U.S. patent application number 17/525433 was filed with the patent office on 2022-03-03 for cell engineering using rnas.
The applicant listed for this patent is Boehringer Ingelheim International GmbH. Invention is credited to Lore FLORIN, Angelika HAUSSER, Hitto KAUFMANN, Monilola OLAYIOYE, Michaela STROTBEK.
Application Number | 20220064690 17/525433 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064690 |
Kind Code |
A1 |
FLORIN; Lore ; et
al. |
March 3, 2022 |
CELL ENGINEERING USING RNAs
Abstract
The invention concerns the field of cell culture technology. It
concerns RNA having a specific sequence, expression vectors
encoding said RNA, production host cell lines comprising said RNA,
and methods of producing recombinant biopharmaceutical products
using engineered host cell with altered levels of said RNAs, such
as small non-coding RNAs, preferably microRNAs (miRNAs). The
invention also relates to engineered host cells with altered levels
in one or more of said RNAs. Those cell lines have improved
secretion and/or growth characteristics in comparison to control
cell lines.
Inventors: |
FLORIN; Lore; (Redwood City,
CA) ; KAUFMANN; Hitto; (Stuttgart, DE) ;
HAUSSER; Angelika; (Stuttgart, DE) ; OLAYIOYE;
Monilola; (Ulm, DE) ; STROTBEK; Michaela;
(Asperg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim International GmbH |
Ingelheim am Rhein |
|
DE |
|
|
Appl. No.: |
17/525433 |
Filed: |
November 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15727056 |
Oct 6, 2017 |
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17525433 |
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14400610 |
Nov 12, 2014 |
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PCT/EP2013/061465 |
Jun 4, 2013 |
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15727056 |
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International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 15/11 20060101 C12N015/11; C12N 15/113 20060101
C12N015/113; C07K 14/62 20060101 C07K014/62; C07K 16/28 20060101
C07K016/28; C12N 9/08 20060101 C12N009/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 6, 2012 |
EP |
12171110.5 |
Claims
1. (canceled)
2. A mammalian expression vector comprising a microRNA selected
from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
3. The mammalian expression vector of claim 2, wherein the vector
comprises a polynucleotide sequence encoding the microRNA.
4. The mammalian expression vector of claim 2, wherein said
microRNA leads to an increase in the production and/or secretion of
a therapeutic protein of interest in a mammalian expression
system.
5. (canceled)
6. The mammalian expression vector of claim 2, further comprising a
selection marker gene.
7. The mammalian expression vector of claim 6, wherein the
selection marker gene is an amplifiable selection marker gene.
8. The mammalian expression vector of claim 7, wherein the
amplifiable selection marker gene is a glutamine synthetase gene or
a dihydrofolate reductase gene.
9. The mammalian expression vector of claim 2, wherein the microRNA
is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO:
11, and SEQ ID NO: 16.
10. The mammalian expression vector according to claim 2,
characterised in that it comprises a combination of several
identical or different microRNAs selected from the group consisting
of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
and SEQ ID NO:20.
11. The mammalian expression vector of claim 10, comprising a
polynucleotide sequence encoding a combination of two or more of
the microRNAs, wherein the RNAs are identical or different.
12. The mammalian expression vector according to claim 10, wherein
the microRNAs are selected from the group consisting of: SEQ ID NO:
6, SEQ ID NO: 11, and SEQ ID NO: 16.
13. The mammalian expression vector of claim 2, further comprising
at least one gene of interest.
14-17. (canceled)
18. A mammalian cell comprising one or more microRNAs selected from
one or more of the group consisting of: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein the
cell is stably transfected with an expression vector encoding said
one or more microRNA.
19-21. (canceled)
22. The mammalian cell of claim 18, wherein the microRNA is
selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 11,
and SEQ ID NO: 16.
23. The mammalian cell of claim 18, comprising a mammalian
expression vector comprising a polynucleotide sequence encoding a
microRNA selected from the group consisting of: SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
24. The mammalian cell of claim 18, further stably expressing a
protein of interest.
25. The mammalian cell of claim 18, wherein the cell is a rodent or
a human cell.
26. The mammalian cell of claim 25, wherein the rodent cell is a
hamster cell.
27. The mammalian cell of claim 25, wherein the human cell is a
HEK-293 cell, a PER.C6 cell or a CAP cell.
28-51. (canceled)
52. The mammalian expression vector of claim 2, wherein the
expression vector is a plasmid or a viral vector.
53. The mammalian cell of claim 18, wherein the expression vector
is a plasmid or a viral vector.
54. A CHO cell comprising one or more microRNAs selected from the
group consisting of: miR-1287 (SEQ ID NO:6), miR-1978 (SEQ ID
NO:11), and miR-557 (SEQ ID NO:16).
55. The CHO cell of claim 54, wherein the CHO cell further stably
expresses a protein of interest.
56. The CHO cell of claim 55, wherein the protein of interest is a
recombinant protein.
57. The CHO cell of claim 54, wherein the cell is stably
transfected with an expression vector encoding said one or more
microRNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/727,056 filed Oct. 6, 2017, which is continuation of
U.S. patent application Ser. No. 14/400,610 filed Nov. 12, 2014,
incorporated herein by reference in its entirety, which is the
National Stage of International Application No. PCT/EP2013/061465
filed Jun. 4, 2013.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII file, created
on Jun. 4, 2013, is named
01-2818-US-3-2021-11-12-sequence-listing.txt and is 8 KB in
size.
TECHNICAL FIELD
[0003] The invention relates to the field of cell culture
technology. It concerns RNA, expression vectors encoding said RNA,
production host cell lines comprising said RNA, and methods of
producing recombinant biopharmaceutical products using engineered
host cells with altered levels of RNA, preferably small non-coding
RNA, especially microRNAs (miRNAs). The invention also relates to
engineered host cells with altered levels in one or more RNAs,
preferably small non-coding RNAs, especially microRNAs. Those cell
lines have improved secretion and/or growth characteristics in
comparison to control cell lines.
BACKGROUND
[0004] Improving titers of therapeutic proteins in production and
thus making processes more efficient is a clear goal in industry.
More efficient processes can lead to reduced costs and shortened
timelines to supply protein material for clinical studies and
markets. As overall yields in production processes are determined
on the one hand by cell specific productivity of the individual
cell as well as the number of viable cells present in the process,
strategies to improve production efficiency usually aim to increase
either of these two parameters, without negatively affecting the
other.
[0005] However, one of the challenges associated with modifying the
behaviour of cells to achieve favourable phenotypes for the
production of recombinant proteins is the complex nature of
intra-cellular regulating circuits. Targeting the expression of one
gene or protein may not be sufficient to alter the phenotype of a
cell unless it is a rate-limiting factor in a critical pathway or
it is a transcription factor with the potential to alter expression
of a whole set of target genes.
[0006] This explains the current interest in microRNAs (also
referred to as "miRNAs") as a potential opportunity to engineer
networks of genes in order to achieve complex phenotypic changes in
mammalian cells (Muller et al, 2008).
[0007] miRNAs are small (.about.22 nt), non-coding RNAs that
regulate gene expression at the level of mRNA degradation and
protein translation. How miRNAs contribute to the regulation of
cell phenotypes is subject of extensive research. Apparently, the
mechanism is quite complex as each miRNA can regulate multiple,
even up to 100 genes and hundreds of miRNA genes are predicted to
be present in mammals. For example, the human genome is estimated
to contain 700-1,000 miRNA genes and in silico predictions suggest
that as many as 30-50% of all proteins may be sensitive to miRNA
effects, establishing them as a significant layer of control within
the cell.
[0008] The first microRNA was discovered in C. elegans in 1993 and
over the last years, microRNAs have been associated with various
processes including development, proliferation, differentiation and
cell death. In 2007, Gammel et al. isolated and sequenced the first
microRNA from CHO cells (Gammel et al, 2007) and since then,
several studies linked microRNAs with different states or phases in
production processes (Gammel et al, 2007; Clarke et al, 2011;
Johnson et al, 2011).
[0009] The ability of RNAs, especially microRNAs, to influence gene
expression is now recognized as a fundamental mechanism of
regulation in cells. In contrast to manipulation (over-expression,
knock-out or knock-down) of one or several individual genes in a
cell, which in several cases turned out not to be sufficiently
effective, microRNAs through their ability of targeting .about.100
target genes in a cell, could provide an elegant and highly
efficient solution to engineer cell behavior.
[0010] One striking advantage of using RNAs, such as miRNAs,
instead of functional proteins is that they do not compete for the
translational machinery that is required to express the recombinant
therapeutic protein product. miRNAs are short, of nucleic acid
nature and can be expressed from simple genetic constructs. Thus,
the translation apparatus is not overloaded and the cell not
burdened with additional energy consumption for gene transcription
and protein synthesis.
[0011] In the literature, there are reports indicating that the
functional role of individual microRNAs may be cell- or
tissue-specific. For example, antisense inhibition of miR-21 and
miR-24 have been reported to result in increased growth rates of
HeLa cells, whereas other reports found that their over-expression
supported tumour cell growth.
[0012] Only recently, a group at the University of Dublin published
a first report on a microRNA engineering approach in CHO producer
cells where they showed that in transient experiments,
over-expression of cgr-miR-7 resulted in higher levels of a
reporter protein (increased production normalised per cell);
however, the total yield was not increased due to a concomitant
reduction in cell numbers (Barron et al., 2011). Also, they did not
demonstrate stable transfections, which would be a requirement for
application in industrial processes, nor was the production of a
therapeutically relevant protein analyzed.
[0013] In the same year, Lin et al (2011) inhibited the expression
of two endogenous microRNAs in CHO and in one case saw a mild
increase in IgG production.
[0014] Another group from the University of Vienna analysed
transient transfection with CHO miRNAs (cgr-miR-17, cgr-miRNA-221,
cgr-miR-21, and cgr-miR-210) in CHO cells and found that
over-expression of cgr-miR-17 increased cell growth. However, they
did not observe an increase in specific productivity and did not
demonstrate an effect for stably expressed miRNAs. Furthermore,
none of these publications used human miRNAs.
[0015] Hence, there is a need for improving recombinant protein
production in mammalian cells, by increasing the specific
productivity and the total yield (i.e., titer or concentration) of
the protein, which is generally applicable and not dependent on the
individual cell line or protein to be produced. It is an objective
of the present invention to provide heterologous human microRNAs to
engineer producer cell behaviour, wherein over-expression (not
inhibition) of the specific microRNAs has a positive impact on cell
productivity.
[0016] It was found that the RNAs, particularly the microRNAs
according to the present invention, exert a positive role on
protein production and/or secretion in more than one cell line.
Surprisingly, the RNAs (microRNAs) provided herein show a
functional benefit not only in CHO cells (which are used for
screening), but also in other rodent and human cell lines.
SUMMARY OF THE INVENTION
[0017] The above objects are solved by the RNAs, expression
vectors, mammalian cells, methods and uses according to the present
invention. By virtue of the RNAs provided herein, it is now
possible to engineer mammalian cells to improve their cell
productivity and/or cell growth. This results in a higher yield of
the protein of interest produced by these cells. The use of the
RNAs provided herein is particularly suited for engineering protein
producer cell lines, and for the generation of improved host cell
lines for the production of proteins of interest. Further, the
invention relates to the modification of mammalian producer cells
to increase the levels of specific RNAs (listed in FIG. 1B),
preferably of small non-coding RNAs, more preferably microRNAs, to
modulate cell productivity and/or cell growth. The invention is
based on functional screening of a library of human microRNAs in
antibody-producing CHO cells, which resulted in 20 RNAs,
reproducibly allowing for improved IgG product titers when
overexpressed (e.g. transiently or stably) in producer cell lines,
such as CHO cells. Furthermore, it is demonstrated herein that the
RNA-mediated, specifically miRNA-mediated, production improvement
is not product-dependent, but increases specific production rates
for example in an antibody producing cell and also in HSA (human
serum albumin) expressing cells (exemplified in FIG. 4).
Engineering the production host cells by transiently or stably
increasing the level of one or more of these 20 selected RNAs
allows to achieve higher productivities and/or secretion without
negatively affecting growth characteristics.
[0018] In a first aspect the present invention relates to a
ribonucleic acid (RNA) selected from the group consisting of: SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID
NO:20, wherein said RNA leads to an increase in the production
and/or secretion of a therapeutic protein of interest from a
mammalian cell.
[0019] In second aspect the present invention relates to a
mammalian expression vector comprising a RNA selected from the
group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20, wherein said vector comprises a
polynucleotide sequence encoding the RNA. In a preferred
embodiment, the RNA encoded by the expression vector is a
non-coding RNA, more preferably the non-coding RNA is a miRNA. The
RNA encoded by the mammalian expression vector of the invention
leads to an increase in the production and/or secretion of a
therapeutic protein of interest in a mammalian expression system.
Although not strictly mandatory the protein of interest or the RNA
may further be operably linked to an amplifiable selection marker.
Alternatively the expression vector may comprise a selection marker
gene, wherein the selection marker gene may be an amplifiable
selection marker gene, such as a glutamine synthetase gene or a
dihydrofolate reductase gene. In a preferred embodiment of this
aspect, the RNA is selected from the group consisting of: SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, and SEQ ID NO: 19. In a more preferred embodiment
the RNA is miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO:6),
miR-1978 (SEQ ID NO:11), or miR-557 (SEQ ID NO:16). In a more
preferred embodiment the RNA miR-1287 (SEQ ID NO:6), miR-1978 (SEQ
ID NO:11), or miR-557 (SEQ ID NO:16). In an even more preferred
embodiment the RNA is miR-1287 (SEQ ID NO:6). In another even more
preferred embodiment, the RNA is miR-557 (SEQ ID NO:16).
[0020] Although not mandatory the mammalian expression vector of
the present invention may comprise a combination of several
identical or different RNAs selected from the group consisting of:
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and
SEQ ID NO:20. Specifically, the expression vector may comprise a
polynucleotide sequence encoding a combination of two or more of
the RNAs, wherein said RNAs may be identical or different. In a
preferred embodiment the RNAs are selected from the group
consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ
ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In
another preferred embodiment the RNAs are selected from the group
consisting of: miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO:6),
miR-1978 (SEQ ID NO:11) and miR-557 (SEQ ID NO:16). In certain
embodiments the mammalian expression vector of the invention
further comprises at least one gene of interest. Preferably the RNA
or the RNA encoded by the expression vector is a non-coding RNA,
more preferably the non-coding RNA is a miRNA.
[0021] The invention further relates to a mammalian expression
vector comprising a polynucleotide sequence encoding any of the
RNAs of the first aspect. The expression vector may also comprise a
polynucleotide sequence encoding a combination of two or more of
the RNAs of the first aspect, wherein the RNAs may be identical or
different.
[0022] In another aspect the present invention relates to a
mammalian cell comprising one or more RNAs selected from one or
more of the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein typically the RNA
is a heterologous or transfected RNA. In a preferred embodiment the
cell is stably transfected with an expression vector encoding said
one or more RNA. Preferably, the one or more RNAs of this aspect
are non-coding RNAs, more preferably the non-coding RNAs are
miRNAs.
[0023] In a preferred embodiment the one or more RNA is selected
from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID
NO: 19, preferably the RNA is selected from the group consisting
of: miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO:6), miR-1978 (SEQ
ID NO:11), and miR-557 (SEQ ID NO:16). In a more preferred
embodiment the one or more RNA is miR-1287 (SEQ ID NO:6), miR-1978
(SEQ ID NO:11), miR-557 (SEQ ID NO:16) or a combination thereof,
even more preferably miR-1287 (SEQ ID NO:6) and/or miR-557 (SEQ ID
NO:16). In another embodiment the mammalian cell may comprise the
mammalian expression vector of the invention. In yet another
embodiment the mammalian cell may further stably express a protein
of interest. Although the mammalian cell of the invention may be a
rodent or a human cell, in a preferred embodiment the cell is a
rodent cell, more preferably a hamster cell and even more
preferably a CHO cell, such as a CHO-DG44. In certain embodiments
the human cell is a HEK-293 cell, a PER.C6 or a CAP cell.
[0024] In yet another aspect the invention relates to a method of
developing a stably transfected mammalian cell comprising the
following steps:
[0025] (a) transfecting at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 into the mammalian cell,
[0026] (b) cultivating said cell for an initial period of time in
the presence of selective pressure, and
[0027] (c) selecting a high-producing transfected cell.
[0028] Preferably the RNA is a non-coding RNA, more preferably the
non-coding RNA is a miRNA. In a preferred embodiment the mammalian
cell in step (a) is stably transfected with an expression vector
encoding said RNA, wherein the expression vector may be the
expression vector of the invention. In one embodiment the mammalian
cell in step (a) is a producer host cell comprising at least one
expression vector comprising at least one gene of interest.
[0029] In yet another aspect the invention relates to a method of
producing a protein of interest, characterised by the following
steps:
[0030] (a) transfecting at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 and at least one expression vector
comprising at least one gene of interest into a mammalian cell,
[0031] (b) selecting a highly-productive transfected cell, and
[0032] (c) cultivating the highly-productive transfected cell
obtained in step (b) under conditions which allow expression of the
gene(s) of interest, and optionally
[0033] (d) harvesting and purifying the protein of interest.
[0034] Preferably the RNA is a non-coding RNA, more preferably, the
non-coding RNA is a miRNA. In a preferred embodiment the
transfection step (a) comprises transfecting an expression vector
encoding said RNA. In a more preferred embodiment the mammalian
cell in step (a) is stably transfected with the expression vector
encoding said RNA, wherein the expression vector may be the
expression vector of the invention. In one embodiment the
expression vector comprising at least one gene of interest of step
(a) may also encode the at least one RNA of step (a).
Alternatively, transfecting the RNA into the mammalian cell may be
done after, prior to or simultaneously to transfecting the gene of
interest.
[0035] In yet another related aspect, the invention relates to a
method of preparing and selecting a recombinant mammalian cell,
comprising the following steps:
[0036] (a) transfecting a mammalian cell with genes that encode at
least a protein/product of interest and a selectable marker,
wherein the cell is co-transfected with at least one RNA selected
from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20,
[0037] (b) selecting a cell with co-integrated genes by cultivating
the cell in the presence of a selective agent, and
[0038] (c) cultivating the cell under conditions which enable
expression of the (different) genes.
[0039] Preferably, the RNA is a non-coding RNA, more preferably the
non-coding RNA is a miRNA. In one embodiment the selectable marker
confers resistance to neomycin, puromycin, bleomycin, zeocin or
blasticidin. In an alternative embodiment the selectable marker may
be an amplifiable selectable marker and the method further
comprises an additional step (b') between steps (b) and (c),
comprising amplifying the co-integrated genes by cultivating the
cell in the presence of an agent, which allows the amplification of
the amplifiable selectable marker gene. The amplifiable selectable
marker gene may encode the amplifiable selectable markers DHFR or
GS.
[0040] In a preferred embodiment the mammalian cell in step (a) is
stably transfected with the expression vector encoding said at
least one RNA, wherein the expression vector may be the expression
vector of the invention. Transfecting the RNA into the mammalian
cell may be done after, prior to or simultaneously to transfecting
the gene of interest. The protein of interest in any one of the
above methods of the inventions may be a recombinant therapeutic
protein, such as an antibody or an Fc-fusion protein.
[0041] In one embodiment of any of the methods of the invention,
the production and/or secretion of the protein of interest is
increased by 10%, 20%, 50%, 100%, 200%, 400% compared to a control
cell, which is not transfected with at least one RNA selected from
the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20. In yet another aspect, the
invention relates to a use of a RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 as a production-promoting element for
the preparation of a recombinant protein of interest in a mammalian
cell. In one embodiment the RNA of the invention is used for
increasing the production and/or secretion of a protein of interest
from a mammalian cell.
