Vectors for protein production

Abstract

The present invention relates to vectors and their use in developing host cell lines for producing proteins of interest, and in particular to vectors utilizing weak promoters to drive selectable markers.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 775,194, filed on December 4, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to vectors and their use in developing host cell lines for producing proteins of interest, and in particular to vectors utilizing weak promoters to drive selectable markers. Background Art

[0004] Therapeutic protein drugs are an important class of medicines serving patients who are most in need of novel therapies. Recently approved recombinant protein therapeutics have been developed to treat a variety of clinical indications, including cancer, autoimmunity / inflammatory conditions, exposure to infectious agents, and genetic disorders. Recent advances in protein engineering technologies allow drug developers and manufacturers to fine-tune and exploit the desirable functional properties of a protein of interest while maintaining (and in some cases improving) product safety or efficacy, or both.

[0005] The manufacture and production of therapeutic proteins is a highly complex process. For example, a typical protein drug may include more than 5,000 key process steps, many times the number of steps required to manufacture a small molecule drug.

[0006] Similarly, the size of protein therapeutics, including monoclonal antibodies and large proteins or fusion proteins, can be several orders of magnitude larger than small molecule drugs with a molecular weight exceeding 100 kDa. In addition, protein therapeutics exhibit complex secondary and tertiary structures that must be maintained. Protein therapeutics cannot be synthesized entirely by chemical processes and must be manufactured in living cells or organisms; therefore, the choice of cell lines, species of origin, and culture conditions will affect the properties of the final product. Moreover, most biologically active proteins require post-translational modifications, which may be impaired when heterologous expression systems are used. In addition, since the product is synthesized by cells or organisms, a complex purification process is involved. In addition, virus removal processes are also implemented, such as removing viral particles by using filters or resins, and inactivation steps using low pH or detergents to prevent serious safety issues of protein raw materials being contaminated by viruses. Given the complexity of therapeutic proteins in terms of their huge molecular size, post-translational modifications, and the various biological materials involved in the manufacturing process, the ability to enhance specific functional attributes while maintaining product safety and efficacy achieved through protein engineering strategies is highly desirable.

[0007] Although it is not easy to integrate novel strategies and methods to modify protein drug products, the potential therapeutic advantages have driven the increased use of such strategies in the drug development process. A variety of protein engineering platform technologies are currently being used to increase the circulation half-life, targeting, and functionality of novel therapeutic protein drugs, as well as to improve yield and product purity. For example, protein conjugation and derivatization methods, including Fc fusion, albumin fusion, and PEGylation, are currently being used to extend the circulation half-life of drugs.

[0008] Protein drugs (biologics) are expensive and time consuming to produce. There is a need in the art for more efficient tools and methods to produce this important class of drugs. Summary of the invention

[0009] The present invention relates to vectors and their use in developing host cell lines for producing proteins of interest, and in particular to vectors utilizing weak promoters to drive selectable markers.

[0010] Thus, in some preferred embodiments, the present invention provides a vector for expressing a protein of interest, the vector comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence and a nucleic acid sequence encoding a protein of interest operably linked to a second promoter sequence, wherein the first promoter sequence has been altered to have reduced promoter activity compared to the unaltered or wild-type form of the first promoter sequence.

[0011] In some preferred embodiments, the first promoter sequence that has been altered to have reduced promoter activity compared to the unaltered or wild-type form of the first promoter sequence is a viral self-inactivating (SIN) long terminal repeat (LTR) promoter sequence. In some preferred embodiments, the SIN LTR promoter sequence has at least 95% identity to SEQ ID NO: 3. In some preferred embodiments, the SIN LTR promoter sequence is SEQ ID NO: 3.

[0012] In some preferred embodiments, the selectable marker is glutamine synthetase (GS). In some preferred embodiments, the selectable marker is dihydrofolate reductase (DHFR).

[0013] In some embodiments, the vector comprises a single poly A signal sequence operably associated with a selectable marker and a nucleic acid encoding a protein of interest. In other embodiments, the vector comprises a first poly A signal sequence operably associated with a selectable marker and a second poly A signal sequence operably associated with a nucleic acid encoding a protein of interest.

[0014] In some preferred embodiments, the protein of interest is selected from the group consisting of: Fc fusion proteins, enzymes, albumin fusions, growth factors, protein receptors, single chain antibodies (scFv), single chain Fc (scFv-Fc), bifunctional antibodies and mini antibodies (scFv-CH3), Fab, single chain Fab (scFab), immunoglobulin heavy chains and immunoglobulin light chains. In some preferred embodiments, the protein of interest is an Fc fusion protein.

[0015] 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.

[0016] In some preferred embodiments, the invention provides host cells comprising vectors as 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 comprises about 1, 20, 50 to 1000 copies of the vector. In some preferred embodiments, the host cell comprises about 10 to 200 copies of the vector. In some preferred embodiments, the host cell comprises about 10 to 100 copies of the vector. In some preferred embodiments, the host cell comprises about 20 to 100 copies of the vector.

[0017] In some preferred embodiments, the host cell also comprises at least a second vector, which encodes and allows expression of a second protein of interest, and wherein the second vector does not include a selection marker. In some preferred embodiments, the host cell also comprises at least a second vector, which encodes and allows expression of a second protein of interest, and wherein the second vector comprises a selection marker different from the selection marker in the first vector. In some preferred embodiments, the first protein of interest in the first vector is one of an immunoglobulin heavy chain or light chain, and the second protein in the second vector is another of an immunoglobulin heavy chain 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 comprises about 1, 20, 50 or 100 to 1000 copies of the second vector. In some preferred embodiments, the host cell comprises about 10 to 200 copies of the second vector. In some preferred embodiments, the host cell comprises about 10 to 100 copies of the second vector. In some preferred embodiments, the host cell comprises about 20 to 100 copies of the second vector.

[0018] In some preferred embodiments, the invention provides a host cell culture comprising a host cell as described above. In some preferred embodiments, the culture produces 1 to 50 g / L / day of the protein of interest. In some preferred embodiments, the culture produces 2 to 10 g / L / day of the protein of interest.

[0019] In some preferred embodiments, the present invention provides a method for producing a protein of interest, the method comprising culturing a host cell as described above and purifying the protein of interest from the host cell culture.

[0020] In some preferred embodiments, the present invention provides an infectious retroviral particle comprising the following elements in 5' to 3' order: 1) 5'LTR; 2) retroviral packaging region; 3) nucleic acid encoding a selection marker; 4) internal promoter; 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter; and 6) SIN 3'LTR. In some preferred embodiments, the 5'LTR is a MoMuSV LTR or a SIN LTR. In some embodiments, the 3'LTR comprises a poly A signal sequence. In some embodiments, the packaging region comprises multiple potential translation start sites. In some preferred embodiments, the selection marker is GS. In some preferred embodiments, the internal promoter is a CMV promoter. In some embodiments, the particle comprises a single poly A signal sequence downstream of the nucleic acid encoding the protein of interest. In some embodiments, the particle comprises a first poly A signal sequence operably associated with the selection marker and a second poly A signal sequence operably associated with the nucleic acid encoding the protein of interest.

[0021] Other embodiments provide a plasmid comprising the following elements in 5' to 3' order: 1) 5' LTR (e.g., SIN LTR); 2) packaging region; 3) selection marker (e.g., GS); 4) internal promoter (e.g., CMV promoter); 5) nucleic acid sequence encoding a protein of interest operably linked to the internal promoter; and 6) poly A signal sequence. In some embodiments, the plasmid comprises a single poly A signal sequence downstream of the nucleic acid encoding the protein of interest.

[0022] Additional embodiments provide a system comprising: a) a first vector comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence and a nucleic acid sequence encoding a first protein of interest operably linked to a second promoter sequence, wherein the first promoter sequence has been altered to have reduced promoter activity compared to an unaltered or wild-type form of the first promoter sequence; and b) a second vector comprising a nucleic acid sequence encoding a second protein of interest operably linked to a promoter sequence, and wherein the second vector does not include a selectable marker.

[0023] In some preferred embodiments, the present invention provides a method for producing a protein of interest, the method comprising: transducing or transfecting one or more host cells with an infectious retroviral particle, plasmid or vector system as described above, developing a host cell line that expresses the protein of interest from the one or more host cells; culturing the host cells under conditions that allow the host cell line to produce the protein of interest; and purifying the protein of interest from the host cell culture.

[0024] In some preferred embodiments, the present invention provides infectious retroviral particles or plasmids as described above for use in transducing one or more host cells to produce a protein of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 . Diagram of the full-length MMLV construct.

[0026] Figure 2 .Diagram of the SIN MMLV LTR construct.

[0027] Figure 3 . Sequence of the full-length MMLV construct (SEQ ID NO: 1).

[0028] Figure 4 .Sequence of SIN MMLV LTR construct (SEQ ID NO: 2).

[0029] Figure 5 . Comparison of pooled cell line titers between full-length MMLV constructs and SIN MMLV LTR constructs.

[0030] Figure 6 . Comparison of pooled cell line titers between approaches using full-length MMLV constructs and SIN MMLV LTR constructs.

[0031] Figure 7 .SIN LTR sequence (SEQ ID NO:3).

[0032] Figure 8 . Map of the proviral plasmid construct (SEQ ID NO: 4).

[0033] Figure 9. Sequence of proviral plasmid construct (SEQ ID NO: 4).

[0034] definition

[0035] To facilitate understanding of the present invention, a number of terms are defined below.

[0036] As used herein, the term "host cell" refers to any eukaryotic cell (eg, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

[0037] As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortalized phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell populations maintained in vitro, including oocytes and embryos.

[0038] As used herein, the term "vector" refers to any genetic element that is capable of replication and can transfer gene sequences between cells when associated with appropriate control elements, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc. Thus, the term includes cloning and expression vehicles as well as viral vectors.

