RU2820218C2 - Vectors for protein production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to expression vectors for producing proteins of interest. Invention discloses an expression vector with a weak promoter SIN LTR, which controls expression of a selectable marker.

EFFECT: presence in the structure of the expression vector of such a weak promoter, which is in a functional relationship with a selectable marker, improves ability of cell culture to produce higher titres and provides high specific productivity of protein of interest.

14 cl, 9 dwg, 7 tbl, 6 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to US Provisional Application No. 62/775194, filed December 4, 2018, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to vectors and their use for developing host cell lines for the production of proteins of interest and, in particular, to vectors that use a weak promoter to drive a selectable marker.

BACKGROUND OF THE ART

Therapeutic protein drugs are an important class of drugs that help patients who most need new therapies. Approved recombinant protein therapeutics have recently been developed to treat a wide range of clinical indications, including cancers, autoimmune/inflammatory diseases, infectious agents, and genetic disorders. Recent advances in protein engineering technologies allow drug developers and manufacturers to precisely regulate and exploit the desired functional characteristics of proteins of interest while maintaining (and in some cases increasing) the safety or efficacy of the product, or both.

The production and production of therapeutic proteins are highly complex processes. For example, a typical protein drug may involve more than 5,000 critical process steps, many times the number required to produce a small molecule drug.

Likewise, protein therapeutics, which contain monoclonal antibodies as well as large or fusion proteins, can be orders of magnitude larger in size than small molecule drugs, with molecular weights exceeding 100 kDa. Additionally, protein therapeutics exhibit complex secondary and tertiary structures that must be maintained. Protein therapeutics cannot be completely synthesized by chemical processes and must be produced in living cells or organisms; therefore, the choice of cell line, species origin, and culture conditions together influence the characteristics of the final product. Moreover, most biologically active proteins require post-translational modifications, which can be disrupted when using heterologous expression systems. In addition, because products are synthesized by cells or organisms, complex purification processes are involved. In addition, virus purification processes such as removal of viral particles using filters or resins, and inactivation steps using low pH or detergents are used to prevent serious safety problems due to viral contamination of protein drug substances. Given the complexity of therapeutic proteins in terms of their large molecular size, post-translational modifications, and the diversity of biological materials involved in their production process, the ability to enhance specific functional attributes while maintaining product safety and efficacy achieved through protein engineering strategies is highly desirable.

Although the integration of new strategies and approaches for protein drug modification is not trivial, the potential therapeutic benefits have prompted the increased use of such strategies in the drug development process. A number of protein engineering platform technologies are currently being used to increase circulation half-life, target and functionality of new protein therapeutics, and to increase product yield and product purity. For example, protein conjugation and derivatization techniques, including Fc-fusion, albumin-fusion, and PEGylation, are currently used to prolong the half-life of a drug from the bloodstream.

Producing a protein pharmaceutical (biological) is expensive and time consuming. The art requires more efficient tools and processes to produce this important class of drugs.

SUMMARY OF THE INVENTION

The present invention relates to vectors and their use for developing host cell lines for the production of proteins of interest and, in particular, to vectors that use a weak promoter to drive a selectable marker.

Accordingly, in some preferred embodiments, the present invention provides vector(s) for expression of a protein of interest comprising a nucleic acid sequence encoding a selectable marker in operative association with a first promoter sequence that has been modified to reduce promoter activity relative to an unmodified or wild-type version of the first promoter sequence, and a nucleic acid sequence encoding the protein of interest is operably linked to the second promoter sequence.

In some preferred embodiments, the first promoter sequence that has been modified to reduce promoter activity relative to the unmodified or wild-type version of the first promoter sequence is a viral Self-Inactivating (SIN) Long Terminal Repeat promoter sequence. LTR). In some preferred embodiments, the SIN LTR promoter sequence is at least 95% identical to SEQ ID NO:3. In some preferred embodiments, the SIN LTR promoter sequence is SEQ ID NO:3.

In some preferred embodiments, the selectable marker is glutamine synthetase (GS). In some preferred embodiments, the selectable marker is dihydrofolate reductase (DHFR).

In some embodiments, the vector contains a single poly-A signal sequence in operative association with a selectable marker and a nucleic acid encoding the protein of interest. In other embodiments, the vector comprises a first poly-A signal sequence in operative association with a selectable marker and a second poly-A signal sequence in operative association with a nucleic acid encoding the protein of interest.

In some preferred embodiments, the protein of interest is selected from the group consisting of an Fc fusion protein, an enzyme, an albumin fusion protein, a growth factor, a protein receptor, a single chain antibody (scFv), a single chain antibody-Fc (scFv-Fc), a diabody, and a minibody ( scFv-CH3), Fab, single chain Fab (scFab), immunoglobulin heavy chain and immunoglobulin light chain. In some preferred embodiments, the protein of interest is an Fc fusion protein.

In some embodiments, the vector is a plasmid. In some preferred embodiments, the vector is a viral vector. In some preferred embodiments, the vector is a retroviral vector. In some preferred embodiments, the vector is a lentiviral vector.

In some preferred embodiments, the present invention provides host cell(s) containing the vector described above. In some preferred embodiments, the host cell line is a GS knockout cell line. In some preferred embodiments, the host cell line is a DHFR knockout cell line. In some preferred embodiments, the host cell line is a Chinese Hamster Ovary (CHO) cell line. In some preferred embodiments, the host cell line is a HEK 293 or CAP cell line. In some preferred embodiments, the host cell contains from about 1, 20, 50 to 1000 copies of the vector. In some preferred embodiments, the host cell contains from about 10 to 200 copies of the vector. In some preferred embodiments, the host cell contains from about 10 to 100 copies of the vector. In some preferred embodiments, the host cell contains from about 20 to 100 copies of the vector.

In some preferred embodiments, the host cell further comprises at least a second vector that encodes and provides expression of a second protein of interest, wherein said second vector does not include a selectable marker. In some preferred embodiments, the host cell further comprises at least a second vector that encodes and provides expression of a second protein of interest, wherein said second vector includes a selectable marker that is different from a selectable marker in the first vector. In some preferred embodiments, the first protein of interest in the first vector is one of an immunoglobulin heavy or light chain, and the second protein in the second vector is another of an immunoglobulin heavy or light chain. In some preferred embodiments, the first protein of interest is an immunoglobulin heavy chain and the second protein of interest is an immunoglobulin light chain. In some preferred embodiments, the host cell contains from about 1, 20, 50, or 100 to 1000 copies of the second vector. In some preferred embodiments, the host cell contains from about 10 to 200 copies of the second vector. In some preferred embodiments, the host cell contains from about 10 to 100 copies of the second vector. In some preferred embodiments, the host cell contains from about 20 to 100 copies of the second vector.

In some preferred embodiments, the present invention provides a host cell culture containing the host cells described above. In some preferred embodiments, the culture produces from 1 to 50 grams/liter/day of the protein of interest. In some preferred embodiments, the culture produces 2 to 10 grams/liter/day of the protein of interest.

In some preferred embodiments, the present invention provides a method for producing a protein of interest, comprising culturing the host cells described above and purifying the protein of interest from the host cell culture.

In some preferred embodiments, the present invention provides an infectious retroviral particle comprising the following elements in order 5' to 3': 1) 5' LTR; 2) retroviral packaging region; 3) a nucleic acid encoding a selectable marker; 4) internal promoter; 5) a nucleic acid sequence encoding the protein of interest, which is operably linked to the internal promoter; and 6) 3’ SIN LTR. In some preferred embodiments, the 5'LTR is a MoMuSV LTR or a SIN LTR. In some embodiments, the 3' LTR contains a poly-A signal sequence. In some embodiments, the packaging region contains a plurality of potential translation initiation sites. In some preferred embodiments, the selectable marker is GS. In some preferred embodiments, the internal promoter is a CMV promoter. In some embodiments, the particle contains a single poly-A signal sequence after a nucleic acid encoding the protein of interest. In some embodiments, the particles comprise a first poly-A signal sequence in operative association with a selectable marker and a second poly-A signal sequence in operative association with a nucleic acid encoding the gene of interest.

In additional embodiments, a plasmid is provided containing the following elements in order 5' to 3': 1) 5' LTR (eg, SIN LTR); 2) packaging area; 3) selectable marker (for example, GS); 4) internal promoter (eg, CMV promoter); 5) a nucleic acid sequence encoding the protein of interest, which is operably linked to the internal promoter; and 6) poly-A signal sequence. In some embodiments, the plasmid contains one poly-A signal sequence after the nucleic acid encoding the protein of interest.

In additional embodiments, a system is provided, comprising a) a first vector containing a nucleic acid sequence encoding a selectable marker in operative association with a first promoter sequence that has been altered to reduce promoter activity relative to an unaltered or wild-type version of the first promoter sequence, and a nucleic acid sequence encoding a first protein of interest is operably linked to a second promoter sequence; and b) a second vector containing a nucleic acid sequence encoding a second protein of interest operably linked to a promoter sequence, wherein the second vector does not include a selectable marker.

In some preferred embodiments, the present invention provides a method for producing a protein of interest, comprising: transducing or transfecting a host cell or cell with an infectious retroviral particle, plasmid or vector system described above, developing a host cell line that expresses the protein of interest from that host cell or cells; culturing host cells under conditions such that the protein of interest is produced by the host cell line; and purifying the protein of interest from the host cell culture.

In some preferred embodiments, the present invention provides an infectious retroviral particle or plasmid as described above for use in transducing a host cell or cells to produce a protein of interest.

DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 - Map of the full-size MMLV design.

FIG. 2 - SIN LTR MMLV design map.

FIG. 3 - Sequence of full-length MMLV design (SEQ ID NO:1).

FIG. 4 - SIN LTR MMLV design sequence (SEQ ID NO:2).

FIG. 5 - Comparison of pooled cell line titer between the full-length MMLV construct and the SIN LTR MMLV construct.

FIG. 6 - Comparison of pooled cell line titer between processes using the full-length MMLV construct and the SIN LTR MMLV construct.

FIG. 7 - Sequence SIN LTR (SEQ ID NO:3).

FIG. 8 - Map of proviral plasmid construct (SEQ ID NO: 4).

FIG. 9 - Sequence of the proviral plasmid construct (SEQ ID NO: 4).

DEFINITIONS

To facilitate understanding of the present invention, definitions for a number of terms are provided below.

As used herein, the term “host cells” refers to any eukaryotic cell (eg, mammalian cells, avian cells, amphibian cells, plant cells, fish cells and insect cells), regardless of their location in vitro or in vivo.

As used herein, the term “cell culture” refers to any in vitro cell culture. The term includes continuous cell lines (eg, with an immortal phenotype), primary cell cultures, terminal cell lines (eg, untransformed cells), and any other cell populations maintained in vitro, including eggs and embryos.

As used herein, the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication when associated with appropriate regulatory elements and that can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles as well as viral vectors.

As used herein, the term “multiplicity of infection” or MOI refers to the ratio of integrating vectors:host cells used during transfection or transduction of host cells. For example, if 1,000,000 vectors are used to transduce 100,000 host cells, then the multiplicity of infection is 10. The use of this term is not limited to transduction events, but includes the introduction of the vector into the host using methods such as lipofection, microinjection, phosphate deposition calcium and electroporation.

As used herein, the term “genome” refers to the genetic material (eg, chromosomes) of an organism.

The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which by one of ordinary skill in the art would be considered desirable for any reason (e.g., treatment of a disease, acquisition of improved traits, expression of a protein of interest in a cell -host, ribozyme expression, etc.). Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (for example, reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences that are not encode an mRNA or protein product (eg, promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).

As used herein, the term “protein of interest” refers to the protein encoded by the nucleic acid of interest.

As used herein, the terms “encoding nucleic acid molecule,” “encoding DNA sequence,” “encoding DNA,” “encoding RNA sequence,” and “encoding RNA” refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a chain of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a DNA or RNA sequence encodes an amino acid sequence.