[0042] In yet another aspect, the invention relates to a use of the
mammalian cell of the invention for producing a protein of
interest, wherein the protein of interest may be a recombinant
therapeutic protein, such as an antibody, an antibody fragment, an
antibody fusion protein, an antibody conjugate or an Fc-fusion
protein. In yet another aspect, the invention relates to the use of
the RNA of the invention for the production of a non-human
transgenic animal, preferably a mammal. Preferably, the RNA in any
of the uses of the invention is a non-coding RNA, more preferably
the non-coding RNA is a miRNA.
[0043] Generally, the RNAs (microRNAs) provided in this invention
increase the yield of a protein of interest, such as a secreted
therapeutic protein, in production processes based on eukaryotic
cells by increasing the productivity of the cell, and/or cell
growth. This reduces the cost of goods of such processes and at the
same time it reduces the number of batches that need to be produced
to generate the material needed for research studies, diagnostics,
clinical studies or market supply of a therapeutic protein. Thus,
the microRNAs, mammalian cells, methods and uses provided herein
find application in the production of recombinant biological
products, especially recombinant therapeutic protein products.
[0044] The invention may furthermore speed up drug development
since the generation of sufficient amounts of material for
pre-clinical studies is a critical work package with regard to
overall development timelines.
[0045] The RNAs (miRNAs), cells and methods of the invention can
further be used for the generation of one or several specific
proteins for either diagnostic purposes, research purposes (target
identification, lead identification, lead optimization) or
manufacturing therapeutic proteins either on the market or in
clinical development.
DESCRIPTION OF THE FIGURES
[0046] FIG. 1A: FIRST MICRORNA SCREEN IN BIWA4-PRODUCING CHO
CELLS
[0047] CHO-DG44 cells secreting a human IgG1 antibody (BIWA4) are
transiently transfected with a human mimic microRNA library
consisting of 879 human microRNAs according to the official
microRNA database mirbase. Antibody concentrations in the
supernatant of the transfected cells are determined and
consequently any positive effect on the antibody titer can be
correlated with the expression of a specific mature microRNA.
[0048] FIG. 1B: HITS OF THE MICRORNA LIBRARY SCREEN.
[0049] 20 miRNAs, namely, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, are defined as hits, when
they show more than 1.4 fold increased antibody titer on day 3 or 4
after transfection compared to control (mean of two independent
experiments, see last column) and additionally more than 1.3 fold
increased antibody titers in each of the two screens. Sequences,
accession numbers and names of the miRNAs as stored in the mirbase
database are listed.
[0050] FIG. 2: SECONDARY SCREEN IN BIWA4-PRODUCING CHO CELLS.
[0051] CHO-DG44 cells stably secreting an IgG1 antibody (BIWA4) are
transfected with each of the 20 miRNA hits (see FIG. 1B) in
quadruplicates. Cell density and viability are determined by cell
counting and trypane blue exclusion. Antibody concentrations in the
supernatants are determined by ELISA analysis on days 1-4. Specific
productivity is calculated, normalized to the control on the
respective day and is shown for day 3 (white bar) and day 4 (black
bar). As a negative control a non targeting siRNA (siLacZ-FITC) and
as a positive control, a siRNA targeting the IgG1 antibody
(siLC-104) are used (n=4, error bars=SEM).
[0052] FIG. 3: TRANSIENT EXPRESSION OF MICRORNAS IN BIBH1-PRODUCING
CHO CELLS
[0053] CHO-DG44 cells stably secreting an IgG1 antibody (BIBH1) are
transfected with each of the 20 validated miRNA hits (see FIG. 1B)
in quadruplicates. Cell density and viability are determined by
cell counting and trypane blue exclusion. Antibody concentrations
in the supernatant are detected on days 1-4 by ELISA analysis.
Specific productivity is calculated, normalized to the control on
the respective day and is shown for day 3 (white bar) and day 4
(black bar). As a negative control a non targeting siRNA
(siLacZ-FITC) and as a positive control a siRNA targeting the IgG1
antibody (siLC-104) are used (n=1, error bars=SEM of
quadruplicates).
[0054] FIG. 4: TRANSIENT EXPRESSION OF MICRORNAS IN HSA-PRODUCING
CELLS
[0055] CHO-DG44 cells stably secreting human serum albumin (HSA)
are transfected with each of the 20 validated miRNA hits (see FIG.
1B) in quadruplicates. Cell density, viability and antibody
concentrations in the supernatant are detected on day 1-4 by cell
counting and trypane blue exclusion or ELISA analysis,
respectively. Specific productivity is calculated, normalized to
the control on the respective day and is shown for day 3 (white
bar) and day 4 (black bar). As a negative control a non targeting
siRNA (siLacZ-FITC) and as a positive control a siRNA targeting
human serum albumin (siHSA) are used (n=2, error bars=SEM).
[0056] FIG. 5: QUANTIFICATION OF MICRORNA EXPRESSION IN
BIWA4-PRODUCING CHO CELLS BY QPCR
[0057] qPCR analysis of expression levels of microRNA miR-557 in
CHO-DG44-derived producer cells after transient transfection with
either a human microRNA-encoding plasmid or the mature microRNA.
The white bars correspond to the sense strand of miR-557, the black
bars correspond to the antisense strand (miR-557*). Relative
expression is calculated and plotted in comparison to control
samples (empty vector and siLacZ-transfected cells,
respectively).
[0058] FIG. 6: EFFECT OF MICRORNA COMBINATIONS ON IGG
PRODUCTION
[0059] CHO-DG44 cells stably secreting an IgG1 (BIWA4) are
transiently transfected with a combination of two validated miRNA
hits (every possible combination of these five miRNAs: hsa-miR-557,
hsa-miR-1271, hsa-miR-1275, hsa-miR-1287 and hsa-miR-1978) in
duplicates. Samples containing a single microRNA are adjusted to a
final RNA concentration of 1 .mu.M by adding mimic miRNA negative
control. Cell density and antibody concentration in the supernatant
are determined on day 1-4 by cell counting or trypane blue
exclusion and ELISA analysis, respectively, and specific
productivity is calculated. Shown are the specific productivities
at day 4 after transfection. Transfection efficiency is monitored
by flow cytometry of siLacZ-FITC transfected cells and ELISA
analysis of the supernatant of siLC-104 (targeting the light chain
of the antibody) transfected cells. As negative controls a non
targeting siRNA (siLacZ-FITC) and a mimic miRNA are used (error
bars=SEM of duplicates).
[0060] FIG. 7: TRANSIENT EXPRESSION OF MICRORNAS IN
INSULIN-SECRETING INS1 CELLS
[0061] The rat cell line INS-1, which endogenously secrets insulin,
is transiently transfected by nucleofection with 5 screen hit
miRNAs. Insulin assay is performed with basal conditions and
insulin secretion is induced using 20 mM glucose. The insulin
concentration in the supernatant is determined by ELISA analysis.
Cell density is measured by crystal violet staining and insulin
concentrations are normalized to the cell density. Shown are the
results of basal (white bar) and induced (black bar) samples. As
negative control a non targeting siRNA (siLacZ) is used (n=1, error
bars=SEM of duplicates).
[0062] FIG. 8: TRANSIENT EXPRESSION OF MICRORNAS IN HEK293
FLPIN
[0063] HEK293 FlpIN cells stably expressing a secretable form of
HRP (ssHRP-flag) are transfected by lipofection with 5 miRNAs
(hsa-miR-1287, hsa-miR-183, hsa-miR-557, hsa-miR-612 and
hsa-miR-644). Two days after transfection ssHRP-flag expression is
induced by doxycyclin and the activity of secreted HRP is measured
the next day. Medium is changed and HRP activity in the supernatant
is assessed by luminescence measurement after 5 hours. The results
are normalized to miRNA neg. control. Shown are the relative
luminescence units normalized for cell density (n=4, error bars
SEM).
[0064] FIG. 9: QUANTIFICATION OF STABLE MICRORNA OVEREXPRESSION IN
BIWA4-PRODUCING CHO CELLS BY FLOW CYTOMETRY
[0065] CHO-DG44 cells stably secreting an IgG1 (BIWA4) were stably
transfected with a GFP-containing expression vector further
encoding (A) hsa-miR-557 (pcDNA6.2-GW/emGFP-miR557-miR557), (B)
hsa-miR-1287 (pcDNA6.2-GW/emGFP-miR1287-miR1287), and (C) both
miRNAs (hsa-miR-557 and hsa-miR-1287;
pcDNA6.2-GW/emGFP-miR557-miR1287). GFP-positive cells were enriched
by FACS and living cells were analyzed by flow cytometry analysis
after cultivation for the indicated times (21 days, dotted line; 51
days, solid line) after sorting. As a negative control,
untransfected cells (grey filled peak) without GFP expression were
used.
[0066] FIG. 10: QUANTIFICATION OF STABLE MICRORNA OVEREXPRESSION IN
BIWA4-PRODUCING CHO CELLS BY QPCR
[0067] CHO-DG44 cells stably secreting an IgG1 (BIWA4) were stably
transfected with an expression vector encoding either hsa-miR-557
(pcDNA6.2-GW/emGFP-miR557-miR557) or hsa-miR-1287
(pcDNA6.2-GW/emGFP-miR1287-miR1287) or encoding for both miRNAs
(hsa-miR-557 and hsa-miR-1287) in combination
(pcDNA6.2-GW/emGFP-miR557-miR1287). GFP-positive cells were
enriched by FACS and RNA was extracted to measure levels of the
mature hsa-miR-557 (A) or hsa-miR-1287 (B) microRNA by qPCR
analysis. Cells transfected with the mature miRNA (hsa-miR-557 or
hsa-miR1287) served as a positive control. For the positive
control, RNA was extracted one day after transient transfection.
Relative expression was calculated by normalizing to the reference
RNU6B.
[0068] FIG. 11: STABLE EXPRESSION OF MICRORNA COMBINATIONS IN
BIWA4-PRODUCING CHO CELLS
[0069] CHO-DG44 cells stably secreting an IgG1 (BIWA4) were stably
transfected with a combination of two validated miRNA hits
(pcDNA6.2-GW/emGFP-miR557-miR1287) or a negative control miRNA
expression plasmid (pcDNA6.2-GW/emGFP-neg. control-miRNA) and
enriched for GFP positive cells by FACS. Stable pools (3
independent pools for the miRNA combination miR557-miR1287 and 4
independent pools for the neg. control miRNA) were grown in
fed-batch cultures. Cell density and antibody concentration in the
supernatant were determined on day 3-7 by cell counting with
trypane blue exclusion and ELISA analysis, respectively, and
specific productivity was calculated. A representative experiment
is shown and the data correspond to the mean of the independent
pools for product concentration (BIWA4) (A), specific productivity
(B) and viable cell density (C). Error bars represent SEM. The
experiment was repeated three times. The parental cell line
(CHO-DG44 secreting BIWA4) served as an additional control.
[0070] FIG. 12: SINGLE CLONE OF BIWA4-PRODUCING CHO CELLS STABLY
EXPRESSING A MICRORNA COMBINATION
[0071] CHO-DG44 stably secreting an IgG1 (BIWA4) were stably
transfected with miRNA expression vectors (pcDNA6.2-GW/emGFP) and
single clones were generated by limited dilution. Either a
combination of 2 validated miRNA hits (-miR557-miR1287-clone) or
the respective empty vector control expressing GFP (-control-clone)
were used during fed-batch cultures. Three independent pools stably
expressing a neg. control miRNA (pcDNA6.2-GW/emGFP-neg.
control-miRNA) served as further controls. Cell density and
antibody concentration in the supernatant were determined on day
3-11 by cell counting with trypane blue exclusion and ELISA
analysis, respectively, and specific productivity was calculated.
Shown is the specific productivity (A) and product concentration
(B) of the single clone expressing the miR557-miR1287 combination
compared to the control clone and the mean of the three independent
pools (neg. control-miRNA).
[0072] FIG. 13A: GLYCOSYLATION ANALYSIS OF BIWA4 ANTIBODY PRODUCED
IN CHO CELLS STABLY EXPRESSING MICRORNAS OR MICRORNA
COMBINATIONS
[0073] CHO-DG44 cells stably secreting an IgG1 (BIWA4) were stably
transfected with miRNA expression plasmids encoding for miRNAs or
miRNA combinations (hsa-miR557, hsa-miR1287, hsa-miR1978, and their
combinations). The composition of the Fc-glycosylation of the IgG
(BIWA4) produced in these cell lines was analysed. The glycans were
first released from the purified antibody by enzymatic digestion
with PNGase F. Glycans were purified, labelled with a fluorescent
dye and separated by microchip-based CGE. The percentages of the
glyco-forms present were calculated from the chromatographic peak
areas shown in FIG. 13B and indicated by arrows. Abbreviations, A2,
biantennary; G, galactose; F, fucose; Man, mannose.
[0074] FIG. 13B: CHROMATOGRAPHIC PEAK AREAS FROM GLYCOSYLATION
ANALYSIS OF BIWA4 ANTIBODY PRODUCED IN CHO CELLS STABLY EXPRESSING
MICRORNAS OR MICRORNA COMBINATIONS.
[0075] FIG. 14: TRANSIENT EXPRESSION OF MICRORNAS IN HELA CELLS
[0076] HeLa cells were transiently transfected with different
miRNAs (hsa-miR-125a-3p, hsa-miR-1978 and hsa-miR557) followed by
ssHRP-FLAG transfection two days later. The next day, the medium
was changed and the activity of secreted HRP in the supernatant was
measured after 4 h (white bars) and 6 h (black bars) by
luminescence measurement. The results were normalized to miRNA neg.
control and are shown as relative luminescence units (RLU) (A)
(n=4, error bars SEM). Furthermore, endogenous IL-8 secretion was
measured two days after transient miRNA (hsa-miR-125a-3p,
hsa-miR-1271, hsa-miR-185*, hsa-miR-193b*, hsa-miR-1978,
hsa-miR-299-3p and hsa-miR557) transfection. Medium was changed and
collected after 6 h (white bars) and 24 h (black bars).
Supernatants were analysed by ELISA and results were normalized to
miRNA neg. control samples (B) (n=3, error bars SEM). Cells
transfected without RNA (mock) or untransfected cells as shown
served as additional controls.
DETAILED DESCRIPTION OF THE INVENTION
[0077] CHO cells are commonly used for the production of
therapeutic proteins. Genetic engineering approaches have attempted
to optimize the productivity of these cells by expressing specific
cDNAs. Naturally existing non-coding RNAs regulate cell fate by
modulating the expression of a whole set of target proteins, which
may possibly result in a super-secretory phenotype when
over-expressed in CHO producer cells. To exploit the power of
non-coding RNAs and to identify those that positively affect
secretion of a heterologous therapeutic protein, CHO-DG44 cells
stably expressing a protein, for example a Fc-containing protein,
such as an antibody, are transiently transfected by nucleofection
with a human microRNA mimic library consisting of 879 microRNAs.
microRNAs that (i) increased the IgG1 titer in the supernatant more
than 1.3-fold on day 3 or 4 compared to control cells in one
experiment and (ii) increased the mean IgG1 titer on day 3 or 4 of
more than 1.4-fold in two experiments are provided by the present
invention. Given that the host cell already produces high amounts
of heterologous protein, a further increase in productivity of
>30% is highly significant and surprising.
[0078] The (micro)RNAs of the present invention specifically
enhance the expression and secretion of immunoglobulin molecules as
well as of other recombinant proteins, e.g. therapeutic proteins
such as human serum albumin (HSA). Surprisingly, the (micro)RNAs of
the present invention also exert a positive effect on the specific
productivity of HSA-secreting CHO cells on days 3 and 4 post
transfection. Thus, the present invention provides 20 (micro) RNAs
that surprisingly function in a product-independent manner.
[0079] Remarkably, the combined transfection of two different
miRNAs provided herein enhanced specific productivity on day 4
compared to singly transfected CHO-DG44 cells in some cases. For
example, co-transfection of miR-557 and miR-1287 clearly had a
positive effect in increasing the productivity compared to both
microRNAs alone. Thus, with certain combinations of microRNAs, it
is possible to achieve an additive or even synergistic effect on
the enhancement of secretory capacity of a cell producing a protein
of interest, preferably a therapeutic protein, which can result in
enhanced specific productivity, enhanced product titer or both.
Preferred combinations of different RNAs are miR-557 (SEQ ID NO:16)
and miR-1287 (SEQ ID NO:6), miR-557 (SEQ ID NO:16) and miR-1978
(SEQ ID NO:11), miRNA-1271 (SEQ ID NO: 3) and miR-1978 (SEQ ID
NO:11) or miR-1287 (SEQ ID NO: 6) and miRNA-1978 (SEQ ID NO:11),
more preferably miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID
NO:6).
[0080] Surprisingly, the microRNAs provided herein (e.g.,
hsa-miR-183, hsa-miR-125-3p, hsa-miR-557, hsa-miR-1271,
hsa-miR-1275, hsa-miR-1287) also exert a positive effect on basal
and glucose-stimulated insulin secretion of rat cells (such as INS1
cells, see FIG. 7). Several of them also led to enhanced secretion
from human cells (HEK293, see e.g. FIG. 8). Thus, unexpectedly the
microRNAs of the present invention positively affect the secretion
of endogenous proteins not only in CHO, but also in other cells of
rodent origin such as rat and in cells of human origin and hence
function in a species- and product-independent manner.
[0081] The present invention further shows that stable
transfectants can be generated. Thus, the present invention further
relates to mammalian cells stably expressing the RNAs of the
invention and methods of developing such cells. Those stably
transfected miRNA transgene host cells may be subjected to batch or
fed-batch fermentations. In each of these settings, overexpression
of the non-coding RNAs of the invention (e.g., miR-577 and
miR-1287, hsa-miR-183, hsa-miR-125-3p, hsa-miR-1271, hsa-miR-1275
or miR-1978) lead to increased protein production and/or secretion,
for example to increased antibody production and/or secretion. The
non-coding RNAs of the present invention are able to enhance the
specific production capacity of the cells grown in serial cultures
or in bioreactor batch or fed batch cultures.
[0082] In one embodiment, the present invention provides a cell
comprising (i) a plasmid encoding one chain of an antibody and
containing a DHFR cassette for amplification, (ii) a plasmid
encoding the other chain of an antibody and containing a neomycin
resistance cassette, and (iii) a plasmid encoding a microRNA of the
present invention, preferably miR-577 and miR-1287, hsa-miR-183,
hsa-miR-125-3p, hsa-miR-1271, hsa-miR-1275 or miRNA-1978 (see Table
1B) and containing a puromycin resistance cassette. The
overexpression of the non-coding RNAs of the present invention
leads to increased antibody titers, indicating that the non-coding
RNAs of the present invention are able to enhance the specific
production capacity of the cells, especially of those grown in
serial cultures or in bioreactor batch or fed batch cultures.
[0083] To explore whether transient expression of the 20 microRNAs
according to the invention also enhances the secretion of IgG4
molecules, CHO cells stably expressing IgG4 are transiently
transfected with each of the 20 microRNAs as described in example 2
and their specific productivity is determined. All 20 microRNAs
exert a positive effect on the specific productivity of
IgG4-secreting CHO cells on days 3 and/or 4 post transfection. Thus
the microRNAs of the present invention function in a
product-independent manner.
[0084] Furthermore, the miRNAs of the invention do not affect
glycosylation of the protein of interest. Glycosylation of
recombinant proteins can have a profound impact on the half-life,
activity and immunogenicity of the biotherapeutic protein drug. As
the exact mechanism of action for the microRNAs used in the present
invention is currently not or only incompletely understood, the
effect of those microRNAs used for cell engineering on protein
glycosylation was analysed and was found to have no unexpected
negative side-effect on the glycosylation of the protein of
interest. Thus the microRNAs provided herein do not affect
glycosylation of the protein of interest, such as in the Fc-domain
of an antibody.