[0039] As used herein, the term "multiplicity of infection" or "MOI" refers to the vector: host cell integration ratio used during transfection or transduction of host cells. For example, if 1,000,000 vectors are used to transduce 100,000 host cells, the multiplicity of infection is 10. The use of this term is not limited to events involving transduction, but encompasses the introduction of vectors into a host by methods such as lipofection, microinjection, calcium phosphate precipitation, and electroporation.

[0040] As used herein, the term "genome" refers to the genetic material (eg, chromosomes) of an organism.

[0041] The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA) whose manipulation is deemed desirable by one of ordinary skill in the art for any reason (e.g., treating a disease, conferring improved quality, expressing a protein of interest in a host cell, expressing a ribozyme, etc.). Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences that do not encode mRNA or protein products (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.).

[0042] As used herein, the term "protein of interest" refers to a protein encoded by a nucleic acid of interest.

[0043] As used herein, the terms "nucleic acid molecule encoding ...," "DNA sequence encoding ...," "DNA encoding ...," "RNA sequence encoding ...," and "RNA encoding ..." refer to the sequence or order 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, the DNA or RNA sequence encodes the amino acid sequence.

[0044] As used herein, the term "promoter", "promoter element" or "promoter sequence" refers to a DNA sequence that, when linked to a nucleotide sequence of interest, is capable of controlling transcription of the nucleotide sequence of interest into mRNA. Although not required, the promoter is usually located 5' (i.e., upstream) of the nucleotide sequence of interest that it controls transcription into mRNA, and provides a site for specific binding of RNA polymerase and other transcription factors to initiate transcription.

[0045] As used herein, the term "altered" when used in relation to a promoter refers to a promoter having an altered nucleic acid sequence compared to a reference wild-type sequence. For example, a sequence promoter may be altered by deleting certain promoter and / or enhancer elements.

[0046] Transcriptional control 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 in yeast, insect and mammalian cells and viruses (similar control elements, i.e., promoters, have also been found in prokaryotes). The choice of specific promoters and enhancers depends on the cell type to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a wide host range, while others work in a limited group of cell types (for review, see Voss et al., Trends Biochem. Sci., 11:287

[1986] ; and Maniatis et al., above). For example, the SV40 early gene enhancer is very active in a variety of cell types from many mammalian species and has been widely used to express proteins in mammalian cells (Dijkema et al., EMBO J. 4:761

[1985] ). Two other examples of promoter / enhancer elements that are active in a wide range of mammalian cell types are the promoter / enhancer element from the human elongation factor 1 alpha 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 the long terminal repeat 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] ).

[0047] As used herein, the term "promoter / enhancer" refers to a DNA segment containing sequences that can provide promoter and enhancer functions (i.e., functions provided by promoter elements and enhancer elements, see the discussion of these functions above). For example, the long terminal repeats of retroviruses contain promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer / promoter is an enhancer / promoter that is naturally connected to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is an enhancer / promoter that is placed in juxtaposition with a gene by means of genetic manipulation (i.e., molecular biotechnology, such as cloning and recombination) so that transcription of the gene is directed by the connected enhancer / promoter.

[0048] As used herein, the term "long terminal repeat" of "LTR" refers to a transcriptional control element located in or separated from the U3 region 5' and 3' of a retroviral genome. As is known in the art, long terminal repeats can be used as control elements in retroviral vectors, or separated from retroviral genomes and used to control expression from other types of vectors.

[0049] As used herein, the terms "complementary" or "complementarity" are used to refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Complementarity can be "partial", in which only some of the bases of the nucleic acids match according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0050] When used with respect to nucleic acids, the terms "homology" and "percent identity" 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 the hybridization of a fully complementary sequence with a target nucleic acid sequence, and is referred to using the functional term "substantially homologous". The inhibition of hybridization of a fully complementary sequence with a target sequence can be examined using hybridization assays (Southern or Northern blotting, solution hybridization, etc.) under low stringency conditions. Substantially homologous sequences or probes (i.e., oligonucleotides that can hybridize with another oligonucleotide of interest) will compete with fully homologous sequences and inhibit their binding (i.e., hybridization) to the target sequence under low stringency conditions. This is not to say that low stringency conditions allow nonspecific binding; low stringency conditions require that the binding of two sequences to each other is a specific (i.e., selective) interaction. The absence of nonspecific binding can be tested by using a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding, the probe will not hybridize with the second non-complementary target.

[0051] As used herein, the terms "in operable combination", "in operable order" and "operably linked" refer to nucleic acid sequences being linked in a manner that produces a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The terms also refer to amino acid sequences being linked in a manner that produces a functional protein.

[0052] As used herein, the term "selectable marker" refers to a gene encoding an enzyme activity, or other protein that confers the ability to grow in a medium that would otherwise lack an essential nutrient; additionally, a selectable marker can confer resistance to antibiotics or drugs on cells expressing the selectable marker.

[0053] 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 a viral G glycoprotein, that can bind to the surface of a host cell and facilitate entry of the viral particle 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 Oncovirinae (e.g., Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), and mouse mammary tumor virus (MMTV), Spumavirinae, and lentiviruses (e.g., human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus; see, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference).

[0054] 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 effectively transfer genes into host cells by utilizing a viral infection process. Exogenous or heterologous genes cloned (i.e., inserted using molecular biology techniques) into the retroviral genome can be effectively delivered to host cells susceptible to retroviral infection. By well-known gene manipulation, the replication ability of the retroviral genome can be destroyed. The resulting replication-defective vector can be used to introduce new genetic material into cells, but they cannot replicate. Helper viruses or packaging cell lines can be used to allow vector particles to assemble and flow out of cells. This type of retroviral vector comprises a replication-defective retroviral genome, which contains a nucleic acid sequence encoding at least one gene of interest (i.e., a polycistronic nucleic acid sequence can encode more than one gene of interest), 5' retroviral long terminal repeats (5'LTR); and 3' retroviral long terminal repeats (3'LTR).

[0055] The term "pseudotyped retroviral vector" refers to a retroviral vector that contains a heterologous membrane protein. The term "membrane-associated protein" refers to a protein associated with the membrane surrounding the viral particle (e.g., viral envelope glycoproteins or the G protein of Rhabdoviridae viruses such as VSV, Piry, Chandipura, and Mokola); these membrane-associated proteins mediate entry of viral particles into host cells. The membrane-associated protein may bind to a specific cell surface protein receptor, as with retroviral envelope proteins, or the membrane-associated protein may interact with the phospholipid components of the plasma membrane of the host cell, as with the G protein derived from members of the Rhabdoviridae family.

[0056] The term "heterologous membrane-associated protein" refers to a membrane-associated protein that is derived from a virus that is not a member of the same viral class or family as the virus from which the nucleocapsid protein of the vector particle is derived. "Viral class or family" refers to the taxonomic rank of a class or family assigned by the International Committee on Taxonomy of Viruses.

[0057] 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 arthritis-encephalitis virus) that is capable of integrating into non-dividing cells (see, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, which are incorporated herein by reference).

[0058] The term "pseudotyped lentiviral vector" refers to a lentiviral vector containing a heterologous membrane protein, such as a viral envelope glycoprotein or the G protein of a Rhabdoviridae virus such as VSV, Piry, Chandipura and Mokola.

[0059] As used herein, the term "transposon" refers to a transposable element (e.g., Tn5, Tn7, and Tn10) that can be moved or transposed from one position in a genome to another. Typically, transposition is controlled by a transposase. As used herein, the term "transposon vector" refers to a vector encoding a nucleic acid of interest flanking the transposon ends. Examples of transposon vectors include, but are not limited to, those described in U.S. Pat. Nos. 6,027,722, 5,958,775, 5,968,785, 5,965,443, and 5,719,055 (all of which are incorporated herein by reference).

[0060] As used herein, the term "adeno-associated virus (AAV) vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. The AAV vector may completely or partially delete one or more of the AAV wild-type genes, preferably the rep and / or cap genes, but retain functional flanking ITR sequences.

[0061] AAV vectors can be constructed using recombinant techniques known in the art to include one or more heterologous nucleotide sequences flanked by functional AAV ITRs at both ends (5' and 3'). In the practice of the present invention, the AAV vector may include at least one AAV ITR and a suitable promoter sequence upstream of the heterologous nucleotide sequence, and at least one AAV ITR downstream of the heterologous sequence. "Recombinant AAV vector plasmid" refers to a type of recombinant AAV vector, in which the vector comprises a plasmid. Typically, as with the AAV vector, 5' and 3' ITRs are flanked by the selected heterologous nucleotide sequence.

[0062] AAV vectors may also include transcription sequences, such as polyadenylation sites, as well as selection markers or reporter genes, enhancer sequences, and other control elements that allow for induction of transcription. Such control elements are described above.

[0063] As used herein, the term "AAV virion" refers to a complete virus particle. AAV virions can be wild-type AAV virions (comprising a linear single-stranded AAV nucleic acid genome associated with an AAV capsid, i.e., a protein coat) or recombinant AAV virions (as described below). In this regard, single-stranded AAV nucleic acid molecules (sense / coding strands or antisense / anti-coding strands, as those terms are generally defined) can be packaged into AAV virions; sense strands and antisense strands have equal infectivity.

[0064] As used herein, the term "recombinant AAV virion" or "rAAV" is defined as an infectious, replication-defective virus composed of an AAV protein shell encapsidating (i.e., surrounded by a protein coat) a heterologous nucleotide sequence which is in turn flanked on the 5' and 3' sides by AAV ITRs. Many techniques for constructing recombinant AAV virions are known in the art (see, e.g., U.S. 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] ; Muzyczka, Current Topics in Microbiol. and Immunol. 158:97-129

[1992] ; Kotin, Human Gene Therapy 5:793-801

[1994] ; Shelling and Smith, Gene Therapy 5:793-801

[1995] ). 1:165-169

[1994] ; and Zhou et al., J. Exp. Med. 179:1867-1875

[1994] , which are incorporated herein by reference in their entireties).

