Improvement of Protein Production Using miRNA Technology

Abstract

The present invention relates to the use of miRNA technology for improving the recombinant production of a polypeptide of interest in a host cell. An expression cassette is provided that produces an miRNA that targets and downregulates a host cell protein that interferes with the production of the polypeptide of interest.

Description

Technical Field

[0001] The present invention relates to the field of recombinant protein production. The present invention provides methods and means for reducing the expression of host cell proteins that interfere with the production of a protein of interest by using artificial miRNAs that target host cell proteins. In particular, expression cassettes for each miRNA comprising an intron sequence having a template for pri-miRNA, vectors and host cells comprising said expression cassettes, and their use for producing a protein of interest are provided.

Background Art

[0002] The generation of recombinant cell lines for producing secreted proteins requires transfection of a DNA vector into host cells and enrichment of stable transfectants using a selectable marker. Secreted proteins can often affect cell parameters such as growth, viability and / or productivity that require cell line engineering methods to achieve stable cell lines with high expression.

[0003] Similarly, the quality of secreted proteins can be affected by endogenous cell-derived factors. In many cases, endogenously expressed genes such as cell surface receptors, enzymes or proteases can be identified as the root cause of unwanted effects (see, for example, WO 2014 / 097113 A2). Each unwanted effect includes enzymatic cleavage of the polypeptide chain of the recombinant protein, such as digestion of the entire protein or amino acid clipping, i.e., removal of one or several amino acids from the N-terminus or C-terminus of the protein of interest. Other effects are unwanted post-translational modifications - or removal of desired modifications. Furthermore, endogenous gene products of the host cell can specifically interact with the protein of interest. Such interactions can recruit the protein of interest from the supernatant of the cell culture, making it difficult to remove the endogenous protein during purification, or can lead to activation of signal transduction pathways within the host cell that result in decreased cell growth, viability and / or productivity. In other cases, the endogenous gene product may simply have chemical and physical properties very similar to the protein of interest, making it difficult to develop a purification process that efficiently removes host cell proteins without reducing the yield of the protein of interest. This is particularly relevant for therapeutic proteins, which require high purity and low residual levels of host cell proteins in the final product to obtain and maintain marketing approval. Thus, endogenous gene products of the host cell can significantly interfere with the recombinant production of the protein of interest. In view of the above, there is a need to provide strategies for reducing the unwanted effects of host cell proteins on the recombinant production of the protein of interest. SUMMARY OF THE INVENTION

[0004] The inventors have found that the use of a specific expression cassette encoding an artificial miRNA is very effective in specifically targeting host cell proteins that interfere with the production of a polypeptide of interest. Using this approach, the expression of interfering host cell proteins can be significantly reduced, thereby improving the yield and / or purity of the polypeptide of interest. The reduction of interfering endogenous gene products using miRNA technology according to the present invention requires only minimal genetic manipulation. The pri-miRNA template sequence can be easily introduced into a plasmid already available for the expression of the polypeptide of interest, or using a standard vector, the miRNA template can be introduced into an established production host cell.

[0005] Furthermore, in most cases, knocking down the interfering host cell protein to residual expression levels is sufficient to significantly improve the production of the polypeptide of interest. For example, a complete knockout of an interfering gene product in a host cell, such as by genetic manipulation of the host cell's genome, is much more complex and can have an adverse effect on the viability and effectiveness of the host cell.

[0006] Accordingly, in a first aspect, the present invention provides an expression cassette for expressing an miRNA in a host cell, the expression cassette comprising an intron sequence containing a template sequence of a pri-miRNA, wherein the pri-miRNA is processed in the host cell to form an miRNA suitable for targeting a gene product of the host cell that interferes with and / or regulates the production of a polypeptide of interest recombinantly expressed in the host cell, and the miRNA comprises a passenger strand and a guide strand having an artificial sequence.

[0007] In a second aspect, the present invention provides a vector nucleic acid for transfection of a host cell, comprising the expression cassette according to the first aspect.

[0008] In a third aspect, the present invention provides a host cell comprising the expression cassette of the first aspect or the vector nucleic acid of the second aspect, which is capable of recombinantly expressing a polypeptide of interest.

[0009] In a fourth aspect, the present invention provides a method for producing a polypeptide of interest in a host cell, comprising: (a) providing a host cell according to the third aspect; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, processing the polypeptide of interest, and providing a method wherein the polypeptide of interest may be encoded, optionally, on the same vector nucleic acid as the pri-miRNA, particularly within the same expression cassette.

[0010] In a fifth aspect, the present invention provides a method for producing the host cell according to the third aspect, comprising: (a) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid contains a coding sequence for the polypeptide of interest either within the expression cassette expressing the miRNA or within an additional expression cassette; or (b) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid does not contain a coding sequence for the polypeptide of interest, and introducing an additional vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, comprising introducing different vector nucleic acids into the host cell simultaneously or sequentially in any order.

[0011] In a sixth aspect, the present invention provides the use of the expression cassette of the first aspect or the vector nucleic acid of the second aspect or the host cell of the third aspect for the production of a polypeptide of interest.

[0012] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and the appended claims. However, it should be understood that the following description, the appended claims, and specific examples showing preferred embodiments of the present application are merely presented by way of illustration. Various modifications and changes within the spirit and scope of the present invention will readily become apparent to those skilled in the art from reading the following.

[0013] Definition As used herein, the following expressions are preferably intended to have the meanings set forth below, except to the extent that the context in which they are used indicates otherwise.

[0014] As used herein, the expression "comprising" includes, in addition to its literal meaning, the expressions "consisting essentially of" and "consisting of", and specifically refers to these. Thus, the expression "comprising" refers to embodiments in which the "comprising" subject of specifically listed elements does not include further elements, as well as embodiments in which the "comprising" subject of specifically listed elements may include and / or actually includes further elements. Similarly, the expression "having" should be understood as the expression "comprising", and also includes the expressions "consisting essentially of" and "consisting of", and is specifically referred to. The term "consisting essentially of" refers, where possible, to embodiments in which the subject includes, in addition to the specifically listed elements of which the subject essentially consists, further elements of 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less.

[0015] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, as well as ribonucleic acid and deoxyribonucleic acid. It may include naturally occurring nucleotides as well as synthetic nucleotides, and may be modified naturally or synthetically, for example by methylation, 5'- and / or 3'-capping. In certain embodiments, the nucleic acid refers to double-stranded deoxyribonucleic acid.

[0016] The term "expression cassette" refers in particular to a nucleic acid construct that enables and can regulate the expression of a coding nucleic acid sequence and / or a template nucleic acid sequence introduced therein. An expression cassette may contain a promoter, a ribosome binding site, an enhancer and other regulatory elements that regulate the transcription of a gene or the translation of mRNA. The exact structure of the expression cassette can vary depending on the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' untranslated sequences that are each involved in the initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, etc. More specifically, the 5' non-transcribed expression control sequence includes a promoter region containing a promoter sequence for the transcriptional control of an operably linked nucleic acid. The expression cassette may also contain an enhancer sequence or an upstream activator sequence. Some expression cassettes are used only for the transcription of a template nucleic acid sequence into an RNA product such as pri-miRNA. Such expression cassettes do not necessarily contain regulatory elements for translation.

[0017] According to the present application, the template nucleic acid is understood as DNA that is transcribed into a functional RNA product or its precursor, in particular pri-miRNA. A functional RNA product has, in particular, alone or in combination with other RNA products and / or proteins, a biological activity, such as the activity of an miRNA that interferes (in combination with the proteins of RISC) with the expression of a target gene.

[0018] According to the present invention, the term "promoter" refers to a nucleic acid sequence that is located upstream (5') of the nucleic acid sequence to be expressed and controls the expression of the sequence by providing a recognition and binding site for RNA-polymerase. A "promoter" may include additional recognition and binding sites for additional factors involved in the regulation of gene transcription. A promoter can control the transcription of a prokaryotic gene or a eukaryotic gene. Further, a promoter can be "inducible", i.e., capable of initiating transcription in response to an inducer, or "constitutive" if transcription is not controlled by an inducer. A gene under the control of an inducible promoter is not expressed or is expressed only to a low extent in the absence of the inducer. In the presence of the inducer, the gene is switched on or the transcription level increases. This is generally mediated by the binding of specific transcription factors.

[0019] The term "vector" is used herein in its most general sense and includes any intermediate vehicle for a nucleic acid that enables the nucleic acid to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to extrachromosomal genetic material that can replicate independently of chromosomal DNA, usually a circular DNA double-stranded construct. The vectors according to the present invention can exist in circular or linearized form. The "vector nucleic acid" as used herein is the nucleic acid forming the vector or the nucleic acid part of the vector.

[0020] The terms "5'" and "3'" are conventions used to describe features of nucleic acid sequences related to either the position of genetic elements and / or the direction (5' to 3') of an event, such as transcription by RNA polymerase or translation by ribosomes proceeding in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Conventionally, DNA sequences, genetic maps, vector maps, and RNA sequences are depicted 5' to 3' from left to right, or the 5' to 3' direction is indicated by an arrow that points in the 3' direction. Thus, 5' (upstream) indicates a genetic element positioned towards the left hand side when following this convention, and 3' (downstream) indicates a genetic element positioned towards the right hand side.

[0021] "Polypeptide" or "polypeptide chain" refers to a molecule that includes a polymer of amino acids linked together by peptide bonds. Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., having 2 to 49 amino acids). In particular, a polypeptide or polypeptide chain can be part of a protein consisting of two or more polypeptide chains. Polypeptides include any active or bioactive protein and / or peptide. A polypeptide can be a pharmaceutically or therapeutically active compound, or a research tool used in assays and the like.

