Improved 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. Expression cassettes are provided that produce miRNAs that target and down-regulate host cell proteins that interfere with the production of polypeptides 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 host cell protein expression that interferes with the production of target proteins using artificial miRNAs targeting host cell proteins. In particular, expression cassettes of corresponding miRNAs containing intron sequences of pri-miRNA templates, vectors and host cells containing the expression cassettes, and their use for producing target proteins are provided. Background Art

[0002] The generation of recombinant cell lines for the production of secreted proteins requires transfection of DNA vectors into host cells and the use of selection markers to enrich for stable transfectants. Secreted proteins may affect cellular parameters such as growth, viability and / or productivity, which often requires cell line engineering approaches to achieve high-expressing stable cell lines.

[0003] Similarly, the quality of secretory proteins can be affected by cell-derived intrinsic factors. Usually, endogenously expressed genes, such as cell surface receptors, enzymes or proteases, can be identified as the root cause of undesirable effects (see, for example, WO2014 / 097113 A2). The corresponding undesirable effects include the enzymatic cleavage of the polypeptide chain of the recombinant protein, such as the digestion of the whole protein or the amino acid shearing, i.e., removing one or more amino acids from the N or C-terminal of the target protein. Other effects are unwanted post-translational modifications or removal of required modifications. In addition, the endogenous gene product of the host cell may interact specifically with the target protein. Such interactions may raise the target protein from the supernatant of the cell culture, making it difficult to remove endogenous proteins in the purification process, or even causing the activation of the signal transduction pathway in the host cell, thereby causing the growth of the cell, the vigor and / or the productivity to reduce. In other cases, the endogenous gene product may only have the chemical and physical properties highly similar to the target protein, which makes it difficult to develop a purification method to effectively remove host cell proteins without reducing the output of the target protein. This is particularly relevant for therapeutic proteins, where high purity and low residual levels of host cell proteins in the final product are prerequisites for obtaining and maintaining marketing authorization.

[0004] Therefore, the endogenous gene products of the host cell may significantly interfere with the recombinant production of the target protein. In view of the above situation, it is necessary to provide strategies to reduce the adverse effects of host cell proteins on the recombinant production of the target protein. Summary of the invention

[0005] The inventors of the present invention have found that some expression cassettes encoding artificial miRNA are very effective in specific targeting to produce host cell proteins that interfere with the target polypeptide. Using this method, the expression of interfering host cell proteins can be significantly reduced, thereby the output and / or purity of the target polypeptide can be improved. Using miRNA technology to reduce interfering endogenous gene products according to the present invention only requires minimal genetic engineering. The pri-miRNA template sequence can be easily introduced into an existing plasmid to express the target polypeptide, or a standard vector can be used to introduce the miRNA template into the production host cell that has been established.

[0006] In addition, in most cases, knocking down the interfering host cell protein to a residual expression level is sufficient to significantly improve the production of the polypeptide of interest. Complete knockout of the interfering gene product of the host cell (e.g., by genetic engineering of the host cell genome) is much more complicated and may have a negative impact on the viability and efficacy of the host cell.

[0007] Therefore, in a first aspect, the present invention relates to an expression cassette for expressing a miRNA in a host cell, comprising an intron sequence containing a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in the host cell to form a miRNA targeting a gene product of the host cell, which gene product of the host cell interferes with the production of a polypeptide of interest recombinantly expressed in the host cell and / or regulates the 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.

[0008] In a second aspect, the present invention provides a vector nucleic acid for transfecting a host cell, comprising an expression cassette according to the first aspect.

[0009] In a third aspect, the present invention provides a host cell comprising an expression cassette according to the first aspect or a vector nucleic acid according to the second aspect, wherein the host cell is capable of recombinantly expressing a polypeptide of interest.

[0010] In a fourth aspect, the present invention provides a method for producing a target polypeptide in a host cell, comprising the following steps:

[0011] (a) providing a host cell according to the third aspect;

[0012] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0013] (c) obtaining the target polypeptide from the cell culture; and

[0014] (d) optionally processing the polypeptide of interest;

[0015] The polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, in particular, encoded in the same expression cassette.

[0016] In a fifth aspect, the present invention provides a method for producing a host cell according to the third aspect, comprising the following steps:

[0017] (a) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid comprises a coding sequence for a polypeptide of interest in an expression cassette for expressing miRNA or in another expression cassette; or

[0018] (b) introducing the vector nucleic acid according to the second aspect into the host cell, wherein the vector nucleic acid does not comprise the coding sequence of the polypeptide of interest, and introducing another vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, wherein the different vector nucleic acids can be introduced into the host cell simultaneously or successively in any order.

[0019] In a sixth aspect, the present invention provides use of the expression cassette according to the first aspect, or the vector nucleic acid according to the second aspect, or the host cell according to the third aspect for producing a polypeptide of interest.

[0020] From the following description and the appended claims, other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art. However, it should be understood that the following description, the appended claims and the specific examples indicating the preferred embodiments of the present application are given by way of illustration only. By reading the following, various changes and modifications within the spirit and scope of the disclosed invention will become apparent to those skilled in the art.

[0021] definition

[0022] As used herein, the following expressions are generally intended to preferably have the meanings as described below, unless otherwise indicated in the context in which they are used.

[0023] In addition to its literal meaning, the expression "comprises" as used herein also includes and refers in particular to the expressions "consisting essentially of" and "consisting of". Thus, the expression "comprising" refers to embodiments in which the subject matter "comprising" the specifically listed elements does not comprise further elements, as well as embodiments in which the subject matter "comprising" the specifically listed elements may and / or does encompass further elements. Likewise, the expression "having" is to be understood as the expression "comprising", and also includes and refers in particular to the expressions "consisting essentially of" and "consisting of". Where possible, the term "consisting essentially of" refers in particular to embodiments in which the subject matter comprises 20% or less, in particular 15% or less, 10% or less, or especially 5% or less of further elements in addition to the specifically listed elements in which the subject matter consists essentially of.

[0024] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, ribonucleic acids and deoxyribonucleic acids. It may contain naturally occurring and synthetic nucleotides and may be modified naturally or synthetically (e.g., by methylation, 5'- and / or 3'-capping). In a specific embodiment, the nucleic acid refers to double-stranded deoxyribonucleic acid.

[0025] The term "expression cassette" refers in particular to a nucleic acid construct capable of initiating and regulating the expression of a coding nucleic acid sequence and / or a template nucleic acid sequence introduced therein. The expression cassette may include a promoter, a ribosome binding site, an enhancer, and other control elements regulating gene transcription or mRNA translation. The exact structure of the expression cassette may vary according to species or cell type, but generally includes 5'-untranscribed and 5'- and 3'-untranslated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. More particularly, the 5'-untranscribed expression control sequence includes a promoter region that includes a promoter sequence for transcription control of an operably connected nucleic acid. The expression cassette may also include an enhancer sequence or an upstream activator sequence. Some expression cassettes are only used to transcribe the template nucleic acid sequence into an RNA product, such as pri-miRNA. Such an expression cassette does not necessarily include a regulatory element for translation.

[0026] According to the present application, template nucleic acid is understood to be a DNA that is transcribed into a functional RNA product or its precursor, in particular a pri-miRNA. The functional RNA product alone or in combination with other RNA products and / or proteins has a biological activity, for example, the activity of miRNA (in combination with RISC proteins) interfering with target gene expression.

[0027] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of a nucleic acid sequence to be expressed, and controls the expression of the sequence by providing recognition and binding sites for RNA-polymerase."Promoter" may include other recognition and binding sites for other factors involved in gene transcription regulation. Promoters may control the transcription of prokaryotic or eukaryotic genes. In addition, promoters may be "inducible", i.e., initiate transcription in response to an inducing agent, or may be "constitutive" if transcription is not controlled by an inducing agent. If there is no inducing agent, the gene under the control of an inducible promoter is not expressed or is only expressed to a small extent. In the presence of an inducing agent, the gene is turned on or the transcription level increases. This is usually mediated by the combination of specific transcription factors.

[0028] As used herein, the term "vector" is used in its most general sense, and comprises any intermediate medium of nucleic acid, which can, for example, introduce the nucleic acid into prokaryotic and / or eukaryotic cells, and is integrated into the genome when appropriate. Such vectors are preferably replicated and / or expressed in cells. Vectors include plasmids, phagemids, phages or viral genomes. As used herein, the term "plasmid" generally relates to the construct of extrachromosomal genetic material, generally circular DNA duplexes, which can replicate independently of chromosomal DNA. According to the present invention, the vector can exist in a circular or linearized form. As used herein, "vector nucleic acid" is a nucleic acid forming a vector or a nucleic acid portion of a vector.

[0029] The term "5'" and "3'" convention is used to describe nucleic acid sequence features related to genetic element position and / or event direction (5' to 3'), such as being transcribed by RNA polymerase or translated by ribosomes in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). By convention, DNA sequences, gene maps, vector cards and RNA sequences are drawn from left to right with 5' to 3', or indicated with arrows in the 5' to 3' direction, wherein the arrow points to the 3' direction. Therefore, when following this convention, 5' (upstream) indicates the genetic element positioned toward the left-hand side, and 3' (downstream) indicates the genetic element positioned toward the right-hand side.

[0030] "Polypeptide" or "polypeptide chain" refers to a molecule comprising 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 2-49 amino acids). In particular, a polypeptide or polypeptide chain can be part of a protein composed of two or more polypeptide chains. Polypeptides include proteins and / or peptides having any activity or biological activity. Polypeptides can be pharmaceutically or therapeutically active compounds, or research tools for use in assays, etc.

[0031] A target amino acid sequence is "derived" from or "corresponds to" a reference amino acid sequence if it is 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% identical to the reference amino acid sequence over its entire length. In a specific embodiment, a target amino acid sequence "derived" from or "corresponds to" a reference amino acid sequence is 100% identical to the reference amino acid sequence over its entire length. Similarly, a target nucleotide sequence is "derived" from or "corresponds to" a reference nucleotide sequence if it is 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% identical to the reference nucleotide sequence over its entire length. In a specific embodiment, a target nucleotide sequence "derived" from or "corresponds to" a reference nucleotide sequence is 100% identical to the reference nucleotide sequence over its entire length. Preferably, the "identity" of an amino acid sequence or a nucleotide sequence according to the invention is determined over the entire length of the reference sequence.

