbaI 1.5 .mu.l; dH.sub.2O up to
10 .mu.l; incubated at 37.degree. C. for 1 hr. If performing at lab
scale (<5 .mu.g), the reaction is cleaned up using Invitrogen's
PURELINK.TM. PCR Micro Kit (Carlsbad, Calif.) per manufacturer's
instructions. Larger scale purifications may need to be done with a
product that has a larger load capacity such as Invitrogen's
standard PURELINK.TM. PCR Kit (Carlsbad, Calif.). Following the
cleanup, the linearized vector is quantified using the NanoDrop and
analyzed to confirm linearization using agarose gel
electrophoresis.
[0365] As a non-limiting example, G-CSF may represent the
polypeptide of interest. Sequences used in the steps outlined in
Examples 1-5 are shown in Table 2. It should be noted that the
start codon (ATG) has been underlined in each sequence of Table
2.
TABLE-US-00002 TABLE 2 G-CSF Sequences SEQ ID NO Description 1
cDNAs equence:
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCT
GCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTG
CCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAG
ATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGGTGAGTGAGTGTGCCAC
CTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCA
TCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGC
TGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCC
CTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGA
CGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGG
CCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCC
AGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAG
GTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 2 cDNA having T7 polymerase
site and Xba restriction site:
TTGGACCCTCGTACAGAAGCTAATACGACTCACTATA
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCT
GCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTG
CCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAG
ATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGGTGAGTGAGTGTGCCAC
CTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCA
TCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGC
TGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCC
CTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGA
CGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGG
CCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCC
AGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAG
GTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGC
GGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGG
TCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3 Optimized
sequence; containing T7 polymerase site and Xba restriction site
TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAA
AAGAAGAGTAAGAAGAAATATAAGAGCCACC
ATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCG
ACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGG
AGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTG
CGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCT
TGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGG
CAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGC
AAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAG
CTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGG
GATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCG
CGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTT
TGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGT
CTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCA 4 mRNA sequence
(transcribed) CUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG
CCACCA AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUG
UCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAA
GCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGG
CACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUU
UGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUG
GACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGA
UGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCC
GGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGC
CACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCG
CAGCCGUGAGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUC
CCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGG
CCGCUCGAGCAUGCAU
Example 2
PCR for cDNA Production
[0366] PCR procedures for the preparation of cDNA are performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times.KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 uM) 0.75 .mu.l; Reverse Primer (10 uM)
0.75 .mu.l; Template cDNA 100 ng; and dH.sub.2O diluted to 25.0
.mu.l. The reaction conditions are at 95.degree. C. for 5 min. and
25 cycles of 98.degree. C. for 20 sec, then 58.degree. C. for 15
sec, then 72.degree. C. for 45 sec, then 72.degree. C. for 5 min.
then 4.degree. C. to termination.
[0367] The reverse primer of the instant invention incorporates a
poly-T.sub.120 for a poly-A.sub.120 in the mRNA. Other reverse
primers with longer or shorter poly(T) tracts can be used to adjust
the length of the poly(A) tail in the mRNA.
[0368] The reaction is cleaned up using Invitrogen's PURELINK.TM.
PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions
(up to 5 .mu.g). Larger reactions will require a cleanup using a
product with a larger capacity. Following the cleanup, the cDNA is
quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the cDNA is the expected size. The cDNA
is then submitted for sequencing analysis before proceeding to the
in vitro transcription reaction.
Example 3
In Vitro Transcription (IVT)
[0369] The in vitro transcription reaction generates mRNA
containing modified nucleotides or modified RNA. The input
nucleotide triphosphate (NTP) mix is made in-house using natural
and un-natural NTPs.
[0370] A typical in vitro transcription reaction includes the
following:
TABLE-US-00003 1. Template cDNA 1.0 .mu.g 2. 10x transcription
buffer (400 mM Tris-HCl 2.0 .mu.l pH 8.0, 190 mM MgCl.sub.2, 50 mM
DTT, 10 mM Spermidine) 3. Custom NTPs (25 mM each) 7.2 .mu.l 4.
RNase Inhibitor 20 U 5. T7 RNA polymerase 3000 U 6. dH.sub.20 Up to
20.0 .mu.l. and 7. Incubation at 37.degree. C. for 3 hr-5 hrs.
[0371] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase is then used to
digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA is purified using Ambion's MEGACLEAR.TM.
Kit (Austin, Tex.) following the manufacturer's instructions. This
kit can purify up to 500 .mu.g of RNA. Following the cleanup, the
RNA is quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred.
Example 4
Enzymatic Capping of mRNA
[0372] Capping of the mRNA is performed as follows where the
mixture includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.2O up to 72
.mu.l. The mixture is incubated at 65.degree. C. for 5 minutes to
denature RNA, and then is transferred immediately to ice.
[0373] The protocol then involves the mixing of 10.times. Capping
Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl.sub.2)
(10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl Methionine
(2.5 .mu.l); RNase Inhibitor (100 U); 2'-O-Methyltransferase (400
U); Vaccinia capping enzyme (Guanylyl transferase) (40 U);
dH.sub.2O (Up to 28 .mu.l); and incubation at 37.degree. C. for 30
minutes for 60 .mu.g RNA or up to 2 hours for 180 .mu.g of RNA.
[0374] The mRNA is then purified using Ambion's MEGACLEAR.TM. Kit
(Austin, Tex.) following the manufacturer's instructions. Following
the cleanup, the RNA is quantified using the NANODROP.TM.
(ThermoFisher, Waltham, Mass.) and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred. The RNA product may also be
sequenced by running a reverse-transcription-PCR to generate the
cDNA for sequencing.
Example 5
PolyA Tailing Reaction
[0375] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This is done by
mixing Capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2)(12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.2O up to 123.5 .mu.l and incubation at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction may be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 .mu.g). Poly-A Polymerase is preferably a recombinant
enzyme expressed in yeast.
[0376] For studies performed and described herein, the poly-A tail
is encoded in the IVT template to comprise 160 nucleotides in
length. However, it should be understood that the processivity or
integrity of the Poly-A tailing reaction may not always result in
exactly 160 nucleotides. Hence Poly-A tails of approximately 160
nucleotides, e.g, about 150-165, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164 or 165 are within the scope of the invention.
Example 6
Formulation of Modified mRNA Using Lipidoids
[0377] 5'-capping of modified RNA may be completed concomitantly
during the in vitro-transcription reaction using the following
chemical RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3''-O-Me-m7G(5')ppp(5') G [the
ARCA cap]; G(5)ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A;
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA may be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-.beta.-methyl. Cap 2 structure may be generated
from the Cap 1 structure followed by the 2'-.beta.-methylation of
the 5'-antepenultimate nucleotide using a 2'-O methyl-transferase.
Cap 3 structure may be generated from the Cap 2 structure followed
by the 2'-O-methylation of the 5'-preantepenultimate nucleotide
using a 2'-O methyl-transferase. Enzymes are preferably derived
from a recombinant source.
[0378] When transfected into mammalian cells, the modified mRNAs
have a stability of between 12-18 hours or more than 18 hours,
e.g., 24, 36, 48, 60, 72 or greater than 72 hours.
Example 7
Capping
[0379] A. Protein Expression Assay
[0380] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 1) containing the ARCA (3' O-Me-m7G(5')ppp(5')G) cap analog or
the Cap1 structure can be transfected into human primary
keratinocytes at equal concentrations. 6, 12, 24 and 36 hours
post-transfection the amount of G-CSF secreted into the culture
medium can be assayed by ELISA. Synthetic mRNAs that secrete higher
levels of G-CSF into the medium would correspond to a synthetic
mRNA with a higher translationally-competent Cap structure.
[0381] B. Purity Analysis Synthesis
[0382] mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 1)
containing the ARCA cap analog or the Cap1 structure crude
synthesis products can be compared for purity using denaturing
Agarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs
with a single, consolidated band by electrophoresis correspond to
the higher purity product compared to a synthetic mRNA with
multiple bands or streaking bands. Synthetic mRNAs with a single
HPLC peak would also correspond to a higher purity product. The
capping reaction with a higher efficiency would provide a more pure
mRNA population.
[0383] C. Cytokine Analysis
[0384] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 1) containing the ARCA cap analog or the Cap1 structure can be
transfected into human primary keratinocytes at multiple
concentrations. 6, 12, 24 and 36 hours post-transfection the amount
of pro-inflammatory cytokines such as TNF-alpha and IFN-beta
secreted into the culture medium can be assayed by ELISA. Synthetic
mRNAs that secrete higher levels of pro-inflammatory cytokines into
the medium would correspond to a synthetic mRNA containing an
immune-activating cap structure.
[0385] D. Capping Reaction Efficiency
[0386] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 1) containing the ARCA cap analog or the Cap1 structure can be
analyzed for capping reaction efficiency by LC-MS after capped mRNA
nuclease treatment. Nuclease treatment of capped mRNAs would yield
a mixture of free nucleotides and the capped 5'-5-triphosphate cap
structure detectable by LC-MS. The amount of capped product on the
LC-MS spectra can be expressed as a percent of total mRNA from the
reaction and would correspond to capping reaction efficiency. The
cap structure with a higher capping reaction efficiency would have
a higher amount of capped product by LC-MS.
Example 8
Formulation of Modified mRNA Using Lipidoids
[0387] Modified mRNAs (mmRNA) are formulated for in vitro
experiments by mixing the mmRNA with the lipidoid at a set ratio
prior to addition to cells. In vivo formulation may require the
addition of extra ingredients to facilitate circulation throughout
the body. To test the ability of these lipidoids to form particles
suitable for in vivo work, a standard formulation process used for
siRNA-lipidoid formulations was used as a starting point. Initial
mmRNA-lipidoid formulations may consist of particles composed of
42% lipidoid, 48% cholesterol and 10% PEG, with further
optimization of ratios possible. After formation of the particle,
mmRNA is added and allowed to integrate with the complex. The
encapsulation efficiency is determined using a standard dye
exclusion assays.
Materials and Methods for Examples 9-13
[0388] A. Lipid Synthesis
[0389] Six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,
C12-200 and DLin-MC3-DMA, were synthesized by methods outlined in
the art in order to be formulated with modified RNA. DLin-DMA and
precursorswere synthesized as described in Heyes et. al, J. Control
Release, 2005, 107, 276-287. DLin-K-DMA and DLin-KC2-DMA and
precursors were synthesized as described in Semple et. al, Nature
Biotechnology, 2010, 28, 172-176. 98N12-5 and precursor were
synthesized as described in Akinc et. al, Nature Biotechnology,
2008, 26, 561-569.
[0390] C12-200 and precursors were synthesized according to the
method outlined in Love et. al, PNAS, 2010, 107, 1864-1869.
2-epoxydodecane (5.10 g, 27.7 mmol, 8.2 eq) was added to a vial
containing Amine 200 (0.723 g, 3.36 mmol, 1 eq) and a stirring bar.
The vial was sealed and warmed to 80.degree. C. The reaction was
stirred for 4 days at 80.degree. C. Then the mixture was purified
by silica gel chromatography using a gradient from pure
dichloromethane (DCM) to DCM:MeOH 98:2. The target compound was
further purified by RP-HPLC to afford the desired compound.
[0391] DLin-MC3-DMA and precursors were synthesized according to
procedures described in WO 2010054401 herein incorporated by
reference in its entirety. A mixture of dilinoleyl methanol (1.5 g,
2.8 mmol, 1 eq), N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1
eq), DIPEA (0.73 mL, 4.2 mmol, 1.5 eq) and TBTU (1.35 g, 4.2 mmol,
1.5 eq) in 10 mL of DMF was stirred for 10 h at room temperature.
Then the reaction mixture was diluted in ether and washed with
water. The organic layer was dried over anhydrous sodium sulfate,
filtrated and concentrated under reduced pressure. The crude
product was purified by silica gel chromatography using a gradient
DCM to DCM:MeOH 98:2. Subsequently the target compound was
subjected to an additional RP-HPLC purification which was done
using a YMC-Pack C4 column to afford the target compound.
[0392] B. Formulation of Modified RNA Nanoparticles
[0393] Solutions of synthesized lipid,
1,2-distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids,
Alabaster, Ala.), cholesterol (Sigma-Aldrich, Taufkirchen,
Germany), and
.alpha.-[3'-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-w-methoxy-p-
olyoxyethylene (PEG-c-DOMG) (NOF, Bouwelven, Belgium) were prepared
at concentrations of 50 mM in ethanol and stored at -20.degree. C.
The lipids were combined to yield molar ratio of 50:10:38.5:1.5
(Lipid:DSPC:Cholesterol:PEG-c-DOMG) and diluted with ethanol to a
final lipid concentration of 25 mM. Solutions of modified mRNA at a
concentration of 1-2 mg/mL in water were diluted in 50 mM sodium
citrate buffer at a pH of 3 to form a stock modified mRNA solution.
Formulations of the lipid and modified mRNA were prepared by
combining the synthesized lipid solution with the modified mRNA
solution at total lipid to modified mRNA weight ratio of 10:1,
15:1, 20:1 and 30:1. The lipid ethanolic solution was rapidly
injected into aqueous modified mRNA solution to afford a suspension
containing 33% ethanol. The solutions were injected either manually
(MI) or by the aid of a syringe pump (SP) (Harvard Pump 33 Dual
Syringe Pump Harvard Apparatus Holliston, Mass.).
[0394] To remove the ethanol and to achieve the buffer exchange,
the formulations were dialyzed twice against phosphate buffered
saline (PBS), pH 7.4 at volumes 200-times of the primary product
using a Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc.
Rockford, Ill.) with a molecular weight cutoff (MWCO) of 10 kD. The
first dialysis was carried at room temperature for 3 hours and then
the formulations were dialyzed overnight at 4.degree. C. The
resulting nanoparticle suspension was filtered through 0.2 .mu.m
sterile filter (Sarstedt, Numbrecht, Germany) into glass vials and
sealed with a crimp closure.
[0395] C. Characterization of formulations
[0396] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern,
Worcestershire, UK) was used to determine the particle size, the
polydispersity index (PDI) and the zeta potential of the modified
mRNA nanoparticles in 1.times.PBS in determining particle size and
15 mM PBS in determining zeta potential.
[0397] Ultraviolet-visible spectroscopy was used to determine the
concentration of modified mRNA nanoparticle formulation. 100 .mu.L
of the diluted formulation in 1.times.PBS was added to 900 .mu.L of
a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the
absorbance spectrum of the solution was recorded between 230 nm and
330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman
Coulter, Inc., Brea, Calif.). The modified RNA concentration in the
nanoparicle formulation was calculated based on the extinction
coefficient of the modified RNA used in the formulation and on the
difference between the absorbance at a wavelength of 260 nm and the
baseline value at a wavelength of 330 nm.
