Method for selecting nucleic acid sequences

Abstract

This provides a method for targeted integration of donor vectors into specific, predefined genomic locations in isolated eukaryotic host cells. [Solution] The vector and host cell together contain nucleic acid components that enable the selection of cells into which the donor vector has been incorporated at a predefined genomic location in the host cell. More specifically, this method results in the selection of a sequence-optimized nucleic acid sequence variant. Such an optimized nucleic acid sequence variant may contain a sequence-optimized expression vector component for subsequent use in recombinant protein production.

Description

Technical Field

[0001] The present invention relates to the field of cell-based methods that utilize targeted integration of a donor vector into a specific predefined genomic position of the genome of a eukaryotic host cell, wherein said vector and the host cell comprise nucleic acid components that enable selective selection of those cells having integrated the donor vector into the predefined genomic position of the host cell genome, and optional detection and elimination of cells that have undergone any additional random integration events into other parts of the genome. The present invention particularly relates to the use of such targeted integration systems for evaluating libraries of nucleic acid sequence variants with the aim of identifying optimized nucleic acid sequence variants therefrom. Such optimized nucleic acid sequence variants can subsequently be used in expression systems for recombinant protein production or other biotechnological applications.

Background Art

[0002] Optimization of expression cassettes for commercial production of proteins Over the past three decades, recombinant protein therapeutics have evolved from a novelty to a dominant position among commercially available drugs. Recombinant production of therapeutic proteins exceeds a market size of 100 billion USD annually and plays an important role in the global economy as well as in advanced healthcare. Classes of therapeutic proteins include recombinant proteins (insulin, growth factors, cytokines and blood factors), vaccines (antigens, VLPs) and monoclonal antibodies. By far the dominant format is monoclonal antibodies[1, 2]. Through the continuous progress of protein engineering and synthetic biology, the classes of therapeutic proteins have become highly diversified, with rapid growth in the development of engineered protein formats such as bispecific and multispecific antibodies[3, 4]. While some recombinant proteins can be produced in unicellular microorganisms such as E. coli, for more complex proteins including both traditional and emerging classes of monoclonal antibodies, Chinese Hamster Ovary (CHO) cells are the dominant host for production[2].

[0003] In today's industry, the primary approach to generating cell lines that produce high-performance therapeutic proteins involves introducing recombinant protein genes into the genome of host CHO cell lines via a random integration approach, and then selecting / screening individual cells that have the gene integrated at a copy number-active genomic site, resulting in a phenotype capable of producing sufficiently high transcription and simultaneously supporting high protein translation and secretion. This is a highly focused and time-consuming process with high inherent uncertainty and biological variability. The duration of a typical process ranges from 3 to 12 months, depending on the host cell growth, the level of automation performed, and the endpoint (e.g., if long-term clonal stability assessment is included).

[0004] One fundamental limitation associated with random integration approaches is low sampling, which translates to cellular diversity in the transfected cell pool. Only about 0.1–1% of transfected cells integrate recombinant DNA. Furthermore, this subpopulation is highly heterogeneous in terms of the integration site, copy number, and integrity of the integrated DNA. Adding to the inherent overall phenotypic diversity of CHO cells, which is unique to CHO cells due to high genomic and epigenomic plasticity, finding highly productive clones is, in a sense, extremely difficult. This is also why high variability in protein production from a stable, non-clonal pool is commonly observed (probabilistic sampling of phenotypic diversity).

[0005] This insufficient sampling and high biological noise also make it difficult to compare different gene cassette designs of therapeutic protein candidates for expression optimization. Comparing multiple variants via the parallel generation of stable pools is a highly demanding task, and high biological noise would make the results unreliable. The use of simultaneous transfection of variant libraries is hampered by the fact that random incorporation typically results in the incorporation of multiple copies of the expression vector, and therefore any cells generated through such a workflow will typically contain copies incorporated from one or more gene cassette designs. Improvement of protein expression by cell line manipulation strategies based on random incorporation of effector genes is hampered for the same reasons.

[0006] One possible major improvement over all of the above limitations is to utilize targeted insertion (SDI) of the gene of interest (GOI). In such a scenario, a pre-identified genomic location known to support high and stable transcription is used as the target of the GOI. The use of a smart combination of pre-transfected sequences and vector designs, including the use of co-transfected nucleic acid enzymes such as nucleases or recombinases, facilitates targeted insertion and ensures that all cells in culture contain the correctly inserted GOI and therefore have high transcription rates. This would significantly reduce the number of clones required for screening operations in cell line development (CLD) and reduce biological noise in comparing gene cassette design or cell line manipulation efforts. Several technical solutions for targeted insertion have been described in the art [5-8]. Nevertheless, challenges remain.

[0007] The Flp-In® system (also known as flipper jelly recombinase, based on Flp recombinase)[9] for targeted integration is an example of a solution that utilizes a single recombinase-recognition sequence in combination with its recombinase to enable targeted integration at a predefined genomic location. After recombinase activation, the complete expression vector is integrated at the recombinase-recognition sequence. Integration at the recombinase-recognition site inactivates one selection marker and activates a second selection marker, thus allowing selection of cells with the correct integration event. The main drawbacks of this solution are: (i) the absence of a mechanism to detect or remove cells with additional copies of the expression vector incorporated due to random integration events; (ii) the absence of a mechanism to remove sequence regions, such as plasmid backbone sequences and active selection marker genes, which may result in negative GOI expression; (iii) the method having questionable mobility in the selection of selection markers, as activation of the selection markers during integration leads to the fusion of extra amino acids at the N-terminus, which may affect their functionality; and (iv) the selection is based on antibiotic resistance, which requires a long time and introduces a potential bias based on the specific growth rate in cells with positive integration.

[0008] To avoid the presence of sequences that may have a negative impact on GOI expression after target integration, different solutions for cassette exchange reactions at predefined genomic locations have been described in the art [5-8]. An example of such a solution is disclosed by Rentschler

[10] . The predefined genomic locations both utilize an active selection marker gene (GFP) adjacent to two orthogonal recombinase recognition sequences that are targets of the same recombinase. Similarly, a GOI in the expression vector is adjacent to two recombinase recognition sequences, which match the two present in the genome. During recombinase operation, a cassette exchange may occur between the selection marker cassette and the GOI cassette. Cells undergoing the cassette exchange can be selected by the absence of GFP expression. Disadvantages of this type of solution are that (i) there is no mechanism to detect or remove cells that have additional copies of the expression vector integrated by a random integration event, and (ii) the selection of cells undergoing the cassette exchange must be delayed to allow for GFP degradation / dilution, as the selection is based on the initial absence of the active gene product. In addition to extending the workflow, this also introduces potential biases based on the unique growth rates of cells with positive incorporation, as mentioned above.

[0009] Haghighat-Khah RE et al. disclose a two-step site-specific cassette exchange system in insects, namely the Aedes aegypti mosquito and the Plutella xylostella moth

[11] . This exchange system utilizes phiC31 recombinase for the integration of an expression vector at a predefined genomic site, followed by the use of a second recombinase (Cre or Flp) for the expression of a plasmid backbone sequence. However, the exchange system by Haghighat-Khah RE et al. does not provide a means to distinguish between targeted integration and random integration events. In addition, it does not provide a means to remove selected marker genes.

[0010] Yuan, Y et al. disclose a recombinase-based method for generating transgenic cells without selection markers and vector scaffolds using PhiC31-mediated gene delivery to a naturally occurring pseudoattP sequence in the genome of target cells

[12] . Selection of cells in which integration occurred was achieved via the presence of an active eGFP expression cassette in the expression vector, and an att-B-TK fusion gene, which is inactivated upon targeted integration, was used as a negative selection marker to eliminate random integration events in a second selection step. The selection system and plasmid bacterial scaffolds were then excised by using two other recombinases, Cre and Dre. Significant drawbacks of the method disclosed by Yuan, Y et al. for the adoption of recombinant protein production application based on integration to a single predefined genomic site are: (i) since inactive TK genes arise from both scenarios, the method does not provide a means to distinguish cells integrated only at the predefined site from cells integrated at random pseudoattP sites; (ii) the first selection step cannot be performed until the transient expression of the selection marker disappears, which is time-consuming and introduces a potential bias based on the specific growth rate among cells with positive integration; and (iii) the first selection step does not distinguish between desired integration, integration at a pseudoattP site, or random integration events.

[0011] Parthiban, K et al. disclose a cassette exchange method based on nuclease-directed integration of full-length IgG-formatted antibody genes into mammalian cells to generate a large repertoire of enriched cells for cells containing one antibody gene per cell

[13] . Selection of cells in which integration occurs at the desired genomic site is based on activation of blastosidin-resistant genes by a naturally occurring endogenous promoter at the selected genomic site. Disadvantages of this solution include (i) the absence of a mechanism to detect or remove cells that have incorporated additional copies of the expression vector due to random integration events, and (ii) the absence of a mechanism to remove selection marker cassettes that may have a negative impact on GOI expression.

[0012] In particular, for applications based on the integration of nucleic acid sequence libraries, there is a critical need to develop methods that can have higher integration efficiency, as this also requires a greater diversity of libraries that can be efficiently evaluated.

[0013] Therefore, identifying improved expression systems for recombinant protein production remains essential in this field. In particular, there is an urgent need for novel methods that can efficiently incorporate donor vectors, enable rapid and accurate selection of cells that incorporate insertions at the correct locations in the host cell genome, and simultaneously allow for the removal of cells that incorporate additional copies at random genomic locations, in order to enable improved means for optimizing the expression cassette of candidate therapeutic proteins.

[0014] Nucleic acid sequence optimization All proteins have a higher potential for expression, ultimately determined by their amino acid sequence, and different sequences can lead to significant differences in expression potential. Therefore, even slight changes in the amino acid sequence of a candidate therapeutic protein outside its target interaction surface can be critical to improving its expression level. When amino acid changes cannot be introduced due to the risk of clinical complications, promoters of different strengths may be required to circumvent overwhelming cellular mechanisms. In addition, many studies conducted over the last 10-20 years (see [14-19] for some) have shown that the expression level of a given protein (with a fixed amino acid sequence) can vary considerably based on the use of different sequence components in the vector (5'-UTR sequence, signal peptide sequence, synonymous coding nucleotide sequence, and 3'-UTR sequence), and that high-performance combinations are at least partially protein-dependent.

[0015] Traditionally, sequence components have been cloned from natural sources. However, there are good reasons why natural sequence elements are not considered optimal for the best expression of a single defined protein under bioprocess conditions. After all, they have evolved within the entire organism, with all the constraints that come with this. With the increasing diversification of therapeutic proteins being studied and the rapid advancements in synthetic biology (which enable the construction and evaluation of novel sequence variants that do not exist in nature and have bp precision), the sequence-based design space for protein expression is overwhelming.

[0016] Consequently, novel methods for identifying optimized sequence variants of nucleic acid sequences that encode or influence target proteins are also being continuously explored, and such methods play a crucial role in the potential for the overall expression of target proteins in recombinant cell systems.

[0017] Therefore, there remains significant untapped potential for improving the function of commercially available protein-producing or recombinant proteins by enabling a greater assessment of the diversity of sequence variants in the final production system, which includes both the vector components and the nucleic acid sequences encoding the target protein, before selecting the final sequence combination for production. [Overview of the project] [Means for solving the problem]

[0018] The aforementioned problems are resolved or at least mitigated by the provision of methods and means further presented herein.

[0019] This disclosure provides a novel solution for recombinant protein production utilizing site-directed integration (SDI) of a single-copy donor vector into a predefined genomic location in an isolated eukaryotic host cell. The SDI-based system of this disclosure is based on a unique and inventive combination of well-established nucleic acid components for efficient integration of the donor vector into a dedicated target site in the host cell. The method provides specific positive selection of host cells into which the donor vector has been integrated into a dedicated predefined genomic location. The method also provides negative selection, which involves detecting any cells in which an undesirable integration event has occurred at another location in the host cell genome and removing them by optional selection. This two-step selection method is unique and will be particularly useful in the field of recombinant protein production, as it enables efficient and unbiased evaluation of nucleic acid sequence variants that affect recombinant protein production or function.

[0020] As previously stated herein, a key element for enabling advanced applications such as improved cell line development, flexible cell line engineering, and simultaneous probing of gene construct libraries is increased control over recombinant gene integration into host cell lines and better control over their copy numbers. This is provided herein. Although the putative hotspot locations have been identified, the SDI system should be applicable to any eukaryotic cell system, including mammalian cells such as human cells; therefore, the method presented herein was first established using Chinese hamster ovary (CHO) cells.

[0021] More specifically, the present disclosure provides a novel solution for nucleic acid sequence optimization for use in recombinant protein production utilizing the aforementioned site-directed integration (SDI) system that enables integration of a single copy of a donor vector into a predefined genomic position of an isolated eukaryotic host cell. The sequence optimization method comprises generating a library of nucleic acid sequence variants of donor vector components, such as promoter, IRES or enhancer sequences, or nucleic acid sequences encoding a protein of interest, to identify optimized nucleic acid sequence variants therefrom, followed by targeted integration of said nucleic acid variants into a population of host cells.

[0022] Accordingly, in a first aspect, the present invention relates to a method for selecting an optimized sequence nucleic acid sequence from a plurality of nucleic acid sequence variants, wherein said optimized sequence nucleic acid sequence corresponds to a eukaryotic cell having a defined phenotype, and said method comprises: i) providing a population of eukaryotic cells, each cell comprising a. nucleic acid sequence I1 comprising a recognition site for a first DNA enzyme; b. nucleic acid sequence E1 comprising a recognition site for a second DNA enzyme; and c. a promoter nucleic acid sequence; said step comprising a predefined genomic locus comprising the above: ii) providing a plurality of donor vectors, each donor vector comprising a. nucleic acid sequence I2; b. nucleic acid sequence E2 comprising the recognition site for said second DNA enzyme; c. a nucleic acid sequence encoding a first selection marker; and d. a nucleic acid sequence region comprising a nucleic acid sequence variant said step of providing a plurality of donor vectors comprising the above: iii) contacting the plurality of donor vectors with the population of cells in the presence of a first DNA enzyme, wherein the presence of the first DNA enzyme enables recombination between the nucleic acid sequence I2 of the donor vector and the nucleic acid sequence I1 present at the predefined genomic position of the cells; iv) selecting and isolating cells having a donor vector integrated at a predefined genomic position by detecting expression of a first selection marker in the cells, wherein expression of the first selection marker is activated by a promoter nucleic acid sequence at the predefined genomic position; and v) selecting and isolating cells having a defined phenotype from the cells of step iv), thereby selecting and isolating a sequence-optimized nucleic acid sequence from said nucleic acid sequence variants, wherein said sequence-optimized sequence corresponds to said defined phenotype comprises the steps of:

[0023] In another aspect, there is provided herein a sequence-optimized nucleic acid sequence selected by the method disclosed herein.

[0024] In yet another aspect, use of a sequence-optimized nucleic acid sequence for producing a recombinant protein is provided.