[0085] The production cells derived from microRNA engineered host
cells show higher secretion rates, i.e. productivities and/or
higher titers (see e.g., example 11). Hence, microRNA engineering
can be done after, prior to or simultaneously to introducing the
protein of interest with similar results, thus offering a broad
range of options for applications in pharmaceutical development
processes.
[0086] Furthermore, for some microRNAs, sufficiently high stable
levels of microRNA in the host cell or the producer cell can only
be achieved after introducing a gene amplification step. Thus, in
another embodiment the miRNAs of the present invention is
amplified. Amplification can be performed by placing the microRNA
expression cassette under the control of an amplifiable genetic
selection marker, such as dihydrofolate reductase (DHFR), glutamine
synthetase (GS) or else. The amplifiable selection marker gene can
be on the same expression vector as the miRNA expression cassette.
Alternatively, the amplifiable selection marker gene and the miRNA
expression cassette can be on different expression vectors, but
integrate in close proximity into the host cell's genome. Two or
more vectors that are co-transfected simultaneously, for example,
often integrate in close proximity into the host cell's genome.
Amplification of the genetic region containing the microRNA
expression cassette is then mediated by adding the amplification
agent (e.g., MTX for DHFR or MSX for GS) into the cultivation
medium. In cases where the expression constructs of the protein of
interest also contain an amplifiable selection marker, it is
possible and preferred to use the identical amplifiable marker gene
for both, microRNA and gene of interest to allow for
co-amplification. However, independent amplification of the
microRNA gene is also possible. Sufficiently high stable levels of
microRNA in the host cell or the producer cell may also be
achieved, e.g., by cloning multiple copies of the microRNA encoding
polynucleotide into an expression vector. Cloning multiple copies
of the microRNA encoding polynucleotide into an expression vector
and amplifying the miRNA expression cassette as described above may
further be combined.
[0087] Furthermore, growth and viability profiles of the microRNA
engineered cells of the invention are comparable or only slightly
lower compared to controls. However, the specific productivity of
microRNA engineered cells is consistently higher compared to
non-engineered cell lines (see e.g. examples 12, 15 and 16). This
results in an overall benefit of the microRNA engineering approach
according to the invention for industrial therapeutic protein
production processes.
Definitions
[0088] The general embodiments "comprising" or "comprised"
encompass the more specific embodiment "consisting of".
Furthermore, singular and plural forms are not used in a limiting
way.
[0089] As used herein, the singular forms "a", "an" and "the"
designate both the singular and the plural, unless expressly stated
to designate the singular only.
[0090] Terms used in the course of the present invention have the
following meaning.
[0091] The term "ribonucleic acid" or "RNA" describes a molecule
consisting of a sequence of nucleotides, which are built of a
nucleobase, a ribose sugar, and a phosphate group. RNAs are usually
single stranded molecules and can exert various functions. The term
ribonucleic acid specifically comprises small non-coding RNA such
as microRNA. The specific RNAs claimed in the present invention are
listed in FIG. 1B with miroRNA number (following the nomination
assigned by Dharmacon) and MIMAT number and may also be referred to
as non-coding RNA or microRNA of the invention. Further examples of
ribonucleic acids are tRNA and hRNA.
[0092] The terms "microRNA" or "miRNA" are used interchangeably
herein. microRNAs are small, about 22 nucleotide-long (typically
between 19 and 25 nucleotides in length) non-coding RNAs. microRNAs
are encoded in the genome of eukaryotic cells and are typically
transcribed by RNA Polymerase III as long primary transcripts that
are then processed in several steps first into .about.70 nt-long
hairpin-loop structures and subsequently into the .about.22 nt RNA
duplex. The active mature strand is then loaded into the
RNA-induced silencing complex (RISC) in order to block translation
of target proteins or degradation of their respective mRNAs. The
specific microRNAs claimed in the present invention are listed in
FIG. 1B with microRNA number (following the nomination assigned by
Dharmacon) and MIMAT number and were taken from the human microRNA
library obtained from Dharmacon (CS-001010 mimic microRNA library;
lot number 09167). The term "microRNA" as used herein therefore
relates to endogenous human miRNAs, but also encompasses other
sequences identified from said library. The prefix "hsa" indicates
the human origin of a microRNA, but may be omitted in the context
of the present invention. The sequences provided herein as SEQ ID
NOs: 1 to 20 represent the sequence of the mature miRNA.
[0093] tRNAs or `transfer RNAs` are 73 to 93 nucleotide long RNA
molecules with a defined secondary structure which function as
adaptors between a codon on the mRNA and its encoded amino acid
thus playing a fundamental role in protein synthesis.
[0094] The term "derivative" as used in the present invention means
a polypeptide molecule or a nucleic acid molecule, which is at
least 70% identical in sequence with the original sequence or its
complementary sequence. Preferably, the polypeptide molecule or
nucleic acid molecule is at least 80% identical in sequence with
the original sequence or its complementary sequence. More
preferably, the polypeptide molecule or nucleic acid molecule is at
least 90% identical in sequence with the original sequence or its
complementary sequence. Most preferred is a polypeptide molecule or
a nucleic acid molecule, which is at least 95% identical in
sequence with the original sequence or its complementary sequence
and displays the same or a similar effect on secretion as the
original sequence.
[0095] Sequence differences may be based on differences in
homologous sequences from different organisms. They might also be
based on targeted modification of sequences by substitution,
insertion or deletion of one or more nucleotides or amino acids,
preferably 1, 2, 3, 4, 5, 7, 8, 9 or 10. Deletion, insertion or
substitution mutants may be generated using site-specific
mutagenesis and/or PCR-based mutagenesis techniques.
[0096] "Host cells" in the meaning of the present invention are
eukaryotic cells, preferably mammalian cells, most preferably
rodent cells such as hamster cells. Preferred cells are BHK21, BHK
TK.sup.-, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, and CHO-DG44 cells or
the derivatives/progenies of any of such cell line. Particularly
preferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21, and even more
preferred are CHO-DG44 and CHO-DUKX cells. Most preferred are
CHO-DG44 cells. In a specific embodiment of the present invention
the host cells are murine myeloma cells, preferably NS0 and Sp2/0
cells or the derivatives/progenies of any of such cell line.
Non-limiting examples of mammalian cells which can be used in the
meaning of this invention are also summarized in Table 1. However,
derivatives/progenies of those cells, other mammalian cells,
including but not limited to human, mice, rat, monkey, and rodent
cell lines, can also be used in the present invention, particularly
for the production of biopharmaceutical proteins. Even other
eukaryotic cells, including but not limited to yeast, insect and
plant cells, can also be used in the methods and uses as described
herein.
TABLE-US-00001 TABLE 1 Eukaryotic production cell lines CELL LINE
ORDER NUMBER NS0 ECACC No. 85110503 Sp2/0-Ag14 ATCC CRL-1581 BHK21
ATCC CCL-10 BHK TK.sup.- ECACC No. 85011423 HaK ATCC CCL-15
2254-62.2 (BHK-21 derivative) ATCC CRL-8544 CHO ECACC No. 8505302
CHO wild type ECACC 00102307 CHO-K1 ATCC CCL-61 CHO-DUKX ATCC
CRL-9096 (= CHO duk.sup.-, CHO/dhfr.sup.-) CHO-DUKX B11 ATCC
CRL-9010 CHO-DG44 (Urlaub et al., 1983) CHO Pro-5 ATCC CRL-1781 V79
ATCC CCC-93 B14AF28-G3 ATCC CCL-14 HEK 293 ATCC CRL-1573 COS-7 ATCC
CRL-1651 U266 ATCC TIB-196 HuNS1 ATCC CRL-8644 CHL ECACC No.
87111906 CAP.sup.1 Wolfel et al.(2011) PER.C6 .RTM. Pau et al.
(2001)/Crucell H4-II-E ATCC CRL-1548 ECACC No.87031301 Reuber
(1961), Pitot (1964) H4-II-E-C3 ATCC CRL-1600 H4TG ATCC CRL-1578
H4-II-E DSM A003129 H4-II-Es DSM ACC3130 .sup.1CAP (CEVEC's
Amniocyte Production) cells are an immortalized cell line based on
primary human amniocytes. They were generated by transfection of
these primary cells with a vector containing the functions El and
pIX of adenovirus 5. CAP cells allow for competitive stable
production of recombinant proteins with excellent biologic activity
and therapeutic efficacy as a result of authentic human
posttranslational modification.
[0097] Host cells are most preferred, when being established,
adapted, and completely cultivated under serum free conditions, and
optionally in media, which are free of any protein/peptide of
animal origin. Commercially available media such as Ham's F12
(Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's
Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium
(MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma),
CD-CHO (Invitrogen, Carlsbad, Calif.), CHO-S-Invitrogen),
serum-free CHO Medium (Sigma), and protein-free CHO Medium (Sigma)
are exemplary appropriate nutrient solutions. Any of the media may
be supplemented as necessary with a variety of compounds,
non-limiting examples of which are hormones and/or other growth
factors (such as insulin, transferrin, epidermal growth factor,
insulin like growth factor), salts (such as sodium chloride,
calcium, magnesium, phosphate), buffers (such as HEPES),
nucleosides (such as adenosine, thymidine), glutamine, glucose or
other equivalent energy sources, antibiotics and trace elements.
Any other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. In
the present invention the use of serum-free medium is preferred,
but media supplemented with a suitable amount of serum can also be
used for the cultivation of host cells. For the growth and
selection of genetically modified cells expressing the selectable
gene a suitable selection agent is added to the culture medium.
[0098] The term "protein" is used interchangeably with "amino acid
residue sequences" or "polypeptide" and refers to polymers of amino
acids of any length. These terms also include proteins that are
post-translationally modified through reactions that include, but
are not limited to, glycosylation, acetylation, phosphorylation or
protein processing. Modifications and changes, for example fusions
to other proteins, amino acid sequence substitutions, deletions or
insertions, can be made in the structure of a polypeptide while the
molecule maintains its biological functional activity. For example
certain amino acid sequence substitutions can be made in a
polypeptide or its underlying nucleic acid coding sequence and a
protein can be obtained with the same properties.
[0099] The term "polypeptide" means a sequence with more than 10
amino acids and the term "peptide" means sequences with up to 10
amino acids in length. However, the terms might be used
interchangeably.
[0100] The present invention is suitable to generate host cells for
the production of biopharmaceutical polypeptides/proteins. The
invention is particularly suitable for the high-yield expression of
a large number of different genes of interest by cells showing
enhanced cell productivity.
[0101] "Gene of interest" (GOI), "selected sequence", "gene that
encodes a product/protein of interest" or "product gene" have the
same meaning herein and refer to a polynucleotide sequence of any
length that encodes a product of interest or "protein of interest",
also mentioned by the term "desired product". The selected sequence
can be full length or a truncated gene, a fusion or tagged gene,
and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a
cDNA. It can be the native sequence, i.e. naturally occurring
form(s), or can be mutated or otherwise modified as desired. These
modifications include codon optimizations to optimize codon usage
in the selected host cell, humanization, fusion or tagging. The
selected sequence can encode a secreted, cytoplasmic, nuclear,
membrane bound or cell surface polypeptide.
[0102] The "protein of interest" or "desired protein" includes
proteins, polypeptides, fragments thereof, peptides, all of which
can be expressed in the selected host cell. Proteins of interest
are preferably therapeutic proteins. Proteins of interest can be
for example antibodies, enzymes, cytokines, lymphokines, adhesion
molecules, receptors and derivatives or fragments thereof, and any
other polypeptides that can serve as agonists or antagonists and/or
have therapeutic or diagnostic use. Examples for a desired
protein/polypeptide are also given below. In the case of more
complex molecules such as monoclonal antibodies, the GOI encodes
one or both of the two antibody chains.
[0103] The term "producing" or "highly producing", "production",
"production and/or secretion", "producing" or "production cell" as
used herein relates to the production of the protein or product of
interest. An "increased production and/or secretion" relates to the
expression of the protein of interest or the product of interest
and means an increase in specific productivity, increased titer or
both. Preferably the specific productivity and the titre are
increased. Increased titer as used herein relates to an increased
concentration in the same volume, i.e., an increase in total yield.
The produced protein of interest may be, for example, a secreted,
cytoplasmic, nuclear, membrane bound, or a cell surface
polypeptide, preferably, the protein of interest is a secreted
protein.
[0104] The term "antibody" refers to a protein consisting of one or
more polypeptides substantially encoded by immunoglobulin genes.
The recognized immunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon and mu constant regions genes as well
as the myriad immunoglobulin variable region genes. As used herein,
the term "antibody" includes a polyclonal, monoclonal, bi-specific,
multi-specific, human, humanized, or chimeric antibody. The terms
"antibody" and "immunoglobulin" are used interchangeably and are
used to denote, without being limited thereto, glycoproteins having
the structural characteristics noted above for immunoglobulins.
[0105] The term "antibody" is used herein in its broadest sense and
specifically covers single monoclonal antibodies (including agonist
and antagonist antibodies) and antibody compositions with
polyepitopic specificity. The term "antibody" specifically covers
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.
bispecific antibodies) and antibody fragments (such as Fv, Fab,
Fab', F(ab)2 or other antigen-binding subsequences of antibodies).
Preferably, they contain or are modified to contain at least the
portion of the CH2 domain of the heavy chain immunoglobulin
constant region comprising the N-linked glycosylation site.
Exemplary antibodies within the scope of the present invention
include but are not limited to anti-CD20, anti-CD33, anti-CD37,
anti-CD40, anti-CD44, anti-CD52, anti-HER2/neu (erbB2), anti-EGFR,
anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2 or anti-IgE
antibodies.
[0106] The term "monoclonal antibody" (mAb) as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies based on the amino acid sequence. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each mAb is directed against a single determinant on
the antigen. In addition to their specificity, the mAbs are
advantageous in that they can be synthesized by cell culture
(hybridomas, recombinant cells or the like) uncontaminated by other
immunoglobulins. The mAbs herein include chimeric, humanized and
human antibodies. "Chimeric antibodies" are antibodies, wherein
light and/or heavy chain genes have been constructed, typically by
genetic engineering, from immunoglobulin variable and constant
regions of different species, such as mouse and human. Or
alternatively, whose heavy chain genes are belonging to a
particular antibody class or subclass while the remainder of the
chain is from another antibody class or subclass of the same or
another species. Also covered are fragments of such antibodies,
preferably fragments that contain or are modified to contain at
least one CH2 domain. For example, the variable segments of the
genes from a mouse monoclonal antibody may be joined to human
constant segments, such as gamma 1 and gamma 3. A typical
therapeutic chimeric antibody is thus a hybrid protein composed of
the variable or antigen-binding domain from a mouse antibody and
the constant or effector domain from a human antibody (e.g. ATCC
Accession No. CRL 9688 secretes an anti-Tac chimeric antibody),
although other mammalian species may be used.
[0107] The term "humanized antibodies" according to the present
invention refers to specific chimeric antibodies, immunoglobulin
chains or fragments thereof (such as Fv, Fab, Fab', F(ab)2 or other
antigen-binding subsequences of antibodies), which contain minimal
sequence derived from non-human immunoglobulin. Preferably they
contain or are modified to contain at least the portion of the CH2
domain of the heavy chain immunoglobulin constant region comprising
the N-linked glycosylation site. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a complementary-determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by the
corresponding non-human residues. Furthermore, humanized antibodies
can comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. These
modifications are made to further refine and maximize antibody
performance. In general, the humanized antibody will comprise at
least one, and typically two, variable domains, in which all or
substantially all off the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the
framework regions are those of a human immunoglobulin consensus
sequence. Preferably, the humanized antibody also comprises at
least a portion of an immunoglobulin constant region, typically
that of a human immunoglobulin.
[0108] Humanized antibody: comprising a human framework region and
one or more CDRs from a non-human (usually a mouse or rat)
antibody. Adjustments in framework amino acids might be required to
keep antigen binding specificity, affinity and or structure of
domain.
[0109] The term "CH2 domain" according to the present invention is
meant to describe the CH2 domain of the heavy chain immunoglobulin
constant region comprising the N-linked glycosylation site. In
defining an immunoglobulin CH2 domain reference is made to
immunoglobulins in general and in particular to the domain
structure of immunoglobulins as applied to human IgG1 by Kabat, E.
A. (Kabat, 1988; Kabat et al., 1991). Accordingly, immunoglobulins
are generally heterotetrameric glycoproteins of about 150 kDa,
composed of two identical light and two identical heavy chains.
Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulins isotypes.
Each heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has an amino terminal variable
domain (VH) followed by carboxy terminal constant domains (CH).
Each light chain has a variable N-terminal domain (VL) and a
C-terminal constant domain (CL).
[0110] Depending on the amino acid sequence of the constant domain
of the heavy chains, antibodies can be assigned to different
classes. There are five major classes: IgA, IgD, IgE, IgG and IgM.
The heavy chain constant domains that correspond to the different
classes of antibodies are called alpha, delta, epsilon, gamma and
mu domains, respectively. The mu chain of IgM contains five domains
(VH, CHmu1, CHmu2, CHmu3 and CHmu4). The heavy chain of IgE also
contains five domains while the heavy chain of IgA has four
domains. The immunoglobulin class can be further divided into
subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2.
[0111] The subunit structures and three-dimensional configuration
of different classes of immunoglobulins are well known. Of these
IgA and IgM are polymeric and each subunit contains two light and
two heavy chains. The heavy chain of IgG contains a polypeptide
chain lying between the CHgamma1 and CHgamma2 domains known as the
hinge region. The alpha chain of IgA has a hinge region containing
an O-linked glycosylation site. The mu and epsilon chains do not
have a sequence analogous to the hinge region of the gamma and
alpha chains, however, they contain a fourth constant domain
lacking in the other in the other immunoglobulin classes.
[0112] A "CH2 domain" therefore is an immunoglobulin heavy chain
constant region domain. The Fc region of a full antibody usually
comprises two CH2 domains and two CH3 domains. According to the
present invention, the CH2 domain is preferably the CH2 domain of
one of the five immunoglobulin classes indicated above. Preferred
are mammalian immunoglobulin CH2 domains such as primate or murine
immunoglobulin with the primate and especially human immunoglobulin
CH2 domains being preferred. The amino acid sequences of
immunoglobulin CH2 domains are known or are generally available to
the skilled artisan (Kabat et al., 1991). A preferred
immunoglobulin CH2 domain within the context of the present
invention is a human IgG and preferably from IgG1, IgG2, IgG3,
IgG4, more preferably a human IgG1 and IgG3 and even more preferred
a human IgG1. Using the numbering system of Edelman (Edelman et
al., 1969), the immunoglobulin CH2 domain preferably begins at
amino acid position equivalent to glutamine 233 of human IgG1 and
extends through amino acid equivalent to lysine 340 (Ellison and
Hood, 1982).
[0113] With respect to human antibody molecules reference is made
to the IgG class in which an N-linked oligosaccharide is attached
to the amide side chain of Asn 297 of the beta-4 bend to the inner
face of the CH2 domain of the Fc region. Preferably, the antibody
or Fc-fusion protein contains or is modified to contain at least a
CH2 domain. The CH2 domain is a CH2 domain of an immunoglobulin
having a single N-linked oligosaccharide of a human IgG CH2 domain.
The CH2 domain is preferably the CH2 domain of human IgG1.
[0114] The term "Proteins of interest", "products of interest" or
"desired proteins" as used herein include, but are not limited to
antibodies and Fc-fusion proteins, all of which can be expressed in
the host cells of the invention. Furthermore, desired proteins or
proteins of interest can be for example enzymes, cytokines,
lymphokines, adhesion molecules, receptors and derivatives or
fragments thereof, and any other polypeptides and scaffolds that
can serve as agonists or antagonists and/or have therapeutic or
diagnostic use. The "product of interest" may also be an antisense
RNA.
