Antiviral cells and uses thereof

Abstract

The present invention provides a mammalian cell line that has been genetically engineered to make it resistant to viral entry and / or replication; and provides a method for using the cell line to reduce or prevent viral contamination of a bioproduction system.

Description

[0001] This application is a divisional application with the same title as the parent invention. The parent invention's Chinese application number is 201580022279.6, its international application number is PCT / US2015 / 018474, and its application date is March 3, 2015. Technical Field

[0002] This invention relates to engineered mammalian cell lines with antiviral properties and the use of said cell lines to reduce or prevent viral contamination in bioproduction systems. Background Technology

[0003] The use of recombinant therapeutic proteins for treating a wide range of diseases or conditions, such as cancer and autoimmune diseases, is increasing. However, large-scale production of these protein therapeutics remains a challenge. For example, reliable high yields must be achieved in industrial manufacturing processes so that downstream processes produce extremely pure products, allowing only trace amounts of contaminants to preferably be contaminant-free.

[0004] The use of animal-free culture media has significantly reduced the incidence of foreign viral contamination. Furthermore, procedures such as ultrafiltration, short-time high-temperature treatment, and / or UVC irradiation of bulk materials have further reduced the contamination rate. However, the risk of viral contamination remains. A contamination incident can be catastrophic for manufacturers in terms of product loss, temporary withdrawal from the market, and costly decontamination. Therefore, mammalian cell lines with increased resistance to viral infection are needed. Attached Figure Description

[0005] Figure 1 The temporal course of MVM virus infection, as determined by Southern blot analysis, is presented. The relative amounts of MVM virus DNA detected at 24, 48, 72, and 96 hours were plotted against wild-type cells (CHOZN GS- / -), Slc35A1 KO, COSMC KO, COSMC KO clone F07, COSMC KO clone G03, and COSMC KO clone H05. Clone H05 has a 12 bp in-frame deletion (i.e., no frameshift mutation).

[0006] Figure 2 The time course of MVM virus infection as detected by plaque assay is shown. Plots are of pfu / mL detected at 24, 48, 72, and 96 hours for wild-type cells (CHOZN), Slc35A1 KO, COSMC KO, COSMC KO clone F07, COSMC KO clone G03, and COSMC KO clone H05.

[0007] Figure 3The effect of MMV infection on cell growth is shown. The graph shows the number of viable cells (cells / ml) in wild-type (2E3) (A) and COSMC KO clone F07 (B) cells over a 120-hour period in the absence of MVM virus (solid line) and in the presence of MOI 1 or 8 (broken line).

[0008] Figure 4 The timeline of cell-associated virus (CAV) infection following MMV infection is presented. The plot shows the MVM virus genome copy number (vgc) per cell (based on the assumption that each infected cell can produce 2 x 10^6 copies / cells). 4 vgc), as detected by qPCR over a 120-hour period in the presence of MVM virus in MOI 1(A) or 8(B) against wild-type (2E3) (achieved) and COSMC KO clone F07 (disconnected) cells.

[0009] Figure 5 The time course of MMV replication in the indicated cell lines is shown. The plots are of vgc / samples at 0 and 21 hours post-infection with MVM virus at MOI 0.3 (A) or 0.03 (B).

[0010] Figure 6 The temporal progression of reovirus 3 replication in the indicated cell lines is presented. The plot shows the vgc / sample associated with wild-type cells (2E3) at 0 and 24 hours post-Reo-3 infection.

[0011] Figure 7 Growth assays for cell growth were presented in the absence (UN) or presence (IN) of MVM virus. Figure A shows the viable cell density (VCD) of wild-type (GS), Slc35A1 KO, COSMC KO clone F07, and COSMC KO clone G03 over 10 days. Figure B shows the VCD of wild-type (GS), IgG-producing clones 71H1 and 71C3 derived from COSMC KO clone F07 over 8 days.

[0012] Figure 8 Growth assays of cells in the absence (UN) or presence (IN) of MVM virus are presented. The plots show VCDs of wild-type (GS), St3Gal4 KO clone 7D10, St3Gal4 KO clone 1B08, and St3Gal4 KO clone 1B10 over 9 days. Summary of the Invention

[0013] In various aspects of this disclosure, mammalian cell lines engineered to inhibit or prevent viral entry and / or viral replication within cells are provided. In some embodiments, the mammalian cell lines are genetically modified such that the entry and / or replication of at least one virus is reduced or eliminated compared to an unmodified parental cell line. In various embodiments, the mammalian cell lines disclosed herein comprise at least one modified chromosomal sequence. In other embodiments, the modified chromosomal sequence is inactivated, such that the cell lines do not produce or produce reduced levels of encoded protein products.

[0014] In one embodiment, the cell line comprises at least one inactivated chromosomal sequence encoding a Core 1 enzyme chaperone (COSMC). In another embodiment, all copies of the chromosomal sequence encoding COSMC are inactivated and the cell line does not produce COSMC. In an alternative embodiment, the cell line comprises at least one inactivated chromosomal sequence encoding a solute carrier family 35 (CMP-sialic acid transporter) member A1 (Slc35A1). In another embodiment, all copies of the chromosomal sequence encoding Slc35A1 are inactivated and the cell line does not produce Slc35A1. In yet another embodiment, the cell line comprises at least one inactivated chromosomal sequence encoding a Core 1 elongase (C1GalT1). In another embodiment, all copies of the chromosomal sequence encoding C1GalT1 are inactivated and the cell line does not produce C1GalT1. In yet another embodiment, the cell line comprises at least one inactivated chromosomal sequence encoding St3β-galactosidase α-2,3-sialic acid transferase 1 (St3Gal1). In another embodiment, all copies of the chromosome sequence encoding St3Gal1 are inactivated and the cell line does not produce St3Gal1. In an alternative embodiment, the cell line comprises at least one inactivated chromosome sequence encoding St3β-galactosidase α-2,3-sialyltransferase 2 (St3Gal2). In another embodiment, all copies of the chromosome sequence encoding St3Gal2 are inactivated and the cell line does not produce St3Gal2. In yet another embodiment, the cell line comprises at least one inactivated chromosome sequence encoding St3β-galactosidase α-2,3-sialyltransferase 3 (St3Gal3). In another embodiment, all copies of the chromosome sequence encoding St3Gal3 are inactivated and the cell line does not produce St3Gal3. In yet another embodiment, the cell line comprises at least one inactivated chromosome sequence encoding St3β-galactosidase α-2,3-sialyltransferase 4 (St3Gal4). In another embodiment, all copies of the chromosome sequence encoding St3Gal4 are inactivated and the cell line does not produce St3Gal4. In an alternative embodiment, the cell line comprises at least one inactivated chromosome sequence encoding St3β-galactosidase α-2,3-sialyltransferase 5 (St3Gal5). In another embodiment, all copies of the chromosome sequence encoding St3Gal5 are inactivated and the cell line does not produce St3Gal5. In yet another embodiment, the cell line comprises at least one inactivated chromosome sequence encoding St3β-galactosidase α-2,3-sialyltransferase 6 (St3Gal6).In another embodiment, all copies of the chromosome sequence encoding St3Gal6 are inactivated and the cell line does not produce St3Gal6. In an alternative embodiment, the cell line comprises inactivated chromosome sequences encoding any two of St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and St3Gal6. In yet another alternative embodiment, the cell line comprises inactivated chromosome sequences encoding any two of St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and St3Gal6. In other embodiments, cell lines comprising inactivated chromosomal sequences encoding COSMC, Slc35A1, C1GalT1, St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 further comprise sequences encoding mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine transferase 1 (Mgat1) and mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase 1 (Mgat1). Inactivated chromosomal sequences of α-1,2-N-acetylglucosamine transferase 2 (Mgat2), mannosyl (α-1,4-)-glycoprotein β-1,4-N-acetylglucosamine transferase 3 (Mgat3), mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosamine transferase 4 (Mgat4), and / or mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosamine transferase 5 (Mgat5). The mammalian cell lines containing the modified chromosomal sequences disclosed herein exhibit increased resistance to viral entry and / or viral replication compared to unmodified parental cell lines.In yet another embodiment, a cell line comprising an inactivated chromosomal sequence encoding St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 is further engineered to overexpress at least one sialyltransferase responsible for producing 2,6-linked sialyltransferases (e.g., St6β-galactosamide α-2,6-sialyltransferase 1 (St6Gal1), St6β-galactosamide α-2,6-sialyltransferase 2 (St6Gal2), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 1 (St6GalNac1 ...amide 1,3)-N-acetylgalactosamide 1 (St6GalNac1), St6(α-N-acetyl-ceramide-2,3-β-galactosamide 1,3)-N-acetylgalactosamide 1 (St6GalNac1), St6(α-N-acetyl-ceramide-2,3-β-galactosamide 1,3)-N-acetylgalactosamide 1,3-galactosamide 1,3) Lactosyl-1,3)-N-acetylgalactosamide 2 (St6GalNac2), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 3 (St6GalNac3), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 4 (St6GalNac4), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 5 (St6GalNac5) and / or St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 6 (St6GalNac6).

[0015] In some embodiments, the mammalian cell line is a non-human cell line. In other embodiments, the mammalian cell line is the Chinese hamster ovary (CHO) cell line. In many specific embodiments, the cell line is a CHO cell line comprising inactivated chromosomal sequences encoding COSMC, Slc35A1, C1GalT1, St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, St3Gal6, or combinations thereof.

[0016] In some embodiments, the mammalian cell lines disclosed herein exhibit resistance to the entry and / or replication of viruses selected from parvoviruses, reoviruses, rotaviruses, influenza viruses, adeno-associated viruses, caliciviruses, parainfluenza viruses, rubella viruses, coronaviruses, noroviruses, encephalocardiitis viruses, polyomaviruses, or combinations thereof. In some embodiments, the parvovirus is mouse parvovirus (MVM), mouse parvovirus type 1, mouse parvovirus type 2, mouse parvovirus type 3, porcine parvovirus 1, bovine parvovirus 1, human parvovirus B19, human parvovirus 4, human parvovirus 5, or combinations thereof. In other embodiments, the reovirus is mammalian reovirus 3, mammalian ororeovirus, avian ororeovirus, or combinations thereof.

[0017] In various embodiments, the mammalian cell lines disclosed herein are prepared by modifying at least one chromosomal sequence using a targeted endonuclease-mediated genome modification technique. The targeted endonuclease may be a zinc finger nuclease, a CRISPR / Cas endonuclease, a transcription activator-like effector (TALE) nuclease, a broad-spectrum nuclease, a site-specific endonuclease, or an artificially targeted DNA double-strand break inducer. In many specific embodiments, the targeted endonuclease is a pair of zinc finger nucleases.

[0018] In some embodiments, the mammalian cell lines disclosed herein also comprise at least one nucleic acid encoding a recombinant protein selected from antibodies, antibody fragments, vaccines, growth factors, cytokines, hormones, coagulation factors, or another therapeutic protein.

[0019] Another aspect of this disclosure covers a method for reducing or preventing viral contamination of recombinant protein products, the method comprising: obtaining an antiviral mammalian cell line as disclosed herein, and expressing the recombinant protein product in the cell line.

[0020] Another aspect of this disclosure provides a method for reducing the risk of viral contamination in a bioproduction system, wherein the method includes providing an antiviral mammalian cell line as disclosed herein for use in the bioproduction system.

[0021] Another aspect of this disclosure covers a composition comprising an antiviral mammalian cell line as disclosed herein and at least one virus, wherein the cell line exhibits resistance to infection caused by said at least one virus.

[0022] Other aspects and iterations of this disclosure are described in more detail below. Detailed description

[0023] This disclosure provides mammalian cell lines engineered to inhibit or prevent viral entry and / or viral replication in the cell line. The engineered cell lines with antiviral properties can be genetically modified to contain modified (e.g., inactivated) chromosomal sequences. Methods for producing recombinant proteins using the cell lines disclosed herein are also provided, wherein the recombinant protein product is substantially free from viral contamination. Therefore, using cell lines resistant to viral infection reduces or eliminates the risk of viral contamination of the bioproduction system and the resulting protein product.

[0024] (I) Antiviral cell lines

[0025] One aspect of this disclosure covers mammalian cell lines engineered to possess viral resistance. In other words, the cell lines disclosed herein exhibit increased resistance to infections caused by at least one virus compared to unmodified parental cell lines. More specifically, viral entry and / or viral replication are reduced or eliminated in the engineered cell lines disclosed herein compared to unmodified parental cell lines. In some embodiments, the mammalian cell line is genetically modified and contains at least one modified chromosomal sequence. Generally, the modified sequence contains a mutation. In many specific embodiments, the modified chromosomal sequence is inactivated (or knocked out), such that the cell line does not produce protein-coding products.

[0026] Generally, resistance (or susceptibility) to viral infection can be determined by comparing the response of engineered mammalian cell lines to viral exposure with the response of unmodified (unengineered) parental cells to the same viral challenge. Viral infection and / or viral proliferation in cell lines can be analyzed using a variety of techniques. Non-limiting examples of suitable techniques include nucleic acid assays (e.g., Southern blotting for detecting the presence of specific viral nucleic acids, PCR or RT-PCR for detecting viral nucleic acids, sequencing, etc.), antibody-based techniques (e.g., Western immunoblotting using antiviral protein antibodies, ELISA, etc.), bioassays (e.g., dot assays, cytopathic effect assays, etc.), and microscopy techniques (e.g., electron microscopy for detecting viral particles, etc.). In some embodiments, viral infection and / or proliferation in engineered mammalian cell lines can be reduced by at least about 10%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 90%, at least about 99%, or more than about 99% relative to unmodified parental cells. In many specific implementation schemes, the engineered mammalian cell lines are resistant to viral infection, meaning that viruses cannot enter and / or multiply in the engineered mammalian cell lines.

[0027] The mammalian cell lines disclosed herein can be engineered in a variety of different ways to confer viral resistance. In some embodiments, the cell line may be modified to reduce or eliminate viral entry via cell surface receptors. In other embodiments, the cell line may be engineered to express molecules that inhibit or block specific viral proteins involved in replication and / or infectivity. In still other embodiments, the cell line may be engineered to overexpress specific cellular antiviral proteins. In some embodiments, the engineering may be genetic engineering, wherein the genome or chromosome sequence is modified. In other words, the cell line is genetically modified. In other embodiments, the engineering may be epigenetic engineering or extrachromosomal engineering.