The term "promoter", "promoter element" or "promoter sequence" as used herein refers to a DNA sequence that is then ligated to a nucleotide sequence of interest, capable of regulating the transcription of the nucleotide sequence of interest into mRNA. A promoter is usually, but not necessarily, located at the 5' end (i.e., upstream) of the nucleotide sequence of interest, the transcription of which into mRNA is regulated by it, and provides a site for specific binding by RNA polymerase and other transcription factors to initiate transcription.

As used herein, the term “altered,” when applied to a promoter, refers to promoters that have an altered nucleic acid sequence compared to the wild-type reference sequence. For example, the promoter sequence may be changed by deleting certain promoter and/or enhancer elements.

Transcriptional regulation signals in eukaryotes include “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes from yeast, insect and mammalian cells, and viruses (similar regulatory elements, i.e. promoters, are also found in prokaryotes). The choice of specific promoter and enhancer depends on the type of cell that needs to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a wide host range, while others function in a limited subset of cell types (for review, see Voss et al., Trends Biochem. Sci., 11:287 [1986]; and Maniatis et al., higher). For example, the early gene enhancer SV40 is highly active in a wide range of cell types in a variety of mammalian species and has found widespread use for protein expression in mammalian cells (Dijkema et al., EMBO J. 4:761 [1985]). Two other examples of promoter/enhancer elements active in a wide range of mammalian cell types come from the elongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91: 217 [1990]; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]) and long terminal repeats of Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [ 1982]) and human cytomegalovirus (Boshart et al., Cell 41:521 [1985]).

As used herein, the term “promoter/enhancer” refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions (ie, functions provided by a promoter element and an enhancer element, see above for discussion of these functions). For example, long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer/promoter is one that is naturally associated in the genome with a given gene. An "exogenous" or "heterologous" enhancer/promoter is one that is placed adjacent to a gene through genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of that gene is controlled by the associated enhancer /promoter.

As used herein, the term “long terminal repeat” or LTR refers to transcriptional regulatory elements located in or derived from the U3 region of the 5' and 3' directions of the retroviral genome. As is known in the art, long terminal repeats can be used as regulatory elements in retroviral vectors, or isolated from the retroviral genome and used to regulate the expression of other types of vectors.

As used herein, the terms “complementary” or “complementarity” refer to polynucleotides (ie, a sequence of nucleotides) linked by base pairing rules. For example, the sequence "5'-A-G-T-3'" is complementary to the sequence "3'-T-C-A-5'". Complementarity can be "partial" when only some of the nucleic acid bases match according to the base pairing rules. Or there may be “complete” or “total” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is especially important in amplification reactions as well as detection methods that depend on binding between nucleic acids.

The terms "homology" and "percentage identity" when applied to nucleic acids refer to the degree of complementarity. There may be partial homology (i.e. partial identity) or complete homology (i.e. complete identity). A partially complementary sequence is a sequence that at least partially inhibits a fully complementary sequence upon hybridization to a target nucleic acid sequence, and refers to the use of the functional term "substantially homologous". Inhibition of hybridization of a fully complementary sequence to a target sequence can be tested using a hybridization assay (Southern or Northern blot, solution hybridization method, etc.) under low stringency conditions. A substantially homologous sequence or probe (ie, an oligonucleotide that is capable of hybridizing to another oligonucleotide of interest) will compete for binding or inhibit (ie, hybridize) a fully homologous sequence to a target sequence under low stringency conditions. This does not mean that low stringency conditions are such that nonspecific binding is allowed; low stringency conditions require that the binding of two sequences to each other must be a specific (i.e., selective) interaction. The absence of nonspecific binding can be studied by using a second target that lacks even a partial degree of complementarity (eg, less than about 30% identity); in the absence of nonspecific binding, the probe will hybridize to a second noncomplementary target.

The terms “in functional combination,” “in a functional order,” and “operably linked,” as used herein, refer to the linkage of nucleic acid sequences such that the nucleic acid molecule is capable of directing the transcription of a given gene and/or the synthesis of the desired protein molecule produced. The term also refers to the linking of amino acid sequences in such a way that a functional protein is produced.

As used herein, the term “selectable marker” refers to a gene encoding an enzymatic activity or other protein that allows it to grow in a medium lacking what would otherwise be an essential nutrient; in addition, the selectable marker may confer resistance to an antibiotic or drug in the cell in which the selectable marker is expressed.

As used herein, the term “retrovirus” refers to a retroviral particle that is capable of entering a cell (i.e., the particle contains a membrane-associated protein, such as an envelope protein or viral glycoprotein G, that can bind to the surface of a host cell and facilitate entry viral particles into the cytoplasm of the host cell) and integrate the retroviral genome (as a double-stranded provirus) into the genome of the host cell. The term “retrovirus” includes the subfamily Oncovirinae (e.g., Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), and mouse mammary tumor virus (MMTV), Spumavirinae, and Lentivirinae (e.g., human immunodeficiency virus, simian immunodeficiency virus, infectious anemia in horses and goat and sheep arthritis-encephalitis virus, see, for example, US patents No. 5994136 and 6013516, both of which are incorporated herein by reference).

As used herein, the term “retroviral vector” refers to a retrovirus that has been modified to express a gene of interest. Retroviral vectors can be used to efficiently transfer genes into host cells using a viral infection process. Foreign or heterologous genes cloned (ie, inserted using molecular biological techniques) into the retroviral genome can be efficiently delivered to host cells that are susceptible to retroviral infection. Thanks to well-known genetic manipulations, the replicative capacity of the retroviral genome can be destroyed. Defective vectors obtained as a result of replication can be used to introduce new genetic material into a cell, but they are incapable of replication. A helper virus or a packaging cell line can be used to ensure the assembly and release of vector particles from the cell. Such retroviral vectors contain a replication-defective retroviral genome containing a nucleic acid sequence encoding at least one gene of interest (i.e., a polycistronic nucleic acid sequence may encode more than one gene of interest), a 5' retroviral long terminal repeat (5' LTR) ; and 3' retroviral long terminal repeat (3' LTR).

The term "pseudotyped retroviral vector" refers to a retroviral vector containing a heterologous membrane protein. The term “membrane-associated protein” refers to a protein (eg, viral envelope glycoprotein or viral G proteins in the family Rhabdoviridae, such as VSV, Piri, Chandipura and Mokola) that is associated with the membrane surrounding the viral particle; these membrane-associated proteins mediate the entry of viral particles into the host cell. A membrane-associated protein can bind to specific cell surface protein receptors, as in the case of retroviral envelope proteins, or a membrane-associated protein can interact with a phospholipid component of the host cell plasma membrane, as in the case of G proteins derived from members of the family Rhabdoviridae.

The term “heterologous membrane-associated protein” refers to a membrane-associated protein derived from a virus that is not a member of the same class or family of viruses from which the nucleocapsid protein of the vector particle is derived. "Class or family of viruses" refers to the taxonomic rank of the class or family as defined by the International Committee on Taxonomy of Viruses.

As used herein, the term “lentiviral vector” refers to a retroviral vector derived from the Lentiviridae family (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine and sheep arthritis-encephalitis virus) that is capable of integrating into nondividing cells (See, for example, US Patent Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

The term “pseudotyped lentiviral vector” refers to a lentiviral vector containing a heterologous membrane protein (eg, the viral envelope glycoprotein or G proteins of viruses of the Rhabdoviridae family, such as VSV, Piri, Chandipura and Mokola).

As used herein, the term “transposon” refers to transposable elements (eg, Tn5, Tn7 and Tn10) that can move or be transferred from one position to another in the genome. In general, transposition is controlled by a transposase. The term "transposon vector" as used herein refers to a vector encoding a nucleic acid of interest flanked by the terminal ends of a transposon. Examples of transposon vectors include, but are not limited to, those described in US Pat. No. 6,027,722; 5958775; 5968785; 5965443 and 5719055, all of which are incorporated herein by reference.

As used herein, the term “adeno-associated viral (AAV) vector” refers to a vector derived from an adeno-associated virus serotype, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. etc., AAV vectors may have one or more wild-type AAV genes deleted in whole or in part, preferably the rep and/or cap genes, but retain functional ITR flanking sequences.

AAV vectors can be constructed using recombinant methods that are known in the art to include one or more heterologous nucleotide sequences flanked at both ends (5' and 3') by functional AAV ITRs. In a practice of the present invention, the AAV vector may include at least one AAV ITR and a suitable promoter sequence located upstream of the heterologous nucleotide sequence, and at least one AAV ITR located downstream of the heterologous sequence. “AAV recombinant vector plasmid” refers to one type of recombinant AAV vector, wherein the vector contains a plasmid. As with AAV vectors, in general, the 5' and 3' ITRs flank the selected heterologous nucleotide sequence.

The AAV vector may also include transcriptional sequences, such as a polyadenylation site, as well as selectable marker or reporter genes, enhancer sequences and other regulatory elements to induce transcription. Such control elements are described above.

As used herein, the term “AAV virion” refers to the complete viral particle. The AAV virion can be a wild-type AAV viral particle (containing a linear single-stranded AAV nucleic acid genome associated with an AAV capsid, i.e., a protein shell) or a recombinant AAV viral particle (described below). In this regard, single-stranded AAV nucleic acid molecules (either the sense/coding strand or the antisense/non-coding strand, as those terms are generally defined) can be packaged into the AAV virion; both sense and antisense strands are equally infectious.

As used herein, the term “recombinant AAV virion” or “rAAV” is an infectious, replication-defective virus consisting of an AAV protein environment encapsidating (i.e., surrounding with a protein shell) a heterologous nucleotide sequence, which in turn is flanked by 5 ' and 3' using ITR AAV. A number of methods are known in the art for constructing recombinant AAV virions (see, for example, US Pat. No. 5,173,414; WO 92/01070; WO 93/03769; Lebkowski et al., Molec. Cell. Biol. 8:3988-3996 [1988 ]; Vincent et al., Vaccines 90 [1990] (Cold Spring Harbor Laboratory Press); Carter, Current Opinion in Biotechnology 3:533-539 [1992]; [1992]; Kotin, Human Gene Therapy 5:793-801 [1994]; Schelling and Smith, Gene Therapy 1:165-169 [1994]; and Zhou et al., J. Exp. Med. [1994], all of which are incorporated herein by reference.

Suitable nucleotide sequences for use in AAV vectors (and, in fact, any of the vectors described herein) include any functional appropriate nucleotide sequence. Thus, the AAV vectors of the present invention may include any desired gene encoding a protein that is defective or absent from the genome of the target cell or that encodes a non-native protein having a desired biological or therapeutic effect (e.g., antiviral function), or the sequence may correspond to a molecule having antisense or ribozyme function. Suitable genes include those used to treat inflammatory diseases, autoimmune, chronic and infectious diseases, including disorders such as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia; various blood disorders, including various anemias, thalassemias and hemophilia; genetic defects such as cystic fibrosis, Gaucher disease, insufficient activity of adenosine deaminase (ADA), emphysema, etc. A number of antisense oligonucleotides (eg, short oligonucleotides complementary to sequences near the translation initiation site (AUG codon) of mRNA) have been described in the art and are used in antisense therapy for cancer and viral diseases. (See, for example, Han et al., Proc. Natl. Acad. Sci. USA 88:4313-4317 [1991]; Uhlmann et al., Chem. Rev. 90:543-584 [1990]; Helene et al. ., Biochim. Biophys. 1049:99-125 [1990]; Proc. Natl. Sci. USA 85:7079-7083 [1989]; -449 [1987]). For a discussion of suitable ribozymes, see, for example, Cech et al. (1992) J. Biol. Chem. 267:17479-17482 and US Pat. No. 5,225,347, which are incorporated herein by reference.

As used herein, the term “purified” refers to molecules of both nucleic acid and amino acid sequences that have been removed from their normal environment, isolated or separated. Therefore, the term “isolated nucleic acid sequence” is a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are normally associated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vectors and their use for developing host cell lines for the production of proteins of interest and, in particular, to vectors that use a weak promoter to drive a selectable marker.