[0398] QUANT-IT.TM. RIBOGREEN.RTM. RNA assay (Invitrogen
Corporation Carlsbad, Calif.) was used to evaluate the
encapsulation of modified RNA by the nanoparticle. The samples were
diluted to a concentration of approximately 5 .mu.g/mL in TE buffer
(10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 .mu.L of the diluted
samples were transferred to a polystyrene 96 well plate, then
either 50 .mu.L of TE buffer or 50 .mu.L of a 2% Triton X-100
solution was added. The plate was incubated at a temperature of
37.degree. C. for 15 minutes. The RIBOGREEN.RTM. reagent was
diluted 1:100 in TE buffer, 100 .mu.L of this solution was added to
each well. The fluorescence intensity was measured using a
fluorescence plate reader (Wallac Victor 1420 Multilablel Counter;
Perkin Elmer, Waltham, Mass.) at an excitation wavelength of
.about.480 nm and an emission wavelength of .about.520 nm. The
fluorescence values of the reagent blank were subtracted from that
of each of the samples and the percentage of free modified RNA was
determined by dividing the fluorescence intensity of the intact
sample (without addition of Triton X-100) by the fluorescence value
of the disrupted sample (caused by the addition of Triton
X-100).
[0399] D. In Vitro Incubation
[0400] Human embryonic kidney epithelial (HEK293) and
hepatocellular carcinoma epithelial (HepG2) cells (LGC standards
GmbH, Wesel, Germany) were seeded on 96-well plates (Greiner
Bio-one GmbH, Frickenhausen, Germany) and plates for HEK293 cells
were precoated with collagen type1. HEK293 were seeded at a density
of 30,000 and HepG2 were seeded at a density of 35,000 cells per
well in 100 .mu.l cell culture medium. For HEK293 the cell culture
medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mM
Sodiumpyruvate and 1.times. non-essential amino acids (Biochrom AG,
Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich,
Munich, Germany) and for HepG2 the culture medium was MEM (Gibco
Life Technologies, Darmstadt, Germany), 10% FCS adding 2 mM
L-Glutamine, 1 mM Sodiumpyruvate and 1.times. non-essential amino
acids (Biochrom AG, Berlin, Germany. Formulations containing
mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5; poly-A tail of
approximately 160 nucleotides not shown in sequence; 5' cap, Cap1)
were added in quadruplicates directly after seeding the cells and
incubated. The mCherry cDNA with the T7 promoter, 5' untranslated
region (UTR) and 3' UTR used in in vitro transcription (IVT) is
given in SEQ ID NO: 6.
[0401] Cells were harvested by transferring the culture media
supernatants to a 96-well Pro-Bind U-bottom plate (Beckton
Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with
1/2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with
respective supernatants and fixed by adding one volume PBS/2% FCS
(both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck,
Darmstadt, Germany). Samples then were submitted to a flow
cytometer measurement with a 532 nm excitation laser and the 610/20
filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson
GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI)
of all events and the standard deviation of four independent wells
are presented in for samples analyzed.
Example 9
Purification on Nanoparticle Formulations
[0402] Nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in
HEK293 and HepG2 were tested to determine if the mean fluorescent
intensity (MFI) was dependent on the lipid to modified RNA ratio
and/or purification. Three formulations of DLin-KC2-DMA and two
formulations of 98N12-5 were produced using a syringe pump to the
specifications described in Table 3. Purified samples were purified
by SEPHADEX.TM. G-25 DNA grade (GE Healthcare, Sweden). Each
formulation before and after purification (aP) were tested at
concentration of 250 ng modified RNA per well in a 24 well plate.
The percentage of cells that are positive for the marker for FL4
channel (% FL4-positive) when analyzed by the flow cytometer for
each formulation and the background sample are shown in FIGS. 3A
and 3B, and the MFI of the marker for the FL4 channel for each
formulation and the background sample are shown in FIGS. 4A and 4B.
The formulations which had been purified had a slightly higher MFI
than those formulations tested before purification.
TABLE-US-00004 TABLE 3 Formulations Formulation # Lipid Lipid/RNA
wt/wt Mean size (nm) NPA-001-1 DLin-KC2-DMA 10 155 nm PDI: 0.08
NPA-001-1 aP DLin-KC2-DMA 10 141 nm PDI: 0.14 NPA-002-1
DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-1 aP DLin-KC2-DMA 15 125
nm PDI: 0.12 NPA-003-1 DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-1
aP DLin-KC2-DMA 20 104 nm PDI: 0.06 NPA-005-1 98N12-5 15 127 nm
PDI: 0.12 NPA-005-1 aP 98N12-5 15 134 nm PDI: 0.17 NPA-006-1 98N12
20 126 nm PDI: 0.08 NPA-006-1 aP 98N12 20 118 nm PDI: 0.13
Example 10
Concentration Response Curve
[0403] Nanoparticle formulations of 98N12-5 (NPA-005) and
DLin-KC2-DMA (NPA-003) were tested at varying concentrations to
determine the MFI of FL4 or mCherry (mRNA sequence shown in SEQ ID
NO: 5; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) over a range of doses. The formulations
tested are outlined in Table 4. To determine the optimal
concentration of nanoparticle formulations of 98N12-5, varying
concentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng,
0.1 ng and 0.01 ng per well) were tested in a 24-well plate of
HEK293, and the results of the FL4 MFI of each dose are shown in
FIG. 5A. Likewise, to determine the optimal concentration of
nanoparticle formulations of DLin-KC2-DMA, varying concentrations
of formulated modified RNA (250 ng 100 ng, 10 ng, 1.0 ng, 0.1 ng
and 0.01 ng per well) were tested in a 24-well plate of HEK293, and
the results of the FL4 MFI of each dose are shown in FIG. 5B.
Nanoparticle formulations of DLin-KC2-DMA were also tested at
varying concentrations of formulated modified RNA (250 ng, 100 ng
and 30 ng per well) in a 24 well plate of HEK293, and the results
of the FL4 MFI of each dose are shown in FIG. 5C. A dose of 1
ng/well for 98N12-5 and a dose of 10 ng/well for DLin-K2-DMA were
found to resemble the FL4 MFI of the background.
[0404] To determine how close the concentrations resembled the
background, we utilized a flow cytometer with optimized filter sets
for detection of mCherry expression, and were able to obtain
results with increased sensitivity relative to background levels.
Doses of 25 ng/well, 0.25 ng/well, 0.025 ng/well and 0.0025 ng/well
were analyzed for 98N12-5 (NPA-005) and DLin-K2-DMA (NPA-003) to
determine the MFI of mCherry. As shown in Table 5, the
concentration of 0.025 ng/well and lesser concentrations are
similar to the background MFI level of mCherry which is about
386.125.
TABLE-US-00005 TABLE 4 Formulations Formulation # NPA-003 NPA-005
Lipid DLin-KC2- 98N12-5 DMA Lipid/RNA 20 15 wt/wt Mean size 114 nm
106 nm PDI: 0.08 PDI: 0.12
TABLE-US-00006 TABLE 5 Concentration and MFI MFI mCherry
Formulation NPA-003 NPA-005 25 ng/well 11963.25 12256.75 0.25
ng/well 1349.75 2572.75 0.025 ng/well 459.50 534.75 0.0025 ng/well
310.75 471.75
Example 11
Manual Injection and Syringe Pump Formulations
[0405] Two formulations of DLin-KC2-DMA and 98N12-5 were prepared
by manual injection (MI) and syringe pump injection (SP) and
analyzed along with a background sample to compare the MFI of
mCherry (mRNA shown in SEQ ID NO: 5; poly-A tail of approximately
160 nucleotides not shown in sequence; 5' cap, Cap1) of the
different formulations. Table 5 shows that the syringe pump
formulations had a higher MFI as compared to the manual injection
formulations of the same lipid and lipid/RNA ratio.
TABLE-US-00007 TABLE 5 Formulations and MFI Lipid/ Method of
Formulation RNA Mean size formu- # Lipid wt/wt (nm) lation MFI
Untreated N/A N/A N/A N/A 674.67 Control NPA-002 DLin-KC2- 15 140
nm MI 10318.25 DMA PDI: 0.11 NPA-002-2 DLin-KC2- 15 105 nm SP
37054.75 DMA PDI: 0.04 NPA-003 DLin-KC2- 20 114 nm MI 22037.5 DMA
PDI: 0.08 NPA-003-2 DLin-KC2- 20 95 nm SP 37868.75 DMA PDI: 0.02
NPA-005 98N12-5 15 127 nm MI 11504.75 PDI: 0.12 NPA-005-2 98N12-5
15 106 nm SP 9343.75 PDI: 0.07 NPA-006 98N12-5 20 126 nm MI
11182.25 PDI: 0.08 NPA-006-2 98N12-5 20 93 nm SP 5167 PDI: 0.08
Example 12
mCherry Fluorescence of Formulations
[0406] Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,
C12-200 and DLin-MC3-DMA were incubated at a concentration of 60
ng/well or 62.5 ng/well in a plate of HEK293 and 62.5 ng/well in a
plate of HepG2 cells for 24 hours to determine the MFI of mCherry
(mRNA shown in SEQ ID NO: 5; poly-A tail of approximately 160
nucleotides not shown in sequence; 5' cap, Cap1) for each
formulation. The formulations tested are outlined in Table 6 below.
As shown in FIG. 6A for the 60 ng/well and FIGS. 6B, 6C, 6D, and 6E
for the 62.5 ng/well, the formulation of NPA-003 and NPA-018 have
the highest mCherry MFI and the formulations of NPA-008, NPA-010
and NPA-013 are most the similar to the background sample mCherry
MFI value.
TABLE-US-00008 TABLE 6 Formulations Formulation # Lipid Lipid/RNA
wt/wt Mean size (nm) NPA-001 DLin-KC2-DMA 10 155 nm PDI: 0.08
NPA-002 DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-2 DLin-KC2-DMA 15
105 nm PDI: 0.04 NPA-003 DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-2
DLin-KC2-DMA 20 95 nm PDI: 0.02 NPA-005 98N12-5 15 127 nm PDI: 0.12
NPA-006 98N12-5 20 126 nm PDI: 0.08 NPA-007 DLin-DMA 15 148 nm PDI:
0.09 NPA-008 DLin-K-DMA 15 121 nm PDI: 0.08 NPA-009 C12-200 15 138
nm PDI: 0.15 NPA-010 DLin-MC3-DMA 15 126 nm PDI: 0.09 NPA-012
DLin-DMA 20 86 nm PDI: 0.08 NPA-013 DLin-K-DMA 20 104 nm PDI: 0.03
NPA-014 C12-200 20 101 nm PDI: 0.06 NPA-015 DLin-MC3-DMA 20 109 nm
PDI: 0.07
Example 13
In Vivo Formulation Studies
[0407] Mice (n=5) are administered intravenously a single dose of a
formulation containing a modified mRNA and a lipid. The modified
mRNA administered to the mice is selected from G-CSF (mRNA shown in
SEQ ID NO: 4; poly-A tail of approximately 160 nucleotides not
shown in sequence; 5' cap, Cap1), erythropoietin (EPO) (mRNA shown
in SEQ ID NO: 7; poly-A tail of approximately 160 nucleotides not
shown in sequence; 5' cap, Cap1), Factor IX (mRNA shown in SEQ ID
NO: 8; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) or mCherry (mRNA sequence shown in SEQ ID
NO: 5; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1). The erythropoietin cDNA with the T7
promoter, 5' untranslated region (UTR) and 3' UTR used in in vitro
transcription (IVT) is given in SEQ ID NO: 9.
[0408] Each formulation also contains a lipid which is selected
from one of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 or
DLin-MC3-DMA. The mice are injected with 100 ug, 10 ug or 1 ug of
the formulated modified mRNA and are sacrificed 8 hours after they
are administered the formulation. Serum from the mice administered
formulations containing human G-CSF modified mRNA are measured by
specific G-CSF ELISA and serum from mice administered human Factor
IX modified RNA is analyzed by specific Factor IX ELISA or
chromogenic assay. The liver and spleen from the mice administered
with mCherry modified mRNA are analyzed by immunohistochemistry
(IHC) or fluorescence-activated cell sorting (FACS). As a control,
a group of mice are not injected with any formulation and their
serum and tissue are collected analyzed by ELISA, FACS and/or
IHC.
Example 14
In Vitro and In Vivo Expression
[0409] A. A. In Vitro Expression in Human Cells Using Lipidoid
Formulations
[0410] The ratio of mmRNA to lipidoid used to test for in vitro
transfection is tested empirically at different lipidoid:mmRNA
ratios. Previous work using siRNA and lipidoids have utilized
2.5:1, 5:1, 10:1, and 15:1 lipidoid:siRNA wt:wt ratios. Given the
longer length of mmRNA relative to siRNA, a lower wt:wt ratio of
lipidoid to mmRNA may be effective. In addition, for comparison
mmRNA were also formulated using RNAIMAX.TM. (Invitrogen, Carlsbad,
Calif.) or TRANSIT-mRNA (Minis Bio, Madison, Wis.) cationic lipid
delivery vehicles. The ability of lipidoid-formulated Luciferase
(IVT cDNA sequence as shown in SEQ ID NO: 10), green fluorescent
protein (GFP) (IVT cDNA sequence as shown in SEQ ID NO: 11), G-CSF
(mRNA sequence shown in SEQ ID NO: 4; poly-A tail of approximately
160 nucleotides not shown in sequence; 5' cap, Cap1), and EPO mmRNA
(mRNA sequence shown in SEQ ID NO: 7; poly-A tail of approximately
160 nucleotides not shown in sequence; 5' cap, Cap1) to express the
desired protein product can be confirmed by luminescence for
luciferase expression, flow cytometry for GFP expression, and by
ELISA for G-CSF and Erythropoietin (EPO) secretion.
[0411] B. In Vivo Expression Following Intravenous Injection
[0412] Systemic intravenous administration of the formulations are
created using various different lipidoids including, but not
limited to, 98N12-5, C12-200, and MD1.
[0413] Lipidoid formulations containing mmRNA are injected
intravenously into animals. The expression of the modified mRNA
(mmRNA)-encoded proteins are assessed in blood and/or other organs
samples such as, but not limited to, the liver and spleen collected
from the animal. Conducting single dose intravenous studies will
also allow an assessment of the magnitude, dose responsiveness, and
longevity of expression of the desired product.