[0025] In still another aspect, there is provided an isolated eukaryotic cell having a defined phenotype corresponding to a sequence-optimized nucleic acid sequence obtainable by the method disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] [Figure 1] Fig. 1 is a schematic diagram showing the general concept of a method for targeted integration of a plurality of donor vectors into predefined genomic positions of a plurality of eukaryotic host cells via the use of at least two DNA enzymes having orthogonal specificity. Following targeted integration of a plurality of donor vectors into predefined positions of a plurality of cells, the defined phenotype is isolated / enriched, which is then used. SOI = sequence of interest. [Figure 2-1] Fig. 2 is a schematic diagram showing the general concept of a method for targeted integration of a donor vector into a predefined genomic position of a host cell via the use of at least two DNA enzymes having orthogonal specificity. [Figure 2-2] It is a continuation of Fig. 2-1. [Figure 3] This figure shows a schematic diagram of the landing pad design (nucleic acid sequence located at a predefined genomic location in the host cell genome) and matching donor vectors for LP1P1 and HyClone LP2P2 cell lines. [Figure 4] This figure illustrates an example of a flow cytometry plot at 7 days post-transfection compared to an untransfected control (NC). Cell density in the plot, representing the enriched major population, is visualized by alternating black and white regions (20% of total cells in each region). The top column shows FACS data from untransfected control (NC) cultures of HyClone CHO cells. The middle column shows FACS data from a randomized integrated control (RI) based on HyClone CHO cells (lacking LP) transfected with donor vector B only (without PhiC31). The bottom column shows FACS data from HyClone CHO LP2P2 cell lines transfected with PhiC31 and donor vector B (SDI). The gates (B, D, F) in the middle plots of each column represent sets based on the untransfected control and report the percentage of cells activating the selection marker higher than the background. [Figure 5] This figure shows a schematic diagram of the changes in the landing pad that are predicted to occur via the landing pad cell line and donor vector used, as well as the activity of PhiC31(1) and Cre(2). [Figure 6] This figure shows flow cytometry plots of the SDI population 7 days after transfection with a Cre recombinase variant compared to a negative false transfection control. Cell density in the plots with enriched major populations is visualized by alternating black and white regions (20% of total cells in each region). The top panel shows plots of false transfection lacking the nucleic acid molecule encoding Cre recombinase, the middle panel shows plots of populations transfected with Cre recombinase expression plasmids, and the bottom panel shows plots of populations transfected with synthetic Cre recombinase mRNA. [Figure 7] This figure shows a schematic diagram of the changes expected to occur in the landing pad via the landing pad cell line and donor vector used, as well as the activity of PhiC31 recombinase (1) and Cre recombinase (2). [Figure 8] This figure shows flow cytometry plots from the process performed according to Figure 7. The density of cells in the plots containing the concentrated main population is visualized by alternating black and white regions (20% of the total cells in each region). The left plot in the upper panel shows the population after the second eGFP (green fluorescent protein) positive screening. The middle plot in the upper panel shows the population 7 days after Cre recombinase transfection. The right plot in the upper panel shows the population after eGFP negative screening performed by gate E after Cre recombinase transfection. The lower panel shows plots of the population 7 days after transfection of the screening cells from step 2 using DNA donor vector B. [Figure 9] This figure shows schematic diagrams of the landing pad cell lines and donor vectors used. GSx = glutamine synthase gene variant. [Figure 10] This figure shows flow cytometry plots of cell population generation using donor vector SDI. The density of cells in the plot with the enriched major population is visualized by alternating black and white regions (20% of total cells in each region). The upper panel shows plots of populations after G418 selection, RFP (red fluorescent protein) positive FACS sorting, and transfection with synthetic Cre recombinase mRNA. eGFP histograms are shown for both the RFP-negative subpopulation (corresponding to integration in the landing pad using Cre recombinase-mediated excision of TagRFP-T) and the RFP-positive subpopulation (corresponding to failure of Cre recombinase-mediated excision of TagRFP-T, which can be caused by off-target integration or cleavage-type integration in the landing pad). The lower panel shows plots of the final SDI pool generated using FACS sorting of RFP-negative / GFP-positive cells from the upper panel. [Figure 11-1] This figure shows the first selection marker of the donor vector linked to the gene encoding the second DNA enzyme via an IRES element. Both the first selection marker and the second DNA enzyme are activated upon integration at a predefined genomic location. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12-1] This figure shows a case where a predefined genomic region also includes the expression cassette of the first DNA enzyme, and when a donor vector is inserted at a predefined genomic position, the recognition site of the second DNA enzyme becomes adjacent to it, and is therefore positioned so that it can be removed in the presence of the second DNA enzyme. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1] This figure illustrates a targeted integration variant that uses gene editing enzymes to catalyze the integration of a donor vector into a predefined genomic location in the host cell genome. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14-1] This figure shows a targeted integration variant that catalyzes integration at a predefined genomic location in the host cell genome using recombinase-mediated cassette exchange (RMCE). [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15-1] This figure shows a targeted integration variant that uses a single recombinase recognition site pair to catalyze the integration of a donor vector at a predefined genomic location in the host cell. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 16-1] This diagram illustrates the use of a single recombinase recognition site pair to catalyze integration at a predefined genomic location, functionally fusing promoter P1, located at the predefined genomic location, to the 5' portion of a split intron. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 17a]This figure shows the transient expression of nine different constructs. The plots show the percentage of cells in the gate for good transient expression of eGFP and mTagBFP2. [Figure 17b] This figure shows the mean fluorescence intensity (MFI) for eGFP expression in logarithmic phase cultures of the construct, with B1, B3, and C1 removed. [Figure 17c] This figure shows the titer measurements of Fc fusion proteins using CEDEX after batch culture of constructs excluding B1, B3, and C1. [Figure 18] This figure shows the correlation between average single-cell fluorescence and bulk titer measurements of Fc-eGFP. [Figure 19] This figure shows schematic diagrams of the landing pad cell lines and donor vectors used. FC-eGFPx = FC-eGFP gene cassette variant. [Figure 20] This figure shows flow cytometry data of cell populations generated using four different DNA donor vectors. The density of cells in the plot with the enriched major population is visualized by alternating black and white regions (20% of the total cells in each region). [Figure 21] This figure shows schematic diagrams of the landing pad cell lines and donor vectors used. FC-eGFPx = a signal peptide containing FC-eGFP gene cassette variants with different 3'-UTRs. The TagBFP2 cassette contains a single peptide and is maintained consistently across all variants. [Figure 22] This figure compares the mean eGFP signal from flow cytometry data generated using cell samples from individual batch cultures of each type, calculated from ViaCore 8K+ titer measurements for Qp and log phase. Panel (a) shows a direct comparison of each vector variant to the value standardized to vector variant 524. Panel (b) shows a correlation graph between Qp and eGFP flow cytometry data. [Figure 23]This figure shows the design of a small library of control DNA donor vectors (pGE0506-pGE0508) and DNA donor vectors with 29 different codons encoding 18 different amino acids and a stop codon at position aa299 in the glutamine synthase gene. GSx = glutamine synthase gene variant. PGK = promoter. [Figure 24] This figure shows the growth of a single-copy integrated cell pool (generated by transfection with PhiC31 and donor vectors 506-509, followed by two selection steps) in glutamine-free medium in the absence of MSX (0 mm MSX) or using 10 μM MSX (10 mm MSX). [Figure 25] This figure shows the growth graph of single cells cloned from a pre-selected pool of 10 μM MSX SDI GS variants (generated using vectors 506, 507, and 509). Individual clones are classified as growing if their cell confluence reaches 30% or more (per well in a 96-well culture plate) 14 days after single-cell cloning. [Figure 26] This diagram illustrates single-cell signal generation through the fusion of a fluorescent protein with a target protein encoded by a single gene. FP1 = fluorescent protein 1, FP2 = fluorescent protein 2. [Figure 27] This diagram illustrates single-cell signal generation by fusing a fluorescent protein to a target protein encoded by one or more genes. AAV = adeno-associated virus, VP = viral protein. [Figure 28] This figure shows single-cell signal generation through the interaction between a target surface-presented protein and a fluorescently labeled target entity. F1 = fluorescent portion 1, F2 = fluorescent portion 2. [Modes for carrying out the invention]

[0027] This disclosure is described in more detail herewith in connection with the accompanying drawings and some non-limiting embodiments.

[0028] definition Details of this disclosure are described below. Any materials and methods similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred materials and methods are described. All words and terms used herein will be deemed to have the same meanings as those usually assigned to those skilled in the art unless otherwise evident from the context.

[0029] A composition "containing" one or more of the enumerated components may also contain other components that are not specifically enumerated.

[0030] The singular forms "a" and "an" are likely to be used to include plural forms as well.

[0031] "Expression" is used to mean the production of a protein from a gene, and in this specification includes the process of the "central dogma," namely the successful action of transcription, translation, and protein folding to reach the active state of the protein.

[0032] As defined herein, “expression vector” is a vector containing a nucleic acid sequence for achieving protein expression from the vector when present in a host cell. In this specification, an expression vector is used, for example, to introduce a specific gene of interest into a cell and then to direct the cellular mechanism for protein synthesis to produce the target protein encoded by the gene of interest. An expression vector may contain an “expression cassette,” which contains a nucleic acid sequence for promoting protein expression. In addition, the vector may contain other nucleic acid sequence elements or components.

[0033] As used herein, “donor vector” is a vector, preferably a DNA vector, comprising nucleic acid elements or components for promoting the integration of the vector into a predefined genomic location of an isolated eukaryotic host cell. The donor vector has nucleic acid sequences that facilitate a recombination event, comprising a nucleic acid sequence present at a predefined genomic location of the host cell, a target nucleic acid sequence optionally encoding a target protein, a recognition site for a second DNA enzyme, and a nucleic acid sequence encoding a first choice marker. Optionally, it may also contain an expression cassette for the second choice marker. “Donor vector” may also sometimes be referred to as “vector” in this specification. “Donor vector” may sometimes be in the form of an expression vector, such as when the donor vector includes an expression cassette encoding a second choice marker. More specifically, the donor vector described herein contains at least nucleic acid sequence I2 for recombination with I1 present at a predefined genomic location of the eukaryotic cell. In addition, it contains the target nucleic acid sequence, also referred to herein as the target gene (“GOI”) if the target nucleic acid encodes a target protein. It also includes nucleic acid sequence E2 containing a recognition site for a second DNA enzyme, which allows for the excision of a portion of the vector skeleton once stable integration of the donor vector occurs at a predefined genomic location in the host cell. It also includes a nucleic acid sequence encoding a first choice marker (SM1), whose expression is achieved only when the donor vector is integrated at the correct location at the predefined genomic location in the host cell. Finally, the donor vector optionally includes an expression cassette encoding a second choice marker (SM2). After the action of the second DNA enzyme, the second choice marker will be expressible and able to direct the cell only if a random integration event of the vector occurs and is used in the second round of selection of this method. The donor vector is preferably a DNA donor vector, but is not limited thereto. DNA donor vectors are sometimes abbreviated as "DDV".

[0034] An "expression cassette" is a nucleic acid component that forms part of an expression vector containing all the elements necessary for the initiation of transcription and translation of the target protein. The target gene encoding the target protein also forms part of the expression cassette. The expression cassette contains, for example, promoters and enhancer sequences essential for transcription initiation, as well as other sequences that promote transcription. Sometimes, the term "integrated cassette" is used herein to refer to a nucleic acid sequence derived from a donor vector that remains at a predefined genomic location after the action of a second DNA enzyme. An "integrated cassette" may include an "expression cassette".

[0035] In this specification, the “single” gene of interest refers to the nucleic acid component required to produce the protein of interest, and since the protein of interest may contain multiple polypeptide chains, it may also refer to multiple genes of interest present in the same expression cassette. An expression cassette containing multiple genes of interest may utilize individual promoters to achieve transcription of each individual gene, or two or more genes may be transcribed as a common mRNA with individual genes separated by IRES elements. This is consistent with the fact that whenever “a” is used in this specification, it may refer to multiple genes. An example of an expression cassette containing more than one gene of interest is when an antibody is to be expressed from a gene of interest, for example, light-chain and heavy-chain antibodies may exist as separate genes in the expression cassette.

[0036] An "intron" is a nucleic acid sequence that is transcribed and then removed by RNA splicing during the production of the final RNA product. Introns are non-coding regions of RNA transcripts, or DNA that codes for them, and are removed by pretranslational splicing.

[0037] In this specification, a promoter functionally fused to the 5' portion of a split intron means that transcription of the 5' portion of the split intron is brought about by the promoter. In this specification, the 5' portion of a split intron is defined to include a splice donor site sequence (e.g., GT). In this specification, the 3' portion of a split intron may be defined to include (i) a splice branch site sequence, (ii) a Py-rich sequence region, and (iii) a splice acceptor site sequence (e.g., AG).

[0038] Transcription involves the conversion of DNA to RNA by cellular mechanisms. A “transcriptional regulatory sequence” is a segment of nucleic acid sequence that has the ability to increase or decrease the final expression of a particular gene; that is, the sequence has the ability to control the transcription of the gene. Examples of transcriptional regulatory sequences include promoters and enhancers.

[0039] The untranslated region ("UTR") refers to one of two sections at each end of the coding sequence on the mRNA strand. The 5' end is called the 5'UTR, and the 3' end is called the 3'UTR.

[0040] As referred to herein, upstream open reading frames (uORFs) are open reading frames (ORFs) located within the 5' untranslated region (5'UTR) of an mRNA molecule. uORFs are commonly involved in the regulation of gene expression in eukaryotes. Translation of uORFs typically inhibits the downstream expression of primary ORFs (open reading frames), and therefore, when presented, leads to a reduction in protein expression. Approximately half of human genes contain these regions.

[0041] An intrasequence ribosome entry site ("IRES") is an RNA element that enables translation initiation in a cap-dependent manner. IRES elements are often referred to as distinct regions of an RNA molecule that can recruit eukaryotic ribosomes into mRNA. While the location of IRES elements is often in the 5' UTR region, it can occur at other locations in mRNA.

[0042] A plasmid is a small, circular extrachromosomal DNA molecule found in bacteria that can replicate independently within a cell. Plasmids are often used as vectors for molecular cloning, i.e., to deliver and introduce selected DNA into a host cell. Plasmids are constructed from specific and essential elements and may contain genes that are homogeneous or heterogeneous in the bacterial host cell. Plasmids always contain, for example, the replication origin of a bacterium, and most frequently, genes for specific antibiotic resistance.

[0043] As used herein, “target nucleic acid sequence” may be defined as a nucleic acid sequence that a person wishes to incorporate into a cell to affect the functionality of said cell. It may include a target gene ("GOI") that codes for a target protein.

[0044] As used herein, “recombinant” proteins refer to proteins produced from expression cassettes introduced into cells via expression vectors. Techniques for producing recombinant proteins are well known to those skilled in the art.

[0045] A "promoter" is a region of DNA that initiates gene transcription when RNA polymerase binds to it. Promoters are located near the transcription start site of a gene.

[0046] As used herein, “host cell” refers to a eukaryotic cell that is intended to be transformed by, or has been transformed by, a donor vector disclosed herein.

[0047] "Isolated cell," "isolated host cell," or "isolated eukaryotic host cell" refers to a cell that has been isolated from its natural environment, meaning it does not contain any additional components that may occur in nature and is no longer part of that natural environment.

[0048] Where used herein, cellular “phenotype” refers to the observable (physical) characteristics or traits of a cell. The term includes the morphology, physical form and / or structure of a cell. It may also include its developmental processes, its biochemical and / or physiological properties, its behavior, and / or the behavior of any products, such as the production of proteins or their measurable quantities.

[0049] In this specification, “predefined genomic location,” sometimes referred to as a “landing pad” (abbreviated as “LP”), or rather as a predefined genomic location containing a landing pad sequence, is intended to refer to a location, or nucleic acid location, characterized by a specific nucleic acid sequence in the host cell genome. A predefined genomic location may also be referred to herein as a “safe harbor site” and / or “recombination site.” At a predefined genomic location in the host cell, a recombination event occurs between nucleic acid sequences l1 and l2, facilitated by the presence of a first DNA enzyme, thereby initiating the expression of a first selection marker and demonstrating a successful integration event. Essentially, a predefined genomic location includes a nucleic acid sequence containing the recognition site of the first DNA enzyme, a nucleic acid sequence containing the recognition site of the second DNA enzyme, and a promoter nucleic acid sequence.

[0050] In this specification, when “targeted integration” is referred to, it is intended to mean the integration or introduction of one nucleic acid sequence element or component into another nucleic acid element or component, thereby facilitating such a recombination event between sequences, thereby generating a hybrid sequence from the original sequence. Such an integration event is triggered by the presence of an enzyme-recognized nucleic acid sequence in any one or more of the nucleic acid sequence elements or components that form the basis of the recombination.

[0051] An "enzyme recognition site" refers to a specific combination of nucleotides in a nucleic acid sequence. This combination is recognized by a specific enzyme that promotes its binding, and that enzyme then initiates action at the recognition site, such as a recombination event between the two sequences.