[0115] Especially, desired proteins/polypeptides or proteins of
interest are for example, but not limited to insulin, insulin-like
growth factor, hGH, tPA, cytokines, such as interleukines (IL),
e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon
(IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor
necrosis factor (TNF), such as TNF alpha and TNF beta, TNF gamma,
TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, VEGF, and single domain
antibodies (camelid derived antibodies). Also included is the
production of erythropoietin or any other hormone growth factors
and any other polypeptides that can serve as agonists or
antagonists and/or have therapeutic or diagnostic use.
[0116] A preferred protein of interest is an antibody or a fragment
or derivative thereof. Thus, the invention can be advantageously
used for production of antibodies such as monoclonal, polyclonal,
multispecific antibodies, or fragments thereof which comprise a CH2
domain, Fc and Fc'-fragments, heavy and light immunoglobulin chains
and/or their constant fragments. Furthermore, the method for
producing a (recombinant) protein according to the invention can be
advantageously used for production of antibodies such as
monoclonal, polyclonal, multispecific antibodies, or fragments
thereof which comprise a CH2 domain, Fc and Fc'-fragments, heavy
and light immunoglobulin chains and/or their constant fragments as
well as Fc-fusion proteins.
[0117] "Fc-fusion proteins" are defined as proteins which contain
or are modified to contain at least the portion of the CH2 domain
of the heavy chain immunoglobulin constant region comprising the
single N-linked glycosylation site. According to the Kabat EU
nomenclature (Kabat et al., 1991) this N-linked glycosylation site
is at position Asn297 in an IgG1, IgG2, IgG3 or IgG4 antibody.
[0118] The other part of the fusion protein can be the complete
sequence or any part of the sequence of a natural or modified
heterologous protein or a composition of complete sequences or any
part of the sequence of a natural or modified heterologous protein.
The immunoglobulin constant domain sequences may be obtained from
any immunoglobulin subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1
or IgA2 subtypes or classes such as IgA, IgE, IgD or IgM.
Preferentially they are derived from human immunoglobulin, more
preferred from human IgG and even more preferred from human IgG1
and IgG3. Non-limiting examples of Fc-fusion proteins are MCP1-Fc,
ICAM-Fc, EPO-Fc and scFv fragments or the like coupled to the CH2
domain of the heavy chain immunoglobulin constant region comprising
the N-linked glycosylation site. Fc-fusion proteins can be
constructed by genetic engineering approaches by introducing the
CH2 domain of the heavy chain immunoglobulin constant region
comprising the N-linked glycosylation site into another expression
construct comprising for example other immunoglobulin domains,
enzymatically active protein portions, or effector domains. Thus,
an Fc-fusion protein according to the present invention comprises
also a single chain Fv fragment linked to the CH2 domain of the
heavy chain immunoglobulin constant region comprising e.g. the
N-linked glycosylation site.
[0119] Furthermore, antibody fragments include e.g. "Fab fragments"
(Fragment antigen-binding=Fab). Fab fragments consist of the
variable regions of both chains, which are held together by the
adjacent constant region. These may be formed by protease
digestion, e.g. with papain, from conventional antibodies, but
similar Fab fragments may also be produced by genetic engineering.
Further antibody fragments include F(ab')2 fragments, which may be
prepared by proteolytic cleavage with pepsin. Using genetic
engineering methods it is possible to produce shortened antibody
fragments which consist only of the variable regions of the heavy
(VH) and of the light chain (VL). These are referred to as Fv
fragments (Fragment variable=fragment of the variable part). Since
these Fv-fragments lack the covalent bonding of the two chains by
the cysteines of the constant chains, the Fv fragments are often
stabilised. It is advantageous to link the variable regions of the
heavy and of the light chain by a short peptide fragment, e.g. of
10 to 30 amino acids, preferably 15 amino acids. In this way a
single peptide strand is obtained consisting of VH and VL, linked
by a peptide linker. An antibody protein of this kind is known as a
single-chain-Fv (scFv). Examples of scFv-antibody proteins are
known to the person skilled in the art.
[0120] In recent years, various strategies have been developed for
preparing scFv as a multimeric derivative. This is intended to
lead, in particular, to recombinant antibodies with improved
pharmacokinetic and biodistribution properties as well as with
increased binding avidity. In order to achieve multimerisation of
the scFv, scFv were prepared as fusion proteins with
multimerisation domains. The multimerisation domains may be, e.g.
the CH3 region of an IgG or coiled coil structure (helix
structures) such as Leucin-zipper domains. However, there are also
strategies in which the interaction between the VH/VL regions of
the scFv are used for the multimerisation (e.g. dia-, tri- and
pentabodies). By diabody the skilled person means a bivalent
homodimeric scFv derivative. The shortening of the Linker in a scFv
molecule to 5-10 amino acids leads to the formation of homodimers
in which an inter-chain VH/VL-superimposition takes place.
Diabodies may additionally be stabilised by the incorporation of
disulphide bridges. Examples of diabody-antibody proteins are known
to the person skilled in the art.
[0121] By minibody the skilled person means a bivalent, homodimeric
scFv derivative. It consists of a fusion protein which contains the
CH3 region of an immunoglobulin, preferably IgG, most preferably
IgG1 as the dimerisation region which is connected to the scFv via
a Hinge region (e.g. also from IgG1) and a Linker region. Examples
of minibody-antibody proteins are known to the person skilled in
the art.
[0122] By triabody the skilled person means a trivalent
homotrimeric scFv derivative. In said scFv derivatives the VH-VL
domains are fused directly without a linker sequence, which leads
to the formation of trimers.
[0123] By "scaffold proteins" the skilled person means any
functional domain of a protein that is coupled by genetic cloning
or by co-translational processes with another protein or part of a
protein that has another function.
[0124] The skilled person will also be familiar with so-called
miniantibodies which have a bi-, tri- or tetravalent structure and
are derived from scFv. The multimerisation is carried out by di-,
tri- or tetrameric coiled coil structures.
[0125] The protein of interest, especially the antibody, antibody
fragment or Fc-fusion protein, is preferably recovered/isolated
from the culture medium as a secreted polypeptide, or it can be
recovered/isolated from host cell lysates if expressed without a
secretory signal. It is necessary to purify the protein of interest
from other recombinant proteins and host cell proteins to obtain
substantially homogenous preparations of the protein of interest.
As a first step, cells and/or particulate cell debris are removed
from the culture medium or lysate. Further, the product of interest
is purified from contaminant soluble proteins, polypeptides and
nucleic acids, for example, by fractionation on immunoaffinity or
ion-exchange columns, ethanol precipitation, reverse phase HPLC,
Sephadex chromatography, and chromatography on silica or on a
cation exchange resin such as DEAE. Methods for purifying a
heterologous protein expressed by host cells are known in the
art.
[0126] By definition any sequences or genes introduced into a host
cell are called "heterologous sequences", "heterologous genes",
"heterologous RNAs" or "transgenes" or "recombinant gene" with
respect to the host cell, even if the introduced sequence, RNA or
gene is identical to an endogenous sequence, RNA or gene in the
host cell. A "heterologous" or "recombinant" protein or RNA is thus
a protein or RNA expressed from a heterologous sequence or gene. In
a preferred embodiment, the introduced sequence, RNA or gene is not
identical to an endogenous sequence, RNA or gene of the host cell
in question, although embodiments where it is identical are also
contemplated in connection with the present invention.
[0127] The term "recombinant" is used interchangeably with the term
"heterologous" throughout the specification of this present
invention, especially in the context with protein and RNA
expression. Thus, a "recombinant" protein is a protein expressed
from a heterologous sequence.
[0128] "Heterologous gene sequences" or "heterologous sequences"
can be introduced into a target cell by using an "expression
vector", preferably a eukaryotic, and even more preferably a
mammalian expression vector. Methods used to construct vectors are
well known to the person skilled in the art and described in
various publications. In particular techniques for constructing
suitable vectors, including a description of the functional
components such as promoters, enhancers, termination and
polyadenylation signals, selection markers, origins of replication,
and splicing signals, are reviewed in considerable details in
(Sambrook et al., 1989) and references cited therein. Vectors may
include but are not limited to plasmid vectors, phagemids, cosmids,
artificial/mini-chromosomes (e.g. ACE), or viral vectors such as
baculovirus, retrovirus, adenovirus, adeno-associated virus, herpes
simplex virus, retroviruses and bacteriophages. The eukaryotic
expression vectors will typically contain also prokaryotic
sequences that facilitate the propagation of the vector in bacteria
such as an origin of replication and antibiotic resistance genes
for selection in bacteria. A variety of eukaryotic expression
vectors, containing a cloning site into which a polynucleotide can
be operably linked, are well known in the art and some are
commercially available from companies such as Stratagene, La Jolla,
Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis. or BD
Biosciences Clontech, Palo Alto, Calif. Usually expression vectors
also comprise an expression cassette encoding a selectable marker,
allowing selection of host cells carrying said expression
marker.
[0129] In the present invention the expression vectors are also
used for introducing "heterologous sequences" or "polynucleotide
sequences" encoding RNAs, preferably non-coding RNAs, more
preferably miRNAs, into a host cell. Such expression vectors may
comprise genomic microRNA sequences for transient or stable
expression of miRNAs in cells, specifically in mammalian cells,
even more specifically in CHO cells. Preferably, said expression
vector is a mammalian expression vector. Means for cloning genomic
microRNA into an expression vector are known to the person skilled
in the art. They include, but are not limited to cloning genomic
microRNA sequences with approximately 300 bp flanking regions into
a mammalian expression vector, such as pBIP-1, operably linked to a
promoter, preferably a strong promoter, such as a CMV promoter or
any other strong promoter known to work in the host cell.
[0130] Alternatively, one or more microRNAs may be cloned as
polynucleotides encoding engineered pre-miRNA sequences (i.e. short
hairpins) into a mammalian expression vector, such as
pcDNA6.2-GW/miR or pcDNA6.2-GW/EmGFP-miR from Invitrogen (see
manual BLOCK-iT.TM. Pol II miR RNAi Expression Vector Kits). Said
vector may encode one or more copies of the same or different
miRNAs. In brief, the mature miRNA sequence is cloned into a given
sequence encoding an optimized hairpin loop sequence and 3' and 5'
flanking regions derived from the murine miRNA mir-155
(Lagos-Quintana et al., 2002). The flanking regions are present on
the vector and a DNA oligonucleotide is designed, which encodes the
miRNA sequence, the mentioned loop and the antisense sequence of
the respective mature miRNA with a two nucleotide depletion to
generate a internal loop in the hairpin stem. Furthermore,
overhangs are added for cloning at both ends. Hairpin structure may
be analyzed using the online tool mfold (M. Zuker. Mfold web server
for nucleic acid folding and hybridization prediction. Nucleic
Acids Res. 31 (13), 3406-3415, 2003). DNA strands are annealed and
ligated into the 3'-UTR of emerald GFP reporter protein gene as
described by the manufacturer. A vector containing more than one
miRNA may be generated applying the chaining method. The negative
control miRNA (supplied by the manufacturer) and the siLacZ may be
used as appropriate negative controls. Alternative vectors that may
be used in the present invention for miRNA expression, without
being limited thereto, are pCMV-MIR (Origene), pmR-ZsGreen1
(clontech) and shMIMIC lentiviral miRNA vector
(ThermoScientific).
[0131] In a preferred embodiment the expression vector comprises at
least one nucleic acid sequence, which is a regulatory sequence
necessary for transcription and translation of nucleotide sequences
that encode a peptide/polypeptide/protein of interest or necessary
for transcription of nucleotide sequences that encode a RNA,
preferably a non-coding RNA, more preferably a miRNA.
[0132] The term "expression" as used herein refers to transcription
and/or translation of a heterologous nucleic acid sequence within a
host cell. The level of expression of a desired product/protein of
interest in a host cell may be determined on the basis of either
the amount of corresponding mRNA that is present in the cell, or
the amount of the desired polypeptide/protein of interest encoded
by the selected sequence as in the present examples. For example,
mRNA transcribed from a selected sequence can be quantified by
Northern blot hybridization, ribonuclease RNA protection, in situ
hybridization to cellular RNA or by PCR. Proteins encoded by a
selected sequence can be quantitated by various methods, e.g. by
ELISA, by Western blotting, by radioimmunoassays, by
immunoprecipitation, by assaying for the biological activity of the
protein, by immunostaining of the protein followed by FACS analysis
or by homogeneous time-resolved fluorescence (HTRF) assays. The
level of expression of a non-coding RNA, such as a miRNA can be
quantified by PCR, such as qPCR.
[0133] "Transfection" as used in the present invention relates to
the introduction of genetic material, into a mammalian host cell,
wherein the mammalian host cell may be transiently transfected or
stably transfected. The genetic material may be an expression
vector comprising a gene of interest or a polynucleotide sequence
encoding a non-coding RNA, such as a miRNA. Alternatively, mature
miRNAs may be transiently transfected into a host cell. Typically,
miRNAs are transfected as double stranded RNAs. It is also possible
to transfect an antisense strand RNA, which is chemically modified
to prevent RISC loading or to transfect a hairpin RNA.
[0134] Transfection of eukaryotic host cells with a polynucleotide
or expression vector, resulting in genetically modified cells or
transgenic cells, can be performed by any method known in the art
(see e.g. (Sambrook et al., 1989)). Transfection methods include,
but are not limited to liposome-mediated transfection, calcium
phosphate co-precipitation, electroporation, nucleofection,
nucleoporation, microporation, polycation (such as
DEAE-dextran)-mediated transfection, protoplast fusion, viral
infections and microinjection. Preferably, the transfection is a
stable transfection. The transfection method that provides optimal
transfection frequency and expression of the heterologous genes in
the particular host cell line and type is favoured. Suitable
methods can be determined by routine procedures. For stable
transfectants the constructs are either integrated into the host
cell's genome or an artificial chromosome/mini-chromosome or
located episomally so as to be stably maintained within the host
cell. Thus, the stably transfected sequences actually remain in the
genome of the cell and its daughter cells. Typically, this involves
the use of a selectable marker gene and the gene of interest or the
polynucleotide sequence encoding the RNA is integrated together
with the selectable marker gene. In some cases the entire
expression vector integrates into the cell's genome, in other cases
only parts of the expression vector integrate into the cell's
genome. Cells "stably expressing" a protein of interest or a RNA
are stably transfected with a gene of interest encoding said
protein of interest or with a polynucleotide sequence encoding said
RNA. Thus, the sequences encoding the protein of interest or RNA
remain in the genome of the cell and its daughter cells. However,
"stably expressing a protein of interest" also includes an
endogenous protein, if the protein of interest is endogenous to the
cell (e.g., insulin secretion by INS-1 cells).
[0135] A "selectable marker gene" or "selection marker gene" is a
gene which encodes a selectable marker and allows the specific
selection of cells which contain this gene, typically by the
addition of a corresponding "selecting agent" to the cultivation
medium. As an illustration, an antibiotic resistance gene may be
used as a positive selectable marker. Only cells which have been
transformed with this gene are able to grow in the presence of the
corresponding antibiotic and are thus selected. Untransformed
cells, on the other hand, are unable to grow or survive under these
selection conditions. There are positive, negative and bifunctional
selectable markers. Positive selectable markers permit the
selection and hence enrichment of transformed cells by conferring
resistance to the selecting agent or by compensating for a
metabolic or catabolic defect in the host cell. By contrast, cells
which have received the gene for the selectable marker can be
selectively eliminated by negative selectable markers. An example
of this is the thymidine kinase gene of the Herpes Simplex virus,
the expression of which in cells with the simultaneous addition of
acyclovir or gancyclovir leads to the elimination thereof. The
selectable marker genes useful in this invention also include the
amplifiable selectable markers. The literature describes a large
number of selectable marker genes including bifunctional
(positive/negative) markers (see for example WO 92/08796 and WO
94/28143). Examples of selectable markers which are useful in the
present invention include, but are not limited to the genes of
aminoglycoside phosphotransferase (APH), hygromycine
phosphostransferase (HYG), dihydrofolate reductase (DHFR),
thymidine kinase (TK), glutamine synthetase, asparagin synthetase
and genes which confer resistance to neomycin (G418/Geneticin),
puromycin, histidinol D, bleomycin, phleomycin, blasticidin and
zeocin. Also included are genetically modified mutants and
variants, fragments, functional equivalents, derivatives,
homologues and fusions with other proteins or peptides, provided
that the selectable marker retains its selective qualities. Such
derivatives display considerable homology in the amino acid
sequence in the regions or domains, which are deemed to be
selective.
[0136] Selection may also be made by fluorescence activated cell
sorting (FACS) using for example a cell surface marker, bacterial
.beta.-galactosidase or fluorescent proteins (e.g. green
fluorescent proteins (GFP) and their variants from Aequorea
victoria and Renilla reniformis or other species; red fluorescent
proteins, fluorescent proteins and their variants from
non-bioluminescent species (e.g. Discosoma sp., Anemonia sp.,
Clavularia sp., Zoanthus sp.) to select for recombinant cells.
[0137] The term "selection agent" or "selective agent" refers to a
substance that interferes with the growth or survival of a cell,
unless a certain selectable marker gene product is present in the
cell which alleviates the effect of the selection agent. For
example, to select for the presence of an antibiotic resistance
gene like APH (aminoglycoside phosphotransferase) in a transfected
cell the antibiotic Geneticin (G418) is used.
[0138] The term "modified neomycin-phosphotransferase (NPT)" covers
all the mutants described in WO2004/050884, particularly the mutant
D227G (Asp227Gly), which is characterised by the substitution of
aspartic acid (Asp, D) for glycine (Gly, G) at amino acid position
227 and particularly preferably the mutant F240I (Phe240Ile), which
is characterised by the substitution of phenylalanine (Phe, F) for
isoleucine (Ile, I) at amino acid position 240.
[0139] The "amplifiable selectable marker gene" usually codes for
an enzyme, which is needed for the growth of eukaryotic cells under
certain cultivation conditions. For example, the amplifiable
selectable marker gene may code for dihydrofolate reductase (DHFR)
or glutamine synthetase (GS). In this case the gene is amplified,
if a host cell transfected therewith is cultivated in the presence
of the selecting agent methotrexate (MTX) or methionine
sulphoximine (MSX), respectively. Sequences linked to the
amplifiable selectable marker gene (i.e., sequences physically
proximal thereto) are co-amplified together with the amplifiable
selectable marker gene. Said co-amplified sequences may be
introduced on the same expression vector or on separate
vectors.
[0140] The following Table 2 gives non-limiting examples of
amplifiable selectable marker genes and the associated selecting
agents, which may be used according to the invention. Suitable
amplifiable selectable marker genes are also described in an
overview by Kaufman (Kaufman, 1990).
TABLE-US-00002 TABLE 2 Amplifiable selectable marker genes
Amplifiable selectable marker gene Accession number Selecting agent
dihydrofolate reductase (DHFR) M19869 (hamster) methotrexate (MTX)
E00236 (mouse) metallothionein D10551 (hamster) cadmium M13003
(human) M11794 (rat) CAD (carbamoylphosphate M23652 (hamster)
N-phosphoacetyl-L-aspartate synthetase:aspartate D78586 (human)
transcarbamylase: dihydroorotase) adenosine-deaminase K02567
(human) Xyl-A- or adenosine, M10319 (mouse) 2'deoxycoformycin AMP
(adenylate)-deaminase D12775 (human) adenine, azaserin, coformycin
J02811(rat) UMP-synthase J03626 (human) 6-azauridine, pyrazofuran
IMP 5'-dehydrogenase J04209 (hamster) mycophenolic acid J04208
(human) M33934 (mouse) xanthine-guanine- X00221 (E. coli)
mycophenolic acid with limiting phosphoribosyltransferase xanthine
mutant HGPRTase or mutant J00060 (hamster) hypoxanthine,
aminopterine and thymidine-kinase M13542, K02581 (human) thymidine
(HAT) J00423, M68489 (mouse) M63983 (rat) M36160 (Herpes virus)
thymidylate-synthetase D00596 (human) 5-fluorodeoxyuridine M13019
(mouse) L12138 (rat) P-glycoprotein 170 (MDR1) AF016535 (human)
several drugs, e.g. adriamycin, J03398 (mouse) vincristin,
colchicine ribonucleotide reductase M124223, K02927 (mouse)
aphidicoline glutamine-synthetase (GS) AF150961 (hamster)
methionine sulphoximine (MSX) U09114, M60803 (mouse) M29579 (rat)
asparagine-synthetase M27838 (hamster) .beta.-aspartylhydroxamate,
albizziin, M27396 (human) 5'azacytidine U38940 (mouse) U07202 (rat)
argininosuccinate- synthetase X01630 (human) canavanin M31690
(mouse) M26198 (bovine) ornithine-decarboxylase M34158 (human)
.alpha.-difluoromethylornithine J03733 (mouse) M16982 (rat)
HMG-CoA-reductase L00183, M12705 (hamster) compactin M11058 (human)
N-acetylglucosaminyl-transferase M55621 (human) tunicamycin
threonyl-tRNA-synthetase M63180 (human) borrelidin
Na.sup.+K.sup.+-ATPase J05096 (human) ouabain M14511 (rat)
[0141] According to the invention a preferred amplifiable
selectable marker gene is a gene which codes for a polypeptide with
the function of GS or DHFR.
