WO2019180607A1 - A vector, a host cell and a method thereof - Google Patents

A vector, a host cell and a method thereof Download PDF

Info

Publication number
WO2019180607A1
WO2019180607A1 PCT/IB2019/052213 IB2019052213W WO2019180607A1 WO 2019180607 A1 WO2019180607 A1 WO 2019180607A1 IB 2019052213 W IB2019052213 W IB 2019052213W WO 2019180607 A1 WO2019180607 A1 WO 2019180607A1
Authority
WO
WIPO (PCT)
Prior art keywords
mrna
corresponds
cricetulus griseus
recombinant
vector
Prior art date
Application number
PCT/IB2019/052213
Other languages
French (fr)
Inventor
Taslimarif SAIYED
Paresh Brijalal PATEL
Rabindranath MUKHOPADHYAYA
Original Assignee
Centre For Cellular And Molecular Platforms
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre For Cellular And Molecular Platforms filed Critical Centre For Cellular And Molecular Platforms
Publication of WO2019180607A1 publication Critical patent/WO2019180607A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • the present disclosure relates to a vector comprising at least one nucleotide encoding ShRNA.
  • the disclosure further relates to a novel recombinant cell in which there is complete or partial knockdown of at least one protease.
  • the disclosure also relates to a method /enhancing the yield of recombinant protein in a host cell. .
  • recombinant host cell for production of recombinant protein such as biopharmaceutical proteins assumes great significance because degradative enzymes such as proteases degrade and bring down the amount of expressed recombinant proteins.
  • degradative enzymes such as proteases degrade and bring down the amount of expressed recombinant proteins.
  • copious screening of individual cell lines is done in order to identify cell clones that show the expression stability necessary for large scale production. This screening practice for eliminating clones that stably express recombinants prolongs the development of biotechnological production processes. Even when using highly stringent selection systems that favor survival of high expressing cells under the used selection conditions, finding suitable production clone within the surviving population which combines a high expression rate with good growth and stability characteristics is difficult.
  • the present disclosure intends to address the limitations encountered in recombinant protein production wherein the proteases in the host cell degrades the expressed protein.
  • the present disclosure relates to a method for enhancing recombinant protein production by causing complete knockdown or partial knockdown of protease in the host to enhance protein production.
  • Another object of the present disclosure is to provide a novel recombinant cell which upon stable transfection or transformation with a polynucleotide encoding product of interest, such as recombinant protein expresses the product of interest with improved stability characteristics.
  • Another object of the present disclosure is to provide a vector comprising nucleotide sequence corresponding to ShRNA, that is capable of causing complete or partial protease knockout in a cell transformed or transfected with the said vector.
  • Another object of the present disclosure is to provide a vector comprising plurality of nucleotide sequences corresponding to plurality of ShRNA, that is capable of causing complete or partial protease knockdown in a cell transformed or transfected with the said vector.
  • FIGURE 1 illustrates schematic representation of the work flow to enhance production of bio- therapeutic (EPO) from an existing producer (CHO-EPO) clone.
  • FIGURE 2 illustrate enhanced production of erythropoietin in a recombinant cell when compared to parental cell.
  • FIGURE 3 illustrates Enhanced production of EPO from CHO-EPO clones.
  • FIGURE 4 illustrates enhanced production of erythropoietin in a recombinant cell having a knockdown of protease I and protease II, respectively.
  • FIGURE 5 illustrates the structure of ShRNA.
  • the present disclosure relates to a recombinant vector comprising nucleotide sequence encoding ShRNA comprising section A to B, B to C and C to D represented by structure 1.
  • the recombinant vector as mentioned above the section A to B comprises about 17 nucleotides to 22 nucleotides, corresponding to sequence from the gene having accession number selected from a group comprising XM_003508l l3. l, NM 001243986.1, XM_003506664.3, CM_003503315.3, XM_003496727.3,
  • the section B to C in the structure of the ShRNA comprises about 7 to 9 nucleotides selected from a group comprising A, T, C and G; and the section C to D in the structure of ShRNA corresponds to reverse complementary sequence of the nucleotide sequence present in the section A to B, wherein the said gene sequence belong to proteases.
  • the shRNA is cloned into a lentiviral construct
  • the present disclosure further relates to a recombinant host cell comprising the vector as mentioned above
  • the host cell as mentioned above has complete or partial knockdown of at least one protease.
  • protease sequence is selected from a group having accession number XM_003508l l3.
  • l corresponding to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986.
  • the host cell is selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK, PER.C6, yeast and E. coli.
  • CHO Chinese hamster ovary
  • NS0, Sp2/0 murine myeloma
  • HEK HEK
  • PER.C6 yeast
  • E. coli E. coli
  • the host cell is capable of enhanced yield of undegraded recombinant protein between about 10% to 300%.
  • the present disclosure further relates to a method of enhancing the recombinant protein production comprising expressing the protein of interest in the host cell mentioned above.
  • the method enhances the recombinant protein production by about 10% to 300%.
  • the present disclosure is based on the unexpected/surprising finding that transfecting or transforming a host cell with a vector comprising at least one nucleotide encoding ShRNA, wherein the said vector causes complete or partial knockdown of protease in a host cell to obtain a novel recombinant host cell capable of higher yield of undegraded recombinant protein.
  • the vector comprising at least one nucleotide encoding ShRNA is a recombinant vector that causes complete or partial knockdown of at least one protease in a cell transformed or transfected with the said vector to yield a recombinant host cell.
  • transfection or transforming the host cell with a vector comprising at least one nucleotide sequence encoding ShRNA allows for significant improvement in yield of recombinant production of a product of interest.
  • the vector comprises plurality of nucleotide sequence encoding ShRNA.
  • plurality of nucleotide sequences encoding to plurality of ShRNA causes expression of plurality of ShRNA.
  • the vector comprises lentiviral construct.
  • the structure of ShRNA in the said vector is represented by Structure 1
  • the protease which are targeted by ShRNA expressed by the vector is selected from a group comprising elastase II, cathepsin-G, granzyme B, thrombin, proprotein convertase subtilisin, furin, lactotransferrin, plasminogen activator tissue type, plasminogen activator urokinase, chymase, kallikerein 1, complement factor I, coagulation factor XI, coagulation factor VII, plasminogen, myelobastin and HtrA serine peptidase 2.
