Vectors for protein manufacture

Abstract

To provide vectors, to provide their use to develop host cell lines for production of a protein of interest, and in particular to provide vectors which utilize a weak promoter to drive a selectable marker.SOLUTION: Provided is a vector for expression of a protein of interest, the vector comprising a nucleic acid sequence encoding a selectable marker in operable association with a first promoter sequence that has been altered to reduce promoter activity as compared to a non-altered or wild-type version of the first promoter sequence, and a nucleic acid sequence encoding the protein of interest operably linked to a second promoter sequence.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 775,194, filed December 4, 2018, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to vectors and their use for developing host cell lines for producing proteins of interest, and in particular to vectors which utilize a weak promoter to drive a selectable marker. [Background technology]

[0003] Therapeutic protein drugs are a prime class of drug recipients, with patients most in need of novel treatments. Recently approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancer, autoimmunity / inflammation, pathogen exposure, and genetic disorders. Recent advances in protein modification technology are enabling drug developers and manufacturers to fine-tune and harness the desired functional properties of a protein of interest while maintaining (and in some cases improving) the safety or efficacy, or both, of their products.

[0004] The manufacturing and production of therapeutic proteins is a highly complex process: for example, a typical protein drug may involve over 5,000 clinical process steps, which often exceeds the number required to manufacture a small molecule drug.

[0005] Similarly, protein therapeutics, including monoclonal antibodies and large or fusion proteins, can be several orders of magnitude larger in size than small molecule drugs, with molecular weights exceeding 100 kDa. Furthermore, protein therapeutics exhibit complex secondary and tertiary structures that must be maintained. Protein therapeutics cannot be fully synthesized by chemical processes and must be produced in living cells or organisms. Thus, the choice of cell line, species of origin, and culture conditions all affect the characteristics of the final product. Furthermore, most biologically active proteins require post-translational modifications that can be compromised when heterologous expression systems are used. Furthermore, because the product is synthesized by cells or organisms, a complex purification process is involved. Furthermore, virus clearance processes, such as removal of viral particles by using filters or resins, and inactivation steps by using low pH or detergents, are implemented to prevent viral contamination, which is a serious safety issue for protein drug substances. Considering the large molecular size, post-translational modifications, and complexity of therapeutic proteins relative to the various biological substances involved in the manufacturing process, the ability to improve certain functional attributes while maintaining the safety and efficacy of the product achieved by protein modification is highly desirable.

[0006] Although incorporating new methods and approaches to modify protein drug products is not a trivial matter, potential therapeutic benefits have led to the increasing use of such methods in drug discovery.A number of protein modification platform technologies are currently being used to increase the circulating half-life, targeting, and functionality of new therapeutic protein drugs, as well as increasing manufacturing yield and product purity.For example, protein conjugation and derivatization approaches, including Fc fusion, albumin fusion, and PEGylation, are currently being used to extend the circulating half-life of drugs.

[0007] The production of protein pharmaceuticals (biologics) is expensive and time consuming, and there is a need in the art for more efficient tools and processes for producing this important class of drugs. [Summary of the invention]

[0008] The present invention relates to vectors and their use for developing host cell lines for producing proteins of interest, and in particular to vectors which utilize a weak promoter to drive a selectable marker.

[0009] Thus, in some preferred embodiments, the present invention provides vector(s) for expressing a protein of interest comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence that has been modified to have reduced promoter activity compared to the unmodified, i.e. wild-type, version of the first promoter sequence, and a nucleic acid sequence encoding a protein of interest operably linked to a second promoter sequence.

[0010] In some preferred embodiments, the first promoter sequence that has been modified to reduce promoter activity compared to the unmodified, i.e. wild-type, version 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 is at least 95% identical to SEQ ID NO:3. In some preferred embodiments, the SIN LTR promoter sequence is SEQ ID NO:3.

[0011] In some preferred embodiments, the selectable marker is glutamine synthetase (GS). In some preferred embodiments, the selectable marker is dihydrofolate reductase (DHFR).

[0012] In some embodiments, the vector comprises a single polyA signal sequence operably associated with the selectable marker and the nucleic acid encoding the protein of interest, hi other embodiments, the vector comprises a first polyA signal sequence operably associated with the selectable marker and a second polyA signal sequence operably associated with the nucleic acid encoding the protein of interest.

