CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to US Provisional Application No. 62/436,714, filed on December 20, 2016, the content of which is hereby incorporated by reference herein in its entirety.

BACKGROUND

Chinese hamster ovary (CHO) cells are commonly used for producing therapeutic proteins with proper posttranslational modifications such as glycosylation. Traditional random integration cell line development (CLD) method for generating high-producer cells is a time-consuming and labor-intensive process that requires screening of many cells. A basic goal in the development of cell lines for protein expression is to express the protein with high productivity and stability over many generations. Targeted integration (TI) of a transgene into an active and stable chromosomal region is desired for stable expression of recombinant proteins. Ideally, the expression titer and stability of a target integrated cell line should depend mostly on the integration site. Hence, it would only require screening hundreds of cells for high productive clones by using TI-CLD strategy.

SUMMARY

In one aspect, provided herein is an engineered cell. The cell contains an exogenous nucleic acid molecule inserted in the genome of the engineered cell, wherein the engineered cell is obtained by a process that includes introducing into a host cell a construct for inserting the exogenous nucleic acid molecule into a target site within an expression-enhancing sequence in the genome of the host cell, the expression-enhancing sequence being at least 80% identical to a sequence selected from SEQ ID NOs: 1-16 or a fragment thereof. For example, the expression-enhancing sequence can be selected from SEQ ID NOs: 1-16.

In some embodiments, the construct is a homology recombination construct that includes the exogenous nucleic acid molecule flanked by a first homology arm and a second homology arm, the first homology arm being homologous to a sequence upstream of the target site and the second homology arm being homologous to a sequence downstream of the target site.

In some embodiments, the expression-enhancing sequence is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. In some embodiment, the host cell is a CHO cell.

The engineered cell can contains the exogenous nucleic acid molecule at the target site. Alternatively or in addition, the engineered cell contains the exogenous nucleic acid molecule at an off-target site, wherein the engineered cell expresses a higher level of the exogenous nucleic acid molecule as compared to a control cell.

In some embodiments, the exogenous nucleic acid encodes a polypeptide.

In another aspect, described herein is a method of producing an engineered cell that contains an exogenous nucleic acid molecule. The method includes introducing into a host cell a construct for inserting the exogenous nucleic acid molecule into a target site within an expression-enhancing sequence in the genome of the host cell, the expression-enhancing sequence being at least 80% identical to a sequence selected from SEQ ID NOs: 1-16 or a fragment thereof, whereby the exogenous nucleic acid is inserted into a genomic site in the host cell to produce the engineered cell. The exogenous nucleic acid can encode a polypeptide.

In some embodiments, the construct is a homology recombination construct that includes the exogenous nucleic acid molecule flanked by a first homology arm and a second homology arm, the first homology arm being homologous to a sequence upstream of the target site and the second homology arm being homologous to a sequence downstream of the target site.

In some embodiments, the host cell is a CHO cell.

In some embodiments, the expression-enhancing sequence is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

In some embodiments, the method can further include, after the introducing step, selecting an engineered cell that expresses a higher level of the nucleic acid molecule as compared to a control cell.

In yet another aspect, described herein is a construct for inserting an exogenous nucleic acid molecule into a target site within an expression-enhancing sequence in the genome of a host cell, the expression-enhancing sequence being at least 80% identical to a sequence selected from SEQ ID NOs: 1-16 or a fragment thereof. For example, the construct can be a homology recombination construct including a first homology arm that is homologous to a sequence upstream of the target site and a second homology arm that is homologous to a sequence downstream of the target site. In some embodiments, the expression-enhancing sequence is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

In some embodiments, the construct further includes an exogenous nucleic acid molecule flanked by the first homology arm and the second homology arm. The construct can further include a promoter operable linked to the exogenous nucleic acid molecule.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a sequence that includes positions 1001-5537 of SEQ ID NO: 7. In a randomly integrated clone, the sequence between the two black boxes was deleted and the pCHO 1.0 vector with Herceptin gene was inserted therein. Two PAM sequences are boxed and two CRISPR targeting sequences are highlighted in grey. Two homology arms used for target integration of a gene are shown in bold font. The underlined sequences are primer sequences.

Fig. 2 is a sequence that includes positions 1100-5208 of SEQ ID NO: 9. The black box shows the site of integration of a Herceptin gene in a randomly integrated clone. A PAM sequence is boxed. Two homology arms used for target integration of a gene are shown in bold font. The underlined sequences are primer sequences.