[0022] When the target amino acid sequence shares identity with a reference amino acid sequence over its entire length of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99%, the target amino acid sequence is "derived from" or "corresponds to" the reference amino acid sequence. In certain embodiments, a target amino acid sequence that is "derived from" or "corresponds to" a reference amino acid sequence is 100% identical to the reference amino acid sequence over its entire length. Similarly, when the target nucleotide sequence shares identity of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% with a reference nucleotide sequence over its entire length, the target nucleotide sequence is "derived from" or "corresponds to" the reference nucleotide sequence. In certain embodiments, a target nucleotide sequence that is "derived from" or "corresponds to" a reference nucleotide sequence is 100% identical to the reference nucleotide sequence over its entire length. "Identity" of an amino acid sequence or nucleotide sequence is preferably determined according to the present invention over the entire length of the reference sequence.

[0023] As used herein, the abbreviation "microRNA" or "miRNA" refers to a single-stranded non-coding RNA molecule that plays a role in RNA silencing and post-transcriptional regulation of gene expression. miRNAs generally consist of 19 to 24 nucleotides, particularly about 22 nucleotides, especially 22 nucleotides. miRNA molecules can silence mRNAs containing complementary nucleotide sequences. Silencing of the target mRNA can occur by cleavage of the mRNA, destabilization of the mRNA, or interference with translation of the mRNA. Silencing of the target mRNA results in a decrease or elimination of the production of the protein encoded by the target mRNA. It is generally understood in the art that miRNAs associate with dicer and argonaute proteins to form an RNA-induced silencing complex (RISC) that binds to the target mRNA.

[0024] miRNAs are produced by transcription of the miRNA precursor (pri-miRNA) from the template DNA sequence. The pri-miRNA contains a hairpin stem-loop structure with a double-stranded stem having a loop connected to one side and single-stranded 5' and 3' extensions adjacent to the other side. The double-stranded stem contains the guide strand that forms the miRNA upon processing and the passenger strand that is essentially complementary to the guide strand. In particular, the passenger strand and the guide strand are complementary to each other except for the nucleotide pair at the end of the hairpin stem-loop structure, i.e., the nucleotide pair of the passenger and guide strands that is farthest from the loop structure. The guide strand and the passenger strand generally have lengths of about 19-24 nucleotides, particularly 22 nucleotides, respectively. The remaining portion of the pri-miRNA is referred to herein as the miRNA scaffold. Thus, from 5' to 3', the pri-miRNA includes: (i) a 5' miRNA scaffold stem consisting of the 5' portion of the stem structure up to the 5' single-stranded extension and the passenger strand; (ii) the passenger strand; (iii) the miRNA scaffold loop; (iv) the guide strand; (v) a 3' miRNA scaffold stem consisting of the 3' portion of the stem structure following the guide strand and the 3' single-stranded extension. Also, the positions of the passenger strand and the guide strand may be interchanged.

[0025] The pri-miRNA is processed by cleaving the 5' and 3' miRNA scaffold stems, resulting in a hairpin structure called pre-miRNA. From 5' to 3', the pre-miRNA consists of the passenger strand, the miRNA scaffold loop, and the guide strand; also, the positions of the passenger strand and the guide strand may be interchanged. Then, the loop structure is cleaved and the resulting RNA duplex is separated into two single-stranded RNA molecules, the guide strand and the passenger strand. The guide strand complementary to the target mRNA molecule forms the RISC, where the passenger strand generally has no function.

[0026] The cells referred to in this specification are, in particular, host cells. According to the present invention, the term "host cell" relates to any cell that can be transformed or transfected with an exogenous nucleic acid. Mammalian cells, such as cells derived from humans, mice, hamsters, pigs, goats or primates, are particularly preferred. The cells can be derived from a number of tissue types and can include primary cells and cell lines. The nucleic acid can be present in the host cell in the form of a single copy or two or more copies, and in one embodiment, is expressed in the host cell. The host cell refers particularly to cells present in cell culture, particularly cells that do not exist in a living multicellular organism.

[0027] The term "pharmaceutical composition" or "pharmaceutical formulation" refers particularly to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises an active compound or a salt or prodrug thereof, together with a carrier, diluent or pharmaceutical excipient, such as a buffer, preservative and tonicity modifier.

[0028] The numbers presented in this specification can, in certain embodiments, be understood as approximate numbers. In particular, these numbers may preferably be up to 10% higher and / or lower, particularly up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and / or lower. In certain embodiments, the numbers presented in this specification are not approximate numbers and can only vary within the range of inaccuracy of technical measurements.

[0029] The numerical ranges described in this specification include the numbers defining the ranges. The headings provided in this specification do not limit the various aspects or embodiments of the present invention that can be read by referring to the specification as a whole. According to one embodiment, the subject matter described in this specification, in the case of a method, includes specific steps, or in the case of a composition, includes specific components, and refers to the subject matter consisting of each step or component. It is preferred to select and combine the preferred aspects and embodiments described in this specification, and the specific subject matter resulting from each combination of the preferred embodiments also belongs to this disclosure.

[0030] Detailed Description of the Invention The present invention is based on the development of a new vector construct for use in knocking down a target host cell protein. The endogenous proteins of a host cell sometimes interfere with the production of a protein of interest in said host cell. In order to prevent this interference, each host cell protein must be removed from the host cell line. In many cases, gene knockout has been performed for this purpose, but this is very time-consuming, labor-intensive, and costly. The present invention differs from this approach in that it acts at the RNA level to effect knockdown of the mRNA encoding the interfering host cell protein. Generally, complete knockout of each host cell gene is not required since minimal expression should result in a similar desired phenotype. Furthermore, knockout can result in unwanted cell compensatory effects. Thus, strong knockdown represents a preferred technique for inactivating interfering host cell genes. In the past, shRNA molecules were encoded in separate vectors and expressed using a strong polymerase-III promoter. Using the new approach according to the present invention, a normal polymerase-II promoter is sufficient and the pri-miRNA can be present on the same vector and even within the same expression cassette as the protein of interest produced in the host cell. This approach also shows high knockdown efficiency at the pool level already during the development of the production cell line, which allows for a very rapid assessment of knockdown efficacy.

[0031] 1. Expression Cassette for miRNA Expression In view of the above, in a first aspect, the present invention is an expression cassette for expressing miRNA in a host cell, the expression cassette comprising an intron sequence comprising a template sequence of pri-miRNA, wherein the pri-miRNA is processed in the host cell to form a miRNA that targets a gene product of the host cell that interferes with and / or regulates the production of a polypeptide of interest recombinantly expressed in the host cell, and wherein the miRNA comprises a passenger strand and a guide strand having an artificial sequence.

[0032] This expression cassette can be used to knockdown the target gene product of a host cell. Thereby, interference with the recombinant production of the polypeptide of interest of the gene product and / or regulation of the polypeptide of interest can be reduced or prevented. The miRNA can specifically target any host cell gene product that interferes with the production of the polypeptide of interest in the desired form. The gene product of the host cell targeted by the miRNA is generally referred to herein as the "interfering gene product".

[0033] It is understood that the miRNA produced from the expression cassette is at least partially complementary to the "underlying" RNA of the interfering gene product, can bind to it, and initiate its silencing. Thus, in embodiments where the interfering gene product is a protein or polypeptide or includes these, the underlying RNA is in particular the mRNA or pre-mRNA encoding the interfering gene product. In embodiments where the interfering gene product is an RNA or includes an RNA, the underlying RNA is in particular said RNA or its precursor. Silencing can occur by degradation of the targeted RNA or by preventing the targeted mRNA from being translated.

[0034] Since the expression cassette contains the template sequence of the pri-miRNA within the intron sequence, it can contain additional sequences for the expression of other products, such as a coding sequence for the production of the polypeptide of interest, a coding sequence for the production of a selectable marker, and template sequences for other RNA products, in particular other pri-miRNAs. Alternatively, the expression cassette can be used only for the production of miRNAs that target the interfering gene product.

[0035] 1.1 Elements of the Expression Cassette The expression cassette contains the template sequence of the pri-miRNA within the intron sequence. When expressed, a pre-mRNA containing the intron sequence is formed. Subsequently, the intron sequence is spliced from the pre-mRNA, thereby forming the pri-miRNA, which is then further processed to ultimately provide the miRNA. The formed pre-mRNA need not contain any sequence encoding a polypeptide.

[0036] In certain embodiments, the expression cassette further comprises a polymerase II promoter. This promoter is operably linked to the template sequence of the pri-miRNA and controls its expression. The promoter can be any RNA polymerase II promoter suitable for gene expression in the host cell, particularly the host cell used for the expression of the polypeptide of interest. In certain embodiments, the promoter is suitable for expression in eukaryotic host cells, particularly mammalian host cells such as CHO cells. For example, the promoter can be selected from the group consisting of the cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF1A) promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, BROAD3 promoter, mouse rosa26 promoter, pCEFL promoter, chicken beta-actin promoter (CBA), beta-actin promoter combined with the CMV early enhancer (CAGG), alpha-1-antitrypsin promoter, and inducible promoters such as the tetracycline-inducible promoter (e.g., pTRE), and the vanillic acid-inducible promoter. In certain embodiments, the promoter is the CMV promoter or the SV40 promoter, particularly the CMV promoter.

[0037] In certain embodiments, the expression cassette further comprises a terminator. The terminator is operably linked to the template sequence of the pri-miRNA and controls its expression. As used herein, the term "terminator" refers particularly to a transcriptional terminator that terminates the transcription of DNA into RNA by RNA polymerase II.

[0038] The template sequence of the pri-miRNA is located particularly between the promoter and the terminator of the expression cassette.

[0039] In certain embodiments, the expression cassette comprises, for example, a coding sequence encoding a polypeptide of interest or a selectable marker. In these embodiments, the expression cassette may further comprise a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). An intron sequence containing the template sequence of the pri-miRNA may be present within the 5'UTR, 3'UTR, or the coding sequence. In particular, the intron sequence is present within the 5'UTR or 3'UTR, particularly within the 5'UTR. In alternative embodiments, the expression cassette does not comprise a coding sequence encoding a polypeptide.

[0040] An intron sequence containing the template sequence of the pri-miRNA particularly comprises a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA. By these splice donor and acceptor sites, the pri-miRNA is spliced from the pre-mRNA after transcription.