[0032] As used herein, "microRNA", abbreviated as "miRNA", is a single-stranded non-coding RNA molecule that plays a role in RNA silencing and post-transcriptional regulation of gene expression. MiRNA is generally composed of 19 to 24 nucleotides, particularly about 22 nucleotides, especially 22 nucleotides. MiRNA molecules can silence mRNAs comprising complementary nucleotide sequences. The silencing of target mRNA may occur by cutting of mRNA, destabilization of mRNA, or blocking of mRNA translation. The silencing of target mRNA reduces or eliminates the production of proteins encoded by the target mRNA. It is generally understood in the art that miRNA associates with dicer and argonaute proteins to form an RNA-induced silencing complex (RISC) that binds to the target mRNA.

[0033] MiRNA is produced by transcribing the template DNA sequence into a miRNA precursor (pri-miRNA).Pri-miRNA comprises a hairpin stem-loop structure, wherein the double-stranded stem is connected to the loop on one side, and the other side is flanked by single-stranded 5' and 3' extensions.The double-stranded stem comprises a guide strand (forming miRNA after processing) and a passenger strand (substantially complementary to the guide strand).Especially, except for the nucleotide pairs at the ends of the hairpin stem-loop structure, i.e., the nucleotide pairs of the passenger strand and the guide strand farthest from the loop structure, the passenger strand and the guide strand are complementary to each other.Guide strand and passenger strand are usually each having a length of about 19 to 24 nucleotides, especially 22 nucleotides.The remainder of pri-miRNA is referred to as miRNA scaffold in this article. From 5' to 3', the pri-miRNA thus comprises (i) a 5'-miRNA scaffold stem, consisting of a 5' single-stranded extension and the 5' portion of the stem structure up to the passenger strand; (ii) the passenger strand; (iii) the miRNA scaffold loop; (iv) the guide strand; and (v) the 3'-miRNA scaffold stem, consisting of the 3' portion of the stem structure following the guide strand and the 3' single-stranded extension. The positions of the passenger strand and the guide strand can also be switched.

[0034] The pri-miRNA is processed by cleaving the 5' and 3' miRNA scaffold stems, generating a hairpin structure called pre-miRNA. From 5' to 3', the pre-miRNA consists of a passenger strand, a miRNA scaffold loop, and a guide strand; the positions of the passenger strand and guide strand can also be switched. The loop structure is then cleaved, and the resulting RNA duplex is separated into two single-stranded RNA molecules, the guide strand and the passenger strand. The guide strand, which is complementary to the targeted mRNA molecule, forms the RISC, while the passenger strand generally has no function.

[0035] Cells mentioned herein are particularly host cells. According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. Particularly preferred are mammalian cells, such as cells of humans, mice, hamsters, pigs, goats or primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Nucleic acids can be present in host cells in the form of a single copy or two or more copies, and are expressed in host cells in one embodiment. Host cells particularly refer to cells present in cell culture, especially cells that are not present in living multicellular organisms.

[0036] The term "pharmaceutical composition" or "pharmaceutical formulation" refers in particular 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, and a carrier, diluent or pharmaceutical excipient, such as a buffer, a preservative and a tonicity regulator.

[0037] The numbers given herein may be understood in certain embodiments as approximate numbers. In particular, the numbers may preferably be up to 10% higher and / or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and / or lower. In particular embodiments, the numbers given herein are not approximate numbers and may only vary within the inaccuracy of technical measurements.

[0038] Numerical ranges described herein include numbers that define ranges. The titles provided herein are not limitations on the various aspects or embodiments of the present invention, which can be read with reference to the entire specification. According to one embodiment, the subject matter described herein as comprising certain steps in the case of a method or as comprising certain components in the case of a composition refers to the subject matter consisting of the corresponding steps or components. Preferably, the preferred aspects and embodiments described herein are selected and combined, and the specific subject matter produced by the corresponding combinations of the preferred embodiments also belongs to the present disclosure. DETAILED DESCRIPTION

[0039] The present invention is based on the development of a novel vector construct for knocking down a target host cell protein. Endogenous proteins of a host cell sometimes interfere with the production of a target protein in the host cell. In order to prevent this interference, one or more corresponding host cell proteins must be removed from the host cell system. Typically, gene knockout is performed for this purpose, which is very time-consuming, labor-intensive and costly. The present invention is different from this method because it works at the RNA level, resulting in the knockdown of mRNA encoding interfering host cell proteins. Typically, it is not necessary to completely knock out the corresponding host cell gene because minimal expression should produce a similar desired phenotype. In addition, knockout may cause undesirable cell compensation effects. Therefore, strong knockdown represents a preferred technique for inactivating interfering host cell genes. In the past, shRNA molecules were encoded on a separate vector and expressed using a strong polymerase III promoter. Using the novel method according to the present invention, a normal polymerase II promoter is sufficient, and pri-miRNA can be present on the same vector as the target protein produced in the host cell, or even in the same expression cassette. In the development process of producing cell lines, this method also shows high knockdown efficiency at the pool level, so that the knockdown efficacy can be evaluated very quickly.

[0040] 1. Expression cassette for expressing miRNA

[0041] In view of the above situation, in the first aspect, the present invention provides an expression cassette for expressing miRNA in a host cell, which comprises an intron sequence containing a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in the host cell to form a miRNA targeting a gene product of the host cell, and the gene product of the host cell interferes with the production of a target polypeptide recombinantly expressed in the host cell and / or regulates the target polypeptide recombinantly expressed in the host cell; and wherein the miRNA comprises a passenger chain and a guide chain having an artificial sequence.

[0042] This expression cassette can be used to knock down the targeted gene product of the host cell. Therefore, the interference of the gene product to the recombinant production of the target polypeptide and / or the regulation of the target polypeptide can be reduced or prevented. The miRNA can specifically target any host cell gene product that hinders the target polypeptide from being produced in the desired form. The gene product of the host cell targeted by the miRNA is generally referred to as "interfering gene product" in this article.

[0043] It should be understood that the miRNA produced from the expression cassette is at least partially complementary and can bind and initiate the silencing of the "basic" RNA of the interfering gene product. Therefore, in the embodiment where the interfering gene product is or comprises a protein or polypeptide, the basic RNA is particularly an mRNA or a precursor mRNA encoding the interfering gene product. In the embodiment where the interfering gene product is or comprises RNA, the basic RNA is particularly the RNA or its precursor. Silencing may occur by the degradation of the targeting RNA or by preventing the targeting mRNA from being translated.

[0044] Since the expression cassette contains the template sequence of the pri-miRNA in the intron sequence, it may contain further sequences for expressing other products, such as coding sequences for producing a polypeptide of interest, coding sequences for producing a selectable marker, and template sequences for other RNA products (especially other pri-miRNAs). Alternatively, the expression cassette may be specifically used to produce miRNAs targeting interfering gene products.

[0045] 1.1 Elements of the expression cassette

[0046] The expression cassette contains the template sequence of the pri-miRNA within the intron sequence. After expression, a pre-mRNA containing the intron sequence is formed. The intron sequence is then spliced ​​from the pre-mRNA to form the pri-miRNA, which is then further processed to ultimately provide the miRNA. The pre-mRNA formed need not contain any sequence encoding a polypeptide.

[0047] In certain embodiments, the expression cassette further comprises a polymerase II promoter. This promoter is functionally connected to the template sequence of the pri-miRNA and controls the expression of the pri-miRNA. The promoter can be any RNA polymerase II promoter suitable for expressing genes in a host cell, especially a host cell for expressing a target polypeptide. In certain embodiments, the promoter is suitable for expression in a eukaryotic host cell, particularly a mammalian host cell, such as a CHO cell. For example, the promoter can be 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 rosa 26 promoter, a pCEFL promoter, a chicken β-actin promoter (CBA), a β-actin promoter coupled to a CMV early enhancer (CAGG), an alpha-1-antitrypsin promoter, and an inducible promoter, such as a tetracycline inducible promoter (e.g., pTRE) and a vanillic acid inducible promoter. In a particular embodiment, the promoter is a CMV promoter or a SV40 promoter, in particular a CMV promoter.

[0048] In certain embodiments, the expression cassette further comprises a terminator. The terminator is functionally connected to the template sequence of the pri-miRNA and controls the expression of the pri-miRNA. The term "terminator" as used herein refers to a transcription terminator that terminates transcription of DNA into RNA, especially by RNA polymerase II.

[0049] The template sequence of the pri-miRNA is particularly located between the promoter and the terminator of the expression cassette.

[0050] In certain embodiments, the expression cassette comprises a coding sequence encoding, for example, a polypeptide of interest or a selective marker. In these embodiments, the expression cassette may further comprise a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). The intron sequence of the template sequence comprising the pri-miRNA may be present in the 5'UTR, 3'UTR or the coding sequence. In particular, the intron sequence is present in the 5'UTR or 3'UTR, especially in the 5'UTR. In an alternative embodiment, the expression cassette does not comprise a coding sequence encoding a polypeptide.

[0051] The intron sequence containing the template sequence of the pri-miRNA specifically contains a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA. Using these splice donor and acceptor sites, the pri-miRNA is spliced ​​out from the pre-mRNA after transcription.

[0052] In certain embodiments, the intron sequence comprises two or more template sequences of pri-miRNA. In these embodiments, the intron sequence comprises a splicing donor site upstream of the template sequence of the first pri-miRNA (i.e., the most 5' template sequence), and a corresponding splicing acceptor site downstream of the template sequence of the last pri-miRNA (i.e., the most 3' template sequence). Adjacent template sequences in the intron sequence can be separated from each other by a spacer sequence. Such a spacer sequence particularly forms an RNA stem-loop structure, such as the sequence of SEQ ID NO:22.

[0053] In certain embodiments, the expression cassette comprises only one intron sequence with one or more template sequences of a pri-miRNA. In alternative embodiments, the expression cassette comprises two or more intron sequences with one or more template sequences of a pri-miRNA.

[0054] The pri-miRNAs present in two or more template sequences in the same or different intron sequences are particularly different from each other. In a particular embodiment, each miRNA produced by the pri-miRNA targets different interfering gene products. In an alternative embodiment, each miRNA produced by the pri-miRNA targets the same interfering gene product. In an even further embodiment, some miRNAs produced by the pri-miRNA target the same interfering gene product, while other miRNAs produced by the pri-miRNA target different interfering gene products. The miRNAs targeting the same interfering gene product particularly bind to different parts of the interfering gene product RNA, especially mRNA or pre-mRNA.