[0028] (a) Antiviral mechanism

[0029] (i) Disruption of cell surface receptors

[0030] In some embodiments, the mammalian cell line is engineered to contain altered cell surface receptors. Viruses can enter cells by specifically attaching to complementary receptors on the cell surface. For many viruses, these cell surface receptors comprise a glycan structure linked to a protein (or lipid). Terminal glycans in sialic acid or its derivatives act as receptors for many viruses (Matrosovich et al., 2013, Top Curr Chem, DOI:10.1007 / 128_2013_466). Therefore, glycoproteins containing O-linked or N-linked glycans with terminal sialic acid residues can act as cell surface receptors for many viruses. Sialic acid refers to derivatives of neuraminic acid and includes, for example, N-acetylneuraminic acid (Neu5Ac or NANA) and N-hydroxyacetylneuraminic acid (Neu5Gc or NGNA).

[0031] In some embodiments, the cell line is engineered to contain glycoproteins lacking terminal sialic acid residues. Terminal sialic acid residues can be eliminated by deletion (i.e., knockout) or alteration of enzymes and / or proteins involved in glycan chain synthesis. Suitable targets include enzymes or proteins involved in the synthesis of O-linked glycans, enzymes or proteins involved in the synthesis of N-linked glycans, and / or enzymes or proteins involved in the synthesis or transport of sialic acid.

[0032] In some implementations, the cell line may lack at least one of the target enzymes or proteins mentioned above (or any combination of the targets mentioned above). As used herein, “lack” means a reduced or undetectable level of the target enzyme or protein, or a reduced or undetectable activity of the target enzyme or protein. The amount or activity of the target enzyme or protein can be reduced or eliminated by modifying at least one chromosomal sequence encoding the target protein or enzyme. For example, the chromosomal sequence can be modified to contain the deletion of at least one nucleotide, the insertion of at least one nucleotide, the substitution of at least one nucleotide, or a combination thereof. Thus, deletions, insertions, and / or substitutions can shift the reading frame of the chromosomal sequence so that no protein product is produced (i.e., the chromosomal sequence is inactive). Alternatively, deletions, insertions, and / or substitutions in the modified chromosomal sequence may result in the production of an altered protein product. Modification of the chromosomal sequence of interest can be achieved using the targeted endonuclease-mediated genome editing techniques detailed in section (III)(a) below. In the case of inactivation of a chromosomal sequence encoding a target enzyme or protein, the engineered cell line produces a lower level of the target enzyme or protein. In other cases where all copies of the chromosomal sequence encoding the target enzyme or protein are inactivated, the engineered cell line does not produce the target enzyme or protein (i.e., the cell line is knocked out or KO). In yet another embodiment, the level of the target enzyme or protein may be reduced or eliminated using an RNA interference-mediated mechanism detailed in section (III)(b) below.

[0033] In some embodiments, the level of the targeted enzyme or protein can be reduced by at least about 5%, about 5% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90%, or about 90% to about 100%. In other embodiments, the level of the targeted enzyme or protein can be reduced to a level undetectable using standard techniques in the art (e.g., Western immunoblotting, ELISA enzyme assays, etc.).

[0034] In some embodiments, the cell line may lack enzymes or proteins involved in O-linked glycosylation. For example, the cell line may lack core 1 elongase (also known as core 1 synthase glycoprotein-N-acetylgalactosamide 3-β-galactosidase 1 or C1GalT1), core 1 enzyme companion protein (also known as C1GalT1-specific companion protein or COSMC), or both. COSMC promotes the folding, stability, and activity of C1GalT1, thereby catalyzing the transfer of galactose residues to O-linked N-acetylgalactosamide (GalNAc) residues on a serine or threonine residue of the protein. In many specific embodiments, the cell line lacks C1GalT1, COSMC, or both. This deficiency may be due to inactivated chromosomal sequences encoding C1GalT1 and / or COSMC, resulting in lower levels of C1GalT1 and / or COSMC proteins or no production of C1GalT1 and / or COSMC proteins. In some cases, at least one chromosomal sequence encoding C1GalT1 and / or COSMC is inactivated. In other cases, all copies of the chromosomal sequence encoding C1GalT1 and / or COSMC are inactivated, resulting in the cell line lacking C1GalT1 and / or COSMC proteins.

[0035] In other embodiments, the cell line may lack at least one sialyltransferase (ST). This sialyltransferase may be a sialyltransferase that adds sialic acid to galactose in an α-2,3-linked conformation, a sialyltransferase that adds sialic acid to galactose or N-acetylgalactosamide in an α-2,6-linked conformation, or a sialyltransferase that adds sialic acid to other sialic acid units in an α-2,8-linked conformation. Suitable non-restrictive examples of sialyltransferases include St3β-galactoside α-2,3-sialyltransferase 1 (St3Gal1), St3β-galactoside α-2,3-sialyltransferase 2 (St3Gal2), St3β-galactoside α-2,3-sialyltransferase 3 (St3Gal3), St3β-galactoside α-2,3-sialyltransferase 4 (St3Gal4), St3β-galactoside α-2,3-sialyltransferase 5 (St3Gal5), St3β-galactoside α-2,3-sialyltransferase 6 (St3Gal6), St 6β-galactoside α-2,6-sialyltransferase 1 (St6Gal1), St6β-galactoside α-2,6-sialyltransferase 2 (St6Gal2), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactamide 1 (St6GalNac1), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactamide 2 (St6GalNac2 ...2 (St6GalNac2), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactamide 2 (St6GalNac2), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-acetylgalactamide 2 (St6GalNac2), St6(α-N-acetyl-cer Acylgalactosamide 3 (St6GalNac3), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 4 (St6GalNac4), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 5 (St6GalNac5), St6(α-N-acetyl-ceramide-2,3-β-galactosyl-1,3)-N-acetylgalactosamide 6 (St6GalNac6), St8α-N-acetyl-ceramide α-2,8 - Sialyltransferase 1 (St8Sia1), St8α-N-acetyl-neuraminidine α-2,8-sialyltransferase 2 (St8Sia2), St8α-N-acetyl-neuraminidine α-2,8-sialyltransferase 3 (St8Sia3), St8α-N-acetyl-neuraminidine α-2,8-sialyltransferase 4 (St8Sia4), St8α-N-acetyl-neuraminidine α-2,8-sialyltransferase 5 (St8Sia5) or St8α-N-acetyl-neuraminidine α-2,8-sialyltransferase 6 (St8Sia6).

[0036] In many specific embodiments, the cell line may lack at least one α-2,3-sialyltransferase (e.g., St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6). This deficiency may be due to an inactivated chromosomal sequence encoding at least one α-2,3-sialyltransferase, resulting in lower levels of that at least one α-2,3-sialyltransferase or no production of that α-2,3-sialyltransferase. In some cases, at least one chromosomal sequence encoding that at least one α-2,3-sialyltransferase may be inactivated. In other cases, all copies of the chromosomal sequence encoding that at least one α-2,3-sialyltransferase may be inactivated, resulting in the cell line lacking that at least one α-2,3-sialyltransferase. In other words, the chromosomal sequences encoding St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 may be knocked out.

[0037] In some cases where the cell line contains at least one inactivated chromosomal sequence encoding at least one α-2,3-sialyltransferase (e.g., St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6), the cell line can be further engineered to overexpress at least one 2,6-linked sialyltransferase responsible for producing it (e.g., St6Gal1, St6Gal2, St6GalNac1, St6GalNac2, St6GalNac3, St6GalNac4, St6GalNac5, and / or St6GalNac6). Therefore, such cell lines can contain glycoproteins with a reduced number (or absence) of terminal 2,3-linked sialyl residues and an increased number of terminal 2,6-linked sialyl residues.

[0038] In other embodiments, the cell line may lack at least one enzyme or protein involved in sialic acid synthesis or transport. Examples of enzymes or proteins involved in sialic acid synthesis or transport include, but are not limited to, glucosamine (UDP-N-acetyl)-2-epiisomerase / N-acetylmannosamine kinase (GNE), N-acetylneuraminic acid synthase (NANS), N-acetylneuraminic acid phosphatase (NANP), cytidine monophosphate N-acetylneuraminic acid synthase (CMAS), and cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH), and solute carrier family 35 (CMP-sialic acid transporter) member A1 (Slc35A1). In some embodiments, the cell line may lack Slc35A1, which transports CMP-sialic acid to the Golgi apparatus. In some cases, at least one chromosomal sequence encoding Slc35A1 may be inactivated. In other cases, all copies of the chromosomal sequence encoding Slc35A1 may be inactivated, making the cell lack the Slc35A1 protein.

[0039] In another embodiment, the cell line may lack at least one enzyme or protein involved in N-glycosylation. In some cases, the enzyme or protein involved in N-glycosylation may be an N-acetylglucosamine transferase that adds GlcNAc residues to the β-linked mannose residues of an N-linked glycan. Examples include mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosamine transferase 1 (Mgat-1), mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase 2 (Mgat-2), mannosyl(α-1,4-)-glycoprotein β-1,4-N-acetylglucosamine transferase 3 (Mgat-3), mannosyl(α-1,3-)-glycoprotein β-1,4-N-acetylglucosamine transferase 4 (Mgat-4), and mannosyl(α-1,6-)-glycoprotein β-1,6-N-acetylglucosamine transferase 5 (Mgat-5). In other cases, the enzyme or protein involved in N-glycosylation can be a galactosyltransferase, which adds a galactose residue in a β-1,4 bond to the GlcNAc residue of an N-linked glycan. The galactosyltransferase can be UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 1 (B4GalT1), UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 2 (B4GalT2), UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 3 (B4GalT3), UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 4 (B4GalT4), UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 5 (B4GalT5), UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 6 (B4GalT6), or UDP-Gal:βGlcNAc β 1,4-galactosyltransferase polypeptide 7 (B4GalT7). In some cases, the cell line may lack at least one N-acetylglucosamine transferase and / or galactosyltransferase. In some iterations, at least one chromosomal sequence encoding at least one N-acetylglucosamine transferase and / or galactosyltransferase may be inactivated. In other cases, all copies of the chromosomal sequence encoding the at least one N-acetylglucosamine transferase and / or galactosyltransferase may be inactivated, resulting in the cell line lacking the at least one N-acetylglucosamine transferase and / or galactosyltransferase.

[0040] (ii) Interfering with viral proteins

[0041] In other embodiments, the mammalian cell line may be engineered to express molecules that inhibit or block viral replication and / or infectivity. For example, the cell line may be engineered to stably express at least one RNA interference (RNAi) agent targeting a specific viral protein involved in replication and / or infectivity. Non-limiting examples of suitable viral proteins include non-structural proteins such as NS1 or NS2, and capsid proteins such as VP1 or VP2. The RNAi agent binds to the target transcript and prevents protein expression by mediating transcript cleavage or disrupting transcript translation.

[0042] In some embodiments, the RNAi agent may be a short interfering RNA (siRNA). Generally, siRNA comprises a double-stranded RNA molecule ranging from about 15 to about 29 nucleotides in length, or more generally, from about 19 to about 23 nucleotides in length. In many specific embodiments, the siRNA may be about 21 nucleotides in length. The siRNA may optionally further comprise one or two single-stranded overhangs, such as 3' overhangs at one or both ends. The siRNA may be formed from two RNA molecules hybridized together, or alternatively, may be generated from short hairpin RNA (shRNA) (see below). In some embodiments, the two strands of the siRNA may be completely complementary, such that there are no mismatches or bulges in the duplex formed between the two sequences. In other embodiments, the two strands of the siRNA may be substantially complementary, such that there are one or more mismatches and / or bulges in the duplex formed between the two sequences. In some embodiments, one or both of the 5' ends of the siRNA may have phosphate groups, while in other embodiments, one or both of the 5' ends may lack phosphate groups.

[0043] One strand of the siRNA, referred to as the "antisense strand" or "guide strand," includes a portion capable of hybridizing with the target transcript. In some embodiments, the antisense strand of the siRNA may be completely complementary to a region of the target transcript, i.e., it hybridizes with the target transcript without a single mismatch or bulge along the entire length of the siRNA. In other embodiments, the antisense strand may be substantially complementary to the target region, i.e., one or more mismatches and / or bulges may be present in the duplex formed by the antisense strand and the target transcript. Typically, the siRNA targets the exon sequence of the target transcript. Those skilled in the art will be familiar with procedures, algorithms, and / or commercial services for designing siRNAs targeting target transcripts.

[0044] In other embodiments, the RNAi agent may be short hairpin RNA (shRNA). Generally, shRNA is an RNA molecule comprising at least two complementary portions of a double-stranded structure of sufficient length to mediate RNA interference (as described above) and at least one single-stranded portion forming a loop connecting the shRNA region forming the double strand. This structure may also be referred to as a stem-loop structure, where the stem is the double-stranded portion. In some embodiments, the double-stranded portions of the structure may be completely complementary, such that there are no mismatches or bulges in the double-stranded region of the shRNA. In other embodiments, the double-stranded portions of the structure may be substantially complementary, such that one or more mismatches and / or bulges may be present in the double-stranded portion of the shRNA. The loop of the structure may be about 1 to about 20 nucleotides long, specifically about 6 to about 9 nucleotides long. The loop may be located at the 5' or 3' end of a region complementary to the target transcript (i.e., the antisense portion of the shRNA).

[0045] The shRNA may further include a dangling splint at the 5' or 3' end. This optional splint may be about 1 to about 20 nucleotides long, or more specifically, about 2 to about 15 nucleotides long. In some embodiments, the splint may contain one or more U residues, for example, between about 1 and about 5 U residues. In some embodiments, the 5' end of the shRNA may have a phosphate ester group. Generally, shRNA is processed into siRNA via conserved cellular RNAi mechanisms. Therefore, shRNA is a precursor to siRNA and similarly capable of inhibiting the expression of target transcripts complementary to a portion of the shRNA (i.e., the antisense portion of the shRNA). Those skilled in the art will be familiar with the available resources for designing and synthesizing shRNA. An exemplary example is... shRNA (Sigma-Aldrich).

[0046] siRNA or shRNA can be expressed in vivo using RNAi expression constructs. Suitable constructs include plasmid vectors, phagemids, granules, artificial / miniature chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated virus vectors, etc.). In one embodiment, the RNAi expression construct can be a plasmid vector (e.g., pUC, pBR322, pET, pBluescript, and variants thereof). The RNAi expression construct can contain two promoter control sequences, each operatively linked to an appropriate coding sequence, such that two independent complementary siRNA strands can be transcribed. The two promoter control sequences can be in the same or opposite orientation. In another embodiment, the RNAi expression vector can contain a promoter control sequence that drives the transcription of a single RNA molecule containing two complementary regions, thereby forming shRNA from the transcript. Generally, the promoter control sequence will be an RNA polymerase III (PolIII) promoter, such as the U6 or H1 promoter. In other embodiments, an RNA polymerase II (PolII) promoter control sequence can be used (some examples are given below). RNAi expression constructs can contain additional sequence elements, such as transcription termination sequences, optional marker sequences, etc. RNAi expression constructs can be introduced into cell lines of interest using standard procedures. The RNAi expression construct can be integrated into the chromosome of the cell line for stable expression. Alternatively, the RNAi expression construct can be expressed extrachromosomally (e.g., in an appendage form) for stable expression in the cell line.