[0013] In some preferred embodiments, the protein of interest is selected from the group consisting of an Fc fusion protein, an enzyme, an albumin fusion, a growth factor, a protein receptor, a single chain antibody (scFv), a single chain Fc (scFv-Fc), a diabody, and a minibody (scFv-CH3), a Fab, a single chain Fab (scFab), an immunoglobulin heavy chain, and an immunoglobulin light chain. In some preferred embodiments, the protein of interest is an Fc fusion protein.

[0014] 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.

[0015] In some preferred embodiments, the present invention provides a host cell(s) comprising the vector described above. In some preferred embodiments, the host cell line is a GS knockout cell line. In some preferred embodiments, the host cell line is a DHFR knockout cell line. In some preferred embodiments, the host cell line is a Chinese Hamster Ovary (CHO) cell line. In some preferred embodiments, the host cell line is a HEK293 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.

[0016] In some preferred embodiments, the host cell further comprises at least a second vector encoding a second protein of interest and allowing expression of the second protein of interest, the second vector not comprising a selection marker. In some preferred embodiments, the host cell further comprises at least a second vector encoding a second protein of interest and allowing expression of the second protein of interest, the second vector comprising 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 the other 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 100-1000 copies of the second vector. In some preferred embodiments, the host cell comprises about 10-200 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.

[0017] In some preferred embodiments, the present invention provides a host cell culture comprising the host cell described above. In some preferred embodiments, the culture produces 1-50 grams / liter / day of the protein of interest. In some preferred embodiments, the culture produces 2-10 grams / liter / day of the protein of interest.

[0018] In some preferred embodiments, the present invention provides a process for the production of a protein of interest comprising culturing a host cell as described above and purifying the protein of interest from the host cell culture medium.

[0019] In some preferred embodiments, the invention provides infectious retroviral-type particles comprising the following elements in 5' to 3' order: 1) a 5' LTR, 2) a retroviral-type packaging region, 3) a nucleic acid encoding a selectable marker, 4) an internal promoter, 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter, and 6) a 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 polyA signal sequence. In some embodiments, the packaging region comprises multiple, potential translation initiation sites. In some preferred embodiments, the selectable marker is GS. In some preferred embodiments, the internal promoter is a CMV promoter. In some embodiments, the particle comprises a single polyA signal sequence downstream of the nucleic acid encoding the protein of interest. In some embodiments, the particle comprises a first polyA signal sequence operably associated with the selectable marker and a second polyA signal sequence operably associated with the nucleic acid encoding the gene of interest.

[0020] Further embodiments provide 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., a GS), 4) an internal promoter (e.g., a CMV promoter), 5) a nucleic acid sequence encoding a protein of interest operably linked to the internal promoter, and 6) a polyA signal sequence. In some embodiments, the plasmid comprises a single polyA signal sequence downstream of the nucleic acid encoding the protein of interest.

[0021] A further embodiment provides a system comprising: a) a first vector comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence that has been modified to have reduced promoter activity compared to an unmodified, i.e., wild-type, version of the first promoter sequence, and a nucleic acid sequence encoding a first protein of interest operably linked to the second promoter sequence; and b) a second vector comprising a nucleic acid sequence encoding a second protein of interest operably linked to the promoter sequence, wherein the second vector does not comprise a selectable marker.

[0022] In some preferred embodiments, the present invention provides a method for producing a protein of interest comprising transducing or transfecting a host cell(s) with an infectious retroviral-type particle, plasmid or vector system as described above and developing a host cell line expressing said protein of interest from said host cell(s), culturing said host cells under conditions such that said protein of interest is produced by said host cell line, and purifying said protein of interest from the host cell culture.

