CN118931847B

CN118931847B - Mammalian cells that highly express EPO protein

Info

Publication number: CN118931847B
Application number: CN202411426389.6A
Authority: CN
Inventors: 秦晓红; 张焱凤; 米立志; 郭佳; 段佳宁; 尚庆斌
Original assignee: Tianjin Yipu Biotechnology Co ltd
Current assignee: Tianjin Yipu Biotechnology Co ltd
Priority date: 2024-10-14
Filing date: 2024-10-14
Publication date: 2025-01-24
Anticipated expiration: 2044-10-14
Also published as: CN118931847A

Abstract

The present invention relates generally to the field of biotechnology, and in particular to mammalian cells that highly express EPO protein. The cell has inserted at the 3 rd intron of the Mast2 gene a gene expression cassette containing one or more copies of the EPO gene and is capable of stably expressing the EPO protein. The gene insertion site provided by the invention has the advantages of high expression quantity and stable passage.

Description

Mammalian cells highly expressing EPO protein

Technical Field

The present invention relates generally to the field of biotechnology, and in particular to mammalian cells that highly express EPO protein.

Background

Erythropoietin (Erythropoietin, EPO) is a highly sialylated glycoprotein hormone secreted by the kidney to act on erythroid progenitors in bone marrow to promote proliferation and differentiation and increase the concentration of hemoglobin in the body. Recombinant EPO protein was expressed from Chinese Hamster Ovary (CHO) cells and rapidly became the most efficacious bioengineered drug worldwide since 1989 when developed and marketed by the american security bioengineering company.

CHO cells are widely used mammalian expression systems in the biopharmaceutical field that produce recombinant proteins with post-translational modifications similar to those of humanized cells, about 70% of the recombinant protein drug being produced by CHO cells. Glycosylation modification of recombinant proteins of EPO after translation is critical, directly affecting their half-life and immunogenicity in humans.

The traditional method of constructing a cell line expressing a foreign protein is to randomly insert it into the genome of the cell. Because of the randomness of the integration site and the gene copy number, the risks of copy number loss and the like exist in the process of long-term passage of industrial production. Because of the great difference of expression quantity among the randomly integrated monoclonal cell strains, a great deal of manpower, material resources and time are required for screening the monoclonal cell strains.

Disclosure of Invention

The invention comprises the following technical scheme:

The first object of the present invention is to provide a mammalian cell in which one or more copies of the EPO gene are inserted into the gene expression cassette at the 3 rd intron of the post 2 gene and which is capable of stably expressing EPO protein.

The gene insertion site provided by the invention has the advantages of high expression quantity and stable passage.

In some embodiments, the copy number is 1, 2, 3, or more.

In some embodiments, the gene expression cassette further comprises one or more elements selected from the group consisting of:

i) A promoter;

ii) a signal peptide coding sequence;

iii) A leader sequence coding sequence;

iv) a terminator;

v) a marker gene;

vi) polyadenylation sequences.

The marker gene in the present invention is broad, and includes the gene used for screening (e.g., antibiotic resistance gene) and the marker gene, etc. Commonly used marker genes are fragments such as nucleic acids for the production of fluorescent proteins. The fluorescent protein may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

The green fluorescent protein can be common GFP, or modified GFP genes, such as enhanced GFP gene EGFP, etc., the blue fluorescent protein can be EBFP, azuritc, tagBFP, the yellow fluorescent protein can be EYFP, ypct, phiYFP, the orange fluorescent protein can be mKO, mOrange, mBanana, etc., and the red fluorescent protein can be TagRFP, mRuby, mCherry, mKate, etc.

In some embodiments, the amino acid sequence of the epo gene is set forth in SEQ ID NO. 1.

In some embodiments, the mammalian cell is a cell line.

In some embodiments, the mammalian cell is a CHO-K1 cell line.

In some embodiments, the insertion position of the gene expression cassette is located by an upstream LP-homology arm as shown in SEQ ID NO. 2 and a downstream LP-homology arm as shown in SEQ ID NO. 3.