[0047] In other embodiments, the cell line can be engineered to stably express dominant-negative forms of viral proteins involved in replication and / or infectivity. The dominant-negative form of the protein is altered or mutated to make it outperform or suppress the wild-type protein. Non-limiting examples of suitable proteins include viral non-structural proteins, such as NS1 or NS2, and viral capsid proteins, such as VP1 or VP2. In many specific embodiments, the cell line can be engineered to express dominant-negative forms of one or more NS1 proteins.

[0048] Dominant-negative proteins, relative to wild-type proteins, can have deletions, insertions, and / or substitutions (Lagna et al., 1998, Curr. TopicsDev.Biol, 36:75-98). Deletions, insertions, and / or substitutions can occur at the N-terminus, C-terminus, or internal locations of the protein. Methods for generating mutant proteins (by site-directed mutagenesis, PCR-based mutagenesis, random mutagenesis, etc.) are well known in the art, as are methods for identifying those with dominant-negative effects. Cell lines can be transfected with expression constructs (see above) containing a sequence encoding a dominant-negative protein, wherein the coding sequence is operatively linked to a PolII promoter control sequence for expression. The promoter control sequence can be constitutive, regulatory, or tissue-specific.

[0049] Suitable constitutive promoter control sequences include, but are not limited to, the cytomegalovirus immediate early promoter (CMV), the simian virus (SV40) promoter, the adenovirus major late promoter, the Rous sarcoma virus (RSV) promoter, the mouse mammary tumor virus (MMTV) promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor (ED1)-α promoter, the ubiquitin promoter, the actin promoter, the microtubule promoter, the immunoglobulin promoter, fragments thereof, or combinations thereof. Examples of suitable regulatory promoter control sequences include, but are not limited to, those regulated by heat shock, metals, steroids, antibiotics, or alcohols. Non-restricted examples of tissue-specific promoters include the B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase 1 promoter, endothelial glycoprotein promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter. The promoter sequence can be wild-type or can be modified to achieve more efficient or effective expression.

[0050] This expression construct may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), optional marker sequences (e.g., antibiotic resistance genes), origin of replication, etc. Additional information can be found in "Current Protocols in Molecular Biology," Ausubel et al., John Wiley & Sons, New York, 2003; or "Molecular Cloning: A Laboratory Manual," Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0051] (iii) Overexpression of cellular proteins involved in antiviral responses

[0052] In many alternative embodiments, the mammalian cell line can be engineered to overexpress cellular proteins involved in the host cell's antiviral response. Non-limiting examples of proteins involved in the antiviral response include double-stranded RNA-activated protein kinase R (PKR, also known as eukaryotic translation initiation factor 2-α kinase 2 or Eif2ak2), receptor-interacting protein kinase 2 (RIPK2), interferons (e.g., type I and type II), interleukins (e.g., IL-1 and IL-6), tumor necrosis factor α, interferon regulator 1, STAT, p53, activating transcription factor 3, NF-κB, eukaryotic initiation factor 2 (eIF2), inhibitor of apoptosis proteins (IAP), and zinc finger antiviral proteins (ZAP). In many specific embodiments, the cell line can be engineered to overexpress PKR.

[0053] Overexpression can be achieved by introducing one or more exogenous copies of the nucleic acid sequence encoding the protein of interest. The sequence encoding the cellular protein of interest is generally operatively linked to a PolII promoter control sequence (see above). Multiple copies of this coding sequence can be tandemly linked and placed under the control of a single promoter control sequence. The sequence encoding the protein of interest can be introduced into the cell line as part of an expression construct (see above). Therefore, the expression construct can be inserted into a chromosomal location, or alternatively, it can be extrachromosomal (e.g., epigenetic) for stable expression.

[0054] Overexpression can also be achieved by modifying the promoter control sequence of the endogenous chromosomal sequence encoding the protein of interest. For example, the endogenous promoter control sequence can be modified by inserting at least one exogenous “strong” promoter control sequence (i.e., one with high affinity for RNA polymerase and / or related factors) (examples of which are presented above). Alternatively, the sequence of the endogenous promoter control sequence can be modified to mimic a “strong” promoter control sequence. The endogenous chromosomal sequence can be modified using targeted endonuclease-mediated genome modification techniques detailed in section (III) below.

[0055] (b) Cell type

[0056] The antiviral cell lines disclosed herein are mammalian cell lines. In some embodiments, cell lines resistant to viral infection may be derived from: Chinese hamster ovary (CHO) cells; mouse myeloma NS0 cells; young hamster kidney (BHK) cells; mouse embryonic fibroblast 3T3 cells (NIH3T3); mouse B-cell lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2 / 0 cells; mouse embryonic mesenchymal C3H-10T1 / 2 cells; mouse carcinoma CT26 cells; mouse prostate DuCuP cells; mouse mammary EMT6 cells; mouse liver cancer Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse cardiac myocardial MyEnd cells; mouse kidney RenCa cells; mouse pancreatic RIN-5F cells; and mouse melanoma cells. X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatocellular carcinoma (HTC); buffalo rat liver BRL3A cells; canine kidney cells (MDCK); canine mammary gland (CMT) cells; rat osteosarcoma D17 cells; rat monocyte / macrophage DH82 cells; monkey kidney SV-40 transformed fibroblasts (COS7) cells; monkey kidney CVI-76 cells; African green monkey kidney (VERO-76) cells; human embryonic kidney cells (HEK293, HEK293T); human cervical cancer cells (HELA); human lung cells (W138); human hepatocytes (HepG2); human U2-OS osteosarcoma cells, human A549 cells, human A-431 cells, or human K562 cells. A comprehensive list of mammalian cell lines is available at the American Center for Type Culture Collection (ATCC, Manassas, VA). In other embodiments, the virus-resistant cell line is a non-human mammalian cell line. In other embodiments, the virus-resistant cell line is a non-human, non-mouse mammalian cell line. In some embodiments, the virus-resistant cell line is a CHO cell line. Numerous CHO cell lines are available from the ATCC. Suitable CHO cell lines include, but are not limited to, CHO-K1 cells and their derivatives.

[0057] In various embodiments, the cell line may lack glutamine synthase (GS), dihydrofolate reductase (DHFR), hypoxanthine-guanine phosphoribosyltransferase (HPRT), or a combination thereof. For example, the chromosomal sequences encoding GS, DHFR, and / or HPRT may be inactivated. In many specific embodiments, all chromosomal sequences encoding GS are inactivated in the cell line.

[0058] (c) Virus

[0059] Engineered mammalian cell lines with viral resistance can resist a variety of mammalian viruses. The virus can be a DNA virus or an RNA virus, and can be enveloped or non-enveloped (“naked”). Non-limiting examples of viruses that can bind to sialic acid receptors and enter mammalian cells include parvovirus, reovirus, rotavirus, influenza virus, adeno-associated virus, calicivirus, parainfluenza virus, rubella virus, coronavirus, norovirus, encephalocarditis virus, and polyomavirus. In some embodiments, the engineered mammalian cell lines are resistant to infection caused by at least one parvovirus. Non-limiting examples of suitable parvoviruses include mouse parvovirus (MVM) (also known as mouse parvovirus (MMV) or rodent proparvovirus 1), mouse parvovirus type 1 (MPV-1), mouse parvovirus type 2 (MPV-2), mouse parvovirus type 3 (MPV-3), porcine parvovirus 1, bovine parvovirus 1, and human parvoviruses (e.g., human parvovirus B19, human parvovirus 4, human parvovirus 5, etc.). In many specific implementations, the parvovirus can be MVM. In other implementations, the virus can be a reovirus, such as mammalian reovirus 3, mammalian ororeovirus, avian ororeovirus, etc.

[0060] In some implementations, engineered mammalian cell lines resistant to viral infectivity may also be resistant to infections caused by organisms in the order Mycoplasma. Specifically, the cell lines disclosed herein may be resistant to infections caused by Mycoplasma or Spiroplasma.

[0061] (d) Optional nucleic acids encoding recombinant proteins

[0062] In some embodiments, mammalian cell lines resistant to viral infection may further comprise at least one nucleic acid encoding a recombinant protein. Generally, the recombinant protein is heterologous, meaning that the protein is not natural to the cell. The recombinant protein may be, but is not limited to, a therapeutic protein selected from: antibodies, antibody fragments, monoclonal antibodies, humanized antibodies, humanized monoclonal antibodies, chimeric antibodies, IgG molecules, IgG heavy chains, IgG light chains, IgA molecules, IgD molecules, IgE molecules, IgM molecules, vaccines, growth factors, cytokines, interferons, interleukins, hormones, coagulation (or clotting) factors, blood components, enzymes, therapeutic proteins, nutritional proteins, functional fragments or functional variants of any of the above, or fusion proteins comprising any of the above proteins and / or their functional fragments or variants.

[0063] In some embodiments, the nucleic acid encoding the recombinant protein may be linked to sequences encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), and / or glutamine synthase (GS), such that HPRT, DHFR, and / or GS can be used as amplifiable optional markers. The nucleic acid encoding the recombinant protein may also be linked to sequences encoding at least one antibiotic resistance gene and / or sequences encoding marker proteins such as fluorescent proteins. In some embodiments, the nucleic acid encoding the recombinant protein may be part of an expression construct. As detailed elsewhere, the expression construct or vector may contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), optional marker sequences, origin of replication, etc. Additional information can be found in Ausubel et al., 2003, ibid. and Sambrook & Russell, 2001, ibid.

[0064] In some embodiments, the nucleic acid encoding the recombinant protein can be located extrachromosomally. That is, the nucleic acid encoding the recombinant protein can be transiently expressed by plasmids, granules, artificial chromosomes, mini-chromosomes, or another extrachromosomal construct. In other embodiments, the nucleic acid encoding the recombinant protein can be integrated chromosomally into the cell's genome. This integration can be random or targeted. Therefore, the recombinant protein can be stably expressed. In some iterations of this embodiment, the nucleic acid sequence encoding the recombinant protein can be operatively ligated to an appropriate heterologous expression control sequence (i.e., a promoter). In other iterations, the nucleic acid sequence encoding the recombinant protein can be placed under the control of an endogenous expression control sequence. The nucleic acid sequence encoding the recombinant protein can be integrated into the genome of a cell line using homologous recombination, targeted endonuclease-mediated genome editing, viral vectors, transposons, plasmids, and other well-known methods. Additional guidance can be found in Ausubel et al., 2003, ibid. and Sambrook & Russell, 2001, ibid.

[0065] (e) Composition

[0066] In some embodiments, an engineered mammalian cell line exhibiting viral resistance may be part of a composition that further comprises at least one virus. The composition thus comprises the engineered cell line disclosed herein (optionally further comprising nucleic acid encoding a recombinant protein) and a virus, wherein the entry and / or replication of the at least one virus is reduced or eliminated in the engineered mammalian cell line. Therefore, the cells in the composition are capable of propagation, but the virus in the composition is unable to propagate because its entry and / or replication within the cells is reduced or eliminated. The composition may further comprise at least one cell growth medium to support the growth of the engineered mammalian cell line. In some cases, the cell growth medium is an animal-free medium.

[0067] (f) Exemplary implementation

[0068] In many specific embodiments, the virus-resistant mammalian cell line is the CHO cell line. This antiviral CHO cell line is resistant to infections caused by mouse parvovirus (MVM) (also known as mouse parvovirus (MMV) or rodent proparvovirus 1) and / or mammalian reovirus 3. Specifically, the genetically modified CHO cell line exhibits enhanced resistance to MVM or reovirus 3 infection compared to the unmodified parental CHO cell line. In some embodiments, the unmodified parental cell line is the CHO(GS- / -) cell line.

[0069] The antiviral CHO cell line contains at least one inactivated chromosomal sequence encoding COSMC, Slc35A1, C1GalT1, St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6. In some embodiments, in the CHO cell line, all copies of the chromosomal sequences encoding COSMC, Slc35A1, C1GalT1, St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 are inactivated or knocked out. In other embodiments, CHO cells comprising inactivated chromosomal sequences encoding COSMC, Slc35A1, C1GalT1, St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 further comprise inactivated chromosomal sequences encoding Mgat1, Mgat2, Mgat3, Mgat4, and / or Mgat5. In other embodiments, CHO cells comprising inactivated chromosomal sequences encoding St3Gal1, St3Gal2, St3Gal3, St3Gal4, St3Gal5, and / or St3Gal6 are further engineered to overexpress St6Gal1, St6Gal2, St6GalNac1, St6GalNac2, St6GalNac3, St6GalNac4, St6GalNac5, and / or St6GalNac6.

[0070] (II) Methods for reducing or preventing viral contamination

[0071] Another aspect of this disclosure covers methods for reducing or preventing viral contamination of recombinant protein products or reducing the risk of viral contamination in bioproduction systems. Generally, the method includes providing an engineered mammalian cell line in which the entry and / or proliferation of at least one virus is reduced or eliminated, and using said cell line to produce the recombinant protein. The engineered mammalian cell line is detailed in section (I) above. The engineered mammalian cell line exhibits resistance to viral infection compared to an unmodified parental cell line. The engineered mammalian cell line exhibits resistance to the virus described in section (I)(c). Suitable recombinant proteins are described in section (I)(d). Means for producing or manufacturing recombinant proteins are well known in the art (see, for example, "Biopharmaceutical Production Technology", Subramanian (ed.), 2012, Wiley-VCH; ISBN: 978-3-527-33029-4). In many specific implementations, the engineered mammalian cell line is genetically modified to contain at least one modified chromosome sequence, making the cell line resistant to viral infection.

[0072] Generally, using the engineered mammalian cell lines disclosed herein can reduce the ability of viruses to replicate in fermenters or other bioproduction containers, resulting in trace levels of replicable viruses, or ideally, levels undetectable by industry best practices. Suitable methods include nucleic acid detection methods (e.g., Southern blotting for detecting viral nucleic acids, PCR or RT-PCR for detecting viral nucleic acids, sequencing, etc.), antibody-based techniques (e.g., Western immunoblotting using antiviral protein antibodies, ELISA, etc.), and microscopy techniques (e.g., cytopathic effect assays, electron microscopy for detecting viral particles, etc.).