[0065] Suitable nucleotide sequences for AAV vectors (and indeed, any vector described herein) include any functionally relevant nucleotide sequences. Thus, the AAV vectors of the present invention may contain any desired gene encoding a protein that is defective or missing in the genome of the target cell, or encoding a non-natural protein with a desired biological or therapeutic effect (e.g., antiviral function), or the sequence may correspond to a molecule with antisense or ribozyme function. Suitable genes include genes for the treatment of the following diseases: inflammatory diseases, autoimmune, chronic and infectious diseases, including conditions such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, etc.; various blood diseases, including various anemias, thalassemias, and hemophilia; genetic defects, such as cystic fibrosis, Gaucher's Disease, adenosine deaminase (ADA) deficiency, emphysema, etc. Many antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) that can be used for reverse therapy against cancer and viral diseases have been described in the art (see, e.g., 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. Acta. 1049:99-125

[1990] ; Agarwal et al., Proc. Natl. Acad. Sci. USA 85:7079-7083

[1989] ; and Heikkila et al., Nature 328:445-449

[1987] ). For a discussion of suitable ribozymes, see, e.g., Cech et al. (1992) J. Biol. Chem. 267: 17479-17482 and U.S. Pat. No. 5,225,347, which are incorporated herein by reference.

[0066] As used herein, the term "purified" refers to a molecule, whether a nucleic acid sequence or an amino acid sequence, that is removed, separated or isolated from its normal environment. Thus, an "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

[0067] The present invention relates to vectors and their use in developing host cell lines for producing proteins of interest, and in particular to vectors utilizing weak promoters to drive selectable markers.

[0068] In some preferred embodiments, the expression system of the present invention utilizes an expression vector comprising a nucleic acid sequence encoding a protein of interest (i.e., a therapeutic protein or other protein desired to be produced) operably associated with additional nucleic acid sequences having various functions. Thus, for example, the 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, vectors include, but are not limited to, retroviral vectors, chromosomal DNA sequences, non-chromosomal DNA sequences, and synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral DNA, such as vaccinia, adenovirus, fowlpox virus, and pseudorabies). It is contemplated that any vector may be used as long as it is replicable and viable in the host. In some preferred embodiments, the vector is a retroviral vector as described in: U.S. Patent No. 6,852,510 and No. 7,332,333 and U.S. Patent Publication No. 200402335173 and No. 20030224415, all of which are incorporated herein by reference in their entirety. In some particularly preferred embodiments, the vector is a pseudotyped retroviral vector. In some preferred embodiments of the present invention, the mammalian expression vector comprises an origin of replication, a suitable promoter and enhancer and any necessary ribosome binding site, a polyadenylation site, a splice donor and acceptor site, a transcription termination sequence and a non-5' flanking non-transcribed sequence. In other embodiments, a DNA sequence and a polyadenylation site derived from SV40 splicing can be used to provide the required non-transcribed gene elements.

[0069] In some preferred embodiments, the vector is a retroviral vector. The organization of many retroviral genomes is well known in the art, and this allows the retroviral genome to adapt to produce retroviral vectors. The generation of the recombinant retroviral vector carrying a gene of interest is usually completed in two stages.

[0070] First, the gene of interest is inserted into a retroviral vector that contains sequences required for efficient expression of the gene of interest (including promoter and / or enhancer elements that can be provided by the viral long terminal repeat (LTR) or 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 sequence required for reverse transcription (+PBS)), and viral LTR. The LTR contains sequences required for viral genomic RNA association, reverse transcriptase and integrase functions, and sequences involved in directing the expression of genomic RNA to be packaged in viral particles. For safety reasons, many recombinant retroviral vectors lack functional copies of genes necessary for viral replication (these essential genes are deleted or disabled); therefore, the resulting virus is called replication-defective.

[0071] Secondly, after constructing the recombinant vector, the vector DNA is introduced into the packaging cell line. The packaging cell line provides the proteins (i.e., virally encoded gag, pol and env proteins) required for trans-packaging the viral genomic RNA into the virus particles with the desired host range. The host range is controlled in part by the type of envelope gene product expressed on the surface of the virus particles. The packaging cell line can express parental monotropic, dual tropic or heterotropic envelope gene products. Alternatively, the packaging cell line may lack the sequence encoding the viral envelope (env) protein. In this case, the packaging cell line packages the viral genome into particles lacking membrane-associated proteins (e.g., env proteins). In order to produce virus particles containing membrane-bound proteins that allow viruses to enter cells, a packaging cell line (VSV) containing a retroviral sequence is transfected with a sequence encoding membrane-bound proteins (e.g., the G protein of vesicular stomatitis virus). Then, the transfected packaging cells will produce virus particles containing membrane-associated proteins expressed by the transfected packaging cell line; these virus particles are referred to as pseudotyped virus particles, which contain viral genomic RNA derived from a virus encapsidated by the envelope protein of another virus.

[0072] The retroviral vector of the present invention can be further modified to include additional regulatory sequences. As described above, the retroviral vector of the present invention includes the following elements in operable association: a) 5'LTR; b) packaging signal; c) 3'LTR and d) nucleic acid encoding a protein of interest located between the 5' and 3'LTRs. In some embodiments of the present invention, when it is desired to transcribe from an internal promoter, the nucleic acid of interest can be arranged in the opposite direction to the 5'LTR. Suitable internal promoters include, but are not limited to, alpha-lactalbumin promoter, CMV promoter (human or monkey) and thymidine kinase promoter.

[0073] In other embodiments of the invention, where secretion of a protein of interest is desired, the vector is modified by including a signal peptide sequence operably associated with the protein of interest. The sequences of several suitable signal peptides are known to those of skill in the art, including but not limited to signal peptide sequences derived from tissue plasminogen activator, human growth hormone, lactoferrin, α-casein, and α-lactalbumin.

[0074] In other embodiments of the invention, the vector is modified by incorporating an RNA export element 3' or 5' into the nucleic acid sequence encoding the protein of interest (see, e.g., U.S. Pat. Nos. 5,914,267, 6,136,597, and 5,686,120; and WO99 / 14310, all incorporated herein by reference). It is contemplated that the use of an RNA export element may allow for high-level expression of the protein of interest without incorporating splicing signals or introns into the nucleic acid sequence encoding the protein of interest.

[0075] In other embodiments, the vector further comprises at least one internal ribosome entry site (IRES) sequence. Several suitable IRES sequences are available, including but not limited to sequences derived from foot-and-mouth disease virus (FDV), encephalomyocarditis virus, and poliovirus. The IRES sequence can be inserted between two transcription units (e.g., nucleic acids encoding different proteins of interest or subunits of multi-subunit proteins such as antibodies) to form a polycistronic sequence, thereby transcribing the two transcription units from the same promoter.

[0076] The most commonly used recombinant retroviral vector is derived from the amphotropic Moloney murine leukemia virus (MoMLV) (see, e.g., Miller and Baltimore Mol. Cell. Biol. 6:2895

[1986] ). The MoMLV system has several advantages: 1) this particular retrovirus can infect many different cell types, 2) established packaging cell lines can be used to produce recombinant MoMLV viral particles, and 3) the transferred genes are permanently integrated into the target cell chromosomes. The established MoMLV vector system comprises a DNA vector containing a small portion of the retroviral sequence (e.g., the viral long terminal repeat or "LTR" and the packaging or "psi" signal) and a packaging cell line. The gene to be transferred is inserted into the DNA vector. The viral sequences present on the DNA vector provide the signals required for the insertion or packaging of the vector RNA into the viral particles and for the expression of the inserted gene. The packaging cell line provides the proteins required for particle assembly (Markowitz et al., J. Virol. 62:1120

[1988] ).

[0077] In some preferred embodiments, the retroviral vector is pseudotyped and, for example, the G protein of VSV is used as a membrane-associated protein. Unlike retroviral envelope proteins that bind to specific cell surface protein receptors to enter cells, the VSV G protein interacts with the phospholipid components of the plasma membrane (Mastromarino et al., J. Gen. Virol. 68: 2359

[1977] ). Since VSV entry into cells does not depend on the presence of specific protein receptors, VSV has an extremely wide host range. Pseudotyped retroviral vectors with VSV G protein have a modified host range unique to VSV (i.e., they can infect cells of almost all vertebrate, invertebrate and insect species). Importantly, VSV G pseudotyped retroviral vectors can be concentrated 2000 times or more by ultracentrifugation without significant loss of infectivity (Burns et al. Proc. Natl. Acad. Sci. USA 90: 8033

[1993] ).

[0078] When a viral G protein is used as a heterologous membrane-associated protein within a virion, the present invention is not limited to the use of a VSV G protein (see, e.g., U.S. Pat. No. 5,512,421, which is incorporated herein by reference). The G proteins in viruses of the genus Vesiculovirus other than VSV (e.g., Piry and Chandipura viruses) 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 proteins of Piry and Chandipura viruses can be used in place of the VSV G protein to pseudotype virions. In addition, the VSV G protein of viruses within the genus Lyssavirus, such as rabies virus and Mokola virus, exhibits a high degree of conservation (amino acid sequence as well as functional conservation) with the VSV G protein. For example, the Mokola virus G protein has been shown to function similarly to the VSV G protein (i.e., mediate membrane fusion) and can therefore be used in place of the VSV G protein. G proteins are used to pseudotype viral particles (Mebatsion et al., J. Virol. 69:1444

[1995] ). Viral particles can be pseudotyped using Piry, Chandipura, or Merckola G proteins as described in Example 2, except that a plasmid containing a sequence encoding Piry, Chandipura, or Merckola G protein under the transcriptional control of an appropriate promoter element (e.g., the CMV intermediate early promoter; a number of expression vectors containing the CMV IE promoter can be used, such as the pcDNA3.1 vector (Invitrogen)) is used instead of pHCMV-G. Sequences encoding other G proteins from other members of the Rhabdoviridae family can be used; sequences encoding a variety of Rhabdoviridae G proteins are available from the GenBank database.