[0142] The term "transformation" or "to transform", "transfection"
or "to transfect" as used herein means any introduction of a
nucleic acid sequence into a cell, resulting in genetically
modified, recombinant, transformed or transgenic cells. The
introduction can be performed by any method known in the art.
Methods include but are not limited to lipofection,
electroporation, polycation (such as DEAE-dextran)-mediated
transfection, protoplast fusion, viral infections and
microinjection or may be carried out by means of the calcium
method, electroshock method, intravenous/intramuscular injection,
aerosol inhalation or an oocyte injection. The transformation may
result in a transient or stable transformation of the host cells.
The term "transfection" or "to transfect", "transformation" or "to
transform" also means the introduction of a viral nucleic acid
sequence in a way which is for the respective virus the naturally
one. The viral nucleic acid sequence needs not to be present as a
naked nucleic acid sequence but may be packaged in a viral protein
envelope. Thus, the term relates not only to the method which is
usually known under the term "transfection" or "to transfect",
"transformation" or "to transform". Transfection methods that
provide optimal transfection frequency and expression of the
introduced nucleic acid are favoured. Suitable methods can be
determined by routine procedures. For stable transfectants the
constructs are either integrated into the host cell's genome or an
artificial chromosome/mini-chromosome or located episomally so as
to be stably maintained within the host cell.
[0143] The term "microRNA engineered cells" or "microRNA engineered
host cells" or "cells engineered to exhibit increased levels of
microRNAs" as used herein relates to mammalian cells, such as CHO
cells, transfected with any one of the RNAs, preferably non-coding
RNAs, more preferably miRNAs of the invention or an expression
vector encoding any one of the RNAs, preferably non-coding RNAs,
more preferably miRNAs of the invention as shown in Table 1B.
Further encompassed are combination of two or more RNAs, preferably
non-coding RNAs, more preferably miRNAs of the invention as shown
in Table 1B, wherein the two or more miRNAs may be the same or
different. Preferably, the mammalian cells are stably transfected
with an expression vector encoding at least one miRNA of the
invention. Such cells may also be referred to as stably microRNA
engineered host cells.
Embodiments
[0144] In a first aspect, the invention concerns a ribonucleic acid
(RNA) selected from the group consisting of: SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, wherein
said RNA leads to an increase in the production and/or secretion of
a protein of interest from a eukaryotic cell, preferably from a
mammalian cell. Preferably said RNA is a small non-coding RNA such
as a micro ribonucleic acid (miRNA). More preferably said RNA is a
miRNA. In one embodiment the invention concerns a micro ribonucleic
acid (miRNA) selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20,
wherein said miRNA leads to an increase in the production and/or
secretion of a therapeutic protein of interest from a mammalian
cell.
[0145] The RNAs may lead to an increase in the production and/or
secretion of a protein of interest in a species-independent manner.
Non-limiting examples thereof are miR-125a-3p (SEQ ID NO: 1),
miR-1271 (SEQ ID NO: 3), miR-1275 (SEQ ID NO: 4), miR-183 (SEQ ID
NO: 8) and miR-557 (SEQ ID NO: 16). Some preferred RNAs therefore
comprise: miR-125a-3p (SEQ ID NO: 1), miR-1271 (SEQ ID NO: 3),
miR-1275 (SEQ ID NO: 4), miR-183 (SEQ ID NO: 8) and miR-557 (SEQ ID
NO: 16). In another preferred embodiment the RNA is selected from
the group consisting of RNA-125a-3p (SEQ ID NO:1), miR-1271 (SEQ ID
NO:3), miR-1287 (SEQ ID NO:6), miR-183 (SEQ ID NO: 8), miR-185*
(SEQ ID NO:9), miR-193b* (SEQ ID NO:10), miR-1978 (SEQ ID NO:11),
miR-365* (SEQ ID NO:14), miR-557 (SEQ ID NO:16), miR-612 (SEQ ID
NO:17), miR-644a (SEQ ID NO:18) and miR-885-3p (SEQ ID NO:19), more
preferably the RNA is selected from the group consisting of:
miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO: 6), miR-183 (SEQ ID
NO:8), miR-185* (SEQ ID NO: 9), miR-1978 (SEQ ID NO:11), miR-365*
(SEQ ID NO:14), miR-557 (SEQ ID NO:16), miR-612 (SEQ ID NO:17),
miR-644a (SEQ ID NO:18) and miR-885-3p (SEQ ID NO: 19), more
preferably from the group consisting of miR-1271 (SEQ ID NO: 3),
miR-1287 (SEQ ID NO:6), miR-1978 (SEQ ID NO:11) and miR-557 (SEQ ID
NO:16). In one embodiment the RNA is miR-1978 (SEQ ID NO:11). In a
preferred embodiment the RNA is miR-1287 (SEQ ID NO:6) or miR-557
(SEQ ID NO:16). The RNA of the invention may be an isolated RNA,
preferably an isolated non-coding RNA, more preferably an isolated
miRNA.
[0146] The invention also relates to combinations of two or more
RNAs, preferably non-coding RNAs, more preferably miRNAs.
Particularly preferred combinations of two RNAs are miR-557 (SEQ ID
NO:16) and miR-1287 (SEQ ID NO:6), miR-557 (SEQ ID NO:16) and
miR-1978 (SEQ ID NO:11), miRNA-1271 (SEQ ID NO: 3) and miR-1978
(SEQ ID NO:11) or miR-1287 (SEQ ID NO: 6) and miRNA-1978 (SEQ ID
NO:11), with miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID NO:6)
being the most preferred combination.
[0147] In one embodiment of this aspect the protein of interest is
a recombinant protein. Preferably, the protein of interest is a
therapeutic protein, as described above. In a specific embodiment
the protein of interest is an antibody.
[0148] In a second aspect, the invention concerns a mammalian
expression vector comprising a ribonucleic acid (RNA) selected from
the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20.
[0149] In one embodiment a mammalian expression vector is provided
that encodes at least one ribonucleic acid (RNA) selected from the
group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20. Preferably the mammalian expression
vector comprises a polynucleotide sequence that comprises the at
least one RNA.
[0150] In certain embodiments the RNA is a micro ribonucleic acid
(miRNA) selected from the group consisting of: SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. The skilled
person will recognize that the RNA of this aspect is encoded by a
polynucleotide sequence. Hence, the expression vector comprises a
polynucleotide sequence encoding said RNA. An expression vector
according to the present invention may also comprise a
polynucleotide sequence encoding any of the RNAs of the first
aspect. Typically, the polynucleotide sequence is a DNA sequence
encoding a pre-miRNA, which is intracellularly processed to a
mature miRNA. Expression of said RNA leads to an increase in the
production and/or secretion of a protein of interest in a mammalian
expression system. The protein of interest may be a recombinant
protein, preferably a therapeutic protein as described above, more
preferably the protein of interest is an antibody.
[0151] The expression vectors of the present invention may further
comprise a selectable marker gene, such as an antibiotic resistance
gene or an amplifiable marker gene. In a specific embodiment the
expression vector comprises an amplifiable selection marker gene,
such as a glutamine synthetase gene or a dihydrofolate reductase
gene. The amplifiable selection marker gene, may be operably linked
to the polynucleotide sequence encoding the RNA. To be operably
linked, the polynucleotide sequence encoding the RNA and the
amplifiable selection marker gene may be located on the same
vector. In some embodiments, the expression vector of the invention
may also comprise a gene of interest. In a further embodiment
either the protein of interest or the RNA is operably linked to an
amplifiable selection marker, such as glutamine synthetase or
dihydrofolate reductase. Typically, the gene of interest and the
polynucleotide sequence encoding the RNA in the expression vector
of the invention are operably linked to a promoter and/or a
terminator. The gene of interest or the polynucleotide sequence
encoding the RNA operably linked to a promoter and/or a terminator
may also be referred to as an expression cassette.
[0152] In a preferred embodiment the RNA is selected from the group
consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, more
preferably the RNA is selected from the group consisting of:
miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO: 6), miR-183 (SEQ ID
NO:8), miR-185* (SEQ ID NO: 9), miR-1978 (SEQ ID NO:11), miR-365*
(SEQ ID NO:14), miR-557 (SEQ ID NO:16), miR-612 (SEQ ID NO:17),
miR-644a (SEQ ID NO:18) and miR-885-3p (SEQ ID NO: 19) and more
preferably the RNA is miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID
NO:6), miR-1978 (SEQ ID NO:11) or miR-557 (SEQ ID NO:16). In one
embodiment the RNA is miR-1978 (SEQ ID NO:11). In another preferred
embodiment the RNA is miR-1287 (SEQ ID NO:6) or miR-557 (SEQ ID
NO:16). In yet another embodiment, the RNA is miR-125a-3p (SEQ ID
NO: 1), miR-1271 (SEQ ID NO: 3), miR-1275 (SEQ ID NO: 4), miR-183
(SEQ ID NO: 8) or miR-557 (SEQ ID NO: 16), which are non-limiting
examples or RNAs that function in a species-independent manner. In
yet another embodiment the mammalian expression vector of the
invention comprises a combination of several identical or different
RNAs selected from the group consisting of: SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. As the
skilled person will understand, the expression vector according to
the invention comprising a combination of several identical or
different RNAs, comprises a polynucleotide sequence encoding a
combination of several, i.e., two or more, of the RNAs, wherein the
RNAs may be identical or different. An expression vector according
to the invention may also comprise a polynucleotide sequence
encoding a combination of two or more of the RNAs of the first
aspect, wherein the RNAs may be identical or different. Typically,
the polynucleotide sequence is a DNA sequence. Several identical or
different RNAs may be two, two or more, three or more, etc. copies
of the same RNA or of different RNAs and any combination thereof,
i.e., one RNA and a different RNA, two identical RNAs, two
identical and one different RNAs, three identical RNAs, three
different RNAs, etc. Typically, a combination of several identical
or different RNAs are two identical or two different RNAs.
Preferably, the several identical or different RNAs are selected
from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID
NO: 19. More preferably the RNAs are selected from the group
consisting of: miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO: 6),
miR-183 (SEQ ID NO:8), miR-185* (SEQ ID NO: 9), miR-1978 (SEQ ID
NO:11), miR-365* (SEQ ID NO:14), miR-557 (SEQ ID NO:16), miR-612
(SEQ ID NO:17), miR-644a (SEQ ID NO:18) and miR-885-3p (SEQ ID NO:
19), most preferably, the RNAs are miR-1271 (SEQ ID NO: 3),
miR-1287 (SEQ ID NO: 6), miR-1978 (SEQ ID NO: 11), miR-557 (SEQ ID
NO: 16) or a combination thereof, even more preferably the RNAs are
miR-557 (SEQ ID NO:16) and/or miR-1287 (SEQ ID NO:6).
[0153] In a specific embodiment the vector comprises a
polynucleotide sequence encoding a combination of several miR-1978s
(SEQ ID NO: 11) RNAs. The combination of several miRNA-1978s (SEQ
ID NO:11) may be two, three or even more miRNA-1978s (SEQ ID
NO:11), preferably two miRNA-1978s (SEQ ID NO:11). In a specific
embodiment the mammalian expression vector comprises a combination
of several identical RNAs, preferably two identical RNAs. Preferred
is a combination of several miR-1978s (SEQ ID NO: 11) RNAs. In a
preferred embodiment the mammalian expression vector encodes a
combination of several identical miR-557s (SEQ ID NO: 16),
preferably two miR-557s or a combination of several identical
miR-1287s (SEQ ID NO:6), preferably two miR-1287s. In another
preferred embodiment the mammalian expression vector encodes a
combination of miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID NO:6),
miR-557 (SEQ ID NO:16) and miR-1978 (SEQ ID NO:11), miRNA-1271 (SEQ
ID NO: 3) and miR-1978 (SEQ ID NO:11) or miR-1287 (SEQ ID NO: 6)
and miRNA-1978 (SEQ ID NO:11), preferably a combination of miR-557
(SEQ ID NO:16) and miR-1287 (SEQ ID NO:6).
[0154] Preferably, the RNA comprised in or encoded by any of the
expression vectors of the invention is a small non-coding RNA such
as a micro ribonucleic acid (miRNA). Preferably said non-coding RNA
is a miRNA. The expression vector may further comprise at least one
gene of interest. Typically, the polynucleotide sequences encoding
the RNA and/or the gene of interest are operably linked to a
promoter and/or a terminator. A gene of interest and the
polynucleotide sequence encoding the RNA operably linked to a
promoter and/or a terminator may also be referred to as an
expression cassette. In yet another aspect the invention concerns a
mammalian cell comprising one or more RNAs selected from one or
more of the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, preferably the RNA is
selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:
11, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and
SEQ ID NO: 19, more preferably the RNA is selected from the group
consisting of: miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO: 6),
miR-183 (SEQ ID NO:8), miR-185* (SEQ ID NO: 9), miR-1978 (SEQ ID
NO:11), miR-365* (SEQ ID NO:14), miR-557 (SEQ ID NO:16), miR-612
(SEQ ID NO:17), miR-644a (SEQ ID NO:18) and miR-885-3p (SEQ ID NO:
19) and more preferably the RNA is selected from the group
consisting of: miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO:6),
miR-1978 (SEQ ID NO:11), and miR-557 (SEQ ID NO:16). In one
embodiment the RNA is miR-1978 (SEQ ID NO: 11). In another
preferred embodiment the RNAs are miR-1287 (SEQ ID NO:6) and/or
miR-557 (SEQ ID NO:16).
[0155] Further, the one or more RNAs comprised in the mammalian
cell of the invention may be a combination of RNAs, preferably a
combination of two RNAs. Preferably said two RNAs are miR-557 (SEQ
ID NO:16) and miR-1287 (SEQ ID NO:6), miR-557 (SEQ ID NO:16) and
miR-1978 (SEQ ID NO:11), miRNA-1271 (SEQ ID NO: 3) and miR-1978
(SEQ ID NO:11) or miR-1287 (SEQ ID NO: 6) and miRNA-1978 (SEQ ID
NO:11), more preferably miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID
NO:6).
[0156] It will be understood that the one or more RNAs are
typically heterologous in respect of said mammalian cell, i.e.,
they (or the sequences encoding them) have been introduced, i.e,
transfected into said cell. Preferably, the RNA of any of the
mammalian cells of the invention is a non-coding RNA and more
preferably the non-coding RNA is a miRNA. Thus, the mammalian cell
of the invention further concerns a mammalian cell
comprising/transfected with one or more miRNAs, preferably
heterologous miRNAs, selected from the group consisting of: SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID
NO:20, preferably the miRNA is selected from the group consisting
of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, more preferably
the miRNA is selected from the group consisting of: miR-1271 (SEQ
ID NO: 3), miR-1287 (SEQ ID NO: 6), miR-183 (SEQ ID NO:8), miR-185*
(SEQ ID NO: 9), miR-1978 (SEQ ID NO:11), miR-365* (SEQ ID NO:14),
miR-557 (SEQ ID NO:16), miR-612 (SEQ ID NO:17), miR-644a (SEQ ID
NO:18) and miR-885-3p (SEQ ID NO: 19), and even more preferably the
miRNA is selected from the group consisting of: miR-1271 (SEQ ID
NO: 3), miR-1287 (SEQ ID NO:6), miR-1978 (SEQ ID NO:11), or miR-557
(SEQ ID NO:16). In one embodiment the miRNA is miR-1978s (SEQ ID
NO: 11) RNAs. In another preferred embodiment the mammalian cell
comprises one or more of miR-1287 (SEQ ID NO:6) and/or miR-557 (SEQ
ID NO:16). Further, the one or more miRNAs comprised in the
mammalian cell of the invention may be a combination of miRNAs,
preferably a combination of two miRNAs. Preferably, said two miRNAs
are miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID NO:6), miR-557 (SEQ
ID NO:16) and miR-1978 (SEQ ID NO:11), miRNA-1271 (SEQ ID NO: 3)
and miR-1978 (SEQ ID NO:11) or miR-1287 (SEQ ID NO: 6) and
miRNA-1978 (SEQ ID NO:11), more preferably miR-557 (SEQ ID NO:16)
and miR-1287 (SEQ ID NO:6).
[0157] Any one of the mammalian cells of the invention is
preferably transfected with said one or more RNAs. More preferably,
the mammalian cell of the invention is stably transfected with an
expression vector encoding said RNA, such as any of the expression
vectors of the present invention. Thus, the mammalian cell of the
present invention may be a stably miRNA engineered mammalian cell.
The mammalian cell may further express a protein/product of
interest, wherein the protein of interest may be endogenously
expressed by the host cell or the host cell is further transiently
or stably transfected with an expression vector encoding the
protein/product of interest. Preferably the mammalian cell is
further stably transfected with an expression vector encoding the
protein/product of interest. Sufficiently high stable levels of
said RNA in the host cell or the producer cell may be achieved,
e.g., by cloning multiple copies of the RNA encoding polynucleotide
into the expression vector. The mammalian host cell stably
transfected with one or more RNAs of the invention may be subjected
to batch or fed-batch fermentations.
[0158] The one or more RNA of the invention lead to an increase in
the production and/or secretion of a protein of interest, further
expressed by said mammalian cell. Preferably said protein of
interest is a recombinant protein, more preferably a therapeutic
protein, even more preferably a therapeutic antibody as described
above.
[0159] In one embodiment the production and/or secretion of the
protein of interest by the mammalian cell of the invention is
increased by 10%, 20%, 50%, 100%, 200%, 400% compared to a control
cell, which is not transfected with at least one RNA selected from
the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20. The invention further concerns a
mammalian cell comprising the mammalian expression vector of the
invention as described above. Also provided herein is the mammalian
cell of the invention, wherein the RNA, preferably the miRNA, is
encoded by the expression vector according to the invention.
[0160] Preferably, the mammalian cell of the invention is a rodent
or a human cell. In a specific embodiment the human cell is a
HEK-293 cell, a PER.C6 or a CAP cell. More preferably, the
mammalian cell is a rodent cell, even more preferably a CHO cell,
and most preferably a CHO-DG44 cell.
[0161] The human RNA or microRNA provided herein can be transiently
or stably introduced into pre-existing producer cell lines
secreting a therapeutic protein of interest. Alternatively, the RNA
or miRNA can either individually or in combination be introduced or
over-expressed in a host cell line to generate a superior host cell
that is an miRNA engineered host cell as basis for producer cell
line development.
[0162] It should be pointed out that miRNA-based engineering
approaches of the invention are also well suited to be combined
with classical cell engineering approaches, since they do not add
to the metabolic burden of the engineered cell. For example, an
engineering approach with proven benefit on secretion (e.g.
over-expression of a transgene such as XBP-1 or BiP or selected
product quality attributes) could be combined with a (mi)RNA-based
engineering approach to address additional features of cell
behavior.