[0414] In one embodiment, lipidoid based formulations of 98N12-5,
C12-200, MD1 and other lipidoids, are used to deliver luciferase,
green fluorescent protein (GFP), mCherry fluorescent protein,
secreted alkaline phosphatase (sAP), human G-CSF, human Factor IX,
or human Erythropoietin (EPO) mmRNA into the animal. After
formulating mmRNA with a lipid, as described previously, animals
are divided into groups to receive either a saline formulation, or
a lipidoid-formulation which contains one of a different mmRNA
selected from luciferase, GFP, mCherry, sAP, human G-CSF, human
Factor IX, and human EPO. Prior to injection into the animal,
mmRNA-containing lipidoid formulations are diluted in PBS. Animals
are then administered a single dose of formulated mmRNA ranging
from a dose of 10 mg/kg to doses as low as 1 ng/kg, with a
preferred range to be 10 mg/kg to 100 ng/kg, where the dose of
mmRNA depends on the animal body weight such as a 20 gram mouse
receiving a maximum formulation of 0.2 ml (dosing is based no mmRNA
per kg body weight). After the administration of the mmRNA-lipidoid
formulation, serum, tissues, and/or tissue lysates are obtained and
the level of the mmRNA-encoded product is determined at a single
and/or a range of time intervals. The ability of
lipidoid-formulated Luciferase, GFP, mCherry, sAP, G-CSF, Factor
IX, and EPO mmRNA to express the desired protein product is
confirmed by luminescence for the expression of Luciferase, flow
cytometry for the expression of GFP and mCherry expression, by
enzymatic activity for sAP, or by ELISA for the section of G-CSF,
Factor IX and/or EPO.
[0415] Further studies for a multi-dose regimen are also performed
to determine the maximal expression of mmRNA, to evaluate the
saturability of the mmRNA-driven expression (by giving a control
and active mmRNA formulation in parallel or in sequence), and to
determine the feasibility of repeat drug administration (by giving
mmRNA in doses separated by weeks or months and then determining
whether expression level is affected by factors such as
immunogenicity). An assessment of the physiological function of
proteins such as G-CSF and EPO are also determined through
analyzing samples from the animal tested and detecting increases in
granulocyte and red blood cell counts, respectively. Activity of an
expressed protein product such as Factor IX, in animals can also be
assessed through analysis of Factor IX enzymatic activity (such as
an activated partial thromboplastin time assay) and effect of
clotting times.
[0416] C. In Vitro Expression Following Intramuscular and/or
Subcutaneous Injection
[0417] The use of lipidoid formulations to deliver
oligonucleotides, including mRNA, via an intramuscular route or a
subcutaneous route of injection needs to be evaluated as it has not
been previously reported. Intramuscular and/or subcutaneous
injection of mmRNA are evaluated to determine if mmRNA-containing
lipidoid formulations are capabable to produce both localized and
systemic expression of a desired proteins.
[0418] Lipidoid formulations of 98N12-5, C12-200, and MD1
containing mmRNA selected from luciferase, green fluorescent
protein (GFP), mCherry fluorescent protein, secreted alkaline
phosphatase (sAP), human G-CSF, human factor IX, or human
Erythropoietin (EPO) mmRNA are injected intramuscularly and/or
subcutaneously into animals. The expression of mmRNA-encoded
proteins are assessed both within the muscle or subcutaneous tissue
and systemically in blood and other organs such as the liver and
spleen. Single dose studies allow an assessment of the magnitude,
dose responsiveness, and longevity of expression of the desired
product.
[0419] Animals are divided into groups to receive either a saline
formulation or a formulation containing modified mRNA. Prior to
injection mmRNA-containing lipidoid formulations are diluted in
PBS. Animals are administered a single intramuscular dose of
formulated mmRNA ranging from 50 mg/kg to doses as low as 1 ng/kg
with a preferred range to be 10 mg/kg to 100 ng/kg. A maximum dose
for intramuscular administration, for a mouse, is roughly 1 mg
mmRNA or as low as 0.02 ng mmRNA for an intramuscular injection
into the hind limb of the mouse. For subcutaneous administration,
the animals are administered a single subcutaneous dose of
formulated mmRNA ranging from 400 mg/kg to doses as low as 1 ng/kg
with a preferred range to be 80 mg/kg to 100 ng/kg. A maximum dose
for subcutaneous administration, for a mouse, is roughly 8 mg mmRNA
or as low as 0.02 ng mmRNA.
[0420] For a 20 gram mouse the volume of a single intramuscular
injection is maximally 0.025 ml and a single subcutaneous injection
is maximally 0.2 ml. The optimal dose of mmRNA administered is
calculated from the body weight of the animal. At various points in
time points following the administration of the mmRNA-lipidoid,
serum, tissues, and tissue lysates is obtained and the level of the
mmRNA-encoded product is determined. The ability of
lipidoid-formulated luciferase, green fluorescent protein (GFP),
mCherry fluorescent protein, secreted alkaline phosphatase (sAP),
human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA
to express the desired protein product is confirmed by luminescence
for luciferase expression, flow cytometry for GFP and mCherry
expression, by enzymatic activity for sAP, and by ELISA for G-CSF,
Factor IX and Erythropoietin (EPO) secretion.
[0421] Additional studies for a multi-dose regimen are also
performed to determine the maximal expression using mmRNA, to
evaluate the saturability of the mmRNA-driven expression (achieved
by giving a control and active mmRNA formulation in parallel or in
sequence), and to determine the feasibility of repeat drug
administration (by giving mmRNA in doses separated by weeks or
months and then determining whether expression level is affected by
factors such as immunogenicity). Studies utilizing multiple
subcutaneous or intramuscular injection sites at one time point,
are also utilized to further increase mmRNA drug exposure and
improve protein production. An assessment of the physiological
function of proteins, such as GFP, mCherry, sAP, human G-CSF, human
factor IX, and human EPO, are determined through analyzing samples
from the tested animals and detecting a change in granulocyte
and/or red blood cell counts. Activity of an expressed protein
product such as Factor IX, in animals can also be assessed through
analysis of Factor IX enzymatic activity (such as an activated
partial thromboplastin time assay) and effect of clotting
times.
Example 15
Split Dose Studies
[0422] Studies utilizing multiple subcutaneous or intramuscular
injection sites at one time point were designed and performed to
investigate ways to increase mmRNA drug exposure and improve
protein production. In addition to detection of the expressed
protein product, an assessment of the physiological function of
proteins was also determined through analyzing samples from the
animal tested.
[0423] Surprisingly, it has been determined that split dosing of
mmRNA produces greater protein production and phenotypic responses
than those produced by single unit dosing or multi-dosing
schemes.
[0424] The design of a single unit dose, multi-dose and split dose
experiment involved using human erythropoietin (EPO) mmRNA (mRNA
sequence shown in SEQ ID NO: 7; poly-A tail of approximately 160
nucleotides not shown in sequence; 5' cap, Cap1) administered in
buffer alone. The dosing vehicle (F. buffer) consisted of 150 mM
NaCl, 2 mM CaCl.sub.2, 2 mM Na.sup.+-phosphate (1.4 mM monobasic
sodium phosphate; 0.6 mM dibasic sodium phosphate), and 0.5 mM
EDTA, pH 6.5. The pH was adjusted using sodium hydroxide and the
final solution was filter sterilized. The mmRNA was modified with
5meC at each cytosine and pseudouridine replacement at each uridine
site.
[0425] Animals (n=5) were injected IM (intramuscular) for the
single unit dose of 100 ug. For multi-dosing, two schedules were
used, 3 doses of 100 ug and 6 doses of 100 ug. For the split dosing
scheme, two schedules were used, 3 doses at 33.3 ug and 6 doses of
16.5 ug mmRNA. Control dosing involved use of buffer only at 6
doses. Control mmRNA involved the use of luciferase mmRNA (IVT cDNA
sequence shown in SEQ ID NO: 10) dosed 6 times at 100 ug. Blood and
muscle tissue were evaluated 13 hrs post injection.
[0426] Human EPO protein was measured in mouse serum 13 h post I.M.
single, multi- or split dosing of the EPO mmRNA in buffer. Seven
groups of mice (n=5 mice per group) were treated and evaluated. The
results are shown in Table 7.
TABLE-US-00009 TABLE 7 Split dose study Avg. Polypeptide pmol/mL
per unit Dose Dose of Total human drug Splitting Group Treatment
mmRNA Dose EPO (pmol/ug) Factor 1 Human EPO mmRNA 1 .times. 100 ug
100 ug 14.3 .14 1 2 Human EPO mmRNA 3 .times. 100 ug 300 ug 82.5
.28 2 3 Human EPO mmRNA 6 .times. 100 ug 600 ug 273.0 .46 3.3 4
Human EPO mmRNA 3 .times. 33.3 ug 100 ug 104.7 1.1 7.9 5 Human EPO
mmRNA 6 .times. 16.5 ug 100 ug 127.9 1.3 9.3 6 Luciferase mmRNA 6
.times. 100 ug 600 ug 0 -- -- 7 Buffer Alone -- -- 0 -- --
[0427] The splitting factor is defined as the product per unit drug
divided by the single dose product per unit drug (PUD). For example
for treatment group 2 the value 0.28 or product (EPO) per unit drug
(mmRNA) is divided by the single dose product per unit drug of
0.14. The result is 2. Likewise, for treatment group 4, the value
1.1 or product (EPO) per unit drug (mmRNA) is divided by the single
dose product per unit drug of 0.14. The result is 7.9.
Consequently, the dose splitting factor (DSF) may be used as an
indicator of the efficacy of a split dose regimen. For any single
administration of a total daily dose, the DSF should be equal to 1.
Therefore any DSF greater than this value in a split dose regimen
is an indication of increased efficacy.
[0428] To determine the dose response trends, impact of injection
site and impact of injection timing, studies are performed. In
these studies, varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and
values in between are used to determine dose response outcomes.
Split dosing for a 100 ug total dose includes three or six doses of
1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug, or values and total doses equal to
administration of the total dose selected.
[0429] Injection sites are chosen from the limbs or any body
surface presenting enough area suitable for injection. This may
also include a selection of injection depth to target the dermis
(Intradermal), epidermis (Epidermal), subcutaneous tissue (SC) or
muscle (IM). Injection angle will vary based on targeted delivery
site with injections targeting the intradermal site to be 10-15
degree angles from the plane of the surface of the skin, between
20-45 degrees from the plane of the surface of the skin for
subcutaneous injections and angles of between 60-90 degrees for
injections substantially into the muscle.
Example 16
Dose Response and Injection Site Selection and Timing
[0430] To determine the dose response trends, impact of injection
site and impact of injection timing, studies are performed
following the protocol outlined in Example 15. In these studies,
varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in
between are used to determine dose response outcomes. Split dosing
for a 100 ug total dose includes three or six doses of 1.6 ug, 4.2
ug, 8.3 ug, 16.6 ug, or values and total doses equal to
administration of the total dose selected.
[0431] Injection sites are chosen from the limbs or any body
surface presenting enough area suitable for injection. This may
also include a selection of injection depth to target the dermis
(Intradermal), epidermis (Epidermal), subcutaneous tissue (SC) or
muscle (IM). Injection angle will vary based on targeted delivery
site with injections targeting the intradermal site to be 10-15
degree angles from the plane of the surface of the skin, between
20-45 degrees from the plane of the surface of the skin for
subcutaneous injections and angles of between 60-90 degrees for
injections substantially into the muscle. RNAIMAX.TM.
Example 17
Routes of Administration
[0432] Further studies were performed to investigate dosing using
different routes of administration. Following the protocol outlined
in Example 15, 4 mice per group were dosed intramuscularly (I.M.),
intravenously (IV) or subcutaneously (S.C.) by the dosing chart
outlined in Table 8. Serum was collected 13 hours post injection
from all mice, tissue was collected from the site of injection from
the intramuscular and subcutaneous group and the spleen, liver and
kidneys were collected from the intravenous group. The results from
the intramuscular group are show in FIG. 7A and the subcutaneous
group results are shown in FIG. 7B.
TABLE-US-00010 TABLE 8 Dosing Chart Total Dosing Group Treatment
Route Dose of mmRNA Dose Vehicle 1 Lipoplex-human EPO mmRNA I.M. 4
.times. 100 ug + 30% 4 .times. 70 ul Lipoplex Lipoplex 2
Lipoplex-human EPO mmRNA I.M. 4 .times. 100 ug 4 .times. 70 ul
Buffer 3 Lipoplex-human EPO mmRNA S.C. 4 .times. 100 ug + 30% 4
.times. 70 ul Lipoplex Lipoplex 4 Lipoplex-human EPO mmRNA S.C. 4
.times. 100 ug 4 .times. 70 ul Buffer 5 Lipoplex-human EPO mmRNA
I.V. 200 ug + 30% 140 ul Lipoplex Lipoplex 6 Lipoplexed-Luciferase
mmRNA I.M. 100 ug + 30% 4 .times. 70 ul Lipoplex Lipoplex 7
Lipoplexed-Luciferase mmRNA I.M. 100 ug 4 .times. 70 ul Buffer 8
Lipoplexed-Luciferase mmRNA S.C. 100 ug + 30% 4 .times. 70 ul
Lipoplex Lipoplex 9 Lipoplexed-Luciferase mmRNA S.C. 100 ug 4
.times. 70 ul Buffer 10 Lipoplexed-human EPO mmRNA I.V. 200 ug +
30% 140 ul Lipoplex Lipoplex 11 Formulation Buffer I.M. 4x multi
dosing 4 .times. 70 ul Buffer
Example 18
In Vivo Delivery of Modified mRNA
[0433] Modified RNA was delivered to C57/BL6 mice intramuscularly,
subcutaneously, or intravenously to evaluate the bio-distribution
of modified RNA using luciferase. A formulation buffer used with
all delivery methods contained 150 mM sodium chloride, 2 mM calcium
chloride, 2 mM Na+-phosphate which included 1.4 mM monobasic sodium
phosphate and 0.6 mM of dibasic sodium phosphate, and 0.5 mM
ethylenediaminetetraacetic acid (EDTA) was adjusted using sodium
hydroxide to reach a final pH of 6.5 before being filtered and
sterilized. A 1.times. concentration was used as the delivery
buffer. To create the lipoplexed solution delivered to the mice, in
one vial 50 .mu.g of RNA was equilibrated for 10 minutes at room
temperature in the delivery buffer and in a second vial 10 .mu.l
RNAiMAX.TM. was equilibrated for 10 minutes at room temperature in
the delivery buffer. After equilibrium, the vials were combined and
delivery buffer was added to reach a final volume of 100 .mu.l
which was then incubated for 20 minutes at room temperature.