[0079] In some preferred embodiments, the vector is a lentiviral vector. Slow viruses (e.g., equine infectious anemia virus, caprine arthritis-encephalitis virus, human immunodeficiency virus) are a subfamily of retroviruses that can be integrated into non-dividing cells. The lentiviral genome and proviral DNA have three genes found in all retroviruses: gag, pol, and env, which are flanked by two LTR sequences. The gag gene encodes internal structural proteins (e.g., matrix proteins, capsid proteins, and nucleocapsid proteins); the pol gene encodes reverse transcriptase, protease, and integrase proteins; and the pol gene encodes viral envelope glycoproteins. The 5' and 3' LTRs control the transcription and polyadenylation of viral RNA. Additional genes in the lentiviral genome include vif, vpr, tat, rev, vpu, nef, and vpx genes.

[0080] A variety of lentiviral vectors and packaging cell lines are known in the art and can be used in the present invention (see, e.g., U.S. Pat. Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference). In addition, VSV G protein has also been used to pseudotype retroviral vectors based on human immunodeficiency virus (HIV) (Naldini et al., Science 272:263

[1996] ). Therefore, VSV G protein can be used to produce a variety of pseudotyped retroviral vectors, and is not limited to MoMLV-based vectors. Lentiviral vectors can also be modified as described above to contain various regulatory sequences (e.g., signal peptide sequences, RNA export elements, and IRES). After the lentiviral vectors are produced, they can be used to transfect host cells as described above for retroviral vectors.

[0081] In some preferred embodiments, the vector is an adeno-associated virus (AAV) vector. AAV is a human DNA parvovirus belonging to the genus Dependovirus. The AAV genome is composed of a linear single-stranded DNA molecule containing about 4680 bases. The genome includes an inverted terminal repeat sequence (ITR) at each end, and the ITR cis-acts as a DNA replication origin and a viral packaging signal. The internal non-repetitive portion of the genome includes two large open reading frames, referred to as AAVrep and cap regions. These regions encode viral proteins involved in virion replication and packaging. A family of at least four viral proteins, Rep 78, Rep 68, Rep 52 and Rep 40 (named according to their apparent molecular weight) are synthesized from the AAV rep region. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3 (for a detailed description of the AAV genome, see, e.g., Muzyczka, Current Topics Microbiol. Immunol. 158:97-129

[1992] ; Kotin, Human Gene Therapy 5:793-801

[1994] ).

[0082] AAV needs to be co-infected with an unrelated helper virus, such as adenovirus, herpes virus or vaccinia, for productive infection. In the absence of such co-infection, AAV establishes a latent state by inserting its genome into the host cell chromosome. Subsequent infection with the helper virus rescues the integrated copy, which can then replicate to produce infectious viral offspring. Unlike unpseudotyped retroviruses, AAV has a wide host range and can replicate in cells of any species, as long as it is co-infected with a helper virus that also reproduces in the species. Therefore, for example, human AAV will replicate in canine cells co-infected with canine adenovirus. In addition, unlike retroviruses, AAV is not associated with any human or animal disease, does not seem to change the biological properties of the host cell after integration, and can be integrated into non-dividing cells. It has also been recently discovered that AAV can be site-specifically integrated into the host cell genome.

[0083] In view of the above properties, many recombinant AAV vectors for gene delivery have been developed (see, e.g., U.S. Pat. Nos. 5,173,414; 5,139,941; WO 92 / 01070 and WO 93 / 03769, all of which are incorporated herein by reference; Lebkowski et al., Molec. Cell. Biol. 8:3988-3996

[1988] ; Carter, Current Opinion in Biotechnology 3:533-539

[1992] ; Muzyczka, Current Topics in Microbiol. and Immunol. 158:97-129

[1992] ; Kotin, (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] ).

[0084] Recombinant AAV virions can be produced in suitable host cells transfected by AAV helper plasmids and AAV vectors. AAV helper plasmids generally include AAV rep and cap coding regions, but lack AAV ITR. Therefore, helper plasmids can neither replicate nor package themselves. AAV vectors generally include selected genes of interest defined by AAV ITRs that provide viral replication and packaging functions. Auxiliary plasmids and AAV vectors with selected genes are introduced into suitable host cells by transient transfection. Then, the transfected cells are infected with helper viruses (e.g., adenoviruses), which can transactivate the AAV promoter present in the transcription and translation of the guidance AAV rep and cap regions on the helper plasmids. Recombinant AAV virions with selected genes are formed, and the recombinant AAV virions can be purified from the preparation. Once AAV vectors are produced, they can be used to transfect (see, e.g., U.S. Patent No. 5,843,742, incorporated herein by reference) host cells with desired infection multiplicity to produce host cells with high copy numbers. As will be appreciated by those skilled in the art, AAV vectors may also be modified as described above to contain various regulatory sequences (eg, signal peptide sequences, RNA export elements, and IRES).

[0085] In some preferred embodiments, the vector is a transposon-based vector. A transposon is a removable genetic element that can be moved or transposed from one position in the genome to another. The transposition within the genome is controlled by a transposase encoded by the transposon. Many examples of transposons are known in the art, including but not limited to Tn5 (see, e.g., de laCruz et al., J.Bact.175:6932-38

[1993] , Tn7 (see, e.g., Craig, Curr.TopicsMicrobiol.Immunol.Immunol.204:27-48

[1996] ; and Tn10 (see, e.g., Morisato and Kleckner, Cell 51:101-111

[1987] ). The ability of transposons to integrate into the genome has been used to create transposon vectors (see, e.g., U.S. Pat. Nos. 5,719,055; 5,968,785; 5,958,775; and 6,027,722; all of which are incorporated herein by reference.) Because transposons are not infectious, transposon vectors are introduced into host cells via methods known in the art (e.g., electroporation, lipofection, or microinjection). Thus, the ratio of transposon vectors to host cells can be adjusted to provide a desired multiplicity of infection to produce high copy number host cells of the present invention.

[0086] Transposon vectors suitable for the present invention generally comprise a nucleic acid encoding a protein of interest inserted between two transposon insertion sequences. Some vectors also comprise a nucleic acid sequence encoding a transposase. In these vectors, one of the insertion sequences is between the transposase and the nucleic acid encoding the protein of interest, thereby not being incorporated into the genome of the host cell during the recombination process. Alternatively, a transposase can be provided by a suitable method (e.g., liposome transfection or microinjection). As will be appreciated by those skilled in the art, transposon vectors can also be modified as described above to contain various regulatory sequences (e.g., signal peptide sequences, RNA export elements, and IRES).

[0087] In some preferred embodiments, the vector includes a selection marker. Suitable selection markers include, but are not limited to, glutamine synthetase (GS), dihydrofolate reductase (DHFR), etc. These genes are described in U.S. Patent Nos. 5,770,359, 5,827,739, 4,399,216, 4,634,665, 5,149,636, and 6,455,275; all of these patents are incorporated herein by reference. In some particularly preferred embodiments, the selection marker is GS, and has a sequence sharing at least 80%, 90%, 95%, 99% or 100% identity with the sequence of position 1789 to 2910 of SEQ ID NO:2. In some preferred embodiments, the selection marker utilized is compatible with a host cell line lacking the production of an enzyme encoded by a selection 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 provides resistance to antibiotics for cells expressing the protein. Suitable antibiotic resistance markers include genes that provide resistance to neomycin (neomycin resistance gene (neo)), hygromycin (hygromycin B phosphotransferase gene), puromycin (puromycin N-acetyltransferase), etc.

[0088] In some embodiments, the nucleotide sequence encoding the selected marker is operably connected to a promoter sequence. In some particularly preferred embodiments, the promoter sequence is for example changed by a sudden change so that the promoter activity is reduced compared to the unmodified form of the promoter. These promoters can be described as weak promoters, because the expression of the gene connected to the promoter occurs at a low level (relative to a high level). Compared with the gene regulated by a weak promoter, the gene regulated by a strong promoter produces more mRNA, thereby produces more product proteins. Therefore, the present invention preferably uses the promoter that has been changed to produce a selected marker with less mRNA than comparable unmodified promoters.

[0089] In some preferred embodiments, the promoter operably connected to the selection marker is a long terminal repeat (LTR) from a retrovirus or a lentivirus. In some particularly preferred embodiments, the promoter is an LTR that has been changed by removing all or part of the U3 region of the LTR. In some embodiments, the promoter operably connected to the selection marker is a self-inactivating (SIN) LTR. Suitable SIN LTRs are known in the art. In some preferred embodiments, the SIN LTR of the present invention preferably has at least 80%, 90%, 95%, 99% or 100% identity with SEQ ID NO: 3, and compared with the promoter activity when there is no deletion in the U3 region, the SIN LTR most preferably has a deletion therein that reduces the promoter activity in the U3 region.

[0090] It should be understood that in the case where the vector is a retroviral vector, the vector is constructed so that the SIN LTR is the 3' LTR of the retroviral vector sequence. Since the U3 deletion is copied to the 5' and 3' LTRs during the reverse transcription process, the integrated SIN vector only contains the LTR with the U3 deletion. Figure 2 As can be seen, the vector contains a SIN 3'LTR. When reverse transcribed, the hCMV-MOMuSV LTR depicted in the vector map is replaced by the SIN 3'LTR depicted in the vector map, so that when the vector is integrated into the host cell chromosome, the SIN LTR is operably linked to the depicted GScDNA and drives its expression.