[0163] In another aspect, the invention concerns a method of/for
developing a (high-producing) stably transfected mammalian cell,
comprising the following steps:
[0164] (a) transfecting at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 into the mammalian cell,
[0165] (b) cultivating said cell for an initial period of time in
the presence of selective pressure, and
[0166] (c) selecting a high-producing transfected cell.
[0167] The skilled person will know that transfecting the at least
one RNA into the mammalian cell comprises transfecting an
expression vector encoding the at least one RNA into said mammalian
cell, wherein the expression vector is stably transfected. Hence,
the expression vector comprises a polynucleotide sequence encoding
said RNA. The stably transfected mammalian cell is a mammalian cell
that is stably transfected with at least one RNA of the invention
and may also be referred to as stably RNA or miRNA engineered host
cell. The expression vector may be the expression vector of the
invention as described above. Typically, the expression vector
further comprises a selectable marker gene as described above,
wherein the selectable marker gene may be an amplifiable selectable
marker gene. Sufficiently high stable levels of said RNA in the
host cell or the producer cell may be achieved, e.g., by cloning
multiple copies of the RNA encoding polynucleotide into the
expression vector. The term "high-producing transfected cell" as
used herein relates to the production of a protein/product of
interest at high specific productivity and/or at high titers,
wherein the protein is preferably secreted. The mammalian cell used
in this aspect may endogenously express the protein/product of
interest. Alternatively, the mammalian cell stably transfected in
step (a) may be a producer host cell already comprising at least
one expression vector comprising a gene of interest. This may be
any new or established cell line developed for stably expressing
the protein of interest. Alternatively, the RNA in this aspect can
either individually or in combination be introduced or
over-expressed in a host cell line to generate a superior host cell
as basis for producer cell line development. The mammalian cell
stably transfected in step (a) may also be selected for a
high-producing transfected host cells in step (c) by transiently or
stably transfecting the cell of step (b) with an expression vector
encoding the protein of interest. The skilled person will
understand that cultivating said cell in step (b) in the presence
of selective pressure for an initial period of time, serves to
enrich for the stably transfected host cells of step (a). For
protein production, the high-producing transfected cell of step (c)
may be subjected to batch or fed-batch fermentation.
[0168] In one embodiment of the above aspect, the invention
concerns a method of/for developing a high-producing stably
transfected mammalian (host) cell (line), comprising the following
steps:
[0169] (a) transfecting a polynucleotide sequence encoding at least
one miRNAs selected from the group consisting of: SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 into the
mammalian cell,
[0170] (b) cultivating said cell (for an initial period) in the
presence of selective pressure (thus to enrich for the stably
transfected (host) cell of step (a)), and
[0171] (c) selecting a high-producing transfected (host) cell.
[0172] A specific example of such a cell development method is
provided in Example 11.
[0173] In a specific embodiment of this aspect the cell in step a)
is transfected with the vector of the invention as described
above.
[0174] In a further embodiment the cell in step a) is a producer
host cell comprising a vector encoding for a gene of interest.
[0175] In yet another aspect, the invention provides a method
of/for producing a protein of interest, comprising the following
steps:
[0176] (a) transfecting at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 and at least one expression vector
comprising at least one gene of interest into a mammalian cell,
[0177] (b) selecting a highly-productive transfected cell,
[0178] (c) cultivating the highly-productive transfected cell
obtained in step (b) under conditions which allow expression of the
gene(s) of interest, and
[0179] (d) harvesting and purifying the protein of interest.
[0180] The skilled person will know that transfecting the at least
one RNA into the mammalian cell may comprise transfecting an
expression vector encoding at least one of said RNAs into said
mammalian cell, wherein the expression vector is preferably stably
transfected. Hence, the expression vector comprises a
polynucleotide sequence encoding said RNA. Transfecting the at
least one RNA or the expression vector encoding the at least one
RNA may be done after, prior to or simultaneously to transfecting
the gene of interest. Alternatively, the expression vector
comprising the at least one gene of interest of step (a) may also
encode the at least one RNA of step (a).
[0181] In certain embodiments the mammalian cell in step (a) is
transfected with any of the expression vectors of the present
invention, preferably the mammalian cell in step (a) is stably
transfected with any of the expression vectors of the present
invention.
[0182] The production cells derived from RNA or microRNA engineered
host cells show higher production and/or secretion rates, i.e.
specific productivity and/or higher titers. The RNA or microRNA
engineering of step (a) can either be done after, prior to or
simultaneously to introducing the gene of interest with similar
results, thus offering a broad range of options for applications in
pharmaceutical development processes. Sufficiently high stable
levels of RNA in the host cell or the producer cell may be
achieved, e.g., by cloning multiple copies of the RNA encoding
polynucleotide into an expression vector. Cultivating the cells
under conditions which enable expression of the genes of interest
according to step (c) may comprise subjecting the cell to batch or
fed-batch fermentations.
[0183] In a specific embodiment of this aspect, the polynucleotide
sequence encoding said RNA is integrated into the mammalian
expression vector comprising the at least one gene of interest.
Hence, the expression vector comprises a polynucleotide sequence
encoding the RNA of the invention and the gene of interest.
[0184] Thus, the invention further concerns a method of/for
producing a protein of interest, characterised by the following
steps:
[0185] (a) integrating at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 into a mammalian expression vector
comprising at least one gene of interest,
[0186] (b) transfecting a mammalian cell with said expression
vector,
[0187] (c) selecting a highly-productive transfected cell,
[0188] (d) cultivating the highly-productive transfected cell
obtained in step (c) under conditions which allow expression of the
gene(s) of interest, and optionally
[0189] (e) harvesting and purifying the protein of interest.
[0190] The invention also concerns a method of producing a
heterologous protein of interest in a mammalian cell comprising
[0191] a) transfecting said mammalian cell with the RNA as
described above or a vector as described above, and
[0192] b) effecting the expression of said protein of interest.
[0193] The invention further concerns a method of/for preparing and
selecting a recombinant mammalian cell, comprising the following
steps:
[0194] (a) transfecting a mammalian cell with genes that encode at
least for a protein/product of interest and an amplifiable
selectable marker, such as DHFR or GS and wherein the (host) cell
is (co-) transfected with at least one RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20,
[0195] (b) selecting a cell with (co-)integrated genes by
cultivating the cell in the presence of a selective agent, such as
e.g. in a hypoxanthine/thymidine-free or glutamine-free medium,
and
[0196] (b') amplifying these (co-)integrated genes by cultivating
the cell in the presence of an agent which allows the amplification
of at least the amplifiable selectable marker gene, such as e.g.
methotrexate or MSX,
[0197] (c) cultivating the cell under conditions which enable
expression of the (different) genes.
[0198] In an alternative embodiment the invention concerns a method
of/for preparing and selecting a recombinant mammalian cell,
characterised by the following steps:
[0199] (a) transfecting a mammalian cell with genes that encode at
least for a protein/product of interest and a selectable marker,
such as a selectable marker conferring resistance to neomycin,
puromycin, bleomycin, zeocin or blasticidin and wherein the (host)
cell is (co-)transfected with at least one RNA selected from the
group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and SEQ ID NO:20,
[0200] (b) selecting a cell with (co-)integrated genes by
cultivating the cells in the presence of a selective agent, and
[0201] (c) cultivating the cell under conditions which enable
expression of the (different) genes.
[0202] The skilled person will recognize that transfecting the at
least one RNA into the mammalian cell according to step (a) of any
of the methods of the invention comprises transfecting an
expression vector encoding at least one of said RNAs into said
mammalian cell, wherein preferably the expression vector is stably
transfected. Hence, the expression vector comprises a
polynucleotide sequence encoding said RNA. In certain embodiments
the expression vector may be the expression vector of the invention
as described above. Sufficiently high stable levels of said RNA in
the host cell or the producer cell may be achieved, e.g., by
cloning multiple copies of the RNA encoding polynucleotide into the
expression vector. In the context of the invention the expression
vector encoding the RNA of the invention may further comprise a
gene of interest. The expression vector encoding the RNA of the
invention may also comprise genes that encode at least a
protein/product of interest and a selectable marker. Alternatively,
the expression vector encoding the RNA of the invention may be
transfected after, prior to or simultaneously to transfection of
the at least one gene of interest, i.e., the genes that encode a
protein/product of interest. Cultivating the cells under conditions
which enable expression of the genes according to step (c) may
comprise subjecting the cell to batch or fed-batch
fermentations.
[0203] In a specific embodiment of any of the above methods of the
invention the production and/or secretion of the protein of
interest is increased by 10%, 20%, 50%, 100%, 200%, 400% compared
to a control cell, which is not transfected with at least one RNA
selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
[0204] In a specific embodiment of any of the above methods of the
invention the proportion of high producers/high producer cells is
increased up to two-fold, three-fold, four-fold, five-fold,
six-fold, seven-fold or ten-fold or more than two-fold, more than
three-fold, more than four-fold, more than five-fold, more than
seven-fold or more than ten-fold. The increase is preferably
measured in relation to a control cell which is not transfected
with at least one RNA selected from the group consisting of: SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID
NO:20. A high producer cell is a cell, which produces the
protein/product of interest with a high titer and/or a high
specific cellular productivity, preferably in an amount sufficient
to enable a commercially viable biopharmaceutical production
process.
[0205] In a preferred embodiment of any of the above methods of the
invention said RNA is a small non-coding RNA such as a micro
ribonucleic acid (miRNA). More preferably said RNA is a miRNA. In a
certain embodiments of any of the methods of the invention the at
least one ribonucleic acid (RNA) is selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20.
[0206] In a further embodiment of any of the methods of the
invention the at least one RNA is SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, or a combination thereof. Preferably the RNA is selected
from the group consisting of: miR-1271 (SEQ ID NO: 3), miR-1287
(SEQ ID NO: 6), miR-183 (SEQ ID NO:8), miR-185* (SEQ ID NO: 9),
miR-1978 (SEQ ID NO:11), miR-365* (SEQ ID NO:14), miR-557 (SEQ ID
NO:16), miR-612 (SEQ ID NO:17), miR-644a (SEQ ID NO:18) and
miR-885-3p (SEQ ID NO: 19), or a combination thereof, more
preferably the at least one RNA is miR-1271 (SEQ ID NO: 3),
miR-1287 (SEQ ID NO: 6), miR-1978 (SEQ ID NO: 11), miR-557 (SEQ ID
NO: 16) or a combination thereof. Most preferably the RNA is
miR-1287 (SEQ ID NO: 6) and/or miR-557 (SEQ ID NO: 16).
[0207] The at least one transfected RNA may be a combination of
RNAs, preferably a combination of two RNAs. Preferably said two
RNAs are miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID NO:6), miR-557
(SEQ ID NO:16) and miR-1978 (SEQ ID NO:11), miRNA-1271 (SEQ ID NO:
3) and miR-1978 (SEQ ID NO:11) or miR-1287 (SEQ ID NO: 6) and
miRNA-1978 (SEQ ID NO:11), more preferably miR-557 (SEQ ID NO:16)
and miR-1287 (SEQ ID NO:6).
[0208] In yet another embodiment of any of the methods of the
invention, the RNA is miR-125a-3p (SEQ ID NO: 1), miR-1271 (SEQ ID
NO: 3), miR-1275 (SEQ ID NO: 4), miR-183 (SEQ ID NO: 8) or miR-557
(SEQ ID NO: 16), which are non-limiting examples of RNAs that
function in a species-independent manner.
[0209] In a specific embodiment of any of the methods of the
present invention said methods may further comprise the following
additional step: harvesting and purifying the protein of
interest.
[0210] The protein of interest in any of the methods of the
invention may be a recombinant protein, preferably a therapeutic
protein as described above, such as an antibody or an Fc-fusion
protein.
[0211] The mammalian cells used in any of the methods of the
invention may be a rodent or a human cell. In one embodiment the
cell is a human cell and the human cell may be, but is not limited
to a HEK-293 cell, a PER.C6 or a CAP cell. Preferably, the cell
used in any of the above methods is a rodent cell, more preferably
a CHO cell, most preferably a CHO-DG44 cell.
[0212] In another aspect, the invention concerns the use of a RNA
selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 as a
production-promoting element for the preparation of a
product/protein of interest. Thus, the product/protein of interest
may be a therapeutical protein or a biopharmaceutical product that
is intended as a medicament for medical use. Alternatively the
invention also concerns the use of a RNA selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, and SEQ ID NO:20 for increasing the production and/or
secretion of a product of interest from a mammalian cell.
[0213] Preferably, the product of interest according to any of the
uses of the invention is a protein of interest, more preferably the
protein of interest is a recombinant protein, such as a therapeutic
protein or an antibody as described herein. In a particularly
preferred embodiment, the protein of interest is an antibody or
antibody fragment or antibody fusion protein or an antibody
conjugate or antibody Fc-fusion protein.
[0214] The invention further concerns the use of a RNA selected
from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 for the production of a
non-human transgenic animal, preferably a mammal.
[0215] In one embodiment the invention provides a use of the
mammalian cell of the invention for producing a protein of
interest, wherein preferably said mammalian cell is a stably miRNA
engineered cell. The RNA according to any of the uses of the
invention is preferably a small non-coding RNA such as a micro
ribonucleic acid (miRNA). More preferably said RNA is a miRNA.
[0216] In one embodiment of any of the above uses the ribonucleic
acid (RNA) is selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID
NO:20.
[0217] In another embodiment of any of the above uses the RNA is
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. Preferably the RNA
is miR-1271 (SEQ ID NO: 3), miR-1287 (SEQ ID NO: 6), miR-1978 (SEQ
ID NO: 11), or miR-557 (SEQ ID NO: 16), more preferably the RNA is
selected from the group consisting of: miR-1271 (SEQ ID NO: 3),
miR-1287 (SEQ ID NO: 6), miR-183 (SEQ ID NO:8), miR-185* (SEQ ID
NO: 9), miR-1978 (SEQ ID NO:11), miR-365* (SEQ ID NO:14), miR-557
(SEQ ID NO:16), miR-612 (SEQ ID NO:17), miR-644a (SEQ ID NO:18) and
miR-885-3p (SEQ ID NO: 19). Most preferably the RNA is miR-1287
(SEQ ID NO: 6) and/or miR-557 (SEQ ID NO: 16).
[0218] Further, the RNA in any of the uses of the invention may be
a combination of RNAs, preferably a combination of two RNAs.
Preferably said two RNAs are miR-557 (SEQ ID NO:16) and miR-1287
(SEQ ID NO:6), miR-557 (SEQ ID NO:16) and miR-1978 (SEQ ID NO:11),
miRNA-1271 (SEQ ID NO: 3) and miR-1978 (SEQ ID NO:11) or miR-1287
(SEQ ID NO: 6) and miRNA-1978 (SEQ ID NO:11), more preferably
miR-557 (SEQ ID NO:16) and miR-1287 (SEQ ID NO:6).
[0219] In another embodiment of any of the above uses, the RNA is
miR-125a-3p (SEQ ID NO: 1), miR-1271 (SEQ ID NO: 3), miR-1275 (SEQ
ID NO: 4), miR-183 (SEQ ID NO: 8) or miR-557 (SEQ ID NO: 16), which
are non-limiting examples of RNAs that function in a
species-independent manner. Preferably, any of the cells according
to any of the uses of the invention is a rodent or a human cell. In
one embodiment the human cell is a HEK-293 cell, a PER.C6 or a CAP
cell. Preferably, the cell used in any of the above uses is a CHO
cell, most preferably a CHO-DG44 cell.
[0220] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology,
molecular biology, cell culture, immunology and the like which are
in the skill of one in the art. These techniques are fully
disclosed in the current literature. All publications and patent
applications mentioned in this specification are indicative of the
level of skill of those skilled in the art to which this invention
pertains. All publications and patent applications cited herein are
hereby incorporated by reference in their entirety in order to more
fully describe the state of the art to which this invention
pertains. The invention generally described above will be more
readily understood by reference to the following example, which is
hereby included merely for the purpose of illustration of certain
embodiments of the present invention and is not intended to limit
the invention in any way.
[0221] Materials and Methods
[0222] Cell Lines
TABLE-US-00003 Designation Description Species CHO/BIWA4
CHO-DG44-based producer cell clone Hamster secreting an IgG1
product CHO/BIBH1 CHO-DG44-based producer cell clone Hamster
secreting an IgG1 product CHO/HSA CHO-based cell pool producing
human Hamster Albumin HEK293 t-rex Human kidney cells transfected
to secret a Human flp-IN horse-raddish-peroxidase reporter protein
cells/ssHRP- flag HeLa cells Human cervix epithelial adenocarcinoma
cells Human INS-1 Rat insulinoma cells endogenously secreting Rat
insulin
[0223] Human MicroRNA Library
[0224] The human microRNA library is obtained from Dharmacon
(CS-001010 mimic microRNA library; lot number 09167).
[0225] Cell Culture of Suspension Cells
[0226] Suspension cultures of mAb producing CHO-DG44 cells (Urlaub
et al., 1986) and stable transfectants thereof are incubated in a
BI proprietary chemically defined, serum-free media. Seed stock
cultures are sub-cultivated every 2-3 days with seeding densities
of 3.times.10.sup.5-2.times.10.sup.5 cells/mL respectively. Cells
are grown in T-flask (Greiner). T-flasks are incubated in
humidified incubators (Varolab) at 37.degree. C. and 5% CO.sub.2.
The cell concentration and viability is determined by trypan blue
exclusion using a counting chamber.
[0227] Cell Culture of INS-1 Cells
[0228] INS-1 cells are cultured in RPMI 1640, 10% FCS, 10 mM HEPES,
50 .mu.M 2-Mercaptoethanole, 1 mM Na-Pyruvate, 2 mM Glutamate in
humidified incubators at 37.degree. C. and 5% CO.sub.2. Insulin
secreted from INS-1 cells is determined using the rat insulin ELISA
80-INSRT-E01 (alpco), following the manufacturer's
instructions.
[0229] Cell Culture of HEK293 FlpIN Cells and Hela Cells
[0230] HEK293, FlpIN and HeLa cells were cultured in DMEM or
RPMI1640 (Life Technologies), respectively, both supplemented with
10% FCS in humidified incubators at 37.degree. C. and 5% CO.sub.2.
Cells were subcultivated by trypsinization every 3 to 4 days when
reaching confluency.
[0231] Fed-Batch Cultivation
[0232] Cells are seeded at 3.times.10.sup.5 cells/ml into 125 ml
shake flasks (Corning) in 30 ml of BI-proprietary production medium
without antibiotics or MTX. The cultures are agitated at 120 rpm in
37.degree. C. and 5% CO.sub.2 in minitron incubator (Infors) which
is reduced to 2% following day 3. BI-proprietary feed solution is
added daily and pH is adjusted to pH 7.0 using NaCO.sub.3 as
needed. Cell densities and viability are determined by trypan-blue
exclusion using an automated counting chamber TC10 (Biorad).
Cumulative specific productivity is calculated as product
concentration analysed by ELISA at the given day divided by the
"integral of viable cells" (IVC) until that time point.
[0233] Transient Expression of Human MicroRNAs in CHO Producer
Cells--MicroRNA Screen
[0234] CHO-DG44 cells stably secreting an IgG1 antibody are
cultivated in BI proprietary chemically defined, serum-free medium
(Boehringer-Ingelheim) supplemented with 500 .mu.g/mL G418 (Gibco,
Life technologies) and 400 nM MTX (Sigma-Aldrich, Germany) and are
subcultivated every 2 or 3 days with a seeding density of
3.times.10.sup.5 cells/mL or 2.times.10.sup.5 cells/mL,
respectively.