A second object of the present invention is to provide a nucleic acid construct comprising an upstream LP-homology arm as shown in SEQ ID NO. 2, a gene expression cassette of an epo gene as defined above, and a downstream LP-homology arm as shown in SEQ ID NO. 3, which are sequentially linked.

In some embodiments, the nucleic acid construct further comprises attP and attB sites, and the gene sequence of BxB1 recombinase.

Preferably, attP has the nucleotide sequence shown in SEQ ID NO. 5.

Preferably attB has the nucleotide sequence shown in SEQ ID NO. 6.

It is a third object of the present invention to provide a vector comprising the nucleic acid construct as described above.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolated virus (e.g., SV 40), insect baculovirus.

A fourth object of the present invention is to provide a gene editing combination product comprising:

a) A carrier as described above, and

B) A nucleic acid cleavage system that targets a specific sequence that recognizes the 3 rd intron of the Mast2 gene and enables insertion of a nick by the nucleic acid construct in a form of homologous recombination.

In some embodiments, the nucleic acid cleavage system is a CRISPR/Cas9 system.

In some embodiments, the sgRNA sequence of the nucleic acid cleavage system is set forth in SEQ ID NO. 4.

A fifth object of the present invention is to provide a method for preparing EPO protein, comprising:

i) Culturing mammalian cells as described above to express the EPO protein;

ii) disrupting the cells and isolating the EPO protein therefrom.

For step ii), the recombinant protein may be isolated and purified by various isolation methods using its physical, chemical and other properties, if desired. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The cell provided by the invention can be used for stably and highly expressing long-acting recombinant EPO protein.

The construction method is preferably to insert the heterologous DNA into a pre-verified locus precisely by site-directed integration technology, so that the mammalian cell line stably expresses the exogenous gene. Specific integration platforms allow for the controlled insertion of DNA into stable genomic sites, with wide application in synthetic biology, recombinant protein production and biological manufacturing. The long-acting recombinant epo gene with optimized codons is accurately integrated at a site with stable high expression in a cell genome by utilizing a fixed-point integration technology, so that the screening process of monoclonal cell strains can be simplified, and the screening efficiency can be improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 schematic representation of knock-in donor LP doser.

FIG. 2 shows the results of flow analysis of CHO-K1 LP-m2 steady state cell monoclonal cell lines;

the horizontal axis shows mCherry red fluorescence detected by PE-A channel. The light color represents the fluorescence peak pattern of CHO-K1 control cells and the dark color is the fluorescence peak pattern of LP-m2 steady-state cell monoclonal cell lines.

FIG. 3 is a schematic representation of the integration of payload with a genome-specific site landing pad.

FIG. 4 is a schematic diagram of the payload and BxB1 lentiviral vector.

FIG. 5 shows the detection results of EPO expression level of a monoclonal cell strain screened by a traditional method through CHO-K1 LP-m2 site-specific integration.

FIG. 6 stability test results of CHO-K1 LP-m2 site-directed integration and EPO expression by monoclonal cell lines screened by conventional methods;

After 10 passages are continuously transferred and cultured for 6 days, EPO expression is detected by a Western blot method, lanes 1-11 are monoclonal cell strains screened by a traditional method, and lanes 12-14 are EPO cell strains of single gene copies obtained by CHO-K1 LP-m2 site-specific integration.

FIG. 7 shows the measurement results of EPO expression levels of different gene copy numbers in CHO-K1 LP-m2 cells;

the EPO expression is detected by using a Western blot method, wherein a lane 1 is an EPO expression cell strain with single gene copy, and a lane 2 is an EPO expression cell strain with triple gene copy.