[0073] (III) Methods for preparing antiviral cell lines

[0074] Another aspect of this disclosure provides a method for preparing antiviral cells with altered cell surface receptors as detailed in sections (I)(a)(i) above. Chromosomal sequences encoding enzymes or proteins involved in glycan synthesis can be knocked down or deleted using various techniques to generate antiviral cell lines. In some embodiments, the antiviral cell line can be prepared using a genome modification method targeting endonucleases. In other embodiments, the antiviral cell line can be prepared using an RNA interference-mediated mechanism. In still other embodiments, the antiviral cell line can be prepared using a site-specific recombination system, random mutagenesis, or other methods known in the art.

[0075] (a) Targeted endonuclease-mediated genome editing

[0076] Targeted endonucleases can be used to modify (i.e., inactivate or alter) specific chromosomal sequences of interest. A specific chromosomal sequence can be inactivated by introducing a targeted endonuclease that targets the specific chromosomal sequence or a nucleic acid encoding the targeted endonuclease into the cell. In one embodiment, the targeted endonuclease recognizes and binds to the specific chromosomal sequence and introduces a double-strand break that can be repaired by a non-homologous end joining (NHEJ) repair process. Because NHEJ is error-prone, deletions, insertions, and / or substitutions of at least one nucleotide can occur, thereby disrupting the reading frame of the chromosomal sequence and preventing the production of a protein product. In another embodiment, the targeted endonuclease can also be used to alter the chromosomal sequence via homologous recombination by co-introducing a polynucleotide with substantial sequence identity to a portion of the targeted chromosomal sequence. The double-strand break introduced by the targeted endonuclease is repaired by a homologous-directed repair process, thereby exchanging the chromosomal sequence with the polynucleotide in a manner that alters or modifies the chromosomal sequence.

[0077] Various targeted endonucleases can be used to modify chromosomal sequences of interest. These targeted endonucleases can be naturally occurring or engineered proteins. Suitable targeted endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), CRISPR / Cas endonucleases, transcription activator-like effector (TALE) nucleases (TALENs), broad-spectrum nucleases, site-specific endonucleases, and artificially targeted DNA double-strand break inducers.

[0078] (i) Zinc finger nucleases

[0079] In many specific implementations, the targeted endonuclease can be a zinc finger nuclease (ZFN). A ZFN binds to a specific target sequence and introduces a double-strand break into that target sequence. Typically, a ZFN comprises a DNA-binding domain (i.e., the zinc finger) and a cleavage domain (i.e., the nuclease), each described below.

[0080] DNA-binding domainDNA-binding domains, or zinc fingers, can be engineered to recognize and bind to any selected nucleic acid sequence. See, for example, Beerli et al., (2002) Nat. Biotechnol. 20:135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nat. Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416; Zhang et al., (2000) J. Biol. Chem. 275(43):33850-33860; Doyon et al., (2008) Nat. Biotechnol. 26:702-708; and Santiago et al., (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. Engineered zinc finger binding domains can exhibit novel binding specificity compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising dinucleotide, triplet, and / or tetranucleotide sequences and individual zinc finger amino acid sequences, wherein each dinucleotide, triplet, or tetranucleotide sequence associates with one or more amino acid sequences of a zinc finger that binds to a specific triplet or tetranucleotide sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated herein by reference in their entirety. As an example, the algorithm described in U.S. Patent 6,453,242 can be used to design zinc finger binding domains to target pre-selected sequences. Alternative methods, such as using a rationally designed non-degenerate recognition cipher table, can also be used to design zinc finger binding domains to target specific sequences (Sera et al., (2002) Biochemistry 41:7074-7081). Publicly available web-based tools for identifying potential target sites in DNA sequences and designing zinc finger binding domains are well known in the art. For example, tools for identifying potential target sites in DNA sequences are available at http: / / www.zincfingertools.org. Tools for designing zinc finger binding domains are available at http: / / zifit.partners.org / ZiFiT. (See also Mandell et al., (2006) Nuc. Acid Res. 34: W516-W523; Sander et al., (2007) Nuc. Acid Res. 35: W599-W605.)

[0081] The zinc finger binding domain can be designed to recognize and bind DNA sequences from about 3 nucleotides to about 21 nucleotides in length. In one embodiment, the zinc finger binding domain can be designed to recognize and bind DNA sequences from about 9 to about 18 nucleotides in length. Generally, the zinc finger binding domain of the zinc finger nuclease used herein includes at least three zinc finger recognition regions or zinc fingers, each of which binds 3 nucleotides. In one embodiment, the zinc finger binding domain includes four zinc finger recognition regions. In another embodiment, the zinc finger binding domain includes five zinc finger recognition regions. In yet another embodiment, the zinc finger binding domain includes six zinc finger recognition regions. The zinc finger binding domain can be designed to bind any suitable target DNA sequence. See, for example, U.S. Patent Nos. 6,607,882, 6,534,261, and 6,453,242, the disclosure of which is incorporated herein by reference in its entirety.

[0082] Exemplary methods for selecting zinc finger recognition regions include phage presentation and two-hybrid systems, described in U.S. Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568; and WO 98 / 37186, WO 98 / 53057, WO 00 / 27878, WO 01 / 88197, and GB 2,338,237, all of which are incorporated herein by reference in their entirety. Furthermore, enhancements to the binding specificity of zinc finger binding domains have been described, for example, in WO 02 / 077227, the entire disclosure of which is incorporated herein by reference.

[0083] Zinc finger binding domains and methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art and are detailed in, for example, U.S. Patent No. 7,888,121, which is incorporated herein by reference in its entirety. Zinc finger recognition regions and / or multi-finger zinc finger proteins may use suitable linker sequences, including linkers of five or more amino acids in length linked together. See U.S. Patent Nos. 6,479,626, 6,903,185, and 7,153,949, the disclosure of which is incorporated herein by reference in its entirety, for non-limiting examples of linker sequences of six or more amino acids in length. The zinc finger binding domains described herein may include combinations of suitable linkers between individual zinc fingers of a protein.

[0084] In some embodiments, the zinc finger nuclease further includes a nuclear localization signal or sequence (NLS). The NLS is an amino acid sequence that facilitates the zinc finger nuclease protein targeting the cell nucleus, thereby introducing double-strand breaks at target sequences in the chromosome. Nuclear localization signals are known in the art. See, for example, Makkerh et al., (1996) Current Biology 6:1025-1027.

[0085] Fracturing domain Zinc finger nucleases also include a cleavage domain. The cleavage domain portion of the zinc finger nuclease can be derived from any endonuclease or exonuclease. Non-restrictive examples of endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, New England Biolabs Catalog or Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., S1 nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease). See also Linn et al. (ed.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) can be used as a source of the cleavage domain.

[0086] The cleavage domain can also originate from the enzyme or a portion thereof, as described above, which requires dimerization of the cleavage activity. Cleavage may require two zinc finger nucleases, as each nuclease contains a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may contain two monomers to form the active enzyme dimer. As used herein, an "active enzyme dimer" is an enzyme dimer capable of cleaving nucleic acid molecules. The two cleavage monomers can originate from the same endonuclease (or a functional fragment thereof), or the monomers can originate from different endonucleases (or functional fragments thereof).

[0087] When two cleavage monomers are used to form an active enzyme dimer, the recognition sites of the two zinc finger nucleases are preferably arranged such that the binding of the two zinc finger nucleases to their respective recognition sites positions the cleavage monomers relative to each other in a spatial orientation that allows the cleavage monomers to form an active enzyme dimer (e.g., by dimerization). As a result, the adjacent edges of the recognition sites can be spaced about 5 to about 18 nucleotides apart. For example, the adjacent edges can be spaced about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides apart. However, it should be understood that any integer number of nucleotides or nucleotide pairs can be interposed between the two recognition sites (e.g., about 2 to about 50 nucleotide pairs or more). The adjacent edges of the recognition sites of the zinc finger nucleases (such as those described in detail herein, for example) can be spaced 6 nucleotides apart. Generally, the cleavage sites are located between the recognition sites.

[0088] Restriction endonucleases (restriction enzymes) are found in many species and are capable of sequence-specific binding to DNA (at recognition sites) and causing DNA cleavage at or near the binding site. Some restriction enzymes (e.g., type IIS) cleave DNA at sites farther from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes double-strand cleavage of DNA at a site 9 nucleotides away from its recognition site on one strand and 13 nucleotides away from its recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994; and Li et al., (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al., (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al., (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al., (1994b) J. Biol. Chem. 269:31978-31982. Therefore, zinc finger nucleases may comprise a cleavage domain from at least one IIS-type restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Exemplary IIS-type restriction enzymes are described, for example, in International Publication WO 07 / 014,275, the disclosure of which is incorporated herein by reference in its entirety. Additional restriction enzymes also contain separable binding and cleaving domains, and these are also covered by this invention. See, for example, Roberts et al., (2003) Nucleic Acids Res. 31:418-420.

[0089] An exemplary IIS-type restriction enzyme in which the cleavage domain and binding domain are separable is FokI. This particular enzyme is an active dimer (Bitinaite et al., (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575). Therefore, for the purposes of this disclosure, the FokI enzyme portion used in zinc finger nucleases is considered as a cleavage monomer. Thus, for targeted double-strand cleavage using the FokI cleavage domain, two zinc finger nucleases (each containing a FokI cleavage monomer) can be used to reconstruct the active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers can also be used.

[0090] In some embodiments, the cleavage domain comprises one or more engineered cleavage monomers capable of reducing or preventing homodimerization. As a non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets affecting the dimerization of the FokI cleavage hemidomain. Exemplary engineered FokI cleavage monomers forming specific heterodimers comprise a pair, wherein the first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of FokI, and the second cleavage monomer includes mutations at amino acid residue positions 486 and 499.

[0091] Therefore, in one embodiment of the engineered cleavage monomer, the mutation at amino acid position 490 replaces Glu(E) with Lys(K); the mutation at amino acid residue 538 replaces Iso(I) with Lys(K); the mutation at amino acid residue 486 replaces Gln(Q) with Glu(E); and the mutation at position 499 replaces Iso(I) with Lys(K). Specifically, the engineered cleavage monomer can be prepared by mutating position 490 from E to K and position 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer named "E490K:I538K", and by mutating position 486 from Q to E and position 499 from I to K in another cleavage monomer to produce an engineered cleavage monomer named "Q486E:I499K". The engineered cleavage monomer described above is a specific heterodimer mutant in which aberrant cleavage is minimized or eliminated. Suitable methods can be used to prepare engineered cleavage monomers, for example by site-directed mutagenesis of wild-type cleavage monomers (FokI) as described in U.S. Patent No. 7,888,121, the entire contents of which are incorporated herein by reference.

[0092] Other structural domains.In some embodiments, the zinc finger nuclease further comprises at least one nuclear localization sequence (NLS). The NLS is an amino acid sequence that facilitates the zinc finger nuclease protein targeting the cell nucleus, thereby introducing a double-strand break at a target sequence in the chromosome. Nuclear localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem., 2007, 282:5101-5105). For instance, in one embodiment, the NLS may be a single sequence, such as PKKKRKV (SEQ ID NO:1) or PKKKRRV (SEQ ID NO:2). In another embodiment, the NLS may be a dichotomous sequence. In yet another embodiment, the NLS may be KRPAATKKAGQAKKKK (SEQ ID NO:3). The NLS may be located at the N-terminus, C-terminus, or internal location of the protein.

[0093] In another embodiment, the zinc finger nuclease may further comprise at least one cell-penetrating domain. In one embodiment, the cell-penetrating domain may be a cell-penetrating peptide sequence derived from the HIV-1 TAT protein. As an example, the TAT cell-penetrating sequence may be GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:4). In another embodiment, the cell-penetrating domain may be a cell-penetrating peptide sequence TLM derived from human hepatitis B virus (PLSSIFSRIGDPPKKKRKV, SEQ ID NO:5). In another embodiment, the cell-penetrating domain may be MPG (GALFLGWLGAAGSTMGAPKKKRKV, SEQ ID NO:6; or GALFLGFLGAAGSTMGAWSQPKKKRKV, SEQ ID NO:7). In another embodiment, the cell-penetrating domain may be Pep-1 (KETWWETWWTEWSQPKKKRKV, SEQ ID NO:8), VP22, a cell-penetrating peptide derived from herpes simplex virus, or a polyarginine peptide sequence. The cell-penetrating domain can be located at the N-terminus, C-terminus, or internal location of the zinc finger nuclease.

[0094] In other embodiments, the zinc finger nuclease may further comprise at least one labeling domain. Non-limiting examples of labeling domains include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the labeling domain may be a fluorescent protein. Suitable non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami). Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric). Kusabira-Orange, mTangerine, tdTomato, or any other suitable fluorescent protein. In another embodiment, the tagging domain may be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, glutathione S-transferase (GST), chitosan-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotinylate carboxyl carrier protein (BCCP), and calmodulin. The tagging domain may be located at the N-terminus, C-terminus, or internal position of the zinc finger nuclease.

[0095] This marker domain can be linked to a zinc finger nuclease via a 2A peptide (Szymczak et al., 2004, Nat. Biotechnol., 589(5):589-94). This 2A peptide was initially characterized in positive-sense RNA viruses that produce multiproteins that are “cleaved” during translation into mature individual proteins. More specifically, the 2A peptide region (~20 amino acids) mediates “cleavage” at its own C-terminus, releasing itself from the downstream region of the multiprotein. Generally, the 2A peptide sequence is capped with glycine and proline residues. During the translation of the 2A peptide, the ribosome pauses after the glycine residue, releasing the nascent polypeptide chain. Translation resumes, and the proline residue of the 2A sequence becomes the first amino acid of the downstream protein.

[0096] (ii) CRISPR / Cas endonuclease

[0097] In other embodiments, the targeted endonuclease may be a CRISPR / Cas endonuclease. CRISPR / Cas endonucleases are RNA-guided endonucleases derived from the CRISPR / Cas system. Bacteria and archaea have evolved RNA-based adaptive immune systems that use CRISPR (clustered, regularly spaced short palindromic repeats) and Cas (CRISPR-associated) proteins to detect and eliminate invading viruses or plasmids. CRISPR / Cas endonucleases can be programmed to introduce target-site-specific double-strand breaks by providing target-specific synthetic guide RNA (Jinek et al., 2012, Science, 337:816-821).

[0098] Nucleotide endonucleaseThe CRISPR / Cas endonuclease can be derived from type I, type II, or type III CRISPR / Cas systems. Non-restrictive examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), and Cse3. (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966.