FIG. 3 is a graph showing the expression (mg/L/copy) of a Herceptin gene inserted at a Tenm3 site in CHO-S cells. The graph is based on the data shown in Table 2 below.

Fig. 4 is a graph showing the expression (mg/L/copy) of a Herceptin gene inserted at a Tenm3 site in DXB 11 cells. The graph is based on the data shown in Table 3 below. 10P, 50P, and 100P each indicate the concentrations of puromycin and MTX used to select the cells. 10P: ^g/ml puromycin and ΙΟΟηΜ MTX; 50P: 50μg/ml puromycin and 500nM MTX; 100P: 100μg/ml puromycin and ΙΟΟΟηΜ MTX.

Fig. 5 is a graph showing the expression titer (mg/L) of individual clones of DXB 11 cells with a Herceptin gene inserted at a Tenm3 site.

Fig. 6 is a graph showing the expression titer (mg/L) of individual clones of DXB 11 cells with a Herceptin gene inserted at a Sival site.

Fig. 7 is a graph showing the specific productivity (QP; mg/10 ⁶ cells/day) of individual clones of DXB11 cells with a Herceptin gene inserted at a Sival site. DETAILED DESCRIPTION

It was unexpectedly discovered that a gene inserted at a site (i.e., a target site) within certain genomic sequences in CHO cells exhibited enhanced expression. Further, it was found that a gene inserted into an off-target genomic site via a homologous recombination construct designed to specifically insert the gene into one of these genomic sequences also exhibited increased expression. Therefore, these genomic sequences are expression- enhancing sequences.

As used herein, the term "site" in "insertion site", "genomic site", and "target site" refers to a region including one or more nucleotides (e.g., 1 to 500 nucleotides).

The term "exogenous nucleic acid molecule" refers to a nucleic acid molecule that is located at a site in a cell that is not the natural site for the nucleic acid molecule. For example, the nucleic acid molecule may naturally exist in the cell at a different site.

Alternatively, the nucleic acid molecule may originate from a different cell.

Unless otherwise stated, the CHO genome referenced herein refers to the Chinese hamster Jul. 2013 Assembly (C_griseus_vl.0/criGril).

The expression-enhancing sequence can be selected from a sequence that is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99%) identical to a sequence selected from SEQ ID NOs:l-16 or a fragment thereof (e.g., 100-2000, 150-1500, 200-2000, 100-500, 250-500, 200-750, 500-1000, 500-1500, 800-1500, 1000-1500, or 1000-2000 nucleotides). A target site for inserting an exogenous nucleic acid molecule can be located anywhere within or near (e.g., within 500 nucleotides upstream or downstream) the expression-enhancing sequence. In some embodiments, the target site is within positions 1-500, 200-500, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 100-2000, 500-2000, 700-2000, 1000-2000, 500- 3000, 1000-3000, 1500-3000, 2000-3000, 2500-3000, 500-4000, 1000-4000, 2000-4000, 1500-5000, 2500-5000, 3500-5000, 4500-5000, 2000-6000, 3000-6000, 4500-6000, 5000- 6000, or 5500-6000 within SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15.

These expression-enhancing sequences can be used to produce an engineered cell that highly expresses one or more exogenous nucleic acid molecules inserted within the genome of the engineered cell. In some embodiments, the engineered cell is produced by integrating an exogenous nucleic acid into the genome of a CHO cell. The engineered cell exhibits a higher (e.g., one or more folds) expression level of the exogenous nucleic acid molecule as compared to a control cell. A "control cell" can be a cell containing the same nucleic acid molecule inserted at a different site or by random integration. For example, a control cell can be generated by randomly integrating a pCHO 1.0 vector containing the nucleic acid molecule into the genome of a CHO host cell. The CHO Consortium has also identified various potential genomic sites. A control cell can be produced by specifically inserting the nucleic acid molecule into one of these sites. The expression level can be measured at the mRNA level or protein level. As an engineered cell or control cell can contain more than one copy of the inserted nucleic acid molecule, the comparison can be normalized by determining the expression level per copy.

Whether a target site within or near one of the expression-enhancing sequences enhances expression of a nucleic acid molecule can be determined by a skilled practitioner in the art. The precise location of the target site within or near an expression-enhancing sequence is not critical as long as the site can enhance expression and permit stable integration of a nucleic acid molecule. The site selection also depends on the genome editing technique used to insert the gene.