Luciferin was administered by intraperitoneal injection (IP) at 150
mg/kg to each mouse prior to imaging during the plateau phase of
the luciferin exposure curve which was between 15 and 30 minutes.
To create luciferin, 1 g of D-luciferin potassium or sodium salt
was dissolved in 66.6 ml of distilled phosphate buffer solution
(DPBS), not containing Mg2+ or Ca2+, to make a 15 mg/ml solution.
The solution was gently mixed and passed through a 0.2 .mu.m
syringe filter, before being purged with nitrogen, aliquoted and
frozen at -80.degree. C. while being protected from light as much
as possible. The solution was thawed using a waterbath if luciferin
was not dissolved, gently mixed and kept on ice on the day of
dosing.
[0434] Whole body images were taken of each mouse 2, 8 and 24 hours
after dosing. Tissue images and serum was collected from each mouse
24 hours after dosing. Mice administered doses intravenously had
their liver, spleen, kidneys, lungs, heart, peri-renal adipose
tissue and thymus imaged. Mice administered doses intramuscularly
or subcutaneously had their liver, spleen, kidneys, lungs,
peri-renal adipose tissue, and muscle at the injection site. From
the whole body images the bioluminescence was measured in photon
per second for each route of administration and dosing regimen.
[0435] A. Intramuscular Administration
[0436] Mice were intramuscularly (I.M.) administered either
modified luciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 10)
(Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),
lipoplexed modified granulocyte colony-stimulating factor (G-CSF)
mRNA (mRNA sequence shown in SEQ ID NO: 4; poly-A tail of
approximately 160 nucleotides not shown in sequence; 5' cap, Cap1)
(Lipoplex-Cytokine) or the formation buffer at a single dose of 50
.mu.g of modified RNA in an injection volume of 50 .mu.l for each
formulation in the right hind limb and a single dose of 5 .mu.g of
modified RNA in an injection volume of 50 .mu.l in the left hind
limb. The bioluminescence average for the luciferase expression
signals for each group at 2, 8 and 24 hours after dosing are shown
in FIG. 8A for the left hind limb and FIG. 8B for the right hind
limb. The bioluminescence showed a positive signal at the injection
site of the 5 .mu.g and 50 .mu.g modified RNA formulations
containing and not containing lipoplex.
[0437] B. Subcutaneous Administration
[0438] Mice were subcutaneously (S.C.) administered either modified
luciferase mRNA (Naked-Luc), lipoplexed modified luciferase mRNA
(Lipoplex-luc), lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or
the formation buffer at a single dose of 50 .mu.g of modified mRNA
in an injection volume of 100 .mu.l for each formulation. The
bioluminescence average for the luciferase expression signals for
each group at 2, 8 and 24 hours after dosing are shown in FIG. 8C.
The bioluminescence showed a positive signal at the injection site
of the 50 .mu.g modified mRNA formulations containing and not
containing lipoplex.
[0439] C. Intravenous Administration
[0440] Mice were intravenously (I.V.) administered either modified
luciferase mRNA (Naked-Luc), lipoplexed modified luciferase mRNA
(Lipoplex-luc), lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or
the formation buffer at a single dose of 50 .mu.g of modified mRNA
in an injection volume of 100 .mu.l for each formulation. The
bioluminescence average for the luciferase expression signal in the
spleen from each group at 2 hours after dosing is shown in FIG. 8D.
The bioluminescence showed a positive signal in the spleen of the
50 .mu.g modified mRNA formulations containing lipoplex.
Example 19
In Vivo Delivery Using Lipoplexes
[0441] A. Human EPO Modified RNA Lipoplex
[0442] A formulation containing 100 .mu.g of modified human
erythropoietin mRNA (mRNA sequence shown in SEQ ID NO: 7; poly-A
tail of approximately 160 nucleotides not shown in sequence; 5'
cap, Cap1) (EPO; fully modified 5-methylcytosine;
N1-methylpseudouridine) was lipoplexed with 30% by volume of
RNAIMAX.TM. (Lipoplex-h-Epo-46; Generation 2 or Gen2) in 50-70 uL
delivered intramuscularly to four C57/BL6 mice. Other groups
consisted of mice receiving an injection of the lipoplexed modified
luciferase mRNA (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID
NO: 10) which served as a control containing 100 .mu.g of modified
luciferase mRNA was lipoplexed with 30% by volume of RNAIMAX.TM. or
mice receiving an injection of the formulation buffer as negative
control at a dose volume of 65ul. 13 hours after the intramuscular
injection, serum was collected from each mouse to measure the
amount of human EPO protein in the mouse serum by human EPO ELISA
and the results are shown in FIG. 9.
[0443] B. Human G-CSF Modified RNA Lipoplex
[0444] A formulation containing 100 .mu.g of one of the two types
of modified human G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 4;
poly-A tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) (G-CSF fully modified with 5-methylcytosine and
pseudouridine (G-CSF) or G-CSF fully modified with 5-methylcytosine
and N1-methyl-pseudouridine (G-CSF-N1) lipoplexed with 30% by
volume of RNAIMAX.TM. and delivered in 150 uL intramuscularly
(I.M), in 150 uL subcutaneously (S.C) and in 225 uL intravenously
(I.V) to C57/BL6 mice. Three control groups were administered
either 100 .mu.g of modified luciferase mRNA (IVT cDNA sequence
shown in SEQ ID NO: 10) intramuscularly (Luc-unsp I.M.) or 150
.mu.g of modified luciferase mRNA intravenously (Luc-unsp I.V.) or
150 uL of the formulation buffer intramuscularly (Buffer I.M.). 6
hours after administration of a formulation, serum was collected
from each mouse to measure the amount of human G-CSF protein in the
mouse serum by human G-CSF ELISA and the results are shown in FIG.
10.
[0445] C. Human G-CSF Modified RNA Lipoplex Comparison
[0446] A formulation containing 100 .mu.g of either modified human
G-CSF mRNA lipoplexed with 30% by volume of RNAIMAX.TM. with a
5-methylcytosine (5mc) and a pseudouridine (.psi.) modification
(G-CSF-Gen1-Lipoplex), modified human G-CSF mRNA with a 5mc and
.psi. modification in saline (G-CSF-Gen1-Saline), modified human
G-CSF mRNA with a N1-5-methylcytosine (N1-5mc) and a .psi.
modification lipoplexed with 30% by volume of RNAIMAX.TM.
(G-CSF-Gen2-Lipoplex), modified human G-CSF mRNA with a N1-5mc and
.psi. modification in saline (G-CSF-Gen2-Saline), modified
luciferase with a 5mc and .psi. modification lipoplexed with 30% by
volume of RNAIMAX.TM. (Luc-Lipoplex), or modified luciferase mRNA
with a 5mc and .psi. modification in saline (Luc-Saline) was
delivered intramuscularly (I.M.) or subcutaneously (S.C.) and a
control group for each method of administration was giving a dose
of 80 uL of the formulation buffer (F. Buffer) to C57/BL6 mice. 13
hours post injection serum and tissue from the site of injection
were collected from each mouse and analyzed by G-CSF ELISA to
compare human G-CSF protein levels. The results of the human G-CSF
protein in mouse serum from the intramuscular administration are
shown in FIG. 11A, and the subcutaneous administration results are
shown in FIG. 11B.
[0447] D. mCherry Modified RNA Lipoplex Comparison
[0448] Intramuscular and Subcutaneous Administration
[0449] A formulation containing 100 .mu.g of either modified
mCherry mRNA (mRNA sequence shown in SEQ ID NO: 5; poly-A tail of
approximately 160 nucleotides not shown in sequence; 5' cap, Cap1)
lipoplexed with 30% by volume of RNAIMAX.TM. or modified mCherry
mRNA in saline is delivered intramuscularly and subcutaneously to
mice. A formulation buffer is also administered to a control group
of mice either intramuscularly or subcutaneously. The site of
injection on the mice may be collected 17 hours post injection for
sectioning to determine the cell type(s) responsible for producing
protein.
[0450] Intravitreal Administration
[0451] A formulation containing 10 .mu.g of either modified mCherry
mRNA lipoplexed with RNAIMAX.TM., modified mCherry mRNA in a
formulation buffer, modified luciferase mRNA lipoplexed with
RNAMAX.TM., modified luciferase mRNA in a formulation buffer can be
administered by intravitreal injection (IVT) in rats in a dose
volume of 5 .mu.l/eye. A formulation buffer is also administered by
IVT to a control group of rats in a dose volume of 5 .mu.l/eye.
Eyes from treated rats can be collected after 18 hours post
injection for sectioning and lysating to determine whether mmRNA
can be effectively delivered in vivo to the eye and result in
protein production, and to also determine the cell type(s)
responsible for producing protein in vivo.
[0452] Intranasal Administration
[0453] A formulation containing 100 .mu.g of either modified
mCherry mRNA lipoplexed with 30% by volume of RNAIMAX.TM., modified
mCherry mRNA in saline, modified luciferase mRNA lipoplexed with
30% by volume of RNAIMAX.TM. or modified luciferase mRNA in saline
is delivered intranasally. A formulation buffer is also
administered to a control group intranasally. Lungs may be
collected about 13 hours post instillation for sectioning (for
those receiving mCherry mRNA) or homogenization (for those
receiving luciferase mRNA). These samples will be used to determine
whether mRNA can be effectively delivered in vivo to the lungs and
result in protein production, and to also determine the cell
type(s) responsible for producing protein in vivo.
Example 20
In Vivo Delivery Using Varying Lipid Ratios
[0454] Modified mRNA was delivered to C57/BL6 mice to evaluate
varying lipid ratios and the resulting protein expression.
Formulations of 100 .mu.g modified human EPO mRNA (mRNA sequence
shown in SEQ ID NO: 7; poly-A tail of approximately 160 nucleotides
not shown in sequence; 5' cap, Cap1) lipoplexed with 10%, 30% or
50% RNAIMAX.TM., 100 .mu.g modified luciferase mRNA (IVT cDNA
sequence shown in SEQ ID NO: 10) lipoplexed with 10%, 30% or 50%
RNAIMAX.TM. or a formulation buffer were administered
intramuscularly to mice in a single 70 .mu.l dose. Serum was
collected 13 hours post injection to undergo a human EPO ELISA to
determine the human EPO protein level in each mouse. The results of
the human EPO ELISA, shown in FIG. 12, show that modified human EPO
expressed in the muscle is secreted into the serum for each of the
different percentage of RNAIMAX.TM..
Example 21
Intramuscular and Subcutaneous In Vivo Delivery in Mammals
[0455] Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO:
7; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) formulated in saline was delivered to
either C57/BL6 mice or Sprague-Dawley rats to evaluate the dose
dependency on human EPO production. Rats were intramuscularly
injected with 50 .mu.l of the modified human EPO mRNA (h-EPO),
modified luciferase mRNA (Luc) (IVT cDNA sequence shown in SEQ ID
NO: 10) or the formulation buffer (F.Buffer) as described in the
dosing chart Table 9.
[0456] Mice were intramuscularly or subcutaneously injected with 50
.mu.l of the modified human EPO mRNA (h-EPO), modified luciferase
mRNA (Luc) or the formulation buffer (F.Buffer) as described in the
dosing chart Table 10. 13 hours post injection blood was collected
and serum was analyzed to determine the amount human EPO for each
mouse or rat. The average and geometric mean in pg/ml for the rat
study are also shown in Table 9.
TABLE-US-00011 TABLE 9 Rat Study Geometric- Avg. mean Group Dose
R#1 R#2 R#3 R#4 R#5 R#6 pg/ml pg/ml h-EPO G#1 150 .mu.g 61.8 86.3
69.9 55.2 59 74.2 67.7 67.1 h-EPO G#2 100 .mu.g 69.4 77.8 48.2 17.6
101.9 161.5 79.4 66.9 h-EPO G#3 50 .mu.g 143.6 60.9 173.4 145.9
61.5 23.9 101.5 85.4 h-EPO G#4 10 .mu.g 7.8 11.8 30.9 36.2 40.6
150.3 46.3 31.2 h-EPO G#5 1 .mu.g 9.1 35.8 -- 46.2 18.1 34.1 28.7
25.4 Luc G#6 100 .mu.g 34.1 36.5 13.5 13.7 -- -- 24.5 22.4 F.Buffer
G#7 -- 14.7 18.5 21.2 20.3 -- -- 18.7 18.5
TABLE-US-00012 TABLE 10 Mouse Study Average Level in serum Route
Treatment Group Dose pg/ml IM h-EPO 1 100 .mu.g 96.2 IM h-EPO 2 50
.mu.g 63.5 IM h-EPO 3 25 .mu.g 18.7 IM h-EPO 4 10 .mu.g 25.9 IM
h-EPO 5 1 .mu.g 2.6 IM Luc 6 100 .mu.g 0 IM F.Buffer 7 -- 1.0 SC
h-EPO 1 100 .mu.g 72.0 SC Luc 2 100 .mu.g 26.7 SC F.Buffer 3 --
17.4
Example 22
Duration of Activity after Intramuscular In Vivo Delivery in
Rats
[0457] Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO:
7; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) formulated in saline was delivered to
Sprague-Dawley rats to determine the duration of the dose response.
Rats were intramuscularly injected with 50 .mu.l of the modified
human EPO mRNA (h-EPO), modified luciferase mRNA (IVT cDNA sequence
shown in SEQ ID NO: 10) (Luc) or the formulation buffer (F.Buffer)
as described in the dosing chart Table 11. The rats were bled 2, 6,
12, 24, 48 and 72 hours after the intramuscular injection to
determine the concentration of human EPO in serum at a given time.
The average and geometric mean in pg/ml for this study are also
shown in Table 11.