[0055] The expression cassette can comprise the coding sequence of the coded polypeptide. The coding sequence can encode the target polypeptide or the selective marker in particular. The coding sequence is preferably functionally connected to the polymerase II promoter and terminator of the expression cassette. In the embodiment in which the expression cassette comprises the template sequence of pri-miRNA and the coding sequence of the target polypeptide, the expression of these two elements is connected. Therefore, the host cell comprising the expression cassette and showing the target polypeptide of high expression level also has the miRNA of high expression level simultaneously. This method significantly simplifies clone development and selection. In the embodiment in which the expression cassette comprises the template sequence of pri-miRNA and the coding sequence of the selective marker, the expression of miRNA is related to the expression of the selective marker. Therefore, by increasing the selection pressure in the clone selection process, the miRNA level also can increase.

[0056] The selectable marker may be selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, bleomycin and blasticidin. In certain embodiments, the selectable marker is folate receptor (FAR).

[0057] 1.2 miRNA

[0058] The expression cassette comprises a template sequence of the pri-miRNA. The pri-miRNA produced from the expression cassette may have any structure suitable for host cell processing to obtain a functional miRNA targeting an interfering gene product of the host cell. The functional miRNA specifically induces a reduction in the level of an interfering gene product in the host cell.

[0059] In certain embodiments, the pri-miRNA comprises a passenger strand and a guide strand. The guide strand particularly comprises or is composed of a miRNA formed after the host cell processes the pri-miRNA. In addition, the pri-miRNA may comprise a miRNA scaffold loop and / or a miRNA scaffold stem, especially a 5'miRNA scaffold stem and a 3'miRNA scaffold stem. In a particular embodiment, from 5' to 3', 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. In an alternative embodiment, from 5' to 3', the pri-miRNA comprises a 5'miRNA scaffold stem, a guide strand, a miRNA scaffold loop, a passenger strand, and a 3'miRNA scaffold stem. An embodiment in which the passenger strand is located upstream of the guide strand is preferred.

[0060] The passenger strand and guide strand of pri-miRNA have artificial sequences. In this respect, artificial sequences refer to sequences that are not found as passenger strands or guide strands in naturally occurring miRNAs. In particular, the sequences of passenger strands and guide strands are not found in naturally occurring miRNAs. In certain embodiments, the guide strand comprises and is particularly composed of sequences designed to reduce the expression of interfering gene products. In particular, the sequence of the guide strand is complementary to a portion of an mRNA encoding a gene product of a host cell targeted by the miRNA.

[0061] In certain embodiments, one or more scaffold sequences of a pri-miRNA or pre-miRNA are derived from naturally occurring pri-miRNAs, in particular naturally occurring pri-miRNAs in mammals, in particular humans. In particular embodiments, all scaffold sequences of a pri-miRNA are derived from naturally occurring pri-miRNAs, in particular naturally occurring pri-miRNAs in mammals, in particular humans. In particular, all scaffold sequences of a pri-miRNA are derived from the same naturally occurring pri-miRNA. The scaffold sequence of a pri-miRNA particularly comprises a 5' miRNA scaffold stem, a miRNA scaffold loop, and a 3' miRNA scaffold stem. Suitable naturally occurring pri-miRNAs from which scaffold sequences 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, miRNA scaffold loop, and 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 a particular embodiment, the naturally occurring pri-miRNA from which the scaffold sequence is derived is miR-30A.

[0062] In particular embodiments, all scaffold sequences of the pri-miRNA have at least 80%, particularly at least 90%, particularly at least 95% nucleotide sequence identity over their entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA has at least 80%, particularly at least 85%, particularly at least 90% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA has at least 80%, particularly at least 90%, particularly at least 95% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In certain embodiments, the miRNA scaffold loop of the pri-miRNA has at least 60%, particularly at least 70%, particularly at least 75% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In these embodiments, the naturally occurring pri-miRNA may be particularly miR-30A.

[0063] In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 1-7 or a derivative thereof. In particular, the 5'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence having 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 a nucleotide sequence of any one of SEQ ID NOs: 1-7, in particular any one of SEQ ID NOs: 1-4, in particular SEQ ID NO: 1.

[0064] In certain embodiments, the miRNA scaffold loop of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 8-10 or a derivative thereof. In particular, the miRNA scaffold loop of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 8-10 or a sequence having at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably 100% nucleotide sequence identity thereto. In particular, the miRNA scaffold loop of the pri-miRNA consists of a nucleotide sequence of any one of SEQ ID NOs: 8-10, in particular SEQ ID NO: 8.

[0065] In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 11-17 or a derivative thereof. In particular, the 3'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 11-17 or a sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity thereto. In particular, the 3'miRNA scaffold stem of the pri-miRNA consists of a nucleotide sequence of any one of SEQ ID NOs: 11-17, in particular any one of SEQ ID NOs: 11-14, in particular SEQ ID NO: 11.

[0066] In certain embodiments, the template sequence of the pri-miRNA comprises at least one recognition site, especially two recognition sites, of a DNA restriction enzyme. In particular, the two recognition sites are used for different DNA restriction enzymes and produce different overhangs after cutting. The two recognition sites are preferably flanked on both sides by the pre-miRNA portion of the pri-miRNA - it comprises a guide strand, a passenger strand and a miRNA scaffold loop. In particular, one of the recognition sites is located within a sequence that is transcribed into a 5' miRNA scaffold stem, and the other recognition site is located within a sequence that is transcribed into a 3' miRNA scaffold stem. In a particular embodiment, the recognition site is located within a sequence that is transcribed into the single-stranded portion of the 5' and 3' miRNA scaffold stems. The recognition site is particularly a unique recognition site within an expression cassette, particularly within the entire vector comprising the expression cassette.

[0067] 1.3 Target genes

[0068] The miRNA expressed by the expression cassette is used to target a gene product of the host cell ("interfering gene product"), which interferes with the production of a target polypeptide recombinantly expressed in the host cell and / or regulates the target polypeptide recombinantly expressed in the host cell. The interfering gene product can be any gene product that interferes with the production of a target polypeptide and / or regulates the target polypeptide. Exemplary interfering gene products are, for example, selected from the group consisting of

[0069] (i) Protease capable of cleaving the target polypeptide

[0070] (ii) Proteins involved in post-translational modification of the target polypeptide

[0071] (iii) Receptor or binding partner of the polypeptide of interest

[0072] (iv) Proteins that are difficult to separate from the target polypeptide

[0073] (v) Proteins involved in the folding and / or secretion of the polypeptide of interest

[0074] (vi) Proteins involved in the transport of components necessary for the production or modification of the polypeptide of interest

[0075] (vii) Proteins involved in the degradation of target polypeptides

[0076] (viii) a protein having at least 70%, in particular at least 80% sequence identity with the polypeptide of interest over its entire length, or an endogenous homologue of the polypeptide of interest.

[0077] The expression of miRNA reduces the production of interfering gene products in the host cell, thereby reducing or eliminating the interference with the production of the target polypeptide and / or the regulation of the target polypeptide. The interfering gene products are particularly endogenous gene products of the host cell.

[0078] In certain embodiments, the interfering gene product is a protease capable of cleaving a polypeptide of interest. In these embodiments, expression of the miRNA specifically reduces cleavage of the polypeptide of interest by the targeted protease.

[0079] In certain embodiments, the interfering gene product is a protein that participates in the post-translational modification of the target polypeptide. For example, the interfering gene product can be a transferase that can catalyze the post-translational modification of the target polypeptide. Exemplary post-translational modifications include acetylation, acylation, sulfation, phosphorylation, alkylation, hydroxylation, amidation, carboxylation, palmitoylation, myristoylation and prenylation. In these embodiments, the expression of miRNA particularly reduces the amount of post-translational modification catalyzed by the targeted transferase.

[0080] In a further embodiment, the interfering gene product can be an enzyme that can catalyze the post-translational modification of the target polypeptide or the removal of a chemical group. The interfering gene product can for example be a lytic enzyme, for example a lipase, a phosphatase or a glycosidase. In these embodiments, the expression of miRNA has especially increased the amount of the corresponding post-translational modification or the chemical group that will be removed by the interfering gene product.

[0081] In further embodiments, the interfering gene product can be a protein that participates 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, expression of the miRNA particularly reduces the extent of glycosylation motifs produced or supported by the interfering gene product.

[0082] In certain embodiments, the interfering gene product is a receptor or binding partner of the target polypeptide. Receptors and binding partners include any gene product that binds to the target polypeptide and thus interferes with its production. This particularly also includes gene products of other species, such as in embodiments where the host cell and the target polypeptide do not belong to the same species (e.g., human polypeptides are expressed in CHO host cells), especially homologues of the natural receptors or binding partners of the target polypeptide. Receptors and binding partners may interfere with the production of the target polypeptide, for example, by isolating the target polypeptide from the cell culture medium, thereby reducing its yield in the culture supernatant, or by reducing host cell proliferation or survival, because the binding of the target polypeptide to the receptor or binding partner activates the signaling pathway in the host cell.

[0083] In certain embodiments, the interfering gene product is a protein that is difficult to separate from the target polypeptide. The corresponding interfering gene product for example comprises a gene product combined with the target polypeptide and a gene product with a chemical and / or physical property similar to the target polypeptide. In this respect, exemplary chemical and / or physical properties are molecular size, total charge, charge distribution, pI value, hydrophobicity and the combination with capture ligands such as protein A or protein G. In these embodiments, the interfering gene product can not use standard purification methods to separate well from the target polypeptide. For example, the interfering gene product and the target polypeptide may have similar binding and / or elution characteristics on one or more chromatographic matrices. In such embodiments, removing the interfering gene product from the product solution causes a significant loss of the target polypeptide, thereby causing the low output of the target polypeptide of purification.

[0084] 2. Vectors and host cells

[0085] In a second aspect, the present invention provides a vector nucleic acid for transfecting a host cell, comprising an expression cassette according to the first aspect. The vector can be any vector suitable for transfecting a host cell. In certain embodiments, the vector is a plasmid. In other embodiments, the vector is a viral vector.

[0086] In addition to the expression cassette, the vector nucleic acid may also include other elements. For example, the vector nucleic acid may include an origin of replication (ORI), a coding sequence encoding a polypeptide of interest, a selectable marker gene and / or an antibiotic resistance gene.

[0087] In certain embodiments, the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest. In particular embodiments in which the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest, the expression cassette according to the first aspect of the invention does not comprise a coding sequence for the polypeptide. In these embodiments, the vector nucleic acid particularly comprises another expression cassette containing a selective marker gene. In alternative embodiments in which the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest, the expression cassette according to the first aspect of the invention comprises a coding sequence encoding a selective marker.