The engineered cell described herein can be obtained by a process that includes introducing into a host cell a construct for inserting the exogenous nucleic acid molecule into a target site within an expression-enhancing sequence in the genome of the host cell.

Although methods and constructs can be used to specifically insert a nucleic acid molecule within one of the expression-enhancing sequences, off-site insertions can nevertheless occur. It was found that engineered cells containing such off-site insertions also exhibited increased expression of the inserted nucleic acid molecules. Without intending to be bound by theory, it is believed that such off-site insertions carry the homology arms (or fragments thereof) in the homology recombination constructs, which are derived from the expression-enhancing sequences. Therefore, the engineered cell described herein can have an exogenous nucleic acid molecule inserted at a target site within one of the expression- enhancing sequences or at a different site.

Various methods can be used to insert an exogenous nucleic acid molecule at a genomic site. Such methods include homologous directed repair, non-homologous end- joining, zinc-finger nuclease (ZFN)-based method, TALEN (Transcription Activator-Like Effector Nuclease)-based method, and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 method. A homology recombination (HR) construct for insertion of an exogenous nucleic acid molecule at a target site within or near one of the expression-enhancing sequences described herein can be designed. The construct includes a first homology arm that is homologous to a sequence upstream of the target site and a second homology arm that is homologous to a sequence downstream of the target site. Each homology arm can include, for example, 200 to 1500 nucleotides (e.g., 200-250, 200-400, 250-500, 300-500, 400-600, 450-650, 500-800, 550-750, 650-900, 800-1000, 950-1200, or 1000-1500 nucleotides). The HR construct can further include multiple cloning sites between the two homology arms such that a gene to be inserted into the genome can be ligated into the construct. Alternatively, an HR construct containing the gene flanked by the two homologous sequences can be constructed using techniques known in the art, e.g., PCR.

The HR construct can be used in a TALEN or CRISPR/Cas9 system to insert a nucleic acid molecule into the genome of a cell.

A target site may be selected depending on the genome editing method used.

TALEN and CRISPR/Cas9 methods both work by introducing a double- stranded DNA break in the genome at a target site. Based on the selected site, an HR construct harboring the nucleic acid molecule to be inserted at the target site can be designed and constructed.

TALEN utilizes a chimeric nuclease that contains an artificial DNA-binding domain of transcription activator-like effector (TALE) proteins and the catalytic domain of restriction endonuclease Fokl. As the code of DNA recognition by TALE proteins has been deciphered, an artificial DNA-binding domain for recognition of any DNA sequence can be designed. To minimize off-site effects, TALEN method can use a pair of chimeric nucleases that each recognizes a sequence on either side of the double-stranded DNA break site. A skilled practitioner would be able to design a TALEN construct directed at the selected site.

CRISPR/Cas9 requires a gRNA specific to the targeted site and the endonuclease Cas9. The target site may be any sequence (about 20 nucleotides) that is unique compared to the rest of the genome and is immediately upstream of a Protospacer Adjacent Motif (PAM). Upon binding of the Cas9/gRNA complex to the target site, Cas9 cleaves the DNA. Two exemplary PAMs within SEQ ID NO: 7 are shown in Fig. 1 and an exemplary PAM within SEQ ID NO: 9 is shown in Fig. 2. A skilled practitioner would be able to design a

CRISPR/Cas9 construct directed at a target site. The exogenous nucleic acid to be inserted can include a sequence encoding a polypeptide operably linked to a promoter that is functional in the engineered cell. The promoter sequence can be endogenous to the coding sequence. In some embodiments, the coding sequence is operably linked to a heterologous promoter sequence. Expression of the exogenous nucleic acid molecule can be further optimized using techniques known in the art. For example, expression can be further enhanced by linking the nucleic acid molecule to a strong promoter and/or one or more transcription enhancer elements.

Integration of the exogenous nucleic acid molecule into the genome of a cell can be verified using methods known in the art. The engineered cells can be cultured under suitable conditions to express the nucleic acid molecule. Whether the engineered cell exhibits enhanced expression can also be determined using methods known in the art, e.g., ELISA, or RT-PCR.