[0052] As used herein, the term "DNA enzyme" is defined as an enzyme that acts on DNA, such as by cutting DNA fragments or by cutting DNA and incorporating it into another DNA sequence. This term includes, but is not limited to, enzymes such as Crisps / Cas9, recombinases, integrases, and nucleases.

[0053] As used herein, the “first DNA enzyme” may be functionally defined as an enzyme that can be involved in the integration of a donor vector at a predefined genomic location in a host cell in the methods disclosed herein. The function of the first DNA enzyme is to introduce a nucleic acid sequence into a predefined genomic region, rather than to remove it. The first DNA enzyme may be one specific enzyme, or it may be a different enzyme when used in the methods disclosed herein. This is, for example, when the integration of the donor vector is sequential and thus repeated multiple times to introduce multiple copies / variants of the target / donor vector nucleic acid sequence into a predefined genomic location in the host cell, or when reversible integration of the target nucleic acid sequence is performed. Examples of “first DNA enzymes” for use in the context of the present method are given elsewhere herein.

[0054] As used herein, the “second DNA enzyme” can be functionally defined as an enzyme that may be involved in excising a nucleic acid sequence region from a predefined genomic location incorporating a donor vector in the method disclosed herein, wherein the nucleic acid sequence region is adjacent to a specific sequence recognized by the second DNA enzyme. When the second DNA enzyme recognizes the sequences, it will cleave the nucleic acid sequence components between these sequences. Examples of “second DNA enzymes” for use in the context of this method are given elsewhere herein.

[0055] "In the presence of the first DNA enzyme" and / or "in the presence of the second DNA enzyme" means that the first and / or second DNA enzymes are provided in any form described herein, for example, as proteins expressed from a donor vector, separate expression vectors, expression cassettes present in the cellular genome, synthetic mRNA, etc. "In the presence of" is intended to mean that the function of the first and / or second DNA enzymes is brought about by any suitable method disclosed herein.

[0056] As used herein, a “selection marker” is a marker (first selection marker) that can indicate, for example, in this context, that a specific event has occurred, that the integration of a donor vector has occurred at a predefined genomic location in the host cell. A selection marker is often a fluorescent protein expressed by the host cell once the donor vector has been integrated at the correct site in the host cell genome. The expression of a fluorescent protein can be detected, for example, by FACS (fluorescence-activated cell sorting). Other possible selection markers are described elsewhere in this specification.

[0057] The “first selection marker,” also abbreviated herein as “SM1,” can be defined as a silent, inactive, or promoter-less selection marker when present in a donor vector. The first selection marker contains a non-coding segment that fits with a promoter located at a predefined genomic site. When the donor vector is incorporated into the correct position at the predefined genomic site, the first selection marker can be expressed, as it now has a promoter for initiating transcription. Once the selection marker is expressed, a population of cells expressing the first selection marker can be selected as positive for stable integration of the donor vector at the predefined genomic site. The first selection marker may also be referred to herein as a “reporter.” Examples of suitable first selection markers are provided elsewhere herein.

[0058] The “second selection marker,” also abbreviated herein as “SM2,” is an optimal characteristic of the donor vector disclosed herein and can be defined as a non-silent active and / or functional selection marker when present in the donor vector. The selection marker is encoded as part of an expression cassette, i.e., the selection marker is transiently expressed upon cell entry and will subsequently promote stable expression in the genome, independently of the site of introduction. In most aspects of the methods presented herein, the second selection marker is a negative selection marker, meaning that cells expressing this marker are preferably not used for recombinant protein production, as these cells (and) incorporate the donor vector at locations other than the predefined genomic site.

[0059] Detailed explanation A method utilizing a specific site-directed integration (SDI) system for targeting and detectable integration of a donor vector to a predefined genomic location in an isolated eukaryotic cell is provided herein. In addition, the method allows for the identification of random integration events of the donor vector to other parts of the eukaryotic host cell genome other than the predefined location.

[0060] This combination provides a “double” selection of a population of cells in which the donor vector is positively integrated at a target site (a predefined genomic location) in the host cell genome, preferably in the absence of additional random integration of the donor vector at other locations in the host cell genome, thereby providing an optimized system for evaluating nucleic acid variants that contribute to subsequent recombinant protein expression or recombinant protein function (see Figure 2). The method in this embodiment uses a combined selection strategy based on positive (integration at a predefined genomic location) and subsequent negative (absence of random integration events) selection of a population of cells to significantly enrich cells that integrate a single copy of the donor vector-derived insertion cassette only at a predefined genomic location. This property is important for applications where a library of donor vectors containing nucleic acid variants is integrated into a population of host cells, as it ensures a “one-to-one” correlation between the cellular phenotype and a specific nucleic acid sequence variant and eliminates the biological noise arising from integration at fluctuating genomic locations.

[0061] The comprehensive solution is based on the integration of so-called "landing pad" (LP) sequences at predefined genomic locations in selected host cells for their ability to support high transcription and their long-term stability. The landing pads are designed in conjunction with matching donor vectors, enabling controlled integration at predefined sites and direct selection of cells in which only the desired integration occurs. In this specification, predefined genomic locations and landing pads / landing pad sequences may be used interchangeably.

[0062] However, more specifically, the SDI system is used herein to introduce a library of nucleic acid sequence variants of nucleic acid sequences into a population of host cells derived from a population of donor vectors by targeted incorporation (see Figure 1). The system has two types of DNA enzyme recognition sequences, (i) Integration of multiple donor vectors containing nucleic acid sequence variants at predefined genomic locations in multiple eukaryotic host cells, (ii) Selection of multiple cells to incorporate a single copy of a donor vector at a predefined genomic location using at least one or possibly two orthogonal selection steps, and (iii) Optional removal of undesirable sequences from the donor vector at predefined genomic locations, and (iv) A second selection step based on a defined phenotype of eukaryotic cells incorporating nucleic acid sequence variants at predefined genomic locations. To make this possible, two different DNA enzymes, such as a specific recombinase, are used in combination.

[0063] SDI methods, which form part of a complete nucleic acid sequence variant optimization method, can be performed in a variety of ways, all of which include the same common key properties.

[0064] A typical implementation of this SDI method is outlined in Figure 2. While methods based on the use of a single donor vector and a single isolated eukaryotic host cell are illustrated herein, the same concept applies to the use of multiple donor vectors (containing multiple nucleic acid sequence variants collectively) for targeted integration into a population of isolated eukaryotic host cells (so that many nucleic acid sequence variants are integrated in different cells).

[0065] The predefined genomic location of the isolated eukaryotic cell includes (i) a nucleic acid sequence I1 containing the recognition site of a first DNA enzyme, (ii) a nucleic acid sequence E1 containing the recognition site of a second DNA enzyme, and (iii) a promoter nucleic acid sequence P1 containing the start transcription site. I1, E2, and P1 are formed with either two symmetrical 5'-3' orientations O1=[I1, P1 with 3'-5' orientation, E1] or O2=[E1, P1 with 5'-3' orientation, I1]. The donor vector includes (i) a nucleic acid sequence I2 that promotes recombination using I1 in the presence of the first DNA enzyme, (ii) a first choice marker gene (SM1) lacking a promoter, (iii) the recognition site E2 of the second DNA enzyme, (iv) an integration cassette IC, and optionally (v) an active expression cassette of a second choice marker gene (SM2). SM1, SM2 (if present), E2, and IC are formed in one of two symmetrical clockwise orientations: O3 = [I2, IC, E2, SM2, SM1 with counterclockwise orientation] or O4 = [I2, SM1, SM2, E2, IC with clockwise orientation]. Nucleic acid sequence elements present at predefined genomic locations and in the donor vector are always formed in one of two matching orientations: (a) O1 / O3 or (b) O2 / O4.

[0066] The integration of a complete or partial donor vector into a predefined genomic location in the isolated eukaryotic cell is achieved by introducing the donor vector into the cell in the presence of a first DNA enzyme, the presence of which enables recombination between nucleic acid sequence I2 of the donor vector and nucleic acid sequence I1 present at the predefined genomic location in the cell.

[0067] Integration at a predefined genomic location is located at the SM1 gene, so that P1 can achieve transcription of the SM1 gene and therefore achieve expression of the SM1 gene product. Consequently, cells with a complete or partial donor vector integrated at a predefined genomic location can be selected and isolated by using SM1 expression as a positive selection criterion.

[0068] Optionally, undesirable sequences that could potentially negatively impact the intended functionality of isolated cells can be specifically removed from predefined genomic locations during a supplementation step that leaves only the integration cassette (IC) and the remaining sequences from I1, I2, E1, and E2.

[0069] Upon insertion of a complete or partial donor vector at a predefined genomic location, two nucleic acid sequences E1 and E2 become adjacent to the plasmid backbone sequence (i.e., the sequence for plasmid propagation in bacteria) and the expression cassettes for SM1 and SM2 (if present) (see Figure 2). In the presence of the second DNA enzyme, this sequence region where E1 and E2 are adjacent is excised from the predefined genomic location via the second DNA enzyme acting on E1 and E2. Cells from which the region where E1 and E2 are adjacent can be selected and isolated in a negative selection process based on the absence of SM1 expression (if SM2 is not present in the original donor vector) and / or the absence of SM2 expression (if SM2 is present in the original donor vector). In addition to achieving the removal of unwanted sequences, any cells that achieve SM1 activation (via a nonspecific mechanism) after integration outside a predefined genomic location will not have adjacent SM1 cells in E1 and E2, and therefore will not be selected in the negative selection step based on SM1 expression. Thus, this supplemental selection step always increases the specificity of isolating cells that have integrated a complete or partial donor vector at a predefined genomic location.

[0070] Using SM2 present in the donor vector, an improved selection process can be performed after the action of the second DNA enzyme. Since SM2 is provided as an active expression cassette, any copy of the donor vector integrated into an undesired genomic location will result in SM2 expression. However, importantly, since E1 is present only at a predefined genomic location, such integration events will not lead to an SM2 expression cassette adjacent to E1 and E2. Therefore, after the action of the second DNA enzyme, which leads to the excision of a sequence region adjacent to E1 and E2, cells that have integrated a single copy of the integration cassette (IC) at a predefined genomic location and only at that location can be selected and isolated in a negative selection process based on the absence of SM2 expression.

[0071] An embedded cassette (IC) typically contains an expression cassette for the target gene (GOI), but the application of the method is not limited to that.

[0072] Further examples of specific and general SDI methods are illustrated using a single donor vector and a single isolated eukaryotic cell, showing only one of two possible symmetrical orientations of key sequence elements present in the donor vector and located at a predefined genomic location.

[0073] One specific implementation of the design concept is outlined in Figure 5, focusing on the landing pad (LP1P1) and DNA donor vector. The results of experiments conducted based on this implementation are also described and discussed in the experimental section of Example 2. This implementation is merely an example of one method of performing the SDI portion of the present invention, but is not intended to be limited thereto. For clarity, for applications to nucleic acid sequence variant optimization, many donor vectors are specifically incorporated into a population of isolated eukaryotic host cells.

[0074] Therefore, in one execution as described in Figure 5, the eukaryotic host cell line contains, at a predefined genomic location, the first recombinase recognition sequence (attP1) for the recombinase PhiC31 recombinase, a 3' to 5' oriented promoter, and the second recombinase recognition sequence (loxP) for the recombinase Cre recombinase.

[0075] PhiC31 recombinase is a DNA recombinase derived from the Streptomyces phage φC31. This enzyme can mediate recombination between two nucleic acid sequences, attB and attP. Cre recombinase is also a site-specific recombinase used in this system to subsequently excise the selection system and the plasmid bacterial skeleton. Thus, once the initial selection has been made, Cre recombinase can be described as "clearing" the vector skeleton from unhelpful sequences. Both PhiC31 recombinase and Cre recombinase are well-known enzymes used in site-specific recombination ([5,6]).

[0076] The matching DNA donor vector comprises a first choice marker lacking a promoter encoded in a counterclockwise orientation (here exemplified by RFP, a red fluorescent protein), a matching PhiC31 recombinase recognition sequence (attB1), an expression cassette containing a nucleic acid sequence encoding the target protein, a supplemental recombinase recognition sequence for Cre recombinase (loxP), a complete functional expression cassette for the second choice marker (optionally, here exemplified by FC-eGFP), and a plasmid backbone (including sequences for bacterial transmission, etc.).

[0077] Simultaneous gene transfer of a DNA donor vector and the vector into eukaryotic host cells for PhiC31 expression, including a predefined genomic location of the landing pad (LP) sequence, will lead to the integration of the donor vector at the LP via PhiC31-mediated recombination of attP1 and attB2 in a fraction of the transfected cells. During integration at the predefined genomic location, a promoter-free selection marker will be positioned to be activated by the promoter located at the predefined genomic location. The activity of the first selection marker can then be used to select cells that have undergone integration at the LP (using FACS in the case of RFP). Appropriate selection should generate a pool of cells in which the majority of cells have a single copy integrated at the LP. However, the cell fraction is expected to have additional copies integrated via off-target integration mechanisms such as DNA repair-mediated random integration at genomic pseudo-attP sequences and PhiC31-mediated integration. A second recombinase-mediated step was designed to address these phenomena and simultaneously enable the removal (i.e., "cleanup") of the selection marker cassette and plasmid backbone at predefined genomic locations.

[0078] Because a predefined genomic location contains a loxP sequence, and the DNA donor vector also contains a strategically placed loxP sequence, an integration event at the predefined genomic location will involve both selection markers (as well as other unwanted sequence elements such as the plasmid backbone) with two loxP sequences adjacent to each other. In contrast, most off-target events should not lead to selection markers adjacent to loxP (some random integration events of the ligated donor vector may lead to an adjacent second selection marker gene, but this should be very rare). By using a second transfection of a vector encoding Cre recombinase, the region adjacent to the loxP sequence can be excised from the genome of the corresponding cell. Cells having a single copy integrated at the predefined genomic location (lacking off-target integration) and having unwanted sequence elements to be removed can be selected via the absence of selection marker activity (absence of eGFP activity using FACS). This is also called selection by negative selection.

[0079] Non-limiting examples of the first DNA enzyme for use in the methods defined herein are DNA recombinases, such as PhiC31 or Bxb1 recombinase, as described elsewhere herein. When used as the first DNA enzyme, a characteristic feature of the recombinase is that it introduces a predefined genomic region rather than removing a nucleic acid sequence region.

[0080] Non-limiting examples of secondary DNA enzymes for use in the methods defined herein are DNA recombinases, such as PhiC31 recombinase, Bxb1 recombinase, Cre recombinase, and Dre recombinase, as described elsewhere herein. When used as a secondary DNA enzyme, a characteristic feature of a recombinase is that it removes a predefined genomic region rather than introducing a nucleic acid sequence region.

[0081] The nucleic acid sequence I1 containing the recognition site of the first DNA enzyme may be the attP or attB site of a PhiC31 or Bxb1 recombinase located at the aforementioned predefined genomic location, or may be otherwise exemplified herein depending on the first DNA enzyme being used in this context. For example, it may also be the loxP site of a Cre recombinase, or may be otherwise exemplified herein.

[0082] Nucleic acid sequence I2 may be the attB or attP site (recognition site) of the PhiC31 or Bxb1 recombinase present in the donor vector, or, as otherwise exemplified herein, depending on which first DNA enzyme is used in this context. For example, it may also be the loxP site of Cre recombinase, or, as otherwise exemplified herein.

[0083] Nucleic acid sequence E1 may be the loxP site of Cre recombinase or the roxP site of Dre recombinase. It may also be the attP or attB site of PhiC31 or Bxb1 recombinase, or may be otherwise exemplified herein.

[0084] Nucleic acid sequence E2 may be the loxP site of Cre recombinase or the roxP site of Dre recombinase. It may also be the attP or attB site of PhiC31 or Bxb1 recombinase, or otherwise may be exemplified herein.

[0085] If the first DNA enzyme is PhiC31 recombinase, then the second DNA enzyme is not PhiC31 recombinase. The same applies to any other first and second DNA enzymes; that is, the first and second DNA enzymes are never identical in the same SDI system.