  • the section A to B comprises about 17 nucleotides to 22 nucleotides, corresponding to protease sequence from the gene having accession number selected from a group comprising XM_003508l l3.l, NM 001243986.1, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3,
  • the section B to C in the structure of the ShRNA comprises about 7 to 9 nucleotides selected from a group comprising A, T, C and G; and the section C to D in the structure of ShRNA corresponds to reverse complementary sequence of the nucleotide sequence present in the section A to B.
  • the illustrated structure of ShRNA upon expression through the vector causes complete knockdown or partial knockdown of proteases having gene sequence accession number CM_003508113.1, NM_001243986.1, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3, XM_007611030.2, CM_003510196.1,
  • l corresponds to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986.
  • l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603. l corresponds to Cricetulus griseus myeloblastin (LOC100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, throm
  • the ShRNA upon expression through the vector in a cell causes complete or partial knockdown of at least one protease through techniques such including but is not limited to expression of zinc finger nucleases (ZFN), expression of engineered homing endonucleases, expression of transcription activator-like effector nuclease, expression of clustered regularly interspaced short palindromic repeats (CRISPR), expression of ShRNA, expression of SiRNA, expression of antisense RNA and miRNA.
  • ZFN zinc finger nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the present disclosure further relates to a recombinant cell which is produced by causing partial or complete knockdown of at least one protease of the host cell.
  • the protease that are partially or completely knockdown in a host cell to obtain recombinant host cell is selected from a group comprising proteases having gene sequence accession number CM_003508113.1, NM_00l243986.l, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3, XM_007611030.2,
  • l corresponds to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986.
  • l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603. l corresponds to Cricetulus griseus myeloblastin (LOC100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, throm
  • the host cell is a prokaryote or a eukaryote.
  • the host is cell line selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK or PER.C6.
  • CHO Chinese hamster ovary
  • NS0, Sp2/0 murine myeloma
  • HEK HEK
  • PER.C6 PER.C6
  • the host cell includes but is not limited to mammalian cell, insect cell, yeast cell or E. coli.
  • the host cell selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK, PER.C6, and other mammalian cell lines, are industry standard cell lines and are not prone to infection by any pathogen.
  • the recombinant cell is selected from a group comprising Chinese hamster ovary (CHO) cell line, murine myeloma (NS0, Sp2/0) cell line, HEK, PER.C6, other mammalian cell lines, yeast and E. coli, which has partial or complete knockdown of at least one of the protease described above.
  • CHO Chinese hamster ovary
  • NS0, Sp2/0 murine myeloma
  • HEK HEK
  • PER.C6 other mammalian cell lines
  • yeast and E. coli which has partial or complete knockdown of at least one of the protease described above.
  • the recombinant cell enhances yield between about 10% to 300% increase in the recombinant protein production.
  • the recombinant cell causes about 10%, about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200% , about 220% , about 240%, about 260%, about 280% or about 300% increase in the recombinant protein production.
  • the recombinant protein is selected from a group comprising antibodies, hormones, enzymes, virus like particles, vaccines and biosimilars like but not limited to Tocilizumab, Bevacizumab, Alemtuzumab, Trastuzumab, Adalimumab, Denosumab, Rituximab, Golimumab, Ustekinumab, Omalizumab, Ipilimumab, Ibritumomabtiuxetan, Alefacept, Rilonacept, Etanercept, Belatacept, Abatacept, Follitropin-b, Follitropin alfa, FH, Osteogenic Protein- 1, Choriogonadotropin a, Thyrotropin a, Darbepoetin a, Interferon b-la, Epoetin b, Epoetin a, Interferon b-la, Antihemophilic Factor, Factor
  • the complete knockdown or partial knockdown of the protease described above causes enhanced production of therapeutic protein.
  • the present disclosure further relates to a method for enhancing/increasing the yield of recovered recombinant protein.
  • the method comprises expression of recombinant protein in the recombinant cell described above, wherein in the recombinant cell there is complete knockdown or partial knockdown of plurality of proteases that enhances or increases the yield of recombinant protein.
  • the method of the present disclosure reduces the cost that is involved in producing recombinant protein as there is no need of adding protease inhibitor(s) externally.
  • the productivity loss during prolonged culturing of the recombinant cell is considerably reduced.
  • the method significantly enhances the recombinant protein yield from the same amount of biomass without altering any media component or without addition of separate cocktail of protease inhibitor(s) or without causing modification to the downstream processing methods, thereby rendering the method more cost-efficient
  • the method of the present disclosure causes about 10% to 300 % increase in the production of the recombinant protein.
  • the method causes about 10%, about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200% , about 220% , about 240%, about 260%, about 280% or about 300% increase in the recombinant protein production.
  • Figures 2 and 4 illustrates the enhancement in the recombinant protein, EPO production, wherein there is increase in protein production in the recombinant cells which has undergone complete or partial knockdown of at least one protease.
  • the Figure 3 illustrates the enhanced production of EPO in the recombinant cell clones of CHO cell lines, the individual recombinant cell clones were analyzed for their enhanced production of EPO.
  • the recombinant CHO cell line enhances the production of recombinant protein including but not limited to erythropoietin (EPO) by at least 10%, preferably at least 300%, more preferably at least 28%
  • EPO erythropoietin
  • elsastase-II is knockdown by the plurality of ShRNA expressed by the above described vector, thereby enhancing the production of the recombinant protein in the cell line.
  • Example 1 Enhanced production of Erythropoietin
  • CHO-K1 cells were used as a host for erythropoietin (EPO) expression.
  • the CHO-K1 cells expressing the EPO were stably transduced with ShRNA against two different proteases (protease 1 (Elastase II) and protease 2 (Granzyme B)), separately.
  • the EPO in the culture supernatant was checked by ELISA.
  • the stable cells expressing the ShRNA against the said proteases showed increase in the EPO expression (23% for protease 1 and 28% for protease 2) in the culture supernatant as compared to the control (illustrated in figure 4). Clonal selection could enhance the yield to about 300%.