TABLE-US-00013 TABLE 11 Dosing Chart Avg. Geometric- Group Dose R#1
R#2 R#3 R#4 R#5 R#6 R#7 pg/ml mean pg/ml h-EPO 2 hour 100 .mu.g
60.0 62.4 53.6 33.2 68.6 66.4 72.8 59.6 58.2 h-EPO 6 hour 100 .mu.g
66.4 102.5 45.6 78.1 56.8 122.5 8.1 68.6 55.8 h-EPO 12 hour 100
.mu.g 132.9 55.1 89.0 80.1 85.6 105.6 63.3 87.4 84.5 h-EPO 24 hour
100 .mu.g 51.1 76.3 264.3 142.4 77.6 73.5 75.0 108.6 95.3 h-EPO 48
hour 100 .mu.g 96.3 59.0 85.7 82.6 63.5 80.3 -- 77.9 77.0 h-EPO 72
hour 100 .mu.g 46.3 66.9 73.5 57.3 136.7 110 69.7 80.1 75.8 Luc 24,
48 and 72 hour 100 .mu.g 60.2 38.5 48.8 46.1 3.6 26.1 -- 37.2 29.2
F.Buffer 24, 48 and 72 hour -- 50.0 10.0 80.9 54.7 -- -- -- 48.9
10.4
Example 23
In Vitro Transfection of VEGF-A
[0458] Human vascular endothelial growth factor-isoform A (VEGF-A)
modified mRNA (mRNA sequence shown in SEQ ID NO: 12; poly-A tail of
approximately 160 nucleotides not shown in sequence; 5' cap, Cap1)
was transfected via reverse transfection in Human Keratinocyte
cells in 24 multi-well plates. Human Keratinocytes cells were grown
in EPILIFE.RTM. medium with Supplement S7 from Invitrogen
(Carlsbad, Calif.) until they reached a confluence of 50-70%. The
cells were transfected with 0, 46.875, 93.75, 187.5, 375, 750, and
1500 ng of modified mRNA (mmRNA) encoding VEGF-A which had been
complexed with RNAIMAX.TM. from Invitrogen (Carlsbad, Calif.). The
RNA:RNAIMAX.TM. complex was formed by first incubating the RNA with
Supplement-free EPILIFE.RTM. media in a 5.times. volumetric
dilution for 10 minutes at room temperature. In a second vial,
RNAIMAX.TM. reagent was incubated with Supplement-free EPILIFE.RTM.
Media in a 10.times. volumetric dilution for 10 minutes at room
temperature. The RNA vial was then mixed with the RNAIMAX.TM. vial
and incubated for 20-30 minutes at room temperature before being
added to the cells in a drop-wise fashion.
[0459] The fully optimized mRNA encoding VEGF-A transfected with
the Human Keratinocyte cells included modifications during
translation such as natural nucleoside triphosphates (NTP),
pseudouridine at each uridine site and 5-methylcytosine at each
cytosine site (pseudo-U/5mC), and N1-methyl-pseudouridine at each
uridine site and 5-methylcytosine at each cytosine site
(N1-methyl-Pseudo-U/5mC). Cells were transfected with the mmRNA
encoding VEGF-A and secreted VEGF-A concentration (pg/ml) in the
culture medium was measured at 6, 12, 24, and 48 hours
post-transfection for each of the concentrations using an ELISA kit
from Invitrogen (Carlsbad, Calif.) following the manufacturers
recommended instructions. These data, shown in Table 12, show that
modified mRNA encoding VEGF-A is capable of being translated in
Human Keratinocyte cells and that VEGF-A is transported out of the
cells and released into the extracellular environment.
TABLE-US-00014 TABLE 12 VEGF-A Dosing and Protein Secretion 6 hours
12 hours 24 hours 48 hours Dose (ng) (pg/ml) (pg/ml) (pg/ml)
(pg/ml) VEGF-A Dose Containing Natural NTPs 46.875 10.37 18.07
33.90 67.02 93.75 9.79 20.54 41.95 65.75 187.5 14.07 24.56 45.25
64.39 375 19.16 37.53 53.61 88.28 750 21.51 38.90 51.44 61.79 1500
36.11 61.90 76.70 86.54 VEGF-A Dose Containing Pseudo-U/5mC 46.875
10.13 16.67 33.99 72.88 93.75 11.00 20.00 46.47 145.61 187.5 16.04
34.07 83.00 120.77 375 69.15 188.10 448.50 392.44 750 133.95 304.30
524.02 526.58 1500 198.96 345.65 426.97 505.41 VEGF-A Dose
Containing N1-methyl-Pseudo-U/5mC 46.875 0.03 6.02 27.65 100.42
93.75 12.37 46.38 121.23 167.56 187.5 104.55 365.71 1025.41 1056.91
375 605.89 1201.23 1653.63 1889.23 750 445.41 1036.45 1522.86
1954.81 1500 261.61 714.68 1053.12 1513.39
Example 24
In Vivo Studies of Factor IX
[0460] Human Factor IX mmRNA (mRNA shown in SEQ ID NO: 8; poly-A
tail of approximately 160 nucleotides not shown in sequence; 5'
cap, Cap1) (Gen1; fully modified 5-methycytosine and pseudouridine)
formulated in saline was delivered to mice via intramuscular
injection. The results demonstrate that Factor IX protein was
elevated in serum as measured 13 hours after administration.
[0461] In this study, mice (N=5 for Factor IX, N=3 for Luciferase
or Buffer controls) were intramuscularly injected with 50 .mu.l of
the Factor IX mmRNA (mRNA sequence shown in SEQ ID NO: 8; poly-A
tail of approximately 160 nucleotides not shown in sequence; 5'
cap, Cap1), Luciferase (cDNA sequence for IVT shown in SEQ ID NO:
10) or the formulation buffer (F.Buffer) at 2.times.100 ug/mouse.
The mice were bled at 13 hours after the intramuscular injection to
determine the concentration of human the polypeptide in serum in
pg/mL. The results revealed that administration of Factor IX mmRNA
resulted in levels of 1600 pg/mL at 13 hours as compared to less
than 100 pg/mL of Factor IX for either Luciferase or buffer control
administration.
Example 25
Multi-Site Administration: Intramuscular and Subcutaneous
[0462] Human G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 4;
poly-A tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) modified as either Gen1 or Gen2 (5-methylcytosine
(5mc) and a pseudouridine (.psi.) modification, G-CSF-Gen1; or
N1-5-methylcytosine (N1-5mc) and a .psi. modification, G-CSF-Gen2)
and formulated in saline were delivered to mice via intramuscular
(IM) or subcutaneous (SC) injection. Injection of four doses or
2.times.50 ug (two sites) daily for three days (24 hrs interval)
was performed. The fourth dose was administered 6 hrs before blood
collection and CBC analysis. Controls included Luciferase (cDNA
sequence for IVT shown in SEQ ID NO: 10) or the formulation buffer
(F.Buffer). The mice were bled at 72 hours after the first mmRNA
injection (6 hours after the last mmRNA dose) to determine the
effect of mmRNA-encoded human G-CSF on the neutrophil count. The
dosing regimen is shown in Table 13 as are the resulting neutrophil
counts (thousands/uL). Asterisks indicate statistical significance
at p<0.05.
[0463] For intramuscular administration, the data reveal a four
fold increase in neutrophil count above control at day 3 for the
Gen1 G-CSF mmRNA and a two fold increase for the Gen2 G-CSF mmRNA.
For subcutaneous administration, the data reveal a two fold
increase in neutrophil count above control at day 3 for the Gen2
G-CSF mmRNA.
TABLE-US-00015 TABLE 13 Dosing Regimen Dose Vol. Dosing Neutrophil
Gr. Treatment Route N = Dose (.mu.g/mouse) (.mu.l/mouse) Vehicle
Thous/uL 1 G-CSF (Gen1) I.M 5 2 .times. 50 ug (four doses) 50 F.
buffer 840* 2 G-CSF (Gen1) S.C 5 2 .times. 50 ug (four doses) 50 F.
buffer 430 3 G-CSF (Gen2) I.M 5 2 .times. 50 ug (four doses) 50 F.
buffer 746* 4 G-CSF (Gen2) S.C 5 2 .times. 50 ug (four doses) 50 F.
buffer 683 5 Luc (Gen1) I.M. 5 2 .times. 50 ug (four doses) 50 F.
buffer 201 6 Luc (Gen1) S.C. 5 2 .times. 50 ug (four doses) 50 F.
buffer 307 7 Luc (Gen2) I.M 5 2 .times. 50 ug (four doses) 50 F.
buffer 336 8 Luc (Gen2) S.C 5 2 .times. 50 ug (four doses) 50 F.
buffer 357 9 F. Buffer I.M 4 0 (four doses) 50 F. buffer 245 10 F.
Buffer S.C. 4 0 (four doses) 50 F. buffer 509 11 Untreated -- 4 --
312
Example 26
Intravenous Administration
[0464] Human G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 4;
poly-A tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) modified with 5-methylcytosine (5mc) and a
pseudouridine (.psi.) modification; or having no modifications and
formulated in 10% lipoplex (RNAIMAX.TM.) were delivered to mice at
a dose of 50 ug RNA and in a volume of 100 ul via intravenous (IV)
injection at days 0, 2 and 4. Neutrophils were measured at days 1,
5 and 8. Controls included non-specific mammalian RNA or the
formulation buffer alone (F.Buffer). The mice were bled at days 1,
5 and 8 to determine the effect of mmRNA-encoded human G-CSF to
increase neutrophil count. The dosing regimen is shown in Table 14
as are the resulting neutrophil counts (thousands/uL; K/uL).
[0465] For intravenous administration, the data reveal a four to
five fold increase in neutrophil count above control at day 5 with
G-CSF mmRNA but not with unmodified G-CSF mRNA or non-specific
controls. Blood count returned to baseline four days after the
final injection. No other changes in leukocyte populations were
observed.
[0466] An asterisk indicates statistical significance at p<0.001
compared to buffer.
TABLE-US-00016 TABLE 14 Dosing Regimen Dose Vol. Dosing Neutrophil
Gr. Treatment N = (.mu.l/mouse) Vehicle K/uL 1 G-CSF (Gen1) 5 100
10% lipoplex 2.91 Day 1 2 G-CSF (Gen1) 5 100 10% lipoplex 5.32* Day
5 3 G-CSF (Gen1) 5 100 10% lipoplex 2.06 Day 8 4 G-CSF (no 5 100
10% lipoplex 1.88 modification) Day 1 5 G-CSF (no 5 100 10%
lipoplex 1.95 modification) Day 5 6 G-CSF (no 5 100 10% lipoplex
2.09 modification) Day 8 7 RNA Control 5 100 10% lipoplex 2.90 Day
1 8 RNA Control 5 100 10% lipoplex 1.68 Day 5 9 RNA Control 4 100
10% lipoplex 1.72 Day 8 10 F. Buffer 4 100 10% lipoplex 2.51 Day 1
11 F. Buffer 4 100 10% lipoplex 1.31 Day 5 12 F. Buffer 4 100 10%
lipoplex 1.92 Day 8
Example 27
Saline Formulation: Intramuscular Administration
[0467] Human G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 4;
poly-A tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) and human EPO mmRNA (mRNA sequence shown in SEQ ID
NO: 7; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1); G-CSF mmRNA (modified with
5-methylcytosine (5mc) and .psi. pseudouridine (.psi.)) and EPO
mmRNA (modified with N1-5-methylcytosine (N1-5mc) and .psi.
modification), were formulated in saline and delivered to mice via
intramuscular (IM) injection at a dose of 100 ug.
[0468] Controls included Luciferase (IVT cDNA sequence shown in SEQ
ID NO: 10) or the formulation buffer (F.Buffer). The mice were bled
at 13 hours after the injection to determine the concentration of
the human polypeptide in serum in pg/mL (G-CSF groups measured
human G-CSF in mouse serum and EPO groups measured human EPO in
mouse serum). The data are shown in Table 15.
TABLE-US-00017 TABLE 15 Dosing Regimen Average Protein Dose Product
Vol. Dosing pg/mL, Group Treatment N = (.mu.l/mouse) Vehicle serum
G-CSF G-CSF 5 50 Saline 19.8 G-CSF Luciferase 5 50 Saline 0.5 G-CSF
F. buffer 5 50 F. buffer 0.5 EPO EPO 5 50 Saline 191.5 EPO
Luciferase 5 50 Saline 15.0 EPO F. buffer F. buffer 4.8
Example 28
EPO Multi-Dose/Multi-Administration
[0469] Studies utilizing multiple intramuscular injection sites at
one time point were designed and performed.
[0470] The design of a single multi-dose experiment involved using
human erythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO:
7; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) or G-CSF (mRNA sequence shown in SEQ ID NO:
4; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) administered in saline. The dosing vehicle
(F. buffer) was used as a control. The EPO and G-CSF mmRNA were
modified with 5-methylcytosine at each cytosine and pseudouridine
replacement at each uridine site.
[0471] Animals (n=5), Sprague-Dawley rats, were injected IM
(intramuscular) for the single unit dose of 100 ug (delivered to
one thigh). For multi-dosing 6 doses of 100 ug (delivered to two
thighs) were used for both EPO and G-CSF mmRNA. Control dosing
involved use of buffer at a single dose. Human EPO blood levels
were evaluated 13 hours post injection.
[0472] Human EPO protein was measured in rat serum 13 hours post
I.M. Five groups of rats were treated and evaluated. The results
are shown in Table 16.
TABLE-US-00018 TABLE 16 Multi-dose study Avg. Pg/mL human Dose of
Total EPO, Group Treatment mmRNA Dose serum 1 Human EPO mmRNA 1
.times. 100 ug 100 ug 143 2 Human EPO mmRNA 6 .times. 100 ug 600 ug
256 3 G-CSF mmRNA 1 .times. 100 ug 100 ug 43 4 G-CSF mmRNA 6
.times. 100 ug 600 ug 58 5 Buffer Alone -- -- 20
Example 29
Signal Sequence Exchange Study
[0473] Several variants of mmRNAs encoding human Granulocyte colony
stimulating factor (G-CSF) (mRNA sequence shown in SEQ ID NO: 4;
poly-A tail of approximately 160 nucleotides not shown in sequence;
5' cap, Cap1) were synthesized using modified nucleotides
pseudouridine and 5-methylcytosine (pseudo-U/5mC). These variants
included the G-CSF constructs encoding either the wild-type N
terminal secretory signal peptide sequence
(MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 13), no secretory
signal peptide sequence, or secretory signal peptide sequences
taken from other mRNAs. These included sequences where the wild
type GCSF signal peptide sequence was replaced with the signal
peptide sequence of either: human .alpha.-1-anti trypsin
(MMPSSVSWGILLLAGLCCLVPVSLA; SEQ ID NO: 14), human Factor IX
(MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR; SEQ ID NO: 15),
human Prolactin (MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 16), or human
Albumin (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 17).
[0474] ng of modified mRNA encoding each G-CSF variant was
transfected into HEK293A (293A in the table), mouse myoblast (MM in
the table) (C2C12, CRL-1772, ATCC) and rat myoblast (RM in the
table) (L6 line, CRL-1458, ATCC) cell lines in a 24 well plate
using 1 ul of Lipofectamine 2000 (Life Technologies), each well
containing 300,000 cells. The supernatants were harvested after 24
hrs and the secreted G-CSF protein was analyzed by ELISA using the
Human G-CSF ELISA kit (Life Technologies). The data shown in Table
17 reveal that cells transfected with G-CSF mmRNA encoding the
Albumin signal peptide secrete at least 12 fold more G-CSF protein
than its wild type counterpart.