[0086] In this specification, the first selection marker (SM1) of the donor vector may be ligated to a gene encoding the second DNA enzyme via an IRES element or the amino acid sequence of SM1, such that both the first selection marker and the second DNA enzyme are activated upon integration at a predefined genomic location, and the second DNA enzyme is fused by a self-cleaving peptide. This is illustrated in Figure 11. This ensures the presence of the second DNA enzyme, provided that the donor vector is integrated at a predefined genomic location and further introduction of the nucleic acid vector does not need to be carried out using the steps of the method. SM1 expression can be advanced until the intracellular concentration of the second DNA enzyme reaches a level high enough to promote the nuclear localization and excision of the adjacent sequence regions of E1 and E2. With a positive selection step at the appropriate timing, cells undergoing integration at the predefined genomic location will contain levels of SM1 that enable positive selection.

[0087] In this specification, the predefined genomic location may also include an expression cassette of the first DNA enzyme, such that, upon insertion of the donor vector at the predefined genomic location, the recognition site of the second DNA enzyme is adjacent to it and removed from the predefined genomic region via the action of the second DNA enzyme. This is illustrated in Figure 12. This should further simplify the method and improve the potential for high insertion efficiency. Since the expression cassette is removed during a later step of the method, there is no cell source consumed in the expression of the first DNA enzyme in the final isolated cells, and any negative consequences of the long-term presence of the first DNA enzyme are avoided.

[0088] Therefore, the first DNA enzyme may be brought about by expression from a predefined genomic location or by introduction into a cell in any form that results in the transient presence of the first DNA enzyme in the cell. This includes the introduction of the isolated protein itself, the introduction of a separate expression plasmid containing an expression cassette for the first DNA enzyme, the presence of an active expression cassette for the first DNA enzyme in the donor vector, or the introduction of synthetic mRNA encoding the first DNA enzyme.

[0089] The second DNA enzyme may be provided as an isolated protein itself, which may be expressed from a separate expression vector or expression cassette on a plasmid, or from synthetic mRNA encoding the second DNA enzyme. It may also be expressed from a donor vector once integrated into a predefined genomic location, as already described.

[0090] As previously stated herein, all aspects of this disclosure enable mobility in the selection of selection markers without making any changes to predefined genomic locations. SM1 may be selected from the group of (i) antibiotic resistance genes, (ii) metabolic enzyme genes such as GS or DHFR, (iii) fluorescent protein genes, or (iv) cell surface markers such as CD4 or CD10. SM2 may be selected from the group of (i) toxic product-producing enzymes such as TK, (ii) fluorescent protein genes, or (iii) cell surface markers such as CD4 or CD10.

[0091] Preferably, both selection markers are selected from the group of (i) fluorescent protein genes or (ii) cell surface markers, enabling a rapid selection process via methods such as FACS or MACS.

[0092] If the selection marker is a fluorescent protein, the expression of the first or second selection marker can be detected, for example, by using FACS. If the selection marker is an antibiotic resistance gene, integration can be detected by culturing cells in the presence of the corresponding antibiotic. If the cells survive in a medium to which the antibiotic has been added, the donor vector has been successfully integrated.

[0093] As described herein, recombinases are useful for excising nucleic acid sequences adjacent to appropriate nucleic acid regions (E1 and E2) in the host cell genome. Since some portions of nucleic acid sequences introduced at predefined genomic locations become superfluous once integration and selection have occurred, this is simply a "cleanup" process in the host cell genome. Their presence can also consume cellular energy. Excision of nucleic acid sequences means that a second DNA enzyme has the ability to cut and remove nucleic acid sequence portions from the host cell genome by binding to nucleotides, i.e., specific combinations of E1 and E2. The presence of nucleic acid sequences E1 and E2 at predefined genomic locations is, in principle, evidence that stable integration of the donor vector is occurring there.

[0094] By integration at a predefined genomic location, the expression cassette encoding the second choice marker (if any) is positioned in the donor vector such that E1 and E2 are adjacent to it. However, if the donor vector is integrated outside the predefined genomic location, E1 and E2 will not be adjacent to the expression cassette encoding the second choice marker.

[0095] Therefore, if the expression of a second selection marker (SM2) among cells in a cell population can be detected, for example, by FACS after the action of a second DNA enzyme, this means that an unwanted integration event of the donor vector is occurring at a different location in the cell. By removing such cells, selection (by negative selection) is possible for cells in which donor vector integration occurs only at a predefined genomic location.

[0096] In Figure 9 and Example 4, the generation of an SDI cell pool is described using two consecutive selection steps, namely, a step of removing nucleic acid sequences that no longer serve a purpose in the cell, in addition to a second DNA enzyme insertion step. In this example, an antibiotic resistance gene was used as the first selection marker (SM1). The second round of selection was performed using Cre recombinase to excise the nucleic acid sequences adjacent to the loxP nucleic acid region at each terminal. The presence of random insertion events was detected by a double-positive signal GFP / RFP (green / red fluorescent protein) using FACS. Cells were sorted based on the positive / negative GFP / RFP signal. This additional step results in the removal of cells that may incorporate one donor vector at a predetermined genomic location, but may randomly incorporate a second or further donor vector at a random non-target location in the host cell genome.

[0097] As previously stated herein, the first DNA enzyme may be a recombinase. The first DNA enzyme may be a mixture of different DNA enzymes, such as recombinases, as long as none of the DNA enzymes in the first DNA enzyme are the same as those in the second DNA enzyme.

[0098] In this specification, there may be more recognition sites, such as two or more recognition sites, than one of the first DNA enzymes present in the donor vector. This also means that there may also be more recognition sites, such as two or more recognition sites, than one of the first DNA enzymes present at predefined genomic locations. An example of such a system utilizing a recombinase is shown in Figure 14. Figure 14 shows a recombinase-mediated cassette exchange (RMCE) for catalyzing integration at a predefined genomic location. Modified variants thereof, as shown in Figures 2, 11-12 and 16, are also included in this disclosure.

[0099] Therefore, in the example in Figure 14, the predefined genomic locations include, in the order of 5' to 3' sequences, (i) the first recognition site of the first recombinase enzyme (I1a); (ii) the second recognition site of the first recombinase enzyme (I1b); (iii) promoter P1 having a 3' to -5' orientation; and (v) the recognition site of the second recombinase enzyme.

[0100] In this example, the donor vector includes, in 5'-3' sequence order: (i) the third recognition site of the first recombinase enzyme (I2a), (ii) an embedded cassette (IC), here exemplified by an expression cassette for the gene of interest (GOI), (iii) the recognition site E2 of the second recombinase enzyme, (iv) an expression cassette for the second choice marker (SM2), (v) the gene for the first choice marker (SM1) encoded in a 3'-5' direction, and (vi) the fourth recognition site of the first recombinase enzyme (I2b).

[0101] The introduction of the donor vector and the first recombinase into a population of cells results in (a) integration of the integration cassette in the donor vector, i.e., the sequence regions adjacent to the third and fourth recombinase recognition sites at predefined genomic locations for cell fractionation (see Figure 14b, panel (ii)) and (b) off-target genomic integration of the donor vector for cell fractionation (outside the predefined genomic locations) (see Figure 14b, panel (iii)).

[0102] Integration at a predefined genomic location results in the formation of an active expression cassette for SM1 (see Figure 14b, panel (ii)). Furthermore, after integration at the predefined genomic location, the two recognition sites of the second recombinase enzyme are adjacent to both SM1 and SM2.

[0103] Off-target event integration (see Figure 14b, panel (iii)) typically does not lead to SM1 activation but integrates an active SM2 whose two recognition sites on the second recombinase enzyme are not adjacent.

[0104] Cells undergoing integration at a predefined genomic location differ from cells that do not undergo integration events (see Figure 14b, panel (i)) and cells that undergo only off-target integration events via SM1 activity. Therefore, SM1 activity can be used to select cells undergoing integration at the LP.

[0105] In addition to integration at a predefined genomic location, the recombinase activity of the second recombinase is introduced in cells selected for SM1 activity to remove cells undergoing off-target integration events. For integration at LP, this results in the excision of both SM1 and SM2, and therefore their corresponding activities. For off-target integration events, this reaction cannot occur, and SM2 activity remains. As a result, cells undergoing only the desired targeted integration event at a predefined genomic location can be selected from cells undergoing multiple integration events via the absence of SM2 activity.

[0106] The predefined genomic locations for the final selected cells (see Figure 14c) do not include the SM2 expression cassette, the activated SM1 expression cassette, or any remaining sequences derived from the donor vector, except for the sequences generated via E1 and E2(E) recombination.

[0107] The first recombinase enzyme can be selected from the group consisting of (i) serine recombinase or (ii) tyrosine recombinase.

[0108] The first to fourth recombinase recognition sites are, (a) Using one matching recognition site of serine recombinase such as PhiC31 [I1a=I1b=attP or attB and I2a=I2b=attB or attP] or Bxb1 [I1a=I1b=Bxb1 attP or Bxb1 attB and I2a=I2b=Bxb1 attB or Bxb1 attP], I1a=I1b and I2a=I2b, (b) Selecting two different matching recognition site pairs using mutated recognition pairs of serine recombinase such as PhiC31 (I1a and I1b = different attP variants or attB variants; I2a and I2b = different attB variants or attP variants), and (c) For tyrosine recombinase, I1a=I2a and I1b=I2b are existing mutated recognition site variant pairs such as Cre (LoxP1 and LoxP2 selected from available mutated loxP pairs), Dre (rox1 and rox2 selected from available mutated rox pairs), or FLP (FRT1 and FRT2 ​​selected from available mutated FRT pairs). This can be selected.

[0109] Unlike the first recombinase enzyme, the second recombinase enzyme can be selected from the group consisting of (i) serine recombinase or (ii) tyrosine recombinase.

[0110] The recognition sites E1 and E2 of the second recombinase enzyme may be identical in sequence, as exemplified by (i) E1=E2=loxP or a mutated variant thereof for use with Cre recombinase, (ii) E1=E2=rox or a mutated variant thereof for use with Dre recombinase, or (iii) E1=E2=FRT or a mutated variant thereof for use with FLP (flippase) recombinase.

[0111] The recognition sites E1 and E2 of the second recombinase enzyme may have different sequences, as exemplified by (i) E1=attP and E2=attB or mutated variants thereof for use with PhiC31 recombinase, or (ii) E1=Bxb1 attP and E2=Bxb1 attB or mutated variants thereof for use with Bxb1 recombinase.

[0112] Further recognition sites of the first DNA enzyme may be referred to herein as variants of I1, namely I1a and I1b, and variants of I2, namely I2a and I2b, etc.

[0113] therefore, (a) I1 comprises two recombinase recognition site variants I1a and I1b; and (b) I2 comprises two recombinase recognition site variants I2a and I2b; and (c) I1a has the ability to recombine using I2a, and I1b has the ability to recombine using I2b in the presence of the first DNA enzyme. A method is also provided herein.

[0114] Sometimes, I1a is identical to I2a, and I1b is identical to I2b. I1a, I1b, I2a, and I2b may each be selected from loxP, rox, or FRT or their variants, and the first DNA enzyme may each be selected from the group consisting of Cre recombinase, Dre recombinase, and FLP recombinase[5].

[0115] In this specification, (a) I1 comprises a single recombinase recognition site; and (b) I2 comprises a single recombinase recognition site; and (c) I1 and I2 have the ability to recombine in the presence of the first DNA enzyme. Another method will be provided.

[0116] The recombinase recognition site composed of I1 may also have a different sequence from the recombinase recognition site composed of I2. The recombinase recognition site provided herein may be selected from attB, attP, Bxb1 attP, Bxb1 attB, or variants thereof. The recombinase may be PhiC31 or Bxb1 recombinase or a variant thereof.

[0117] Any variant or mutant of a recognition site / DNA enzyme would be a functionally equivalent variant or mutant. Those skilled in the art would construct and generate such functionally equivalent variants or mutants.

[0118] An example of using a single recombinase recognition site pair to catalyze integration at a predefined genomic location is shown in Figure 15. The modified variants described in Figures 2, 11-12, and 16 are also included in this disclosure.

[0119] In this example, the predefined genomic locations include, in 5'-3' sequence order, (i) the first recognition site of the first recombinase enzyme (I1); (ii) promoter P1 with a 3'-5' orientation; and (iii) the recognition site of the second recombinase enzyme.

[0120] In this example, the donor vector includes, in 5'-3' sequence order: (i) the second recognition site of the first recombinase enzyme (I2); (ii) an integration cassette (IC), here exemplified by an expression cassette for the gene of interest (GOI); (iii) the second recognition site E2 of the second recombinase enzyme; (iv) an expression cassette for the second choice marker (SM2); and (v) the gene for the first choice marker (SM1), encoded in a 3'-5' direction.

[0121] The introduction of the donor vector and the first recombinase into a population of LP cells results in (a) integration of the donor vector in the LP for LP cell fractionation (see Figure 15b, panel (ii)) and (b) off-target genomic integration of the donor vector for cell fractionation (outside a predefined genomic location) (see Figure 15b, panel (iii)).

[0122] Integration at a predefined genomic location results in the formation of an active expression cassette for SM1 (see Figure 15b, panel (ii)). Furthermore, after integration at the predefined genomic location, the two recognition sites E1 and E2 of the second recombinase enzyme are adjacent to both SM1 and SM2.

[0123] Off-target event integration (see Figure 15b, panel (iii)) typically does not lead to SM1 activation but integrates an active SM2 that is not adjacent to the recognition site of the second recombinase enzyme.

[0124] Cells undergoing integration at a predefined genomic location are distinct from cells that do not undergo integration events (see Figure 15b, panel (i)) and cells that undergo only off-target integration events via SM1 activity. Therefore, SM1 activity can be used to select cells undergoing integration at a predefined genomic location.

[0125] In addition to integration at a predefined genomic location, the recombinase activity of the second recombinase is introduced in cells selected for SM1 activity to remove cells undergoing off-target integration events. For integration at a predefined genomic location, this results in the excision of both SM1 and SM2, and therefore their corresponding activities. Due to off-target integration phenomena, this reaction cannot occur, and SM2 activity remains. As a result, cells undergoing only the desired target integration events in LP can be selected from LP cells undergoing multiple integration events via the absence of SM2 activity.

[0126] The predefined genomic locations for the final selected cells (see Figure 15c) do not include the SM2 expression cassette, the activated SM1 expression cassette, or any remaining sequences derived from the donor vector, except for the sequences generated via recombination of I1 and I2 (I12) and E1 and E2 (E).

[0127] The first recombinase enzyme can be selected from the group of serine recombinases such as PhiC31 and Bxb1. The first and second recombinase recognition sites (I1 and I2) can be selected using recognition sites that match the selected recombinase according to (a) I1=attP variant and I2=attB variant or (b) I1=attB variant and I2=attP variant.

[0128] Unlike the first recombinase enzyme, the second recombinase enzyme can be selected from the group consisting of (i) serine recombinase or (ii) tyrosine recombinase.

[0129] The recognition sites E1 and E2 of the second recombinase enzyme may be identical in sequence, as exemplified by (i) E1=E2=loxP or a mutated variant thereof for use with Cre recombinase, (ii) E1=E2=rox or a mutated variant thereof for use with Dre recombinase, or (iii) E1=E2=FRT or a mutated variant thereof for use with FLP recombinase.

[0130] The recognition sites E1 and E2 of the second recombinase enzyme may have different sequences, as exemplified by (i) E1=attP and E2=attB or their mutated variants for use with PhiC31 recombinase, or (ii) E1=Bxb1 attP and E2=Bxb1 attB.

[0131] The method described in Figure 16 is also provided, which exemplifies a single recombinase recognition site pair for catalyzing integration at a predefined genomic location, where promoter P1, located at the predefined genomic location, is functionally fused to the 5' portion of the split intron. The method described previously, but modified by Figure 16 (i.e., using a split intron design), is also encompassed by this disclosure.

[0132] In this example, the predefined genomic location further includes the 5' portion of an intron having a 3'-5' orientation and a functional sequence region F1 having a 3'-5' orientation between the first recognition site of the first recombinase enzyme and the promoter P1 having a 3'-5' orientation.