Abstract

The present disclosure relates to a vector comprising at least one nucleotide encoding ShRNA. The disclosure further relates to a novel recombinant cell in which there is complete or partial knockdown of at least one protease. The said recombinant cell causes enhanced production of recombinant protein by about 10% to 300%. The disclosure further relates to a method of enhancing the recombinant protein production, wherein the enhancement of the produced recombinant protein is about 10% to 300%.

Description

“A VECTOR, A HOST CELL AND A METHOD THEREOF”
TECHNICAL FIELD
The present disclosure relates to a vector comprising at least one nucleotide encoding ShRNA. The disclosure further relates to a novel recombinant cell in which there is complete or partial knockdown of at least one protease. The disclosure also relates to a method /enhancing the yield of recombinant protein in a host cell. .
BACKGROUND OF THE DISCLOSURE
The market for recombinant protein production, particularly biopharmaceutical continues to grow at a high rate as biopharmaceuticals have become more and more important for today’s medicinal need. Currently, an increasing number of recombinant proteins, such as biopharmaceuticals is produced in both prokaryotic and eukaryotic cells. Successful and high production of recombinants arenthus crucial. The time to generate host cells producing recombinant protein of interest is an essential component of the scale-up process to increase the yields. Furthermore, considering the production costs for recombinant proteins, such as biopharmaceuticals and any other essential recombinant products it is important to have high yield stably expressed in host cells expressing these recombinant proteins.
Generation of recombinant host cell for production of recombinant protein, such as biopharmaceutical proteins assumes great significance because degradative enzymes such as proteases degrade and bring down the amount of expressed recombinant proteins. Usually copious screening of individual cell lines is done in order to identify cell clones that show the expression stability necessary for large scale production. This screening practice for eliminating clones that stably express recombinants prolongs the development of biotechnological production processes. Even when using highly stringent selection systems that favor survival of high expressing cells under the used selection conditions, finding suitable production clone within the surviving population which combines a high expression rate with good growth and stability characteristics is difficult.
The present disclosure intends to address the limitations encountered in recombinant protein production wherein the proteases in the host cell degrades the expressed protein. SUMMARY OF THE DISCLOSURE
It is an object of the present disclosure to improve recombinant production of a product, particular recombinant protein of interest in a host, such as prokaryotic cell and eukaryotic cell. Accordingly, the present disclosure relates to a method for enhancing recombinant protein production by causing complete knockdown or partial knockdown of protease in the host to enhance protein production.
Another object of the present disclosure is to provide a novel recombinant cell which upon stable transfection or transformation with a polynucleotide encoding product of interest, such as recombinant protein expresses the product of interest with improved stability characteristics.
Another object of the present disclosure is to provide a vector comprising nucleotide sequence corresponding to ShRNA, that is capable of causing complete or partial protease knockout in a cell transformed or transfected with the said vector. Another object of the present disclosure is to provide a vector comprising plurality of nucleotide sequences corresponding to plurality of ShRNA, that is capable of causing complete or partial protease knockdown in a cell transformed or transfected with the said vector.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
FIGURE 1 illustrates schematic representation of the work flow to enhance production of bio- therapeutic (EPO) from an existing producer (CHO-EPO) clone.
FIGURE 2 illustrate enhanced production of erythropoietin in a recombinant cell when compared to parental cell.
FIGURE 3 illustrates Enhanced production of EPO from CHO-EPO clones.
FIGURE 4 illustrates enhanced production of erythropoietin in a recombinant cell having a knockdown of protease I and protease II, respectively. FIGURE 5 illustrates the structure of ShRNA.
DETAILED DESCRIPTION
The present disclosure relates to a recombinant vector comprising nucleotide sequence encoding ShRNA comprising section A to B, B to C and C to D represented by structure 1.
Figure imgf000004_0001
In an embodiment of the present disclosure, the recombinant vector as mentioned above the section A to B comprises about 17 nucleotides to 22 nucleotides, corresponding to sequence from the gene having accession number selected from a group comprising XM_003508l l3. l, NM 001243986.1, XM_003506664.3, CM_003503315.3, XM_003496727.3,
XM_007611030.2, XM 003510196.1, XM_0076528l9.2, XM_003507462.1,
XM_003504087.2, XM_007643683.l, XM 003502603.1, XM_003502077.2,