In some preferred embodiments, the expression systems of the present invention use an expression vector that includes a nucleic acid sequence encoding a protein of interest (i.e., a therapeutic protein or other protein to be produced) in operational relationship with additional nucleic acid sequences, performing various functions. Thus, for example, a nucleic acid sequence of interest may be included in any of a variety of expression vectors for expressing a polypeptide. In some embodiments of the present invention, the vector includes, but is not limited to, retroviral vectors, chromosomal, non-chromosomal sequences, and synthetic DNA sequences (e.g., SV40 derivatives, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from combinations plasmids and phage DNA, and viral DNA such as from vaccinia virus, adenovirus, fowlpox-diphtheria virus and pseudorabies viruses). It is understood that any vector may be used as long as it is replicable and viable in the host. In some preferred embodiments, the vectors are retroviral vectors, as described in US Pat. Nos. 6,852,510 and 7,332,333 and US Patent Publications. No. 200402335173 and 20030224415, all of which are incorporated herein by reference in their entirety. In some particularly preferred embodiments, the vectors are pseudotyped retroviral vectors. In some preferred embodiments of the present invention, the mammalian expression vectors include an origin of replication, a suitable promoter and enhancer, as well as any necessary ribozyme binding sites, a polyadenylation site, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences . In other embodiments, DNA sequences derived from the SV40 slice sequence and the polyadenylation site may be used to provide the desired non-transcribed genetic elements.

In some preferred embodiments, the vectors are retroviral vectors. The genome architecture of numerous retroviruses is well known in the art, and this allows the retroviral genome to be adapted for the production of retroviral vectors. Production of a recombinant retroviral vector carrying the gene of interest is typically achieved in two steps.

First, the gene of interest is inserted into a retroviral vector that contains the sequences necessary for efficient expression of the gene of interest (including promoter and/or enhancer elements, which may be provided by viral long terminal repeats (LTRs) or an internal promoter/enhancer and associated splicing signals ), sequences required for efficient packaging of viral RNA into infectious virions (e.g., packaging signal (Psi), tRNA primer binding site (-PBS), 3' regulatory sequences required for reverse transcription (+PBS)) and viral LTRs. LTRs contain sequences required for the binding of viral genomic RNA, reverse transcriptase and integrase functions, and sequences involved in controlling the expression of genomic RNA requiring packaging into viral particles. For safety reasons, many recombinant retroviral vectors lack functional copies of genes that are required for viral replication (these essential genes are either deleted or inactive); therefore the resulting virus is said to be replication defective.

Second, after constructing the recombinant vector, the vector DNA is introduced into the packaging cell line. Packaging cell lines provide the proteins required in trans for packaging viral genomic RNA into viral particles having the desired host range (ie, the virus-encoded gag, pol, and env proteins). The host range is controlled in part by the type of envelope gene product expressed on the surface of the virus particle. Packaging cell lines can express ecotropic, amphotropic, or xenotropic envelope gene products. Alternatively, the packaging cell line may lack sequences encoding the viral envelope (env) protein. In this case, the packaging cell line will package the viral genome into particles that lack membrane-associated protein (eg, env protein). To produce viral particles containing a membrane-associated protein that will allow virus entry into a cell, a packaging cell line containing retroviral sequences is transfected with sequences encoding the membrane-associated protein (eg, vesicular stomatitis virus (VSV) G protein). The transfected packaging cell will then produce viral particles that contain the membrane-associated protein expressed by the transfected packaging cell line; these viral particles, which contain viral genomic RNA derived from one virus, encapsidated by the envelope proteins of another virus, are called pseudotyped viral particles.

The retroviral vector of the present invention can be further modified to include additional regulatory sequences. As described above, the retroviral vectors of the present invention include the following elements in functional connection: a) 5' LTR; b) packing signal; c) a 3' LTR; and d) a nucleic acid encoding a protein of interest located between the 5' and 3' LTR. In some embodiments of the present invention, the nucleic acid of interest may be placed in the opposite orientation to the 5' LTR when transcription from an internal promoter is desired. Suitable internal promoters include, but are not limited to, alpha-lactalbumin promoter, CMV (human or simian) promoter, and thymidine kinase promoter.

In other embodiments of the present invention, in which secretion of a protein of interest is desired, the vectors are modified to include a signal peptide sequence in operative association with the protein of interest. The sequences of several suitable signal peptides are known to those skilled in the art, including, but limited to, those derived from tissue plasminogen activator, human growth hormone, lactoferrin, alpha-casein and alpha-lactalbumin.

In other embodiments of the present invention, the vectors are modified by introducing an RNA export element (see, for example, US Pat. Nos. 5,914,267; 6,136,597 and 5,686,120; and WO99/14310, all of which are incorporated herein by reference) in either the 3' or 5' to the nucleic acid sequence encoding the protein of interest. It is understood that the use of an RNA export element allows for high levels of expression of the protein of interest without introducing splice signals or introns into the nucleic acid sequence encoding the protein of interest.

In still other embodiments, the vector further comprises at least one internal ribosome entry site (IRES) sequence. Sequences of several suitable IRESs are available, including, but limited to, those derived from foot-and-mouth disease virus (FDV), encephalomyocarditis virus, and poliovirus. An IRES sequence can be inserted between two transcription units (eg, nucleic acids encoding different proteins of interest or subunits of a multisubunit protein, such as an antibody) to form a polycistronic sequence such that the two transcription units are transcribed from the same promoter.

The most widely used recombinant retroviral vectors come from the amphotropic Moloney murine leukemia virus (MoMLV) (see, for example, Miller and Baltimore Mol. Cell. Biol. 6:2895 [1986]). The MoMLV system has several advantages: 1) this specific retrovirus can infect many different cell types, 2) stable packaging cell lines are available to produce recombinant MoMLV virus particles, and 3) the transferred genes are permanently integrated into the chromosome of the target cell. Persistent MoMLV vector systems include a DNA vector containing a small portion of the retroviral sequence (eg, viral long terminal repeat, or LTR, and packaging or psi signal) and a packaging cell line. The gene that needs to be transferred is inserted into the DNA vector. The viral sequences found on the DNA vector provide the signal necessary for insertion or packaging of the vector RNA into the viral particle and for expression of the inserted gene. The packaging cell line provides the proteins required for particle assembly (Markowitz et al., J. Virol. 62:1120 [1988]).

In some preferred embodiments, the retroviral vectors are pseudotyped and, for example, use the VSV G protein as the membrane-associated protein. Unlike retroviral envelope proteins, which bind to a specific cell surface protein receptor to promote cell entry, the VSV G protein interacts with the phospholipid component of the plasma membrane (Mastromarino et al., J. Gen. Virol. 68:2359 [1977]) . Because VSV entry into cells does not depend on the presence of specific protein receptors, VSV has an extremely wide host range. Pseudotyped retroviral vectors carrying the VSV G protein have altered host range properties of VSV (ie, they can infect most of all vertebrate, invertebrate and insect cell types). It is important to note that VSV G-pseudotyped retroviral vectors can be concentrated 2000-fold or more by ultracentrifugation without significant loss of infectivity (Burns et al. Proc. Natl. Acad. Sci. USA 90:8033 [1993]).

The present invention is not limited to the use of the VSV G protein, in which the viral G protein is used as a heterologous membrane-associated protein on the viral particle (see, for example, US Pat. No. 5,512,421, which is incorporated herein by reference). G proteins of Vesiculovirus viruses other than VSV, such as Piri and Chandipur viruses, which are highly homologous to the VSV G protein and, like the VSV G protein, contain covalently linked palmitic acid (Brun et al. Intervirol. 38:274 [1995] and Masters et al., Virol. 171:285 (1990]) Thus, the G protein of Piri and Chandipura viruses can be used instead of the VSV G protein to pseudotype viral particles. In addition, the G protein of VSV viruses within the genus Lyssa. viruses such as rabies and Mokola viruses exhibit a high degree of conservation (amino acid sequence conservation as well as functional conservation) with the VSV G proteins. For example, the Mokola virus G protein has been shown to function in a manner similar to the VSV G protein (i.e. . mediates membrane fusion) and can therefore be used in place of the VSV G protein to pseudotype viral particles (Mebatsion et al., J. Virol. 69:1444 [1995]). Viral particles can be pseudotyped using either G protein. Piri, Chandipur, or Mokola proteins as described in Example 2, except that plasmid-containing sequences encoding any of the Piri, Chandipur, or Mokola G proteins are used and are under the transcriptional control of a suitable promoter element (e.g., the CMV immediate-early promoter ; Numerous expression vectors containing the CMV IE promoter are available, such as the pcDNA3.1 vector (Invitrogen) instead of pHCMV-G. Sequences encoding other G proteins derived from other members of the family Rhabdoviridae may be used; sequences encoding numerous rhabdovirus G proteins available in the Genbank database.

In some preferred embodiments, the vectors are lentiviral vectors. Lentiviruses (eg, equine infectious anemia virus, goat and sheep arthritis-encephalitis virus, human immunodeficiency virus) are a subfamily of retroviruses that are capable of integration into nondividing cells. The lentiviral genome and proviral DNA typically contain three genes found in retroviruses: gag, pol and env, which are flanked by two LTR sequences. The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes reverse transcriptase, protease and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs control transcription and polyadenylation of viral RNA. Additional genes in the lentiviral genome include the vif, vpr, tat, rev, vpu, nef, and vpx genes.

A variety of lentiviral vectors and packaging cell lines are known in the art and find use herein (see, for example, US Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference). In addition, the VSV G protein is also used to pseudotype human immunodeficiency virus (HIV)-based retroviral vectors (Naldini et al., Science 272:263 [1996]). Thus, the VSV G protein can be used to create a variety of pseudotyped retroviral vectors, and these are not limited to MoMLV-based vectors. Lentiviral vectors can also be modified, as described above, to contain various regulatory sequences (eg, signal peptide sequences, RNA export elements and IRES). Once lentiviral vectors are produced, they can be used to transfect host cells as described above for retroviral vectors.

In some preferred embodiments, the vectors are adeno-associated vectors (AAV). AAV is a human DNA parvovirus that belongs to the genus Dependovirus. The AAV genome consists of a linear, single-stranded DNA molecule that contains approximately 4680 bases. The genome includes inverted terminal repeats (ITRs) at each end that function in cis as origins of DNA replication and as packaging signals for the virus. The internal part of the genome without repeats includes two large open reading frames, known respectively as the rep and cap regions of AAV. These regions encode viral proteins involved in virion replication and packaging. A family of at least four viral proteins is synthesized in the rep region of AAV, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The cap region of AAV encodes at least three proteins, VP1, VP2 and VP3 (for a detailed description of the AAV genome, see, for example, Muzyczka, Current Topics Microbiol. Immun. 158:97-129 [1992]; Kotin, Human Gene Therapy 5:793-801 [1994]).

AAV requires coinfection with an unrelated helper virus, such as adenovirus, herpesvirus, or vaccinia virus, to establish a productive infection. In the absence of such coinfection, AAV enters a latent state by inserting its genome into the host cell chromosome. Subsequent infection with a helper virus unlocks the integrated copy, which can then replicate to produce infectious viral progeny. Unlike non-pseudotyped retroviruses, AAV has a wide host range and is capable of replicating in cells from any species, provided there is coinfection with a helper virus that will also replicate in such species. Thus, for example, human AAV will replicate in canine cells coinfected with canine adenovirus. In addition, unlike retroviruses, AAV is not associated with any human or animal disease, does not appear to alter the biological properties of the host cell after integration, and is capable of integrating into nondividing cells. Recently, it was also discovered that AAV is capable of site-specific integration into the host cell genome.

In light of the properties described above, a number of recombinant AAV gene delivery vectors have been developed (see, for example, US Pat. Nos. 5,173,414; 5,139,941; WO 92/01070 and WO93/03769, both incorporated herein by reference; Lebkowski et al., Molec Cell Biol 8:3988-3996 [1988]; Current Opinion in Biotechnology 3:533-539 [1992]; (1994) Human Gene Therapy 5:793-801; Shelling and Smith, Gene Therapy 1:165-169 [1994]; and Zhou et al., J. Exp Med 179:1867-1875 [1994]).