Further, as the expression-enhancing sequences can exert an expression-enhancing effect whether they are at their native genomic loci or at different loci, they can be included in expression vectors for transient expression of genes. For example, an expression vector can contain a gene and one or more expression-enhancing sequences. If more than one expression-enhancing sequences are included, they can be arranged in tandem with or without spacers between them. The vector can be introduced into a host cell to transiently express the gene.

Various host cells known in the art can be used to generate the engineered cells described herein. Such host cells can include any mammalian cells. Preferably, the host cells are CHO cells.

The engineered cells described herein can be used in various commercial and experimental applications. In particular, the cells can be employed for producing therapeutic proteins.

The specific example below is to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent.

EXAMPLE

We previously generated two CHO cell lines, 3C8 and 3G7, by randomly integrating a pCHO 1.0 vector containing the Herceptin gene into the genome of CHO-S host cells. 3C8 and 3G7 respectively harbor 12 and 5 copies of the gene and produce 3g/L and 2.5g/L of the gene product. The integration sites in these two cell lines were analyzed. See Table 1 below. The Srxnl, Adh5, Asphd/Josd2, Tenm3, Sival, Synel, Smarccl, and Rsgl9 sites were located within SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, respectively.

Table 1. Integration sites of 3C8 and 3G7 cell lines

The Tenm3 integration site in the 3C8 cell line is shown in Fig. 1. In the 3C8 cell line, the sequence between the two black boxes was deleted and the pCHO 1.0 vector with Herceptin gene was inserted therein. The integration site within Sival in the 3G7 cell line is shown in Fig. 2.

We tested the Tenm3 integration site by inserting a Herceptin gene into the genome of CHO host cells using CRISPR. Referring to Fig. 1, the two CRISPR targeting sequences used are highlighted in grey and the PAMs are boxed. The upstream and downstream homology sequences used for integrating the Herceptin gene are shown in bold font in Fig. 1.

A CRISPR vector and a homology recombination donor vector were introduced into CHO-S and DXB11 host cells. The cells were sorted and recovered 48 hours after transfection. Different concentrations of puromycin (10, 50 or 100 μg/ml) and MTX (100, 500, or 1000 nM) were used to selected cells containing integrated genes. See Tables 2 and 3 below. FACS or limited dilution was used to select single cells to establish single cell cultures. Integration of the Herceptin gene into the pre-selected site was verified using the T7E1 assay and junction-PCR assay.

Expression of the inserted Herceptin gene in the pooled CHO cells selected by puromycin and MTX was assayed using ELISA. We generated three controls by inserting the gene into each of three active integration sites previously identified by the CHO

Consortium (control 1, control 2, and control 3). The expression titer per copy (mg/L/copy) of the gene inserted within the Tenm3 integration site was significantly higher than the three controls. See, Table 2, Table 3, Fig. 3, and Fig. 4.

Table 2. Targeted integration at Tenm3 site in CHO-S host cells

Table 3. Targeted integration at Tenm3 site in DXBll host cells

We tested single clones derived from DXB 11 cells in 6-day batch cultures and found that they exhibited enhanced expression of the inserted gene. See Fig. 5. In particular, the clones DXB11-1E8, DXB11-1E2, and DXB11-1G5 could be cultured for 60 generations without losing the enhanced expression.

We also inserted the Herceptin gene into the Sival, Synel, Smarccl, and Rgsl9 sites in DXBll host cells using CRISPR to generate engineered cells. 750 μg/ml of geneticin without MTX was used to select cells with the desired insertion. This condition selected cells with a low copy number of the insertion. Despite the low copy number, these engineered cells also showed increased expression of the gene as compared to cells generated by random integration (titer = about 80 mg/L). See Table 4. In particular, the selected cell pool generated by targeting the Sival site had a titer of 235 mg/L/copy. Fig. 2 shows the PAM sequence and the two homology arms used to insert the gene into a Sival site.

Table 4. Targeted integration at Sival, Synel, Smarccl, and Rgsl9 sites in DXBl l host cells

Sival Synel Smarccl Rgsl9 mg/L 134.01 117.44 123.71 146.71 mg/L/copy 235 135 169 177 Individual clones from the Sival pool were also tested. As shown in Figs. 6 and 7, these clones all exhibited increased expression of the Herceptin gene. Analysis showed that, among these clones, although some had off-target insertions and some had multiple copies of the insertions, many had only one copy of an on-target insertion.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.