XM_0035l 1129.3, XM_003502604. l, XM_003502079.l and XM_003497300. l; the section B to C in the structure of the ShRNA comprises about 7 to 9 nucleotides selected from a group comprising A, T, C and G; and the section C to D in the structure of ShRNA corresponds to reverse complementary sequence of the nucleotide sequence present in the section A to B, wherein the said gene sequence belong to proteases.
In another embodiment of the present disclosure, the shRNA is cloned into a lentiviral construct
The present disclosure further relates to a recombinant host cell comprising the vector as mentioned above
In another embodiment of the present disclosure, the host cell as mentioned above has complete or partial knockdown of at least one protease.
In another embodiment of the present disclosure, wherein the protease sequence is selected from a group having accession number XM_003508l l3. l corresponding to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986. l corresponds to Cricetulus griseus furin, paired basic amino acid cleaving enzyme (Furin), mRNA; XM_003506664.3 corresponds to Cricetulus griseus lactotransferrin (Ltf), mRNA; XM_0035033l5.3 corresponds to Cricetulus griseus plasminogen activator, tissue type (Plat), mRNA; XM_003496727.3 corresponds to Cricetulus griseus plasminogen activator, urokinase (Plau), transcript variant X2, mRNA; XM_0076l 1030.2 corresponds to Cricetulus griseus chymase (LOC 100767563), mRNA; XM_003510196.1 corresponds to Cricetulus griseus kallikrein 1 (Klkl), mRNA; XM_0076528l9.2 corresponds to Cricetulus griseus complement factor I (Cfi), transcript variant XI, mRNA; XM_003507462.l corresponds to Cricetulus griseus coagulation factor XI (Fl l), mRNA; XM_003504087 corresponds to Cricetulus griseus coagulation factor VII (F7), mRNA; XM_007643683.l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603.l corresponds to Cricetulus griseus myeloblastin (LOC 100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC 100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, thrombin (F2), transcript variant XI, mRNA.
In an embodiment of the present disclosure, the host cell is selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK, PER.C6, yeast and E. coli.
In an embodiment of the present disclosure, the host cell is capable of enhanced yield of undegraded recombinant protein between about 10% to 300%.
The present disclosure further relates to a method of enhancing the recombinant protein production comprising expressing the protein of interest in the host cell mentioned above.
In an embodiment of the present disclosure, the method enhances the recombinant protein production by about 10% to 300%.
The present disclosure is based on the unexpected/surprising finding that transfecting or transforming a host cell with a vector comprising at least one nucleotide encoding ShRNA, wherein the said vector causes complete or partial knockdown of protease in a host cell to obtain a novel recombinant host cell capable of higher yield of undegraded recombinant protein.
In an embodiment, the vector comprising at least one nucleotide encoding ShRNA is a recombinant vector that causes complete or partial knockdown of at least one protease in a cell transformed or transfected with the said vector to yield a recombinant host cell.
In an embodiment of the present disclosure, transfection or transforming the host cell with a vector comprising at least one nucleotide sequence encoding ShRNA allows for significant improvement in yield of recombinant production of a product of interest.
In another embodiment of the present disclosure, the vector comprises plurality of nucleotide sequence encoding ShRNA.
In an embodiment of the present disclosure plurality of nucleotide sequences encoding to plurality of ShRNA, causes expression of plurality of ShRNA.
In an embodiment of the present disclosure, the vector comprises lentiviral construct.
In an embodiment of the present disclosure, the structure of ShRNA in the said vector is represented by Structure 1
Figure imgf000006_0001
In an embodiment of the present disclosure, the protease which are targeted by ShRNA expressed by the vector is selected from a group comprising elastase II, cathepsin-G, granzyme B, thrombin, proprotein convertase subtilisin, furin, lactotransferrin, plasminogen activator tissue type, plasminogen activator urokinase, chymase, kallikerein 1, complement factor I, coagulation factor XI, coagulation factor VII, plasminogen, myelobastin and HtrA serine peptidase 2.
In an embodiment of the present disclosure, in the illustrated structure of ShRNA, the section A to B comprises about 17 nucleotides to 22 nucleotides, corresponding to protease sequence from the gene having accession number selected from a group comprising XM_003508l l3.l, NM 001243986.1, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3,
XM_007611030.2, CM_003510196.1, CM_007652819.2, XM_003507462.l,
XM_003504087.2, XM_007643683. l, XM_003502603.l, XM_003502077.2,
XM_0035l 1129.3, XM_003502604.l, XM_003502079.1 and XM_003497300.l, or any combination thereof; the section B to C in the structure of the ShRNA comprises about 7 to 9 nucleotides selected from a group comprising A, T, C and G; and the section C to D in the structure of ShRNA corresponds to reverse complementary sequence of the nucleotide sequence present in the section A to B.