[0041] In certain embodiments, the intron sequence comprises two or more template sequences for pri-miRNAs. In these embodiments, the intron sequence comprises a splice donor site upstream of the template sequence of the first pri-miRNA, i.e., the most 5'-side template sequence, and a corresponding splice acceptor site downstream of the template sequence of the last pri-miRNA, i.e., the most 3'-side template sequence. Adjacent template sequences within the intron sequence can be separated from each other by a spacer sequence. Such spacer sequences form an RNA stem-loop structure, particularly, such as the sequence of SEQ ID NO: 22.

[0042] In certain embodiments, the expression cassette comprises only one intron sequence having one or more template sequences for pri-miRNAs. In alternative embodiments, the expression cassette comprises two or more intron sequences having one or more template sequences for pri-miRNAs.

[0043] In particular, the pri-miRNAs of two or more template sequences present within the same or different intron sequences are different from each other. In certain embodiments, each miRNA produced from a pri-miRNA targets a different interfering gene product. In alternative embodiments, each miRNA produced from a pri-miRNA targets the same interfering gene product. In still further embodiments, some miRNAs produced from a pri-miRNA target the same interfering gene product, while other miRNAs produced from the pri-miRNA target different interfering gene products. miRNAs that target the same interfering gene product bind to different portions of the RNA of the interfering gene product, particularly, mRNA or pre-mRNA.

[0044] The expression cassette may contain a coding sequence encoding a polypeptide. The coding sequence may in particular encode a polypeptide of interest or a selectable marker. The coding sequence is preferably operably linked to a polymerase II promoter and a terminator of the expression cassette. In embodiments where the expression cassette contains a template sequence for pri-miRNA and a coding sequence for a polypeptide of interest, the expression of these two elements is linked. Thereby, host cells containing the expression cassette and simultaneously showing a high expression level of the polypeptide of interest also have a high expression level of miRNA. The development and selection of clones are significantly simplified by this approach. In embodiments where the expression cassette contains a template sequence for pri-miRNA and a coding sequence for a selectable marker, the expression of miRNA is linked to the selectable marker expression. Thus, by increasing the selection pressure during clone selection, the miRNA level also increases.

[0045] The selectable marker may be selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, zeocin and blasticidin. In certain embodiments, the selectable marker is folate receptor (FAR).

[0046] 1.2 miRNA The expression cassette contains a template sequence for pri-miRNA. The pri-miRNA produced from the expression cassette may have any structure suitable for processing by the host cell in order to obtain a functional miRNA that targets an interfering gene product of the host cell. The functional miRNA in particular induces a decrease in the level of the interfering gene product in the host cell.

[0047] In certain embodiments, the pri-miRNA comprises a passenger strand and a guide strand. The guide strand specifically comprises or consists of an miRNA formed after processing of the pri-miRNA by the host cell. Further, the pri-miRNA may comprise an miRNA scaffold loop and / or an miRNA scaffold stem, particularly a 5'miRNA scaffold stem and a 3'miRNA scaffold stem. In certain embodiments, the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a passenger strand, an miRNA scaffold loop, a guide strand, and a 3'miRNA scaffold stem. In alternative embodiments, the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a guide strand, an miRNA scaffold loop, a passenger strand, and a 3'miRNA scaffold stem. Embodiments in which the passenger strand is located upstream of the guide strand are preferred.

[0048] The passenger strand and the guide strand of the pri-miRNA have artificial sequences. The artificial sequences in this regard refer to sequences not found as the passenger strand or the guide strand in naturally occurring miRNAs. In particular, the sequences of the passenger strand and the guide strand are not found in naturally occurring miRNAs. In certain embodiments, the guide strand comprises, particularly consists of, a sequence designed to reduce the expression of an interfering gene product. In particular, the sequence of the guide strand is complementary to a part of the mRNA encoding the gene product of the host cell to which the miRNA is targeted.

[0049] In certain embodiments, one or more of the scaffold sequences of pri-miRNA or pre-miRNA are derived from naturally occurring pri-miRNA, particularly those that are naturally occurring in mammals, particularly humans. In certain embodiments, all of the scaffold sequences of pri-miRNA are derived from naturally occurring pri-miRNA, particularly those that are naturally occurring in mammals, particularly humans. In particular, all of the scaffold sequences of pri-miRNA are derived from the same naturally occurring pri-miRNA. The scaffold sequence of pri-miRNA particularly includes a 5'miRNA scaffold stem, a miRNA scaffold loop, and a 3'miRNA scaffold stem. Suitable naturally occurring pri-miRNA from which the scaffold sequence can be derived include miR-30A, miR-E, SIBR, eSIBR, miR-1, miR-155, miR-16, miR-16-1, miR-16-2, miR-3G, miRGE, miR100, miR125b, miR-130a, miR-190a, miR-193a, miR-211, miR-26a, miR-340, miR-7-2, miR-96, and miR-44. Thus, in one embodiment, the 5'miRNA scaffold stem, the miRNA scaffold loop, and the 3'miRNA scaffold stem are derived from one or more pre-miRNAs selected from the group consisting of miR-30A, miR-E, SIBR, eSIBR, miR-1, miR-155, miR-16, miR-16-1, miR-16-2, miR-3G, miRGE, miR100, miR125b, miR-130a, miR-190a, miR-193a, miR-211, miR-26a, miR-340, miR-7-2, miR-96, and miR-44. In certain embodiments, the naturally occurring pri-miRNA from which the scaffold sequence is derived is miR-30A.

[0050] In certain embodiments, all of the scaffold sequences of the pri-miRNA share at least 80%, particularly at least 90%, and particularly at least 95% nucleotide sequence identity over their entire length with the corresponding scaffold sequences of naturally occurring pri-miRNA. In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA shares at least 80%, particularly at least 85%, and particularly at least 90% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of naturally occurring pri-miRNA. In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA shares at least 80%, particularly at least 90%, and particularly at least 95% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of naturally occurring pri-miRNA. In certain embodiments, the miRNA scaffold loop of the pri-miRNA shares at least 60%, particularly at least 70%, and particularly at least 75% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of naturally occurring pri-miRNA. In these embodiments, the naturally occurring pri-miRNA can be particularly miR-30A.

[0051] In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence derived therefrom. In particular, the 5'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7, or a sequence sharing at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity therewith. In particular, the 5'miRNA scaffold stem of the pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 1-7, particularly any one of SEQ ID NOs: 1-4, and particularly SEQ ID NO: 1.

[0052] In certain embodiments, the miRNA scaffold loop of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-10 or a sequence derived therefrom. In particular, the miRNA scaffold loop of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-10, or a sequence sharing at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably 100% nucleotide sequence identity therewith. In particular, the miRNA scaffold loop of the pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 8-10, particularly the nucleotide sequence of SEQ ID NO: 8.

[0053] In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 11-17 or a sequence derived therefrom. In particular, the 3'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 11-17, or a sequence sharing at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity therewith. In particular, the 3'miRNA scaffold stem of the pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 11-17, particularly any one of SEQ ID NOs: 11-14, particularly the nucleotide sequence of SEQ ID NO: 11.

[0054] In certain embodiments, the template sequence of the pri-miRNA comprises at least one recognition site, particularly two recognition sites, for a DNA restriction enzyme. In particular, the two recognition sites are for different DNA restriction enzymes and generate different overhangs upon cleavage. The two recognition sites are preferably adjacent to the pre-miRNA portion of the pri-miRNA, including the guide strand, the passenger strand, and the miRNA scaffold loop, on both sides. In particular, one of the recognition sites is located within the sequence transcribed into the 5'miRNA scaffold stem, and the other recognition site is located within the sequence transcribed into the 3'miRNA scaffold stem. In certain embodiments, the recognition sites are located within the sequences transcribed into the single-stranded portions of the 5' and 3'miRNA scaffold stems. The recognition sites are particularly unique recognition sites within the expression cassette, particularly within the entire vector having the expression cassette.

[0055] 1.3 Target Gene The miRNA expressed by the expression cassette is for targeting an interfering gene product, a host cell gene product that interferes with and / or regulates the production of a polypeptide of interest recombinantly expressed in a host cell. The interfering gene product can be any gene product that interferes with and / or regulates the production of the polypeptide of interest. Exemplary interfering gene products are selected from the group consisting of, for example: (i) a protease capable of cleaving the polypeptide of interest (ii) a protein involved in post-translational modification of the polypeptide of interest (iii) a receptor or binding partner of the polypeptide of interest (iv) a protein that is difficult to separate from the polypeptide of interest (v) a protein involved in folding and / or secretion of the polypeptide of interest (vi) a protein involved in the transport of components required for the production or modification of the polypeptide of interest (vii) a protein involved in the degradation of the polypeptide of interest (viii) A protein that shares at least 70%, particularly at least 80% sequence identity with the polypeptide of interest over its entire length, or an endogenous homolog of the polypeptide of interest.

[0056] The expression of miRNA reduces the production of the interfering gene product in the host cell, thereby reducing or eliminating the production of the polypeptide of interest and / or the interference with the regulation of the polypeptide of interest. The interfering gene product is, in particular, an endogenous gene product of the host cell.

[0057] In certain embodiments, the interfering gene product is a protease capable of cleaving the polypeptide of interest. In these embodiments, the expression of miRNA particularly reduces the cleavage of the polypeptide of interest by the targeted protease.

[0058] In certain embodiments, the interfering gene product is a protein involved in the post-translational modification of the polypeptide of interest. For example, the interfering gene product can be a transferase capable of catalyzing the post-translational modification of the polypeptide of interest. Exemplary post-translational modifications include acetylation, acylation, sulfation, phosphorylation, alkylation, hydroxylation, amidation, carboxylation, palmitoylation, myristoylation, and isoprenylation. In these embodiments, the expression of miRNA particularly reduces the amount of post-translational modification catalyzed by the targeted transferase.