[0133] In this example, the donor vector further includes a sequence region located between the first selection marker SM1 having a 3'-5' orientation and the second recognition site of the first recombinase enzyme. The sequence region includes a 3'-5' oriented intron 3' portion, further including (a) a functional sequence region F3 having a 3'-5' orientation and (b) a functional sequence region F2 downstream of a splice acceptor site sequence, in the order of a 5'-3' sequence.

[0134] Upon integration at a predefined genomic location (see Figure 16b, top panel), a complete expression cassette containing a functional intron is formed for the first selection marker SM1. Thus, SM1 expression is activated.

[0135] After off-target integration (see Figure 16b, lower panel), a truncated version of the SM1 expression cassette is integrated.

[0136] By chance, transcription of a cleaved SM1 cassette may occur. This may result from (a) promoter rescue, in which the donor vector is incorporated in such a way that the cleaved SM1 cassette is positioned in-frame with the native promoter present in the cell genome, or (b) cleavage and concatemerization of the donor vector, in which the promoter present in the donor vector is reoriented in-frame with the cleaved SM1 cassette, followed by the incorporation of the resulting concatemer.

[0137] Such random occurrences can reduce the specificity of SM1, which is based on the selection of cells that incorporate the donor vector at a predefined genomic location. Improved specificity can be achieved by using specific combinations of functional sequence regions F1-F3 (see Figure 16b).

[0138] In the initial design of F1-F3, (a) SM1 (when present in the donor vector) lacks the ATG start codon and is directly fused to the 3' intron, and (b) F1 consists of a start transcription site (TSS), a first 5'-UTR region, a Kozak / translation start site, and an ATG start codon, all with 3'-5' orientation (from 3' to 5'). After off-target integration, this means that any integrated SM1 gene lacks the start codon and therefore does not produce functional SM1 protein expression. However, upon integration at a predefined genomic location, a functional expression cassette is formed. During intron splicing, the ATG start codon is directly fused to SM1, leading to proper expression of the SM1 protein.

[0139] In the second design of F1-F3, (a) SM1 contains the ATG start codon, (b) F3 consists of a second 5'-UTR region and a Kozak / translation start site, all having 3'-5' orientation (from 3'-5'), (c) F2 contains at least one short upstream open reading frame (uORF) with 3'-5 orientation, and (d) F1 consists of an initiation transcription site (TSS) and a first 5'-UTR region. After off-target integration, the cleaved SM1 cassette will typically hold one or more uORFs. These uORFs will reduce initiation at the intended SM1 start codon, thereby improving the distinction between off-target integration-based SM1 activation and SM1 activation based on integration at a predefined genomic location. Preferably, a series of multiple uORFs are used and placed at the shortest distance from the SM1 start codon (just downstream of the intron splice branch site).

[0140] Since the optimal 5'-UTR sequence can be used for SM1, the use of split intron design also improves the expression of activated SM1. In designs lacking split introns, the sequences generated via I1 and I2 recombination (see Figure 15) will be included by the SM1 5'-UTR. This leads to a 5'-UTR extended with a potentially suboptimal sequence composition, which can reduce the possible expression levels of SM1 (affecting the specificity in the positive selection process based on SM1). Through the use of split introns described herein, the I1 / I2 recombination product is incorporated into the fully formed intron upon integration at a predefined genomic location (see Figure 16). During cell maturation of SM1 mRNA, the intron is spliced ​​out, and the corresponding SM1 5'-UTR is fully defined by F1 and F2. Thus, the SM1 5'-UTR can be designed with a complete control to optimize SM1 expression for the intended purpose. Variations in the F2-F3 design provide further flexibility to the SM1 expression level during integration in the LP. Extending the length of the 5'-UTR region in F3 reduces SM1 expression, while adding a transcriptional enhancer element in F2 can increase SM1 expression to a higher level than that achieved with the optimal 5'-UTR alone.

[0141] Finally, the use of split intron designs can improve the recombination efficiency between I1 and I2 at predefined genomic locations, as shown in the experimental section of Example 1. One possible explanation for the observed improved integration efficiency is that the 5' portion of the split intron at the predefined genomic location acts as a critical spacer that can avoid / reduce steric interference between the RNA polymerase initiation complex and the copy of the first DNA enzyme (e.g., PhiC31) binding around the start transcription site, and performs this function by binding and manipulating I1. To increase integration efficiency, the aforementioned 5' portion of the split intron can be designed to have a length of at least 50 bp, at least 100 bp, or at least 300 bp.

[0142] Figure 13 illustrates a method for catalyzing the integration of a donor vector at a predefined genomic location using a gene editing enzyme (the first DNA enzyme is a gene editing enzyme). Modifications thereof, as shown in Figures 2 and 11-12, are also included in this disclosure.

[0143] In this example, the predefined genomic locations include, in 5'-3' sequence order: (i) a left homologous arm (LHA), (ii) a recognition / cleavage site (CS) for a gene editing enzyme, (iii) a right homologous arm (RHA) that also functions as the 5' portion of an intron with 3'-5' orientation (i.e., having a splice donor site at the end closer to the promoter), (iv) a promoter P1 with 3'-5' orientation, and (v) a recognition site E1 for a second DNA enzyme.

[0144] In this example, the donor vector includes, in 5'-3' sequence order: (i) the left homologous arm (LHA), (ii) an embedded cassette (IC), exemplified by an expression cassette for the gene of interest (GOI), (iii) the recognition site E2 for the second DNA enzyme, (iv) an expression cassette for the second choice marker (SM2), (v) the gene for the first choice marker (SM1) encoded in a 3'-5' orientation, (vi) a 3'- portion of an intron having a 3'-5' orientation (i.e., having a splice branch site and a splice acceptor site at the end closest to SM1), and (vii) the right homologous arm (RHA), which also functions as a 5' portion of an intron having a 3'-5' orientation.

[0145] The introduction of gene editing enzymes with cleavage specificity for donor vectors and CS into a population of eukaryotic cells results in (a) double-strand breaks at predefined genomic locations in the CS for the eukaryotic cell fraction, (b) integration of LHA and RHA into the donor vector region adjacent to it via homology-directed DNA repair for the eukaryotic cell fraction with double-strand breaks at the CS, and (c) off-target genomic integration of the donor vector (outside the predefined genomic region) for the LP cell fraction.

[0146] Integration at a predefined genomic location results in the formation of an active expression cassette for SM1 (see Figure 13b, panel (ii)). The integration further generates a complete functional intron between promoter P1 and SM1, so that the mature mRNA of SM1 does not contain RHA. Furthermore, after integration at LP, the two recognition sites of the second DNA enzyme are adjacent to both SM1 and SM2.

[0147] Off-target integration (see Figure 13b, panel (iii)) typically does not lead to SM1 activation, but rather integrates an active SM2 whose two recognition sites on the second DNA enzyme are not adjacent.

[0148] Eukaryotic cells that undergo integration at a predefined genomic location differ from cells that do not exhibit integration (see Figure 13b, panel (i)) and cells that only undergo off-target integration events via SM1 activity. Therefore, SM1 activity can be used to select cells that undergo integration at a predefined genomic location.

[0149] In addition to integration at predefined genomic locations, a recombinase activity (a second DNA enzyme) capable of recombining E1 and E2 is introduced into cells selected for SM1 activity to remove cells undergoing off-target integration events. Due to integration at LP, this results in the excision of both SM1 and SM2, and therefore their corresponding activities. Due to off-target integration phenomena, this reaction cannot occur, and SM2 activity remains. As a result, cells undergoing only the desired targeted integration events at predefined genomic locations can be selected from cells undergoing multiple integration events via the absence of SM2 activity.

[0150] The predefined genomic locations for the final selected cells (see Figure 13c) do not include the SM2 expression cassette, the activated SM1 expression cassette, or any remaining sequences derived from the donor vector, except for the sequences generated via E1 and E2(E) recombination.

[0151] Gene editing enzymes may be selected from, but are not limited to, (i) homing endonucleases such as Zn finger nucleases (ZFNs) and meganucleases; (iii) TALENs; or (iv) DNA or RNA guide nuclei such as CRISPR / Cas9.

[0152] The second DNA enzyme has recombinase activity and may be selected from the group consisting of (i) serine recombinase or (ii) tyrosine recombinase.

[0153] E1 and E2 may be identical in sequence, as exemplified by (i) E1=E2=loxP or a mutated variant thereof for use with Cre recombinase, (ii) E1=E2=rox or a mutated variant thereof for use with Dre recombinase, or (iii) E1=E2=FRT or a mutated variant thereof for use with FLP recombinase.

[0154] E1 and E2 may have different sequences, as exemplified by (i) E1=attP and E2=attB for use with PhiC31 recombinase, or (ii) E1=Bxb1 attP and E2=Bxb1 attB.

[0155] Accordingly, a method is provided herein in which the first DNA enzyme is a gene editing enzyme such as a gene editing nuclease. This provides a method wherein (a) I1 comprises a cleavage site of the gene editing nuclease and two sequence regions LHA1 and RHA1; (b) I2 comprises two sequence regions LHA2 and RHA2 homologous to LHA1 and LHA2; and (c) I1 and I2 are capable of recombination in the presence of the first DNA enzyme.

[0156] As already stated, the gene editing enzyme is selected from the group consisting of (i) Zn finger nucleases (ZFN); (ii) homing endonucleases such as meganucleases; (iii) TALENS and (iv) DNA or RNA guide nucleases such as CRISPR / Cas9, but is not limited thereto.

[0157] Nucleic acid sequences E1 and E2 may be the same recombinase recognition site, such as loxP, rox, or FRT, or a variant thereof, provided that E1 and E2 are different from I1 and I2.

[0158] The second DNA enzyme may be selected from the group consisting of Cre recombinase, Dre recombinase, and FLP recombinase, provided that the first DNA enzyme is neither Cre recombinase, Dre recombinase, nor FLP recombinase.

[0159] In the method provided herein, promoter nucleic acid sequence P1 may be functionally fused to the 5' portion of a split intron when incorporated at the predefined genomic location. This is illustrated in Figure 16, which has already been discussed herein. The introduction of a split intron between promoter P1 and I1 (or its variant) at the predefined genomic location results in a “spacer” that minimizes steric hindrance that may arise from the polymerase to the promoter due to interference. The presence of this spacer results in improved expression of the first choice marker (SM1), as shown in the experimental section of Example 1.

[0160] Accordingly, a method is provided herein in which a predefined genome location further includes a functional sequence region F1 having a 3'-5' orientation between the 5' portion of an intron having a 3'-5' orientation and the first recognition site of the first recombinase enzyme and the promoter P1 having a 3'-5' orientation, and the donor vector further includes a sequence region located between the first selection marker SM1 having a 3'-5' orientation and the second recognition site of the first recombinase enzyme. The sequence region includes the 3' portion of an intron having a 3'-5' orientation, further including (a) a functional sequence region F3 having a 3'-5' orientation and (b) a functional sequence region F2 downstream of the splice acceptor site, in the order of a 5'-3' sequence.

[0161] The excised nucleic acid sequence, as already discussed in this specification, (a) A nucleic acid sequence encoding the first choice marker; (b) Promoter nucleic acid sequence P1; and / or (c) A method comprising an expression cassette encoding a second choice marker is also provided herein.

[0162] The above design of the nucleic acid sequence to be excised results in the selection of cells in which the donor vector is randomly incorporated at locations other than predefined genomic locations, based on the expression of a second selection marker (SM2). This means that using such a design, SM2 expression can be positive only for cells that incorporate the donor vector outside the predefined genomic locations after the action of the second DNA enzyme. Thus, the second round of selection can be performed using a negative selection step based on SM2 expression to select cells in which the donor vector is incorporated outside the predefined genomic locations. Removal of the expression cassette encoding the second selection marker is also an improvement to the method, as it conserves energy that could instead be used for cells to produce the protein of interest.

[0163] The first selection marker may be selected from (i) fluorescent proteins and (ii) heterogeneous cell surface markers, in addition to those described elsewhere herein. The use of fluorescent proteins or cell surface markers as selection markers offers certain advantages because selection can be performed using rapid and direct isolation methods (i.e., based on FACS or MACS) as soon as the concentration of the first selection marker is increased above a certain limit (enabling detection of fluorescence above background in FACS and enabling efficient binding to magnetic beads in MACS). This is in contrast to selection markers based on metabolic enzymes or antibiotic resistance genes, which require long-term and indirect isolation strategies, as cells lacking the active selection marker grow too slowly, while cells with the active selection marker grow too slowly.

[0164] The first DNA enzyme may be provided in the form of a plasmid, mRNA, or purified protein, and optionally, the first DNA enzyme may be encoded by and expressed from the donor vector. The first DNA enzyme may also be expressed from an expression cassette encoding the first DNA enzyme located at a predefined genomic location in the cell in step i) of the method disclosed herein.

[0165] As previously stated herein, the donor vector of step ii) may further comprise an expression cassette encoding a second DNA enzyme, the expression of which is activated when the donor vector is incorporated into a predefined genomic location of the cell in step i) in the method disclosed herein.

[0166] The second DNA enzyme may also be provided in the form of a plasmid, mRNA, or purified protein.

[0167] The eukaryotic cells to be used in the methods presented herein may be selected from the group consisting of yeast cells, filamentous fungal cells, plant cells, insect cells, or mammalian cells. Mammalian cells may be, but are not limited to, human, monkey, rodent, or mouse cells. Eukaryotic cells are isolated eukaryotic cells as already described herein. Isolated cells are cells that have been isolated or removed from their natural environment.

[0168] Eukaryotic cells for use in the methods presented herein may be specifically selected based on their suitability for recombinant protein production in a bioreactor. Suitable cells can be selected from the CHO or HEK cell line group.

[0169] Eukaryotic cells for use in the methods presented herein may be specifically selected based on their similarity to cell types found in mammalian species such as humans.

[0170] The eukaryotic cells to be used in the methods presented herein may be selected from a group of cell lines capable of growing in suspension culture.

[0171] Key general theoretical advantages of some general SDI systems that form part of the present invention disclosed herein are: (1) To enable a selection process that minimizes the possibility that isolated cells will not have the desired result, which is a single copy of an integration cassette (containing a nucleic acid sequence variant) integrated at a predefined genomic location and only at that location. This is a critical characteristic because, for the optimization of nucleic acid sequence variants, such as expression cassette design based on transfection of a donor vector mixture containing a library of expression cassette designs, a one-to-one correlation between the cell phenotype and a single corresponding gene cassette design is necessary to ensure the proper selection of nucleic acid sequence variants with the desired characteristics.

[0172] (2) This allows for selection to retain only the sequences that contribute to the productivity of the cell line. There is no cell source that consumes the expression of the selected marker protein, and the risk of unpredictable interference between the nucleic acid sequence variant under investigation and the selected marker cassette is avoided. In other words, the presence of bacterial sequences that may have a negative impact on the long-term expression stability of GOIs can be avoided, thereby increasing the reliability of the method.

[0173] (3) Since the selection markers are not part of a predefined genome location sequence, there is flexibility in the selection of selection markers. The optimal selection marker can be chosen based on the application.

[0174] (4) The desired integration event activates the expression of a first selection marker, enabling the positive selection of cells with integration at a predefined genomic location with high specificity. Using a selection marker such as a fluorescent protein or cell surface marker, this allows for a very short time between transfection and the selection of positively integrated organisms (e.g., using FACS or MACS). Results should be obtained in 2-3 days. This reduces the time required to perform the method. In addition, early isolation of cells with integration at the desired location from cells that have undergone or have not undergone an unwanted integration event may have further advantages, as it minimizes the risk that unwanted cells may proliferate faster than desired cells (and potentially be lost from evaluation). Thus, the efficiency and performance of the method may be improved compared to methods lacking this characteristic.