[0023] In some preferred embodiments, the invention provides infectious retroviral-type particles or plasmids as described above for use in transducing a host cell(s) for the production of a protein of interest. [Brief description of the drawings]

[0024] [Figure 1] Map of full-length MMLV constructs. [Figure 2] Map of the SIN MMLV LTR construct. [Figure 3] Full-length MMLV construct sequence (SEQ ID NO:1). [Figure 4] Sequence of the SIN MMLV LTR construct (SEQ ID NO:2). [Figure 5] Pooled cell line titer comparison of full-length and SIN MMLV LTR constructs. [Figure 6] Titer comparison of pooled cell lines between processes using full-length MMLV constructs and SIN MMLV LTR constructs. [Figure 7] SIN LTR sequence (SEQ ID NO:3). [Figure 8] Proviral plasmid construct map (SEQ ID NO:4). [Figure 9] Proviral plasmid construct sequence (SEQ ID NO:4). [Mode for carrying out the invention]

[0025] definition To facilitate the understanding of this invention, a number of terms are defined below.

[0026] As used herein, the term "host cell" refers to any eukaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

[0027] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, defined cell lines (e.g., non-transformed cells), and any population of cells maintained in vitro, including oocytes and embryos.

[0028] As used herein, the term "vector" refers to any genetic element, e.g., plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replicating and transferring genetic sequences between cells when associated with the proper regulatory elements. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0029] As used herein, the term "multiplicity of infection" or "MOI" refers to the ratio of integrating vectors:host cells used during transfection or transduction of host cells. For example, if 1,000,000 vectors are used to transduce 100,000 host cells, the multiplicity of infection is 10. Use of the term is not limited to events involving transduction, but instead encompasses the introduction of vectors into a host by methods such as lipofection, microinjection, calcium phosphate precipitation, and electroporation.

[0030] As used herein, the term "genome" refers to the genetic material (eg, chromosomes) of an organism.

[0031] The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA) whose manipulation may be deemed desirable by one of skill in the art for any reason (e.g., treating a disease, imparting 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 for structural genes (e.g., reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.) and non-coding regulatory sequences that do not code for an mRNA or protein product (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.).

[0032] As used herein, the term "protein of interest" means a protein encoded by a nucleic acid of interest.

[0033] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, the DNA or RNA sequence codes for an amino acid sequence.

[0034] As used herein, the term "promoter," "promoter element," or "promoter sequence" refers to a DNA sequence that, when ligated to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. Promoters are typically, but not necessarily, located 5' (i.e., upstream) of the nucleotide sequence of interest that they control transcription into mRNA and provide a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.

[0035] As used herein, the term "modified" when used in reference to a promoter refers to a promoter having a modified nucleic acid sequence compared to a reference wild-type sequence. For example, the sequence promoter can be modified by deleting certain promoter and / or enhancer elements.

[0036] Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237,

[1987] ). Promoter and enhancer elements have been isolated from a wide variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (analogous regulatory elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on which cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others function in a limited subset of cell types (for reviews, see Voss, et al., Trends Biochem. Sci., 11:287

[1986] ; and Maniatis, et al., supra). For example, the SV40 early gene enhancer is highly active in a wide variety of cell types from many mammalian species and is widely used for expression of proteins in mammalian cells (Dijkema et al., EMBO J. 4:761

[1985] ). Two other examples of promoter / enhancer elements active in a wide range of mammalian cell types are those derived 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] ), as well as the long terminal repeat of Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777

[1982] ) and the human cytomegalovirus (Boshart et al., Cell 41:521

[1985] ).

[0037] As used herein, the term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of conferring both promoter and enhancer functions (i.e., functions provided by promoter and enhancer elements; see above for a discussion of these functions). For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. An enhancer / promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in proximity to a gene by genetic manipulation (i.e., molecular biology techniques such as cloning and recombination) such that transcription of that gene is driven by the associated enhancer / promoter.

[0038] As used herein, the term "long terminal repeat" of "LTR" refers to a transcription control element located 5' and 3' of the U3 region of a retroviral genome, or isolated from it. As is known in the art, the long terminal repeat can be used as a regulatory element within a retroviral vector, or can be isolated from a retroviral genome to control expression from other types of vectors.

[0039] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) 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", where only some of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. Complementarity is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids.