[0091] In certain preferred embodiments of the present invention, the nucleic acid sequence encoding the protein of interest in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct mRNA synthesis. Promoters suitable for the present invention include, but are not limited to, LTR or SV40 promoter, Escherichia coli lac or trp, phage λP L and P R , T3 and T7 promoters as well as cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase and mouse metallothionein-I promoters and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses.

[0092] As mentioned above, exemplary diagrams of vectors of the present invention are provided in Figure 3 and corresponds to SEQ ID NO: 2. In some preferred embodiments, the vector comprises the following elements in 5' to 3' order:

[0093] -5'LTR (taking hCMV-MoMuSV LTR as an example)

[0094] -Retroviral packaging region

[0095] - Nucleic acid encoding a selectable marker (exemplified by GS cDNA)

[0096] -Internal promoter (using the simian CMV (sCMV) promoter as an example)

[0097] - A nucleic acid sequence encoding a protein of interest operably linked to an internal promoter (using the "anyway" sequence as an example)

[0098] -WPRE sequence

[0099] -SIN 3'LTR.

[0100] In some embodiments, the vector is a plasmid comprising the following elements in 5' to 3' order: 1) a 5' LTR (e.g., a SIN LTR); 2) a packaging region; 3) a selectable marker (e.g., GS); 4) an internal promoter (e.g., a CMV promoter); and 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter.

[0101] In some embodiments, the vector comprises a single poly A signal sequence downstream of the nucleic acid encoding the protein of interest. For example, in some embodiments, the vector comprises the following components in 5' to 3' order: promoter (e.g., SIN)-selection marker-stop codon-promoter (e.g., CMV)-protein of interest-stop codon-polyA.

[0102] In some embodiments, the invention provides host cells and host cell cultures, wherein the host cells express the protein of interest from the above-mentioned vectors. In preferred embodiments, the host cells are mammalian host cells. Many mammalian host cell lines are known in the art. Generally, when these host cells are placed in a culture medium containing appropriate nutrients and growth factors for monolayer culture or suspension culture, they can grow and survive, as described in more detail below. Generally, cells can express a large amount of specific protein of interest and secrete it 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 CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture; see, e.g., Graham et al., J. GenVirol. 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 CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68

[1982] ); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovine kidney cells); CAP (CEVEC's Amniocyte Production) cells; and human liver tumor cell line (Hep G2).

[0103] In some particularly preferred embodiments, the host cells are modified so that they lack or are naturally deficient in an enzyme activity that is necessary for cell growth or survival in the presence of a selection agent and that is provided by a selection marker. For example, Chinese hamster ovary (CHO) cells have been modified to lack GS. In some preferred embodiments where the vector includes a GS selection marker, the host cell line lacks GS. In some particularly preferred embodiments, the host cell line lacking GS is available from Merck KGaA GS - / - Cell lines. In other embodiments, where the selectable marker is, for example, DHFR, the cell line may preferably lack DHFR activity (i.e., DHFR - ). Suitable DHFR- cell lines include, but are not limited to, CHO-DG44 and their derivatives.

[0104] The vector can be introduced into the host cell by any suitable means. In some preferred embodiments, a vector (such as a retroviral vector) encoding a protein of interest has been produced, which can be used for transfection or transduction of host cells. Preferably, the host cell is transfected or transduced with an integration vector with a multiplicity of infection sufficient to cause at least 1, preferably at least 2 or more integration of retroviral vectors. In some embodiments, a multiplicity of infection of 10 to 1,000,000 can be utilized so that the genome of the infected host cell contains 2 to 1000 copies of the integration vector, preferably 5 to 1000 copies of the integration vector, and most preferably 20 to 500 copies of the integration vector. In other embodiments, a multiplicity of infection of 10 to 10,000 is utilized. When utilizing unpseudotyped retroviral vectors to infect, the host cell is incubated together with the culture medium from the retroviral production cell, and the culture medium contains the infectious vector of the required titer (i.e., colony forming unit, CFU). When pseudotyped retroviral vectors are utilized, the vector is concentrated to an appropriate titer by ultracentrifugation and then added to the host cell culture. Alternatively, the concentrated vector may be diluted in a culture medium appropriate for the cell type. Additionally, when it is desired that the host cell express more than one protein of interest, the host cell may be transfected with multiple vectors each containing nucleic acids encoding a different protein of interest.

[0105] In each case, the host cells are exposed to the medium containing the infectious retroviral vector for a sufficient period of time to allow infection and subsequent integration of the vector. In general, the amount of medium used to cover the cells should be kept as small as possible to promote the maximum number of integration events per cell. As a general guideline, the number of colony forming units (cfu) per milliliter should be approximately 10 5 Up to 10 7 cfu / ml, depending on the number of integration events desired.

[0106] In some embodiments, after transfection or transduction, the cells are propagated, then trypsinized and re-plated. Single colonies are then selected to provide cloned cell lines. In further embodiments, cloned cell lines are screened by Southern blot or PCR assay to verify that the desired number of integration events have occurred. It is also contemplated that clone selection allows identification of cell lines that produce high-quality protein. In other embodiments, cells are not cloned after transfection.

[0107] In some embodiments, host cells are transfected with vectors encoding different proteins of interest. Vectors encoding different proteins of interest can be used to transfect cells simultaneously (e.g., exposing host cells to solutions containing vectors encoding different proteins of interest), or transfection can be continuous (e.g., first transfecting host cells with vectors encoding the first protein of interest, and then transfecting host cells with vectors encoding the second protein of interest after a period of time). In some preferred embodiments, host cells are transfected with an integration vector encoding the first protein of interest, a high-expressing cell line containing multiple integration copies of the integration vector is selected (e.g., clone selection is performed), and the selected cell line is transfected with an integration vector encoding the second protein of interest. This process can be repeated to introduce a variety of proteins of interest. In some embodiments, the multiplicity of infection can be manipulated (e.g., increased or decreased) to increase or decrease the expression of the protein of interest. Similarly, different promoters can be used to change the expression of the protein of interest. It is contemplated that these transfection methods can be used to construct host cell lines containing complete exogenous metabolic pathways, or to provide enhanced protein processing capabilities for host cells (e.g., enzymes required for post-translational modification can be provided for host cells).

[0108] In further embodiments, cell lines are continuously transfected with vectors encoding the same gene. In some preferred embodiments, host cells are transfected with an integration vector encoding a protein of interest (e.g., an MOI of about 10 to 1,000,000, preferably 100 to 10,000), a cell line containing a single or multiple integration copies of the integration vector or expressing a high level of the desired protein is selected (e.g., clone selection is performed), and the selected cell line is retransfected with the vector (e.g., an MOI of about 10 to 1,000,0000; preferably 100 to 10,000). In some embodiments, cell lines containing at least two integration copies of the vector are identified and selected. This process can be repeated multiple times until the desired level of protein expression is obtained, and can also be repeated to introduce vectors encoding multiple proteins of interest. Unexpectedly, continuous transfection with the same gene causes an increase in protein production in the cells produced, not just an accumulation.

[0109] The present invention contemplates a variety of continuous transfection procedures. In some embodiments, in the case of utilizing retroviral vectors, a continuous transduction procedure is provided. In a preferred embodiment, a cell collection is subjected to continuous transduction. In these embodiments, the initial host cell collection is contacted with a retroviral vector, preferably with a multiplicity of infection in the range of about 0.5 to about 1000 vectors / host cells. The cells are then cultured for several days in an appropriate culture medium (e.g., with a selection agent). An aliquot of the cells is then taken to determine the number of integrated vectors and frozen for future use. The remaining cells are then contacted with the retroviral vector again, preferably with a multiplicity of infection in the range of about 0.5 to about 1000 vectors / host cells. This process is repeated until cells with the desired number of integrated vectors are obtained. For example, the process can be repeated up to 10 to 20 times or more. In some embodiments, if desired, cells can be cloned and selected after any specific transduction step, but the time to obtain the desired integrated vector copy number can be reduced by utilizing a cell collection in the absence of transduction.

[0110] In some embodiments of the invention, the protein of interest is secreted during host cell culture following transformation of a suitable host strain and growth of the host strain to an appropriate cell density in culture medium. In some preferred embodiments utilizing amplifiable markers, it is contemplated that the transduced host cells are cultured in a culture medium containing a gene inhibitor. Suitable inhibitors include, but are not limited to, methotrexate for inhibiting DHFR and methionine sulfoximine (Msx) or phosphinothricin for inhibiting GS. It is contemplated that as the concentration of these inhibitors in the cell culture system increases, amplifiable markers with higher copy numbers (and therefore one or more genes of interest) or cells containing genes that produce higher insertions are selected.

[0111] Therefore, host cells containing the vectors described above are cultured according to methods known in the art. Suitable culture conditions for mammalian cells are well known in the art (see, e.g., J. Immunol. Methods (1983) 56: 221-234

[1983] , Animal Cell Culture: A Practical Approach 2nd Edition, Rickwood, D. and Hames, BD eds., Oxford University Press, New York

[1992] ).

[0112] Host cell cultures of the present invention are prepared in a culture medium suitable for the specific cells cultivated. Commercially available culture medium such as ActiPro culture medium (HyClone), ExCell Advanced fed-batch culture medium (SAFC), Ham's F10 (Sigma, St.Louis, MO), minimum essential medium (MEM, Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium) (DMEM, Sigma) are exemplary nutrient solutions. Suitable culture medium is also described in U.S. Patent No. 4,767,704, No. 4,657,866, No. 4,927,762, No. 5,122,469, No. 4,560,655 and WO 90 / 03430 and WO 87 / 00195; Their disclosures are incorporated herein by reference. Any of these media may be supplemented as necessary with serum, hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), lipids (e.g., linoleic acid or other fatty acids) and suitable carriers thereof, and glucose or an equivalent energy source. In some preferred embodiments, where a selection marker such as GS is utilized, for example, the media will lack glutamine. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.