[0059] In a further embodiment, the interfering gene product can be an enzyme capable of catalyzing the post-translational modification of the polypeptide of interest or the removal of a chemical group of the polypeptide of interest. The interfering gene product can be, for example, a hydrolase such as lipase, phosphatase, or glycosidase. In these embodiments, the expression of miRNA particularly increases the amount of each post-translational modification or chemical group that would be removed by the interfering gene product.

[0060] In further embodiments, the interfering gene product can be a protein involved in the glycosylation of the polypeptide of interest. Exemplary proteins include glycosyltransferases, glycosidases, and nucleotide sugar transporters, such as fucosyltransferases and sialyltransferases. In these embodiments, the expression of the miRNA reduces, in particular, the degree of glycosylation motifs produced by or with the support of the interfering gene product.

[0061] In certain embodiments, the interfering gene product is a receptor or binding partner of the polypeptide of interest. Receptors and binding partners include any gene product that binds to the polypeptide of interest and thereby interferes with its production. This includes, in particular, in embodiments where the host cell is not of the same species as the polypeptide of interest (e.g., expression of a human polypeptide in a CHO host cell), gene products of other species, in particular homologs of natural receptors or binding partners of the polypeptide of interest. Receptors and binding partners can interfere with the production of the polypeptide of interest, for example, by sequestering the polypeptide of interest from the cell culture medium, thereby reducing its yield in the culture supernatant, or by reducing the growth or survival of the host cell, as binding of the polypeptide of interest to its receptor or binding partner activates signal pathways in the host cell.

[0062] In certain embodiments, the interfering gene product is a protein that is difficult to separate from the polypeptide of interest. Each interfering gene product includes, for example, a gene product that binds to the polypeptide of interest and a gene product that has chemical and / or physical properties similar to those of the polypeptide of interest. Exemplary chemical and / or physical properties in this regard are molecular size, overall charge, charge distribution, pI value, hydrophobicity, and binding to capture ligands such as protein A or protein G. In these embodiments, the interfering gene product cannot be sufficiently separated from the polypeptide of interest, particularly using standard purification methods. For example, the interfering gene product and the polypeptide of interest may have similar binding and / or elution properties on one or more chromatography matrices. In such embodiments, removal of the interfering gene product from the product solution results in a significant loss of the polypeptide of interest and thus a low yield of the purified polypeptide of interest.

[0063] 2. Vector and Host Cell In a second aspect, the present invention provides a vector nucleic acid for transfection of a host cell, comprising the expression cassette described in the first aspect. The vector can be any vector suitable for transfection of a host cell. In certain embodiments, the vector is a plasmid. In other embodiments, the vector is a viral vector.

[0064] The vector nucleic acid may include additional elements in addition to the expression cassette. For example, the vector nucleic acid may include an origin of replication (ORI), a coding sequence encoding the polypeptide of interest, a selectable marker gene, and / or an antibiotic resistance gene.

[0065] In certain embodiments, the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest. In a particular embodiment where the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest, the expression cassette according to the first aspect of the invention does not contain a coding sequence of the polypeptide. In these embodiments, the vector nucleic acid particularly contains a further expression cassette containing a selection marker gene. In an alternative embodiment where the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest, the expression cassette according to the first aspect of the invention contains a coding sequence encoding a selection marker.

[0066] In certain embodiments, the vector nucleic acid contains a coding sequence encoding a polypeptide of interest. The coding sequence encoding the polypeptide of interest may be present within the expression cassette according to the first aspect of the invention or may be present within a further expression cassette. In a particular embodiment, the vector nucleic acid contains at least two expression cassettes, a first expression cassette for expressing the polypeptide of interest and a second expression cassette for expressing a selection marker, and either the first or the second expression cassette is an expression cassette according to the first aspect of the invention. In an alternative embodiment, the vector nucleic acid contains at least three expression cassettes, the first expression cassette is an expression cassette according to the first aspect of the invention, the second expression cassette is for expressing the polypeptide of interest, and the third expression cassette is for expressing a selection marker. If the polypeptide of interest is composed of two or more different polypeptide chains, the different polypeptide chains may be encoded within the same expression cassette or within separate expression cassettes. Additional expression cassettes containing coding sequences of different polypeptide chains of the polypeptide of interest may also be present on the vector nucleic acid.

[0067] In a particular embodiment, two or more expression cassettes of the vector nucleic acid are expression cassettes according to the first aspect of the invention. Each of these expression cassettes may contain the template sequences of the same or different pri-miRNAs, particularly different pri-miRNAs, which may target the same or different interfering gene products of the host cell.

[0068] In a third aspect, the present invention provides a host cell comprising the expression cassette of the first aspect or the vector nucleic acid of the second aspect, which is capable of recombinantly expressing a polypeptide of interest.

[0069] The host cell can be any type of host cell that produces a gene product that interferes with the production of the polypeptide of interest and / or regulates the polypeptide. In certain embodiments, the host cell is known to produce an interfering gene product. The host cell is particularly suitable for producing the polypeptide of interest.

[0070] A host cell "capable of recombinantly expressing a polypeptide of interest" is in particular a host cell comprising a nucleic acid encoding the polypeptide of interest. In particular, the nucleic acid encoding the polypeptide of interest is heterologous to the host cell and has been introduced into the host cell using genetic engineering techniques.

[0071] In certain embodiments, the host cell is a mammalian cell. The host cell can in particular be a rodent cell, a primate cell or a human cell. In certain embodiments, the mammalian cell is selected from the group consisting of, but not limited to, cells derived from mice, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20 and NIH3T3; cells derived from rats, such as PC12, PC12h, GH3, MtT, YB2 / 0 and Y0; cells derived from hamsters, such as BHK, CHO and DHFR gene-deficient CHO; cells derived from monkeys, such as COS1, COS3, COS7, CV1 and Vero; and cells derived from humans, such as Hela, HEK293, CAP, PER-C6 derived from the retina, cells derived from diploid fibroblasts, myeloma cells and HepG2. In a specific embodiment, the host cell is a Chinese hamster ovary (CHO) cell. The host cell may be suitable for suspension culture and / or adherent culture, and can in particular be used in suspension culture.

[0072] In addition to the expression cassette according to the first aspect or the vector nucleic acid according to the second aspect of the present invention, the host cell may contain further exogenous nucleic acids. In particular, the host cell may contain an expression cassette for expressing a polypeptide of interest that is not the expression cassette of the first aspect and is not present on the vector nucleic acid of the second aspect. The expression cassette for the expression of the polypeptide of interest may be present on a further vector nucleic acid or may be integrated into the genome of the host cell.

[0073] In certain embodiments, the coding sequence of the polypeptide of interest is in the host cell (i) within the expression cassette that expresses the miRNA, (ii) within a further expression cassette on the same vector nucleic acid as the expression cassette that expresses the miRNA, or (iii) present on a further vector nucleic acid or in the genome of the host cell.

[0074] 3. Production Method In a fourth aspect, the present invention provides a method for producing a polypeptide of interest in a host cell, comprising: (a) providing a host cell according to the third aspect; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, processing the polypeptide of interest, and providing a method wherein the polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, particularly within the same expression cassette.

[0075] The steps of the method are generally performed in the order shown.

[0076] In certain embodiments, the method comprises, between steps (a) and (b), (a1) inoculating the host cell into a cell culture medium to provide a cell culture, and (a2) further comprising culturing the host cells in the cell culture under conditions that can increase the number of cells in the cell culture.

[0077] Suitable conditions for culturing host cells, increasing their cell numbers, and expressing the polypeptide of interest depend on the particular host cells, vectors, and expression cassettes used in the method. Those skilled in the art can readily determine the appropriate conditions, which are also already known in the art for a plurality of host cells. In certain embodiments, the vector nucleic acid in the host cell comprises one or more selectable marker genes. In these embodiments, the culture conditions of step (a2) and / or (b) may include the presence of the corresponding selection agent in the cell culture medium.

[0078] Obtaining the polypeptide of interest from the cell culture in step (c) particularly includes isolating the polypeptide of interest from the cell culture. In particular, the isolation of the polypeptide of interest refers to the separation of the polypeptide of interest from the remaining components of the cell culture. As used herein, the term "cell culture" particularly includes the cell culture medium and the cells. In certain embodiments, the polypeptide of interest is secreted by the host cells. In these embodiments, the polypeptide of interest is isolated from the cell culture medium. The separation of the polypeptide of interest from the cell culture medium can be carried out, for example, by chromatography methods. Suitable methods and means for isolating the polypeptide of interest are known in the art and can be readily applied by those skilled in the art.

[0079] The resulting polypeptide of interest can optionally be subjected to further processing step (d), for example further purification, modification and / or formulation steps, in order to produce the polypeptide of interest with the desired quality and composition. Such further processing steps and methods are generally known in the art. Suitable purification steps include, for example, affinity chromatography, size exclusion chromatography, anion and / or cation exchange chromatography, hydrophilic interaction chromatography and reverse phase chromatography. Further steps can include virus inactivation, ultrafiltration and diafiltration. The formulation step can include buffer exchange, addition of formulation components, pH adjustment and concentration adjustment. Any combination of these and further steps can be used.

[0080] In certain embodiments, the method for producing the polypeptide of interest further comprises, as step (d) or part of step (d), providing a pharmaceutical formulation comprising the polypeptide of interest. Providing a pharmaceutical formulation comprising the polypeptide of interest, or formulating the polypeptide of interest as a pharmaceutical composition, in particular comprises exchanging the buffer solution or buffer solution components of the composition comprising the polypeptide of interest. Further, this step can include lyophilization of the polypeptide of interest. In particular, the polypeptide of interest is transferred to a composition containing only pharmaceutically acceptable components.

[0081] In certain embodiments, the production of the interfering gene product in the host cell is reduced as compared to the same host cell that does not express the miRNA targeting the interfering gene product. In particular, the amount of the interfering gene product in the host cell is reduced to 50% or less, particularly 20% or less, and even 10% or less as compared to the same host cell that does not express the miRNA targeting the interfering gene product.