[0040] The terms "homology" and "percent identity" when used in relation to nucleic acids refer to the degree of complementarity. There can be partial homology (i.e. partial identity) or complete homology (i.e. complete identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence, which is meant to use the functional term "substantially homologous". Inhibition of hybridization of a completely complementary sequence to a target sequence can be investigated using a hybridization assay (Southern or Northern blot, solution hybridization, etc.) under low stringency conditions. A substantially homologous sequence or probe (i.e. an oligonucleotide that can hybridize to another oligonucleotide of interest) competes with and inhibits the binding (i.e. hybridization) of a completely homologous sequence to a target sequence under low stringency conditions. This does not mean that low stringency conditions are such that they allow non-specific binding. Low stringency conditions require that the binding of two sequences to each other is a specific (i.e. selective) interaction. The absence of non-specific 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 non-specific binding, the probe does not hybridize to the second non-complementary target.

[0041] As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a way that a nucleic acid molecule is produced that is capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The terms also refer to the linking of amino acid sequences in such a way that a functional protein is produced.

[0042] As used herein, the term "selection marker" refers to a gene that encodes an enzymatic activity or other protein that confers the ability to grow in medium lacking what is otherwise an essential nutrient. In addition, a selection marker may confer resistance to an antibiotic or drug on the cell in which the selection marker is expressed.

[0043] As used herein, the term "retrovirus" refers to a retroviral particle that is capable of entering a cell (i.e., the particle contains a membrane-associated protein, such as an envelope protein or viral G glycoprotein, that can bind to the host cell surface and facilitate entry of the viral particle into the host cell cytoplasm) and can integrate the retroviral genome (as a double-stranded provirus) into the genome of the host cell. The term "retrovirus" encompasses Oncovirinae (e.g., Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), and mouse mammary tumor virus (MMTV)), Spumavirinae, and Lentivirinae (e.g., human immunodeficiency virus, simian immunodeficiency virus, 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).

[0044] As used herein, the term "retroviral vector" refers to a retrovirus that has been modified to express a gene of interest. Retroviral vectors can be used to efficiently transfer genes into host cells by taking advantage of the viral infection process. Foreign or heterologous genes cloned (i.e., inserted using molecular biology techniques) into the retroviral genome can be efficiently delivered to host cells susceptible to infection by retroviruses. Well-known genetic manipulations can destroy the replication capacity of the retroviral genome. The resulting replication-deficient vectors allow the introduction of new genetic material into cells, but they are unable to replicate. Helper viruses or packaging cell lines can be used to allow the assembly of vector particles and their escape from the cell. Such retroviral vectors include a replication-deficient retroviral genome that contains a nucleic acid sequence encoding at least one gene of interest (i.e., a polycistronic nucleic acid sequence can encode two or more genes of interest), a 5' retroviral long terminal repeat (5'LTR); and a 3' retroviral long terminal repeat (3'LTR).

[0045] 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 virus particle (e.g., viral envelope glycoprotein or G protein in the Rhabdoviridae family of viruses, such as VSV, Piry, Chandipura, and Mokola). These membrane-associated proteins mediate the entry of virus particles into host cells. Membrane-associated proteins can bind to specific cell surface protein receptors, as in the case of retroviral envelope proteins, or they can interact with the phospholipid components of the plasma membrane of host cells, as in the case of the G protein from members of the Rhabdoviridae family.

[0046] The term "heterologous membrane-associated protein" means a membrane-associated protein derived from a virus that is not a member of the same viral class or family as the nucleocapsid protein of the vector particle is derived from. "Viral class or family" means the taxonomic rank of the class or family assigned by the International Committee on Taxonomy of Viruses.

[0047] 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, both of which are incorporated herein by reference).

[0048] The term "pseudotyped lentiviral vector" refers to a lentiviral vector that contains a heterologous membrane protein (e.g., a viral envelope glycoprotein or G protein in viruses of the Rhabdoviridae family, such as VSV, Piry, Chandipura, and Mokola).

[0049] As used herein, the term "transposon" refers to a transposable element (e.g., Tn5, Tn7, and Tn10) that can move or transpose from one location to another in a genome. Generally, transposition is controlled by a transposase. As used herein, the term "transposon vector" refers to a vector that encodes a nucleic acid of interest flanked by the ends of a transposon. Examples of transposon vectors include, but are not limited to, those described in U.S. Patent 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.

[0050] 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. An AAV vector can be deleted in whole or in part of one or more AAV wild-type genes, preferably the rep and / or cap genes, but retain functional flanking ITR sequences.