Recombinant AAV virions can be produced in a suitable host cell that has been transfected with both an AAV helper plasmid and an AAV vector. The AAV helper plasmid typically includes the AAV rep and cap coding regions but lacks the AAV ITR. Accordingly, the helper plasmid cannot replicate or package itself. The AAV vector typically includes a selected gene of interest associated with AAV ITRs that provide viral replication and packaging functions. Both the helper plasmid and the AAV vector carrying the selected gene are introduced into a suitable host cell by transient transfection. The transfected cell is then infected with a helper virus, such as an adenovirus, which transactivates the AAV promoter located on a helper plasmid that controls transcription and translation of the AAV rep and cap regions. Recombinant AAV virions containing the selected gene are generated and can be purified from the production medium. Once AAV vectors are produced, they can be used to transfect (eg, US Pat. No. 5,843,742, incorporated herein by reference) host cells at the desired multiplicity of infection to produce high copy number host cells. One skilled in the art will appreciate that AAV vectors can also be modified, as described above, to contain various regulatory sequences (eg, signal peptide sequences, RNA export elements, and IRES).

In some preferred embodiments, the vectors are transposon-based vectors. Transposons are mobile genetic elements that can move or be transferred from one position to another in the genome. Transposition within the genome is controlled by the enzyme transposase, which is encoded by this transposon. Many examples of transposons are known in the art, including, but limited to, Tn5 (see, for example, de la Cruz et al., J. Bact. 175: 6932-38 [1993], Tn7 (see, for example, Craig, Curr . Topics Microbiol. Immunol. 204: 27-48 [1996]), and Tn10 (see, for example, Morisato and Kleckner, Cell 51:101-111 [1987]). see, for example, US patents No. 5,719,055; 5,958,775; and 6,027,722; all of which are incorporated herein by reference.) Since transposons are not infectious, transposon vectors are introduced into host cells by methods known in the art (eg , electroporation, lipofection or microinjection). Therefore, the ratio of transposon vectors into host cells can be adjusted to provide the desired multiplicity of infection to obtain high copy number host cells of the present invention.

Transposon vectors suitable for use in the present invention typically include a nucleic acid encoding a protein of interest inserted between two transposon insertion sequences. Some vectors also include a nucleic acid sequence encoding a transposase enzyme. In these vectors, one of the insertion sequences is located between the transposase enzyme and the nucleic acid encoding the protein of interest such that it is not incorporated into the host cell genome during recombination. Alternatively, the transposase enzyme may be provided by a suitable method (eg lipofection or microinjection). One skilled in the art will appreciate that transposon vectors can also be modified, as described above, to contain various regulatory sequences (eg, signal peptide sequences, RNA export elements and IRES).

In some preferred embodiments, the vectors include a selectable marker. Suitable selectable markers include, but are not limited to, glutamine synthase (GS), dihydrofolate reductase (DHFR), and the like. These genes are described in US patent No. 5770359; 5827739; 4399216; 4634665; 5149636 and 6455275; all of which are incorporated herein by reference. In some particularly preferred embodiments, the selectable marker is a GS and has a sequence that shares at least 80%, 90%, 95%, 99%, or 100% sequence identity with the sequence at positions 1789 to 2910 of SEQ ID NO:2. In some preferred embodiments, the selectable marker used is compatible with a host cell line that is defective in producing the enzyme encoded by the selectable marker nucleic acid sequence. Suitable host cell lines are described in more detail below. In other embodiments, the selectable marker is an antibiotic resistance marker, i.e. a gene that produces a protein that allows cells to express that protein with resistance to an antibiotic. Suitable antibiotic resistance markers include genes that confer resistance to neomycin (neomycin resistance gene (neo)), hygromycin (hygromycin B phosphate transferase gene), puromycin (puromycin N-acetyl transferase) and the like.

In some embodiments, the nucleic acid sequence encoding the selectable marker is operably linked to a promoter sequence. In some particularly preferred embodiments, the promoter sequence has been altered, for example by mutation, to reduce promoter activity compared to an unchanged version of the promoter. These promoters may be described as weak promoters, in that expression of the gene associated with the promoter occurs at a low level, as opposed to a high level. Genes regulated by strong promoters produce more mRNA and therefore produce more protein than genes regulated by weak promoters. Thus, the present invention preferably uses a promoter for a selectable marker that has been modified to produce less mRNA than a comparable unmodified promoter.

In some preferred embodiments, the promoter operably linked to the selectable marker is a long terminal repeat (LTR) from a retrovirus or lentivirus. In some particularly preferred embodiments, the promoter is an LTR that has been modified by removing either all or a portion of the U3 region of the LTR. In some embodiments, the promoter operably linked to the selectable marker is a self-inactivating (SIN) LTR. Suitable SIN LTRs are known in the art. In some preferred embodiments, the SIN LTRs of the present invention preferably have at least 80%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:3, and the SIN LTR most preferably contains a deletion in the U3 region, which reduces promoter activity compared to promoter activity if there is no deletion in this U3 region.

It is easy to understand that when the vector is a retroviral vector, the vector is designed such that the SIN LTR is the 3' LTR sequence of the retroviral vector. Due to the fact that the U3 deletion is copied to the 5′ and 3′ LTRs during reverse transcription, integrated SIN vectors contain only U3-deleted LTRs. A map of an exemplary retroviral vector of the present invention is shown as FIG. 2. As you can see, the vector contains a 3’ SIN LTR. After reverse transcription, the hCMV-MOMuSV LTR mapped on the vector is replaced by the 3' SIN LTR mapped on the vector such that when the vector integrates into the host cell chromosome, the SIN LTR functionally binds to it and directs expression of the described GS cDNA.

In some preferred embodiments of the present invention, a nucleic acid sequence encoding a protein of interest in an expression vector is operably linked to corresponding expression regulation sequence(s) (promoter) to control mRNA synthesis. The promoter used in the present invention includes, but is not limited to, the LTR or SV40 promoter, E. coli lac or trp, lambda phage P _L and P _R , T3 and T7 promoter, and cytomegalovirus (CMV) immediate early promoter. herpes simplex virus (HSV) thymidine kinase and mouse metallothionein-I promoters and other promoters known to regulate gene expression in prokaryotic or eukaryotic cells or their viruses.

As stated above, a typical vector map of the present invention is shown in FIG. 3 and corresponds to SEQ ID NO:2. In some preferred embodiments, the vectors include the following element in the 5' to 3' direction:

- 5’ LTR (using the example of hCMV-MoMuSV LTR);

- retroviral packaging region;

- a nucleic acid encoding a selectable marker (for example, GS cDNA);

- internal promoter (using the example of the monkey CMV promoter (sCMV));

- a nucleic acid sequence encoding the protein of interest (using the example of the “anyway” sequence), which is functionally linked to the internal promoter;

- WPRE sequence;

- 3’ SIN LTR.

In some embodiments, the vector is a plasmid containing the following elements in order 5' to 3': 1) 5' LTR (eg, SIN LTR); 2) packaging area; 3) selectable marker (for example, GS); 4) internal promoter (for example, CMV); 5) a nucleic acid sequence encoding the protein of interest, which is operably linked to the internal promoter.

In some embodiments, the vectors contain a single poly-A signal sequence following a nucleic acid encoding the protein of interest. For example, in some embodiments, the vector contains the following components in order 5' to 3': Promoter (eg, SIN) selectable marker - stop codon - promoter (eg, CMV) - protein of interest - stop codon - poly-A.

In some embodiments, the present invention provides host cells and a host cell culture, wherein the host cells express a protein of interest from the vectors described above. In a preferred embodiment, the host cells are mammalian host cells. A number of mammalian host cell lines are known in the art. In general, these host cells are able to grow and survive when grown either in monolayer culture or in suspension culture in a medium containing appropriate nutrients and growth factors, as described in more detail below. Typically, cells are capable of expressing and secreting large amounts of a particular protein of interest into the culture medium. Examples of suitable mammalian host cells include, but are not limited to, Chinese hamster ovary cells (CHO-K1, ATCC CCl-61); bovine mammary epithelial cells (ATCC CRL 10274; bovine mammary epithelial cells); monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; see, for example, Graham et al., J. Gen Virol., 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 [1982]); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP cells (CEVEC from Amniocyte Production) and a human hepatoma line (Hep G2).

In some particularly preferred embodiments, the host cells are modified such that they are defective, or are naturally defective, in enzymatic activity that is required for the cells to grow or survive in the presence of the selection agent provided by the selectable marker. For example, Chinese hamster ovary (CHO) cells have been modified to be GS defective. In some preferred embodiments, in which the vector includes a GS selectable marker, the host cell line is GS defective. In some particularly preferred embodiments, the GS-defective host cell line is a CHOZN® GS ^-/- cell line offered by Merck KGaA. In other embodiments, in which the selectable marker is, for example, DHFR, the cell line may preferably be defective in DHFR activity (ie, is DHFR ^- ). Suitable DHFR cell lines include, but are not limited to, CHO-DG44 and derivatives thereof.

Vectors can be introduced into host cells by any suitable means. In some preferred embodiments, vectors (eg, retroviral vectors) encoding a protein of interest are prepared and can be used to transfect or transduce host cells. Preferably, the host cells are transfected or transduced with integrating vectors at a multiplicity of infection sufficient to obtain integration of at least 1 and preferably at least 2 or more retroviral vectors. In some embodiments, multiplicity of infection values of 10 to 1,000,000 may be used such that the genomes of the infected host cells contain from 2 to 1000 copies of integrated vectors, preferably from 5 to 1000 copies of integrated vectors, and most preferably from 20 to 500 copies of integrated vectors. In other embodiments, MOIs of 10 to 10,000 are used. When non-pseudotyped retroviral vectors are used for infection, host cells are incubated with culture medium from retrovirus-producing cells containing the desired titer (ie, number of colony forming units, CFU) of infectious vectors. When pseudotyped retroviral vectors are used, the vectors are concentrated to the appropriate titer by ultracentrifugation and then added to host cell culture. Alternatively, concentrated vectors can be diluted in a culture medium appropriate for the cell type. In addition, when expression of more than one host cell protein of interest is desired, host cells can be transfected with multiple vectors, each containing a nucleic acid encoding a different protein of interest.

In each case, host cells are placed in a medium containing infectious retroviral vectors for a sufficient period of time to allow infection and subsequent integration of the vectors. In general, the amount of medium used to coat cells should be kept to the minimum possible volume in order to promote the maximum number of integration events per cell. As a general guide, the number of colony-forming units (CFU) per milliliter should be approximately 10 ⁵ to 10 ⁷ CFU/ml, depending on the number of integration events desired.

In some embodiments, following transfection or transduction, the cells are allowed to proliferate and are then trypsinized and subcultured. Individual colonies are then selected to generate clonally selected cell lines. In still further embodiments, clonally selected cell lines are screened by Southern blot or PCR assays to verify that the desired number of integration events have occurred. It is also implied that clonal selection allows for the identification of cell lines that produce the greatest amounts of protein. In other embodiments, cells are not clonally selected following transfection.

In some embodiments, host cells are transfected with vectors encoding various proteins of interest. Vectors encoding different proteins of interest can be used to transfect cells simultaneously (eg, host cells are exposed to a solution containing vectors encoding different proteins of interest), or transfection can be sequential (eg, host cells are first transfected with a vector encoding the first protein of interest). protein, incubated for a period of time and then host cells are transfected with a vector encoding the second protein of interest). In some preferred embodiments, host cells are transfected with an integrating vector encoding a first protein of interest, highly expressed cell lines (e.g., clonally selected) containing multiple integrated copies of the integrating vector are selected, and the selected cell lines are transfected with an integrating vector encoding a second protein of interest. This process can be repeated to introduce multiple proteins of interest. In some embodiments, the multiplicity of infection may be manipulated (eg, increased or decreased) to increase or decrease expression of the protein of interest. Likewise, different promoters can be used to alter the expression of proteins of interest. It is understood that these methods can be used to construct host cell lines containing entire exogenous metabolic pathways, or to provide host cells with increased ability to process proteins (eg, host cells can provide enzymes necessary for post-translational modification).