[0082] The present invention further provides a method for producing a polypeptide of interest in high yield and / or high purity, comprising (a) providing a host cell according to the third aspect; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, comprising the step of processing the polypeptide of interest; There is provided a method in which the polypeptide of interest can optionally be encoded on the same vector nucleic acid as the pri-miRNA, particularly within the same expression cassette.

[0083] The increase in yield and / or purity is determined in comparison to the same method for producing the polypeptide of interest in which the host cell does not contain the expression cassette according to the first aspect of the invention and does not produce an miRNA targeting the interfering gene product of the host cell.

[0084] The present invention further provides a method for increasing the yield and / or increasing the purity of a polypeptide of interest produced by a host cell, comprising: (a1) providing a host cell capable of producing the polypeptide of interest; (a2) introducing the vector nucleic acid according to the second aspect of the present invention into the host cell; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, comprising the step of processing the polypeptide of interest. In certain embodiments, the vector nucleic acid introduced into the host cell in step (a2) does not contain the coding sequence of the polypeptide of interest.

[0085] In a fifth aspect, the present invention is a method for producing a host cell according to the third aspect, comprising: (a) introducing the vector nucleic acid according to the second aspect into the host cell, wherein the vector nucleic acid contains the coding sequence for the polypeptide of interest either within the expression cassette expressing the miRNA or within an additional expression cassette; or (b) A step of introducing a vector nucleic acid according to the second aspect into a host cell, the step in which the vector nucleic acid does not contain the coding sequence of the polypeptide of interest, and a step of introducing a further vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, the method comprising the step of introducing different vector nucleic acids into the host cell simultaneously or sequentially in any order is provided.

[0086] The vector nucleic acid is artificially introduced into the host cell. In particular, the vector nucleic acid is introduced by transfection. Transfection in this regard can be transient or stable, and in particular stable transfection is used. Thus, in certain embodiments, the produced host cell contains the expression cassette according to the first aspect stably integrated into its genome.

[0087] In a sixth aspect, the present invention provides the use of the expression cassette of the first aspect or the vector nucleic acid of the second aspect or the host cell of the third aspect for the production of a polypeptide of interest. The features and embodiments of the method for producing the polypeptide of interest described herein are equally applicable to this use.

[0088] The present invention further provides the use of the expression cassette of the first aspect or the vector nucleic acid of the second aspect for improving the production of a polypeptide of interest by a host cell, the use comprising introducing the expression cassette or the vector nucleic acid into a host cell capable of producing the polypeptide of interest. In certain embodiments, the vector nucleic acid introduced into the host cell does not contain the coding sequence of the polypeptide of interest. Improving the production of the polypeptide of interest may include increasing the yield and / or the purity of the polypeptide of interest.

[0089] The present invention further provides the use of a vector nucleic acid for transfection of a host cell. In a specific aspect, the host cell is a non-human mammalian cell such as a Chinese hamster ovary (CHO) cell.

[0090] 4. Specific Embodiments Hereinafter, specific embodiments of the present invention will be described. These embodiments can be combined with further embodiments, features and examples described in this specification.

[0091] Embodiment 1 An expression cassette for expressing miRNA in a host cell, comprising an intron sequence containing a template sequence of pri-miRNA, wherein the pri-miRNA is processed in the host cell to interfere with the production of a polypeptide of interest recombinantly expressed in the host cell and / or target a gene product of the host cell that regulates the polypeptide of interest, and the miRNA is suitable for forming a miRNA that contains a passenger strand and a guide strand having an artificial sequence.

[0092] Embodiment 2 The expression cassette according to embodiment 1, further comprising a polymerase II promoter and a terminator operably linked to the template sequence of pri-miRNA, wherein the template sequence of pri-miRNA is located between the promoter and the terminator of the expression cassette.

[0093] Embodiment 3 The expression cassette according to embodiment 1 or 2, wherein the template sequence of pri-miRNA is present in the 5'untranslated region, 3'untranslated region or coding sequence of the expression cassette, particularly in the 5'untranslated region or 3'untranslated region.

[0094] Embodiment 4 The expression cassette according to any one of embodiments 1 to 3, wherein the pre-mRNA produced during transcription of the expression cassette contains pri-miRNA, and the miRNA is formed by processing of the pre-mRNA.

[0095] Embodiment 5 The expression cassette according to any one of embodiments 1 to 4, comprising a splice donor site upstream of pri-miRNA and a corresponding splice acceptor site downstream of pri-miRNA.

[0096] Embodiment 6 The expression cassette according to any one of Embodiments 1 to 5, comprising two or more template sequences for pri-miRNA, wherein each miRNA targets the same or different gene products.

[0097] Embodiment 7 The expression cassette according to Embodiment 6, wherein two or more template sequences for pri-miRNA are located within the same intron sequence.

[0098] Embodiment 8 The expression cassette according to Embodiment 7, comprising a splice donor site upstream of the template sequence of the first pri-miRNA and a corresponding splice acceptor site downstream of the template sequence of the last pri-miRNA.

[0099] Embodiment 9 The expression cassette according to any one of Embodiments 6 to 8, wherein adjacent template sequences of the two or more template sequences for pri-miRNA are separated from each other by a spacer sequence that forms an RNA stem-loop structure, for example, the sequence of SEQ ID NO: 22.

[0100] Embodiment 10 The expression cassette according to any one of Embodiments 1 to 9, wherein the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a passenger strand, a miRNA scaffold loop, a guide strand, and a 3'miRNA scaffold stem, or the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a guide strand, a miRNA scaffold loop, a passenger strand, and a 3'miRNA scaffold stem.

[0101] Embodiment 11The expression cassette according to Embodiment 10, wherein the 5'miRNA scaffold stem, the miRNA scaffold loop, and the 3'miRNA scaffold stem are derived from one or more pri-miRNAs selected from the group consisting of miR-30A, miR-E, SIBR, eSIBR, miR-1, miR-155, miR-16, miR-16-1, miR-16-2, miR-3G, miRGE, miR-100, miR-125b, miR-130a, miR-190a, miR-193a, miR-211, miR-26a, miR-340, miR-7-2, miR-96, and miR-44.

[0102] Embodiment 12 The expression cassette according to Embodiment 10 or 11, wherein the 5'miRNA scaffold stem contains any one nucleotide sequence of SEQ ID NOs: 1 to 4, and / or the miRNA scaffold loop contains the nucleotide sequence of SEQ ID NO: 8, and / or the 3'miRNA scaffold stem contains any one nucleotide sequence of SEQ ID NOs: 11 to 14.

[0103] Embodiment 13 The expression cassette according to any one of Embodiments 10 to 12, wherein the 5'miRNA scaffold stem and the 3'miRNA scaffold stem each contain a recognition site for a restriction enzyme.

[0104] Embodiment 14 The expression cassette according to Embodiment 13, wherein the recognition sites in the 5'miRNA scaffold stem and the 3'miRNA scaffold stem are each unique recognition sites.

[0105] Embodiment 15The gene product of the host cell targeted by the miRNA is selected from the group consisting of a protease capable of cleaving the polypeptide of interest, a protein involved in post-translational modification of the polypeptide of interest, a receptor or binding partner of the polypeptide of interest, a protein difficult to separate from the polypeptide of interest, a protein involved in folding and / or secretion of the polypeptide of interest, a protein involved in the transport of components necessary for the production or modification of the polypeptide of interest, a protein involved in the degradation of the polypeptide of interest, a protein sharing at least 70%, particularly at least 80% sequence identity with the polypeptide of interest over its entire length, and an endogenous homolog of the polypeptide of interest. The expression cassette according to any one of Embodiments 1 to 14.

[0106] Embodiment 16 The gene product of the host cell targeted by the miRNA is a protease capable of cleaving the polypeptide of interest. The expression cassette according to any one of Embodiments 1 to 15.

[0107] Embodiment 17 The gene product of the host cell targeted by the miRNA is a transferase capable of catalyzing post-translational modification of the polypeptide of interest, such as acetylation, acylation, sulfation, phosphorylation, alkylation, hydroxylation, amidation, carboxylation, palmitoylation, myristoylation, and isoprenylation. The expression cassette according to any one of Embodiments 1 to 15.

[0108] Embodiment 18 The gene product of the host cell targeted by the miRNA is an enzyme capable of catalyzing post-translational modification or removal of chemical groups of the polypeptide of interest, such as a hydrolase, for example, lipase, phosphatase, or glycosidase. The expression cassette according to any one of Embodiments 1 to 15.

[0109] Embodiment 19The gene product of the host cell targeted by the miRNA is a protein involved in the glycosylation of the polypeptide of interest, particularly a glycosyltransferase, glycosidase, or nucleotide sugar transporter, such as fucosyltransferase or sialyltransferase. The expression cassette according to any one of Embodiments 1 to 15.

[0110] Embodiment 20 The miRNA targets an endogenous gene product of the host cell. The expression cassette according to any one of Embodiments 1 to 19.

[0111] Embodiment 21 The artificial sequence of the guide strand is complementary to a part of the mRNA encoding the gene product of the host cell targeted by the miRNA. The expression cassette according to any one of Embodiments 1 to 20.

[0112] Embodiment 22 The artificial sequence of the passenger strand and / or the guide strand is not found in naturally occurring miRNAs. The expression cassette according to any one of Embodiments 1 to 20.

[0113] Embodiment 23 The promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a ubiquitin C (UBC) promoter, an elongation factor 1 alpha (EF1A) promoter, a phosphoglycerate kinase (PGK) promoter, a Rous sarcoma virus (RSV) promoter, a BROAD3 promoter, a mouse rosa26 promoter, a pCEFL promoter, a chicken β-actin promoter (CBA), a β-actin promoter combined with a CMV early enhancer (CAGG), an α-1-antitrypsin promoter, and inducible promoters such as a tetracycline-inducible promoter (e.g., pTRE) and a vanillic acid-inducible promoter. The expression cassette according to any one of Embodiments 1 to 22.