TABLE-US-00019 TABLE 17 Signal Peptide Exchange 293A MM RM Signal
peptides (pg/ml) (pg/ml) (pg/ml) G-CSF Natural 9650 3450 6050
.alpha.-1-anti trypsin 9950 5000 8475 Factor IX 11675 6175 11675
Prolactin 7875 1525 9800 Albumin 122050 81050 173300 No Signal
peptide 0 0 0
Example 30
Cytokine Study: PBMC
[0475] PBMC Isolation and Culture:
[0476] 50 mL of human blood from two donors was received from
Research Blood Components (lots KP30928 and KP30931) in sodium
heparin tubes. For each donor, the blood was pooled and diluted to
70 mL with DPBS (SAFC Bioscience 59331C, lot 071M8408) and split
evenly between two 50 mL conical tubes. 10 mL of Ficoll Paque (GE
Healthcare 17-5442-03, lot 10074400) was gently dispensed below the
blood layer. The tubes were centrifuged at 2000 rpm for 30 minutes
with low acceleration and braking. The tubes were removed and the
buffy coat PMBC layers were gently transferred to a fresh 50 mL
conical and washed with DPBS. The tubes were centrifuged at 1450
rpm for 10 minutes.
[0477] The supernatant was aspirated and the PBMC pellets were
resuspended and washed in 50 mL of DPBS. The tubes were centrifuged
at 1250 rpm for 10 minutes. This wash step was repeated, and the
PBMC pellets were resuspended in 19 mL of Optimem I (Gibco 11058,
lot 1072088) and counted. The cell suspensions were adjusted to a
concentration of 3.0.times.10 6 cells/mL live cells.
[0478] These cells were then plated on five 96 well tissue culture
treated round bottom plates (Costar 3799) per donor at 50 uL per
well. Within 30 minutes, transfection mixtures were added to each
well at a volume of 50 uL per well. After 4 hours post
transfection, the media was supplemented with 10 uL of Fetal Bovine
Serum (Gibco 10082, lot 1012368)
[0479] Transfection Preparation:
[0480] mRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO:
4; poly-A tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1) (containing either (1) natural NTPs, (2)
100% substitution with 5-methyl cytidine and pseudouridine, or (3)
100% substitution with 5-methyl cytidine and N1-methyl
pseudouridine; mmRNA encoding luciferase (IVT cDNA sequence shown
in SEQ ID NO: 10) (containing either (1) natural NTPs or (2) 100%
substitution with 5-methyl cytidine and pseudouridine) and TLR
agonist R848 (Invivogen tlrl-r848) were diluted to 38.4 ng/uL in a
final volume of 2500 uL Optimem I.
[0481] Separately, 432 uL of Lipofectamine 2000 (Invitrogen
11668-027, lot 1070962) was diluted with 13.1 mL Optimem I. In a 96
well plate nine aliquots of 135 uL of each mmRNA, positive control
(R-848) or negative control (Optimem I) was added to 135 uL of the
diluted Lipofectamine 2000. The plate containing the material to be
transfected was incubated for 20 minutes. The transfection mixtures
were then transferred to each of the human PBMC plates at 50 uL per
well. The plates were then incubated at 37 C. At 2, 4, 8, 20, and
44 hours each plate was removed from the incubator, and the
supernatants were frozen.
[0482] After the last plate was removed, the supernatants were
assayed using a human G-CSF ELISA kit (Invitrogen KHC2032) and
human IFN-alpha ELISA kit (Thermo Scientific 41105-2). Each
condition was done in duplicate.
[0483] Results:
[0484] The ability of unmodified and modified mRNA (mmRNAs) to
produce the encoded protein was assessed (G-CSF production) over
time as was the ability of the mRNA to trigger innate immune
recognition as measured by interferon-alpha production. Use of in
vitro PBMC cultures is an accepted way to measure the
immunostimulatory potential of oligonucleotides (Robbins et al.,
Oligonucleotides 2009 19:89-102).
[0485] Results were interpolated against the standard curve of each
ELISA plate using a four parameter logistic curve fit. Shown in
Tables 18 and 19 are the average from 2 separate PBMC donors of the
G-CSF and IFN-alpha production over time as measured by specific
ELISA.
[0486] In the G-CSF ELISA, background signal from the Lipofectamine
2000 untreated condition was subtracted at each timepoint. The data
demonstrated specific production of human G-CSF protein by human
peripheral blood mononuclear is seen with G-CSF mRNA containing
natural NTPs, 100% substitution with 5-methyl cytidine and
pseudouridine, or 100% substitution with 5-methyl cytidine and
N1-methyl pseudouridine. Production of G-CSF was significantly
increased through the use of modified mRNA relative to unmodified
mRNA, with the 5-methyl cytidine and N1-methyl pseudouridine
containing G-CSF mRNA showing the highest level of G-CSF
production. With regards to innate immune recognition, unmodified
mRNA resulted in substantial IFN-alpha production, while the
modified mRNA largely prevented interferon-alpha production.
TABLE-US-00020 TABLE 18 G-CSF Signal G-CSF signal - 2 Donor Average
pg/mL 2 Hr 4 Hr 8 Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 120.3
136.8 421.0 346.1 431.8 G-CSF (5mC/N1-methyl 256.3 273.7 919.3
1603.3 1843.3 pseudouridine) GCSF (Natural-no modification) 63.5
92.6 129.6 258.3 242.4 Luciferase (5mC/pseudouridine) 4.5 153.7
33.0 186.5 58.0
TABLE-US-00021 TABLE 19 IFN-alpha signal IFN-alpha signal - 2 donor
average pg/mL 2 Hr 4 Hr 8 Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine)
21.1 2.9 3.7 22.7 4.3 G-CSF (5mC/N1-methyl 0.5 0.4 3.0 2.3 2.1
pseudouridine) G-CSF (Natural) 0.0 2.1 23.3 74.9 119.7 Luciferase
(5mC/pseudouridine) 0.4 0.4 4.7 1.0 2.4 R-848 39.1 151.3 278.4
362.2 208.1 Lipofectamine 2000 control 0.8 17.2 16.5 0.7 3.1
Example 31
Quantification in Exosomes
[0487] The quantity and localization of the mmRNA of the present
invention can be determined by measuring the amounts (initial,
timecourse, or residual basis) in isolated exosomes. In this study,
since the mmRNA are typically codon-optimized and distinct in
sequence from endogenous mRNA, the levels of mmRNA are quantitated
as compared to endogenous levels of native or wild type mRNA by
using the methods of Gibbings, PCT/IB2009/005878, the contents of
which are incorporated herein by reference in their entirety.
[0488] In these studies, the method is performed by first isolating
exosomes or vesicles preferably from a bodily fluid of a patient
previously treated with a polynucleotide, primary construct or
mmRNA of the invention, then measuring, in said exosomes, the
polynucleotide, primary construct or mmRNA levels by one of mRNA
microarray, qRT-PCR, or other means for measuring RNA in the art
including by suitable antibody or immunohistochemical methods.
Example 32
Bifunctional mmRNA
[0489] Using the teachings and synthesis methods described herein,
modified RNAs are designed and synthesized to be bifunctional,
thereby encoding one or more cytotoxic protein molecules as well as
be synthesized using cytotoxic nucleosides.
[0490] Administration of the bifunctional modified mRNAs is
effected using either saline or a lipid carrier. Once administered,
the bifunctional modified mRNA is translated to produce the encoded
cytotoxic peptide. Upon degradation of the delivered modified mRNA,
the cytotoxic nucleosides are released which also effect
therapeutic benefit to the subject.
Example 33
Synthesis of Modified mRNA
[0491] Modified mRNA is generated from a cDNA template containing a
T7 RNA-polymerase promoter sequence using a commercially available
T7 RNA polymerase transcription kit (MEGASCRIPT.RTM. High Yield
Transcription KIT, AMBION.RTM., Austin, Tex.; MSCRIPT.TM. mRNA
Production Kit, EPICENTRE.RTM. Biotechnologies, Madison, Wis.). An
in vitro transcription reaction contains between 1-2 .mu.g of
template DNA in the form of a linearized plasmid, PCR product, or
single-stranded oligonucleotide with a double-stranded polymerase
promoter region. The template DNA encodes a strong translation
initiation sequence such as a strong consensus Kozak sequence or an
optimized, high-expression IRES including the EMCV IRES. Reaction
volumes are between 20-40 .mu.l and contain
3'-O-Me-m.sup.7-G(5')ppp(5')G ARCA cap analog (NEW ENGLAND
BIOLABS.RTM.) in addition to an optimized ribonucleotide mixture of
determined modified adenine, guanine, cytidine and uridine
ribonucleotide analogs. Final reaction concentrations for
nucleotide are 6 mM for the cap analog and 1.5-7.5 mM for each of
the other nucleotides. The temperature and duration of the in vitro
transcription reaction are optimized for efficiency, fidelity and
yield. Reactions may be incubated from 3-6 hours and up to 16 hours
at 37.degree. C. Following the in vitro transcription reaction, the
capped mRNA undergoes polyadenylation using a commercially
available poly-A tailing kit (EPICENTRE.RTM. Biotechnologies,
Madison, Wis.). The resulting capped and polyadenylated synthetic
mRNA is then purified by denaturing agarose gel electrophoresis to
confirm production of full-length product and to remove any
degradation products followed by spin column filtration (RNeasy
Kit, Qiagen, Valencia, Calif.; MEGACLEAR.TM. AMBION.RTM., Austin,
Tex.). Purified synthetic mRNAs are resuspended in RNase-free water
containing an RNase inhibitor (RNASIN.RTM. Plus RNase Inhibitor,
Promega, Madison, Wis.), quantified by NANODROP.TM. (Thermo
Scientific, Logan, Utah) and stored at -20.degree. C.
Example 34
Bulk Transfection of Modified mRNA into Cell Culture
[0492] A. Cationic Lipid Delivery Vehicles
[0493] RNA transfections are carried out using RNAIMax (Invitrogen,
Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison, Wis.)
cationic lipid delivery vehicles. RNA and reagent are first diluted
in Opti-MEM basal media (Invitrogen, Carlsbad, Calif.). 100 ng/uL
RNA is diluted 5.times. and 5 .mu.L, of RNAIMax per ug of RNA is
diluted 10.times.. The diluted components are pooled and incubated
15 minutes at room temperature before they are dispensed to culture
media. For TRANSIT-mRNA transfections, 100 ng/uL RNA is diluted
10.times. in Opti-MEM and BOOST reagent is added (at a
concentration of 2 .mu.L, per .mu.g of RNA), TRANSIT-mRNA is added
(at a concentration of 2 .mu.L per .mu.g of RNA), and then the
RNA-lipid complexes are delivered to the culture media after a
2-minute incubation at room temperature. RNA transfections are
performed in Nutristem xenofree hES media (STEMGENT.RTM.,
Cambridge, Mass.) for RiPS derivations, Dermal Cell Basal Medium
plus Keratinocyte Growth Kit (ATCC) for keratinocyte experiments,
and Opti-MEM plus 2% FBS for all other experiments. Successful
introduction of a modified mRNA (mmRNA) into host cells can be
monitored using various known methods, such as a fluorescent
marker, such as Green Fluorescent Protein (GFP). Successful
transfection of a modified mRNA can also be determined by measuring
the protein expression level of the target polypeptide by e.g.,
Western Blotting or immunocytochemistry. Similar methods may be
followed for large volume scale-up to multi-liter (5-10,000 L)
culture format following similar RNA-lipid complex ratios.
[0494] B. Electroporation Delivery of Exogenous Synthetic mRNA
Transcripts
[0495] Electroporation parameters are optimized by transfecting
MRC-5 fibroblasts with in vitro synthetic modified mRNA (mmRNA)
transcripts and measuring transfection efficiency by quantitative
RT-PCR with primers designed to specifically detect the exogenous
transcripts. Discharging a 150 uF capacitor charged to F into
2.5.times.10.sup.6 cells suspended in 50 .mu.l of Opti-MEM
(Invitrogen, Carlsbad, Calif.) in a standard electroporation
cuvette with a 2 mm gap is sufficient for repeated delivery in
excess of 10,000 copies of modified mRNA transcripts per cell, as
determined using the standard curve method, while maintaining high
viability (>70%). Further experiments may reveal that the
voltage required to efficiently transfect cells with mmRNA
transcripts can depend on the cell density during electroporation.
Cell density may vary from 1.times.10.sup.6 cell/50 .mu.l to a
density of 2.5.times.10.sup.6 cells/50 .mu.l and require from 110V
to 145V to transfect cells with similar efficiencies measured in
transcript copies per cell. Large multi-liter (5-10,000 L)
electroporation may be performed similar to large volume flow
electroporation strategies similar to methods described with the
above described constraints (Li et al., 2002; Geng et al.,
2010).
Example 35
Overexpression of Ceramide Transfer Protein to Increase Therapeutic
Antibody Protein Production in Established CHO Cell Lines
[0496] A. Batch Culture
[0497] An antibody producing CHO cell line (CHO DG44) secreting a
humanized therapeutic IgG antibody is transfected a single time
with lipid cationic delivery agent alone (control) or a synthetic
mRNA transcript encoding wild type ceramide transfer protein (CERT)
or a non-phosphorylation competent Ser132A CERT mutant. The
sequences are taught in for example, U.S. Ser. No. 13/252,049, the
contents of which are incorporated herein by reference in their
entirety. CERT is an essential cytosolic protein in mammalian cells
that transfers the sphingolipid ceramide from the endoplasmic
reticulum to the Golgi complex where it is converted to
sphingomyelin (Hanada et al., 2003). Overexpression of CERT
significantly enhances the transport of secreted proteins to the
plasma membrane and improves the production of proteins that are
transported via the secretory pathway from eukaryotic cells thereby
enhancing secretion of proteins in the culture medium. Synthetic
mRNA transcripts are pre-mixed with a lipid cationic delivery agent
at a 2-5:1 carrier:RNA ratio. The initial seeding density is about
2.times.10.sup.5 viable cells/mL. The synthetic mRNA transcript is
delivered after initial culture seeding during the exponential
culture growth phase to achieve a final synthetic mRNA copy number
between 10.times.10.sup.2 and 10.times.10.sup.3 per cell. The basal
cell culture medium used for all phases of cell inoculum generation
and for growth of cultures in bioreactors was modified CD-CHO
medium containing glutamine, sodium bicarbonate, insulin and
methotrexate. The pH of the medium was adjusted to 7.0 with 1 N HCl
or 1N NaOH after addition of all components. Culture run times
ended on days 7, 14, 21 or 28+. Production-level 50 L scale
reactors (stainless steel reactor with two marine impellers) were
used and are scalable to >10,000 L stainless steel reactors
(described in commonly-assigned patent application U.S. Ser. No.