In still further embodiments, cell lines are sequentially transfected with vectors encoding the same gene. In some preferred embodiments, host cells are transfected (e.g., at an MOI of about 10 to 1,000,000, preferably 100 to 10,000) with an integrating vector encoding a protein of interest, cell lines are selected (e.g., clonally selected) containing one or multiple integrated copies of the integrating vector or expressing high levels of the desired protein, and the selected cell lines are retransfected with the same vector (eg, at an MOI of about 10 to 1,000,000, preferably 100 to 10,000). In some embodiments, cell lines containing at least two integrated copies of the vector are identified and selected. This process can be repeated numerous times until the desired level of protein expression is achieved and can also be repeated to introduce vectors encoding multiple proteins of interest. Surprisingly, it was discovered that sequential transfection of the same gene leads to increases in protein production from the resulting cells, which is not simply an additive effect.

The present invention contemplates a variety of sequential transfection procedures. In some embodiments that employ retroviral vectors, sequential transduction procedures are provided. In preferred embodiments, sequential transduction is performed on a pool of cells. In these embodiments, the initial pool of host cells is contacted with retroviral vectors, preferably at a multiplicity of infection ranging from about 0.5 to about 1000 vectors/host cell. The cells are then cultured for several days in a suitable medium (eg, a selection agent). An aliquot of cells is then removed to determine the amount of integrated vectors and frozen for future possible use. The remaining cells are then re-exposed to the retroviral vectors, again preferably at a multiplicity of infection ranging from about 0.5 to about 1000 vectors/host cell. This process is repeated until the desired number of integrated vectors is obtained. For example, the process may be repeated 10 to 20 or more times. In some embodiments, cells can be clonally selected after any particular transduction step if desired, however, using a pool of cells in the absence of transduction results in increased time to obtain the desired number of integrated copies of the vector.

In some embodiments of the present invention, after transforming a suitable host strain and growing the host strain in the medium to an appropriate cell density, the protein of interest is secreted during culture of the host cells. In preferred embodiments in which amplifiable markers are used, it is assumed that the culture of the transduced host cells is in a medium containing an inhibitor of the gene. Suitable inhibitors include, but are not limited to, methotrexate to inhibit DHFR and methionine sulfoximine (Msx) or phosphinothricin to inhibit GS. It is understood that as the concentrations of these inhibitors increase in a cell culture system, cells with more copies of the amplified marker (and thus genes or genes of interest) or cells that contain higher-producing insertions are selected.

Accordingly, host cells containing the vectors as described above are cultured according to methods known in the art. Suitable culture conditions for mammalian cells are well known in the art (see, for example, J. Immunol. Methods (1983) 56:221-234 [1983], Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. Oxford University Press, New York [1992]).

Host cell cultures of the present invention are prepared in a medium suitable for the particular cell being cultured. Examples of nutrient solutions include commercially available media such as ActiPro (HyClone), ExCell Advanced Fed Batch (SAFC), Ham's F10 (Sigma, St. Louis, MO), Minimal Essential Medium (MEM, Sigma) , RPMI-1640 (Sigma) and Dulbecco's modified Eagle's medium (DMEM, Sigma). Suitable media are also described in US Pat. No. 4,767,704; 4657866; 4927762; 5122469; 4560655; and WO 90/03430 and WO 87/00195; the descriptions of which are incorporated herein by reference. Any of these media may be supplemented, as necessary, with serum, hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate salts), buffers (such as HEPES), nucleosides (such as adenosine and thymine), antibiotics (such as gentamicin (gentamicin), trace elements (known as inorganic compounds, usually present in final concentrations in the micromolar range), lipids (such as linoleic acid or other fatty acids), and their suitable carriers, and glucose or an equivalent energy source. In some preferred embodiments in which a selectable marker, such as GS, is used, for example, the medium will be free of glutamine. Any other necessary additives may be included in appropriate concentrations as may be known to those skilled in the art. this field of technology.

The present invention also contemplates the use of various culture systems (eg, Petri dishes, 96-well plates, roller bottles and bioreactors) for transfected host cells. For example, transfected host cells can be cultured in a perfusion system. Perfusion culture refers to providing a continuous flow of culture medium through the culture maintained at high cell densities. The cells are suspended and do not require a solid support to grow on. In general, fresh nutrients can be supplied continuously with concomitant removal of toxic metabolites and, ideally, selective removal of dead cells. All methods of filtration, capture and microencapsulation are suitable for renewing the culture environment in sufficient proportions.

As another example, in some embodiments, a fed-batch culture procedure may be used. In the preferred fed-batch culture of mammalian host cells, cells and culture medium are initially added to the culture vessel, and additional culture nutrients are fed continuously or in discrete additions during the culture process, with or without periodic collection of cells and/or products until culture ceases. A fed-batch culture may include, for example, a semi-continuous fed-batch culture in which the entire culture (including cells and media) is periodically removed and replaced with fresh media. Fed-batch culture differs from simple batch culture, in which all cell culture components (including cells and all culture nutrients) are added to the culture vessel at the beginning of the culture process. Fed-batch culture may differ from perfusion culture in that the supernatant is not removed from the culture vessel during the process (in perfusion culture, cells are maintained in culture, e.g. by filtration, encapsulation, attachment to microcarriers, etc., and culture medium is continuously or periodically introduced and removed from the culture vessel). In some particularly preferred embodiments, batch cultures are maintained in roller bottles.

Additionally, the culture cells can be propagated in accordance with any schedule or practice that may be suitable for the particular host cell and the particular intended production plan. Therefore, the present invention provides a single-step or multi-step culture procedure. In single-step culture, host cells are inoculated into the culture medium and the processes of the present invention are applied during a single phase of cell culture production. Alternatively, a multi-stage culture is provided. In multi-stage culture, cells can be cultured in a number of stages or phases. For example, cells may be grown into the first stage or growth phase of a culture, with the cells often being removed from storage and inoculated into a medium suitable for promoting growth and high viability. The cells can be maintained in the growth phase for a suitable period of time by adding fresh medium to the host cell culture.

Fed-batch or continuous cell culture conditions are developed to enhance the growth of mammalian cells during the growth phase of the cell culture. In the growth phase, cells are grown under conditions and for a period of time that are most suitable for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO ₂ ), and the like, are appropriate for the particular host and will be apparent to one of ordinary skill in the art. In general, adjust the pH to between approximately 6.5 and 7.5 using either an acid (eg CO ₂ ) or a base (eg Na ₂ CO ₃ or NaOH). A suitable temperature range for culturing mammalian cells such as CHO cells is between approximately 30° and 38° C. and a suitable dO ₂ value is between 5-90% air saturation.

Following the polypeptide production phase, the polypeptide of interest is isolated from the culture medium using techniques that are well established in the art. The protein of interest is preferably isolated from the culture medium as a secreted polypeptide (eg, secretion of the protein of interest is controlled by a signal peptide sequence), although it can also be isolated from host cell lysates. At the first stage, the culture medium or lysate is centrifuged to remove debris from cell fragments. The polypeptide is then purified from contaminating soluble proteins and polypeptides, following procedures that are typical for suitable purification procedures: immunoaffinity or ion exchange column fractionation; ethanol precipitation; reverse phase HPLC; chromatography on silica gel or a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE electrophoresis; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and Sepharose and Protein A columns to remove contaminants such as IgG. A protease inhibitor such as phenylmethyl sulfofluoride (PMSF) may also be useful to inhibit proteolytic degradation during purification. In addition, the protein of interest can be fused in frame to a marker sequence that allows for purification of the protein of interest. A non-limiting example of marker sequences includes a hexahistidine marker, which can be supplied by a vector, preferably the pQE-9 vector, and a hemagglutinin (HA) marker. The HA marker corresponds to an epitope derived from the influenza virus hemagglutinin protein (see, for example, Wilson et al., Cell, 37:767 [1984]). One skilled in the art will appreciate that purification methods suitable for the polypeptide of interest may require modification to account for changes in the nature of the polypeptide when expressed in recombinant cell culture.

EXPERIMENTAL PART

The present invention provides a unique route of combining retroviral transduction (referred to herein as GPEx® technology) in combination with a glutamine synthase (GS) knockout CHO cell line system, which has resulted in unexpected improvements in the ability of cells to produce high titers and specific yields of the protein of interest. The GS knockout cell line used in these studies was the CHOZN cell line offered by MilliporeSigma. The GPEx® technology was applied in a similar manner to how it was previously applied to normal CHO cells. However, in the viral expression vector, the GS gene used to select the GS knockout cell line was attenuated by a very weak promoter from the SIN (self-inactivating) LTR present in one version of the GPEx expression vector that we used at Catalent. The combination of such a vector, a derivation process from a normal GPEx cell line, and a GS knockout CHO cell line yielded production levels up to 7 times greater than the traditional GPEx process in an unmodified CHO cell line.

Experiment 1

Two combined cell lines were generated using different viral vectors. Both lines were generated using normal GPEx transduction processes and the GS knockout cell line CHOZN. Both expression vectors are designed to express the Anyway test protein. Anyway is an Fc fusion protein. The only difference between the two expression vectors was that one vector, once inserted into a cell line, could gain control of GS gene expression by the full-length LTR Moloney murine leukemia virus (MMLV), and the other vector could gain control of GS expression by the SIN LTR of MMLV. The gene constructs used to produce retrovector particles are shown in FIG. 1 and 2. Sequences for gene constructs are suggested in FIG. 3 and 4.

Development of the CHOZN cell line

Retrovector production: The expression constructs noted above were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro and pol proteins. The expression plasmid containing the envelope gene was also co-transfected with each of the gene constructs. Cotransfection resulted in the production of high-titer replication-incompetent retrovector, which was concentrated by ultracentrifugation and used for cell transductions ( 1 , 2 ). The use of transduction with retrovectors produced using the above constructs on CHO cells results in duplication of the 3' LTR sequence to the 5' end of the sequence, replacing the hCMV-MoMuSV 5'LTR, and subsequently driving GS gene expression in the pooled cell line.

Transduction of CHOZN cells with a retrovector: Pooled cell lines containing retrovector inserts from each of the above constructs were generated by transducing the parental Chinese hamster ovary cell line CHOZN with a retrovector derived from gene constructs designed to express the Anyway protein. One round of transduction was performed to generate a combined cell line for each of the two constructs. After cell transductions were completed, the cell lines were placed in glutamine-free medium to perform GS selection.

Anyway Fed Batch Production from Two Pooled Cell Populations: After transduction, the pooled cell lines were scaled up to produce in the test fed-batch culture in duplicate in 250 mL shake flasks. Each shake flask was seeded with 300,000 viable cells per ml in a 50 ml working volume of ExCell Advanced Fed-Batch Medium (SAFC) and incubated in a humidified incubator (70-80%) shaking at 110 rpm with 5% CO ₂ and temperature 37°C. During production, cultures were fed every other day, starting on day 3, using one feed addition. Glucose concentrations were monitored daily and added if the level fell below 4 g/L. Cultures were stopped when viability levels became ≤50%.

Results

The results are presented in Table 1 and FIG. 5. These results are unexpected. With similar average gene copy numbers for the two pools, there was a significant difference in titers between the two cell lines pooled. Thus, even with a slightly higher average gene copy number, the full-length LTR or wild-type gene construct gave significantly lower titers, and this translated into a significantly lower specific cell productivity per gene insert. There is selection or competitive advantage for highly expressing cells containing a version of the SIN gene construct.

