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.

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

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%.