In an embodiment of the present disclosure, the illustrated structure of ShRNA upon expression through the vector causes complete knockdown or partial knockdown of proteases having gene sequence accession number CM_003508113.1, NM_001243986.1, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3, XM_007611030.2, CM_003510196.1,
XM_0076528l9.2, XM_003507462.1, XM_003504087.2, XM_007643683.l,
XM_003502603. l, XM_003502077.2, XM_0035l 1129.3, XM_003502604.l,
XM_003502079.1 or XM_003497300.l, or any combination.
In an embodiment of the present disclosure the accession number XM_003508l l3. l corresponds to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986. l corresponds to Cricetulus griseus furin, paired basic amino acid cleaving enzyme (Furin), mRNA; XM_003506664.3 corresponds to Cricetulus griseus lactotransferrin (Ltf), mRNA; XM_0035033 l5.3 corresponds to Cricetulus griseus plasminogen activator, tissue type (Plat), mRNA; XM_003496727.3 corresponds to Cricetulus griseus plasminogen activator, urokinase (Plau), transcript variant X2, mRNA; XM_0076l 1030.2 corresponds to Cricetulus griseus chymase (LOC 100767563), mRNA; XM_003510196.1 corresponds to Cricetulus griseus kallikrein 1 (Klkl), mRNA; XM_0076528l9.2 corresponds to Cricetulus griseus complement factor I (Cfi), transcript variant XI, mRNA; XM_003507462.1 corresponds to Cricetulus griseus coagulation factor XI (Fl 1), mRNA; XM_003504087 corresponds to Cricetulus griseus coagulation factor VII (F7), mRNA; XM_007643683. l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603. l corresponds to Cricetulus griseus myeloblastin (LOC100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, thrombin (F2), transcript variant XI, mRNA
In an embodiment of the present disclosure, the ShRNA upon expression through the vector in a cell causes complete or partial knockdown of at least one protease through techniques such including but is not limited to expression of zinc finger nucleases (ZFN), expression of engineered homing endonucleases, expression of transcription activator-like effector nuclease, expression of clustered regularly interspaced short palindromic repeats (CRISPR), expression of ShRNA, expression of SiRNA, expression of antisense RNA and miRNA.
The present disclosure further relates to a recombinant cell which is produced by causing partial or complete knockdown of at least one protease of the host cell.
In an embodiment of the present disclosure, the protease that are partially or completely knockdown in a host cell to obtain recombinant host cell is selected from a group comprising proteases having gene sequence accession number CM_003508113.1, NM_00l243986.l, XM_003506664.3, XM_0035033 l5.3, XM_003496727.3, XM_007611030.2,
CM_003510196.1, XM_0076528l9.2, XM_003507462.l, XM_003504087.2,
XM_007643683. l, XM_003502603.1, XM_003502077.2, XM_0035l 1129.3,
XM_003502604. l, XM_003502079. l and XM_003497300.1
In an embodiment of the present disclosure the accession number XM_003508l l3. l corresponds to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986. l corresponds to Cricetulus griseus furin, paired basic amino acid cleaving enzyme (Furin), mRNA; XM_003506664.3 corresponds to Cricetulus griseus lactotransferrin (Ftf), mRNA; XM_0035033 l5.3 corresponds to Cricetulus griseus plasminogen activator, tissue type (Plat), mRNA; XM_003496727.3 corresponds to Cricetulus griseus plasminogen activator, urokinase (Plau), transcript variant X2, mRNA; XM_0076l 1030.2 corresponds to Cricetulus griseus chymase (FOC 100767563), mRNA; XM_003510196.1 corresponds to Cricetulus griseus kallikrein 1 (Klkl), mRNA; XM_0076528l9.2 corresponds to Cricetulus griseus complement factor I (Cfi), transcript variant XI, mRNA; XM_003507462.1 corresponds to Cricetulus griseus coagulation factor XI (Fl 1), mRNA; XM_003504087 corresponds to Cricetulus griseus coagulation factor VII (F7), mRNA; XM_007643683. l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603. l corresponds to Cricetulus griseus myeloblastin (LOC100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, thrombin (F2), transcript variant XI, mRNA, in the 1. Chinese hamster Ovary cell or their equivalents in murine myeloma cell lines including NS0, Sp2/0 and HEK or PER.C6
In an embodiment of the present disclosure, the host cell is a prokaryote or a eukaryote.
In an embodiment of the present disclosure, the host is cell line selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK or PER.C6.
In another embodiment of the present disclosure, the host cell includes but is not limited to mammalian cell, insect cell, yeast cell or E. coli.
In an embodiment of the present disclosure, the host cell selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK, PER.C6, and other mammalian cell lines, are industry standard cell lines and are not prone to infection by any pathogen.
In an embodiment of the present disclosure, the recombinant cell is selected from a group comprising Chinese hamster ovary (CHO) cell line, murine myeloma (NS0, Sp2/0) cell line, HEK, PER.C6, other mammalian cell lines, yeast and E. coli, which has partial or complete knockdown of at least one of the protease described above.