60/436,050, filed Dec. 23, 2002, and U.S. Ser. No. 10/740,645). A
data acquisition system (Intellution Fix 32, OSlsoft, LLC, San
Leandro, Calif.) recorded temperature, pH, and dissolved oxygen
(DO) throughout runs. Gas flows were controlled via rotameters. Air
was sparged into the reactor via a submerged frit (5 .mu.m pore
size) and through the reactor head space for CO.sub.2 removal.
Molecular oxygen was sparged through the same frit for DO control.
CO.sub.2 was sparged through same frit as used for pH control.
Samples of cells were removed from the reactor on a daily basis. A
sample used for cell counting was stained with trypan blue (Sigma,
St. Louis, Mo.). Cell count and cell viability determination were
performed via hemocytometry using a microscope. For analysis of
metabolites, additional samples were centrifuged for 20 minutes at
2000 rpm (4.degree. C.) for cell separation. Supernatant was
analyzed for the following parameters: titer, sialic acid, glucose,
lactate, glutamine, glutamate, pH, pO.sub.2, pCO.sub.2, ammonia,
and, optionally, lactate dehydrogenase (LDH). Additional back-up
samples were frozen at -20.degree. C. To measure secreted humanized
IgG antibody titers, supernatant is taken from seed-stock cultures
of all stable cell pools, the IgG titer is determined by ELISA and
divided by the mean number of cells to calculate the specific
productivity. The highest values are the cell pools with the
Ser132A CERT mutant, followed by wild type CERT. In both, IgG
expression is markedly enhanced compared to carrier-alone or
untransfected cells.
Continuous or Batch-Fed Culture
[0498] An antibody producing CHO cell line (CHO DG44) secreting
humanized IgG antibody is transfected with lipid cationic delivery
agent alone (control) or a synthetic mRNA transcript encoding wild
type ceramide transfer protein or a non-phosphorylation competent
Ser132A CERT mutant. Synthetic mRNA transcripts are pre-mixed with
a lipid cationic delivery agent at a 2-5:1 carrier:RNA ratio. The
initial seeding density was about 2.times.10.sup.5 viable cells/mL.
Synthetic mRNA transcript is delivered after initial culture
seeding during the exponential culture growth phase to achieve a
final synthetic mRNA copy number between 10.times.10.sup.2 and
10.times.10.sup.3 per cell. The basal cell culture medium used for
all phases of cell inoculum generation and for growth of cultures
in bioreactors was modified CD-CHO medium containing glutamine,
sodium bicarbonate, insulin and methotrexate. The pH of the medium
was adjusted to 7.0 with 1 N HCl or 1N NaOH after addition of all
components. Bioreactors of 5 L scale (glass reactor with one marine
impeller) were used to obtain maximum CERT protein production and
secreted humanized IgG antibody curves. For continuous or fed-batch
cultures, the culturing run time is increased by supplementing the
culture medium one or more times daily (or continuously) with fresh
medium during the run. In the a continuous and fed-batch feeding
regimens, the cultures receive feeding medium as a
continuously-supplied infusion, or other automated addition to the
culture, in a timed, regulated, and/or programmed fashion so as to
achieve and maintain the appropriate amount of synthetic
mRNA:carrier in the culture. The preferred method is a feeding
regimen of a once per day bolus feed with feeding medium containing
synthetic mRNA:carrier on each day of the culture run, from the
beginning of the culture run to the day of harvesting the cells.
The daily feed amount was recorded on batch sheets.
Production-level 50 L scale reactors (stainless steel reactor with
two marine impellers) were used and are scalable to >10,000 L
stainless steel reactors. A data acquisition system (Intellution
Fix 32) recorded temperature, pH, and dissolved oxygen (DO)
throughout runs. Gas flows were controlled via rotameters. Air was
sparged into the reactor via a submerged frit (5 .mu.m pore size)
and through the reactor head space for CO.sub.2 removal. Molecular
oxygen was sparged through the same frit for DO control. CO.sub.2
was sparged through same frit as used for pH control. Samples of
cells were removed from the reactor on a daily basis. A sample used
for cell counting was stained with trypan blue (Sigma, St. Louis,
Mo.). Cell count and cell viability determination were performed
via hemocytometry using a microscope. For analysis of metabolites,
additional samples were centrifuged for 20 minutes at 2000 rpm
(4.degree. C.) for cell separation. Supernatant was analyzed for
the following parameters: titer, sialic acid, glucose, lactate,
glutamine, glutamate, pH, pO.sub.2, pCO.sub.2, ammonia, and,
optionally, lactate dehydrogenase (LDH). Additional back-up samples
were frozen at -20.degree. C. To measure secreted humanized IgG
antibody titers, supernatant is taken from seed-stock cultures of
all stable cell pools, the IgG titer is determined by ELISA and
divided by the mean number of cells to calculate the specific
productivity. The highest values are the cell pools with the
Ser132A CERT mutant, followed by wild type CERT. In both, IgG
expression is markedly enhanced compared to carrier-alone or
untransfected cells.
Example 36
De Novo Generation of a Mammalian Cell Line Expressing Human
Erythropoietin as a Therapeutic Agent
[0499] A. Batch Culture
[0500] This Example describes the production of human
erythropoietin protein (EPO) from cultured primary CHO cells.
Erythropoietin is a glycoprotein hormone that is required for red
blood cell synthesis. EPO protein may be used as a therapeutic
agent for anemia from cancer, heart failure, chronic kidney disease
and myelodysplasia. Primary CHO cells are isolated and cultured as
described (Tjio and Puck, 1958). Primary CHO cells were then
expanded in modified CD-CHO medium containing glutamine, sodium
bicarbonate, insulin, and methotrexate (see Example 35) using T-75
flasks (Corning, Corning, N.Y.) and 250 and 500 mL spinners
(Bellco, Vineland, N.J.). T-flasks and spinners were incubated at
37.degree. C. in 6% CO.sub.2. After sufficient inoculum was
generated, the culture was transferred into a either a 5 L or a 50
L bioreactor as described above (see Example 35). Synthetic mRNA
transcript encoding the human erythropoietin protein are pre-mixed
with a lipid cationic delivery agent at a 2-5:1 carrier:RNA ratio
in a minimum of 1% total culture volume. The initial seeding
density is about 2.times.10.sup.5 viable cells/mL. The synthetic
mRNA transcript is delivered after initial culture seeding during
the exponential culture growth phase to achieve a final synthetic
mRNA copy number between 10.times.10.sup.2 and 10.times.10.sup.3
per cell. Culture growth and analysis were performed as described
above (see Example 34).
[0501] B. Continuous or Batch-Fed Culture
[0502] A primary CHO cell line derived and expanded as described
above (see Example 36a) is transfected with lipid cationic delivery
agent alone (control) or a synthetic mRNA transcript encoding human
erythropoietin protein. Synthetic mRNA transcripts are pre-mixed
with a lipid cationic delivery agent at a 2-5:1 carrier:RNA ratio.
The initial seeding density was about 2.times.10.sup.5 viable
cells/mL. Synthetic mRNA transcript is delivered after initial
culture seeding during the exponential culture growth phase to
achieve a final synthetic mRNA copy number between
10.times.10.sup.2 and 10.times.10.sup.3 per cell. Culture
conditions were as described above (Example 35a). For continuous or
fed-batch cultures, the culturing run time is increased by
supplementing the culture medium one or more times daily (or
continuously) with fresh medium during the run. In the a continuous
and fed-batch feeding regimens, the cultures receive feeding medium
as a continuously-supplied infusion, or other automated addition to
the culture, in a timed, regulated, and/or programmed fashion so as
to achieve and maintain the appropriate amount of synthetic
mRNA:carrier in the culture. The preferred method is a feeding
regimen of a once per day bolus feed with feeding medium containing
synthetic mRNA:carrier on each day of the culture run, from the
beginning of the culture run to the day of harvesting the cells.
The daily feed amount was recorded on batch sheets.
Production-level 50 L scale reactors (stainless steel reactor with
two marine impellers) were used and are scalable to >10,000 L
stainless steel reactors. Culture growth and analysis were
performed as described herein (see Example 35).
[0503] It is to be understood that the words which have been used
are words of description rather than limitation, and that changes
may be made within the purview of the appended claims without
departing from the true scope and spirit of the invention in its
broader aspects.
[0504] While the present invention has been described at some
length and with some particularity with respect to the several
described embodiments, it is not intended that it should be limited
to any such particulars or embodiments or any particular
embodiment, but it is to be construed with references to the
appended claims so as to provide the broadest possible
interpretation of such claims in view of the prior art and,
therefore, to effectively encompass the intended scope of the
invention.
[0505] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, section headings, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Sequence CWU 1
1
171624DNAHomo sapiens 1atggctggac ctgccaccca gagccccatg aagctgatgg
ccctgcagct gctgctgtgg 60cacagtgcac tctggacagt gcaggaagcc acccccctgg
gccctgccag ctccctgccc 120cagagcttcc tgctcaagtg cttagagcaa
gtgaggaaga tccagggcga tggcgcagcg 180ctccaggaga agctggtgag
tgagtgtgcc acctacaagc tgtgccaccc cgaggagctg 240gtgctgctcg
gacactctct gggcatcccc tgggctcccc tgagcagctg ccccagccag
300gccctgcagc tggcaggctg cttgagccaa ctccatagcg gccttttcct
ctaccagggg 360ctcctgcagg ccctggaagg gatctccccc gagttgggtc
ccaccttgga cacactgcag 420ctggacgtcg ccgactttgc caccaccatc
tggcagcaga tggaagaact gggaatggcc 480cctgccctgc agcccaccca
gggtgccatg ccggccttcg cctctgcttt ccagcgccgg 540gcaggagggg
tcctggttgc ctcccatctg cagagcttcc tggaggtgtc gtaccgcgtt
600ctacgccacc ttgcccagcc ctga 6242829DNAHomo sapiens 2ttggaccctc
gtacagaagc taatacgact cactataggg aaataagaga gaaaagaaga 60gtaagaagaa
atataagagc caccatggct ggacctgcca cccagagccc catgaagctg
120atggccctgc agctgctgct gtggcacagt gcactctgga cagtgcagga
agccaccccc 180ctgggccctg ccagctccct gccccagagc ttcctgctca
agtgcttaga gcaagtgagg 240aagatccagg gcgatggcgc agcgctccag
gagaagctgg tgagtgagtg tgccacctac 300aagctgtgcc accccgagga
gctggtgctg ctcggacact ctctgggcat cccctgggct 360cccctgagca
gctgccccag ccaggccctg cagctggcag gctgcttgag ccaactccat
420agcggccttt tcctctacca ggggctcctg caggccctgg aagggatctc
ccccgagttg 480ggtcccacct tggacacact gcagctggac gtcgccgact
ttgccaccac catctggcag 540cagatggaag aactgggaat ggcccctgcc
ctgcagccca cccagggtgc catgccggcc 600ttcgcctctg ctttccagcg
ccgggcagga ggggtcctgg ttgcctccca tctgcagagc 660ttcctggagg
tgtcgtaccg cgttctacgc caccttgccc agccctgaag cgctgccttc
720tgcggggctt gccttctggc catgcccttc ttctctccct tgcacctgta
cctcttggtc 780tttgaataaa gcctgagtag gaaggcggcc gctcgagcat gcatctaga
8293772DNAHomo sapiens 3ttggaccctc gtacagaagc taatacgact cactataggg
aaataagaga gaaaagaaga 60gtaagaagaa atataagagc caccatggcc ctgcagttgc
tgctttggca ctcggccctc 120tggacagtcc aagaagcgac tcctctcgga
cctgcctcat cgttgccgca gtcattcctt 180ttgaagtgtc tggagcaggt
gcgaaagatt cagggcgatg gagccgcact ccaagagaag 240ctctgcgcga
catacaaact ttgccatccc gaggagctcg tactgctcgg gcacagcttg
300gggattccct gggctcctct ctcgtcctgt ccgtcgcagg ctttgcagtt
ggcagggtgc 360ctttcccagc tccactccgg tttgttcttg tatcagggac
tgctgcaagc ccttgaggga 420atctcgccag aattgggccc gacgctggac
acgttgcagc tcgacgtggc ggatttcgca 480acaaccatct ggcagcagat
ggaggaactg gggatggcac ccgcgctgca gcccacgcag 540ggggcaatgc
cggcctttgc gtccgcgttt cagcgcaggg cgggtggagt cctcgtagcg