[0114] Embodiment 24The expression cassette according to any one of Embodiments 1 to 23, wherein the promoter is a CMV promoter or an SV40 promoter.

[0115] Embodiment 25 The expression cassette according to any one of Embodiments 2 to 24, further comprising a coding sequence of a polypeptide of interest, functionally linked to a polymerase II promoter and a terminator.

[0116] Embodiment 26 The expression cassette according to any one of Embodiments 2 to 24, further comprising a coding sequence of a selectable marker, functionally linked to a polymerase II promoter and a terminator.

[0117] Embodiment 27 The expression cassette according to Embodiment 26, wherein the selectable marker is selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, zeocin and blasticidin.

[0118] Embodiment 28 The expression cassette according to any one of Embodiments 1 to 27, wherein the host cell is a mammalian cell, particularly a human, primate or rodent cell, particularly a human or hamster cell.

[0119] Embodiment 29 The expression cassette according to any one of Embodiments 1 to 28, wherein the host cell is a CHO cell.

[0120] Embodiment 30 A vector nucleic acid for transfection of a host cell, comprising the expression cassette according to any one of Embodiments 1 to 29.

[0121] Embodiment 31 The vector nucleic acid according to Embodiment 30, further comprising an additional expression cassette suitable for expressing a polypeptide of interest.

[0122] Embodiment 32A host cell comprising the expression cassette according to any one of Embodiments 1 to 29 or the vector nucleic acid according to Embodiment 30 or 31, which is capable of recombinantly expressing a polypeptide of interest.

[0123] Embodiment 33 The host cell according to Embodiment 31 or 32, which is a mammalian cell, particularly a human, primate or rodent cell, particularly a human or hamster cell.

[0124] Embodiment 34 The host cell according to Embodiment 33, which is a CHO cell.

[0125] Embodiment 35 The host cell according to any one of Embodiments 32 to 34, wherein the coding sequence of the polypeptide of interest is present in the host cell (i) within the expression cassette expressing the miRNA, (ii) within a further expression cassette on the same vector nucleic acid as the expression cassette expressing the miRNA, or (iii) on a further vector nucleic acid or in the genome of the host cell.

[0126] Embodiment 36 A method for producing a polypeptide of interest in a host cell, comprising: (a) providing a host cell according to any one of Embodiments 32 to 35; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, processing the polypeptide of interest. A method, wherein the polypeptide of interest can optionally be encoded on the same vector nucleic acid as the pri-miRNA, particularly within the same expression cassette.

[0127] Embodiment 37 The method according to Embodiment 36, wherein step (d) comprises providing a pharmaceutical formulation comprising the polypeptide of interest.

[0128] Embodiment 38 A method for increasing the yield and / or purity of a polypeptide of interest produced by a host cell, comprising: (a1) providing a host cell capable of producing a polypeptide of interest; (a2) introducing the vector nucleic acid according to Embodiment 30 into the host cell; (b) culturing the host cell in a cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the polypeptide of interest from the cell culture; and (d) optionally, processing the polypeptide of interest.

[0129] Embodiment 39 A method for producing a host cell according to any one of Embodiments 32 to 35, comprising: (a) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid contains a coding sequence for the polypeptide of interest in either the expression cassette expressing the miRNA or an additional expression cassette; or (b) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid does not contain a coding sequence for the polypeptide of interest, and introducing an additional vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, the method comprising introducing different vector nucleic acids into the host cell simultaneously or sequentially in any order.

[0130] Embodiment 40 Use of the expression cassette according to Embodiments 1 to 29, the vector nucleic acid according to Embodiments 30 or 31, or the host cell according to Embodiments 32 to 35 for the production of a polypeptide of interest.

[0131] Embodiment 41Use of the expression cassette according to Embodiments 1 to 29 or the vector nucleic acid according to Embodiments 30 or 31 for improving the production of a polypeptide of interest, comprising introducing the expression cassette or the vector nucleic acid into a host cell capable of producing the polypeptide of interest.

[0132] Embodiment 42 Use according to Embodiment 41, wherein the host cell comprises a nucleic acid encoding the polypeptide of interest.

[0133] Embodiment 43 Use according to Embodiment 41 or 42, wherein the expression cassette or the vector nucleic acid does not contain the coding sequence of the polypeptide of interest.

[0134] Embodiment 44 Use according to Embodiments 41 to 43, wherein improving the production of the polypeptide of interest comprises increasing the yield and / or the purity of the polypeptide of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0135]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6-1

Figure 6-2

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14-1

Figure 14-2

Figure 15

Figure 16

Figure 17-1

Figure 17-2

Figure 18

Figure 19

Example

[0136] In the following examples, different expression cassettes containing the template sequence of pri-miRNA were used to knockdown target genes in host cells that interfere with the production of the polypeptide of interest. In Examples 1 to 4, the pri-miRNA template is present in the intron sequence within the 5’UTR of the polypeptide of interest or within the 3’UTR of the selectable marker gene, targeting the receptor protein of the host cell to which the polypeptide of interest binds and having an adverse effect on the growth and survival of the host cell. In Example 5, host cells that have already been engineered to produce the polypeptide of interest are further transfected with a vector containing an expression cassette that contains only the pri-miRNA template within the intron sequence but does not contain the coding sequence of the polypeptide. Here, the miRNA targets host cell proteins that are difficult to separate from the polypeptide of interest during the purification process. In Example 6, an alternative miRNA scaffold is used to target the receptor protein. In Example 7, host cells are engineered with an expression cassette that encodes the polypeptide of interest and contains a pri-miRNA cluster within the intron sequence in the 5’UTR. Here, the pri-miRNA cluster contains three pri-miRNAs that each target a host cell protein with proteolytic function. In Example 8, host cells are engineered with two separate expression cassettes that each contain a pri-miRNA cluster but do not contain the coding sequence of the polypeptide. The engineered cells are further transfected with a vector encoding the polypeptide of interest. Here, the miRNA targets host cell proteins that proteolytically degrade the polypeptide of interest.

[0137] Example 1: Vector Design. The intron-miRNA encoding vectors (pCMV01-pCMV12) are based on the standard vector (pCMV) encoding CMV-driven expression of the polypeptide of interest POI1 (Figure 1). The vectors were modified by inserting the miRNA sequence of the intron into two different loci: the miRNA scaffold is placed in the RK intron upstream of the POI1 gene (Figure 2), or in a synthetic intron implemented in the 3’UTR of the selectable marker gene (folate receptor, FAR, Figure 3). The sequence environment selected for the miRNA scaffold for proof of concept is similar to the human miR-30A and miR-E molecules (e.g., Fellmann et al., 2011 Molecular Cell 41, 733-746), and is expected to result in optimal processing of the resulting miRNA sequence. However, the sequence was further modified: i) the EcoRI restriction site was replaced with a BglII restriction site, ii) the sequence downstream of the XhoI restriction site was replaced with a CHO-derived sequence, and iii) additional miR-30A scaffolds were added upstream and downstream of the published sequence. Different miRNA sequences targeting the receptor of POI1: miRNA-1, miRNA-2, miRNA-3, miRNA-4, miRNA-5 and miRNA-sc (scrambled guide strand sequence as a control) were tested. The design of the pri-miRNA is shown in Figure 4.

[0138] Example 2: RT-qPCR Analysis of Stable CHO Pools. To test whether the POI1 receptor-targeting miRNA sequences placed in the intron pri-miRNA result in knockdown of the POI1 receptor mRNA level, as well as improvement of the growth rate and productivity of POI1-producing CHO cells, cells were transfected with vectors pCMV and pCMV01-12. Stable pools were selected using different selectable markers and used for RT-qPCR analysis.

[0139] Quantification of the mRNA levels of the POI1 receptor using RT-qPCR revealed that all miRNA sequences targeting the POI1 receptor downregulated the POI1 receptor mRNA levels when placed in the RK intron. Pools transfected with the control vector (no miRNA; miRNA scramble) showed equivalent levels of POI1 receptor mRNA. The most performant miRNA-1 reduced the POI1 receptor mRNA levels to 3.9% compared to 109% (no miRNA; see Figure 5A). In contrast, when the miRNA was placed in the synthetic intron of the FAR 3’UTR, the knockdown efficiency was less prominent, but miRNA-1 showed a 13.9% decrease in the POI1 receptor mRNA levels (see Figure 5B). The difference can be explained by the locus of the intronic miRNA. When placed in the RK intron, the transcript is driven by the strong CMV promoter, which should simultaneously result in a larger amount of processed miRNA molecules. In contrast, the miRNA expression from the intron in the FAR 3’UTR is driven by the weaker SV40 promoter, resulting in a smaller amount of processed miRNA molecules compared to the CMV promoter. These data also confirm the high knockdown efficiency of miRNA-1 when compared to other miRNA sequences that require stronger expression for efficient knockdown of the POI1 receptor mRNA levels.

[0140] Example 3: Cell Growth and Productivity of Stable CHO Pools. The pools with the best performance (miRNA-1 and miRNA-5, RK intron and FAR intron at both loci) and the "miRNA-free" pool were run in fed-batch mode. Thus, the POI1-producing pools were inoculated into standard 14-day 100 mL shake flask fed-batch cultures, and cell growth, cell viability, and the productivity titer were evaluated at different time points. The pool with normal POI1 receptor mRNA levels (miRNA-free) showed slower cell growth and lower viable cell density compared to the POI1 receptor knockdown pool (Figure 6). The stable CHO pool (miRNA-1 in the RK intron) that showed the strongest POI1 receptor knockdown efficiency also revealed the fastest cell growth and the highest viable cell density (Figure 6). All POI1 receptor knockdown pools grew to a higher viable cell density compared to the control pool. The miRNA-free CHO pool was found to have a maximum titer of 2 g / L. All POI1 receptor knockdown pools showed a higher titer compared to the control. The use of miRNA-1 in the RK intron resulted in the highest titer (3.7 g / L), which is an 85% increase in titer compared to the CHO control pool (miRNA-free).