In an embodiment of the present disclosure, the recombinant cell enhances yield between about 10% to 300% increase in the recombinant protein production.
In an embodiment of the present disclosure, the recombinant cell causes about 10%, about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200% , about 220% , about 240%, about 260%, about 280% or about 300% increase in the recombinant protein production.
In an embodiment of the present disclosure, the recombinant protein is selected from a group comprising antibodies, hormones, enzymes, virus like particles, vaccines and biosimilars like but not limited to Tocilizumab, Bevacizumab, Alemtuzumab, Trastuzumab, Adalimumab, Denosumab, Rituximab, Golimumab, Ustekinumab, Omalizumab, Ipilimumab, Ibritumomabtiuxetan, Alefacept, Rilonacept, Etanercept, Belatacept, Abatacept, Follitropin-b, Follitropin alfa, FH, Osteogenic Protein- 1, Choriogonadotropin a, Thyrotropin a, Darbepoetin a, Interferon b-la, Epoetin b, Epoetin a, Interferon b-la, Antihemophilic Factor, Factor IX, Antihemophilic Factor, Factor VIII, Alteplase, Faronidase, Imiglucerase, agalsidase-b, Hyaluronidase, Alglucosidase alfa, GalNAc 4-sulfatase, Human DNase, Tenecteplase, Efalizumab, Certoilzumab, Anakinra.
In an embodiment of the present disclosure, the complete knockdown or partial knockdown of the protease described above causes enhanced production of therapeutic protein.
The present disclosure further relates to a method for enhancing/increasing the yield of recovered recombinant protein.
In an embodiment of the present disclosure, the method comprises expression of recombinant protein in the recombinant cell described above, wherein in the recombinant cell there is complete knockdown or partial knockdown of plurality of proteases that enhances or increases the yield of recombinant protein.
In an embodiment of the present disclosure, the method of the present disclosure reduces the cost that is involved in producing recombinant protein as there is no need of adding protease inhibitor(s) externally.
In an embodiment of the present disclosure, during the method producing recombinant protein the productivity loss during prolonged culturing of the recombinant cell is considerably reduced.
In an embodiment of the present disclosure, the method significantly enhances the recombinant protein yield from the same amount of biomass without altering any media component or without addition of separate cocktail of protease inhibitor(s) or without causing modification to the downstream processing methods, thereby rendering the method more cost-efficient
In an embodiment of the present disclosure, the method of the present disclosure causes about 10% to 300 % increase in the production of the recombinant protein.
In an embodiment of the present disclosure, the method causes about 10%, about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200% , about 220% , about 240%, about 260%, about 280% or about 300% increase in the recombinant protein production.
In an embodiment, the Figures 2 and 4 illustrates the enhancement in the recombinant protein, EPO production, wherein there is increase in protein production in the recombinant cells which has undergone complete or partial knockdown of at least one protease.
In an embodiment, the Figure 3 illustrates the enhanced production of EPO in the recombinant cell clones of CHO cell lines, the individual recombinant cell clones were analyzed for their enhanced production of EPO.
In an exemplary embodiment of the present disclosure, the recombinant CHO cell line enhances the production of recombinant protein including but not limited to erythropoietin (EPO) by at least 10%, preferably at least 300%, more preferably at least 28% In the CHO cell line, elsastase-II is knockdown by the plurality of ShRNA expressed by the above described vector, thereby enhancing the production of the recombinant protein in the cell line.
Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon the description provided. The embodiments provide various features and advantageous details thereof in the description. Descriptions of well- known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments. The example provided herein is intended merely to facilitate an understanding of ways in which the embodiments provided may be practiced and to further enable those of skilled in the art to practice the embodiments provided. Accordingly, the following example should not be construed as limiting the scope of the embodiments. EXAMPLE
Example 1: Enhanced production of Erythropoietin
CHO-K1 cells were used as a host for erythropoietin (EPO) expression. The CHO-K1 cells expressing the EPO were stably transduced with ShRNA against two different proteases (protease 1 (Elastase II) and protease 2 (Granzyme B)), separately. The EPO in the culture supernatant was checked by ELISA. The stable cells expressing the ShRNA against the said proteases showed increase in the EPO expression (23% for protease 1 and 28% for protease 2) in the culture supernatant as compared to the control (illustrated in figure 4). Clonal selection could enhance the yield to about 300%.