Table 1. Comparison of pooled cell lines for two gene constructs

United cell lines Average copy number of pool genes Final titer (mg/l) PKC/gene copy dt-LTR 19 101 0.0385 SIN-LTR 16 246 0.113

Experiment 2

Based on the previous results, the inventors designed an experiment to compare the way in which we have traditionally implemented the GPEx technology to practice the technology in exactly the same way, but using the SIN-GS vector described above in combination with the GS knockout cell line CHOZN. The processes were kept as similar as possible with only two major differences. The first was that the traditional GPEx uses the original Chinese hamster cell line (GCHO) GPEx®, and the new version uses the CHOZN cell line. The second difference was that there were different gene constructs used to create the retrovector. In the traditional GPEx the construct does not contain the GS gene and in the new version it does not contain the GS gene. All other components of the gene construct were similar. Each construct again expresses the Anyway protein. Once pooled cell lines were completed for each method, they were compared for production in a fed-batch assay.

Development of GCHO and CHOZN cell lines

Retrovector production: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro and pol proteins. The expression plasmid containing the envelope gene was also co-transfected with each of the gene constructs. Cotransfection resulted in the production of high-titer replication-incompetent retrovector, which was concentrated by ultracentrifugation and used for cell transductions ( 1 , 2 ).

Transduction of CHOZN cells with a retrovector: Pooled cell lines containing retrovector inserts from each construct were generated by transducing either the parental CHOZN Chinese hamster ovary cell line or the parental GCHO Chinese hamster ovary cell line with a retrovector derived from either the gene construct containing GS or a gene construct not containing GS, both designed to express the Anyway protein. Three rounds of transduction were performed to create a combined cell line for each of the two constructs in the corresponding cell line. Additional cycles tended to increase the number of insertions in the cell line and subsequently the gene copy number. After completion of each transduction cycle, CHOZN cell-based pools were placed in glutamine-free medium for GS selection. Traditional GPEx cells were not selected as in the normal procedure.

Results

Since the expected copy number increased for both methods with repeated rounds of transduction. However, higher gene copy numbers were observed in each of the new GPEx cell pools compared to traditional GPEx, as shown in Table 2.

Table 2. Comparison of gene copy numbers of pooled cell lines for two different processes

Number of transduction cycles Copy number in pooled cell lines Traditional GPEx New GPEx 1x 15 47 2x 38 69 3x 50 79

Anyway Fed Batch Production from Two Pooled Cell Populations (3 Transduction Cycles): Following transduction, the pooled cell lines were scaled up to produce in the test fed-batch culture in duplicate in 250 mL shake flasks. Each shake flask was seeded with 300,000 viable cells per ml in a working volume of 50 ml ActiPro medium (HyClone) and incubated in a humidified incubator (70-80%) with shaking at 120 rpm with 5% CO ₂ and a temperature of 37 °C ( 34 °C starting from day 6). Cultures were fed six times during production analysis using two different feed additives. Glucose concentrations were monitored daily and added if the level fell below 4 g/L. Cultures were stopped when viability levels became ≤50%.

The results are presented in Table 3 and FIG. 6. And again we observed very unexpected results. Clonal cell lines from the optimized traditional GPEx producing the Anyway product expressed a maximum of 1.8 g/L, and the new GPEx pool exceeded this expression by more than twofold before any clonal selection. The new GPEx pool also contains a higher copy number and also provides greater production per gene insert, as shown in experiment No. 1. Together, these factors resulted in a significant difference in cell specific productivity, with nearly 3-fold greater pg/cell/day for the new GPEx pool. The overall viable cell density was also higher for the new GPEx pool compared to the conventional pool, also contributing to the significant difference in titers observed.

Table 3. Comparison of pooled cell lines for two different processes to produce Anyway

United cell lines Average copy number of pool genes Final titer (g/l) Specific productivity pg/cell/day (PCS) PKC/gene copy Traditional GPEx, 3x 50 0.59 6.6 0.13 New GPEx, 3x 79 4.25 15.2 0.19

Experiments 3 and 4

The above data relates to an Fc fusion produced from a single gene. Experiments 3 and 4 use separate vector transductions for the heavy and light chains (2 transductions for the light chain and 3 transductions for the heavy chain in both experiments) with two different antibodies "Peelaway" and "Yourway" with both GPEx® technology and a new process using SIN LTR to control GS expression.

Retrovector production: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro and pol proteins. The expression plasmid containing the envelope gene was also co-transfected with each of the gene constructs. Cotransfection resulted in the production of high-titer replication-incompetent retrovector, which was concentrated by ultracentrifugation and used for cell transductions ( 1 , 2 ).

Transduction of CHOZN cells with a retrovector: Pooled cell lines containing retrovector inserts from each construct were generated by transducing either the parental CHOZN Chinese hamster ovary cell line or the parental GCHO Chinese hamster ovary cell line with a retrovector derived from the severe and light chains containing GS, or gene constructs not containing GS, designed to express two different antibodies. Two rounds of transduction for the light chain and three rounds of transduction for the heavy chain were performed to generate a pooled cell line for each of the four constructs in each of the corresponding antibody cell lines. Additional cycles tended to increase the number of insertions in the cell line and subsequently the gene copy number. After completion of each transduction cycle, CHOZN cell-based pools were placed in glutamine-free medium for GS selection. Traditional GPEx cells were not selected as in the normal procedure.

Peelaway and Yourway fed-batch production from pooled cell line populations (2 rounds of transduction for the light chain and 3 rounds of transduction for the heavy chain): After transduction, the pooled cell lines were scaled up to produce in the test fed-batch culture in duplicate in shake flasks at 250 ml. Each shake flask was seeded with 300,000 viable cells per ml in a working volume of 50 ml ActiPro medium (HyClone) and incubated in a humidified incubator (70-80%) with shaking at 120 rpm with 5% CO ₂ and a temperature of 37 °C ( 34 °C starting from day 6). Cultures were fed six times during production analysis using two different feed additives. Glucose concentrations were monitored daily and added if the level fell below 4 g/L. Cultures were stopped when viability levels became ≤50%.

Results

The results are presented in Tables 4 and 5. Significant benefits were observed for titer values with the new vector and process.

Table 4. Comparison of pooled cell lines for two different processes to produce Peelaway antibody

United cell lines Final titer (g/l) Specific productivity pg/cell/day (PCS) Traditional GPEx, 5x 0.57 5.4 New GPEx, 5x 1.79 9.6

Table 5. Comparison of Pooled Cell Lines for Two Different Processes to Produce Yourway Antibody

United cell lines Final titer (g/l) Specific productivity pg/cell/day (PCS) Traditional GPEx, 5x 2.09 18.5 New GPEx, 5x 3.66 14.0

Additional copy number analysis was performed on 4 pools of Yourway cells. Pools of cells generated after 1 cycle of light chain transduction and 2 cycles of heavy chain transduction were compared with pools of cells generated after 2 cycles of light chain transduction and 3 cycles of heavy chain transduction. Each of these pooled cell lines was either maintained in continuous growth conditions in glutamine-containing media or placed in glutamine-free media. For each of the four combined cell lines, the number of gene copies to the total number of genes, the number of heavy chain genes, and the number of light chain genes were calculated.

Table 6

United cell line Glutamine +/- Total gene copy number Heavy chain gene copy number Light chain gene copy number LC1x/HC2x + 31 15 18 LC1x/HC2x - 63 17 48 LC2x/HC3x + 64 28 43 LC2x/HC3x - 94 32 65

These data show that even though each of the cells in these pools contains numerous copies of the GS gene attenuated by the SIN LTR, there is significant selection being made for cells with high copy numbers and, according to the data above, those copies that provide high expression levels.

Experiment 5

The Yourway antibody product was used for this experiment. The heavy chain gene construct used to create the cell line was the same as in experiment 4. The light chain gene construct was identical to the heavy chain gene construct in all respects except that it contains the light chain coding sequence and lacks the GS gene . A single transduction was performed for each containing light chain (-GS) and heavy chain (+GS) to create a combined cell line.

Retrovector production: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro and pol proteins. The expression plasmid containing the envelope gene was also co-transfected with each of the gene constructs. Cotransfection resulted in the production of high-titer replication-incompetent retrovector, which was concentrated by ultracentrifugation and used for cell transductions ( 1 , 2 ).

Transduction of CHOZN cells with a retrovector: Pooled cell lines containing retrovector inserts from each of the above constructs were generated by transducing the parental CHOZN Chinese hamster ovary cell line with a retrovector derived from light chain gene constructs without GS and heavy chain constructs containing GS . One round of transduction for the light chain and one round of transduction for the heavy chain were performed to create a combined cell line. After completion of both transduction cycles, the pool of CHOZN cells was divided, half of it was placed in glutamine-free media, and the other half was continuously fed with glutamine during cell expansion and subsequent productivity assessment.

Yourway Fed Batch Production from Pooled Cell Line Populations (1 Light Chain Transduction and 1 Heavy Chain Transduction): Pooled cell lines were scaled up for production in the test fed-batch culture in 250 mL shake flasks. Shake flasks were seeded with 300,000 viable cells per ml in a working volume of 50 ml of ActiPro medium (HyClone) and incubated in a humidified incubator (70-80%) with shaking at 120 rpm with 5% CO ₂ and a temperature of 37 °C (34 °C starting from day 6). Cultures were fed six times during production analysis using two different feed additives. Glucose concentrations were monitored daily and added if the level fell below 4 g/L. Glutamine-supplemented cultures were monitored daily for glutamine content and fed when levels fell below 2 mM. Cultures were stopped when viability levels became ≤50%.

Results

The results are presented in Table 7. Selection by glutamine removal significantly increased the number of heavy chain gene copies in the cell pool. Somewhat surprisingly, the copy number of the light chain also increased, but not to the same extent as the heavy chain. Total antibody production was also significantly higher than that of the unselected culture, reaching the levels shown in Table 5 when performing only two transductions instead of the five performed in this experiment.

Table 7. Comparison of pooled cell lines with and without glutamine selection to produce Yourway antibody. The combined cell line was produced by one light chain transduction (-GS) and one heavy chain transduction (+GS)

United cell lines Average copy number of pool heavy chain genes Average copy number of pool light chain genes Final titer (mg/l) +Glutamine 2.1 12.5 516 - Glutamine 24.1 36.0 3323

Experiment 6

The gene construct was designed and produced to investigate whether the technology would perform similarly using traditional cell transfection methodologies, compared to retrovector transduction. One example of a conventional plasmid transfection construct generated is shown in FIG. 8 (plasmid map) and FIG. 9 (actual plasmid sequence; SEQ ID NO:4).

1. Bleck, G.T. 2005 An alternative method for the rapid generation of stable, high-expressing mammalian cell lines (A Technical Review). Bioprocessing J. Sep/Oct. pp. 1-7.

2. Bleck, G.T., 2010. GPEx® A Flexible Method for the Rapid Generation of Stable, High Expressing, Antibody Producing Mammalian Cell Lines Chapter 4 In: Current Trends in Monoclonal Antibody Development and Manufacturing, Biotechnology: Pharmaceutical Aspects, Edited by: S.J. Shire et al. © 2010 American Association of Pharmaceutical Scientists, DOI 10.1007/978-0-387-76643-0_4.

All publications and patents mentioned in the above description are incorporated herein by reference. Various modifications and variations of the described method and system of this invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, it is intended that various modifications of the described methods of carrying out the present invention, which will be obvious to those skilled in the art, are included within the scope of the following claims.