[0099] In one embodiment, the CRISPR / Cas endonuclease is derived from a type II CRISPR / Cas system. In an exemplary embodiment, the CRISPR / Cas endonuclease is derived from the Cas9 protein. The Cas9 protein may be derived from *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus* spp., *Nocardia dassonvillei*, *Streptococcus spp.*, *Streptococcus aureus*, *Streptococcus spp.*, *Streptococcus rosea*, *Streptococcus aureus*, *Bacillus pyogenes*, *Bacillus subtilis*, *Microbacterium siberianum*, *Lactobacillus bulgaricus*, *Lactobacillus salivarius*, *Microcystis aeruginosa*, *Burkholderia spp.*, *Naphthalene-degrading Polar Monotrophus*, *Polaris spp.*, *Cyclocarya pallida*, *Cyclocarya spp.*, *Microcystis aeruginosa*, *Synechococcus*, *Arabidobacterium arabinose*, *Ammoniacium dansii*, *Extremophila*, etc. The Cas9 protein is derived from thermophilic cellulose-degrading anaerobic bacteria, gold-mining bacteria, Clostridium botulinum, Clostridium difficile, *Gyrophylloides*, thermophilic halophilic anaerobic bacteria, thermophilic propionate symbiotic bacteria, thermophilic acidophilic thiobacilli, ferrooxidizing thiobacilli, purple sulfur bacteria, *Hymenobacter* spp., halophilic nitrosococci, *Nitrosococcus vannamei*, *Pseudomonas aeruginosa*, *Flammulina racemicis*, extreme halophilic methanogens, *Anabaena*, *Nostoc* spp., *Spirulina macrocarpa*, *Spirulina articulata*, *Spirulina* spp. ...

[0100] Generally, CRISPR / Cas proteins contain at least one RNA recognition domain and / or RNA binding domain. The RNA recognition and / or RNA binding domains interact with guide RNA, thereby directing the CRISPR / Cas protein to a specific chromosome or chromosomal sequence (i.e., the target site). The CRISPR / Cas protein may also contain nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains.

[0101] The CRISPR / Cas protein can be derived from wild-type CRISPR / Cas protein, modified CRISPR / Cas protein, or fragments of wild-type or modified CRISPR / Cas protein. The CRISPR / Cas protein can be modified to increase nucleic acid binding affinity and / or specificity, alter enzyme activity, and / or change another property of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the CRISPR / Cas protein can be modified, deleted, or inactivated. The CRISPR / Cas protein can be truncated to remove domains that are not essential for the protein's function. The CRISPR / Cas protein can also be truncated or modified to optimize the activity of the protein or the effector domain fused to the CRISPR / Cas protein.

[0102] In some embodiments, the CRISPR / Cas endonuclease may be derived from wild-type Cas9 protein or a fragment thereof. In other embodiments, the CRISPR / Cas endonuclease may be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein may be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas9 protein that do not involve RNA-guided cleavage may be removed from the protein, making the modified Cas9 protein smaller than the wild-type Cas9 protein.

[0103] Generally, Cas9 proteins contain at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein may contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to cleave single strands, thereby creating double-strand breaks in DNA (Jinek et al., Science, 337:816-821). In one embodiment, the CRISPR-based endonuclease is derived from the Cas9 protein and contains two functional nuclease domains.

[0104] Target sites typically have a length of about 14-15 bp and are identified by the naturally occurring CRISPR / Cas system (Cong et al., Science, 339:819-823). These target sites are not sequence-restricted except that a sequence complementary to the 5' end of the guide RNA (i.e., called the prototypical spacer sequence) immediately follows (3' or downstream) a common sequence. This common sequence is also known as the prototypical spacer adjacent motif (or PAM). Examples of PAMs include, but are not limited to, NGG, NGNNG, and NNAGAAW (where N is defined as any nucleotide and W is defined as A or T). At typical lengths, only about 5-7% of target sites within the target genome will be unique, suggesting that off-target effects can be significant. The length of the target site can be extended by commanding two binding events. For example, CRISPR-based endonucleases can be modified so that they can only cleave one strand of a double-stranded sequence (i.e., converted to a nicking enzyme). Therefore, using a CRISPR-based nicking enzyme in combination with two different guide RNAs will essentially double the length of the target site while still achieving double-strand breaks.

[0105] Therefore, in some embodiments, the Cas9-derived endonuclease can be modified to contain a functional nuclease domain (RuvC-like or HNH-like nuclease domain). For example, the Cas9-derived protein can be modified to delete or mutate one of the nuclease domains, rendering it nonfunctional (i.e., the domain lacks nuclease activity). In some embodiments, one of the nuclease domains is inactive, and the Cas9-derived protein can introduce nicks into double-stranded nucleic acids (such proteins are called "nickases"), but cannot cleave double-stranded DNA. For example, an aspartic-to-alanine (D10A) conversion in the RuvC-like domain converts the Cas9-derived protein into an "HNH" nickase. Similarly, a histidine-to-alanine (H840A) conversion in the HNH domain (in some cases, the histidine is located at position 839) converts the Cas9-derived protein into a "RuvC" nickase. Thus, for example, in one embodiment, the Cas9-derived nickase has an aspartic-to-alanine (D10A) conversion in the RuvC-like domain. In another embodiment, the Cas9-derived nickase has a histidine-to-alanine (H840A or H839A) conversion in the HNH domain. The RuvC-like or HNH-like nuclease domain of the Cas9-derived nickase can be modified using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis and whole-genome synthesis, as well as other methods known in the art.

[0106] Other structural domains.The CRISPR / Cas endonuclease or nickase typically includes at least one nuclear localization signal (NLS). For example, in one embodiment, the NLS may be a single sequence, such as PKKKRKV (SEQ ID NO:1) or PKKKRRV (SEQ ID NO:2). In another embodiment, the NLS may be a dichotomous sequence. In yet another embodiment, the NLS may be KRPAATKKAGQAKKKK (SEQ ID NO:3). The NLS may be located at the N-terminus, C-terminus, or internal location of the protein.

[0107] In some embodiments, the CRISPR / Cas endonuclease or nickase may further include at least one cell-penetrating domain. This cell-penetrating domain may be a cell-penetrating peptide sequence derived from the HIV-1 TAT protein. As an example, the TAT cell-penetrating sequence may be GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:4). In another embodiment, the cell-penetrating domain may be a cell-penetrating peptide sequence TLM derived from human hepatitis B virus (PLSSIFSRIGDPPKKKRKV, SEQ ID NO:5). In yet another embodiment, the cell-penetrating domain may be MPG (GALFLGWLGAAGSTMGAPKKKRKV, SEQ ID NO:6; or GALFLGFLGAAGSTMGAWSQPKKKRKV, SEQ ID NO:7). In a further embodiment, the cell-penetrating domain may be Pep-1 (KETWWETWWTEWSQPKKKRKV, SEQ ID NO:8), VP22, a cell-penetrating peptide derived from herpes simplex virus, or a polyarginine peptide sequence. The cell-penetrating domain can be located at the N-terminus, C-terminus, or internal location of the protein.

[0108] In other embodiments, the CRISPR / Cas endonuclease or nickase may further include at least one labeling domain. Non-limiting examples of the labeling domain include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the labeling domain may be a fluorescent protein. Suitable non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), and red fluorescent proteins (mKate, m...). Kate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred) and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In another embodiment, the tagging domain may be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotinylate carboxyl carrier protein (BCCP), and calmodulin. The tag domain can be located at the N-terminus, C-terminus, or internal location of the protein. The tag domain can be linked to the CRISPR / Cas endonuclease or cleavage enzyme via a 2A peptide (Szymczak et al., 2004, Nat. Biotechnol., 589(5):589-94).

[0109] Guide RNA. The CRISPR / Cas endonuclease is guided to its target site by a guide RNA. The guide RNA interacts with both the CRISPR / Cas endonuclease and the target site within the chromosome, where the CRISPR / Cas endonuclease or nicking enzyme cleaves at least one strand of the double-stranded sequence. The guide RNA can be introduced into the cell along with the CRISPR / Cas endonuclease or the nucleic acid encoding the CRISPR / Cas endonuclease. Alternatively, DNA encoding both the CRISPR / Cas endonuclease and the guide RNA can be introduced into the cell.

[0110] Guide RNAs contain three regions: a first region at the 5' end complementary to the sequence at the target site; a second inner region forming a stem-loop structure; and a third 3' region that remains essentially single-stranded. The first region is different for each guide RNA, allowing each guide RNA to guide a CRISPR / Cas endonuclease or nicking enzyme to a specific target site. The second and third regions (also called scaffold regions) can be identical across all guide RNAs.

[0111] The first region of the guide RNA is complementary to the sequence at the target site (i.e., the prototype spacer sequence), allowing the first region of the guide RNA to base-pair with the sequence at the target site. Generally, there are no mismatches (i.e., perfect complementarity) between the first region of the guide RNA and the sequence at the target site. In various embodiments, the first region of the guide RNA can contain from about 10 nucleotides to more than about 25 nucleotides. For example, the length of the region of the first region of the guide RNA that base-pairs with the target site in the chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides. In an exemplary embodiment, the length of the first region of the guide RNA is about 19 or 20 nucleotides.

[0112] The guide RNA also includes a second region that forms a secondary structure. In some embodiments, this secondary structure includes a stem (or hairpin) and a loop. The lengths of the loop and the stem can vary. For example, the length of the loop can range from about 3 to about 10 nucleotides, and the length of the stem can range from about 6 to about 20 base pairs. The stem may include one or more ridges having 1 to about 10 nucleotides. Thus, the entire length of the second region can range from about 16 to about 60 nucleotides. In one exemplary embodiment, the loop is about 4 nucleotides long, and the stem comprises about 12 base pairs.

[0113] The guide RNA also includes a essentially single-stranded third region at its 3' end. Therefore, this third region is not complementary to any chromosomal sequence in the cell of interest, nor to the rest of the guide RNA. The length of this third region can vary. Generally, it is longer than about 4 nucleotides. For example, its length can range from about 5 to about 60 nucleotides.

[0114] The combined length of the second and third regions (or scaffold) of the guide RNA can range from about 30 to about 120 nucleotides. In one aspect, the combined length of the second and third regions of the guide RNA ranges from about 70 to about 100 nucleotides.

[0115] In some embodiments, the guide RNA comprises a single molecule including all three regions. In other embodiments, the guide RNA may comprise two separate molecules. The first RNA molecule may comprise half of the "stem" of the first region and the second region of the guide RNA. The second RNA molecule may comprise the other half of the "stem" of the second region and the third region of the guide RNA. Thus, in this embodiment, the first RNA molecule and the second RNA molecule each contain complementary nucleotide sequences. For example, in one embodiment, the first RNA molecule and the second RNA each contain a sequence (about 6 to about 20 nucleotides) that base-pairs with another sequence to form a functional guide RNA.

[0116] (iii) Other targeted endonucleases

[0117] In other embodiments, the targeted endonuclease can be a broad-spectrum nuclease. Broad-spectrum nucleases are deoxyribonucleases characterized by long recognition sequences, typically ranging from about 12 to about 40 base pairs. As a result of this requirement, the recognition sequence generally appears only once in any given genome. Among broad-spectrum nucleases, the homing endonuclease family, named LAGLIDADG, has become a valuable tool for genome and genome engineering research (see, for example, Arnould et al., 2011, Protein Eng DesSel, 24(1-2):27-31). Broad-spectrum nucleases can target specific chromosomal sequences by modifying their recognition sequences using techniques well-known to those skilled in the art.

[0118] In another implementation, the targeted endonuclease may be a transcription activator-like effector (TALE) nuclease. TALE is a transcription factor derived from the plant pathogen Xanthomonas that can be readily engineered to bind to novel DNA targets. TALE or a truncated version thereof may be linked to the catalytic domain of an endonuclease, such as FokI, to create a targeted endonuclease known as a TALE nuclease or TALEN (Sanjana et al., 2012, Nat Protoc, 7(1):171-192).

[0119] In other embodiments, the targeted endonuclease may be a site-specific endonuclease. Specifically, the site-specific endonuclease may be a "rare-cut" endonuclease, whose recognition sequence is rarely found in the genome. Alternatively, the site-specific endonuclease may be engineered to cleave at the site of interest (Friedhoff et al., 2007, Methods Mol Biol 352:1110123). Generally, the recognition sequence of the site-specific endonuclease appears only once in the genome. In alternative embodiments, the targeted endonuclease may be an artificially targeted DNA double-strand break inducer.

[0120] (iv) Optional polynucleotides

[0121] Targeted genome modification methods may further include introducing at least one polynucleotide into a cell, the polynucleotide comprising a sequence substantially identical to a sequence on at least one side of a targeted cleavage site, such that double-strand breaks introduced by the targeted endonuclease can be repaired by homology-directed repair; and exchanging the sequence of the polynucleotide with an endogenous chromosomal sequence, thereby modifying the endogenous chromosomal sequence. For example, the polynucleotide comprises a first sequence substantially identical to a sequence on one side of the targeted cleavage site and a second sequence substantially identical to a sequence on the other side of the targeted cleavage site. Alternatively, the polynucleotide comprises a first sequence substantially identical to a sequence on one side of the targeted cleavage site and a second sequence substantially identical to a sequence located remotely from the targeted cleavage site. The sequence located remotely from the targeted cleavage site may be tens, hundreds, or thousands of nucleotides upstream or downstream of the targeted cleavage site.

[0122] The lengths of the first and second sequences in the polynucleotide that have substantial sequence identity with the sequence in the target chromosome sequence can and will vary. Generally, the length of each of the first and second sequences in the polynucleotide is at least about 10 nucleotides. In various embodiments, the length of the polynucleotide sequence having substantial sequence identity with the chromosome sequence can be about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, or more than 100 nucleotides.

[0123] The phrase "substantial sequence identity" means that the sequence in the polynucleotide has at least about 75% sequence identity with the chromosome sequence of interest. In some embodiments, the sequence in the polynucleotide has 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the chromosome sequence of interest.

[0124] The length of the polynucleotide can and will vary. For example, the polynucleotide can range from about 20 nucleotides to about 200,000 nucleotides. In various embodiments, the polynucleotide ranges from about 20 nucleotides to about 100 nucleotides, about 100 nucleotides to about 1000 nucleotides, about 1000 nucleotides to about 10,000 nucleotides, about 10,000 nucleotides to about 100,000 nucleotides, or about 100,000 nucleotides to about 200,000 nucleotides.