Claims

WE CLAIM:
1. A recombinant vector comprising nucleotide sequence encoding ShRNA comprising section A to B, B to C and C to D represented by structure 1.
Figure imgf000013_0001
2. The recombinant vector as claimed in claim 1, the section A to B comprises about 17 nucleotides to 22 nucleotides, corresponding to sequence from the gene having accession number selected from a group comprising XM_003508l l3.l,
NM 001243986.1, XM_003506664.3, CM_003503315.3, XM_003496727.3,
XM_007611030.2, XM 003510196.1, XM_0076528l9.2, XM_003507462.1,
XM_003504087.2, XM 007643683.1, XM_003502603.l, XM_003502077.2,
XM_0035l 1129.3, XM_003502604.l, XM_003502079.l and XM_003497300.l; the section B to C in the structure of the ShRNA comprises about 7 to 9 nucleotides selected from a group comprising A, T, C and G; and the section C to D in the structure of ShRNA corresponds to reverse complementary sequence of the nucleotide sequence present in the section A to B.
3. The recombinant vector as claimed in claim 1 , wherein the vector shRNA is cloned into a lentiviral construct.
4. A recombinant host cell comprising the vector as claimed in claim 1.
5. The recombinant host cell as claimed in claim 4, has complete or partial knockdown of at least one protease.
6. The recombinant cell as claimed in claim 5, wherein the protease sequence is selected from a group having accession number XM_003508l l3.l corresponding to Cricetulus griseus proprotein convertase subtilisin/kexin type 2 (Pcsk2), transcript variant XI, mRNA; NM_00l243986.l corresponds to Cricetulus griseus furin, paired basic amino acid cleaving enzyme (Furin), mRNA; XM_003506664.3 corresponds to Cricetulus griseus lactotransferrin (Ltf), mRNA; XM_0035033l5.3 corresponds to Cricetulus griseus plasminogen activator, tissue type (Plat), mRNA; XM_003496727.3 corresponds to Cricetulus griseus plasminogen activator, urokinase (Plau), transcript variant X2, mRNA; XM_0076l 1030.2 corresponds to Cricetulus griseus chymase (LOC 100767563), mRNA; XM_003510196.1 corresponds to Cricetulus griseus kallikrein 1 (Klkl), mRNA; XM_0076528l9.2 corresponds to Cricetulus griseus complement factor I (Cfi), transcript variant XI, mRNA; XM_003507462.l corresponds to Cricetulus griseus coagulation factor XI (Fl l), mRNA; XM_003504087 corresponds to Cricetulus griseus coagulation factor VII (F7), mRNA; XM_007643683.l corresponds to Cricetulus griseus plasminogen (Plg), transcript variant XI, mRNA; XM_003502603.l corresponds to Cricetulus griseus myeloblastin (LOC100752428), mRNA; XM_003502077.2 corresponds to Cricetulus griseus granzyme B (LOC 100765811), transcript variant X2, mRNA; XM_003511129.3 corresponds to Cricetulus griseus HtrA serine peptidase 2 (Htra2), mRNA; XM_003502604.l corresponds to Cricetulus griseus elastase, neutrophil expressed (Elane), mRNA; XM_003502079.1 corresponds to Cricetulus griseus cathepsin G (LOC 100767270), mRNA; and XM_003497300.l corresponds to Cricetulus griseus coagulation factor II, thrombin (F2), transcript variant XI, mRNA.
7. The recombinant host cell as claimed in claim 5 is selected from a group comprising Chinese hamster ovary (CHO) cell lines, murine myeloma (NS0, Sp2/0) cell lines, HEK, PER.C6, yeast and E.coli.
8. The recombinant host cell as claimed in claim 4 is capable of enhanced yield of undegraded recombinant protein between about 10% to 300%.
9. A method of enhancing the recombinant protein production comprising expressing the protein of interest in the recombinant host cell as claimed in claim 4.
10. The method as claimed in claim 9 enhances the recombinant protein production by about 10% to 300%.
PCT/IB2019/052213 2018-03-19 2019-03-19 A vector, a host cell and a method thereof WO2019180607A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201741033057 2018-03-19
IN201741033057 2018-03-19