[0051] AAV vectors can be constructed by using recombinant techniques known in the art to include one or more heterologous nucleotide sequences flanked at both ends (5' and 3') by functional AAV ITRs. In carrying out the present invention, AAV vectors can include at least one AAV ITR and a suitable promoter sequence located upstream of the heterologous nucleotide sequence, and at least one AAV ITR located downstream of the heterologous sequence. "Recombinant AAV vector plasmid" refers to a type of recombinant AAV vector, where the vector comprises a plasmid. As with AAV vectors in general, the 5' and 3' ITRs flank the selected heterologous nucleotide sequence.

[0052] AAV vectors can also include transcriptional sequences, such as polyadenylation sites and selectable markers or reporter genes, enhancer sequences, and other regulatory elements that allow induction of transcription. Such regulatory elements are described above.

[0053] As used herein, the term "AAV virion" refers to a complete virus particle. AAV virions can be wild-type AAV virus particles (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid, i.e., protein coat) or recombinant AAV virus particles (described below). In this regard, single-stranded AAV nucleic acid molecules (either the sense / coding strand or the antisense / anticoding strand, as these terms are generally defined) can be packaged into AAV. Both the sense and antisense strands are equally infectious.

[0054] As used herein, the term "recombinant AAV virion" or "rAAV" is defined as an infectious, replication-incompetent virus composed of an AAV protein shell that encapsidates (i.e., surrounds) a heterologous nucleotide sequence, which in turn is flanked 5' and 3' by AAV ITRs. Several 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 1:165-169

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

[1994] , all of which are incorporated herein by reference.

[0055] Suitable nucleotide sequences for use in AAV vectors (and indeed any of the vectors described herein) include any functionally related nucleotide sequences. Thus, the AAV vectors of the present invention can include any desired gene that encodes a protein that is missing or absent from the target cell genome, or that encodes a non-native protein with a desired biological or therapeutic effect (e.g., antiviral function), or the sequence can correspond to a molecule with antisense or ribozyme function. Suitable genes include those used to treat genetic defects such as inflammatory diseases, autoimmune diseases, chronic diseases, and infectious diseases, including diseases such as AIDS, cancer, neurological diseases, cardiovascular diseases, and hypercholesterolemia; various blood diseases, including various anemias, thalassemias, and hemophilias; cystic fibrosis, Gaucher's disease, adenosine deaminase (ADA) deficiency, emphysema, and the like. A number of antisense oligonucleotides (e.g., short oligonucleotides that are complementary to sequences surrounding the translation initiation site (AUG codon) of mRNA) that are useful in antisense therapy for cancer and viral diseases have been described in the art (e.g., Han et al., Proc. Natl. Acad. Sci. USA 88:4313-4317

[1991] ; Uhlmann et al., J. Med. 1999, 11:111-112). et al.,Chem.Rev.90:543-584

[1990] , Helene (See, e.g., Cech 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., 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] , which are incorporated herein by reference. See, e.g., et al. (1992) J. Biol. Chem. 267:17479-17482, and U.S. Patent No. 5,225,347.

[0056] As used herein, the term "purified" refers to molecules, either nucleic acid sequences or amino acid sequences, that are removed, isolated, or separated from their normal environment. Thus, an "isolated nucleic acid sequence" is a purified nucleic acid sequence. "Substantially purified" molecules are at least 60%, preferably at least 75%, and more preferably at least 90% free from other components that normally accompany them.

[0057] The present invention relates to vectors and their use for developing host cell lines for producing proteins of interest, and in particular to vectors which utilize a weak promoter to drive a selectable marker.

[0058] In some preferred embodiments, the expression system of the present invention utilizes an expression vector that includes 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 that perform various functions. Thus, for example, the nucleic acid sequence of interest can be included in any one of a variety of expression vectors for expressing a polypeptide. In some embodiments of the present invention, the vector includes, but is not limited to, retroviral vectors, chromosomal, non-chromosomal, and synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, phage DNA; vectors derived from baculovirus, yeast plasmids, combinations of plasmids and phage DNA, and viral DNA such as variola, adenovirus, fowlpox virus, and pseudorabies). It is contemplated that any vector can 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 Nos. 6,852,510 and 7,332,333, and U.S. Patent Application Publication Nos. 200402335173 and 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, the mammalian expression vector contains an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. In other embodiments, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the necessary nontranscribed genetic elements.