[0125] Typically, the polynucleotide is DNA. The DNA can be single-stranded or double-stranded. The polynucleotide can be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear DNA fragment, a PCR fragment, naked nucleic acid, or a complex of nucleic acid with a delivery medium such as liposomes or poloxamer. In some embodiments, the polynucleotide is single-stranded. In many exemplary embodiments, the polynucleotide is a single-stranded oligonucleotide containing fewer than about 200 nucleotides.

[0126] In some embodiments, the polynucleotide further includes a marker. This marker enables screening for targeted integration. In some embodiments, the marker is a restriction endonuclease site. In other embodiments, the marker is a fluorescent protein, a purification tag, or an epitope tag. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), and red fluorescent proteins (mKate, m...). Kate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred) and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the tag may be a purification tag and / or an epitope tag. Exemplary tags include, but are not limited to, glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotinylate carboxyl carrier protein (BCCP), and calmodulin.

[0127] (v) Delivery to cells

[0128] The method involves introducing the targeted endonuclease into cells of interest. The targeted endonuclease can be introduced into cells as a purified isolated protein or a nucleic acid encoding the targeted endonuclease. The nucleic acid can be DNA or RNA. In an embodiment where the nucleic acid is mRNA, the mRNA can be 5' capped and / or 3' polyadenylated. In an embodiment where the nucleic acid is DNA, the DNA can be linear or circular. The DNA can be part of a vector, wherein the encoding DNA can be operatively linked to a suitable promoter. Those skilled in the art will be familiar with suitable vectors, promoters, other control elements, and means of introducing the vector into cells of interest.

[0129] The targeted endonuclease molecule and the aforementioned optional polynucleotides can be introduced into cells through a variety of methods. Suitable delivery methods include microinjection, electroporation, sonication, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic polymer transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipid transfection, puncture transfection, optical transfection, proprietary agents to enhance nucleic acid uptake, and delivery via liposomes, immunoliposomes, virions, or artificial viruses. In one specific embodiment, the targeted endonuclease molecule and polynucleotides are introduced into cells via nuclear transfection.

[0130] In embodiments that introduce more than one targeted endonuclease molecule and more than one polynucleotide into a cell, the molecule may be introduced simultaneously or sequentially. For example, multiple targeted endonuclease molecules, each specific to a target cleavage site (and optional polynucleotide), may be introduced simultaneously. Alternatively, each targeted endonuclease molecule and optional polynucleotide may be introduced sequentially.

[0131] The ratio of the targeted endonuclease molecule to the optional polynucleotide can and will vary. Generally, the ratio of the targeted endonuclease molecule to the polynucleotide is in the range of about 1:10 to about 10:1. In various embodiments, the ratio of the targeted endonuclease molecule to the polynucleotide can be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the ratio is about 1:1.

[0132] (vi) Cell culture

[0133] The method also includes maintaining the cell under suitable conditions such that double-strand breaks introduced by the targeted endonuclease can be repaired by: (i) a non-homologous end joining repair method, in which the chromosomal sequence is modified by the deletion, insertion, and / or substitution of at least one nucleotide; or, optionally, (ii) a homologous directed repair method, in which the chromosomal sequence is modified by sequence exchange with a polynucleotide. In embodiments of introducing a nucleic acid encoding the targeted endonuclease into the cell, the method includes maintaining the cell under suitable conditions such that the cell expresses the targeted endonuclease.

[0134] Generally, the cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and described, for example, in the following literature: Santiago et al., (2008) PNAS 105:5809-5814; Moehle et al., (2007) PNAS 104:3055-3060; Urnov et al., (2005) Nature 435:646-651; and Lombardo et al., (2007) Nat. Biotechnology 25:1298-1306. Those skilled in the art will understand that methods for culturing cells are known in the art and can and will vary depending on the cell type. In all cases, conventional optimization can be used to determine the optimal technique for a particular cell type.

[0135] During this step of the method, the targeted endonuclease recognizes, binds to, and creates a double-strand break at a targeted cleavage site in the chromosome sequence, while during double-strand break repair, at least one nucleotide deletion, insertion, and / or substitution is introduced into the targeted chromosome sequence. In many specific embodiments, the targeted chromosome sequence is inactivated.

[0136] Once it has been confirmed that the chromosome sequence of interest has been modified, single-cell clones can be isolated and genotyped (by DNA sequencing and / or protein analysis). Cells containing a modified chromosome sequence can undergo one or more rounds of additional targeted genomic modification to modify other chromosome sequences (e.g., see Example 1).

[0137] (b) RNA interference

[0138] In another embodiment, the antiviral cell line can be prepared using an RNA interference (RNAi) agent that inhibits the expression of the target mRNA or transcript. The RNAi agent may cause cleavage of the target mRNA or transcript. Alternatively, the RNAi agent may prevent or disrupt the translation of the target mRNA into a protein.

[0139] In some embodiments, the RNAi agent may be a short interfering RNA (siRNA). Generally, siRNA comprises a double-stranded RNA molecule with a length ranging from about 15 to about 29 nucleotides. The length of the siRNA may be about 16-18, 17-19, 21-23, 24-27, or 27-29 nucleotides. In one specific embodiment, the length of the siRNA is about 21 nucleotides. The siRNA may optionally further comprise one or two single-stranded overhangs, such as 3' overhangs at one or both ends. The siRNA may be formed from two RNA molecules hybridized together, or alternatively, may be generated from short hairpin RNA (shRNA) (see below). In some embodiments, the two strands of the siRNA are perfectly complementary, such that there are no mismatches or bulges in the duplex formed between the two sequences. In other embodiments, the two strands of the siRNA are substantially complementary, such that there are one or more mismatches and / or bulges in the duplex formed between the two sequences. In some embodiments, one or both of the 5' ends of the siRNA have phosphate groups, while in other embodiments, one or both of the 5' ends lack phosphate groups. In other embodiments, one or both of the 3' ends of the siRNA have hydroxyl groups, while in other embodiments, one or both of the 5' ends lack hydroxyl groups.

[0140] One strand of the siRNA, referred to as the "antisense strand" or "guide strand," includes a portion capable of hybridizing with the target transcript. In some embodiments, the antisense strand of the siRNA is completely complementary to a region of the target transcript, i.e., it hybridizes with the target transcript without a single mismatch or bulge on a target region that is between about 15 and 29 nucleotides long, preferably at least 16 nucleotides long, and more preferably about 18-20 nucleotides long. In other embodiments, the antisense strand is substantially complementary to the target region, i.e., one or more mismatches and / or bulges may be present in the duplex formed by the antisense strand and the target transcript. Typically, the siRNA targets the exon sequence of the target transcript. Those skilled in the art will be familiar with programs, algorithms, and / or commercial services for designing siRNAs targeting target transcripts. An exemplary example is Rosetta siRNA DesignAlgorithm (Rosetta Inpharmatics, North Seattle, WA) and siRNA (Sigma-Aldrich, St. Louis, MO). This siRNA can be synthesized enzymatically in vitro using methods well-known to those skilled in the art. Alternatively, this siRNA can be chemically synthesized using oligonucleotide synthesis techniques well-known in the art.

[0141] In other embodiments, the RNAi agent may be short hairpin RNA (shRNA). Generally, shRNA is an RNA molecule comprising at least two complementary portions of a double-stranded structure of sufficient length to mediate RNA interference (as described above) and at least one single-stranded portion forming a loop connecting the shRNA region forming the double strand. This structure is also referred to as a stem-loop structure, where the stem is the double-stranded portion. In some embodiments, the double-stranded portions of this structure are perfectly complementary, such that there are no mismatches or bulges in the double-stranded region of the shRNA. In other embodiments, the double-stranded portions of this structure are substantially complementary, such that one or more mismatches and / or bulges exist in the double-stranded portion of the shRNA. The loop of this structure may be about 1 to about 20 nucleotides long, preferably about 4 to about 10 nucleotides long, and more preferably about 6 to about 9 nucleotides long. The loop may be located at the 5' or 3' end of a region complementary to the target transcript (i.e., the antisense portion of the shRNA).

[0142] The shRNA may further include a dangling splint at the 5' or 3' end. This optional splint may be about 1 to about 20 nucleotides long, and more preferably about 2 to about 15 nucleotides long. In some embodiments, the splint contains one or more U residues, for example, between about 1 and about 5 U residues. In some embodiments, the 5' end of the shRNA has a phosphate group, while in other embodiments it does not. In other embodiments, the 3' end of the shRNA has a hydroxyl group, while in other embodiments it does not. Generally, shRNA is processed into siRNA via a conserved cellular RNAi mechanism. Therefore, shRNA is a precursor to siRNA and similarly capable of inhibiting the expression of target transcripts complementary to a portion of the shRNA (i.e., the antisense portion of the shRNA). Those skilled in the art will be familiar with the available resources (as detailed above) for the design and synthesis of shRNA.

[0143] In other embodiments, the RNAi agent may be an RNAi expression vector. Typically, RNAi expression vectors are used for intracellular (in vivo) synthesis of RNAi agents, such as siRNA or shRNA. In one embodiment, a single vector containing two promoters is used to transcribe two independent complementary siRNA strands, each promoter directing the transcription of a single siRNA strand (i.e., each promoter is operatively linked to a template of the siRNA, enabling transcription). The two promoters may be oriented in the same direction, in which case each promoter is operatively linked to a template of one of the complementary siRNA strands. Alternatively, the two promoters may be oriented in opposite directions, thereby side-linking a single template, so that the transcription of the promoter can synthesize two complementary siRNA strands. In another embodiment, the RNAi expression vector may contain a promoter that drives the transcription of a single RNA molecule containing two complementary regions, thereby causing the transcript to form shRNA.

[0144] Those skilled in the art will understand that siRNA and shRNA agents are preferably produced in vivo via transcription of more than one transcription unit. Generally, the promoter used to direct the in vivo expression of one or more siRNA or shRNA transcription units may be a promoter targeting RNA polymerase III (PolIII). Some PolIII promoters, such as U6 or H1 promoters, do not require cis-regulatory elements within the transcribed region and are therefore preferred in some embodiments. In other embodiments, a PolII-targeting promoter may be used to drive the expression of one or more siRNA or shRNA transcription units. In some embodiments, tissue-specific, cell-specific, or inducible PolII promoters may be used.

[0145] Standard recombinant DNA methods can be used to generate constructs that provide templates for the synthesis of siRNA or shRNA and insert them into any of a variety of different vectors suitable for expression in eukaryotic cells. Recombinant DNA techniques are described in Ausubel et al., 2003, ibid. and Sambrook & Russell, 2001, ibid. Those skilled in the art will also understand that vectors may contain additional regulatory sequences (e.g., termination sequences, translation control sequences, etc.) and optional marker sequences. DNA plasmids are known in the art, including those based on pBR322, PUC, etc. Since many expression vectors already contain suitable promoters, it may only be necessary to insert the nucleic acid sequence encoding the RNAi agent of interest at the appropriate position relative to that promoter. Viral vectors can also be used to provide intracellular expression of RNAi agents. Suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, herpesvirus vectors, etc. In one specific embodiment, the RNAi expression vector is an shRNA lentiviral vector or lentiviral particle, such as The one provided in the TRCshRNA product (Sigma-Aldrich).

[0146] The RNAi agent or RNAi expression vector can be introduced into the cell using methods well known to those skilled in the art. Such techniques are described, for example, in the following literature: Ausubel et al., 2003, ibid.; or Sambrook & Russell, 2001, ibid. In some embodiments, the RNAi expression vector (e.g., a viral vector) is stably integrated into the genome of the cell to disrupt Mgat1 expression in subsequent cell generations.

[0147] (c) Site-specific recombination

[0148] In an alternative implementation, site-specific recombination techniques can be used to prepare the virus-resistant cell line. For example, site-specific recombination techniques can be used to delete all or part of the chromosomal sequence of interest, or to introduce single nucleotide polymorphisms (SNPs) into the chromosomal sequence of interest. In one implementation, the Cre-loxP site-specific recombination system, the Flp-FRT site-specific recombination system, or variations thereof, are used to target the chromosomal sequence of interest. Such recombination systems are commercially available, and further teaching of these techniques can be found, for example, in Ausubel et al., 2003, ibid.

[0149] definition

[0150] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions to those skilled in the art of the invention for many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings assigned to them.

[0151] When describing elements of the invention or preferred embodiments thereof, the articles “a,” “an,” “the,” and “the” are intended to indicate the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements may be present in addition to those listed.

[0152] As used in this article, “deficiency” means that the level of the target enzyme or protein is reduced or undetectable, or that the activity of the target enzyme or protein is reduced or undetectable.

[0153] As used in this article, the term "endogenous sequence" refers to a chromosome sequence that is natural to the cell.

[0154] The term "exogenous sequence" refers to a chromosome sequence that is not natural to the cell or has been moved to a different chromosomal location.

[0155] "Genetically modified" cells are cells whose genomes have been modified, that is, cells that contain at least an engineered chromosomal sequence with at least one nucleotide insertion, at least one nucleotide deletion, and / or at least one nucleotide substitution.

[0156] The terms "genome modification" and "genome editing" refer to methods that alter a specific chromosomal sequence to modify that sequence. The chromosomal sequence can be modified to include the insertion, deletion, and / or substitution of at least one nucleotide. The modified chromosomal sequence is inactive and therefore does not produce a product. Alternatively, the chromosomal sequence can be modified to produce an altered product.

[0157] As used herein, “gene” refers to the DNA region (including exons and introns) that encodes a gene product, as well as all DNA regions that regulate the production of gene products, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or the transcribed sequence. Therefore, genes include, but are not limited to, promoter sequences, terminators, translation regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, isolators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0158] The term "heterogeneous" refers to an entity that is not natural to the cell or species of interest.

[0159] The terms "nucleic acid" and "polynucleotide" refer to polymers of deoxyribonucleotides or ribonucleotides in linear or cyclic conformations. For the purposes of this disclosure, these terms should not be considered limiting in terms of polymer length. The term may encompass known analogs of natural nucleotides, as well as nucleotides modified in their base, sugar, and / or phosphate moieties. Generally, analogs of a particular nucleotide have the same base-pairing specificity; that is, an analog of A will pair with a T base. Nucleotides of nucleic acids or polynucleotides can be linked by phosphodiester, thiophosphate, phosphoramide, diaminophosphate bonds, or combinations thereof.

[0160] The term "nucleotide" refers to deoxyribonucleotides or ribonucleotides. The nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or a nucleotide analog. A nucleotide analog is a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modification of the sugar or base moiety of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxy, methyl, phosphoryl, and thiol groups, as well as substitution of the carbon and nitrogen atoms of the base with other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino groups.

[0161] The terms "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues.