Publications (1)

Publication Number Publication Date
WO2019180607A1 true WO2019180607A1 (en) 2019-09-26

Family

ID=67988430

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2019/052213 WO2019180607A1 (en) 2018-03-19 2019-03-19 A vector, a host cell and a method thereof

Country Status (1)

Country Link
WO (1) WO2019180607A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846894B2 (en) * 2004-12-30 2010-12-07 Agency For Science, Technology And Research Chinese hamster apoptosis-related genes
US8273722B2 (en) * 2007-07-13 2012-09-25 Dharmacon, Inc. Enhanced biotherapeutic production using inhibitory RNA
EP2829608A1 (en) * 2013-07-23 2015-01-28 Universität Bielefeld Method for recombinant protein production in mammalian cells
US20170044587A1 (en) * 2014-04-29 2017-02-16 Novartis Ag Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846894B2 (en) * 2004-12-30 2010-12-07 Agency For Science, Technology And Research Chinese hamster apoptosis-related genes
US8273722B2 (en) * 2007-07-13 2012-09-25 Dharmacon, Inc. Enhanced biotherapeutic production using inhibitory RNA
EP2829608A1 (en) * 2013-07-23 2015-01-28 Universität Bielefeld Method for recombinant protein production in mammalian cells
US20170044587A1 (en) * 2014-04-29 2017-02-16 Novartis Ag Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest

Similar Documents

Publication Publication Date Title
Lalonde et al. Therapeutic glycoprotein production in mammalian cells
CN107435051B (en) Cell line gene knockout method for rapidly obtaining large fragment deletion through CRISPR/Cas9 system
Datta et al. An'omics approach towards CHO cell engineering
CN107429255B (en) Methods for introducing multiple expression constructs into eukaryotic cells
Schatz et al. Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature
Ruth et al. Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris
WO2015010114A1 (en) Methods and compositions for producing double allele knock outs
CN105143464B (en) Universal protein overexpression mark and its application comprising RAMP functions
Meents et al. p27Kip1‐mediated controlled proliferation technology increases constitutive sICAM production in CHO‐DUKX adapted for growth in suspension and serum‐free media
CN116904331A (en) Carbon source regulated protein production in recombinant host cells
Hemayatkar et al. Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae
WO2020012446A1 (en) Double knock-out cho cell line method of its generation and producing therapeutic proteins therefrom
BR112020005217A2 (en) methods for genetic engineering of kluyveromyces host cells
Wang et al. Use of CRISPR‐Cas tools to engineer Trichoderma species
Vijayakumar et al. A Systematic review of the potential of Pichia pastoris (Komagataella phaffii) as an alternative host for biologics production
WO2019180607A1 (en) A vector, a host cell and a method thereof
AU588236B2 (en) Novel vector comprising dna sequences from the bombyx mori nuclear polyhedrosis virus, and the use of said vector to produce gene products by genetic engineering
US11739311B2 (en) Gene recombinant vector, genetically engineered strain and preparation method of collagenase
EP0658626B1 (en) Methods for producing protein C
CN105779490A (en) Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody
CA2384122A1 (en) Cyclic depsipeptide synthetase and gene thereof, and mass production system for cyclic depsipeptide
KR101380728B1 (en) Method for mass production of Factor VII/VIIa
EP1554389A2 (en) Method for making recombinant proteins
CN109593697B (en) Recombinant pseudomonas for producing 3-hydroxypropionic acid and construction method thereof
Melchor-Moncada et al. Scale-Up of the Fermentation Process for the Production and Purification of Serratiopeptidase Using Silkworm Pupae as a Substrate

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19772254

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19772254

Country of ref document: EP

Kind code of ref document: A1