[0113] The present invention also contemplates the use of a variety of culture systems (e.g., culture 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 by maintaining a high cell density. The cells are suspended and can continue to grow without the need for a solid support. Typically, fresh nutrients must be provided continuously while removing toxic metabolites and, ideally, selectively removing dead cells. Filtration, interception, and microencapsulation methods are all suitable for renewing the culture environment at a sufficient rate.

[0114] As another example, in some embodiments, a fed-batch culture procedure can be adopted. In a preferred fed-batch culture, first a mammalian host, cells and culture medium are provided to a culture vessel, and other culture nutrients are supplied to the culture continuously or with discontinuous increments during the culture process, and cells and / or products are regularly harvested or not harvested before terminating the culture. Fed-batch culture can include, for example, semi-continuous fed-batch culture, in which the whole culture (including cells and culture medium) is regularly removed and replaced with fresh culture medium. Fed-batch culture is different from simple batch culture, in which all components (including cells and all culture nutrients) used for cell culture are supplied to the culture vessel at the beginning of the culture process. Fed-batch culture can also be distinguished from perfusion culture, in that the difference is that supernatant is not removed from the culture vessel in the process (in perfusion culture, cells are restricted in the culture by, for example, filtering, encapsulation, anchoring to microcarriers, etc., and culture medium is continuously or intermittently introduced into the culture vessel and removed from the culture vessel). In some particularly preferred embodiments, batch cultures are performed in roller bottles.

[0115] In addition, the cells of the culture can be propagated according to any scheme or routine that may be suitable for the specific host cell and the specific production plan under consideration. Therefore, the present invention contemplates single-step or multi-step culture procedures. In single-step culture, the host cell is inoculated into a culture environment, and the method of the present invention is adopted in a single production stage of the cell culture. Alternatively, it is envisioned that multi-stage culture. In multi-stage culture, cells can be cultured in multiple steps or stages. For example, cells can be grown in the first step or growth stage culture, wherein the cells that may be removed from the storage reservoir are inoculated into a culture medium suitable for promoting growth and high viability. By adding fresh culture medium to the host cell culture, cells can be maintained in the growth stage for a suitable period of time.

[0116] Design fed-batch or continuous cell culture conditions to enhance the growth of mammalian cells in the growth phase of cell culture. In the growth phase, cells are grown under conditions and time that maximize growth. For example, temperature, pH value, dissolved oxygen (dO2) and other culture conditions are culture conditions for specific hosts, and it will be apparent to the ordinary skilled technician. Usually, acid (for example, CO2) or alkali (for example, Na2CO3 or NaOH) is used to adjust the pH value to a level between about 6.5 and 7.5. The suitable temperature range for cultivating mammalian cells such as CHO cells is between about 30 ℃ and 38 ℃, and suitable dO2 is 5-90% of air saturation.

[0117] After the polypeptide production stage, the polypeptide of interest is recovered from the culture medium using techniques confirmed in the art. Although the protein of interest can also be recovered from the host cell lysate, it is preferred to recover the protein of interest from the culture medium in the form of secreted polypeptides (for example, the secretion of the protein of interest is directed by a signal peptide sequence). In the first step, the culture medium or lysate is centrifuged to remove granular cell debris. Thereafter, the polypeptide is purified from contaminant soluble proteins and polypeptides, and the following procedures are examples of suitable purification procedures: by fractionation on an immunoaffinity or ion exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or on a cation exchange resin (such as DEAE); chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and protein A agarose gel columns to remove contaminants such as IgG. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) can also be used to inhibit proteolytic degradation during purification. In addition, the protein of interest can be fused in frame with a marker sequence that allows purification of the protein of interest. Non-limiting examples of marker sequences include hexa-histidine tags, which can be provided by a vector, preferably a pQE-9 vector; and hemagglutinin (HA) tags. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (see, e.g., Wilson et al., Cell, 37:767

[1984] ). Those skilled in the art will appreciate that purification methods suitable for the polypeptide of interest may need to be modified to take into account changes in polypeptide properties after expression in recombinant cell culture.

[0118] experiment

[0119] The present invention provides for retroviral transduction (referred to herein as The GS knockout cell line used in these studies is the CHOZN cell line available from MilliporeSigma. The technique was practiced in a similar manner as previously performed in normal CHO cells. However, in the viral expression vector, the GS gene selected for the GS knockout cell line was driven out by a very weak promoter from the SIN (self-inactivating) LTR present in one of the versions of the GPEx expression vector used by Catalent. The combination of the vector, the normal GPEx cell line production method, and the GS knockout CHO cell line produced 7 times higher levels of production than the traditional GPEx method in an unmodified CHO cell line.

[0120] Experiment 1:

[0121] Two pooled cell lines were generated from two different viral vectors. Both cell lines were generated using a conventional GPEx transduction method and the GS knockout cell line CHOZN. Both expression vectors were designed to express the test protein "Anyway". Anyway is an Fc fusion protein. The only difference between the two expression vectors is that one vector, after insertion into the cell line, will have the full-length Moloney murine leukemia virus (MMLV) LTR driving GS gene expression, while the other vector will have the MMLV SIN LTR driving GS expression. Figure 1 and Figure 2 Gene constructs for producing retroviral vector particles are provided in . Figure 3 and 4 The sequences of the gene constructs are provided in .

[0122] CHOZN Cell Line Development

[0123] Production of retroviral vectors: The above expression constructs were introduced into a HEK 293 cell line that constitutively produces MLV gag, pro, and pol proteins. An expression plasmid containing the envelope was also co-transfected with each gene construct. Co-transfection resulted in the production of high titer retroviral vectors that were replication incompetent, which were concentrated by ultracentrifugation and used for cell transduction (1, 2). Retroviral vectors generated from the above constructs were used to transduce CHO cells, resulting in the 3'LTR sequence being replicated to the 5' end of the sequence, replacing the hCMV-MoMuSV 5'LTR, and subsequently controlling the expression of the GS gene in the combined cell lines.

[0124] Transduction of CHOZN cells with retroviral vectors: Pooled cell lines containing retroviral vector inserts from each of the constructs described above were prepared by transducing the CHOZN Chinese Hamster Ovary parental cell line with retroviral vectors made from gene constructs developed to express the Anyway protein. A single transduction cycle was performed to generate a pooled cell line for each of the two constructs. After cell transduction was complete, the cell lines were placed in glutamine-free medium for GS selection.

[0125] Anyway Fed-batch production from two pooled cell populations: After transduction, the pooled cell lines were scaled up in duplicate 250 mL shake flasks in fed-batch studies to increase yield. Each shake flask was inoculated 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 (70-80%) shaking incubator at 110 rpm with 5% CO2 and a temperature of 37°C. Starting from day 3 of production, the cultures were fed with a feed supplement every other day. Glucose was monitored daily and supplemented if the level was below 4 g / L. The culture was terminated when viability was ≤50%.

[0126] result

[0127] The results are shown in Table 1 and Figure 5 These results are surprising. There is a large difference in titer between the two pooled cell lines, given similar average gene copy numbers in both collections. Thus, even with a slightly higher average gene copy number, the wild-type or full-length LTR gene constructs gave significantly lower titers, which translated into significantly lower cell-specific productivity per gene insert. A selection or competitive advantage emerged for high-expressing cells containing the SIN version of the gene construct.

[0128] Table 1. Comparison of pooled cell lines between two gene constructs.

[0129]

[0130] Experiment 2:

[0131] Based on previous results, an experiment was designed to compare the traditional method of practicing the GPEx technique with the method of practicing the technique exactly the same but using the above-mentioned SIN-GS vector in combination with the CHOZN GS knockout cell line. The methods were kept as similar as possible with only two major differences. The first was that traditional GPEx used The parental cell line of the Chinese Hamster Ovary (GCHO) was used, while the new format used the CHOZN cell line. The second difference is that the gene construct used to prepare the retroviral vector is different. For the traditional GPEx, the construct does not contain the GS gene, while for the new format, it contains the GS gene. All other components of the gene construct are similar. Each construct again expresses the Anyway protein. After completing the merged cell lines for each method, their yields were compared in a fed-batch culture analysis.

[0132] GCHO and CHOZN cell line development:

[0133] Production of retroviral vectors: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro, and pol proteins. An expression plasmid containing the envelope was also co-transfected with each gene construct. Co-transfection resulted in the production of high-titer retroviral vectors that were replication-incompetent, concentrated by ultracentrifugation and used for cell transduction (1,2).

[0134] Transduction of CHOZN cells with retroviral vectors: Pooled cell lines containing retroviral vector insertions from each construct were prepared by transducing the CHOZN Chinese Hamster Ovary parental cell line or the GCHO Chinese Hamster Ovary parental cell line with retroviral vectors made from gene constructs containing GS or gene constructs without GS developed for expressing Anyway proteins. Three transduction cycles were performed to generate pooled cell lines for each of the two constructs in the respective cell lines. Additional cycles typically increase the number of inserts in the cell line, thereby increasing the gene copy number. After each transduction cycle was completed, the CHOZN-based cell collection was placed in glutamine-free medium for GS selection. Traditional GPEx cells were not selected as in the normal procedure.

[0135] result

[0136] As expected, both methods increased copy number with repeated transduction cycles. However, significantly higher gene copy numbers were observed in each new GPEx cell pool compared to conventional GPEx, as shown in Table 2.

[0137] Table 2. Gene copy numbers of pooled cell lines compared between two different methods

[0138]

[0139] Anyway Fed-batch production from two pooled cell populations (3 transduction cycles): After transduction, the pooled cell lines were scaled up in duplicate 250 mL shake flasks in fed-batch studies to increase yield. Each shake flask was inoculated with 300,000 viable cells per mL in a 50 mL working volume of ActiPro medium (HyClone) and incubated in a humidified (70-80%) shaking incubator at 120 rpm with 5% CO2 and a temperature of 37°C (34°C starting on day 6). During production, the cultures were fed six times with two different feed supplements. Glucose was monitored daily and supplemented if the level was below 4 g / L. The culture was terminated when viability was ≤50%.