[0088] In certain embodiments, the vector nucleic acid comprises a coding sequence encoding a polypeptide of interest. The coding sequence encoding the polypeptide of interest may be present in an expression cassette according to the first aspect of the present invention, or may be present in another expression cassette. In a particular embodiment, the vector nucleic acid comprises at least two expression cassettes, a first expression cassette for expressing the polypeptide of interest and a second expression cassette for expressing a selective marker, wherein the first or second expression cassette is an expression cassette according to the first aspect of the present invention. In an alternative embodiment, the vector nucleic acid comprises at least three expression cassettes, a first expression cassette is an expression cassette according to the first aspect of the present invention, a second expression cassette for expressing the polypeptide of interest, and a third expression cassette for expressing a selective marker. If the polypeptide of interest is composed of two or more different polypeptide chains, the different polypeptide chains may be encoded in the same expression cassette or in separate expression cassettes. Additional expression cassettes comprising coding sequences of different polypeptide chains of the polypeptide of interest may also be present on the vector nucleic acid.

[0089] In certain embodiments, two or more expression cassettes of the vector nucleic acid are expression cassettes according to the first aspect of the present invention. These expression cassettes can each contain template sequences of the same or different pri-miRNAs, in particular different pri-miRNAs, which can target the same or different interfering gene products of the host cell.

[0090] In a third aspect, the present invention provides a host cell comprising an expression cassette according to the first aspect or a vector nucleic acid according to the second aspect, wherein the host cell is capable of recombinantly expressing a polypeptide of interest.

[0091] The host cell can be any type of host cell that produces a gene product that interferes with the production of the target polypeptide and / or regulates the target polypeptide. In certain embodiments, host cells that produce interfering gene products are known. The host cell is particularly suitable for producing the target polypeptide.

[0092] 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. The nucleic acid encoding the polypeptide of interest is in particular heterologous to the host cell and is introduced into the host cell, in particular using genetic engineering techniques.

[0093] In certain embodiments, the host cell is a mammalian cell. The host cell can be particularly a rodent cell, a primate cell or a human cell. In certain embodiments, the mammalian cell is selected from, but is not limited to, a group consisting of cells derived from the following animals: mice, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20 and NIH3T3; rats, such as PC12, PC12h, GH3, MtT, YB2 / 0 and Y0; hamsters, such as BHK, CHO and DHFR gene-deficient CHO; monkeys, such as COS1, COS3, COS7, CV1 and Vero; and people, such as Hela, HEK293, CAP, retina-derived PER-C6, 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 be particularly used for suspension culture.

[0094] In addition to the expression cassette according to the first aspect of the invention or the vector nucleic acid according to the second aspect of the invention, the host cell may also contain other exogenous nucleic acids. In particular, the host cell may contain an expression cassette for expressing a polypeptide of interest, which is not an expression cassette according to the first aspect and is not present on the vector nucleic acid according to the second aspect. The expression cassette for expressing a polypeptide of interest may be present on another vector nucleic acid or integrated into the genome of the host cell.

[0095] In certain embodiments, the coding sequence of the polypeptide of interest is present in the host cell.

[0096] (i) within an expression cassette for expressing a miRNA,

[0097] (ii) in another expression cassette on the same vector nucleic acid as the expression cassette expressing the miRNA, or

[0098] (iii) on another vector nucleic acid or in the genome of a host cell.

[0099] 3. Generation method

[0100] In a fourth aspect, the present invention provides a method for producing a target polypeptide in a host cell, comprising the following steps:

[0101] (a) providing a host cell according to the third aspect;

[0102] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0103] (c) obtaining the target polypeptide from the cell culture; and

[0104] (d) optionally processing the polypeptide of interest;

[0105] The polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, in particular, encoded in the same expression cassette.

[0106] The steps of the method are generally performed in the order indicated.

[0107] In some embodiments, the method further comprises the following steps between steps (a) and (b):

[0108] (a1) inoculating a cell culture medium with the host cells to provide a cell culture, and

[0109] (a2) cultivating the host cells in cell culture under conditions that allow for an increase in the number of cells in the cell culture.

[0110] The suitable conditions for cultivating host cells, increasing their cell number and expressing target polypeptides depend on the specific host cells, carriers and expression cassettes used in the method. Those skilled in the art can easily determine suitable conditions, and for a variety of host cells, these conditions are also known in the art. In certain embodiments, the carrier nucleic acid in the host cell comprises one or more selective marker genes. In these embodiments, the culture conditions in step (a2) and / or (b) may include the presence of a corresponding selection agent in the cell culture medium.

[0111] Obtaining the target polypeptide from the cell culture in step (c) particularly includes separating the target polypeptide from the cell culture. Separation of the target polypeptide particularly refers to the separation of the target polypeptide from the remaining components of the cell culture. As used herein, the term "cell culture" particularly includes cell culture medium and cells. In certain embodiments, the target polypeptide is secreted by the host cell. In these embodiments, the target polypeptide is separated from the cell culture medium. Separating the target polypeptide from the cell culture medium can be performed, for example, by chromatography. Suitable methods and means for separating the target polypeptide are known in the art and can be easily applied by those skilled in the art.

[0112] The obtained polypeptide of interest may optionally be subjected to additional processing steps (d), such as additional purification, modification and / or formulation steps, to produce a polypeptide of interest having a desired quality and composition. Such additional 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. Additional steps may include viral inactivation, ultrafiltration, and diafiltration. The formulation step may include buffer exchange, formulation component addition, pH adjustment, and concentration adjustment. Any combination of these steps and additional steps may be used.

[0113] In certain embodiments, the method for producing the polypeptide of interest further comprises the step of providing a pharmaceutical formulation comprising the polypeptide of interest as step (d) or as part of step (d). Providing a pharmaceutical formulation comprising the polypeptide of interest or formulating the polypeptide of interest into a pharmaceutical composition particularly comprises exchanging a buffer solution or a buffer solution component of a composition comprising the polypeptide of interest. In addition, this step may comprise lyophilization of the polypeptide of interest. In particular, the polypeptide of interest is transferred to a composition comprising only pharmaceutically acceptable ingredients.

[0114] In certain embodiments, compared to the same host cell that does not express the miRNA targeting the interfering gene product, the generation of the interfering gene product in the host cell is reduced. In particular, compared to the same host cell that does not express the miRNA targeting the interfering gene product, the amount of the interfering gene product in the host cell is reduced to 50% or less, particularly 20% or less or even 10% or less.

[0115] The present invention further provides a method for producing a polypeptide of interest with increased yield and / or increased purity, comprising the following steps:

[0116] (a) providing a host cell according to the third aspect;

[0117] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0118] (c) obtaining the target polypeptide from the cell culture; and

[0119] (d) optionally processing the polypeptide of interest;

[0120] The polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, in particular, encoded in the same expression cassette.

[0121] The increase in yield and / or purity is determined compared to the same method for producing the polypeptide of interest, wherein the host cell does not comprise the expression cassette according to the first aspect of the invention and does not produce a miRNA targeting an interfering gene product of the host cell.

[0122] The present invention further provides a method for increasing the yield and / or purity of a target polypeptide produced by a host cell, comprising the following steps:

[0123] (a1) providing a host cell capable of producing the target polypeptide;

[0124] (a2) introducing the vector nucleic acid according to the second aspect of the present invention into the host cell;

[0125] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0126] (c) obtaining the target polypeptide from the cell culture; and

[0127] (d) optionally processing the polypeptide of interest.

[0128] In certain embodiments, the vector nucleic acid introduced into the host cell in step (a2) does not contain a coding sequence for the polypeptide of interest.

[0129] In a fifth aspect, the present invention provides a method for producing a host cell according to the third aspect, comprising the following steps:

[0130] (a) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid comprises a coding sequence for a polypeptide of interest in an expression cassette for expressing miRNA or in another expression cassette; or

[0131] (b) introducing the vector nucleic acid according to the second aspect into the host cell, wherein the vector nucleic acid does not comprise the coding sequence of the polypeptide of interest, and introducing another vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, wherein the different vector nucleic acids can be introduced into the host cell simultaneously or successively in any order.

[0132] The vector nucleic acid is artificially introduced into the host cell. In particular, the vector nucleic acid is introduced by transfection. In this respect, the transfection can be transient or stable, especially using stable transfection. Therefore, in certain embodiments, the host cell produced comprises an expression cassette according to the first aspect that is stably integrated into its genome.

[0133] In a sixth aspect, the present invention provides the use of the expression cassette according to the first aspect, the vector nucleic acid according to the second aspect, or the host cell according to the third aspect for producing a polypeptide of interest. The features and embodiments of the method for producing the polypeptide of interest described herein also apply to this use.

[0134] The present invention further provides the use of the expression cassette according to the first aspect or the vector nucleic acid according to the second aspect for improving the production of a target polypeptide in a host cell, comprising introducing the expression cassette or the vector nucleic acid into a host cell capable of producing the target polypeptide. In certain embodiments, the vector nucleic acid introduced into the host cell does not comprise the coding sequence of the target polypeptide. Improving the production of the target polypeptide may include increasing the yield and / or purity of the target polypeptide.

[0135] The present invention further provides the use of the vector nucleic acid for transfecting a host cell. In particular, the host cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0136] 4. Specific embodiments

[0137] In the following, particular embodiments of the invention are described. These embodiments may be combined with other embodiments, features and examples described herein.

[0138] Example 1 An expression cassette for expressing miRNA in a host cell, comprising an intron sequence containing a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in the host cell to form a miRNA targeting a gene product of the host cell, and the gene product of the host cell interferes with the production of a target polypeptide recombinantly expressed in the host cell and / or regulates the target polypeptide recombinantly expressed in the host cell; and wherein the miRNA comprises a passenger chain and a guide chain having an artificial sequence.

[0139] Embodiment 2 The expression cassette according to embodiment 1 further comprises a polymerase II promoter and a terminator functionally linked to the template sequence of the pri-miRNA, wherein the template sequence of the pri-miRNA is located between the promoter and the terminator of the expression cassette.

[0140] Embodiment 3 is an expression cassette according to embodiment 1 or 2, wherein the template sequence of the pri-miRNA is present in the 5' untranslated region, 3' untranslated region or coding sequence of the expression cassette, especially in the 5' untranslated region or 3' untranslated region.

[0141] Embodiment 4 The expression cassette according to any one of embodiments 1 to 3, wherein the pre-mRNA produced when the expression cassette is transcribed comprises the pri-miRNA, and wherein the miRNA is formed by processing the pre-mRNA.