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LIST OF SEQUENCES

<110> CATALENT PHARMA SOLUTIONS, LLC

<120> VECTORS FOR PROTEIN PRODUCTION

<130> GALA-37375.601

<150> US 62/775 194

<151> 2018-12-04

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 6217

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 1

gtccggccat tagccatatt attcattggt tatatagcat aaatcaatat tggctattgg

60

ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca

120

ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca

180

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct

240

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta

300

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac

360

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt

420

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag

480

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat

540

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat

600

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc

660

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcaa

720

taaaagagcc cacaacccct cactcggcgc gccagtcttc cgatagactg cgtcgcccgg

780

gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc

840

cttgggaggg tctcctctga gtgattgact acccacgacg ggggtctttc atttgggggc

900

tcgtccggga tttggagacc cctgcccagg gaccaccgac ccaccaccgg gaggtaagct

960

ggccagcaac ttatctgtgt ctgtccgatt gtctagtgtc tatgtttgat gttatgcgcc

1020

tgcgtctgta ctagttagct aactagctct gtatctggcg gacccgtggt ggaactgacg

1080

agttctgaac acccggccgc aaccctggga gacgtcccag ggactttggg ggccgttttt

1140

gtggcccgac ctgaggaagg gagtcgatgt ggaatccgac cccgtcagga tatgtggttc

1200

tggtaggaga cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt

1260

ggaaccgaag ccgcgcgtct tgtctgctgc agcgctgcag catcgttctg tgttgtctct

1320

gtctgactgt gtttctgtat ttgtctgaaa attagggcca gactgttacc actcccttaa

1380

gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg

1440

tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat

1500

ggccgcgaga cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt

1560

cacctggccc gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg

1620

cttttgaccc ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc

1680

ctccatccgc cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc

1740

tttatccagc cctcactcct tctctaggcg ccggaattgc cttccaccat ggccacctca

1800

gcaagttccc acttgaacaa aaacatcaag caaatgtact tgtgcctgcc ccaggtgag

1860

aaagtccaag ccatgtatat ctgggttgat ggtactggag aaggactgcg ctgcaaaacc

1920

cgcaccctgg actgtgagcc caagtgtgta gaagagttac ctgagtggaa ttttgatggc

1980

tctagtacct ttcagtctga gggctccaac agtgacatgt atctcagccc tgttgccatg

2040

tttcgggacc ccttccgcag agatcccaac aagctggtgt tctgtgaagt tttcaagtac

2100

aaccggaagc ctgcagagac caatttaagg cactcgtgta aacggataat ggacatggtg

2160

agcaaccagc acccctggtt tggaatggaa caggagtata ctctgatggg aacagatggg

2220

cacccttttg gttggccttc caatggcttt cctgggcccc aaggtccgta ttactgtggt

2280

gtgggcgcag acaaagccta tggcagggat atcgtggagg ctcactaccg cgcctgcttg

2340

tatgctgggg tcaagattac aggaacaaat gctgaggtca tgcctgccca gtgggagttc

2400

caaataggac cctgtgaagg aatccgcatg ggagatcatc tctgggtggc ccgtttcatc

2460

ttgcatcgag tatgtgaaga ctttggggta atagcaacct ttgaccccaa gcccattcct

2520

gggaactgga atggtgcagg ctgccatacc aactttagca ccaaggccat gcgggaggag

2580

aatggtctga agcacatcga ggaggccatc gagaaactaa gcaagcggca ccggtaccac

2640

attcgagcct acgatcccaa ggggggcctg gacaatgccc gtcgtctgac tgggttccac

2700

gaaacgtcca acatcaacga cttttctgct ggtgtcgcca atcgcagtgc cagcatccgc

2760

attccccgga ctgtcggcca ggagaagaaa ggttactttg aagaccgccg cccctctgcc

2820

aactgtgacc cctttgcagt gacagaagcc atcgtccgca catgccttct caatgagact

2880

ggcgacgagc ccttccaata caaaaactaa agatccctat ggctattggc caggttcaat

2940

actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga

3000

tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata

3060

gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg

3120

ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc

3180

gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg

3240

ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc

3300

ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa

3360

gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga

3420

tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt

3480

tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg

3540

tttagggaac cgccattctg cctggggacg tcggaggagc tcgaaagctt ctagacaatt

3600

gccgccacca tgatgtcctt tgtctctctg ctcctggttg gcatcctatt ccatgccacc

3660

caggccagtg atacaggtag acctttcgta gagatgtaca gtgaaatccc cgaaattata

3720

cacatgactg aaggaaggga gctcgtcatt ccctgccggg ttacgtcacc taacatcact

3780

gttactttaa aaaagtttcc acttgacact ttgatccctg atggaaaacg cataatctgg

3840

gacagtagaa agggcttcat catatcaaat gcaacgtaca aagaaatagg gcttctgacc

3900

tgtgaagcaa cagtcaatgg gcatttgtat aagacaaact atctcacaca tcgacaaacc

3960

aatacaatca tagatgtcgt tctgagtccg tctcatggaa ttgaactatc tgttggagaa

4020

aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg

4080

gaataccctt cttcgaagca tcagcataag aaacttgtaa acccgagacct aaaaacccag

4140

tctgggagtg agatgaagaa gtttttgagc accttaacta tagatggtgt aacccggagt

4200

gaccaaggat tgtacacctg tgcagcatcc agtgggctga tgaccaagaa aaacagcaca

4260

tttgtcaggg tccatgaaaa agacaaaact cacacatgcc caccgtgccc agcacctgaa

4320

ctcctggggg gaccctcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc

4380

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc

4440

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccacgggag

4500

gagcagtaca acagcacata tcgtgtggtc agcgtcctca ccgtcctgca ccaggactgg

4560

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag

4620

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca

4680

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat

4740

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc

4800

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac

4860

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac

4920

aaccactaca cgcagaagag cctctccctg tctcccggga aatgatgaga tctcgagttc

4980

gacatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac

5040

tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt

5100

gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat

5160

gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca

5220

acccccactg gttggggcat tgccaccacc tgtcagctcc tttccggggac tttcgctttc

5280

cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg

5340

gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct

5400

tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct

5460

tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt

5520

ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat

5580

cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct

5640

gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac

5700

tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa

5760

acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag

5820

ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcaggggccaa

5880

gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt

5940

ttccaagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt

6000

tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac

6060

ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa

6120

taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc

6180

tgagtgattg actacccgtc agcgggggtc tttcatt

6217

<210> 2

<211> 6065

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 2

gtccggccat tagccatatt attcattggt tatatagcat aaatcaatat tggctattgg

60

ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca

120

ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca

180

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct

240

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta

300

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac

360

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt

420

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag

480

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat

540

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat

600

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc

660

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcaa

720

taaaagagcc cacaacccct cactcggcgc gccagtcttc cgatagactg cgtcgcccgg

780

gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc

840

cttgggaggg tctcctctga gtgattgact acccacgacg ggggtctttc atttgggggc

900

tcgtccggga tttggagacc cctgcccagg gaccaccgac ccaccaccgg gaggtaagct

960

ggccagcaac ttatctgtgt ctgtccgatt gtctagtgtc tatgtttgat gttatgcgcc

1020

tgcgtctgta ctagttagct aactagctct gtatctggcg gacccgtggt ggaactgacg

1080

agttctgaac acccggccgc aaccctggga gacgtcccag ggactttggg ggccgttttt

1140

gtggcccgac ctgaggaagg gagtcgatgt ggaatccgac cccgtcagga tatgtggttc

1200

tggtaggaga cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt

1260

ggaaccgaag ccgcgcgtct tgtctgctgc agcgctgcag catcgttctg tgttgtctct

1320

gtctgactgt gtttctgtat ttgtctgaaa attagggcca gactgttacc actcccttaa

1380

gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg

1440

tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat

1500

ggccgcgaga cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt

1560

cacctggccc gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg

1620

cttttgaccc ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc

1680

ctccatccgc cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc

1740

tttatccagc cctcactcct tctctaggcg ccggaattgc cttccaccat ggccacctca

1800

gcaagttccc acttgaacaa aaacatcaag caaatgtact tgtgcctgcc ccaggtgag

1860

aaagtccaag ccatgtatat ctgggttgat ggtactggag aaggactgcg ctgcaaaacc

1920

cgcaccctgg actgtgagcc caagtgtgta gaagagttac ctgagtggaa ttttgatggc

1980

tctagtacct ttcagtctga gggctccaac agtgacatgt atctcagccc tgttgccatg

2040

tttcgggacc ccttccgcag agatcccaac aagctggtgt tctgtgaagt tttcaagtac

2100

aaccggaagc ctgcagagac caatttaagg cactcgtgta aacggataat ggacatggtg

2160

agcaaccagc acccctggtt tggaatggaa caggagtata ctctgatggg aacagatggg

2220

cacccttttg gttggccttc caatggcttt cctgggcccc aaggtccgta ttactgtggt

2280

gtgggcgcag acaaagccta tggcagggat atcgtggagg ctcactaccg cgcctgcttg

2340

tatgctgggg tcaagattac aggaacaaat gctgaggtca tgcctgccca gtgggagttc

2400

caaataggac cctgtgaagg aatccgcatg ggagatcatc tctgggtggc ccgtttcatc

2460

ttgcatcgag tatgtgaaga ctttggggta atagcaacct ttgaccccaa gcccattcct

2520

gggaactgga atggtgcagg ctgccatacc aactttagca ccaaggccat gcgggaggag

2580

aatggtctga agcacatcga ggaggccatc gagaaactaa gcaagcggca ccggtaccac

2640

attcgagcct acgatcccaa ggggggcctg gacaatgccc gtcgtctgac tgggttccac

2700

gaaacgtcca acatcaacga cttttctgct ggtgtcgcca atcgcagtgc cagcatccgc

2760

attccccgga ctgtcggcca ggagaagaaa ggttactttg aagaccgccg cccctctgcc

2820

aactgtgacc cctttgcagt gacagaagcc atcgtccgca catgccttct caatgagact

2880

ggcgacgagc ccttccaata caaaaactaa agatccctat ggctattggc caggttcaat

2940

actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga

3000

tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata

3060

gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg

3120

ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc

3180

gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg

3240

ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc

3300

ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa

3360

gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga

3420

tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt

3480

tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg

3540

tttagggaac cgccattctg cctggggacg tcggaggagc tcgaaagctt ctagacaatt

3600

gccgccacca tgatgtcctt tgtctctctg ctcctggttg gcatcctatt ccatgccacc

3660

caggccagtg atacaggtag acctttcgta gagatgtaca gtgaaatccc cgaaattata

3720

cacatgactg aaggaaggga gctcgtcatt ccctgccggg ttacgtcacc taacatcact

3780

gttactttaa aaaagtttcc acttgacact ttgatccctg atggaaaacg cataatctgg

3840

gacagtagaa agggcttcat catatcaaat gcaacgtaca aagaaatagg gcttctgacc

3900

tgtgaagcaa cagtcaatgg gcatttgtat aagacaaact atctcacaca tcgacaaacc

3960

aatacaatca tagatgtcgt tctgagtccg tctcatggaa ttgaactatc tgttggagaa

4020

aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg

4080

gaataccctt cttcgaagca tcagcataag aaacttgtaa acccgagacct aaaaacccag

4140

tctgggagtg agatgaagaa gtttttgagc accttaacta tagatggtgt aacccggagt

4200

gaccaaggat tgtacacctg tgcagcatcc agtgggctga tgaccaagaa aaacagcaca

4260

tttgtcaggg tccatgaaaa agacaaaact cacacatgcc caccgtgccc agcacctgaa

4320

ctcctggggg gaccctcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc

4380

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc

4440

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccacgggag

4500

gagcagtaca acagcacata tcgtgtggtc agcgtcctca ccgtcctgca ccaggactgg

4560

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag

4620

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca

4680

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat

4740

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc

4800

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac

4860

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac

4920

aaccactaca cgcagaagag cctctccctg tctcccggga aatgatgaga tctcgagttc

4980

gacatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac

5040

tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt

5100

gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat

5160

gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca

5220

acccccactg gttggggcat tgccaccacc tgtcagctcc tttccggggac tttcgctttc

5280

cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg

5340

gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct

5400

tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct

5460

tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt

5520

ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat

5580

cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct

5640

gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac

5700

tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accggtcgac

5760

cggtcgaccc tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc

5820

tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct

5880

ccccgagctc aataaaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac

5940

tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg

6000

tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag cggggggtctt

6060

tcatt

6065

<210> 3

<211> 442

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 3

aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc

60

atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac

120

agggtcgacc ggtcgaccgg tcgaccctag agaaccatca gatgtttcca gggtgcccca