[0175] A preferred implementation of a method utilizing a serine recombinase such as PhiC31 or Bxb1 as the primary DNA enzyme in combination with a single matching recombinase recognition sequence pair (i.e., attP / attB) offers the potential for superior integration efficiency. Specifically, in combination with promoter P1, it functionally fuses to the 5' portion of the split intron. The PhiC31 or Bxb1-mediated recombination of their corresponding attP / attB pairs is an irreversible reaction; therefore, theoretically, integration should be limited solely by transfection efficiency and plasmid stability. This contrasts with Cre-based integration or CRISPR / Cas9-based integration, where competitive, unproductive reaction pathways may exist, and for cassette exchange reactions, efficiency is negatively correlated with the size of the integration cassette. Integration efficiency is a critical performance parameter because it directly affects the number of nucleic acid sequence variants, which can be simultaneously evaluated by the method. The integration efficiency for one preferred implementation of the method is illustrated in the experimental section of Example 1.

[0176] Therefore, this disclosure provides a novel and improved method for the efficient and selective targeted incorporation of many nucleic acid sequence variants (e.g., encoding a protein of interest) into a population of isolated eukaryotic host cells. A population of isolated host cells, each selectively incorporating a single copy of a donor vector containing the nucleic acid sequence variant, presents an excellent system for optimizing nucleic acid sequence variants that have an effect on recombinant protein production or protein function and will find use in many different scopes.

[0177] Next, a method for performing a selection step based on a “defined phenotype” of isolated eukaryotic cells incorporating nucleic acid sequence variants at predefined genomic locations is outlined along with its relevant applications. Therefore, the terms “defined phenotype” as intended in this specification are described below. These descriptions are intended to be merely examples, and the invention is not limited thereto.

[0178] Generally, the phenotypic selection process may be based on any measurable characteristics of cells (using current or future analytical technologies) and corresponding measurement results used to specifically target cells for selection. Measurement and subsequent isolation of targeted cells for selection can be carried out by (i) using technologies that can be measured and manipulated at the single-cell level, such as FACS, or by (ii) first isolating single cells in separate compartments, thereby enabling selection by measurement using bulk analytical technologies and subsequent elimination of undesired compartments.

[0179] Alternatively, the phenotypic selection step may be based on any characteristic of the cells that can be used to indirectly select or enrich the phenotype, based on either (i) specific growth under defined growth conditions or (ii) specific binding of cells to a solid phase such as magnetic beads, followed by isolation of bound or unbound cells using MACS.

[0180] Since each individual cell harboring a single nucleic acid sequence variant is carefully selected, selection based on one or more phenotypic features explores whether a particular sequence variant has a negative, neutral, or positive effect on said feature. In any case, a particular nucleic acid sequence variant corresponds to a defined phenotype, meaning that the measured cellular features of cells harboring said variant will exhibit a defined statistical distribution. Selection of cells with measured features above a defined value results in the selection or enrichment of nucleic acid sequence variants that have an effect on said feature. Nucleic acid sequence variants can be selected / enriched for use at any defined measurement interval of defined phenotypic features.

[0181] Therefore, the sequence-optimized nucleic acid sequence according to the present invention should be understood as a sequence corresponding to a defined measurement interval of the selected phenotypic features. The optimized nucleic acid sequence, selected and isolated via the isolation of a defined cellular phenotype, can be identified by sequencing genomic DNA at a predefined genomic location. When genomic DNA is extracted from cloned cells (a population arising from a single cell), standard Sanger sequencing can be used. When genomic DNA is extracted from a diverse population of cells, parallel sequencing methods

[20] can be used.

[0182] One application of this method is to explore nucleic acid sequence variants in donor vectors for their impact on the expression of recombinant proteins having defined amino acid sequences. This has value for the commercial production of recombinant proteins, such as therapeutic proteins, proteins used as reference materials or reagents in diagnostic tests, or proteins used as reagents in research applications. As already eluten, many different functional sequence components have been shown to affect the protein levels that can be obtained at different stages of expression [14-19].

[0183] Sequences that affect the transcription of a target gene (GOI) include the transcription of the promoter (core promoter sequence, which includes the initiation transcription site sequence, the proximal promoter sequence, and transcription enhancer sequences, including their spacing to other promoter components) and the presence (or absence) of sequences that affect local DNA structure in the genome (chromatin modification elements, chromatin insulators, substrate / scaffold binding regions, etc.). In addition to the effects at the transcription level, cytoplasmic mRNA levels are also affected by the efficiency of nuclear export (influenced by the presence, location, and selection of intron sequences) and their cytoplasmic degradation rates (influenced by the selection of 3'-UTRs). Translation initiation is influenced by the selection of 5'-UTR and 3'-UTR sequences, and secretion is influenced by the selection of signal peptide amino acid sequences. In addition to these non-coding sequence elements, nucleic acid sequences used to encode the target protein (POI) (synonymous coding sequence selection) have been shown to have a significant effect on many stages of expression, including polypeptide chain folding via a process called simultaneous translation folding

[19] .

[0184] For multi-chain proteins such as antibodies, the ratio of different chains is crucial for the observed secretion of functional proteins. With advances in nucleic acid synthesis technology that enable base-pair-level control of nucleic acid sequences, the design space for nucleic acid sequences is vast. Therefore, a significant opportunity to improve expression and thus the economics of production can be obtained by optimizing the nucleic acid sequence variants present in donor vectors for given recombinant proteins.

[0185] In this application, the defined phenotype can be defined, for example, by a measurement of the expression of a target protein (i.e., recombinant protein) within a specific range. One method of performing the phenotypic selection step is to perform single-cell cloning (e.g., using FACS or limiting dilution) from a population of cells generated by the SDI portion of the disclosed method, and measure the bulk titer after a defined period of culture. Clones giving titers within a specific range are selected, thereby selecting and isolating the corresponding optimized nucleic acid sequence variants. In this implementation, the diversity of nucleic acid sequence variants that can be examined is limited by the number of clones that can be cultured and evaluated in parallel. Using traditional parallel cultures, such as static culture in microtiter plates, the number of clones is actually limited to approximately 10,000 (100 96-well plates). To ensure that biological and technological variability is covered, this means that a nucleic acid sequence variant library with several hundred variabilities can be efficiently examined. The diversity of the library may be slightly increased by using novel parallel culture and droplet manipulation techniques

[21] or other microsystem-based techniques [22, 23] or evaluation techniques.

[0186] However, to significantly increase the diversity of accessible libraries, technologies that enable single-cell measurements (e.g., FACS) are preferred. To enable evaluation of the expression of the target protein by FACS, the target protein can be genetically fused to a fluorescent protein at one c or n terminus of its polypeptide chain (see Figures 26 and 27a). To standardize technical and biological noise, an additional gene encoding a second fluorescent protein (having a corresponding gene cassette that is maintained constant for all donor vector variants in the aforementioned population of donor vectors) can be utilized, and selection can be made based on the ratio of the release of the two fluorescent proteins (see Figure 26b). For target proteins containing multiple polypeptides, such as viral particles or bispecific antibodies, gene fusion of two different fluorescent proteins to two different polypeptide chains can be performed, and when constructed in close proximity, construction-specific expression signals based on FRET activity between the two fused polypeptides can be enabled (see Figure 27b). Another means of producing modifiable POI expression measurements in FACS analysis is to ensure the tethering of POIs on the cell surface, followed by binding of POIs to a fluorescent detection reagent before FACS sorting, see Figure 28. This can be achieved via fusion with the membrane anchoring domain of the POI [13, 30]. Optionally, the presence of the membrane anchoring domain in the POI can be modulated, i.e., via the use of leaky translation stop codons

[24] . Cells exhibiting a fluorescent signal within a defined range can be isolated in one or optional multiple sequential FACS sorting steps (optionally using different fluorescence ranges). In addition to the increased diversity that can be obtained, methods such as FACS also allow cells to be cultured using culture formats and conditions relevant to production on a large scale before evaluation and sorting.

[0187] The defined phenotype can also be linked to the measurement of other cellular features that are pre-apoptotic stress signaling or specific stress signaling states, such as ER stress, amino acid starvation, or hypoxia.

[0188] The reading of different cellular characteristics in a single cell may potentially be possible through the introduction of engineered, genetically encoded, and reported circuits into isolated eukaryotic host cells. See, for example,

[25] .

[0189] Using the disclosed method for identifying sequence variants corresponding to defined phenotypes, nucleic acid sequence variants can be directly optimized to improve the expression of a given recombinant protein of interest. Data on the performance of different nucleic acid sequence variants obtained by the method can also be used to predict design rules that can be used to select donor vector designs for other relevant or unrelated recombinant proteins. Such design rules may be based, for example, on machine learning or other artificial intelligence techniques. As outlined in Figure 1, the method can also be used to directly isolate cells for downstream applications such as the production of the protein of interest. Finally, the method can be used to isolate optimized nucleic acid sequence variants for direct downstream applications, such as incorporation into donor vectors for cell line development (CLD).

[0190] Another application of the method is to evaluate different recombinant proteins (i.e., differences in amino acid sequences) for their expression in a given type of eukaryotic cell. Performing the method for this application can utilize any of the design examples already described for donor vector sequence optimization. Evaluating different amino acid sequences can be used to screen early therapeutic protein candidates for their manufacturability or developability through the introduction of amino acid sequence diversity at locations outside their functional surface, or for improving their expression in the final intended production host

[27] . The method can also be used to improve the functionality of naive libraries for the discovery of novel therapeutic protein candidates based on target binding (i.e., antibody discovery using naive antibody libraries). Diversity in different scaffold designs can be screened for their impact on expression, both for selecting scaffolds to use in novel library designs and for inferring design rules for library design and construction for a given scaffold.

[0191] In addition to the amino acid sequence of the POI (including the potential signal peptide), the nucleic acid sequence used to encode the POI (i.e., the GOI), and the non-coding sequence used in the donor vector, expression is also influenced by the cellular mechanisms present in isolated eukaryotic cells. The innate diversity in cellular mechanisms between individual cells is typically leveraged by clonal screening campaigns to find the best possible production host for a given protein, which is then introduced using a given donor vector.

[0192] However, to go beyond natural diversity, cell line manipulation approaches can be used

[26] . The challenge lies in the complexity of cells, which current knowledge means that the action of a particular manipulation strategy may often be protein-based and even clonal.

[0193] Therefore, further applications of the methods disclosed herein enable improved means of performing protein and clone-specific cell manipulation. Cell manipulation may be based on the introduction of different effector genes, such as (i) expression cassettes encoding naturally occurring proteins (for overexpression) or proteins not present in the cells used (for the introduction of novel functionality), (2) introduction of genes encoding natural or engineered transcription factors for the regulation of endogenous gene expression, or (3) introduction of genes encoding natural or engineered regulatory RNAs such as miRNAs or lncRNAs for the manipulation of cellular pathways at a general or local level.

[0194] Based on knowledge of bottlenecks regarding the clones and proteins produced, libraries of different nucleic acid construct variants (each containing one or more effector genes) can be designed and constructed as donor vector libraries. Using isolated eukaryotic cells (see Example 3 in the Experimental Section) that already stably express a predetermined recombinant protein, which is a control, and include a predefined genomic location, a population of cells is isolated in which each cell has a single nucleic acid construct variant containing a defined set of effector genes at the predefined genomic location. Phenotypic selection and sequence identification can be performed using the same basic techniques already described.

[0195] In different applications, methods can be used to select recombinant protein sequences with desired functionality. A typical example is screening a library of amino acid sequence variants (encoded by nucleic acid sequence variants) for their binding characteristics (i.e., function) to a specific target molecule or structure (i.e., in antibody discovery, for example).

[0196] Typical libraries are constructed based on the introduction of sequence diversity at key locations of another conserved protein sequence, often called a protein scaffold. Examples of protein scaffolds include the IgG1 scaffold, nanobody scaffold, and Z-domain scaffold [27-29]. To enable screening of variants expressed from single cells, protein scaffold variants can be gene-fused to membrane-anchored domains, allowing for screening by methods called surface presentation and mammalian presentation [13, 30].

[0197] The present invention provides an improved means of performing mammalian presentation based on the characteristics of previously discussed and disclosed SDI methods. In the first part of the method, a library of amino acid sequence variants (all fused to membrane anchor domains) composed of multiple donor vectors of the present disclosure is used to generate a population of isolated eukaryotic cells highly enriched with respect to cells having single amino acid sequence variants from the library at and only at the predefined genomic locations (and thus, single amino acid variants presented on the surface).

[0198] In this application, the defined phenotype can be defined, for example, by a measurement of the binding of the target structure to a surface-presented amino acid variant within a specific range. In a preferred execution, target structures (e.g., proteins) are labeled with fluorophores and incubated with a population of isolated eukaryotic cells having amino acid sequence variants on their surface (see Figure 28). After incubation and washing of the cells, FACS is used to record the amino acid sequence variants to target binding by fluorescence. Cells giving fluorescence readings within a specific range are isolated. Typically, a repeatable FACS isolation step after incubation with reduced concentrations of labeled target is used to enrich high-affinity conjugates. Optionally, the target binding signal can be standardized by the amount of amino acid sequence variant presented in the cells. This can be achieved by incubation with a fluorescently labeled reagent (fluorescence that does not overlap with the target fluorescence) that binds to a conserved epitope (i.e., the FC portion of the IgG1 scaffold) present in all presented amino acid sequence variants (see Figure 28). In another preferred implementation, the target (i.e., the protein) is presented on the surface of a eukaryotic cell that expresses a fluorescent protein in the cytoplasm.

[0199] Another example of the application of methods for selecting recombinant protein sequences with desired functionality is the optimization of the quality of therapeutic proteins. Therefore, selection is based on characteristics such as the glycosylation properties and three-dimensional structure (protein fold) of the recombinant protein. By presenting recombinant protein variants on the cell surface, glycosylation and three-dimensional structure can be evaluated by incubation with fluorescently labeled reagents such as glycoform-specific affinity conjugates and / or structure-sensitive affinity conjugates (e.g., antibody targets), followed by measurement by FACS and isolation. Optionally, the reagent binding signal can be standardized by the amount of recombinant protein variant presented in the cells. See Figure 28.

[0200] Other examples of the application of the method for selecting recombinant proteins having desired function include (i) discovery or optimization of fluorescent proteins using direct isolation of desired variants by FACS; (ii) discovery or optimization of enzymes whose enzymatic activity results in a detectable difference in phenotype, such as an enzyme conferring antibiotic resistance; (iii) discovery or optimization of recombinases for integration of a donor vector into a predefined genomic location by contacting a population of isolated eukaryotic cells having a recombinase variant at the aforementioned predefined genomic location and a second predefined genomic location, and only at that location, with a donor vector and isolating the positive component via positive FACS sorting of cells having an activated first selection marker; or (iv) development and optimization of gene-encoded signaling and / or logic circuits

[25] by incorporating a number of candidate circuit designs that result in a measurable cellular output, followed by selection of a variant having desired performance.

[0201] Therefore, in a first aspect, the present invention relates to a method for selecting a sequence-optimized nucleic acid sequence from a population of nucleic acid sequence variants, wherein the sequence-optimized nucleic acid sequence corresponds to an isolated eukaryotic cell having a defined phenotype, and the method is i) A step of preparing a population of isolated eukaryotic cells, wherein each cell is a. Nucleic acid sequence I1 containing the recognition site of the first DNA enzyme; b. Nucleic acid sequence E1 containing the recognition site of the second DNA enzyme; and c. Promoter nucleic acid sequence; The step includes a predefined genome location: ii) Each of them, a. Nucleic acid sequence I2; b. Nucleic acid sequence E2 containing the recognition site of the second DNA enzyme; c. First choice marker nucleic acid sequences encoding; and d. Nucleic acid sequence region containing a nucleic acid sequence variant, The process of preparing multiple donor vectors, including: iii) A step of bringing multiple donor vectors into contact with a population of cells in the presence of a first DNA enzyme, wherein the presence of the first DNA enzyme enables recombination between nucleic acid sequence I2 of the donor vector and nucleic acid sequence I1 located at a predefined genomic location of the cell; iv) A step of selecting and isolating cells having a donor vector incorporated at a predefined genomic location by detecting the expression of a first selection marker in the cells, wherein the expression of the first selection marker is activated by a promoter nucleic acid sequence at a predefined genomic location; and v) A step of selecting and isolating cells having a defined phenotype from the cells of step iv), thereby selecting and isolating a sequence-optimized nucleic acid sequence from the nucleic acid sequence variant, wherein the sequence-optimized sequence corresponds to the defined phenotype, and the step Includes.