[0140] The results are provided in Table 3 and Figure 6Again, very unexpected results were observed. The optimized traditional GPEx clonal cell line produced a maximum expression of 1.8 g / L of Anyway product and the new GPEx collection exceeded twice that expression before any clonal selection. The new GPEx collection had a higher copy number and a higher yield per gene insert, as seen in experiment #1. These two factors together caused a significant difference in cell-specific productivity, with the new GPEx collection approaching 3 times higher pg / cell / day. The overall viable cell density of the new GPEx collection was also higher compared to the traditional collection, which also contributed to the significant titer differences observed.

[0141] Table 3. Comparison of cell lines between two different methods regarding the pooling of Anyway

[0142]

[0143] Experiments 3 and 4:

[0144] The above data are for Fc fusion proteins produced from a single gene. Experiments 3 and 4 utilized Technology and a novel approach using the SIN LTR to drive GS expression, transduction was performed using two different antibodies, “Peelaway” and “Yourway”, via separate heavy and light chain vector transductions (2 light chains and 3 heavy chains for both experiments).

[0145] Retroviral vector production: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro, and pol proteins. An expression plasmid containing the envelope was also co-transfected with each gene construct. Co-transfection resulted in the production of high-titer retroviral vectors that were replication-incompetent, which were concentrated by ultracentrifugation and used for cell transduction (1,2).

[0146] Transduction of CHOZN cells with retroviral vectors: Pooled cell lines containing retroviral vector insertions from each construct were prepared by transducing the CHOZN Chinese Hamster Ovary parental cell line or the GCHO Chinese Hamster Ovary parental cell line with retroviral vectors made from heavy and light chain gene constructs containing GS or gene constructs without GS developed to express two different antibodies. Two light chain transduction cycles and three heavy chain transduction cycles were performed to generate pooled cell lines for each of the four constructs in each corresponding antibody cell line. Additional cycles typically increase the number of inserts in the cell line, thereby increasing the gene copy number. After each transduction cycle is completed, the CHOZN-based cell pool is placed in glutamine-free medium for GS selection. Traditional GPEx cells are not selected as in normal procedures.

[0147] Peelaway and Yourway were produced from fed-batch of a pooled cell line population (2 light chain transduction cycles and 3 heavy chain transduction cycles): After transduction, the pooled cell lines were scaled up in duplicate 250 mL shake flasks in a fed-batch study to increase yield. Each shake flask was inoculated with 300,000 viable cells per mL in a 50 mL working volume of ActiPro medium (HyClone) and incubated in a humidified (70-80%) shaking incubator at 120 rpm with 5% CO2 and a temperature of 37°C (34°C starting on day 6). During production, the culture was fed six times using two different feed supplements. Glucose was monitored daily and supplemented if the level was below 4 g / L. The culture was terminated when viability was ≤50%.

[0148] result

[0149] The results are presented in Tables 4 and 5. A significant advantage in titer was observed with the new vector and method.

[0150] Table 4. Comparison of cell lines between two different methods regarding the incorporation of Peelaway antibodies

[0151]

[0152] Table 5. Comparison of cell lines between two different methods regarding the pooling of Yourway antibodies

[0153]

[0154] Additional copy number analysis was performed on four Yourway cell pools. A cell pool generated after 1 light chain transduction cycle and 2 heavy chain transduction cycles was compared to a cell pool generated after 2 light chain transduction cycles and 3 heavy chain transduction cycles. Each of these two pooled cell lines was either set up to continue growing in medium containing glutamine or placed in medium lacking glutamine. For each of the four pooled cell lines, gene copy number was calculated for the total number of genes, the number of heavy chain genes, and the number of light chain genes.

[0155] Table 6.

[0156]

[0157] These data suggest that even though each cell in these collections has a large number of copies of the GS gene driven out by the SIN LTR, there is still clear selection for those with greater numbers of copies and, according to the data above, those copies are expressed at higher levels.

[0158] Experiment 5

[0159] For this experiment, Yourway antibody products were used. The heavy chain gene construct used to generate the cell line was the same as that used in Experiment 4. The light chain gene construct was identical to the heavy chain gene construct in all respects, except that it contained the light chain coding sequence and lacked the GS gene. A single transduction was performed for each light chain (-GS) and heavy chain (+GS) containing the reverse transcription vector to generate a pooled cell line.

[0160] Retroviral vector production: Expression constructs were introduced into the HEK 293 cell line, which constitutively produces MLV gag, pro, and pol proteins. An expression plasmid containing the envelope was also co-transfected with each gene construct. Co-transfection resulted in the production of high-titer retroviral vectors that were replication-incompetent, which were concentrated by ultracentrifugation and used for cell transduction (1,2).

[0161] Transduction of CHOZN cells with retroviral vectors: Pooled cell lines containing retroviral vector inserts from each of the constructs described above were prepared by transducing the CHOZN Chinese Hamster Ovary parental cell line with retroviral vectors made from a light chain gene construct without GS and a heavy chain construct containing GS. One light chain transduction cycle and one heavy chain transduction cycle were performed to generate the pooled cell line. After both transduction cycles were completed, the CHOZN-based cell pool was split and half was placed in glutamine-free medium while the other half continued to be supplemented with glutamine during cell expansion and subsequent yield assessment.

[0162] Fed-batch production of Yourway from a pooled cell line population (1 light chain transduction and 1 heavy chain transduction): In fed-batch studies, the pooled cell lines were scaled up in 250 mL shake flasks to increase productivity. Shake flasks were inoculated with 300,000 viable cells per mL in a 50 mL working volume of ActiPro medium (HyClone) and incubated in a humidified (70-80%) shaking incubator at 120 rpm with 5% CO2 and a temperature of 37°C (34°C starting on day 6). During production, the cultures were fed six times with two different feed supplements. Glucose was monitored daily and supplemented if levels were below 4 g / L. Glutamine was monitored daily for cultures supplemented with glutamine and fed if levels were below 2 mM. Cultures were terminated when viability was ≤ 50%.

[0163] result

[0164] The results are presented in Table 7. Selection by removal of glutamine significantly increased the heavy chain gene copy number of the cell pool. Somewhat unexpectedly, the light chain copy number also increased, but not to the extent of the heavy chain. Total antibody production was also significantly higher than in unselected cultures, where only two transductions were performed, rather than the five transductions completed for the experiment, to reach the levels shown in Table 5.

[0165] Table 7. Comparison of pooled cell lines for Yourway antibodies with and without glutamine selection Pooled cell lines were generated from single light chain transduction (-GS) and single heavy chain transduction (+GS).

[0166]

[0167] Experiment 6

[0168] A gene construct was designed and generated to test whether the technique would operate similarly using traditional cell transfection methods compared to retroviral vector transduction. An example of a traditional plasmid transfection construct generated is shown in Figure 8 (plasmid map) and Figure 9 (actual plasmid sequence; SEQ ID NO:4).

[0169] 1.Bleck,GT2005An alternative method for the rapid generation ofstable,high-expressing mammalian cell lines(A Technical Review). BioprocessingJ. September / October pp. 1-7.

[0170] 2. Bleck, G. T., 2010. A Flexible Method for the Rapid Generation of Stable, High Expressing, Antibody Producing Mammalian Cell Lines, Chapter 4: Current Trends in Monoclonal Antibody Development and Manufacturing, Biotechnology: Pharmaceutical Aspects, edited by SJShire et al. 2010 American Association of Pharmaceutical Scientists, DOI 10.1007 / 978-0-387-76643-0_4.