FIG. 8 shows the detection result of EPO expression level of cell lines subjected to CHO-K1 LP-m2 site-specific integration and other site-specific integration;

the EPO expression is detected by using a Western blot method, lanes 1-2 are EPO expression cell strains of single gene copies obtained by fixed-point integration of CHO-K1 LP-m2, and lanes 3-5 are EPO expression cell strains of single gene copies obtained by fixed-point integration of 11 th exons of the CHO-K1 Clcc1 gene.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present invention, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present invention, all references to which this invention pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present invention by reference, but are not limited to being able to implement the present invention. It should be understood that when a reference is made to the description of the invention in conflict with the description, the invention is modified in light of or adaptive to the description of the invention.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

The present application relates to a mammalian cell having one or more copies of an EPO gene inserted into a gene expression cassette at the 3 rd intron of the post 2 gene and capable of stably expressing EPO protein.

In some embodiments, the copy number is 1, 2, 3, or more.

i) A promoter;

ii) a signal peptide coding sequence;

iii) A leader sequence coding sequence;

iv) a terminator;

v) a marker gene;

vi) polyadenylation sequences.

In some embodiments, the mammalian cell is a cell line.

In some embodiments, the mammalian cell is a CHO-K1 cell line.

Preferably, attP has the nucleotide sequence shown in SEQ ID NO. 5.

Preferably attB has the nucleotide sequence shown in SEQ ID NO. 6.

a) A carrier as described above, and

In some embodiments, the nucleic acid cleavage system is a CRISPR/Cas9 system.

i) Culturing mammalian cells as described above to express the EPO protein;

ii) disrupting the cells and isolating the EPO protein therefrom.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

EXAMPLE 1 site-directed integration of CHO-K1 LP-m2 cell construction

To achieve stable expression of foreign genes in mammalian cell lines, precise insertion of heterologous DNA into well-characterized genomic sites is required. The characterized genomic locus supporting the long-term stable expression of the transgene is selected, and a CHO-K1 LP-m2 monoclonal cell line containing a Landing Pad (LP) recombination locus is constructed.

The method comprises the following specific steps:

LP-m2 steady state cells were constructed by CRISPR/Cas9 system. And (3) cutting genomic DNA of a selected site by using the Cas9 protein, inserting LP donor into the cut genome by homologous recombination, and constructing a site-specific integration platform of the exogenous gene. The LP-m2 gRNA/Cas9 plasmid was constructed with pSpCas9 (BB) -2A-Puro (PX 459) V2.0 as vector, and gRNA was introduced before gRNA-scaffold.

LP donor is used as a donor for gene knock-in of the CRISPR/Cas9 system, two ends of the fragment are provided with homologous arms of a genome, and the fragment can be inserted into a target position of the genome based on the homologous sequences. The landing pad of LP donor consists of the CMV promoter, bxB1 recombinase recognition site attP sequence, reporter gene mCherry and SV40 polyA signal sequence in order. mCherry is transcribed under the control of a strong promoter CMV, and red fluorescence can be used to screen LP-m2 steady-state cell monoclonal while also being able to characterize expression of exogenous genes at selected genomic loci.

The LP-m2 gRNA/Cas9 plasmid was co-transfected with purified LP donor fragment at a mass ratio of 1:1 into CHO-K1 cells. Flow sorting was performed on co-transfected CHO-K1 LP-m2 cells based on red fluorescence to obtain mCherry positive cells. The monoclonal cells are separated by limiting dilution method, and monoclonal cell strain which stably expresses red fluorescence is selected.

Example 2 construction of EPO highly expressed cell lines Using site-directed integration techniques

Based on the CHO-K1 LP-m2 steady state cell platform, multiple copies of the epo gene can be inserted into selected sites via BxB1 recombinase mediated, allowing for controlled integration.

The payload design is as follows:

The attB site is placed before the resistance gene, replacing its promoter. When the payload plasmid is co-transferred with the BxB1 recombinase plasmid into the LP-m2 steady state cells, the BxB1 enzyme cleaves the attP and attB sites, allowing the linearized payload to be inserted into the desired location. The resistance gene in payload is expressed using the CMV promoter of the landing pad and exhibits resistance, and the desired polyclonal cells can be screened by drug killing. EPO expression depends on the self-contained promoter and, after integration, the poly a sequence of payload causes downstream mCherry to no longer express and the red fluorescence of the cells to disappear. This can be one of the bases for judging successful integration of the epo gene.