[0235] Cells are transfected via nucleofection one day after
subcultivation (4e5 cells/sample) in 96-well NUCLEOFECTOR
Nucleofection Kit SG (Lonza) containing 1 .mu.M RNA using the Amaxa
96-well Shuttle Device (Lonza) and program 96-DT-133 according to
the manufacturer's instructions. Cells are then seeded with a
density of 3.times.10.sup.5 cells/mL into 4 96-well flat bottom
plates (Greiner). One day after transfection the volume of the
medium is doubled by addition of fresh medium. Supernatants are
collected on days 1-4 post transfection and stored at -20.degree.
C. until antibody measurement by ELISA. As negative controls a
mimic microRNA negative control #1 (Dharmacon) and a non-targeting
siRNA coupled with FITC (siLacZ-FITC) are used. FITC positive cells
are measured by flow cytometry to determine the transfection
efficiency. As a positive control, a siRNA targeting the light
chain of the IgG1 antibody is transfected resulting in decreased
antibody concentrations. The microRNA library is obtained from
Dharmacon (CS-001010 mimic microRNA library) and the siRNAs are
from MWG.
[0236] Antibody concentrations are normalized to the mean values of
all samples contained in the plate and compared with those of the
non-targeting and negative controls.
[0237] Validation Screen
[0238] CHO-DG44 cells are transfected via nucleofection as
described above and seeded into 12-well plates (Greiner). Cell
densities and viability are determined by trypan blue exclusion
using a CEDEX cell quantification system (Roche). Product
concentrations in the supernatant are measured by ELISA.
[0239] Determination of Recombinant Antibody Concentration
[0240] To assess recombinant antibody production in transfected
cells, supernatants are collected from cell cultures at the given
time points. The product concentration is then analyzed by enzyme
linked immunosorbent assay (ELISA). The concentration of secreted
monoclonal antibody product is measured using HRP-conjugated
antibodies against the human Fc fragment (Jackson Immuno Research
Laboratories) and the human kappa light chain (Sigma).
[0241] Transfection of INS-1 Cells
[0242] INS-1 cells are transfected by nucleofection. Cells are
trypsinized and 6.times.10.sup.6 cells/sample are nucleofected with
kit V (Lonza) and 2 .mu.M RNA using program T-27.
[0243] Crystal Violet Assay
[0244] Cells are washed with PBS and 500 .mu.L crystal violet
staining solution (0.1% in 20% methanol) per 24-well are incubated
for 20 minutes at room temperature. The plates are washed, dried
and the dye is dissolved in 200 .mu.L methanol per well by shaking
for 30 minutes. 50 .mu.L are transferred into 96-well plate and
measured with a multiscan reader (Thermo Scientific) at 550 nm.
[0245] Insulin Assay
[0246] Nucleofected INS1E cells are trypsinized and replated into 6
wells of a 24 well plate. One day later, cells are washed twice
with KRB (Krebs Ringer Buffer: 135 mM NaCl, 3.6 mM KCl, 1.5 mM
CaCl.sub.2), 0.5 mM NaH.sub.2PO.sub.4, 0.5 mM MgCl.sub.2, 5 mM
NaHCO.sub.3, 0.1% bovine serum albumin, 10 mM HEPES) 0.5 ml per
well and preincubated for 2 hours in KRB (1 mL per well). KRB is
removed and new KRB with or without 20 mM glucose is added. Cells
are incubated for 15 minutes before the supernatant is collected
and stored at -20.degree. C. Rat insulin concentration is measured
by ELISA (80-INSRT-E01 from alpco).
[0247] Human Albumin ELISA
[0248] Albumin concentrations in cell culture supernatants are
analyzed by ELISA (Bethyl Labs) according to manufacturer's
instructions. As a coating antibody goat anti-human albumin and as
a detection antibody HRP-conjugated goat anti-human albumin is
used. To generate a standard curve a reference serum provided in
the kit is used (Bethyl Labs).
[0249] Interleukin-8 (IL-8) Secretion Assay
[0250] HeLa cells were reverse transfected with microRNAs using
RNAiMAX transfection agent sold under the trademark LIPOFECTAMINE
(Life Technologies) according to manufacturer's instructions for
24-well plates (Greiner Bio-One). Two days post transfection cells
were washed, medium was replaced by fresh cell culture medium and
cell culture supernatants were harvested after 6 and 24 hours. The
concentration of IL-8 in clarified cell supernatants was quantified
by ELISA (human IL-8 ELISA Set, ImmunoTools) according to the
manufacturer's instructions and results were normalized to the
miRNA negative control. Mock-transfected cells served as an
additional control.
[0251] Transient HRP Secretion Assay
[0252] HeLa cells were reverse transfected with microRNAs using
RNAiMAX transfection agent sold under the trademark LIPOFECTAMINE
(Life Technologies) according to manufacturer's instructions
(Greiner Bio-One). Two days later, cells were transfected with a
plasmid encoding ssHRP-FLAG using Mirus Hela Monster (Mirus). After
24 hours, cells were washed and medium was replaced by fresh
serum-free and phenol red-free medium (RPMI1640) and cell culture
supernatants were harvested after 4 and 6 hours. The amount of
ssHRP in clarified cell supernatants was quantified by addition of
ECL reagent (Pierce) and measurement of the luminescence signal
(relative luminescence units) with a luminometer (Tecan) in white
96-well plates (Nunc). Relative luminescence units (RLU) were
normalized to those of the miRNA neg. control. Untransfected and
mock-transfected cells served as additional controls.
[0253] Stable HRP Secretion Assay
[0254] HEK293 FlpIn cells stably secreting ssHRP-Flag are reverse
transfected with microRNAs using RNAiMAX transfection agent sold
under the trademark LIPOFECTAMINE (Invitrogen) according to
manufacturer's instructions in collagen coated 24-well plates
(Greiner Bio-One). Two days post transfection, ssHRP expression is
induced by addition of 20 ng/ml doxycycline (Merck). After 12
hours, medium is replaced by fresh serum-free and phenol red-free
medium and cell culture supernatant is harvested after 5 hours. The
amount of ssHRP in clarified cell supernatants is quantified by
addition of ECL reagent (Pierce) and luminescence signal is
measured with a luminometer (Tecan) in white 96-well plates (Nunc).
Relative luminescence unites are normalized to miRNA neg. control
luminescence signals. To normalize for cell density, a crystal
violet assay is performed after supernatant collection and relative
luminescence units are divided by crystal violet signals.
[0255] Generation of Antibody-Producing Cells
[0256] CHO-K1 or CHO-DG44 cells (Urlaub et al., Cell 1983) are
stably transfected with expression plasmids encoding heavy and
light chains of an IgG1-type antibody. Selection is carried out by
cultivation of transfected cells in the presence of the respective
antibiotics encoded by the expression plasmids. After about 3 weeks
of selection, stable cell populations are obtained and further
cultivated according to a standard stock culture regime with
subcultivation every 2 to 3 days. In a next (optional) step,
FACS-based single cell cloning of the stably transfected cell
populations is carried out to generate monoclonal cell lines.
[0257] Stable Over Expression of MicroRNAs
[0258] Genomic microRNA sequences with approximately 300 bp
flanking regions are subcloned from pCMV-mir vectors (Origene) into
pBIP-1 (BI) containing a CMV promoter and a puromycin resistance
cassette (pBIP-1-mir plasmid). CHO-DG44 cells stably secreting an
IgG1 are transfected with LIPOFECTAMINE 2000 transfection agent and
Plus reagent (Invitrogen) using an optimized protocol with
pBIP-1-mir plasmids and cells are selected with 10 .mu.g/mL
puromycin. Single clones are generated by limited dilution.
Overexpression is verified by qPCR analysis. Alternatively, the
BLOCK-iT.TM. Pol II miR RNAi expression vector kit
(PcDNA6.2-GW/emGFP-miRNA expression system kit) was used for stably
expressing miRNAs. DNA oligonucleotides encoding one or multiple
copies of a specific or of different microRNAs are cloned as short
hairpins into the mammalian expression vector pcDNA6.2.
[0259] For that purpose, DNA oligonucleotides encoding the
respective miRNAs are designed as described in the manual. In
brief, the mature miRNA sequence was embedded in a given sequence
including an optimized hairpin loop sequence and 3' and 5' flanking
regions derived from the murine miRNA mir-155 (Lagos-Quintana et
al., 2002). The flanking regions are present on the vector and a
DNA oligonucleotide is designed, which encodes the miRNA sequence,
the mentioned loop and the antisense sequence of the respective
mature miRNA with a 2 nucleotide depletion to generate a internal
loop in the hairpin stem. Furthermore, overhangs are added for
cloning at both ends. Hairpin structure may be analyzed using the
online tool mfold (M. Zuker. Mfold web server for nucleic acid
folding and hybridization prediction. Nucleic Acids Res. 31 (13),
3406-3415, 2003). DNA strands are annealed and ligated into the
3'-UTR of emerald GFP reporter protein gene as described by the
manufacturer. A vector containing more than one miRNA may be
generated applying the chaining technique. The negative control
miRNA (supplied by the manufacturer) and the siLacZ may be used as
appropriate negative controls.
[0260] The BLOCK-iT.TM. Pol II miR RNAi expression vector kit
(PcDNA6.2-GW/emGFP-miRNA expression system kit) is also used to
express more than one miRNA or one miRNA with multiple copies. In
brief two copies of specific microRNAs (e. g. hsa-miR557,
hsa-miR1287 or miR1978 indicated by
pcDNA6.2-GW/emGFP-miR557-miR557, pcDNA6.2-GW/emGFP-miR1287-miR1287
or pcDNA6.2-GW/emGFP-miR1978-miR1978) or different microRNAs (e.g.
pcDNA6.2-GW/emGFP-miR557-miR1287, pcDNA6.2-GW/emGFP-miR557-miR1978
or pcDNA6.2-GW/emGFP-miR1287-miR1978) are cloned as DNA
oligonucleotides encoding said miRNAs as short hairpins into the
mammalian expression vector pcDNA6.2-GW/emGFP-miRNA (BLOCK-iT.TM.
Pol II miR RNAi expression vector kit, K4936-00 from life
technologies). The oligonucleotide sequences used for cloning of
miRNAs into the vector backbone are as follows:
TABLE-US-00004 hsa-miR-557 forward: 5'- (SEQ ID NO: 21)
TGCTGGTTTGCACGGGTGGGCCTTGTCTGTTTTGGCCAC
TGACTGACAGACAAGGCCCACGTGCAAAC-3' hsa-miR-557 reverse: 5'- (SEQ ID
NO: 22) CCTGGTTTGCACGTGGGCCTTGTCTGTCAGTCAGTGGCC
AAAACAGACAAGGCCCACCCGTGCAAACC-3' hsa-miR-1287 forward: 5'- (SEQ ID
NO: 23) TGCTGTGCTGGATCAGTGGTTCGAGTCGTTTTGGCCACT
GACTGACGACTCGAACCACATCCAGCA-3' hsa-miR-1287 reverse: 5'- (SEQ ID
NO: 24) CCTGTGCTGGATGTGGTTCGAGTCGTCAGTCAGTGGCCA
AAACGACTCGAACCACTGATCCAGCAC-3 hsa-miR-1978 forward: 5'- (SEQ ID NO:
25) TGCTGGGTTTGGTCCTAGCCTTTCTAGTTTTGGCCACTG
ACTGACTAGAAAGGCTAACCAAACC-3' hsa-miR-1978 reverse: 5'- (SEQ ID NO:
26) CCTGGGTTTGGTTAGCCTTTCTAGTCAGTCAGTGGCCAA
AACTAGAAAGGCTAGGACCAAACCC-3'
[0261] The chaining method was used to clone two copies of specific
microRNAs into these constructs as described in the manufacturer's
manual. In brief, the miRNA cassette was excised with the enzymes
BamHI and XhoI. The vector containing already one miRNA was opened
with the enzymes BglII and XhoI. DNA was mixed with orange loading
buffer and was separated in a 1% agarose gel prepared with TAE
buffer, bands were visualized with ethidium bromide and bands of
appropriate size were excised from the gel. Size was verified with
DNA ladder. DNA was eluted with the gel extraction kit. DNA insert
was ligated into the vector using T4 DNA ligase according to
manufacturers instructions. Subsequently, competent E. coli were
transformed with the DNA and plated on agar plates containing
spectinomycin. Colonies were picked and DNA was extracted with a
DNA purification kit, checked by control digest with BamHI and
BglII first, followed by sequencing.
[0262] Stable cell lines were generated by transfection as
described above, selection with 10 .mu.g/mL blasticidin S (Life
Technologies) and FACS enrichment for GFP positive cells
(FACSdiva). Cells were analysed by flow cytometry for GFP
expression and in parallel qPCR analysis was done to monitor miRNA
overexpression. Single clones are generated by limited dilution of
unsorted stable pools. As control vectors either a negative control
miRNA expressing vector (pcDNA6.2-GW/emGFP-neg. control miRNA,
provided by the kit) or an empty vector (pcDNA6.2-GW/emGFP) both
expressing GFP were stably transfected as described.
[0263] Fed-batch cultivations were performed with the different
miRNA-overexpressing stable pools, the control pools and the
parental cells, which are CHO-DG44 cells stably secreting an IgG1.
Furthermore single clones were evaluated during fed-batch
cultivation.
[0264] MicroRNA Expression Measurement by qPCR Analysis
[0265] 2.times.10.sup.5 to 2.times.10.sup.6 cells are used for RNA
extraction with mirVANA miRNA isolation kit (Ambion). As a positive
control CHO-DG44 cells are transiently transfected with mature
microRNA. cDNA is generated with 10 ng RNA using Taqman microRNA
reverse transcription kit (Applied Biosystems) according to the
manufacturer's instructions. qPCR is performed with Taqman microRNA
assays (Applied Biosystems) using a Cfx96 device (Bio-rad). RNU6B
is used as reference. Calculation is done with the single threshold
method and .DELTA..DELTA.Cq values are calculated (Bio-rad CFX
manager software 2.1).
[0266] Antibody Purification
[0267] Cell culture supernatant produced during fed-batch
cultivation was concentrated using 50 kDa Amicon centrifugal filter
units (Millipore). The concentrate containing the antibody was
purified with Protein A HP spin trap columns (GE Healthcare)
according to the manufacturer's instructions. The antibody elution
buffer comprises 0.1 M glycine-HCl neutralized with 1 M Tris-HCl to
pH 7. Amino groups, however, interfere with the downstream
glycosylation analysis. Hence, the buffer was exchanged to PBS
(Gibco) using the same filter units as before. Protein
concentration was determined photometrically by measuring
absorbance at 280 nm using a NANODROP spectrometer
(ThermoScientific) and the protein specific extinction coefficient.
Antibody quality was assessed by standard reducing SDS PAGE. Heavy
and light chains at 50 kDa and 25 kDa were observed, with no other
significant bands present.
[0268] Analysis of the Glycosylation Pattern
[0269] To elucidate the structure and composition of the
Fc-glycosylation of IgGs produced in the miRNA cell lines, the
glycans are released from the purified antibody after reduction by
enzymatic digestion with PNGase F. Glycans are purified,
fluorescently labelled with 2-Aminobenzamide (2-AB) and
fractionated on a HPLC column. The percentages of the glyco-forms
present are calculated from the chromatographic peak area ratios
and allow the qualitative and quantitative verification of the
glycostructures and composition. Alternatively, purified glycans
are labelled with a fluorescent dye and separated by
microchip-based capillary gel electrophoresis (CGE), e.g., using
the PROFILERPRO Glycan Profiling Kit Ver 2 on a LABCHIP GXII
capillary gel electrophoresis (CGE) instrument (Caliper Life
Sciences). Glycostructures may also be analysed by mass
spectrometry.
EXAMPLES
Example 1: First microRNA Screen in BIWA4-Producing CHO Cells
[0270] CHO cells are commonly used for the production of
therapeutic proteins. Genetic engineering approaches have attempted
to optimize the productivity of these cells by expressing specific
cDNAs. Naturally existing non-coding RNAs regulate cell fate by
modulating the expression of a whole set of target proteins, which
may possibly result in a super-secretory phenotype when ectopically
expressed in CHO producer cells. To exploit the power of non-coding
RNAs and identify those that positively affect secretion of a
heterologous protein, CHO-DG44 cells stably expressing an IgG1
(BIWA4) are transiently transfected by nucleofection with a human
microRNA mimic library consisting of 879 microRNAs (FIG. 1).
Antibody concentrations in the supernatant of the transfected cells
are determined on days 3 and 4 post transfection by ELISA. The
experiment is repeated and 20 microRNAs that (i) increased the IgG1
titer in the supernatant more than 1.3-fold on day 3 or 4 compared
to control cells and (ii) increased the mean IgG1 titer of both
experiments on day 3 or 4 more than 1.4-fold are defined as hits.
Given that the host cell already produces high amounts of
heterologous protein, a further increase in productivity of >30%
is highly significant. Names and sequences of the 20 microRNAs are
listed in FIG. 1B.
Example 2: Secondary Screen in BIWA4-Producing CHO Cells
[0271] To validate the hits from the primary screen performed in a
96-well format, a secondary screen with BIWA4-producing CHO cells
is performed in a larger culture format (12-well) with
quadruplicate samples (FIG. 2). In addition to measuring the IgG
titer in the supernatant, cell density and viability are determined
enabling the calculation of the specific productivity. Remarkably,
in the secondary screen, all 20 microRNAs defined as hits in the
primary screen increased the specific productivity of host cells
determined on day 3 and/or 4 post transfection.
Example 3: Transient Expression of miRNAs in BIBH1-Producing CHO
Cells
[0272] To explore whether the increased specific productivity was
specific to BIWA4-producing CHO cells or could equally be seen in
another IgG1-producing CHO cell line, CHO cells stably expressing
another IgG1 (BIBH1) are transiently transfected with each of the
20 microRNAs as described in Example 2 and their specific
productivity is determined (FIG. 3). Surprisingly, all 20 microRNAs
that are confirmed as hits in the secondary screen with
BIWA1-producing CHO cells also increase the specific productivity
of BIBH1-producing CHO cells on day 3 and/or 4 post
transfection.
Example 4: Transient Expression of miRNAs in HSA-Producing CHO
Cells
[0273] To explore whether transient expression of the 20 microRNAs
specifically enhanced the expression and secretion of IgG1
molecules or also elevates the production of other therapeutic
proteins, such as human serum albumin (HSA), CHO cells stably
expressing HSA are transiently transfected with each of the 20
microRNAs as described in example 2 and their specific productivity
is determined (FIG. 4). Surprisingly, all 20 microRNAs also exert a
positive effect on the specific productivity of HSA-secreting CHO
cells on days 3 and 4 post transfection, providing evidence that
the microRNAs function in a product-independent manner.
Example 5: Analysis of miRNA Expression in Transfected
BIWA4-Producing CHO Cells by Quantitative PCR
[0274] We transiently transfect BIWA4 cells with either a plasmid
encoding microRNA hsa-miR-557 (pBIP-1-mir-gen.miR-557) or with the
mature miRNA hsa-miR-557 as described in Example 1. Control cells
are transfected with an empty vector (pBIP-1) or with a control
siRNA (siLacZ). To validate the expression of transiently
transfected microRNAs, we isolate RNA from all cells two days after
transfection and perform qPCR analysis of the mature miRNA sequence
and the antisense strand of the pre-miRNA. Indeed, we detect
strongly increased levels of mature miRNA sequence in cells
transfected with the mature miRNA compared to control cells (FIG.
5). Further, we also detect increased levels of the mature miRNA in
cells transfected with the miRNA-encoding plasmid whereas the level
of the antisense strand is comparable to the controls. This
demonstrates that transfection of both mature and plasmid-encoded
microRNAs leads to increased levels of the respective microRNA in
CHO cells, indicating that CHO cells are able to correctly process
human microRNA precursors into their mature form.
Example 6: Expression of miRNA Combinations in BIWA4-Producing CHO
Cells
[0275] Furthermore, we explore whether a combination of these
microRNAs further boosts the specific productivity of
BIWA4-producing CHO cells.