[0171] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein 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 conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes for carrying out the invention are apparent to those skilled in the art and are intended to be included within the scope of the following claims. Sequence Listing <110> CATALENT PHARMA SOLUTIONS, LLC <120> Vectors for protein production <130> PPI21171043US <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 constructs <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 cgtcgccccgg 780 gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc 840 cttgggagg tctcctctga gtgattgact acccacgacg ggggtctttc atttggggc 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 ccagggtgag 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 2460. caataggac cctgtgaagg aatccgcatg ggagatcatc tctgggtggc ccgtttcatc ttgcatcgag tatgtgaaga ctttggggta atagcaacct ttgaccccaa gcccattcct gggaactgga atggtgcagg ctgccatacc aactttagca ccaaggccat gcgggaggag aatggtctga agcacatcga ggaggccatc gagaactaa gcaagcggca ccggtaccac attcgagcct acgatcccaa ggggggcctg gacaatgccc gtcgtctgac tgggttccac gaaacgtcca acatcaacga cttttctgct ggtgtcgcca atcgcagtgc cagcatccgc attccccgga ctgtcggcca ggttactttg aagaccgccg cccctctgcc aactgtgacc cctttgcagt gacagaagcc atcgtccgca catgccttct caatgagact ggcgacgagc ccttccaata caaaactaa agatccctat ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata 3060. 3120. gccactcccc cattgacgtc aatggtctct fathertggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc 3180 gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg 3240 ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc 3300 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 aaaggaaggga 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 accgagacct 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 ttcttctctct 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 tttctctcc ttgtataaat cctggttgct gtctctttat 5160 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca 5220 accccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 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 ccttcttcc cgcggcctgc tgccggctct gcggcctctt 5520 ccgcgtcttc gccttgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat 5580 cgataaaata aaagattttta tttagtctcc agaaaaaggg gggaatgaaa gacccacct 5640 gtaggtttgg caagctagct tagtaacgc cattttgcaa ggcatggaaa aatacataac 5700 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa 5760 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag 5820 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 5880 gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt 5940 ttccagggtg 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 construct <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 cgtcgccccgg 780 gtacccgtat tcccaataaa gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc 840 cttgggagg tctcctctga gtgattgact acccacgacg ggggtctttc atttggggc 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 ccagggtgag 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 aatggtctga agcacatcga ggaggccatc gagaactaa gcaagcggca ccggtaccac attcgagcct acgatcccaa ggggggcctg gacaatgccc gtcgtctgac tgggttccac gaaacgtcca acatcaacga cttttctgct ggtgtcgcca atcgcagtgc cagcatccgc attccccgga ctgtcggcca ggttactttg aagaccgccg cccctctgcc aactgtgacc cctttgcagt gacagaagcc atcgtccgca catgccttct caatgagact ggcgacgagc ccttccaata caaaactaa agatccctat ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata 3060. 3120. gccactcccc cattgacgtc aatggtctct fathertggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc 3180. gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc 3300 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 aaaggaaggga 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 accgagacct 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 ttcttctctct 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 tttctctccc ttgtataaat cctggttgct gtctctttat 5160 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca 5220 accccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 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 ccttcttcc cgcggcctgc tgccggctct gcggcctctt 5520 ccgcgtcttc gccttgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat 5580 cgataaaata aaagattttta tttagtctcc agaaaaaggg gggaatgaaa gacccacct 5640 gtaggtttgg caagctagct tagtaacgc 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 ataaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac 5940 tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg 6000 tctcgctggt ccttgggagg gtctcctctg agtgattgac tacccgtcag cggggtctt 6060 tcatt 6065 <210> 3 <211> 442 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <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 construct <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 cgacgggggt 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 ttgggggccg 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 atggggctc 3000 ctaataccgc ccatagccac tccccattg acgtcaatgg tcttatata tggtctttcc 3060 tattgacgtc atatgggcgg tcctattgac gtatatggcg cctccccat tgacgtcaat 3120 tacggtaaat ggcccgcctg gctcaatgcc cattgacgtc ataggacca cccaccattg 3180 acgtcaatgg gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca 3240 atgacggtaa atggcccact tggcagtaca tcaattacta ttatagtaa cttggcaagt 3300 acattactat tggagtacg ccaggtaca tggcagtac tcccattgac gtcaatggcg 3360 gtaaatggcc cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat 3420 gacgcaaatg ggcgttccat tgacgtaat gggcggtagg cgtgcctaat gggaggtcta 3480 tataagcaat gctcgtttag ggaaccgcca ttctgctgg ggacgtcgga ggagctcgaa 3540 agcttctaga caatgccgc 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 ccctgtctc cggggaaatga 4920 tgagatctcg agttcgacat cgataatcaa cctctggatt acaaaatttg tgaaagattg 4980 actggtattc ttaactatgt tgctccttt 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 ggaaggagac 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 tagtagttc gccagttaat agtttgcgca acgttgttgc 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 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatcttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataaggggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 8040 taaaaatagg cgtatcacga ggccctttcg tc 8072

Claims

1. A vector for expressing a protein of interest, the vector comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence and a nucleic acid sequence encoding the protein of interest operably linked to a second promoter sequence, the first promoter sequence having been altered to have reduced promoter activity compared to an unaltered or wild-type form of the first promoter sequence, wherein: The first promoter sequence is a viral self-inactivating (SIN) long terminal repeat (LTR) promoter sequence, and the SIN LTR sequence is SEQ ID NO:

3.

2. The carrier according to claim 1, wherein The selectable marker is glutamine synthetase (GS), or wherein the selectable marker is dihydrofolate reductase (DHFR).

3. The vector of claim 1, wherein the vector comprises a single poly A signal sequence operably associated with the selectable marker and the nucleic acid encoding the protein of interest.

4. The vector of claim 3, wherein the vector comprises a first poly A signal sequence operably associated with the selectable marker and a second poly A signal sequence operably associated with the nucleic acid encoding the protein of interest.

5. The vector of claim 1, wherein the protein of interest is selected from the group consisting of: Fc fusion proteins, enzymes, albumin fusions, growth factors, protein receptors, single chain antibodies (scFv), single chain Fc (scFv-Fc), diabodies and miniantibodies (scFv-CH3), Fab, single chain Fab (scFab), immunoglobulin heavy chains, and immunoglobulin light chains. The vector of claim 5 , wherein the protein of interest is an Fc fusion protein.

7. The vector according to claim 1, wherein the vector is a viral vector.

8. The vector according to claim 7, wherein the vector is a retroviral vector.

9. The vector according to claim 1, wherein the vector is a plasmid.

10. A host cell comprising the vector according to any one of claims 1 to 9. The host cell according to claim 10 , wherein the host cell line is a GS knockout cell line.

12. The host cell of claim 10, wherein the host cell line is a DHFR knockout cell line.

13. The host cell of claim 10, wherein the host cell line is selected from the group consisting of: Chinese Hamster Ovary (CHO) cell line, HEK 293 cell line, and CAP cell line.

14. The host cell of claim 10, wherein the host cell comprises 1 to 1000 copies of the vector.

15. The host cell of claim 10, wherein the host cell comprises 10 to 200 copies of the vector.

16. The host cell of claim 10, wherein the host cell comprises 10 to 100 copies of the vector.

17. The host cell of claim 10, wherein the host cell comprises 20 to 100 copies of the vector.

18. The host cell of claim 10, wherein the host cell further comprises at least a second vector encoding and allowing for expression of a second protein of interest, and wherein the second vector does not comprise a selectable marker.

19. The host cell of claim 10, wherein the host cell further comprises at least a second vector encoding and allowing expression of a second protein of interest, and wherein the second vector comprises a selection marker different from the selection marker in the first vector; The first carrier is the carrier according to any one of claims 1 to 9.

20. The host cell of claim 18 or 19, wherein the first protein of interest in the first vector is one of an immunoglobulin heavy chain or light chain, and the second protein in the second vector is the other of an immunoglobulin heavy chain or light chain; in, The first vector is the vector according to any one of claims 1 to 9, and the first protein of interest is the protein of interest expressed by the first vector.

21. The host cell of claim 20, wherein the first protein of interest is an immunoglobulin heavy chain and the second protein of interest is an immunoglobulin light chain.

22. The host cell of claim 18 or 19, wherein the host cell comprises 1 to 1000 copies of the second vector.

23. The host cell of claim 18 or 19, wherein the host cell comprises 10 to 200 copies of the second vector.

24. The host cell of claim 18 or 19, wherein the host cell comprises 10 to 100 copies of the second vector.

25. The host cell of claim 18 or 19, wherein the host cell comprises 20 to 100 copies of the second vector.

26. A host cell culture comprising the host cell according to any one of claims 10 to 25.

27. The host cell culture of claim 26, wherein the culture produces 1 to 150 pg / cell / day of the protein of interest.

28. The host cell culture of claim 26, wherein the culture produces 2 to 50 pg / cell / day of the protein of interest.

29. A method for producing a protein of interest, the method comprising culturing the host cell of any one of claims 10 to 25 and purifying the protein of interest from the host cell culture.

30. An infectious retroviral particle comprising the following elements in 5' to 3' order: 1) 5'LTR; in, The 5'LTR is a SIN LTR, and the sequence of the SIN LTR is SEQ ID NO: 3; 2) Retroviral packaging region; 3) a nucleic acid encoding a selectable marker; 4) internal promoter; 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter; 6) SIN 3'LTR.

31. The infectious retroviral particle according to claim 30, wherein The selectable marker is glutamine synthetase (GS), or wherein the selectable marker is dihydrofolate reductase (DHFR).

32. The infectious retroviral particle of claim 30, wherein the 3' LTR comprises a poly A signal sequence.

33. The infectious retroviral particle of claim 30, wherein the packaging region comprises a plurality of potential translation initiation sites.

34. The infectious retroviral particle of claim 30, wherein the internal promoter is a CMV promoter.

35. The infectious retroviral particle of claim 30, wherein the particle comprises a single poly A signal sequence downstream of the nucleic acid encoding the protein of interest.

36. A plasmid comprising the following elements in 5' to 3' order: 1) 5'LTR; in, The 5'LTR is a SIN LTR, and the sequence of the SIN LTR is SEQ ID NO: 3; 2) Packaging area; 3) Selection markers; 4) internal promoter; 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter; and 6) poly A signal sequence.

37. The plasmid according to claim 36, wherein The selectable marker is glutamine synthetase (GS), or wherein the selectable marker is dihydrofolate reductase (DHFR).

38. The plasmid of claim 36, wherein the packaging region comprises a plurality of potential translation start sites.

39. The plasmid of claim 36, wherein the internal promoter is a CMV promoter.

40. The plasmid of claim 36, wherein the plasmid comprises a single poly A signal sequence downstream of the nucleic acid encoding the protein of interest.

41. A system comprising: a) a first vector, wherein the first vector is the vector according to any one of claims 1 to 9 and expresses a first protein of interest; and b) a second vector comprising a nucleic acid sequence encoding a second protein of interest operably linked to a promoter sequence, and wherein the second vector does not include a selectable marker.

42. The system of claim 41, wherein the first protein of interest in the first vector is one of an immunoglobulin heavy chain or a light chain, and the second protein in the second vector is the other of an immunoglobulin heavy chain or a light chain.

43. A method for producing a protein of interest, the method comprising: transducing or transfecting one or more host cells with an infectious retroviral particle according to any one of claims 30 to 35, a plasmid according to any one of claims 36 to 40, or a system according to claim 41 or 42; developing a host cell line that expresses the protein of interest; culturing the host cell line under conditions such that the host cell line produces the protein of interest; and The protein of interest is purified from the host cell culture.

44. Use of an infectious retroviral particle according to any one of claims 30 to 35, a plasmid according to any one of claims 36 to 40, a system according to claim 41 or 42 for transducing one or more host cells to produce a protein of interest.

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