[0142] Embodiment 5 An expression cassette according to any one of embodiments 1 to 4, comprising a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA.

[0143] Embodiment 6 An expression cassette according to any one of embodiments 1 to 5, comprising two or more template sequences of pri-miRNA, each miRNA targeting the same or different gene products.

[0144] Embodiment 7 The expression cassette according to embodiment 6, wherein the two or more template sequences of the pri-miRNA are located in the same intron sequence.

[0145] Embodiment 8 is an expression cassette according to embodiment 7, which comprises 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.

[0146] Embodiment 9 is an expression cassette according to any one of embodiments 6 to 8, wherein adjacent template sequences of two or more template sequences of the pri-miRNA are separated from each other by a spacer sequence, and the spacer sequence forms an RNA stem-loop structure, such as the sequence of SEQ ID NO: 22.

[0147] Embodiment 10 is an 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 wherein 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.

[0148] Embodiment 11 An 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.

[0149] Embodiment 12 An expression cassette according to embodiment 10 or 11, wherein the 5'miRNA scaffold stem comprises the nucleotide sequence of any one of SEQ ID NOs: 1-4, and / or the miRNA scaffold loop comprises the nucleotide sequence of SEQ ID NO: 8, and / or the 3'miRNA scaffold stem comprises the nucleotide sequence of any one of SEQ ID NOs: 11-14.

[0150] Embodiment 13 An expression cassette according to any one of embodiments 10 to 12, wherein the 5' miRNA scaffold stem and the 3' miRNA scaffold stem each comprise a recognition site for a restriction enzyme.

[0151] Embodiment 14 is an 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.

[0152] Embodiment 15 is an expression cassette according to any one of embodiments 1 to 14, wherein the gene product of the host cell targeted by the miRNA is selected from the group consisting of: a protease capable of cleaving the target polypeptide, a protein involved in the post-translational modification of the target polypeptide, a receptor or binding partner of the target polypeptide, a protein that is difficult to separate from the target polypeptide, a protein involved in the folding and / or secretion of the target polypeptide, a protein involved in the transport of components necessary for the production or modification of the target polypeptide, a protein involved in the degradation of the target polypeptide, a protein having at least 70%, in particular at least 80%, sequence identity with the target polypeptide over its entire length, and an endogenous homolog of the target polypeptide.

[0153] Embodiment 16 is an expression cassette according to any one of embodiments 1 to 15, wherein the gene product of the host cell targeted by the miRNA is a protease capable of cleaving the target polypeptide.

[0154] Embodiment 17 An expression cassette according to any one of embodiments 1 to 15, wherein 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 prenylation.

[0155] Embodiment 18 An expression cassette according to any one of embodiments 1 to 15, wherein the gene product of the host cell targeted by the miRNA is an enzyme capable of catalyzing post-translational modification of the target polypeptide or removal of a chemical group, such as a hydrolase, such as a lipase, phosphatase or glycosidase.

[0156] Embodiment 19 is an expression cassette according to any one of embodiments 1 to 15, wherein the gene product of the host cell targeted by the miRNA is a protein involved in the glycosylation of the target polypeptide, in particular a glycosyltransferase, a glycosidase or a nucleotide sugar transporter, such as a fucosyltransferase or a sialyltransferase.

[0157] Embodiment 20 An expression cassette according to any one of embodiments 1 to 19, wherein the miRNA targets an endogenous gene product of the host cell.

[0158] Embodiment 21 The expression cassette of any one of embodiments 1 to 20, wherein the artificial sequence of the guide strand is complementary to a portion of an mRNA encoding a gene product of the host cell targeted by the miRNA.

[0159] Embodiment 22 An expression cassette according to any one of embodiments 1 to 20, wherein the artificial sequence of the passenger strand and / or the guide strand is not found in a naturally occurring miRNA.

[0160] Embodiment 23 An expression cassette according to any one of embodiments 1 to 22, wherein 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 rosa 26 promoter, a pCEFL promoter, a chicken β-actin promoter (CBA), a β-actin promoter coupled to a CMV early enhancer (CAGG), an alpha-1-antitrypsin promoter, and an inducible promoter, such as a tetracycline-inducible promoter (e.g., pTRE) and a vanillic acid-inducible promoter.

[0161] Embodiment 24 An expression cassette according to any one of embodiments 1 to 23, wherein the promoter is a CMV promoter or a SV40 promoter.

[0162] Embodiment 25 An expression cassette according to any one of embodiments 2 to 24, further comprising a coding sequence for the polypeptide of interest, wherein the coding sequence is functionally linked to the polymerase II promoter and the terminator.

[0163] Embodiment 26 An expression cassette according to any one of embodiments 2 to 24, further comprising a coding sequence for a selectable marker, wherein the coding sequence is functionally linked to the polymerase II promoter and the terminator.

[0164] Embodiment 27 The expression cassette of embodiment 26, wherein the selectable marker is selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, bleomycin and blasticidin.

[0165] Embodiment 28 An 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, especially a human or hamster cell.

[0166] Embodiment 29 An expression cassette according to any one of embodiments 1 to 28, wherein the host cell is a CHO cell.

[0167] Embodiment 30 A vector nucleic acid for transfecting a host cell, comprising the expression cassette according to any one of embodiments 1 to 29.

[0168] Embodiment 31 The vector nucleic acid according to embodiment 30 further comprises an additional expression cassette suitable for expressing the polypeptide of interest.

[0169] Embodiment 32 A host cell comprising an expression cassette according to any one of embodiments 1 to 29 or a vector nucleic acid according to embodiment 30 or 31, wherein the host cell is capable of recombinantly expressing the polypeptide of interest.

[0170] Embodiment 33 The host cell according to embodiment 31 or 32, which is a mammalian cell, particularly a human, primate or rodent cell, especially a human or hamster cell.

[0171] Embodiment 34 The host cell according to embodiment 33, which is a CHO cell.

[0172] Embodiment 35 A 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

[0173] (i) within an expression cassette for expressing a miRNA,

[0174] (ii) in another expression cassette on the same vector nucleic acid as the expression cassette expressing the miRNA, or

[0175] (iii) on another vector nucleic acid or in the genome of said host cell.

[0176] Example 36 A method for producing a polypeptide of interest in a host cell, the method comprising the following steps:

[0177] (a) providing a host cell according to any one of embodiments 32 to 35;

[0178] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0179] (c) obtaining the target polypeptide from the cell culture; and

[0180] (d) optionally processing the polypeptide of interest;

[0181] The polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, in particular, encoded in the same expression cassette.

[0182] Embodiment 37 is a method according to embodiment 36, wherein step (d) comprises providing a pharmaceutical formulation comprising the polypeptide of interest.

[0183] Example 38 A method for increasing the yield and / or purity of a target polypeptide produced by a host cell, the method comprising the following steps:

[0184] (a1) providing a host cell capable of producing the target polypeptide;

[0185] (a2) introducing the vector nucleic acid according to Example 30 into the host cell;

[0186] (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest;

[0187] (c) obtaining the target polypeptide from the cell culture; and

[0188] (d) optionally processing the polypeptide of interest.

[0189] Embodiment 39 A method for producing a host cell according to any one of embodiments 32 to 35, the method comprising the following steps:

[0190] (a) introducing a vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid comprises a coding sequence for a polypeptide of interest in an expression cassette for expressing the miRNA or in another expression cassette; or

[0191] (b) introducing the vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid does not comprise the coding sequence of the target polypeptide, and introducing another vector nucleic acid 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 successively in any order.

[0192] Embodiment 40 Use of the expression cassette according to embodiments 1 to 29, the vector nucleic acid according to embodiment 30 or 31, or the host cell according to embodiments 32 to 35 for producing a polypeptide of interest.

[0193] Embodiment 41 Use of the expression cassette according to embodiments 1 to 29 or the vector nucleic acid according to embodiment 30 or 31 for improving host cells to produce a target polypeptide, comprising introducing the expression cassette or vector nucleic acid into a host cell capable of producing the target polypeptide.

[0194] Embodiment 42 is the use according to embodiment 41, wherein the host cell contains a nucleic acid encoding the polypeptide of interest.

[0195] Embodiment 43 The use according to embodiment 41 or 42, wherein the expression cassette or the vector nucleic acid does not contain the coding sequence of the target polypeptide.

[0196] Embodiment 44 The use according to embodiments 41 to 43, wherein improving the production of the target polypeptide comprises increasing the yield and / or purity of the target polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0197] Figure 1 The vector map of the original pCMV vector used to express the polypeptide of interest comprising heavy and light chains is shown. The vector contains the folate receptor selectable marker gene (FAR), the DHFR selectable marker gene and the ampicillin resistance gene.

[0198] Figure 2 A vector map of a pCMV vector containing a miRNA scaffold encoding miRNA-1 into the RK intron located in the 5'UTR of the CMV-driven POI1 heavy chain transcript (pCMV01) is shown. Intronic miRNAs are highlighted.

[0199] Figure 3 A vector map of a pCMV vector containing a miRNA scaffold encoding miRNA-1 into a synthetic intron located in the 3'UTR of the SV40 driven FAR selectable marker gene transcript (pCMV02) is shown. The intronic miRNA is highlighted.

[0200] Figure 4 The putative mode of action and overall assembly of intronic pri-miRNAs are shown, where the encoded artificial miRNA targeting the POI1 receptor is located in the RK intron. The miRNA scaffold includes two restriction sites (RE1 and RE2) for exchanging miRNA sequences. The miRNA loop structure as well as the regions in the miRNA scaffold are essential for the efficient processing of miRNA molecules. The intronic region ensures the production of two functional RNA molecules: i) a complete mRNA molecule that enables the translation of the POI1 nucleotide sequence into a polypeptide sequence, and ii) a complete miRNA molecule that contains a region complementary to the endogenous mRNA of the POI1 receptor and thereby inhibits POI1 receptor expression.

[0201] Figure 5RT-qPCR results for stable CHO pools expressing POI1 and intronic miRNAs encoding different guide strand sequences targeting POI1 receptor mRNA are shown. In both loci (RK intron and synthetic intron), control pools (no miRNA and miRNA scrambled) showed similar expression profiles of POI1 receptor mRNA levels. Pools expressing miRNA sequences targeting the POI1 receptor in a pri-miRNA scaffold located in the RK intron showed efficient knockdown of POI1 receptor mRNA levels. All miRNAs reduced expression to less than 25%, while miRNA-1 was the most effective, with only 3.9% of POI1 receptor mRNA levels remaining. In contrast, when located in a synthetic intron of the 3'UTR of FAR, miRNA molecules targeting the POI1 receptor were less efficient or even lost their ability to knock down POI1 receptor mRNA levels. In addition, miRNA-1 still showed the strongest reduction in POI1 receptor mRNA levels, down to 13.9%. The mean + / -SD of three stable transfected pools in each case is shown. One of the control pools (no miRNA) was used for normalization (set to 1).