[0162] As used herein, the terms “target site” or “target sequence” refer to a portion of a chromosomal sequence that is to be modified or edited and engineered to recognize and bind to the target endonuclease (provided that sufficient conditions for binding exist).

[0163] The terms "upstream" and "downstream" refer to positions within a nucleic acid sequence relative to a fixed location. Upstream refers to the region 5' (i.e., close to the 5' end of the strand) relative to that location, while downstream refers to the region 3' (i.e., close to the 3' end of the strand) relative to that location.

[0164] As used herein, “viral resistance” refers to the ability of cells to resist viral infection. More specifically, in the engineered cell lines disclosed herein, viral entry and / or viral replication are reduced or eliminated compared to unmodified parental cell lines.

[0165] Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence it encodes, and comparing these sequences with second nucleotide or amino acid sequences. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage of identity. The percentage of identity between two sequences (whether nucleic acid or amino acid sequences) is the exact number of matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. The local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) provides an approximate alignment method for nucleic acid sequences. This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 Supplement 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining the percentage of sequence identity is provided by Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other suitable procedures for calculating the percentage of identity or similarity between sequences are generally known in the art; for example, another alignment procedure is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; strands = two; cutoff value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these procedures can be found on the GenBank website. The expected degree of sequence identity relative to the sequences described in this paper ranges from approximately 80% to 100% and any integer value in between.Typically, the percentage of identity between sequences is at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.

[0166] Since various changes can be made to the cells and methods described above without departing from the scope of the invention, all content contained in the above description and the embodiments given below should be interpreted as illustrative and not limiting. Example

[0167] The following examples illustrate certain aspects of the present invention.

[0168] Example 1: Preparation of CHO cell lines modified with targeted genes

[0169] ZFN-mediated gene modification techniques are employed to inactivate (i.e., knock out) genes encoding enzymes or proteins involved in N-linked or O-linked glycosylation reactions. Generally, proprietary algorithms are used to design pairwise ZFNs targeting specific sites within the coding region of the gene of interest. ZFN expression constructs are prepared using standard procedures, and in vitro transcription, mRNA polyadenylation, and end-capping methods are employed, such as... ZFN mRNA was generated by knocking out the ZFN plasmid DNA as described in the Sigma-Aldrich product information. Briefly, the ZFN plasmid DNA was linearized and purified using phenol / chloroform DNA extraction. MessageMax was used. TM The linearized DNA was capped using the T7 ARCA-Capped Message Transcription Kit (Cell Script Inc.). Poly(A) Polymerase Tailing Kit (EpiCentre) was used to add poly(A) tails. MEGAclear was used. TM A kit (Ambion) was used to purify ZFN mRNA. Parental cells were maintained in suspension culture at [temperature missing]. Cells were cultured in CHO-CD fusion medium (Sigma-Aldrich). One day before transfection, cells were inoculated at 0.5 × 10⁻⁶ cells / mL. 6 Cells / mL were seeded in bioreactor tubes. Typically, each transfection contained 1 × 10⁶ cells / mL in 150 μL of growth medium. 6 One cell and 5 μg ZFN DNA or mRNA. Transfection was performed by electroporation in 0.2 cm tubes at 140 V and 950 μF. Electroporated cells were then placed in 2 mL of growth medium in a 6-well plate under static culture.

[0170] Cells were removed from the culture on days 3 and 10 post-transfection and treated with GeneElute. TM A miniature mammalian genomic DNA preparation kit (Sigma-Aldrich) is used to isolate genomic DNA. For example... The ZFN knockout process described in the product information was validated using a Cel-1 nuclease assay to assess ZFN-induced cleavage. This assay was performed to determine the efficiency of ZFN-mediated gene mutations as previously described (Miller et al., Nat. Biotechnol. 2007, 25:778-785). This assay detects alleles derived from wild-type target loci due to imperfect repair mediated by non-homologous end joining (NHEJ) of ZFN-induced DNA double-strand breaks. PCR amplification of the target region from a pool of ZFN-treated cells produced a mixture of wild-type (WT) and mutant amplicons. Melting and re-annealing of this mixture resulted in mismatch formation between heteroduplexes of the WT and mutant alleles. The DNA “bubbles” formed at the mismatch sites were cleaved by the Surveyor nuclease Cel-1, and the cleavage products were separated by gel electrophoresis.

[0171] After confirming ZFN activity, single-cell cloning was performed on ZFN-transfected cells using limiting dilutions. For this purpose, cells were plated at an approximate density of approximately 0.5 cells / well using a mixture of 80% CHO serum-free cloning medium, 20% conditioned medium, and 4 mM L-glutamine. Clonalness and growth were verified under a microscope on days 7 and 14 post-plate loading. Growing clones were amplified and genotyped by PCR and DNA sequencing.

[0172] Mgat1 KO cell lineAs part of the synthesis of complex N-glycans, Mgat1 adds GlnNac to the Man5GlcNAc2 N-linked polysaccharide structure. A pair of ZFNs was designed to target the 5'-AACAAGTTCAAGTTCccagcaGCTGTGGTAGTGGAGGAC-3' (SEQ ID NO: 9; ZFN binding sites are indicated in uppercase letters and cleavage sites in lowercase letters) of the CHO Mgat1 gene. The parental cell line was CHOK1(GS- / -) (Sigma Aldrich), in which glutamate synthase was knocked out. This parental cell line produces wild-type N-glycan and O-glycan structures. Essentially as detailed above, the CHOK1(GS- / -) cell line was transfected with Mgat1 ZFN DNA. Cel-1 assays confirmed the presence of two cleavage fragments, 220 bp and 197 bp, on days 3 and 10 post-transfection, indicating ZFN activity. The single-cell clone was identified as having a deletion in one allele ranging from 2 bp to 55 bp (the second allele was not detected). This cell line produced a truncated N-linked structure (i.e., Man5NeuAc2 glycoform) capped with five mannose residues and a wild-type O-glycan structure.

[0173] COSMC KO cell line The most common second biosynthetic step in O-glycosylation is core-1 elongation. Core-1 elongation is catalyzed by a single enzyme, C1GalT1 (aka T-synthase), which requires a dedicated chaperone, COSMC, to function. COSMC is an ER protein that appears to specifically bind to T-synthase and ensure its full activity in the Golgi apparatus. To disrupt the COSMC gene, CHOK1 (GS- / -) cells were transfected with DNA or RNA encoding a ZFN designed to target the sequence 5'-GCCTTCTCAGTGTTCCGGAaaagtgTCCTGAACAAGGTGGGAT-3' (SEQ ID NO: 10) in the CHO COSMC gene. Cel-1 nuclease assays confirmed the presence of three cleavage fragments (i.e., 318 bp, 184 bp, and 134 bp) in the ZFN-transfected cells. Single-cell clones with a 4 bp deletion in one allele of COSMC (the second allele was not detected) were isolated. This cell line produced truncated (i.e., immature) O-glycan structures and wild-type N-glycans.

[0174] COSMC / Mgat3 KO cell lineA pair of ZFNs was designed to target the 5'-TTCCTGGACCACTTCCCAcccggtGGCCGGCAGGATGGC-3' (SEQ ID NO: 11) in the coding region of Mgat3. The COSMC KO cell line detailed above was transfected with the ZFN DNA as detailed above. After confirming ZFN cleavage, single-cell clones were isolated. Sequencing showed that the mutant clones had deletions of 9, 10, 11, or 41 bp in the Mgat3 gene. This cell line produced wild-type N-glycans and truncated O-glycans (i.e., similar to the parental cell line).

[0175] COSMC / Mgat3 / Mgat5 KO cell line The COSMC / Mgat 3 KO cell line, as detailed above, was transfected with plasmid DNA encoding a ZFN designed to target the 5'-TTCTGCACTTCACCATCCAgcagcgGACTCAGCCTGAGAGCAGCT-3' (SEQ ID NO:12) coding region of Mgat5. Single-cell clones with a 129 bp deletion in the Mgat5 gene were isolated. This cell line produced N-glycans with fewer side branches and truncated O-glycans.

[0176] Example 2: Viral infection and viral resistance testing

[0177] The above-mentioned CHO cell lines were grown and their ability to support or resist infection after challenge with the prototype MVM virus (virus strain MVMp) was tested. Briefly, cells were grown in appropriate culture medium and MVMp virus was added at a multiplicity of infection (MOI) of 1 or 10, and the infected cells were incubated for 24, 48, or 72 hours prior to assay. As a control, cells were grown and incubated in the absence of virus. Uninfected cells were also treated with the enzyme neuraminidase to remove terminal sialic acid from surface glycans (N-linked and O-linked) and subsequently infected at the indicated MOI.

[0178] The presence of cytopathic effect (CPE) was observed visually in both infected and uninfected cells, and cells were collected by centrifugation at indicated time points. Viral DNA infectivity and yield were screened using Southern blotting analysis of total genomic DNA and Western blotting analysis using antiviral protein antibodies. For Southern blotting, cell pellets from two samples acquired at 24 h were collected and total genomic DNA was isolated. DNA was quantified by Nanodrop spectroscopy, and samples were regularized to ensure equal loading on agarose gels for size fractionation. Size-fractionated DNA was transferred to a charged membrane (Southern blotting) and used... 32P-labeled viral DNA probes detect viral DNA synthesis on this membrane. For specific... 32 Quantification of P-labeled viral double-stranded DNA bands was obtained by phosphorus imaging, and the relative values ​​are reported in Table 1.

[0179]

[0180] For Western blot analysis, cell spheres collected at 24 h were dissolved in SDS buffer, and proteins were separated using SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane, blocked, and immunoblotted against the presence of viral NS1 protein using an anti-NS1 antibody (Western immunoassay). The viral protein levels detected by Western blot are shown in Table 2.

[0181]

[0182]

[0183] When infected with the MVMp viral strain, the parental cell line CHOK1(GS- / -) (i.e., possessing the wild-type glycan structure) exhibited classic infectivity and viral DNA production and protein synthesis as expected. Viral DNA and viral protein products were readily apparent 24 hours post-infection when cells were infected at any MOI. Strong production of viral products was also observed at later time points. As expected, uninfected wild-type cells showed no evidence of infection regardless of time point.

[0184] When wild-type cells were treated with the enzyme neuraminidase (NA) to remove sialic acid from the cell surface and subsequently infected with MVMp, a decrease in viral yield (indicating reduced infection) was also observed (ranging from 5.32-fold at MOI=10 to 7.34-fold at MOI=1). Although NA-treated cells still showed slight susceptibility to infection, CHO cells are known to rapidly regenerate sialic acid (SA) structures after this treatment. Therefore, the observed low infection levels may originate from cells that have regenerated terminal SA glycans and thus provide an entry point for the virus.

[0185] Viral infection of Mgat1 KO cells with inactivated CHO Mgat1 gene and no Mgat1 enzyme production elicited only mild resistance (e.g., a 2.1 to 2.7-fold reduction, as measured by phosphorus imaging of the blot). Because this cell line possesses truncated N-glycans, these results suggest that higher-order N-glycan receptors with 2–3-linked sialic acid may play only a minor role in initial viral capsid binding and viral entry into cells.

[0186] When COSMC KO cells were infected with MVMp virus, the cell line exhibited significant resistance to the virus. The fold change in resistance (compared to wild-type cell lines) ranged from 5.5-fold (MOI = 10) to 190-fold (MOI = 1). Similar results were obtained from additional gene knockout targeting the Mgat3 gene (presumably a pseudogene in CHO cells) and the Mgat5 gene (responsible for higher-order N-connected branching). Western blot analysis yielded similar results, indicating that viral resistance occurred when the O-glycan structure was truncated. These studies reveal that disrupting the sialic acid receptor significantly reduces MVM binding and / or entry into cells.

[0187] Example 3: Generation of other COSMC KO and Slc35A1 KO cell lines

[0188] As described in Example 1 above, novel COSMC KO cell line clones were generated using CHOK1 (GS- / -) cells and a pair of ZFNs designed to target the sequence 5'-GCCTTCTCAGTGTTCCGGAaaagtgTCCTGAACAAGGTGGGAT-3' (SEQ ID NO: 10) in exon 2 of the CHO COSMC gene. Cel-1 nuclease assays confirmed the presence of three cleavage fragments (i.e., 318 bp, 184 bp, and 134 bp) in the ZFN-transfected cells. Five single-cell clones were isolated, and sequencing revealed deletions of 1 to 12 bp, as shown below.

[0189] clone genotype COSMC F07 2bp missing COSMC G03 1bp insertion COSMC H04 1bp insertion COSMC A09 9bp missing COSMC H05 12bp missing

[0190] Clone F07 was further modified using standard procedures to express human IgG. Among the many clones identified, two IgG-producing clones (identified as 71H1 and 71C3) were isolated for further testing (see below).

[0191] The Slc35A1 gene, encoding the nucleotide sugar transporter (CMP-sialic acid transporter), was knocked out from CHOK1 (GS- / -) cells using a ZFN designed to target 5'-AGCTTATACCGTAGCTTTaagataCACAAGGACAACAGCTAAA-3' (SEQ ID NO:13) or a ZFN designed to target 5'-TTCAAGCTATACTGCTTGGCAGTGATGACTCTGGTGGCT-3' (SEQ ID NO:17) in exon 1 of the CHOSlc35A1 gene. Cel-1 nuclease assays confirmed the presence of two cleaved fragments in the ZFN-transfected cells. A single-cell clone (B12) was isolated, and sequencing revealed a 1 bp deletion around the ZFN binding site. The Slc35A1 KO cell line formed N-linked and O-linked glycan structures without terminal sialic acid.

[0192] Staining with biotin-labeled sophora japonica lectin II (MALII; 20 μg / mL) and streptavidin labeled with Alexa Fluor 647 (5 μg / mL) showed a significant reduction in staining in COSMC KO clone F07, COSMC KO clone G03, and Slc35A1 KO cell lines compared to the parental cell lines (indicating the absence of terminal sialic acid residues in the KO cell lines).

[0193] Example 4: Resistance of COSMC KO and Slc35A1 KO cell lines to MMV virus

[0194] Essentially as described in Example 2 above, wild-type (i.e., CHOZNGS- / -), COSMC KO (generated in Examples 1 and 3 above), and Slc35A1 KO (generated in Example 3 above) cells were infected with MVMp virus at an MOI of 0.3. Infection was determined by Southern blotting (essentially as described above) and standard plaque assays.