[0202] The first selection marker may also be abbreviated and referred to herein as "SM1".

[0203] The second selection marker may also be abbreviated and referred to herein as "SM2".

[0204] Therefore, the sequence-optimized nucleic acid sequence corresponds to a eukaryotic cell having a “defined phenotype,” where “defined phenotype” means that cells having specific physical characteristics or behaviors already defined and discussed herein are selected for the purpose of analyzing and selecting the optimized sequence variant. As already defined herein, the phenotypic selection step may generally be based on corresponding measurement results used to specifically target cells for any measurable characteristics and selection of cells (using current or future analytical technologies).

[0205] The use of multiple donor vectors and host cell populations containing different nucleic acid variants means that it is possible to evaluate more than one nucleic acid sequence in the same run. Naturally, not all single cells incorporate different nucleic acid sequence variants, but depending on the method, this allows for a wide range of complex approaches that enable the evaluation of a significant number of nucleic acid sequence variants simultaneously.

[0206] Each of the multiple donor vectors in step ii) e. An expression cassette encoding a second choice marker; A method further comprising the above is also provided herein; The aforementioned method, vi) a step of excising nucleic acid sequences adjacent to nucleic acid sequences E1 and E2 derived from cells selected and isolated in step iv) or step v) in the presence of step iii) or a second DNA enzyme, wherein the presence of the second DNA enzyme enables recombination between nucleic acid sequences E1 and E2, and the presence of an expression cassette encoding a second selection marker in which nucleic acid sequences E1 and E2 are not adjacent in the cell indicates stable integration of the donor vector to a genomic location outside a predefined genomic location in the cell; and vii) Steps to select and isolate cells derived from the process lacking an expression cassette encoding a second selection marker. It also includes.

[0207] A method is also provided herein that further comprises the step of obtaining nucleic acid sequence information from the isolated cells of step iv), v), or vii) by sequencing the sequence-optimized nucleic acid sequences located at predefined genomic locations in the isolated cells. Sequencing of the optimized nucleic acid sequences located at predefined genomic locations can be performed by sequencing methods well known to those skilled in the art

[20] . Sequences are performed and the sequence variant that worked most efficiently in the SDI system according to this disclosure is identified by comparing it with variants that did not work efficiently.

[0208] As previously stated herein, nucleic acid sequence variants of multiple donor vectors may consist of promoter variants, intron variants, transcriptional regulatory sequence variants, DNA structure regulatory sequence variants, 5' untranslated region variants, 3' untranslated region variants, intra-sequence ribosome entry site variants, target gene variants, nucleic acid sequence variants encoding signal peptides, and / or any combination of such variants. Other examples of nucleic acid sequence variant categories can also be anticipated that may be evaluated in the context of this disclosure to identify optimized nucleic acid sequence variants from among multiple nucleic acid sequence variants of a particular sequence category.

[0209] The selection of sequence-optimized nucleic acid sequences by selecting cells having a defined phenotype in step v) includes selection based on one or more of the following phenotypic features of the cells: (i) The presence or expression level of endogenous biomolecules in the cells; (ii) Expression level of the target recombinant protein by the cells; (iii) the growth rate of the cells; and / or (iv) Functionality and methods of recombinant proteins that are the target encoded by the nucleic acid sequence region in the cells are also provided herein.

[0210] The recombinant protein in question may be a recombinant fusion protein, such as a protein fused to a membrane anchoring domain for localization on the cell surface, and / or a fluorescent protein or a protein fused to a fluorescent protein domain. Therefore, the endogenous biomolecule may consist of, but is not limited to, proteins, mRNA, miRNA (microRNA), lncRNA (long non-coding RNA), or metabolites.

[0211] As previously stated herein, the selection of sequence-optimized nucleic acid sequences by selecting cells having a defined phenotype in step v) of the method presented herein may include selection based on the functionality of the recombinant protein of interest. The functionality of the recombinant protein of interest may be measured and determined based on the interaction between the recombinant protein of interest and a target structure, such as a small molecule, DNA molecule, RNA molecule, protein, viral particle, protein complex, exosome, or cell, when it is localized and expressed on the cell surface of the cell, and optionally the target structure is tagged with a fluorescent moiety. Fluorescent moieties for use in the context of the method according to the present invention will be obvious to those skilled in the art.

[0212] More specifically, the recombinant protein of interest may also be an affinity protein candidate, and the level of expression is determined by the presentation of the affinity protein candidate on the cell surface of the cell. The affinity protein candidate may be a single-chain polypeptide fused to a membrane anchor domain, and optionally, the single-chain polypeptide may be selected from the group consisting of Z-scaffold proteins, nanobody scaffold proteins, single-chain fragment variable (scfv) scaffold proteins, finomer scaffold proteins, DARPin scaffold proteins and / or adnectin scaffold proteins [28, 29].

[0213] The affinity protein candidate may also comprise two or more polypeptide chains, such as an antibody, and the nucleic acid sequence variant corresponding to the affinity protein candidate encodes the affinity protein candidate variant, such as an antibody variant, where, for example, one of the two or more polypeptides of the antibody variant is fused to a membrane anchor domain [13, 30].

[0214] A method is also provided which optionally involves exposing an affinity protein candidate to a specific target component, and then detecting the binding of the affinity protein candidate to the specific target component, thereby determining the binding specificity, selectivity, affinity, and / or functionality of the affinity protein candidate.

[0215] The target component may be selected from protein complexes, exosomes, or cells, such as small molecules, DNA molecules, RNA molecules, proteins, or viral particles, and optionally, the target structure is tagged with a fluorescent moiety.

[0216] The affinity protein candidate may be any protein capable of binding to or having affinity for the target component.

[0217] As previously stated herein, the selection of sequence-optimized nucleic acid sequences by selecting cells having a defined phenotype in step v) of the method presented herein may include selection based on the expression level or functionality of the recombinant protein of interest and / or the presence or level of endogenous biomolecules. Such presence or level may be measured at the single-cell level, for example, by using flow cytometry.

[0218] A method is also provided in which the nucleic acid sequence region of a donor vector comprises nucleic acid sequence variants for the expression of one or more recombinant proteins of interest from the donor vector, and the plurality of donor vectors comprises different nucleic acid sequence variants encoding different amino acid sequence variants of the one or more recombinant proteins of interest.

[0219] A method is also provided in which the nucleic acid sequence region of a donor vector contains a nucleic acid sequence variant for the expression of a target recombinant protein from the donor vector, and the nucleic acid sequence variants present in multiple donor vectors contain nucleic acid sequence variants that encode essentially identical amino acid sequence variants of the target recombinant protein. Such a method is expected to identify the target optimal nucleic acid sequence encoding the target protein.

[0220] A method is also provided that includes multiple donor vectors, each containing a nucleic acid sequence region with essentially the same nucleic acid sequence, to encode an essentially identical recombinant protein of interest, wherein the nucleic acid sequence region includes nucleic acid sequence variants of different donor vector components, such as nucleic acid sequence variants of the promoter or enhancer nucleic acid sequence of the donor vector. Such a method is expected to identify the optimal nucleic acid sequence variant for resulting in the expression and secretion of the recombinant protein of interest.

[0221] Therefore, such methods identify sequence-optimized nucleic acid donor vector components for use in recombinant protein expression systems based on eukaryotic cell systems. Accordingly, sequence-optimized nucleic acid sequences selected by the methods disclosed herein are also provided.

[0222] The use of such sequence-optimized nucleic acid sequences for producing recombinant proteins is also provided.

[0223] The use of sequence-optimized nucleic acid sequences selected by the methods disclosed herein for designing further sequence-optimized nucleic acid sequences is also provided. This means generating additional optimized nucleic acid sequence variants using sequence identity information of the sequence-optimized nucleic acid sequences selected by the methods disclosed herein. Thus, this information is used for design purposes for designing additional variants of the selected nucleic acid sequences.

[0224] In a further aspect of this disclosure, isolated eukaryotic cells having a defined phenotype corresponding to a sequence-optimized nucleic acid sequence that can be obtained by the method disclosed herein are provided.

[0225] This disclosure is intended to illustrate different ways of carrying out the invention, and is not intended to limit thereto, as described herein by the following experimental section.

[0226] Experiment section Abbreviation LP = Landing Pad LP1P1=attP1 including landing pad LP2P2=attP2 and landing pad 2 including split intron CHO = Chinese hamster ovary FC-eGFP = Enhanced green fluorescent protein fused to IgG1-derived FC. TagBFP2 = Blue Fluorescent Protein Variant TagRFP-T = Red Fluorescent Protein Variant G418, also known as Genethecin, is a broad-spectrum antibiotic that selectively targets mammalian cells expressing the neomycin resistance gene (NeoR).

[0227] Sequence List The following sequences are used in the experimental section, but this disclosure is not limited to these sequences. Therefore, we also expect that the function of such sequence variants remains essentially the same as that of the original sequences.

[0228] attP1(sequence number 1)

[0229] [ka]

[0230] attB1(sequence number 2)

[0231] [ka]

[0232] attP2 (SEQ ID NO: 3)

[0233]

Chemical Formula

[0234] attB2 (SEQ ID NO: 4)

[0235]

Chemical Formula

[0236] PhiC31 gene (SEQ ID NO: 5)

[0237]

Chemical Formula

[0238] loxP (SEQ ID NO: 6)

[0239]

Chemical Formula

[0240] Cre gene (SEQ ID NO: 7)

[0241]

Chemical Formula

[0242] FC-eGFP gene (SEQ ID NO: 8)

[0243]

Chemical Formula

[0244] FC-TagBFP2 gene (SEQ ID NO: 9)

[0245] [Chem.]

[0246] TagRFP-T gene (SEQ ID NO: 10)

[0247] [Chem.]

[0248] eGFP gene (SEQ ID NO: 11)

[0249] [Chem.]

[0250] TagBFP2 gene (SEQ ID NO: 12)

[0251] [Chem.]

[0252] NeoR gene (SEQ ID NO: 13)

[0253] [Chem.]

[0254] GS gene (SEQ ID NO: 14)

[0255] [Chem.]

[0256] 5' portion of 381 5'UTR (SEQ ID NO: 15)

[0257] [Chem.]

[0258] 462 5'UTR's 5' portion (sequence number 16)

[0259] [ka]

[0260] 464 5'UTR's 5' portion (sequence number 17)

[0261] [ka] [Examples]

[0262] Efficiency of phiC31 recombinase-mediated integration and selective marker activation To investigate integration efficiency, HyClone CHO LP cells and non-LP HyClone CHO control cells were transfected using a combination of PhiC31 recombinase expression plasmids and either donor vector A or B (Figure 3). The donor vector contains expression cassettes for FC-eGFP and FC-TagBFP2, as well as a promoter-less TagRFP-T gene, positioned to be activated upon integration in LP cells.

[0263] Two HyClone-CHO LP variants and matching donor vectors were investigated; see Figure 3. In HyClone-CHO LP1P1, the promoter in LP is located immediately downstream of attP1. In donor vector A, the TagRFP-T gene is located immediately upstream of attB1. In HyClone-CHO LP2P2, the 5' portion of the split intron is located between attP2 in LP and the downstream promoter. In donor vector B, the 3' portion of the split intron is located between attB2 and the upstream TagRFP-T gene. Integration efficiency was assessed by flow cytometry 7 days after transfection by measuring the percentage of cells presenting RFP signaling above background (clearly compared to untransfected controls; see Figure 4).

[0264] Figure 4 illustrates an example of the resulting flow cytometry data for HyClone CHO LP2P2 in comparison to the control. The complete set of results is summarized in Table 1. For LP1P1, only untransfected controls were used, while for LP2P2, both randomized integration controls (RI controls, donor vector only) and pseudo-att integration controls (donor vector + PhiC31 in CHO cell lines lacking LP) were used. The data showed that both LP variants were functional, but the LP2P2 variant, utilizing a split-intron design, gave superior integration efficiency.

[0265] [Table 1] [Examples]

[0266] Efficiency of Cre recombinase-mediated resection of vector skeleton at the integration site HyClone CHO LP1P1 cells were transfected using a donor vector containing a PhiC31 expression plasmid and expression cassettes for FC-eGFP and FC-TagBFP2, positioned to be activated upon integration in LP cells, and a promoter-less TagRFP-T gene (Figure 5). Cells incorporating the donor vector in the landing pad (LP) were enriched by several FACS sorting steps that gated the Tag-RFP-T signal and the average expression of both FC-eGFP and FC-TagBFP2 above the background. The resulting sorted and expanded pool of cells was then transfected a second time using either (a) a Cre recombinase expression plasmid, (b) a synthetic mRNA encoding Cre, or (c) a pseudotransfection solution lacking Cre recombinase encoding a nucleic acid molecule. Seven days after the second transfection, the entire cell population was analyzed by flow cytometry to assess the efficiency of excision of the region where two loxP sites are adjacent.

[0267] A plot of flow cytometry analysis after Cre recombinase transfection can be seen in Figure 6. The data shows an increase in cells not expressing FC-TagBFP2 in the pool treated with two Cre recombinases compared to a sham control. This, in turn, clearly demonstrates correct integration of the donor vector in LP, with two loxP sites on FC-TagBFP2 where the Cre recombinase enzyme can act adjacent to each other. According to the data, the Cre recombinase-catalyzed excision reaction is highly effective, with a yield of at least 80%. [Examples]

[0268] Repetitive integration at the same genomic location using orthogonal attP / attB pairs HyClone CHO LP1P1 cells were transfected using a donor vector (Figure 7, Donor Vector A) containing the PhiC31 expression plasmid and expression cassettes for the attB1 sequence, followed by the attP2 sequence, the 5' portion of the split intron, the promoter, the loxP sequence, and eGFP. Seven days post-transfection, eGFP-positive cells were sorted by FACS, followed by cell expansion. A second, more rigorous sorting of eGFP-positive cells was then performed. After the second positive sorting (Figure 8, Sorting in Step 1), the expanded cells were transfected using synthetic mRNA encoding Cre recombinase. Seven days after Cre recombinase transfection, eGFP-negative cells were sorted using FACS. After expansion, the eGFP-negative cell pool was analyzed by flow cytometry (Figure 8, Sorting in Step 2). Data from the sorting and analysis steps are shown in the upper panel of Figure 8. During these processes, it is assumed that the landing pad in the CHO genome changes as shown in steps (1) and (2) in Figure 7.

[0269] To verify the functionality of the altered landing pads, the eGFP-negative pool obtained after final selection (Figure 8, selection in step 2) was transfected using DNA donor vector B (Figure 7, step 3), and analyzed by flow cytometry 7 days after transfection (Figure 8, bottom panel). The data demonstrate the functionality of the novel landing pads. Finally, cells derived from the eGFP-negative pool were cloned using single-cell selection by FACS, and the landing pad regions of their genomes were amplified by PCR and sequenced. Correct alteration of the landing pads was confirmed in multiple clones by sequencing (complete assurance of the novel landing pad region), indicating that the alteration outlined in Figure 7 was successfully achieved. [Examples]

[0270] Generation of an SDI cell pool using two consecutive selection steps HyClone CHO LP2P2 cells (clones generated in Example 3) were transfused using the PhiC31 recombinase expression plasmid and donor vector constructed according to Figure 9.

[0271] Starting two days after transfection, cells were cultured in the presence of G418, and cells were selected to incorporate the donor vector on the landing pad, thereby introducing the neomycin resistance gene (Neo R The enzyme was activated. After returning to a high viability rate (>98%) following G418 selection, cells were sorted by FACS based on a GFP / RFP double-positive signal. After expansion, the sorted cells were transfected using synthetic mRNA encoding Cre recombinase. Seven days after Cre recombinase transfection, cells were sorted by FACS using a GFP-positive / RFP-negative signal (Figure 10, Gate E). The final SDI pool was analyzed by flow cytometry after expansion.