[0202] Figure 6 Cell viability (A), viable cell density (B) and POI1 titer (C) of pools run in fed-batch mode are shown. The CHO pool expressing miRNA-1 in the RK intron showed the highest viable cell density and the highest POI1 titer. The mean + / - SD of three stable transfected pools is shown in each case.

[0203] Figure 7 Cell line engineering and development strategies for the interfering gene product (IGP) knockdown approach are shown.

[0204] Figure 8 Cell viability (dashed line) and viable cell density of different host cells during optimized fed batch are shown. Compared with the stably transfected control cell lines (POI2 cell line and control gene KD), IGP knockdown pools (IGP_A and IGP_B) showed similar viable cell density. The mean + / -SD of three stable transfected pools in each case is shown. For POI2 and host cell lines, no replication is shown.

[0205] Fig. 9 POI2 titers in different host cells are shown. Under optimized fed-batch conditions, the titers of the IGP KD pool were comparable to the control. The mean + / - SD of three stable transfected pools is shown for each condition. For the POI2 cell line, replicates are not shown.

[0206] Fig.10The relative mRNA expression of POI2, IGP and control genes is shown. On the 10th day of optimized fed batch, RNA was sampled and purified. qPCR was used to quantify POI2, IGP, control gene and GAPDH (housekeeping gene) transcripts. Compared with the control, the two IGP KD pools showed significantly lower IGP transcript levels. The mean value + / -SD of three stable transfection pools in each case is shown. For POI2 and host cell lines, no repetitions are shown. POI2 and host cell lines are used for normalization (set to 1) of POI2, IGP and control genes, respectively.

[0207] Fig.11 IGP protein levels quantified using IGP ELISA are shown. The IGP KD pool shows that IGP protein levels are lower in the non-purified harvest at day 10 of the optimized fed-batch run. One selected pool and POI2 cell line for each case are shown. The POI2 cell line is used for normalization (set to 1).

[0208] Fig.12 POI2 titers of IGP KD pools and single cell clones are shown. The IGP_A encoding pool was used for single cell cloning. 96 clones were inoculated into a 24 dwp standard fed batch and POI2 titers were assessed. As expected, some clones showed higher titers compared to the original pool and POI2 cell line control, while others had lower titers.

[0209] Fig.13 Shown is the IGP mRNA level of IGP KD single cell clone.IGP_A coding pool is used for single cell clone.96 clones are inoculated into 24dwp standard fed batch, and at the 10th day of fed batch, qPCR quantitative IGP mRNA level is used.Compared with POI2 cell line control (normalized to 1), most clones show that IGP transcript levels are significantly reduced.

[0210] Fig.14Shown are the characteristics of the top 3 IGP KD clones compared to the POI2 cell line in a 7L bioreactor run. IGP_A Clone-A and Clone-B were inoculated in two separate bioreactors, while IGP_A Clone-C and the POI2 cell line were inoculated in a single bioreactor. A: On the 14th day of the fed-batch process from the 7L bioreactor, POI2 titers were significantly higher in the IPG KD clones. B: IGP mRNA levels were greatly reduced on the 10th day of the standard fed-batch run in the 7L bioreactor compared to the POI2 cell line (normalized to 1). IGP_A Clone-B showed a more than 100-fold reduction in IGP transcripts. C: IGP protein levels in the non-purified harvest from the 7L bioreactor run showed that the IGP levels of the top 3 clones were below LOQ (2ppm) compared to the POI2 cell line.

[0211] Fig.15 Titers and RT-qPCR results of stable CHO pools expressing POI3 and an intronic miR-3G-derived miRNA scaffold encoding a guide strand sequence targeting the POI1 receptor mRNA are shown. A: POI3 titers are similar to CHO pools that produce POI3 and do not encode miRNA or encode intronic miRNAs with miR-3G-derived miRNA scaffolds targeting the POI1 receptor. B: CHO pools expressing intronic miRNAs derived from miR-3G and targeting the POI1 receptor effectively reduce mRNA levels of the POI1 receptor compared to the no-miRNA control pool. The mean + / - SD of three stable transfected pools in each case is shown. One control pool (no miRNA) was used for normalization (set to 1).

[0212] Fig.16The triple miRNA concept (A) and qPCR data of the transfected CHO pool are shown (B). A: The vector pCMV05 encodes an intronic miRNA cluster with three realized artificial miRNAs, each targeting a different protease. The three miRNAs are located in a single intron, separated by a linker, and encoded in a single expression cassette that drives the expression of the miRNA cluster targeting the protease and the POI4 gene. B: Stable pools were generated with pCMV05-derived vectors that do not encode miRNAs (POI4 pool) or encode triple miRNA clusters targeting protease-5, protease-7 and protease-10 (POI4-triple miR-A pool) or protease-4, protease-9 and protease-11 (POI4-triple miR-B pool). After pool generation, qPCR was used to quantify the expression of the targeted and selected non-targeted proteases and the POI4 gene. The pool expressing the triple miRNA cluster significantly reduced the expression of all three targeted proteases, while the expression of the non-targeted protease and POI4 gene was basically unaffected. Targeted proteases are indicated with arrows. Means + / - SD of two stable transfected pools are shown in each case. One POI4 cell line pool (no miRNA) was used for normalization (set to 1).

[0213] Fig.17 qPCR data of 14-miR concept (A), transfection CHO pool (B) and CHO clone (C) are shown.A: vector pCMV06 encodes two intron miRNA clusters separated in two different expression cassettes, and the expression cassette is encoded in a single vector with hygromycin and puromycin selection markers. Intron miRNA clusters encode 9 and 5 artificial miRNAs targeting different proteases respectively. 9 and 5 miRNAs are each located in a single intron, separated by a joint, and each cluster is encoded in a single expression cassette, which only drives the expression of the intron miRNA cluster targeting protease. B: stable CHO pools expressing pCMV06 are generated, and qPCR is used to evaluate the gene expression of targeted protease and control genes. Compared with the host cell line, all 14 targeted protease genes are significantly reduced, while the control gene expression is not affected. The mean value + / -SD of three stable transfection pools in each case is illustrated. A repeat of the repeated host cell line is normalized (set to 1). C: Generate a monoclonal cell line derived from a CHO pool expressing pCMV06, and show qPCR data for three clones. 14miR clone-A shows a significant knockdown of the protease targeted by the 5miR cluster, but the protease targeted by the 9miR cluster is not knocked down. In contrast, 14miR clone-B and -C show a significant downregulation of all 14 targeted proteases. The host cell line is used for normalization (set to 1).

[0214] Fig.1814miR clones were stably transfected with a vector expressing POI5, and the resulting 14miR clone POI5 pool was inoculated into a standard fed batch to assess the production titer (A) and proteolytic degradation (B) of purified and low pH and time stressed POI5. A: The 14miR clone POI5 pool produced similar POI5 titers compared to the host cell line POI5 pool control. B: POI5 was purified and analyzed by MS on day 0. After 7 days of incubation at pH 5, the purified POI5 was re-analyzed using MS. POI5 material derived from the 14miR clone-A POI5 pool exhibited higher proteolytic degradation compared to the host cell line, however, 14miR clones-B and -C showed lower or no proteolytic degradation of POI5.

[0215] Fig.19 14miR clones were stably transfected with a vector expressing POI6, and the resulting 14miR clone POI6 pool was inoculated into a standard feed batch to evaluate the production titer (A) and proteolytic degradation (B) of purified and low pH and room temperature stressed POI5. A: The 14miR clone POI6 pool produced similar POI6 titers compared to the host cell line POI6 pool control. B: POI6 was purified and analyzed by MS on day 0. After 7 days of incubation at room temperature (RT) at pH 5, the purified POI6 was re-analyzed using MS. Compared to the host cell line, POI6 material derived from the 14miR clone-A POI6 pool exhibited higher proteolytic degradation, however, 14miR clones-B and -C showed significantly lower proteolytic degradation of POI6.

[0216] Examples

[0217] In the following examples, different expression cassettes containing pri-miRNA template sequences are used to knock down target genes that interfere with the production of target polypeptides in host cells. In Examples 1 to 4, the pri-miRNA template is present in the 5'UTR of the target polypeptide or in the intron sequence of the 3'UTR of the selective marker gene, which targets the receptor protein of the host cell that the target polypeptide binds and has a negative impact on the growth and survival of the host cell. In Example 5, the host cell that has been engineered to produce the target polypeptide is further transfected with a vector containing an expression cassette that contains only the pri-miRNA template in the intron sequence, but does not contain the coding sequence of the polypeptide. Here, miRNA targets host cell proteins that are difficult to separate from the target polypeptide during purification. In Example 6, alternative miRNA scaffolds are used to target receptor proteins. In Example 7, the host cell is engineered with an expression cassette that encodes the target polypeptide and contains a pri-miRNA cluster in the intron sequence in the 5'UTR. Here, the pri-miRNA cluster contains three pri-miRNAs, each targeting a host cell protein with proteolytic function. In Example 8, host cells were engineered with two separate expression cassettes, each containing a pri-miRNA cluster but without the coding sequence for a polypeptide. The engineered cells were further transfected with a vector encoding a polypeptide of interest. Here, the miRNA targets a host cell protein that proteolytically degrades the polypeptide of interest.

[0218] Example 1: Vector design.

[0219] The vectors encoding intronic miRNA (pCMV01-pCMV12) are based on the standard vector (pCMV), which encodes the expression of the CMV-driven target polypeptide POI1 ( Figure 1 The vector was modified by inserting intronic miRNA sequences into two different loci: the miRNA scaffold was located in the RK intron upstream of the POI1 gene ( Figure 2 ) or in a synthetic intron implemented in a selectable marker gene (folate receptor, FAR, Figure 3) in the 3'UTR of the published sequence. The sequence context chosen for the miRNA scaffold used for proof-of-concept was similar to human miR-30A and miR-E molecules (e.g. Fellmann et al., 2011, Molecular Cell 41, 733–746) and was expected to optimally process the resulting miRNA sequence. However, the sequence was further modified: i) the EcoRI restriction site was replaced by a BglII restriction site, ii) the sequence downstream of the XhoI restriction site was replaced by a CHO-derived sequence, and iii) an additional miR-30A scaffold was added upstream and downstream of the published sequence. Different miRNA sequences targeting the POI1 receptor were tested: miRNA-1, miRNA-2, miRNA-3, miRNA-4, miRNA-5, and miRNA-sc (with a scrambled guide strand sequence as a control). The design of the pri-miRNAs was as follows: Figure 4 shown.