[0276] For this purpose, CHO-DG44 cells stably secreting an IgG1
(BIWA4) are transiently transfected with a combination of 2
validated miRNA hits (every possible combination of these 5 miRNAs:
hsa-miR-557, hsa-miR-1271, hsa-miR-1275, hsa-miR-1287 and
hsa-miR-1978) in duplicates. Samples containing a single microRNA
are adjusted to a final RNA concentration by adding mimic miRNA
negative control. Transfection efficiency is monitored by flow
cytometry of siLacZ-FITC transfected cells and ELISA analysis of
the supernatant of siLC-104 (targeting the light chain of the
antibody) transfected cells. As negative controls a non targeting
siRNA (siLacZ-FITC) and a mimic miRNA are used (error bars=SEM of
duplicates). Cell density and antibody concentration in the
supernatant are determined on day 1-4 and specific productivity is
calculated (FIG. 6).
[0277] Remarkably, the combined transfection of two different
miRNAs enhanced specific productivity on day 4 compared to singly
transfected CHO-DG44 cells in about 50% of the cases tested: E.g.
co-transfection of miR-557 and miR-1287 has a clearly positive
effect in increasing the productivity compared to both microRNAs
alone. These data show that with certain combinations of microRNAs,
it is possible to achieve an additive (and maybe even synergistic)
effect on the enhancement of secretive capacity of a cell producing
a therapeutic protein of interest.
Example 7: Transient Expression of miRNAs in Insulin-Secreting INS
Cells
[0278] To explore whether transient expression of microRNAs
enhances the secretion of an endogenous protein, INS-1 cells, a rat
insulinoma cell line, which secretes insulin, are transiently
transfected with a subset of the 20 microRNAs (hsa-miR-183,
hsa-miR-125-3p, hsa-miR-557, hsa-miR-1271, hsa-miR-1275,
hsa-miR-1287). Starved cells are stimulated with glucose for 15
minutes or left untreated. Basal and glucose-induced insulin
concentrations in the supernatant of the cells are determined by
ELISA. Surprisingly, all 6 microRNAs exert a positive effect on
basal and glucose-stimulated insulin secretion of INS1 cells on day
3 post transfection (FIG. 7), providing further evidence that these
microRNAs function in a species- and product-independent manner and
also positively affect the secretion of endogenous proteins not
only in CHO, but also in other cells of rodent origin.
Example 8: Transient Expression of miRNAs in ssHRP-Producing Human
HEK293 FlpIN Cells
[0279] To explore whether transient expression of microRNAs
enhances the constitutive secretion of a model cargo protein in
human cells, HEK293FlpIn cells stably and inducibly expressing a
secretable form of horse radish peroxidase (ssHRP) are transiently
transfected with 5 microRNAs by lipofection. Two days post
transfection, ssHRP expression is induced by addition of
doxycycline. 12 hours after induction the amount of ssHRP in the
supernatant of the cells is determined after 5 hours, quantified by
addition of ECL reagent and measurement of the luminescent signal
in plate reader. Luminescence signals are normalized for cell
density. 5 microRNAs (hsa-miR-1287, hsa-miR-183, hsa-miR-557,
hsa-miR-612 and hsa-miR-644) exerted a positive effect on ssHRP
secretion of human HEK293 FlpIn cells (FIG. 8), providing evidence
that the microRNAs function in a species- and product-independent
manner.
Example 9: Stable Expression of a miRNA in BIWA4-Producing CHO
Cells
[0280] CHO-DG44 cells stably expressing an IgG1 (BIWA4) are
transfected with an expression construct encoding a non-coding RNA
(see FIG. 1B) and subsequently subjected to selection to obtain
stable cell pools. The expression construct may contain two or more
copies of the same or different non-coding RNAs. During subsequent
passages, 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 seen in the cell pools harbouring the
non-coding RNAs. IgG expression is markedly enhanced compared to
MOCK or untransfected cells. Very similar results can be obtained
if the stable transfectants are subjected to batch or fed-batch
fermentations (FIG. 11). In each of these settings, overexpression
of non-coding RNAs leads to increased antibody secretion,
indicating that non-coding RNAs are able to enhance the specific
production capacity of the cells grown in serial cultures or in
bioreactor batch or fed batch cultures.
Example 10: Stable Expression and Amplification of a miRNA in
Rituximab-Producing CHO Cells
[0281] Parental CHO-DG44 cells are either sequentially or
concomitantly transfected with three expression plasmids: (i) a
plasmid encoding the light chain of the antibody Rituximab and
containing a DHFR cassette for amplification, (ii) a plasmid
encoding the heavy chain of Rituximab and containing a neomycin
resistance cassette, and (iii) a plasmid encoding a microRNA (see
FIG. 1B) and containing a puromycin resistance cassette. Cells with
stable genomic integration are obtained by puromycin and neomycin
selection and the simultaneous removal of hypoxantin/thymidine.
Then, amplification of the light chain of the antibody is achieved
by the successive increase of the concentration of methothrexate in
the medium. It is expected that the light chain and the microRNA
are co-amplified through indirect mechanisms. During subsequent
passages of stable CHO-DG44 cells expressing Rituximab and the
non-coding RNA, supernatant is taken from seed-stock cultures of
all stable cell pools; the Rituximab titer is determined by ELISA
and divided by the mean number of cells to calculate the specific
productivity. The highest values are seen in the cell pools
harbouring the amplified non-coding RNA. Rituximab expression is
markedly enhanced compared to cells without stable expression of
the non-coding RNA. Very similar results can be obtained if the
stable transfectants are subjected to batch or fed-batch
fermentations. In each of these settings, overexpression of the
non-coding RNA leads to increased antibody titers, indicating that
non-coding RNAs are able to enhance the specific production
capacity of the cells grown in serial cultures or in bioreactor
batch or fed batch cultures.
Example 11: Generation of an Optimized Host Cell for Production of
Therapeutic Proteins
[0282] To be more flexible in the application of microRNA
engineering, we also generate CHO host cells which are stably
engineered to exhibit increased levels of either one or a
combination of the microRNAs provided in the present invention. In
a second step, these engineered host cells and un-engineered
control host cells are then transfected with an expression
construct encoding a protein of interest and productivities and
titers of said protein is then analyzed both in seed-stock cultures
and fed-batch processes.
[0283] The results demonstrate that production cells derived from
microRNA engineered host cells show higher secretion rates, i.e.
productivities as well as in most cases also higher titers. Hence,
we conclude that microRNA engineering can either be done after,
prior to or simultaneously to introducing the protein of interest
with similar results, thus offering a broad range of options for
applications in pharmaceutical development processes.
[0284] Alternatively, sufficiently high stable levels of microRNA
in the host cell or the producer cell can be achieved by cloning
multiple copies of the microRNA into an expression vector.
Example 12: Expression of Therapeutic Proteins from a Secretion
Optimized Host Cell
[0285] To test the production performance of microRNA-engineered
cells generated by either of the methods described in examples 3,
6, 11 or in the detailed description of the invention using
amplification of miRNAs, they are subjected to fed-batch processes
in chemically-defined media to reflect the conditions in industrial
processes. The fed-batch is performed in shake flasks that are
previously demonstrated to be a predictive screening model for
performance in larger scales. The process is run over >7 days.
Cell counts, viabilities and product titers are measured at regular
intervals to monitor the production behavior of microRNA engineered
cells as well as non-engineered cell lines which are included as
controls.
[0286] This experiment shows that growth and viability profiles of
microRNA engineered cells are comparable or only slightly lower
compared to controls. However, the specific productivity of
microRNA engineered cells is consistently higher compared to
non-engineered cell lines which proved the benefit of this microRNA
engineering approaches for industrial therapeutic protein
production processes.
Example 13: Analysis of microRNA Expression in Stably Transfected
BIWA4-Producing CHO Cells by Flow Cytometry
[0287] BIWA4 cells are stably transfected with a plasmid encoding a
GFP cassette plus two microRNA copies
(pcDNA6.2-GW/emGFP-miR557-miR557,
pcDNA6.2-GW/emGFP-miR1287-miR1287) or a combination of the
individual microRNAs (pcDNA6.2-GW/emGFP-miR557-miR1287). After
selection with blasticidin S (encoded on the pcDNA6.2-GW/emGFP
vector) cells are sorted based on their GFP fluorescence. Control
cells are untransfected parental cells. To validate the expression
of stably transfected microRNAs, cells are analyzed by flow
cytometry for GFP expression, which correlates with microRNA
expression, and GFP positive populations can be detected over 51
days (FIG. 9). This shows that CHO cells are able to stably
overexpress human microRNAs for at least 7 weeks.
Example 14: Analysis of microRNA Expression in Stably Transfected
BIWA4-Producing CHO Cells by Quantitative PCR
[0288] BIWA4 cells are stably transfected with a plasmid encoding a
GFP cassette plus two microRNA copies
(pcDNA6.2-GW/emGFP-miR557-miR557,
pcDNA6.2-GW/emGFP-miR1287-miR1287) or the combination of the
individual microRNAs (pcDNA6.2-GW/emGFP-miR557-miR1287). After
selection with blasticidin S (encoded on the pcDNA6.2-GW/emGFP
vector) cells are sorted based on their GFP fluorescence. Cells
transfected with the mature miRNAs (hsa-miR-557 or hsa-miR-1287) as
described in Example 1 are used as positive controls. Cell pools
stably expressing the control vector (pcDNA6.2-GW/emGFP-neg.
control miRNA) and untransfected parental cells (BIWA4) served as
negative controls. To validate the expression of stably transfected
microRNAs, we isolate RNA from all cells and perform qPCR analysis
of the mature miRNA sequence. Increased levels of the mature miRNA
are detected in cells transfected with the miRNA-encoding plasmid,
whereas hardly any signal is detectable in control vector
transfected cells and parental cells (FIG. 10). This demonstrates
that stable genomic integration of plasmid-encoded microRNAs leads
to increased levels of the respective microRNA in CHO cells and
shows that CHO cells are able to correctly process human microRNA
precursors into their mature form.
Example 15: Fed-Batch Cultivation of Stable miRNA Overexpressing
BIWA4-Producing CHO Cells
[0289] CHO-DG44 stably secreting an IgG1 (BIWA4) were stably
transfected with miRNA expression vectors (pcDNA6.2-GW/emGFP) and
single clones were generated by limited dilution. Either a
combination of 2 validated miRNA hits (-miR557-miR1287-clone) or
the respective empty vector control expressing GFP (-control-clone)
were used during fed-batch cultures. Three independent pools stably
expressing a neg. control miRNA (pcDNA6.2-GW/emGFP-neg.
control-miRNA) served as further controls. Cell density and
antibody concentration in the supernatant were determined on day
3-11 by cell counting with trypane blue exclusion and ELISA
analysis, respectively, and specific productivity was calculated.
The highest titres and specific productivity are seen in the cells
co-expressing the non-coding RNAs (hsa-miR-557 and hsa-miR-1287).
IgG expression is markedly enhanced compared to the negative
control (pcDNA6.2-GW/emGFP-neg. control miRNA) or parental cells
(FIG. 11). This proves that stable clones of CHO cells expressing
selected non-coding RNAs have an increased titre and specific
production capacity, without any negative effect on viable cell
density, when grown in fed batch cultures.
Example 16: Stable Single Clones of miRNA Overexpressing
BIWA4-Producing CHO Cells
[0290] CHO-DG44 cells stably expressing an IgG1 (BIWA4) are
transfected with an expression construct encoding non-coding RNAs
(see FIG. 1B) and are subjected to selection to obtain stable cell
pools that are subsequently used to generate single clones. The
expression construct contains a combination of non-coding RNAs
(pcDNA6.2-GW/emGFP-miR557-miR1287). During fed-batch cultivation
supernatant is taken from the single clone; the IgG titer is
determined by ELISA and divided by the mean number of cells to
calculate the specific productivity. The highest values are seen in
the cells co-expressing the non-coding RNAs (hsa-miR-557 and
hsa-miR-1287). IgG expression is markedly enhanced compared to the
negative control pools (pcDNA6.2-GW/emGFP-neg. control miRNA) or
control clone (FIG. 12). This proves that stable single clones of
CHO cells expressing selected non-coding RNAs have an increased
specific production capacity when grown in fed batch cultures.
Example 17: Analysis of Antibody Glycosylation
[0291] To elucidate the structure and composition of the
Fc-glycosylation of IgGs produced in the miRNA cell lines, the
glycans are released from the purified antibody after reduction by
enzymatic digestion with PNGase F. The antibody glycosylation
pattern was analyzed with the PROFILERPRO Glycan Profiling Kit Ver
2 on a LABCHIP GXII capillary gel electrophoresis (CGE) instrument
(Caliper Life Sciences) according to the manufacturer's protocol.
Electropherograms were analyzed by the LABCHIP GX software package
to identify and quantify the individual sugar structures. All
values are normalized to 100% total sugar structures per sample.
The results in FIGS. 13A and 13B show that the microRNAs provided
herein do not affect glycosylation of the protein-of-interest, such
as in the Fc-domain of an antibody.
Example 18: Transient Expression of miRNAs in HeLa Cells
Transiently Expressing ssHRP
[0292] To explore whether transient expression of microRNAs
enhances the constitutive secretion of a model cargo protein in
human cells, HeLa cells are transiently transfected with 3
microRNAs by lipofection. Two days post transfection, ssHRP-FLAG is
transiently transfected and 24 hours later the amount of ssHRP in
the supernatant of the cells is determined after 4 and 6 hours,
quantified by addition of ECL reagent and measurement of the
luminescent signal in plate reader. All three microRNAs
(hsa-miR-125a-3p, hsa-miR-1978 and hsa-miR-557) tested exerted a
positive effect on ssHRP secretion of human Hela cells (FIG. 14A),
providing evidence that the microRNAs function in a species- and
product-independent manner.
Example 19: Transient Expression of miRNAs in HeLa Cells
Endogenously Secreting IL-8
[0293] To explore whether transient expression of microRNAs
enhances the constitutive secretion of an endogenous model cargo
protein in human cells, HeLa cells are transiently transfected with
7 microRNAs by lipofection. Two days post transfection the amount
of IL-8 in the supernatant of the cells is determined after 6 and
24 hours, quantified by ELISA analysis. All 7 microRNAs
(hsa-miR-125a-3p, hsa-miR-1271, hsa-miR-185*, hsa-miR-193b*,
hsa-miR-1978, hsa-miR-299-3p and hsa-miR-557) tested exerted a
positive effect on IL-8 secretion of human Hela cells (FIG. 14B),
providing evidence that the microRNAs function in a species- and
product-independent manner.
TABLE-US-00005 SEQUENCE TABLE SEQ ID NO 1: miR-125a-3p SEQ ID NO 2:
miR-149 SEQ ID NO 3: miR-1271 SEQ ID NO 4: miR-1275 SEQ ID NO 5:
miR-1285 SEQ ID NO 6: miR-1287 SEQ ID NO 7: miR-1293 SEQ ID NO 8:
miR-183 SEQ ID NO 9: miR-185* SEQ ID NO 10: miR-193b* SEQ ID NO 11:
miR-1978 SEQ ID NO 12: miR-23b* SEQ ID NO 13: miR-299-3p SEQ ID NO
14: miR-365* SEQ ID NO 15: miR-450b-3p SEQ ID NO 16: miR-557 SEQ ID
NO 17: miR-612 SEQ ID NO 18: miR-644a SEQ ID NO 19: miR-885-3p SEQ
ID NO 20: miR-892a SEQ ID NO: 21: hsa-miR-557 forward SEQ ID NO:
22: hsa-miR-557 reverse SEQ ID NO: 23: hsa-miR-1287 forward SEQ ID
NO: 24: hsa-miR1287 reverse SEQ ID NO: 25: hsa-miR1978 forward SEQ
ID NO: 26: hsa-miR1978 reverse
REFERENCE LIST
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[0297] Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P.
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Sequence CWU 1
1
26122RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-125a-3p" /organism="Homo sapiens" 1acaggugagg uucuugggag
cc 22223RNAHomo sapienssource1..23/mol_type="unassigned RNA"
/note="miR-149" /organism="Homo sapiens" 2ucuggcuccg ugucuucacu ccc
23322RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-1271" /organism="Homo sapiens" 3cuuggcaccu agcaagcacu ca
22417RNAHomo sapienssource1..17/mol_type="unassigned RNA"
/note="miR-1275" /organism="Homo sapiens" 4gugggggaga ggcuguc
17522RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-1285" /organism="Homo sapiens" 5ucugggcaac aaagugagac cu
22622RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-1287" /organism="Homo sapiens" 6ugcuggauca gugguucgag uc
22722RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-1293" /organism="Homo sapiens" 7uggguggucu ggagauuugu gc
22822RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-183" /organism="Homo sapiens" 8uauggcacug guagaauuca cu
22922RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-185*" /organism="Homo sapiens" 9aggggcuggc uuuccucugg uc
221022RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-193b*" /organism="Homo sapiens" 10cgggguuuug agggcgagau
ga 221121RNAArtificial Sequencesource1..21/mol_type="unassigned
RNA" /note="miR-1978" /organism="Artificial Sequence" 11gguuuggucc
uagccuuucu a 211222RNAHomo sapienssource1..22/mol_type="unassigned
RNA" /note="miR-23b*" /organism="Homo sapiens" 12uggguuccug
gcaugcugau uu 221322RNAHomo sapienssource1..22/mol_type="unassigned
RNA" /note="miR-299-3p" /organism="Homo sapiens" 13uaugugggau
gguaaaccgc uu 221422RNAHomo sapienssource1..22/mol_type="unassigned
RNA" /note="miR-365*" /organism="Homo sapiens" 14agggacuuuc
aggggcagcu gu 221522RNAHomo sapienssource1..22/mol_type="unassigned
RNA" /note="miR-450b-3p" /organism="Homo sapiens" 15uugggaucau
uuugcaucca ua 221623RNAHomo sapienssource1..23/mol_type="unassigned
RNA" /note="miR-557" /organism="Homo sapiens" 16guuugcacgg
gugggccuug ucu 231725RNAHomo
sapienssource1..25/mol_type="unassigned RNA" /note="miR-612"
/organism="Homo sapiens" 17gcugggcagg gcuucugagc uccuu
251819RNAHomo sapienssource1..19/mol_type="unassigned RNA"
/note="miR-644a" /organism="Homo sapiens" 18aguguggcuu ucuuagagc
191922RNAHomo sapienssource1..22/mol_type="unassigned RNA"
/note="miR-885-3p" /organism="Homo sapiens" 19aggcagcggg guguagugga
ua 222021RNAHomo sapienssource1..21/mol_type="unassigned RNA"
/note="miR-892a" /organism="Homo sapiens" 20cacugugucc uuucugcgua g
212168DNAArtificial Sequencesource1..68/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 21tgctggtttg
cacgggtggg ccttgtctgt tttggccact gactgacaga caaggcccac 60gtgcaaac
682268DNAArtificial Sequencesource1..68/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 22cctggtttgc
acgtgggcct tgtctgtcag tcagtggcca aaacagacaa ggcccacccg 60tgcaaacc
682366DNAArtificial Sequencesource1..66/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 23tgctgtgctg
gatcagtggt tcgagtcgtt ttggccactg actgacgact cgaaccacat 60ccagca
662466DNAArtificial Sequencesource1..66/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 24cctgtgctgg
atgtggttcg agtcgtcagt cagtggccaa aacgactcga accactgatc 60cagcac
662564DNAArtificial Sequencesource1..64/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 25tgctgggttt
ggtcctagcc tttctagttt tggccactga ctgactagaa aggctaacca 60aacc
642664DNAArtificial Sequencesource1..64/mol_type="unassigned DNA"
/note="synthetic" /organism="Artificial Sequence" 26cctgggtttg
gttagccttt ctagtcagtc agtggccaaa actagaaagg ctaggaccaa 60accc
64
* * * * *