[0195] like Figure 1 As shown, wild-type cells exhibited high levels of viral DNA, while the Slc35A1 KO and COSMC KO cell lines had very low levels of viral DNA; the COSMC F07 KO and COSMC G03 KO clones had low levels of viral DNA; and the COSMC H05 FO clone with a 12bp deletion had viral DNA levels similar to the parental cell lines. Since the deletion in H05 is in the same frame, it is not surprising that this cell exhibited near-wild-type levels of viral susceptibility. Phage plaque assay results are shown in... Figure 2In the study, Slc35A1 KO, COSMC KO, COSMC F07 KO, and COSMC G03 KO cells showed extremely low viral levels.

[0196] Additional viral resistance tests were performed using wild-type (i.e., CHOZN GS- / -, also known as 2E3), COSMC KO clones F07 and G03, and Slc35A1 KO clone B12. Cells were grown in medium supplemented with 6 mM L-glutamine and infected with MMVp at MOI 1 or 8, and incubated under suitable conditions. Cell samples were collected at 0, 24, 48, 72, 96, and 120 hours; cell viability was determined by trypan blue staining, and MMV quantification was performed by qPCR.

[0197] Cell vitality is presented in Figure 3 In the wild-type cells (i.e., 2E3), cell viability decreased to approximately 50% at 120 hours post-infection (see Figure A), while in COSMC clone F07, at least 93% of the cells remained viable at 120 hours post-infection (see Figure B). Figure 4 The time course of cell-associated virus (viral genome copies (vgc) per cell) following MMV infection with MOI1 (see Figure A) or MOI8 (see Figure B) in wild-type (2E3) and COSMC clone F07 is shown (calculated based on the fact that each infected cell can produce 2 × 10⁻⁶ cells / cells). 4 (hypothesis of VGC). The following presents the fractionation of infected cells under various conditions.

[0198]

[0199] Wild-type (i.e., 2E3), COSMC KO clones F07, G03, and H04, and Slc35A1 KO clone B12 cells were infected with MMVp at an MOI of 0.3 or 0.03 at time -3 hours. At time 0 hours, cells were washed three times with growth medium and then incubated for 21 hours (i.e., a single replication cycle). Viral replication was determined by identifying cell-associated viruses at 0 and 21 hours. Figure 5 As shown, viral replication was reduced in the COSMC KO cell line and eliminated in the Slc35A1 KO cell line.

[0200] Example 5: Resistance of COSMC KO and Slc35A1 KO cell lines to reovirus 3

[0201] Wild-type (i.e., 2E3), COSMC KO clone F07, and Slc35A1 KO clone B12 cells were infected with reovirus 3 at two dilutions within 3 hours. 50=5.6E+07 and 5.6E+06). At time 0 h, cells were washed three times and then incubated for 24 h (i.e., a single replication cycle). Viral replication was determined by identifying cell-associated viruses at 0 and 24 h. At 24 h, the level of cell-associated viruses was significantly reduced in the COSMC KO clone and almost eliminated in the Slc35A1 KO clone (see [link to relevant documentation]). Figure 6 ).

[0202] Example 6: Growth assay of COSMC KO and Slc35A1 KO cell lines

[0203] Wild-type (i.e., CHOZN GS- / -), COSMCKO clones F07 and G03, and Slc35A1 KO clone B12 cells were grown for 8 to 10 days in the absence or presence of MVMp virus (MOI 0.1). Cell growth was monitored by measuring viable cell density (by trypan blue staining) on ​​days 0, 1, 2, 3, 6, 7, 8, and 10. Figure 7 As shown in Figure A, various KO cells exhibited similar growth profiles in the presence or absence of the virus, indicating resistance to viral inoculation. In contrast, wild-type cells displayed classic infectivity, impaired growth, and low cell viability when compared to uninfected cultures.

[0204] Essentially, as detailed above, the cell growth of wild-type and IgG-producing clones (i.e., 71H1 and 71C3) of COSMC KO clone F07 was monitored in the absence or presence of the virus. Figure 7 As shown in Figure B, the IgG-producing KO cell lines also exhibited significant resistance to viral infection. Infected cultures grew at a similar rate to uninfected cultures and reached peak cell density. These data suggest that secretion of cell surface IgG has no significant effect on the degree of viral resistance exhibited by the COSMC KO parent. Although the IgG-producing cell lines grew at a slower rate compared to wild-type cells, both IgG producers showed resistance to viral infection (i.e., there was no difference when compared to uninfected cultures).

[0205] Example 7: Generation and growth assay of the St3Gal4 KO cell line

[0206] Essentially as described in Example 1, St3Gal4KO cells were generated using a ZFN designed to target the 5'-GGCAGCCTCCAGTGTCGTC gttgtgTTGTGGTGGGGA ATGGGC (SEQ ID NO:14) in the CHO St3Gal4 gene. Cel-1 nuclease assays confirmed the presence of three cleavage fragments (i.e., 344 bp, 210 bp, and 135 bp) in the ZFN-transfected cells. Four single-cell clones were isolated, and sequencing revealed the following mutations. St3Gal4KO cells exhibited lower levels of 2-3 linked sialic acid structures.

[0207]

[0208]

[0209] St3Gal4KO (i.e., clones 7D10, 1B8, and 1B10) and wild-type cells were grown in the absence or presence of MVMp virus (MOI 0.1), and cell growth was monitored for 9 days. St3Gal4KO cells also showed significant resistance to viral infection, such as... Figure 8 As shown in the figure, infected KO cultures grew at a similar rate to uninfected KO cultures and reached peak cell density. These studies suggest that disrupting specific 2-3-linked sialic acid structures on the cell surface also appears to significantly reduce MVM entry into these cells.

[0210] Example 8: Generation of St3Gal4 and St3Gal6 double KO cell lines

[0211] St3Gal4KO (i.e., clones 7D10, 1B8, and 1B10) cell lines were used as starting cells to generate cell lines that also contained the knocked-out St3Gal6 gene. ZFN was engineered to target 5'-CGGTACCTCTGATTTTGCTttgccCTATGGGACAAGGCC-3' (SEQ ID NO:15) and gene editing was performed substantially as described in Example 1. Cel-1 nuclease assays confirmed the presence of three cleavage fragments (i.e., 308 bp, 171 bp, and 137 bp) in ZFN-transfected cells. Six single-cell clones were isolated, and sequencing revealed the following mutations.

[0212]

[0213] Example 9: Generation of C1GalT1 KO cell line

[0214] Essentially as described in Example 1, the C1GalT1 gene in CHOK1 (GS- / -) cells was knocked out using a ZFN designed to target 5'-ACCCTCATGCTAGACatttaGATGATAACGAACCCAGTC-3' (SEQ ID NO:16). Cel-1 nuclease assays confirmed the presence of two cleaved fragments (i.e., 223 bp and 148 bp) in the ZFN-transfected cells. Seven single-cell clones were isolated, and sequencing revealed the following mutations.

[0215] C1GalT1 clone Allele 1 genotype Allele 2 genotype 2A8 8bp missing 8bp missing 2A11 5bp missing 90bp insertion 2C12 8bp missing Not detected 2G12 5bp missing Not detected 3D12 8bp missing Not detected 3E6 1bp missing 2bp deletion and 97bp insertion 3F2 5bp missing 5bp deletion and 31bp insertion

[0216] Staining with biotin-labeled MALII and Alexa Fluor 647-labeled streptavidin showed a significant reduction in staining in C1GalT1 KO clone 2C12 compared to the parental cell line.

[0217] Example 10: Generation of a cell line overexpressing St6Gal1

[0218] Because it was assumed that the MVM virus does not bind to α-2,6-linked sialic acid, a CHO cell line overexpressing St6Gal1 was generated. The coding sequence for Chinese hamster St6Gal1 was obtained from GenBank (AB492855) and chogenome.org AQ2 (AFTD01061789 and AFTD01061790). A batch-synthesized Kozak sequence (5'-GCCGCCACCAatg-3'; SEQ ID NO:18) was added to the open reading frame of the 5'-untranslated region (UTR). The synthesized fragment was cloned into the expression vector pJ602 (DNA2.0; Menlo Park, CA). This construct was used to transfect (by electroporation) CHO (GS- / -) host cell lines and IgG-expressing CHO cell lines. Single-cell clones were isolated using a FACSAria™ III cell sorter. For this purpose, cells were stained with FITC-conjugated elderberry lectin (FITC-SNA) that binds to α-2,6-linked sialic acid, and cells with the first 5% fluorescence were seeded and cultured at 1 cell / well. Single-cell clones were analyzed using two-color FACS on a MACS QuantVR analyzer (Miltenyi Biotec, San Diego, CA) after staining with biotin-labeled MALII and Alexa Fluor 647-labeled streptavidin that binds to α-2,3-linked sialic acid. Single-cell clones overexpressing St6Gal1 had a higher FITC to Alexa Fluor ratio compared to untransfected parental cell lines.

[0219] IgG was isolated from two clones overexpressing St6Gal1 (i.e., clones 31 and 32) and from parental cell clones. IgG or total cellular protein extracts were reduced and carboxyamide-methylated according to standard procedures, followed by trypsin treatment overnight (12–16 h) at 37 °C. Trypsin was inactivated by heating at 100 °C for 5 min. Fragment purification was performed using C18 SPE cartridges (Waters, 300 mg packing). After washing with 5% acetic acid (AcOH), peptides / glycopeptides were eluted sequentially in 20% isopropanol / 5% AcOH, 40% isopropanol / 5% AcOH, and 100% isopropanol. The eluent was dried to reduce volume, reconstituted in phosphate buffer containing PNG enzyme F, and incubated overnight at 37 °C. The released glycans were purified using a C18 filter cartridge, fully methylated, and diluted to 1 mM lithium carbonate / 50% MeOH. The solutions were then directly infused into an LTQ Orbitrap Discovery mass spectrometer (Thermo Scientific) at a flow rate of 0.5 mL / min for nanospray ionization. Complete Fourier transform mass spectrometry (FTMS) spectra of each sample were obtained at 30,000 ppm to determine which glycans contained sialic acid. Sialic acid bonding of the sialylated glycans was determined by MSn analysis. Multiple ion selection and fragmentation steps were performed on each sample within an ion trap to break down complex glycans into individual galactoses, and the fragmentation patterns were then observed.

[0220] A striking difference exists between the N-glycan distribution in the GS(- / -) host cell line and the two St6Gal1 overexpressing cell lines (i.e., clones 31 and 32). Specifically, no sialylated glycans were identified in the host cell line due to their low abundance. In contrast, monosialylated, disialylated, trisialylated, and tetrasialylated N-glycans were identified in both St6Gal1 overexpressing cell lines. Specifically, in clone 32, all but one sialylated population exhibited α-2,3-linked and α-2,6-linked N-glycans. sequence list <110> Sigma-Aldridge LLC Lin Nan J. Mascarlenhaals A. Often D. Anianz H. George K. Kaiser <120> Antiviral cells and their uses <130> 047497-469433 <150> US 61 / 947,860 <151> 2014-03-04 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Pro Lys Lys Lys Arg Arg Val 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 4 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 4 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Pro Lys Lys 1 5 10 15 Lys Arg Lys Val 20 <210> 5 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 5 Pro Leu Ser Ser Ile Phe Ser Arg Ile Gly Asp Pro Pro Lys Lys Lys 1 5 10 15 Arg Lys Val <210> 6 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Pro Lys Lys Lys Arg Lys Val 20 <210> 7 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 7 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 <210> 8 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Synthesized <400> 8 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 9 <211> 39 <212> DNA <213> Grey Hamster (Cricetulus griseus) <400> 9 aacaagttca agttcccagc agctgtggta gtggaggac 39 <210> 10 <211> 43 <212> DNA <213> Grey Hamster <400> 10 gccttctcag tgttccggaa aagtgtcctg aacaaggtgg gat 43 <210> 11 <211> 39 <212> DNA <213> Grey Hamster <400> 11 ttcctggacc acttcccacc cggtggccgg caggatggc 39 <210> 12 <211> 45 <212> DNA <213> Grey Hamster <400> 12 ttctgcactt caccatccag cagcggactc agcctgagag cagct 45 <210> 13 <211> 43 <212> DNA <213> Grey Hamster <400> 13 agcttatacc gtagctttaa gatacacaag gacaacagct aaa 43 <210> 14 <211> 43 <212> DNA <213> Cricetulus migratorius <400> 14 ggcagcctcc agtgtcgtcg ttgtgttgtg gtggggaatg ggc 43 <210> 15 <211> 39 <212> DNA <213> Cricetulus migratorius <400> 15 cggtacctct gattttgctt tgccctatgg gacaaggcc 39 <210> 16 <211> 39 <212> DNA <213> Cricetulus migratorius <400> 16 accctcatgc tagacattta gatgataacg aacccagtc 39 <210> 17 <211> 39 <212> DNA <213> Cricetulus migratorius <400> 17 ttcaagctat actgcttggc agtgatgact ctggtggct 39 <210> 18 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 18 gccgccacca atg 13

Claims

1. A method of conferring to a genetically modified mammalian cell line reduced or eliminated entry and / or propagation of a parvovirus as compared to an unmodified parental cell line, comprising the step of inactivating in said cell line a chromosomal sequence encoding mannosyl (a-l,3-) -glycoprotein beta-l,2-N-acetylglucosaminyltransferase 1 (Mgatl), wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said parvovirus is a minute virus of mice (MVM).

2. The method of claim 1, wherein a targeted endonuclease-mediated genome modification technology is used to inactivate said chromosomal sequence encoding Mgatl.

3. The method of claim 2, wherein said targeted endonuclease is a zinc finger nuclease.

4. The method of any one of claims 1 to 3, said cell line comprising at least one nucleic acid encoding a recombinant protein selected from an antibody, an antibody fragment, a vaccine, a cytokine, a hormone, or a coagulation factor.

5. The method of any one of claims 1 to 3, further comprising the step of inactivating in said cell line at least one chromosomal sequence encoding glutamine synthetase.

6. The method of any one of claims 1 to 3, wherein said cell line is used in a biological production system.

7. The method of claim 6, wherein said biological production system is resistant to viral contamination.

8. The method of any one of claims 1 to 3, said cell line comprising at least one nucleic acid encoding a recombinant protein that is a growth factor.

9. A method of reducing the risk of viral contamination of a biological production system, the method comprising: A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

10. A method of reducing or preventing viral contamination of a recombinant protein product, said method comprising: a) obtaining a genetically modified mammalian cell line produced by the method of any one of claims 1 to 4; and b) expressing said recombinant protein product in said genetically modified mammalian cell line, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

11. A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

12. A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

13. A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

14. A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

15. A genetically modified mammalian cell line produced by the method of any one of claims 1 to 7 for use in a biological production system, wherein said cell line is a Chinese hamster ovary (CHO) cell line, and wherein said virus is a minute virus of mice (MVM).

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