[0272] FACS / flow cytometry data can be seen in Figure 10. The additional selection step after Cre recombinase transfection reduces heterogeneity in the pool, as shown by the mean and CV of eGFP signaling for TagRFP-T positive (incorrect integration) and TagRFP-T negative (correct integration) cells. [Examples]

[0273] Evaluation of different 5'-UTR and signal peptide combinations for high expression. The preferred combinations A1, B2, and C3, known to have good expression in previous experiments, are shown in bold. Nine different constructs were created by combining three different 5'-UTRs (A, B, C) and three different signal peptides (1, 2, 3) for Fc-eGFP expression; see Table 2. Each constructed donor plasmid was transfected into the HyClone CHO LP2P2 cell line together with the PhiC31 plasmid for site-directed integration. After 2 days, transient expression of eGFP and mTagBFP2 was analyzed using flow cytometry. The number (%) of cells with good expression of eGFP and mTagBFP2 can be seen in Figure 17a, showing good values ​​for the preferred variants A1, B2, and C3. When the donor vector was inserted into the landing pad (LP), the neomycin selection marker was activated, and therefore, after 2 days, G418 was added as a selection pressure. When all cultures again reached >98% viable cells, the mRNA of the Cre enzyme was transfected into the cells to remove the RFP and neomycin expression cassettes. After one week, single cells were selected for eGPF and mTagBFP2 positive signals and RFP negative signals for each sample. During G418 selection, no cultures with constructs B1, B3, and C1 remained. Batch cultures were performed for each remaining sample, and fluorescence (eGFP and mTagBFP2) was measured in logarithmic phase cultures (Figure 17b, eGFP fluorescence), and FC titer was measured post-culture using CEDEX (Figure 17c, mg / L). The ranking of constructs can be seen in Table 3. A good correlation was found between the fluorescence signal results and FC-based titer measurements; see Figure 18. The results indicate that UTR C and signal peptide 2 give better expression, and that their combination also produced the highest expression.

[0274] [Table 2]

[0275] [Table 3] [Examples]

[0276] Evaluation of 5'-UTR variants by flow cytometry using Fc-eGFP as a single-cell Qp probe. To evaluate the ability to distinguish between different expression levels of relevant target genes at the single-cell level, four different DNA donor vectors were constructed with various gene constructs for a signal peptide containing Fc-eGFP (see Figure 19 for general donor vector designs and Table 4 for variant descriptions). Three of the variants differed only in their 5'-UTR sequences, while the negative control vector had a complete Fc-eGFP cassette containing a missing promoter. All four vectors presented the same signal peptide containing the Fc-TagBFP2 gene cassette as an internal control.

[0277] [Table 4]

[0278] HyClone CHO LP2P2 cells were transfected using either the PhiC31 expression plasmid or one of four DNA donor vectors. Individual transfections and selections were performed for all DNA donor vectors. Starting two days post-transfection, cells were cultured in the presence of G418, and cells were selected to incorporate the donor vector on the landing pad, thereby enabling the neomycin resistance gene (Neo RThe ) was activated. After G418 selection, the cells returned to a high viability (>98%) and were analyzed by flow cytometry. For comparison of different variants, the cell population was gated in silico, as shown in the upper panel in Figure 20. From the total viable cell population, major clusters of RFP-positive cells were selected and their TagBFP2 / eGFP values ​​are plotted. Major clusters of TagBFP2 / eGFP-positive cells were de-gated and used for comparison of different variants. Overlay plots of these gated populations can be seen in the lower panel of Figure 20.

[0279] Data comparing expression produced using different variants demonstrate a wide dynamic range of Fc-eGFP response. Furthermore, rankings based on Fc-eGFP response correlate with the predicted performance of different constructs, with high-efficiency positive controls showing the best response, and negative controls (absence of Fc-eGFP expression cassette) not producing an eGFP response above background. Based on data published in scientific literature

[31] , 462 triple uORF transcription attenuation sequences should produce stronger negative transcription attenuation of downstream genes compared to 464 single uORF transcription attenuation sequences. For the three variants with intact Fc-eGFP cassettes, the signal response for TagBFP2 similarly adds reliability to the eGFP ranking. For negative controls, the TagBFP2 response is elevated, which may be due to the absence of the Fc-eGFP promoter. [Examples]

[0280] Comparison of 3'-UTR variants using flow cytometry and titer evaluation with Fc-eGFP as a single-cell Qp probe. Eight(8) different DNA donor vectors with different 3'-UTR sequences in the Fc-eGFP cassette were constructed (see Figure 21 for general vector designs).

[0281] HyClone CHO LP2P2 cells were transfected using either a PhiC31 recombinase expression plasmid or one of eight DNA donor vectors. Individual transfection and selection were performed for all DNA donor vectors. Seven days after transfection, the initial cell population was FACS sorted based on mRaspberry-positive signals (corresponding to activation of genes without promoters in the landing pad). After expansion, a second, more rigorous mRaspberry-positive FACS sort was performed to obtain a pool of SDI cells for each DNA donor vector variant. After expansion, shaken flask batch cultures were performed at an initial cell density of 0.25 × 10⁶ cells. 6 Cells / ml were started. Live cell density was measured daily using a ViCell instrument. Samples from each culture were analyzed by flow cytometry on day 3 of culture. Titer in the growth medium was measured on day 5 of culture using a ViaCore 8K+ instrument with an IgG1 standard for generating calibration curves.

[0282] Qp (days 0-5) was calculated for each culture and compared with the average eGFP signal of the corresponding flow cytometry data (which surrogates the Qp for day 3). The data can be seen in Figure 22.

[0283] As can be seen, Qp calculated from titer and VCD measurements correlates with flow cytometry data. This, in turn, demonstrates that high-expression cells can be selected / enriched from a pool of cells containing a library of nucleic acid variants (e.g., 3'-UTR variants) using fusions of fluorescent proteins to target proteins, thereby enabling selection / enrichment for high-performance nucleic acid variants. [Examples]

[0284] Evaluation of small libraries of glutamine synthase variants for their efficiency as selection markers Three control DNA donor vectors and one DNA donor vector library were constructed using a general design according to Example 4. The vectors differed only in the sequence at codon 299 of the coding sequence for glutamine synthase (see Figure 23 for details). The positive control, Arg with codon CGA, is known to encode a highly functional enzyme (data not shown). The negative control, Gly with codon GGA, has been shown to function for MSX selection after random incorporation of vectors with this GS variant (however, accompanied by reduced cell growth, which indicates reduced enzyme efficiency, data not shown). To evaluate whether other amino acids / codons at position 299 could produce glutamine synthase with higher efficiency compared to the negative control, libraries containing methionine variants and 29 functional codons representing 18 different amino acids were tested in a site-directed incorporation workflow using HyClone CHO LP2P2.

[0285] HyClone CHO LP2P2 was transfected using three control plasmids and plasmid libraries in separate transfections. A cell pool selected for the integration of a single DNA donor vector copy in the landing pad was generated according to the workflow described in Example 4. As an initial test of GS functionality and as an enrichment step for functional variants in the library, each final pool of cells was cultured in L-glutamine-deficient medium either in the absence of MSX or in the presence of 10 μM MSX. Growth curves can be seen in Figure 24. As can be seen, all variants can grow in the absence of MSX, but the growth rates vary. The positive control grew the fastest, followed by the library. The negative control and methionine variant had the slowest growth rates. In the presence of 10 μM MSX, only the positive control and library grew, and the positive control grew at a faster rate.

[0286] As a selection for functional GS activity, single cells were grown using 10 μM MSX, and then cloned into 96-well static culture plates from library cultures, positive control cultures, and negative control cultures using FACS. Single cells were grown in glutamine-free medium, either in the absence of MSX or in the presence of 5 μM MSX. Wells were monitored for growth using the Solentim plate imaging system. Graphs illustrating the growth of the library compared to the control can be seen in Figure 25.

[0287] As can be seen, clones grow for all variants in the absence of MSX. For cultures using 5 μM MSX, clones do not grow for the negative control, but do grow for the positive control and library-derived clones.

[0288] To identify GS variants that grow in the presence of 5 μM MSX, genomic DNA was extracted from the corresponding wells, and the GS region was amplified by PCR. A total of 96 PCR products from 96 clones were sent to Sanger sequencing, and high-quality sequencing results were obtained for 79 clones. The Sanger sequencing sequences were aligned to the GS variants using Geneious Prime software. A summary of the identified GS variants can be seen in Table 5.

[0289] [Table 5]

[0290] As can be seen, 99% of the identified clones have Arg at position 299, and only one variant has Gly. The fact that 99% of the identified variants correspond to the same amino acid (but with a different codon) in the positive control illustrates the usefulness and accuracy of the SDI system for evaluating libraries of sequence variants in parallel workflows. It should also be noted that all three different Arg codons present in the library are identified from the grown clones, but with distinctly different frequencies, which means that the effect of a single synonymous codon change can be picked up by the methodology.

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Claims

1. A method for selecting a sequence-optimized nucleic acid sequence from multiple nucleic acid sequence variants, The sequence-optimized nucleic acid sequence corresponds to a eukaryotic cell having a defined phenotype, i) A step of preparing a population of isolated eukaryotic cells, wherein each cell is a. Nucleic acid sequence I1 containing the recognition site of the first DNA enzyme; b. Nucleic acid sequence E1 containing the recognition site of the second DNA enzyme; and c. Promoter nucleic acid sequence The step includes a predefined genome location; ii) Each of them, a. Nucleic acid sequence I2; b. Nucleic acid sequence E2 containing the recognition site of the second DNA enzyme; c. A nucleic acid sequence encoding the first choice marker; and d. Nucleic acid sequence region containing a nucleic acid sequence variant A process of preparing multiple donor vectors, including: iii) A step of bringing a plurality of donor vectors into contact with a population of cells in the presence of a first DNA enzyme, wherein the presence of the first DNA enzyme enables recombination between nucleic acid sequence I2 of the donor vector and nucleic acid sequence I1 located at a predefined genomic location in the cell; iv) A step of selecting and isolating cells having a donor vector incorporated at a predefined genomic location by detecting the expression of a first selection marker in the cells, wherein the expression of the first selection marker is activated at the predefined genomic location by the promoter nucleic acid sequence; and v) A step of selecting and isolating cells having a defined phenotype from the cells of step iv), thereby selecting and isolating a sequence-optimized nucleic acid sequence from the nucleic acid sequence variant, wherein the sequence-optimized sequence corresponds to the defined phenotype, Methods that include...

2. Each of the multiple donor vectors in step ii) e. Expression cassette encoding a second choice marker It further includes; vi) a step of excising nucleic acid sequences adjacent to nucleic acid sequences E1 and E2 from cells selected and isolated in step iv) or step v) in the presence of step iii), wherein the presence of the second DNA enzyme enables recombination between nucleic acid sequences E1 and E2, and the presence of an expression cassette encoding a second selection marker in which nucleic acid sequences E1 and E2 are not adjacent in the cell indicates stable integration of the donor vector to a genomic location outside a predefined genomic location in the cell; and vii) Steps to select and isolate cells derived from the process lacking an expression cassette encoding a second selection marker. The method according to claim 1, further comprising:

3. The method according to claim 1 or 2, further comprising the step of obtaining nucleic acid sequence information from the isolated cells in step iv), v), or vii) by sequencing the sequence-optimized nucleic acid sequences located at the predefined genomic locations of the isolated cells.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid sequence variants of the multiple donor vectors consist of promoter variants, intron variants, transcriptional regulatory sequence variants, DNA structure regulatory sequence variants, 5' untranslated region variants, 3' untranslated region variants, intra-sequence ribosome entry site variants, target gene variants, nucleic acid sequence variants encoding signal peptides, and / or any combination of such variants.

5. The selection of a sequence-optimized nucleic acid sequence by the selection of cells having the phenotype defined in step v) of claim 1 is the following phenotypic characteristics of the cells: (i) The presence or expression level of endogenous biomolecules in the cells; (ii) Expression level of the target recombinant protein by the cells; (iii) the growth rate of the cells; and / or (iv) Functionality of the recombinant protein that is the target encoded by the nucleic acid sequence region in the cell The method according to any one of claims 1 to 4, comprising a selection based on one or more of the following.

6. The method according to claim 5, wherein the target recombinant protein is a recombinant fusion protein, such as a protein fused to a membrane anchor domain for localization on the cell surface, and / or a fluorescent protein or a protein fused to a fluorescent protein domain.

7. The method according to claim 5, wherein the endogenous biomolecule constitutes a protein, mRNA, miRNA, lncRNA, or metabolite.

8. The method according to claim 5 or 6, wherein when the functionality of the recombinant protein in question is localized and expressed on the cell surface of the cell, the functionality of the recombinant protein in question is measured and determined based on the interaction between the recombinant protein in question and a target structure such as a small molecule, DNA molecule, RNA molecule, protein, viral particle or other protein complex, exosome or cell, and optionally the target structure is tagged with a fluorescent moiety.

9. The method according to any one of claims 5 to 8, wherein the target recombinant protein is a candidate affinity protein, and the level of expression is determined by the presentation of the candidate affinity protein on the cell surface of the cell.

10. The method according to claim 9, wherein the affinity protein candidate is a single-chain polypeptide fused to a membrane anchor domain, and optionally the single-chain polypeptide is selected from the group consisting of Z-scaffold protein, nanobody scaffold protein, single-chain fragment variable (scfv) scaffold protein, finomer scaffold protein, DARPin scaffold protein, and / or adnectin scaffold protein.

11. The method according to claim 9, wherein the affinity protein candidate comprises two or more polypeptide chains, such as an antibody variant, a nucleic acid sequence variant corresponding to the affinity protein candidate encodes the affinity protein candidate variant, such as an antibody variant, and one of the two or more polypeptides is fused to a membrane anchor domain.

12. The method according to any one of claims 9 to 11, further comprising the step of determining the binding specificity, selectivity, affinity and / or functionality of the affinity protein candidate by bringing a specific target component to the affinity protein candidate, which is optionally exposed to the affinity protein candidate and subsequently detected to bind the affinity protein candidate to the specific target component, labeled with a fluorescent marker.

13. The method according to claim 12, wherein the target component is selected from protein complexes, exosomes, or cells such as small molecules, DNA molecules, RNA molecules, proteins, and viral particles, and optionally the target structure is tagged with a fluorescent moiety.

14. The method according to any one of claims 5 to 13, wherein the expression level or functionality of the recombinant protein of interest and / or the presence or level of endogenous biomolecules are measured at the single-cell level, for example, by using flow cytometry.

15. The method according to any one of claims 1 to 14, wherein the nucleic acid sequence region of a donor vector comprises a nucleic acid sequence variant for the expression of one or more recombinant proteins of interest from the donor vector, and the plurality of donor vectors comprises different nucleic acid sequence variants encoding different amino acid sequence variants of the one or more recombinant proteins of interest.

16. The method according to any one of claims 1 to 14, wherein the nucleic acid sequence region of the donor vector includes a nucleic acid sequence variant for the expression of the target recombinant protein from the donor vector, and the nucleic acid sequence variants present in a plurality of donor vectors include nucleic acid sequence variants encoding essentially identical amino acid sequence variants of the target recombinant protein.

17. The method according to any one of claims 1 to 14, comprising a plurality of donor vectors, each comprising a nucleic acid sequence region containing essentially identical nucleic acid sequences, for encoding essentially identical recombinant proteins of interest, wherein the nucleic acid sequence region comprises nucleic acid sequence variants of different donor vector components, such as nucleic acid sequence variants of the promoter or enhancer nucleic acid sequence of the donor vector.

18. The method according to any one of claims 1 to 17, for identifying sequence-optimized nucleic acid donor vector components for use in recombinant protein expression systems based on eukaryotic cell systems.

19. Sequence-optimized nucleic acid sequences selected by the method described in any one of claims 1 to 18.

20. Use of the sequence-optimized nucleic acid sequence according to claim 19 for producing recombinant proteins.

21. Use of the sequence-optimized nucleic acid sequence according to claim 19 for designing further sequence-optimized nucleic acid sequences.

22. Isolated eukaryotic cells having a defined phenotype corresponding to a sequence-optimized nucleic acid sequence that can be obtained by the method described in any one of claims 1 to 21.

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