[0141] Example 4: Stability of POI1 Receptor Knockdown and POI1 Productivity in CHO Pools. We tested the stability of POI1 receptor knockdown by quantifying POI1 receptor transcript levels using qPCR every four weeks. We also confirmed the POI1 titer levels in batch mode every two weeks. Interestingly, after eight weeks, the pool without POI1 receptor knockdown produced less POI1 compared to the POI1 receptor knockdown pool. This observation could support the hypothesis that knockdown of the POI1 receptor gives a survival signal to the cell pool. By linking miRNA expression to POI1 expression, the gene integration of the pCMV cassette can be stabilized, thus reducing the risk of selecting unstable clones. Also, the qPCR results indicate that the POI1 receptor knockdown remains stable over eight weeks while the other expression cassettes of the pCMV vector do not change.

[0142] Example 5: Generation of Interfering Gene Product (IGP) Knockdown Cell Lines. The strategy for generating the IGP KD cell line is shown in Figure 7. The CHO parental cell line was transfected with a vector encoding the polypeptide of interest POI2, and pools were selected using MTX in a low folic acid medium. The pools were subjected to single cell cloning, and monoclonal cell lines expressing POI2 (referred to as POI2 cell lines) were selected. Subsequently, using the primary seed lot (PSL) of the POI2 cell line, a vector encoding an artificial intron miRNA targeting the interfering gene product IGP was transfected, and a pool was generated using puromycin.

[0143] Two different miRNAs, designated IGP_A and IGP_B, targeting CHO IGP mRNA and both targeting the 3’UTR of the transcript were generated. For pool generation, miRNAs targeting different control genes, the parental POI2 cell line, and the empty parental host cell line (CHO) as a control were included. All samples (triplicate pool generation for the knockdown approach) were inoculated into an optimized fed-batch run, and cell growth, gene expression, and POI2 titer were evaluated on different days (Figures 8 - 10). Also, the IGP protein level at the harvest level was evaluated using IGP ELISA (Figure 11).

[0144] After confirmation of efficient IGP knockdown at the pool generation and mRNA levels, the IGP_A pool was selected for single cell cloning, and 96 growing clones were inoculated into a 24dwp standard fed-batch to evaluate the IGP knockdown efficiency and POI2 productivity (Figures 12 and 13).

[0145] The top 30 clones were further characterized. Based on multiple parameters (USP, DSP, IGP data, POI2 protein characteristics), the top 3 clones were selected and inoculated into a 7L bioreactor. IGP expression was significantly decreased in the IGP knockdown clones, resulting in an increase in the POI2 titer (Figure 14).

[0146] Example 6: Alternative miRNA Scaffolds Alternative miRNA scaffolds were tested for knockdown of the POI1 receptor based on the miR-16-2 and miR-3G sequences (e.g., Watanabe et al., 2016, RNA Biology 13(1), 25-33). For this purpose, miRNA-1 was incorporated into a customized miR-3G scaffold. The artificial miRNA was encoded in an expression cassette encoding POI3 driven by the CMV promoter. Stable CHO pools were generated and the POI3 titer and POI1 receptor mRNA expression were quantified. The control pool (without miRNA) produced a similar titer of POI3 compared to the pool expressing miRNA-1 encoded in the adapted miR-3G scaffold (Figure 15A). Also, the POI1 receptor was efficiently downregulated in the pool expressing miRNA-1 encoded in the adapted miR-3G scaffold (Figure 15B).

[0147] Example 7: Multiplex Knockdown Strategy Using Artificial miRNA Clusters Multiple miRNAs can then be encoded in a single intron, enabling simultaneous knockdown of multiple target genes. This approach was tested for triple knockdown of three different proteases. Three miRNAs, each targeting a different protease, are separated by specific spacer sequences to ensure proper RNA folding and efficient miRNA processing. The triple miRNA cluster is implemented in the intron of a CMV-driven expression cassette driving the expression of POI4 (Figure 16A). Two different triple miRNA clusters were designed for knockdown of three different proteases: triple miR-A targets proteases 5, 7, and 10, and triple miR-B targets proteases 4, 9, and 11.

[0148] A stable CHO pool was generated using a vector that produces POI4 and does not encode any of miRNA, triple miR-A or triple miR-B. The mRNA expressions of POI4, target protease and non-target protease were quantified using RT-PCR and normalized to the pool that produces only POI4 (without miRNA) (Figure 16B). Stable CHO pools encoding triple miR-A and triple miR-B efficiently knockdown specific target proteases, while the expression of irrelevant non-target proteases remains the same as that of the control pool.

[0149] Example 8: Generation of 14miR Protease Knockdown Cell Lines The vector pCMV06 encoding 14miR contains two expression cassettes that drive the expression of intronic miRNA clusters, target nine or five endogenous proteases, and do not contain the coding sequence of the polypeptide of interest (Figure 17A). The selectable markers puromycin and hygromycin are encoded on separate expression cassettes that allow the selection of stable CHO pools using either of the selectable markers. A stable CHO pool expressing pCMV06, called the 14miR pool, was generated using puromycin as the selectable marker. When the expression of target endogenous proteases was measured using qPCR, it was significantly decreased in the 14 miR pools compared to the host cell line (Figure 17B). However, the expression of non-target endogenous control genes remained unaffected. Monoclonal 14 miR clones were generated using single cell cloning, and three clones called 14miR clone-A, -B and -C were further characterized using qPCR (Figure 17C). 14miR clone-A downregulates the protease targeted by the 5miR cluster, assuming the loss of the 9miR expression cassette, but does not downregulate the protease targeted by the 9miR cluster. In contrast, 14miR clones-B and -C significantly decrease the expression of all 14 target endogenous proteases. Most proteases were strongly downregulated by the miRNA cluster, while two to three miRNAs showed moderate knockdown.

[0150] 14 miR clones were stably transfected with vectors encoding POI5 or POI6. Host cell lines were transfected as controls. Stable pools were generated using MTX in low folate medium. The generated 14 miR clone POI5 or POI6 pools and host cell line POI5 or POI6 pools were inoculated into standard feed batches, and the production titers were evaluated on day 14. The 14 miR clone POI5 and POI6 pools showed similar titers of POI5 or POI6 compared to the host cell line pools (Figures 18A and 19A). Variation among clones regarding the productivity of the polypeptide of interest could be observed. Also, POI5 or POI6 was purified using the production harvest, and the purified material was analyzed using mass spectrometry on day 0. The purified substance was exposed to low pH for 7 days at room temperature and re-analyzed using mass spectrometry (Figures 18B and 19B). Proteolysis of POI5 or POI6 after 7 days of treatment was higher in the 14 miR clone-A pool compared to the host cell line pool. In contrast, 14 miR clones -B and -C showed significantly lower proteolysis compared to the host cell line pool.

[0151] Example 9: Materials and Methods 1. Expression Vector Construction The vectors used in the examples consist of the following elements: an hCMV promoter / enhancer driving the expression of individual genes necessary for the construction of the POI construct, a polyadenylation signal (polyA), a folate receptor, DHFR, puromycin and hygromycin resistance genes as selectable markers, an origin of replication from Escherichia coli (CoIE ori) and an ampicillin (amp) resistant β-lactamase gene for amplification in bacteria. Different plasmid settings were evaluated and are shown in more detail in the figures.

[0152] 2. Cell Lines, Culture, Transfection and Selection CHO cell lines were cultured in 24 deep well plates or shake flasks in a non-humidified shaking incubator cabinet at 300 rpm (24 dwp) or 150 rpm (shake flask), 10% CO 2、They were cultured by suspending them in a chemically defined culture medium at 36.5 °C. The cell viability and growth rate were monitored by an automated system (ViCell, Beckman Coulter) or using analytical flow cytometry (CytoFlex, Beckman Coulter). The cells were passaged 2 - 3 times a week into fresh medium and maintained in the logarithmic growth phase.

[0153] The linearized expression vector was transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). The transfection reaction was carried out in a chemically defined culture medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in the exponential growth phase with a cell viability of over 95%. The transfection was performed with 5×10 6 cells per transfection. Immediately after transfection, the cells were transferred to a shaking flask containing a chemically defined culture medium. The cell pool was incubated at 36.5 °C and 10% CO 2 for 48 hours, and then the selection process was initiated.

[0154] As described above, the selection procedure was carried out using the selection markers encoded by the individual expression vectors. Proteins FoIR and DHFR are involved in the same molecular pathway; FolR transports folic acid and the folic acid analogue MTX into the cell, and DHFR converts it into important precursors for purine and methionine synthesis. When combined as a selection principle, a specific and powerful selection regime can be adopted to enrich recombinant cells expressing both recombinant proteins. Puromycin selection is driven by the inhibition of protein synthesis, and the vector encoding the puromycin resistance marker gene enables the cells to survive in the presence of puromycin.

[0155] Forty-eight hours after transfection and growth under low folate conditions, an additional selection pressure was applied by adding 10 nM MTX to the chemically defined culture medium. Alternatively, puromycin was used as the selection agent. Forty-eight hours after transfection, 0.003 mg / mL puromycin was added to the chemically defined culture medium. After pool recovery, cells were frozen in culture medium supplemented with 7.5% DMSO and cell pellets were prepared.

[0156] 3. Gene Expression Analysis by Quantitative Real-Time PCR RNA extraction was performed using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. For real-time qPCR, cDNA was synthesized from 200 ng / μl diluted RNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems), and 10-fold diluted cDNA was analyzed in triplicate using the QuantiFast SYBR Green PCR Kit (Qiagen) or the TaqMan Primer / Probe system and TaqMan Mastermix (Applied Biosystems). GAPDH was amplified as an endogenous control for normalization. Amplification and analysis were performed using the ABI PRISM® 7900HT Sequence Detection System. For calculation of the relative quantity (RQ) of gene expression for sample comparison, the comparative -ΔΔCt 2 method was used to normalize the data.