[0220] Example 2: RT-qPCR analysis of stable CHO pools.

[0221] To test whether the miRNA sequence targeting the POI1 receptor located in the intronic pri-miRNA would lead to knockdown of the POI1 receptor mRNA level and to improved growth rate and productivity of POI1-producing CHO cells, cells were transfected with vectors pCMV and pCMV01-12. Stable pools were selected using different selection markers and used for RT-qPCR analysis.

[0222] Quantification of POI1 receptor mRNA levels using RT-qPCR showed that all miRNA sequences targeting POI1 receptor downregulated POI1 receptor mRNA levels when located in the RK intron. Pools transfected with control vector (no miRNA; miRNA scrambled) showed comparable levels of POI1 receptor mRNA. Figure 5 A) The best performing miRNA-1 reduced POI1 receptor mRNA levels to 3.9%. In contrast, when the miRNA was located in a synthetic intron in the 3'UTR of FAR, the knockdown efficiency was less pronounced, however, miRNA-1 showed a reduction in POI1 receptor mRNA levels to 13.9% (see Figure 5B). The difference could be explained by the locus of the intronic miRNA: when located in the RK intron, the transcript is driven by the strong CMV promoter, which should simultaneously result in a greater amount of processed miRNA molecules. In contrast, expression of the intronic miRNA in the 3'UTR of FAR is driven by the weaker SV40 promoter, resulting in a lower amount of processed miRNA molecules compared to the CMV promoter. These data also confirm the high knockdown efficiency of miRNA-1 compared to other miRNA sequences, which require stronger expression to effectively knockdown POI1 receptor mRNA levels.

[0223] Example 3: Stabilization of cell growth and productivity of CHO pools.

[0224] The best performing pools (miRNA-1 and miRNA-5 in two loci, RK intron and FAR intron) as well as the "no miRNA" pool were run in fed-batch mode. Thus, a standard 14-day 100 mL shake flask fed-batch culture was inoculated with the POI1-producing pool and cell growth, cell viability, and product titer were assessed at different time points. The pool with normal POI1 receptor mRNA levels (no miRNA) showed slower cell growth and lower viable cell density ( ) compared to the POI1 receptor knockdown pool. Figure 6 The stable CHO pool showed the strongest knockdown efficiency of the POI1 receptor (miRNA-1 is in the RK intron), and also showed the fastest cell growth and the highest viable cell density ( Figure 6 ). All POI1 receptor knockdown pools grew to higher viable cell densities compared to the control pool. The CHO pool without miRNA had titers as high as 2 g / L. All POI1 receptor knockdown pools showed higher titers compared to the control. Using miRNA-1 in the RK intron resulted in the highest titer (3.7 g / L), an 85% increase in titer compared to the CHO control pool (no miRNA).

[0225] Example 4: Stability of POI1 receptor knockdown and POI1 productivity in CHO pools.

[0226] We tested the stability of POI1 receptor knockdown by quantifying POI1 receptor transcript levels using qPCR every four weeks. In addition, we checked POI1 titer levels in batch mode every two weeks. Interestingly, after 8 weeks, the pool without POI1 receptor knockdown produced lower amounts of POI1 compared to the POI1 receptor knockdown pool. This observation may confirm the hypothesis that knockdown of POI1 receptor sends a survival signal to the cell pool. Linking miRNA expression to POI1 expression may stabilize the genetic integration of the pCMV cassette, thereby reducing the risk of selecting unstable clones. In addition, qPCR results showed that POI1 receptor knockdown remained stable over 8 weeks, while other expression cassettes of the pCMV vector did not change.

[0227] Example 5: Generation of interfering gene product (IGP) knockdown cell lines.

[0228] IGP KD cell line generation strategy Figure 7 As shown. The CHO parent cell line was transfected with a vector encoding the polypeptide of interest POI2 and pooled using MTX in a low-folate medium. These pools were single-cell cloned and a monoclonal cell line expressing POI2 was selected, referred to as the POI2 cell line. Subsequently, the primary seed batch (PSL) of the POI2 cell line was used to transfect a vector encoding an artificial intronic miRNA targeting the interfering gene product IGP, and puromycin was used to generate a pool.

[0229] Two different miRNAs targeting CHO IGP mRNA were generated, termed IGP_A and IGP_B, both targeting the 3'UTR of the transcript. For pool generation, miRNAs targeting different control genes, the parental POI2 cell line, as well as the empty parental host cell line (CHO) were included as controls. All samples (triplicate pool generation of the knockdown approach) were inoculated into an optimized fed-batch run and cell growth, gene expression, and POI2 titer were assessed on different days ( Figures 8 to 10 In addition, IGP protein levels at harvest level were assessed using IGP ELISA ( Fig.11 ):

[0230] After pool generation and confirmation of efficient IGP knockdown at the mRNA level, the IGP_A pool was selected for single-cell cloning and 96 expanded clones were inoculated into a 24 dwp standard fed-batch to assess IGP knockdown efficiency and POI2 productivity ( Fig.12 and 13 ).

[0231] The top 30 clones were further characterized. Based on a number of parameters (USP, DSP, IGP data, POI2 protein characteristics), the top 3 clones were selected and inoculated into a 7L bioreactor. IGP expression was significantly reduced in the IGP knockdown clones, resulting in an increase in POI2 titer ( Fig.14 ).

[0232] Example 6: Alternative miRNA scaffolds

[0233] Alternative miRNA scaffolds were tested for knockdown of the POI1 receptor based on miR-16-2 and miR-3G sequences (e.g. Watanabe et al., 2016, RNA Biology 13(1), 25-33). To this end, miRNA-1 was implemented into the adapted miR-3G scaffold. The artificial miRNA was encoded in an expression cassette encoding POI3 driven by a CMV promoter. Stable CHO pools were generated and POI3 titers and POI1 receptor mRNA expression were quantified. The control pool (no miRNA) produced similar POI3 titers (P < 0.05) compared to the pool expressing miRNA-1 encoded in the adapted miR-3G scaffold. Fig.15 A). Furthermore, in the pool expressing miRNA-1 encoded in the adapted miR-3G scaffold, POI1 receptor was effectively downregulated ( Fig.15 B).

[0234] Example 7: Multiple knockdown strategy using artificial miRNA clusters

[0235] Subsequently, encoding multiple miRNAs in a single intron enabled simultaneous knockdown of multiple target genes. This approach tested triple knockdown of three different proteases. The three miRNAs (each targeting a different protease) were separated by specific spacer sequences to ensure correct RNA folding and efficient miRNA processing. The triple miRNA cluster was implemented in an intron of a CMV-driven expression cassette driving POI4 expression ( Fig.16 A). Two different triple miRNA clusters were designed to knock down three different proteases: triple miR-A targets proteases 5, 7, and 10, while triple miR-B targets proteases 4, 9, and 11.

[0236] Stable CHO pools were generated using vectors encoding POI4 without miRNA, triple miR-A, or triple miR-B. mRNA expression of POI4, targeted and non-targeted proteases was quantified by RT-PCR and normalized to the pool producing only POI4 (no miRNA) ( Fig.16 B). Stable CHO pools encoding triple miR-A and triple miR-B efficiently knocked down specific target proteases, while expression of irrelevant non-targeted proteases remained similar to the control pool.

[0237] Example 8: Generation of 14miR protease knockdown cell lines

[0238] The vector pCMV06 encoding 14 miR contains two expression cassettes that drive the expression of intronic miRNA clusters targeting 9 or 5 endogenous proteases, but does not have the coding sequence of the target polypeptide ( Fig.17 A). The selection markers puromycin and hygromycin were encoded on separate expression cassettes, allowing selection of stable CHO pools using either selection marker. Using puromycin as a selection marker, a stable CHO pool expressing pCMV06 was generated, referred to as the 14-miR pool. Expression of the targeted endogenous protease was significantly reduced in the 14-miR pool compared to the host cell line, as measured using qPCR ( Fig.17 B). However, the expression of the non-targeted endogenous control gene remained unaffected. Single cell cloning was used to generate monoclonal 14-miR clones, and three clones (referred to as 14miR clones-A, -B, and -C) were further characterized using qPCR ( Fig.17 C). 14miR clone-A down-regulates proteases targeted by the 5miR cluster, but not by the 9miR cluster, assuming the 9miR expression cassette is lost. In contrast, 14miR clones-B and -C significantly reduce the expression of all 14 targeted endogenous proteases. Most proteases are strongly down-regulated by the miRNA cluster, while two to three miRNAs show moderate knockdown.

[0239] 14miR clones were stably transfected with vectors encoding POI5 or POI6. Transfected host cell lines served as controls. Stable pools were generated using MTX in low folate medium. The generated 14miR clone POI5 or POI6 pools and host cell line POI5 or POI6 pools were inoculated into standard fed batches and the production titers were assessed on day 14. Compared to the host cell line pools, the 14miR clone POI5 and POI6 pools showed similar POI5 or POI6 titers ( Fig.18 A and 19A). Inter-clone variation in the productivity of the polypeptide of interest could be observed. In addition, production harvests were used to purify POI5 or POI6 and the purified material was analyzed using mass spectrometry at day 0. The purified material was exposed to low pH for 7 days at room temperature and re-analyzed using mass spectrometry ( Fig.18 B and 19B). Proteolytic degradation of POI5 or POI6 after 7 days of treatment was higher in the 14miR clone-A pool compared to the host cell line pool. In contrast, 14miR clones-B and -C showed significantly lower proteolytic degradation compared to the host cell line pool.

[0240] Example 9: Materials and methods.

[0241] 1. Expression vector construction

[0242] The vector used in the examples consists of the following elements: hCMV promoter / enhancer driving the expression of the various genes required for the assembly of the POI construct, a polyadenylation signal (polyA), a folate receptor, DHFR, puromycin and hygromycin resistance genes as selection markers, an E. coli replication origin (CoIE ori), and a β-lactamase gene providing ampicillin (amp) resistance to initiate amplification in bacteria. Different plasmid setups were evaluated and further details are provided in the figures.