600agccaccttc aatcattttt ggaagtctcg taccgggtgc tgagacatct
tgcgcagccg 660tgagccttct gcggggcttg ccttctggcc atgcccttct
tctctccctt gcacctgtac 720ctcttggtct ttgaataaag cctgagtagg
aaggcggccg ctcgagcatg ca 7724746RNAHomo sapiens 4cucacuauag
ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga gccaccaaug 60gcccugcagu
ugcugcuuug gcacucggcc cucuggacag uccaagaagc gacuccucuc
120ggaccugccu caucguugcc gcagucauuc cuuuugaagu gucuggagca
ggugcgaaag 180auucagggcg auggagccgc acuccaagag aagcucugcg
cgacauacaa acuuugccau 240cccgaggagc ucguacugcu cgggcacagc
uuggggauuc ccugggcucc ucucucgucc 300uguccgucgc aggcuuugca
guuggcaggg ugccuuuccc agcuccacuc cgguuuguuc 360uuguaucagg
gacugcugca agcccuugag ggaaucucgc cagaauuggg cccgacgcug
420gacacguugc agcucgacgu ggcggauuuc gcaacaacca ucuggcagca
gauggaggaa 480cuggggaugg cacccgcgcu gcagcccacg cagggggcaa
ugccggccuu ugcguccgcg 540uuucagcgca gggcgggugg aguccucgua
gcgagccacc uucaaucauu uuuggaaguc 600ucguaccggg ugcugagaca
ucuugcgcag ccgugagccu ucugcggggc uugccuucug 660gccaugcccu
ucuucucucc cuugcaccug uaccucuugg ucuuugaaua aagccugagu
720aggaaggcgg ccgcucgagc augcau 7465854RNAHomo sapiens 5gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug guauccaagg 60gggaggagga
caacauggcg aucaucaagg aguucaugcg auucaaggug cacauggaag
120guucggucaa cggacacgaa uuugaaaucg aaggagaggg ugaaggaagg
cccuaugaag 180ggacacagac cgcgaaacuc aaggucacga aagggggacc
acuuccuuuc gccugggaca 240uucuuucgcc ccaguuuaug uacgggucca
aagcauaugu gaagcauccc gccgauauuc 300cugacuaucu gaaacucagc
uuucccgagg gauucaagug ggagcggguc augaacuuug 360aggacggggg
uguagucacc guaacccaag acucaagccu ccaagacggc gaguucaucu
420acaaggucaa acugcggggg acuaacuuuc cgucggaugg gccggugaug
cagaagaaaa 480cgaugggaug ggaagcguca ucggagagga uguacccaga
agauggugca uugaaggggg 540agaucaagca gagacugaag uugaaagaug
ggggacauua ugaugccgag gugaaaacga 600cauacaaagc gaaaaagccg
gugcagcuuc ccggagcgua uaaugugaau aucaaguugg 660auauuacuuc
acacaaugag gacuacacaa uugucgaaca guacgaacgc gcugagggua
720gacacucgac gggaggcaug gacgaguugu acaaaugaua agcugccuuc
ugcggggcuu 780gccuucuggc caugcccuuc uucucucccu ugcaccugua
ccucuugguc uuugaauaaa 840gccugaguag gaag 8546924DNAHomo sapiens
6tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga
60aaagaagagt aagaagaaat ataagagcca ccatggtatc caagggggag gaggacaaca
120tggcgatcat caaggagttc atgcgattca aggtgcacat ggaaggttcg
gtcaacggac 180acgaatttga aatcgaagga gagggtgaag gaaggcccta
tgaagggaca cagaccgcga 240aactcaaggt cacgaaaggg ggaccacttc
ctttcgcctg ggacattctt tcgccccagt 300ttatgtacgg gtccaaagca
tatgtgaagc atcccgccga tattcctgac tatctgaaac 360tcagctttcc
cgagggattc aagtgggagc gggtcatgaa ctttgaggac gggggtgtag
420tcaccgtaac ccaagactca agcctccaag acggcgagtt catctacaag
gtcaaactgc 480gggggactaa ctttccgtcg gatgggccgg tgatgcagaa
gaaaacgatg ggatgggaag 540cgtcatcgga gaggatgtac ccagaagatg
gtgcattgaa gggggagatc aagcagagac 600tgaagttgaa agatggggga
cattatgatg ccgaggtgaa aacgacatac aaagcgaaaa 660agccggtgca
gcttcccgga gcgtataatg tgaatatcaa gttggatatt acttcacaca
720atgaggacta cacaattgtc gaacagtacg aacgcgctga gggtagacac
tcgacgggag 780gcatggacga gttgtacaaa tgataagctg ccttctgcgg
ggcttgcctt ctggccatgc 840ccttcttctc tcccttgcac ctgtacctct
tggtctttga ataaagcctg agtaggaagg 900cggccgctcg agcatgcatc taga
9247725RNAHomo sapiens 7gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug ggagugcacg 60agugucccgc gugguugugg uugcugcugu cgcucuugag
ccucccacug ggacugccug 120ugcugggggc accacccaga uugaucugcg
acucacgggu acuugagagg uaccuucuug 180aagccaaaga agccgaaaac
aucacaaccg gaugcgccga gcacugcucc cucaaugaga 240acauuacugu
accggauaca aaggucaauu ucuaugcaug gaagagaaug gaaguaggac
300agcaggccgu cgaagugugg caggggcucg cgcuuuuguc ggaggcggug
uugcgggguc 360aggcccuccu cgucaacuca ucacagccgu gggagccccu
ccaacuucau gucgauaaag 420cggugucggg gcuccgcagc uugacgacgu
ugcuucgggc ucugggcgca caaaaggagg 480cuauuucgcc gccugacgcg
gccuccgcgg caccccuccg aacgaucacc gcggacacgu 540uuaggaagcu
uuuuagagug uacagcaauu uccuccgcgg aaagcugaaa uuguauacug
600gugaagcgug uaggacaggg gaucgcugau aagcugccuu cugcggggcu
ugccuucugg 660ccaugcccuu cuucucuccc uugcaccugu accucuuggu
cuuugaauaa agccugagua 720ggaag 72581536RNAHomo sapiens 8gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaau gcagcgcguc 60aacaugauua
uggccgaauc gccgggacuc aucacaaucu gccucuuggg uuaucucuug
120ucggcagaau guaccguguu cuuggaucac gaaaacgcga acaaaauucu
uaaucgcccg 180aagcgguaua acuccgggaa acuugaggag uuugugcagg
gcaaucuuga acgagagugc 240auggaggaga aaugcuccuu ugaggaggcg
agggaagugu uugaaaacac agagcgaaca 300acggaguuuu ggaagcaaua
cguagauggg gaccagugug agucgaaucc gugccucaau 360gggggaucau
guaaagauga caucaauagc uaugaaugcu ggugcccguu uggguuugaa
420gggaagaacu gugagcugga ugugacgugc aacaucaaaa acggacgcug
ugagcaguuu 480uguaagaacu cggcugacaa uaagguagua ugcucgugca
cagagggaua ccggcuggcg 540gagaaccaaa aaucgugcga gcccgcaguc
ccguucccuu gugggagggu gagcguguca 600cagacuagca aguugacgag
agcggagacu guauuccccg acguggacua cgucaacagc 660accgaagccg
aaacaauccu cgauaacauc acgcagagca cucaguccuu caaugacuuu
720acgagggucg uaggugguga ggacgcgaaa cccggucagu uccccuggca
ggugguauug 780aacggaaaag ucgaugccuu uuguggaggu uccauuguca
acgagaagug gauugucaca 840gcggcacacu gcguagaaac aggagugaaa
aucacgguag uggcgggaga gcauaacauu 900gaagagacag agcacacgga
acaaaagcga aaugucauca gaaucauucc acaccauaac 960uauaacgcgg
caaucaauaa guacaaucac gacaucgcac uuuuggagcu ugacgaaccu
1020uuggugcuua auucguacgu caccccuauu uguauugccg acaaagagua
uacaaacauc 1080uucuugaaau ucggcuccgg guacguaucg ggcuggggca
gaguguucca uaaggguaga 1140uccgcacugg uguugcaaua ccucagggug
ccccucgugg aucgagccac uugucugcgg 1200uccaccaaau ucacaaucua
caacaauaug uucugugcgg gauuccauga aggugggaga 1260gauagcugcc
agggagacuc aggggguccc cacgugacgg aagucgaggg gacgucauuu
1320cugacgggaa uuaucucaug gggagaggaa ugugcgauga aggggaaaua
uggcaucuac 1380acuaaagugu cacgguaugu caauuggauc aaggaaaaga
cgaaacucac gugaucagcc 1440agcgcugccu ucugcggggc uugccuucug
gccaugcccu ucuucucucc cuugcaccug 1500uaccucuugg ucuuugaaua
aagccugagu aggaag 15369794DNAHomo sapiens 9caagcttttg gaccctcgta
cagaagctaa tacgactcac tatagggaaa taagagagaa 60aagaagagta agaagaaata
taagagccac catgggagtg cacgagtgtc ccgcgtggtt 120gtggttgctg
ctgtcgctct tgagcctccc actgggactg cctgtgctgg gggcaccacc
180cagattgatc tgcgactcac gggtacttga gaggtacctt cttgaagcca
aagaagccga 240aaacatcaca accggatgcg ccgagcactg ctccctcaat
gagaacatta ctgtaccgga 300tacaaaggtc aatttctatg catggaagag
aatggaagta ggacagcagg ccgtcgaagt 360gtggcagggg ctcgcgcttt
tgtcggaggc ggtgttgcgg ggtcaggccc tcctcgtcaa 420ctcatcacag
ccgtgggagc ccctccaact tcatgtcgat aaagcggtgt cggggctccg
480cagcttgacg acgttgcttc gggctctggg cgcacaaaag gaggctattt
cgccgcctga 540cgcggcctcc gcggcacccc tccgaacgat caccgcggac
acgtttagga agctttttag 600agtgtacagc aatttcctcc gcggaaagct
gaaattgtat actggtgaag cgtgtaggac 660aggggatcgc tgataagctg
ccttctgcgg ggcttgcctt ctggccatgc ccttcttctc 720tcccttgcac
ctgtacctct tggtctttga ataaagcctg agtaggaagg cggccgctcg
780agcatgcatc taga 794101869DNAHomo sapiens 10agcttttgga ccctcgtaca
gaagctaata cgactcacta tagggaaata agagagaaaa 60gaagagtaag aagaaatata
agagccacca tggaagatgc gaagaacatc aagaagggac 120ctgccccgtt
ttaccctttg gaggacggta cagcaggaga acagctccac aaggcgatga
180aacgctacgc cctggtcccc ggaacgattg cgtttaccga tgcacatatt
gaggtagaca 240tcacatacgc agaatacttc gaaatgtcgg tgaggctggc
ggaagcgatg aagagatatg 300gtcttaacac taatcaccgc atcgtggtgt
gttcggagaa ctcattgcag tttttcatgc 360cggtccttgg agcacttttc
atcggggtcg cagtcgcgcc agcgaacgac atctacaatg 420agcgggaact
cttgaatagc atgggaatct cccagccgac ggtcgtgttt gtctccaaaa
480aggggctgca gaaaatcctc aacgtgcaga agaagctccc cattattcaa
aagatcatca 540ttatggatag caagacagat taccaagggt tccagtcgat
gtataccttt gtgacatcgc 600atttgccgcc agggtttaac gagtatgact
tcgtccccga gtcatttgac agagataaaa 660ccatcgcgct gattatgaat
tcctcgggta gcaccggttt gccaaagggg gtggcgttgc 720cccaccgcac
tgcttgtgtg cggttctcgc acgctaggga tcctatcttt ggtaatcaga
780tcattcccga cacagcaatc ctgtccgtgg taccttttca tcacggtttt
ggcatgttca 840cgactctcgg ctatttgatt tgcggtttca gggtcgtact
tatgtatcgg ttcgaggaag 900aactgttttt gagatccttg caagattaca
agatccagtc ggccctcctt gtgccaacgc 960ttttctcatt ctttgcgaaa
tcgacactta ttgataagta tgacctttcc aatctgcatg 1020agattgcctc
agggggagcg ccgcttagca aggaagtcgg ggaggcagtg gccaagcgct
1080tccaccttcc cggaattcgg cagggatacg ggctcacgga gacaacatcc
gcgatcctta 1140tcacgcccga gggtgacgat aagccgggag ccgtcggaaa
agtggtcccc ttctttgaag 1200ccaaggtcgt agacctcgac acgggaaaaa
ccctcggagt gaaccagagg ggcgagctct 1260gcgtgagagg gccgatgatc
atgtcaggtt acgtgaataa ccctgaagcg acgaatgcgc 1320tgatcgacaa
ggatgggtgg ttgcattcgg gagacattgc ctattgggat gaggatgagc
1380acttctttat cgtagatcga cttaagagct tgatcaaata caaaggctat
caggtagcgc 1440ctgccgagct cgagtcaatc ctgctccagc accccaacat
tttcgacgcc ggagtggccg 1500ggttgcccga tgacgacgcg ggtgagctgc
cagcggccgt ggtagtcctc gaacatggga 1560aaacaatgac cgaaaaggag
atcgtggact acgtagcatc acaagtgacg actgcgaaga 1620aactgagggg
aggggtagtc tttgtggacg aggtcccgaa aggcttgact gggaagcttg
1680acgctcgcaa aatccgggaa atcctgatta aggcaaagaa aggcgggaaa
atcgctgtct 1740gataagctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct cccttgcacc 1800tgtacctctt ggtctttgaa taaagcctga
gtaggaaggc ggccgctcga gcatgcatct 1860agagggccc 186911930DNAHomo
sapiens 11tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa
ataagagaga 60aaagaagagt aagaagaaat ataagagcca ccatggtgag caagggcgag
gagctgttca 120ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
aaacggccac aagttcagcg 180tgtccggcga gggcgagggc gatgccacct
acggcaagct gaccctgaag ttcatctgca 240ccaccggcaa gctgcccgtg
ccctggccca ccctcgtgac caccctgacc tacggcgtgc 300agtgcttcag
ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
360ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
tacaagaccc 420gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
catcgagctg aagggcatcg 480acttcaagga ggacggcaac atcctggggc
acaagctgga gtacaactac aacagccaca 540acgtctatat catggccgac
aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 600acaacatcga
ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
660gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
gccctgagca 720aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
gttcgtgacc gccgccggga 780tcactctcgg catggacgag ctgtacaagt
aagctgcctt ctgcggggct tgccttctgg 840ccatgccctt cttctctccc
ttgcacctgt acctcttggt ctttgaataa agcctgagta 900ggaaggcggc
cgctcgagca tgcatctaga 93012716RNAHomo sapiens 12gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug aacuuucucu 60ugucaugggu
gcacuggagc cuugcgcugc ugcuguaucu ucaucacgcu aaguggagcc
120aggccgcacc cauggcggag gguggcggac agaaucacca cgaaguaguc
aaauucaugg 180acguguacca gaggucguau ugccauccga uugaaacucu
uguggauauc uuucaagaau 240accccgauga aaucgaguac auuuucaaac
cgucgugugu cccucucaug aggugcgggg 300gaugcugcaa ugaugaaggg
uuggagugug uccccacgga ggagucgaau aucacaaugc 360aaaucaugcg
caucaaacca caucaggguc agcauauugg agagaugucc uuucuccagc
420acaacaaaug ugaguguaga ccgaagaagg accgagcccg acaggaaaac
ccaugcggac 480cgugcuccga gcggcgcaaa cacuuguucg uacaagaccc
ccagacaugc aagugcucau 540guaagaauac cgauucgcgg uguaaggcga
gacagcugga auugaacgag cgcacgugua 600ggugcgacaa gccuagacgg
ugagcugccu ucugcggggc uugccuucug gccaugcccu 660ucuucucucc
cuugcaccug uaccucuugg ucuuugaaua aagccugagu aggaag 7161330PRTHomo
sapiens 13Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala
Leu Gln1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln
Glu Ala 20 25 30 1425PRTHomo sapiens 14Met Met Pro Ser Ser Val Ser
Trp Gly Ile Leu Leu Leu Ala Gly Leu1 5 10 15 Cys Cys Leu Val Pro
Val Ser Leu Ala 20 25 1546PRTHomo sapiens 15Met Gln Arg Val Asn Met
Ile Met Ala Glu Ser Pro Ser Leu Ile Thr1 5 10 15 Ile Cys Leu Leu
Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His
Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg 35 40 45
1621PRTHomo sapiens 16Met Lys Gly Ser Leu Leu Leu Leu Leu Val Ser
Asn Leu Leu Leu Cys1 5 10 15 Gln Ser Val Ala Pro 20 1724PRTHomo
sapiens 17Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser
Ser Ala1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg 20
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