[0157] 4. Upstream Processing Following selection, the material was generated in either a shake flask fed-batch, 24 deep well plate cultures or an ambr15 bioreactor. The fed-batch cultures were inoculated with a cell seeding density of 4E5 vc / ml (a proprietary feed solution was added from day 3 and the culture temperature was shifted to 33 °C on day 5). During the culture, in-process controls were performed to monitor the concentration of the POI construct. Cell culture samples for RNA isolation were taken on day 10 of the process. The individual cultures were incubated for 14 days. At the end of the culture process, the cells were separated from the culture supernatant by centrifugation and subsequently sterile filtered prior to further downstream processing.

[0158] 5. Protein Quantification Using ELISA The amount of Chinese hamster (CHO) IGP was determined using a sandwich ELISA. Samples were added to microtiter plates coated with an anti-IGP antibody (capture antibody). The bound IGP was then quantified by incubation with a biotinylated anti-IGP antibody (detection antibody), followed by streptavidin-peroxidase and tetramethylbenzidine (TMB) as substrate and measurement of the absorbance at 450 nm. The IGP levels in the samples were calculated based on a CHO IGP standard.

[0159] 6. Purification Method The recombinant protein was purified by chromatography on an Akta avant 25 system (Cytiva). The protein was captured by affinity chromatography under neutral pH conditions and eluted under acidic conditions using 50 mM acetic acid at pH 3.0. All eluates were titrated up to pH 5.0 immediately after elution using 1 M Tris base.

[0160] 7. Sample Processing and Analytical Analysis The sample buffer was exchanged using Amicon Ultra-4 Centrifugal Filter Devices. The pH of the buffer was set to pH 4 using 50 mM acetic acid. The samples were transferred to 1.5 mL Eppendorf tubes and incubated at room temperature for 7 days.

[0161] The deglycosylation of the sample was performed using 1 mg of purified recombinant protein in Tris-HCl buffer at pH 7.5. PNGase F was added and incubated overnight at 37 °C.

[0162] All candidates were analyzed by an LC / MS system (WATERS, Xevo XS). The mobile phase was as follows: (A) 0.1% trifluoroacetic acid (TFA) in milliQ water and 0.09% TFA in acetonitrile. The gradient of mobile phase B was 5% - 50% over 7 minutes, and the total run time for each injection was 10 minutes. The separation of proteolytic products was carried out (at 70 °C) using a Waters BioResolve RP mAb Polyphenyl Column 450 Å, 2.7 μm, 2.1 mm × 150 mm. The column loading for each injection was 1.0 μg for intact analysis and 0.44 μg for reduced analysis. The capillary voltage was set at 1.8 kV, the sampling cone was set at 190 V, and the source offset was set at 30 V for all analyses. The source temperature and desolvation temperature were maintained at 125 °C and 400 °C, respectively. The desolvation gas flow was 800 L / h, the cone gas was 50 L / h, and the nebulizer gas was 6.5 bar. This system was controlled by MassLynx. All data were imported into the Genedata MS Refiner workflow and then processed within the Genedata MS Refiner workflow.

[0163]

Table 1

[0164]

Table 2

Claims

1. An expression cassette for expressing a miRNA in a host cell, comprising an intron sequence containing a template sequence for a prim-miRNA, wherein the prim-miRNA is suitable for forming a miRNA that targets a gene product of the host cell, which is processed in the host cell to interfere with and / or modulate the production of a target polypeptide that is recombinantly expressed in the host cell, and the miRNA comprises a passenger strand and a guide strand having an artificial sequence.

2. An expression cassette according to claim 1, (i) A polymerase II promoter and terminator functionally linked to the template sequence of prim-miRNA, wherein the template sequence of prim-miRNA is located between the promoter and terminator of the expression cassette; and / or (ii) An expression cassette further comprising a splice donor site upstream of the prim-miRNA and a corresponding splice acceptor site downstream of the prim-miRNA.

3. The expression cassette according to claim 1, comprising two or more template sequences for prim-miRNAs, each miRNA targeting the same or different gene products, and the two or more template sequences for prim-miRNAs optionally located within the same intron sequence.

4. The expression cassette according to claim 1, wherein the pri-miRNA comprises a 5' miRNA scaffold stem, a passenger strand, a miRNA scaffold loop, a guide strand, and a 3' miRNA scaffold stem from 5' to 3', the 5' miRNA scaffold stem containing any one nucleotide sequence of SEQ ID NOs: 1 to 4, and / or the miRNA scaffold loop containing the nucleotide sequence of SEQ ID NOs: 8, and / or the 3' miRNA scaffold stem containing any one nucleotide sequence of SEQ ID NOs: 11 to 14.

5. The host cell gene product targeted by the miRNA is (i) selected from the group consisting of a protease capable of cleaving the polypeptide of interest, a protein involved in the post-translational modification of the polypeptide of interest, a receptor or binding partner of the polypeptide of interest, a protein that is difficult to separate from the polypeptide of interest, a protein involved in the folding and / or secretion of the polypeptide of interest, a protein involved in the transport of components necessary for the production or modification of the polypeptide of interest, a protein involved in the degradation of the polypeptide of interest, a protein that shares at least 70%, particularly at least 80%, sequence identity with the polypeptide of interest over its entire length, and an endogenous homolog of the polypeptide of interest; (ii) A protease capable of cleaving the target polypeptide; (iii) A transferase capable of catalyzing post-translational modification of the target polypeptide, such as acetylation, acylation, sulfation, phosphorylation, alkylation, hydroxylation, amidation, carboxylation, palmitoylation, myristoylation, or isoprenylation; (iv) an enzyme capable of catalyzing the post-translational modification of the target polypeptide or the removal of a chemical group of the target polypeptide, such as a hydrolase such as lipase, phosphatase, or glycosidase; or (v) a protein involved in the glycosylation of the polypeptide of the interest, particularly a glycosyltransferase, glycosidase, or nucleotide sugar transporter, such as a fucosyltransferase or sialyltransferase; and / or (vi) The endogenous gene product of the host cell, The expression cassette according to claim 1.

6. The expression cassette according to claim 1, wherein the artificial sequence of the passenger strand and / or the guide strand is not found in naturally occurring miRNAs.

7. The expression cassette according to claim 1, wherein the promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter, Simianvirus 40 (SV40) promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF1A) promoter, phosphoglycerate kinase (PGK) promoter, Roussarcoma virus (RSV) promoter, BROAD3 promoter, mouse rosa26 promoter, pCEFL promoter, chicken β-actin promoter (CBA), β-actin promoter conjugated with CMV early enhancer (CAGG), α-1-antitrypsin promoter, and inducible promoters such as tetracycline-inducible promoters (e.g., pTRE), and vanillic acid-inducible promoters, preferably the CMV promoter or the SV40 promoter.

8. The expression cassette according to claim 2, further comprising a coding sequence for the target polypeptide or a selected marker, functionally linked to the polymerase II promoter and the terminator.

9. The expression cassette according to claim 1, wherein the host cell is a mammalian cell, particularly a human, primate, or rodent cell, particularly a human or hamster cell, preferably a CHO cell.

10. A vector nucleic acid for transfection of host cells, comprising an expression cassette according to any one of claims 1 to 9.

11. The vector nucleic acid according to claim 10, further comprising an additional expression cassette suitable for expressing the target polypeptide.

12. A host cell comprising an expression cassette according to any one of claims 1 to 9, the host cell capable of recombinantly expressing the target polypeptide.

13. A host cell comprising the vector nucleic acid described in Claim 10, the host cell capable of recombinantly expressing the target polypeptide.

14. The host cell according to claim 12, which is a mammalian cell, particularly a human, primate, or rodent cell, particularly a human or hamster cell, preferably a CHO cell.

15. The host cell according to claim 13, wherein the host cell is a mammalian cell, particularly a human, primate, or rodent cell, particularly a human or hamster cell, preferably a CHO cell.

16. A method for producing a target polypeptide in host cells, (a) A step of providing the host cell according to claim 12; (b) A step of culturing host cells in a cell culture under conditions that enable the expression of the target polypeptide; (c) A step of obtaining the target polypeptide from a cell culture; and (d) a step of processing the target polypeptide, if applicable; Includes, A method wherein the target polypeptide may optionally be encoded on the same vector nucleic acid as the prim-miRNA, particularly within the same expression cassette.

17. The method according to claim 16, wherein step (d) includes providing a pharmaceutical formulation comprising the target polypeptide.

18. A method for increasing the yield and / or purity of a target polypeptide produced by a host cell, (a1) A step of providing a host cell capable of producing the target polypeptide; (a2) A step of introducing the vector nucleic acid according to claim 10 into the host cell; (b) A step of culturing the host cells in a cell culture under conditions that enable the expression of the target polypeptide; (c) A step of obtaining the target polypeptide from the cell culture; and (d) a step of processing the target polypeptide, if applicable; A method that includes this.

19. A method for producing host cells according to claim 12, (a) A step of introducing a vector nucleic acid according to a second embodiment into a host cell, wherein the vector nucleic acid contains a coding sequence for the target polypeptide within the expression cassette expressing the miRNA or within a further expression cassette; or (b) A step of introducing a vector nucleic acid according to a second embodiment into a host cell, wherein the vector nucleic acid does not contain the coding sequence of the target polypeptide, and a step of introducing further vector nucleic acids suitable for recombinant expression of the target polypeptide into the host cell, wherein different vector nucleic acids can be introduced into the host cell simultaneously or sequentially in any order; A method that includes this.

20. Use of an expression cassette according to any one of claims 1 to 9 for the production of the polypeptide of the above purpose.

21. Use of the vector nucleic acid according to claim 10 for the production of the target polypeptide.

22. Use of the host cell according to claim 12 for the production of the target polypeptide.

23. Use of an expression cassette according to any one of claims 1 to 7 and 9 for improving the production of a target polypeptide by a host cell, comprising introducing the expression cassette into a host cell capable of producing the target polypeptide.

24. Use of the vector nucleic acid according to claim 10 for improving the production of a target polypeptide by a host cell, comprising introducing the vector nucleic acid into a host cell capable of producing the target polypeptide.

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