[0243] 2. Cell lines, culture, transfection and selection

[0244] CHO cells are suspended in a patented chemically defined culture medium in a 24-well plate or shake flask at 300 rpm (24 dwp) or 150 rpm (shake flask), 10% CO2, 36.5°C non-humidified cabinet shaker. Cell viability and growth rate are monitored by an automated system (ViCell, Beckman Coulter) or using analytical flow cytometry (CytoFlex, Beckman Coulter). Cells are passaged 2-3 times per week to fresh culture medium and maintained in the logarithmic growth phase.

[0245] The linearized expression vector was transfected by electroporation (Amaxa Nucleofection System, Lonza, Germany). The transfection reaction was performed in chemically defined medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in exponential growth phase with a cell viability greater than 95%. 5 x 10 6 Cells were transfected. Immediately after transfection, cells were transferred to shake flasks containing chemically defined medium. Before starting the selection process, the cell pools were incubated at 36.5°C and 10% CO2 for 48 hours.

[0246] As described above, the selection procedure is performed using the selection markers encoded by each expression vector. Both proteins FoIR and DHFR are involved in the same molecular pathway; FolR transports folic acid and the folic acid analog MTX into cells, and DHFR converts it into an important precursor for purine and methionine synthesis. Combining them as a selection principle, a specific strong selection scheme can be used to enrich recombinant cells expressing two recombinant proteins. Puromycin selection is driven by its inhibition of protein synthesis, and the vector encoding the puromycin resistance marker gene enables cells to survive in the presence of puromycin.

[0247] 48 hours after transfection and growth under low folate conditions, additional selection pressure was applied by adding 10 nM MTX to a chemically defined medium. Alternatively, puromycin was used as a selection agent. 48 hours after transfection, 0.003 mg / mL puromycin was added to a chemically defined medium. After the pool was recovered, the cells were frozen in a medium supplemented with 7.5% DMSO, and cell pellets were prepared.

[0248] 3. Gene Expression Analysis by Quantitative Real-time PCR

[0249] RNA extraction was performed using the Qiagen RNeasy micro 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 premix (Applied Biosystems) and 10x diluted cDNA was analyzed in triplicate using the QuantiFast SYBR Green PCR kit (Qiagen) or the TaqMan Primer / Probe system and TaqMan master mix (Applied Biosystems). GAPDH was amplified as an endogenous control for normalization. The ABI Amplification and analysis were performed using the 7900HT Sequence Detection System. To calculate the relative quantity (RQ) of gene expression for sample comparison, a comparative 2 -ΔΔCt method, and the data were normalized.

[0250] 4. Upstream processing

[0251] After selection, material was produced in shake flask fed batch, 24 deep well plate culture or ambr15 bioreactor. Fed batch culture was inoculated with a cell inoculum density of 4E5 vc / ml (a proprietary feed solution was added starting from day 3, and the culture temperature was changed to 33°C on day 5). In-process control was performed during the cultivation to monitor the concentration of the POI construct. Cell culture samples for RNA isolation were collected on day 10 of the process. Individual cultures were cultivated for a period of 14 days. At the end of the cultivation process, cells were separated from the culture supernatant by centrifugation and then sterile filtered before further downstream processing.

[0252] 5. Protein Quantification Using ELISA

[0253] Use sandwich ELISA to measure the amount of Chinese hamster (CHO) IGP. Sample is added in the microtiter plate coated with anti-IGP antibody (capture antibody).Then by incubating with biotinylated anti-IGP antibody (detection antibody), then with streptavidin-peroxidase and tetramethylbenzidine (TMB) as substrate, and measure absorbance at 450nm to quantitatively combine IGP.Based on the IGP level in the CHO IGP standard calculation sample.

[0254] 6. Purification Method

[0255] 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 with 50 mM acetic acid pH 3.0. Immediately after elution, all eluates were titrated to pH 5.0 with 1 M Tris base.

[0256] 7. Sample Processing and Analysis

[0257] The sample buffer was exchanged using an Amicon Ultra-4 centrifugal filter device. The pH of the buffer was set to pH 4 with 50 mM acetic acid. The samples were transferred to 1.5 mL Eppendorf tubes and incubated at room temperature for 7 days.

[0258] Deglycosylation of the samples 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.

[0259] All candidates were analyzed on an LC / MS system (WATERS, Xevo XS). The mobile phases were: (A) 0.1% trifluoroacetic acid (TFA) in miliQ water and (B) 0.09% TFA in acetonitrile. The gradient of mobile phase B was 5% to 50% in 7 minutes, and the total run time for each injection was 10 minutes. A Waters BioResolve RP mAb polyphenyl column ( 2.7μm, 2.1mm x 150mm) was used to separate protein degradation products at 70°C. The column load for each injection was 1.0μg for the complete analysis and 0.44ug for the simplified analysis. For all analyses, the capillary voltage was set to 1.8kV, the sampling cone was set to 190V and the source chamber offset was 30V. The source temperature and desolvation temperature were maintained at 125°C and 400°C, respectively. The desolvation gas flow was 800L / h, the cone gas flow was 50L / h, and the nebulizer gas flow was 6.5Bar. The system was controlled by MassLynx. All data were imported into the Genedata MS Refiner workflow and then processed there.

[0260]

[0261]

[0262]

[0263]

Claims

1. An expression cassette for expressing miRNA in a host cell, comprising an intron sequence containing a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in the host cell to form a miRNA targeting a gene product of the host cell, the gene product of the host cell interferes with the production of a recombinantly expressed polypeptide of interest in the host cell and / or regulates the recombinantly expressed polypeptide of interest in the host cell; and wherein the miRNA comprises a passenger strand and a guide strand having an artificial sequence.

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

3. An expression cassette according to claim 1 or 2, comprising two or more template sequences of pri-miRNA, each miRNA targeting the same or different gene products, wherein the two or more template sequences of the pri-miRNA are optionally located within the same intron sequence.

4. The expression cassette of any one of claims 1 to 3, 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; wherein the 5' miRNA scaffold stem comprises the nucleotide sequence of any one of SEQ ID NOs: 1-4, and / or the miRNA scaffold loop comprises the nucleotide sequence of SEQ ID NO: 8, and / or the 3' miRNA scaffold stem comprises the nucleotide sequence of any one of SEQ ID NOs: 11-14.

5. The expression cassette according to any one of claims 1 to 4, wherein the gene product of the host cell targeted by the miRNA (i) is 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 having a sequence identity of at least 70%, in particular at least 80%, with the polypeptide of interest over its entire length, and an endogenous homologue of the polypeptide of interest; (ii) is a protease capable of cleaving the target polypeptide; (iii) is a transferase capable of catalyzing a post-translational modification of the polypeptide of interest, such as acetylation, acylation, sulfation, phosphorylation, alkylation, hydroxylation, amidation, carboxylation, palmitoylation, myristoylation or prenylation; (iv) is an enzyme capable of catalyzing the post-translational modification or removal of a chemical group of the polypeptide of interest, such as a hydrolase, for example a lipase, phosphatase or glycosidase; or (v) is a protein involved in the glycosylation of the polypeptide of interest, in particular a glycosyltransferase, glycosidase or nucleotide sugar transporter, such as a fucosyltransferase or a sialyltransferase; and / or (vi) is a gene product endogenous to the host cell.

6. The expression cassette according to any one of claims 1 to 5, wherein the artificial sequence of the passenger strand and / or the guide strand is not found in naturally occurring miRNA.

7. The expression cassette of any one of claims 1 to 6, wherein 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 murine rosa 26 promoter, a pCEFL promoter, a chicken β-actin promoter (CBA), a β-actin promoter coupled to a CMV early enhancer (CAGG), an alpha-1-antitrypsin promoter, and an inducible promoter, such as a tetracycline-inducible promoter (e.g. pTRE) and a vanillic acid-inducible promoter; preferably a CMV promoter or a SV40 promoter.

8. The expression cassette according to any one of claims 2 to 7, further comprising a coding sequence for the polypeptide of interest or a selectable marker, the coding sequence being functionally linked to the polymerase II promoter and the terminator.

9. The expression cassette according to any one of claims 1 to 8, wherein the host cell is a mammalian cell, in particular a human, primate or rodent cell, especially a human or hamster cell, preferably a CHO cell.

10. A vector nucleic acid for transfecting a host cell, comprising the 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 polypeptide of interest.

12. A host cell comprising the expression cassette according to any one of claims 1 to 9 or the vector nucleic acid according to claim 10 or 11, wherein the host cell is capable of recombinantly expressing the polypeptide of interest.

13. The host cell according to claim 11 or 12, which is a mammalian cell, in particular a human, primate or rodent cell, especially a human or hamster cell, preferably a CHO cell.

14. A method for producing a polypeptide of interest in a host cell, the method comprising the following steps: (a) providing a host cell according to claim 12 or 13; (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the target polypeptide from the cell culture; and (d) optionally processing the polypeptide of interest; The polypeptide of interest may optionally be encoded on the same vector nucleic acid as the pri-miRNA, in particular, encoded in the same expression cassette.

15. The method of claim 14, wherein step (d) comprises providing a pharmaceutical formulation comprising the polypeptide of interest.

16. A method for increasing the yield and / or purity of a target polypeptide produced by a host cell, the method comprising the following steps: (a1) providing a host cell capable of producing the target polypeptide; (a2) introducing the vector nucleic acid according to claim 10 into the host cell; (b) cultivating the host cell in cell culture under conditions that allow expression of the polypeptide of interest; (c) obtaining the target polypeptide from the cell culture; and (d) optionally processing the polypeptide of interest.

17. A method for producing a host cell according to claim 12 or 13, the method comprising the following steps: (a) introducing the vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid comprises the coding sequence of the polypeptide of interest in an expression cassette for expressing miRNA or in another expression cassette; or (b) introducing the vector nucleic acid according to the second aspect into a host cell, wherein the vector nucleic acid does not comprise the coding sequence of the polypeptide of interest, and introducing another vector nucleic acid suitable for recombinant expression of the polypeptide of interest into the host cell, wherein different vector nucleic acids can be introduced into the host cell simultaneously or successively in any order.

18. Use of the expression cassette according to any one of claims 1 to 9, or the vector nucleic acid according to claim 10 or 11, or the host cell according to claim 12 or 13 for producing a polypeptide of interest.

19. Use of the expression cassette according to any one of claims 1 to 7 and 9 or the vector nucleic acid according to claim 10 for improving host cells to produce a target polypeptide, comprising introducing the expression cassette or vector nucleic acid into a host cell capable of producing the target polypeptide.

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