180

aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct

240

gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg

300

ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag

360

ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac

420

ccgtcagcgg gggtctttca tt

442

<210> 4

<211> 8072

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 4

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca

60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc

180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc

240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat

300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt

360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt catttaaatg aaagacccca

420

cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat

480

aactgagaat agaaaagttc agatcaaggt caggaacaga tggaacaggg tcgaccggtc

540

gaccggtcga ccctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga

600

ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct

660

gctccccgag ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcttccgata

720

gactgcgtcg cccgggtacc cgtattccca ataaagcctc ttgctgtttg catccgaatc

780

gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactaccca cgacggggggt

840

ctttcatttg ggggctcgtc cgggatttgg agacccctgc ccagggacca ccgacccacc

900

accgggaggt aagctggcca gcaacttatc tgtgtctgtc cgattgtcta gtgtctatgt

960

ttgatgttat gcgcctgcgt ctgtactagt tagctaacta gctctgtatc tggcggaccc

1020

gtggtggaac tgacgagttc tgaacacccg gccgcaaccc tgggagacgt cccagggact

1080

ttggggggccg tttttgtggc ccgacctgag gaagggagtc gatgtggaat ccgaccccgt

1140

caggatatgt ggttctggta ggagacgaga acctaaaaca gttcccgcct ccgtctgaat

1200

ttttgctttc ggtttggaac cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg

1260

ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc tgaaaattag ggccagactg

1320

ttaccactcc cttaagtttg accttaggtc actggaaaga tgtcgagcgg atcgctcaca

1380

accagtcggt agatgtcaag aagagacgtt gggttacctt ctgctctgca gaatggccaa

1440

cctttaacgt cggatggccg cgagacggca cctttaaccg agacctcatc acccaggtta

1500

agatcaaggt cttttcacct ggcccgcatg gacacccaga ccaggtcccc tacatcgtga

1560

cctgggaagc cttggctttt gacccccctc cctgggtcaa gccctttgta caccctaagc

1620

ctccgcctcc tcttcctcca tccgccccgt ctctccccct tgaacctcct cgttcgaccc

1680

cgcctcgatc ctccctttat ccagccctca ctccttctct aggcgccgga attgccttcc

1740

accatggcca cctcagcaag ttcccacttg aacaaaaaca tcaagcaaat gtacttgtgc

1800

ctgccccagg gtgagaaagt ccaagccatg tatatctggg ttgatggtac tggagaagga

1860

ctgcgctgca aaacccgcac cctggactgt gagcccaagt gtgtagaaga gttacctgag

1920

tggaattttg atggctctag tacctttcag tctgagggct ccaacagtga catgtatctc

1980

agccctgttg ccatgtttcg ggaccccttc cgcagagatc ccaacaagct ggtgttctgt

2040

gaagttttca agtacaaccg gaagcctgca gagaccaatt taaggcactc gtgtaaacgg

2100

ataatggaca tggtgagcaa ccagcacccc tggtttggaa tggaacagga gtatactctg

2160

atgggaacag atgggcaccc ttttggttgg ccttccaatg gctttcctgg gccccaaggt

2220

ccgtattact gtggtgtggg cgcagacaaa gcctatggca gggatatcgt ggaggctcac

2280

taccgcgcct gcttgtatgc tggggtcaag attacaggaa caaatgctga ggtcatgcct

2340

gcccagtggg agttccaaat aggaccctgt gaaggaatcc gcatgggaga tcatctctgg

2400

gtggcccgtt tcatcttgca tcgagtatgt gaagactttg gggtaatagc aacctttgac

2460

cccaagccca ttcctgggaa ctggaatggt gcaggctgcc ataccaactt tagcaccaag

2520

gccatgcggg aggagaatgg tctgaagcac atcgaggagg ccatcgagaa actaagcaag

2580

cggcaccggt accacattcg agcctacgat cccaaggggg gcctggacaa tgcccgtcgt

2640

ctgactgggt tccacgaaac gtccaacatc aacgactttt ctgctggtgt cgccaatcgc

2700

agtgccagca tccgcattcc ccggactgtc ggccaggaga agaaaggtta ctttgaagac

2760

cgccgcccct ctgccaactg tgaccccttt gcagtgacag aagccatcgt ccgcacatgc

2820

cttctcaatg agactggcga cgagcccttc caatacaaaa actaaagatc cctatggcta

2880

ttggccaggt tcaatactat gtattggccc tatgccatat agtattccat atatgggttt

2940

tcctattgac gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc

3000

ctaataccgc ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc

3060

tattgacgtc atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat

3120

tacggtaaat ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg

3180

acgtcaatgg gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca

3240

atgacggtaa atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt

3300

acattactat tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg

3360

gtaaatggcc cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat

3420

gacgcaaatg ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta

3480

tataagcaat gctcgtttag ggaaccgcca ttctgcctgg ggacgtcgga ggagctcgaa

3540

agcttctaga caattgccgc caccatgatg tcctttgtct ctctgctcct ggttggcatc

3600

ctattccatg ccacccaggc cagtgataca ggtagacctt tcgtagagat gtacagtgaa

3660

atccccgaaa ttatacacat gactgaagga agggagctcg tcattccctg ccgggttacg

3720

tcacctaaca tcactgttac tttaaaaaag tttccacttg acactttgat ccctgatgga

3780

aaacgcataa tctgggacag tagaaagggc ttcatcatat caaatgcaac gtacaaagaa

3840

atagggcttc tgacctgtga agcaacagtc aatgggcatt tgtataagac aaactatctc

3900

acacatcgac aaaccaatac aatcatagat gtcgttctga gtccgtctca tggaattgaa

3960

ctatctgttg gagaaaagct tgtcttaaat tgtacagcaa gaactgaact aaatgtgggg

4020

attgacttca actgggaata cccttcttcg aagcatcagc ataagaaact tgtaaaccga

4080

gacctaaaaa cccagtctgg gagtgagatg aagaagtttt tgagcacctt aactatagat

4140

ggtgtaaccc ggagtgacca aggattgtac acctgtgcag catccagtgg gctgatgacc

4200

aagaaaaaca gcacatttgt cagggtccat gaaaaagaca aaactcacac atgcccaccg

4260

tgcccagcac ctgaactcct ggggggaccc tcagtcttcc tcttcccccc aaaacccaag

4320

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac

4380

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag

4440

acaaagccac gggaggagca gtacaacagc acatatcgtg tggtcagcgt cctcaccgtc

4500

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc

4560

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg

4620

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg

4680

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag

4740

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc

4800

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg

4860

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc cgggaaatga

4920

tgagatctcg agttcgacat cgataatcaa cctctggatt acaaaatttg tgaaagattg

4980

actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct

5040

ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg

5100

ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact

5160

gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc

5220

gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc

5280

cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa

5340

tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc

5400

ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg

5460

gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg

5520

gccgcctccc cgcatcgatg ggggaggcta actgaaacac ggaagggagac aataccggaa

5580

ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt

5640

tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc

5700

ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg

5760

tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cggatccttt

5820

ccactgtacg cgtagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta

5880

tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc

5940

ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg

6000

aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg

6060

tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg

6120

gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa

6180

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc

6240

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc

6300

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag

6360

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct

6420

cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta

6480

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc

6540

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc

6600

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt

6660

gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct

6720

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc

6780

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca

6840

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta

6900

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa

6960

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg

7020

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg

7080

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc

7140

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc

7200

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa

7260

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc

7320

cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg

7380

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc

7440

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat

7500

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg

7560

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc

7620

ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg

7680

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat

7740

gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg

7800

gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg

7860

ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct

7920

catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac

7980

atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta

8040

taaaaatagg cgtatcacga ggccctttcg tc

8072

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Claims (20)

1. A vector for expressing a protein of interest, containing a nucleic acid sequence encoding a selectable marker in functional connection with a first promoter sequence, which is a viral Self-Inactivating (SIN) Long Terminal Repeat (LTR) promoter sequence, and a nucleic acid sequence encoding a protein of interest operably linked to the second promoter sequence. 2. The vector of claim 1, wherein said SIN LTR promoter sequence is at least 95% identical to SEQ ID NO:3, wherein preferably said SIN LTR promoter sequence is SEQ ID NO:3. 3. Vector according to any one of paragraphs. 1, 2, in which the selectable marker is glutamine synthetase (GS) or dihydrofolate reductase (DHFR). 4. Vector according to any one of paragraphs. 1-3, wherein said vector comprises one poly-A signal sequence in operative association with said selectable marker and said nucleic acid encoding a protein of interest, or wherein said vector comprises a first poly-A signal sequence in operative association with said selectable marker and a second poly-A signal sequence in operative association with said nucleic acid encoding a protein of interest. 5. Vector according to any one of paragraphs. 1-4, wherein the protein of interest is selected from the group consisting of an Fc fusion protein, an enzyme, an albumin fusion protein, a growth factor, a protein receptor, a single chain antibody (scFv), a single chain antibody-Fc (scFv-Fc), a diabody, and minibody (scFv-CH3), Fab, single chain Fab (scFab), immunoglobulin heavy chain and immunoglobulin light chain, wherein preferably said protein of interest is an Fc fusion protein. 6. Vector according to any one of paragraphs. 1-5, wherein said vector is a viral vector, preferably a retroviral vector and/or plasmid. 7. A host cell for expressing a protein of interest, comprising a vector according to any one of claims. 1-6, wherein preferably said host cell further comprises at least a second vector that encodes and provides expression of a second protein of interest, and wherein i) said second vector does not include a selectable marker, or ii) said second vector includes a selectable marker different from the selectable marker in the first vector. 8. The host cell according to claim 7, wherein the host cell line is selected from the group consisting of a GS knockout cell line, a Chinese hamster ovary (CHO) cell line, a HEK 293 cell line and a CAP cell line, preferably a knockout cell line DHFR. 9. Host cell according to any one of paragraphs. 7, 8, wherein said host cell contains from about 1 to 1000 copies of the vector, preferably from about 10 to 200 copies of the vector, more preferably from about 10 to 100 copies of the vector, most preferably from about 20 to 100 copies of the vector. 10. Host cell according to any one of paragraphs. 7-9, wherein the first protein of interest in the first vector is one of an immunoglobulin heavy or light chain, and the second protein in the second vector is another of an immunoglobulin heavy or light chain, wherein preferably the first protein of interest is a heavy chain immunoglobulin, and the second protein of interest is an immunoglobulin light chain. 11. Host cell according to any one of paragraphs. 7-10, wherein said host cell contains from about 1 to 1000 copies of the second vector, preferably from about 10 to 200 copies of the second vector, more preferably from about 10 to 100 copies of the second vector, most preferably from about 20 to 100 copies of the second vector . 12. A host cell culture for the production of a protein of interest, comprising host cells according to any one of claims. 7-11, wherein preferably said culture produces from 1 to 150 picograms/cell/day of the protein of interest, more preferably said culture produces from 2 to 50 picograms/cell/day of the protein of interest. 13. Expression system containing: a first vector containing a nucleic acid sequence encoding a selectable marker in functional association with a first promoter sequence, which is a viral Self-Inactivating (SIN) promoter sequence of a Long Terminal Repeat (LTR), and a nucleic acid sequence encoding a first protein of interest operably linked to a second promoter sequence, wherein preferably said first protein of interest is one of an immunoglobulin heavy or light chain; And a second vector containing a nucleic acid sequence encoding a second protein of interest operably linked to a promoter sequence, wherein preferably said second protein of interest is another immunoglobulin heavy or light chain and wherein said second vector does not include a selectable marker. 14. A method for producing a protein of interest, comprising: transduction or transfection of a host cell or host cells with the system of claim 13; developing a host cell line from said transduced or transfected host cell or host cells that express the protein of interest; culturing host cells under conditions such that the protein of interest is produced by said host cell line; And purifying the protein of interest from the host cell culture.

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