To further increase the efficiency of transfection and integration, lentiviruses were used to deliver the payload fragment and BxB1 gene, designed as follows:

The pCDH vector contains a CMV and EF 1a double promoter, and the BxB1 gene is inserted into the EF 1a promoter, while the CMV promoter in the vector is replaced by the payload fragment. Because the payload fragment contains the transcription termination signal PolyA, it is constructed by reverse insertion into the pCDH vector to minimize RNA mistermination during lentiviral packaging. The polyclonal cells successfully integrated with the epo gene are obtained through drug killing screening.

Example 3 sorting and identification of EPO highly expressing cell lines

The epo gene (1 or 3 copies) was integrated into the cell genome using PEI or Lipo6000 transfection. After 48 hours of culture of transfected cells, drug killing screening was performed with 1.5 mg/mL G418, with medium changes every 2-3 days. After culturing for about 7 days, 95% of cells in the negative control group die after falling off under a fluorescence inverted microscope. After further 3 days of culture with medium containing 1.5 mg/mL G418, cell clusters were formed. Transfected CHO-K1 LP-m2 cells were flow sorted based on red fluorescence, red was false positive, and EPO expressing positive cells were colorless due to replacement of the mCherry gene. Colorless monoclonal cells in the 96-well plates are sorted, inoculated into the 48-well plates after being grown, and after being cultured for 6 days, the culture solution is sucked for detecting the expression quantity by using a Western Blotting method. The correlation results are shown in fig. 5-7.

To further verify the effect of the present application, the inventors devised a control. The control was derived from site-directed integration of exon 11 of the Clcc1 gene described in Gaidukov L, Wroblewska L, Teague B, et al. A multi-landing pad DNA integration platform for mammalian cell engineering[J]. Nucleic acids research, 2018, 46(8): 4072-4086., but the relevant expression system was set up using the examples of the present application. The results are shown in FIG. 8, where the insertion sites of the present application are significantly improved compared to the control.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Claims

1. A CHO-K1 cell line, characterized in that a gene expression cassette containing one or more copies of an epo gene is inserted into the third intron of the Mast2 gene, and the cell line can stably express the EPO protein, wherein the amino acid sequence of the epo gene is shown in SEQ ID NO: 1.

2. The CHO-K1 cell line according to claim 1, characterized in that the gene expression cassette further comprises one or more elements selected from the following group:

i) Promoter;

ii) signal peptide coding sequence;

iii) leader coding sequence;

iv) terminator;

v) marker genes;

vi) Polyadenylation sequence.

3. The CHO-K1 cell line according to claim 1 or 2, characterized in that the copy number is 1, 2, 3 or more.

4. A nucleic acid construct, characterized in that it comprises an upstream LP-homologous arm shown in SEQ ID NO: 2, a gene expression cassette of an epo gene, and a downstream LP-homologous arm shown in SEQ ID NO: 3, which are sequentially connected; wherein the gene expression cassette of the epo gene is defined as the gene expression cassette in any one of claims 1 to 3.

5 . The nucleic acid construct according to claim 4 , characterized in that it further comprises attP and attB sites, and a gene sequence of the BxB1 recombinase.

6. A vector, characterized in that it comprises the nucleic acid construct according to claim 4 or 5.

7. A gene editing combination product, characterized in that it comprises:

a) the vector according to claim 6; and

b) a nucleic acid cleavage system that targets and recognizes a specific sequence in the third intron of the Mast2 gene and enables the nucleic acid construct to be inserted into the nick in the form of homologous recombination.

8. The gene editing combination product according to claim 7, characterized in that the nucleic acid cleavage system is a CRISPR/Cas9 system; and its sgRNA sequence is shown in SEQ ID NO:4.

9. A method for preparing EPO protein, comprising:

i) culturing the CHO-K1 cell line according to any one of claims 1 to 3 to express EPO protein;

ii) Disrupt the cells and isolate the EPO protein from them.