[0059] In some preferred embodiments, the vector is a retroviral vector.The genome organization of many retroviruses is well known in the art, which allows retroviral genomes to be adapted to produce retroviral vectors.The production of recombinant retroviral vectors carrying genes of interest is typically achieved in two steps.

[0060] First, a gene of interest is inserted into a retroviral vector that contains sequences necessary for efficient expression of the gene of interest (including promoter and / or enhancer elements, which may be provided by the viral long terminal repeat (LTR) or by an internal promoter / enhancer and associated splicing signals), sequences necessary for efficient packaging of viral RNA into infectious virions (e.g., packaging signal (Psi), tRNA primer binding site (-PBS), 3' regulatory sequences necessary for reverse transcription (+PBS)), and viral LTR. The LTR contains sequences necessary for the association of viral genomic RNA, reverse transcriptase, and integrase functions, as well as sequences involved in directing the expression of genomic RNA to be packaged within viral particles. For safety reasons, many recombinant retroviral vectors lack functional copies of genes essential for viral replication (these essential genes are either deleted or disabled). The resulting virus is therefore said to be replication-incompetent.

[0061] Next, after construction of the recombinant vector, the vector DNA is introduced into a packaging cell line. The packaging cell line provides the proteins required during transport (i.e., virally encoded gag, pol, and env proteins) to package the viral genomic RNA into viral 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 viral particle. The packaging cell line may express an ecotropic, amphotropic, or xenotropic envelope gene product. Alternatively, the packaging cell line may lack sequences encoding viral envelope (env) proteins. In this case, the packaging cell line packages the viral genome into particles that lack membrane-associated proteins (e.g., env proteins). To generate viral particles containing membrane-associated proteins that allow the virus to enter cells, the packaging cell line containing the retroviral sequences is transfected with sequences encoding membrane-associated proteins (e.g., G protein of vesicular stomatitis virus (VSV)). The transfected packaging cells then produce viral particles that contain the membrane-associated proteins expressed by the transfected packaging cell line. These viral particles, which contain the viral genomic RNA from one virus encapsidated by the envelope proteins of another virus, are said to be pseudotyped viral particles.

[0062] The retroviral vector of the present invention can be further modified to include additional regulatory sequences. As mentioned above, the retroviral vector of the present invention comprises 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 5'LTR and 3'LTR. In some embodiments of the present invention, when transcription from an internal promoter is desired, the nucleic acid of interest can be placed in reverse orientation relative to 5'LTR. Suitable internal promoters include, but are not limited to, alpha-lactalbumin promoter, CMV promoter (human or monkey), and thymidine kinase promoter.

[0063] 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. Several suitable signal peptide sequences are known to those of skill in the art, including, but not limited to, those derived from tissue plasminogen activator, human growth hormone, lactoferrin, α-casein, and α-lactalbumin.

[0064] In other embodiments of the invention, the vector is modified by incorporating an RNA export factor (see, e.g., U.S. Pat. Nos. 5,914,267, 6,136,597, and 5,686,120, and WO99 / 14310, all of which are incorporated herein by reference) either 3' or 5' to the nucleic acid sequence encoding the protein of interest. It is contemplated that the use of an RNA export factor allows for high level expression of the protein of interest without incorporating splice signals or introns into the nucleic acid sequence encoding the protein of interest.

[0065] In yet another embodiment, the vector comprises at least one internal ribosome entry site (IRES) sequence. Several suitable IRES sequences are available, including but not limited to those derived from foot and mouth disease virus (FDV), encephalomyocarditis virus, and poliovirus. The IRES sequence can be placed between two transcription units (e.g., nucleic acids encoding different proteins of interest or subunits of a multisubunit protein, such as an antibody) to form a polycistronic sequence, where the two transcription units can be transcribed from the same promoter.

[0066] The most commonly used recombinant retroviral vectors are 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 specific retrovirus is capable of infecting many different cell types, 2) established packaging cell lines are available for the production of recombinant MoMLV viral particles, and 3) the transferred gene is permanently integrated into the chromosome of the target cell. The established MoMLV vector system includes a DNA vector that contains a small portion of retroviral sequences (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 necessary signals for the insertion or packaging of the vector RNA into viral particles and for the expression of the inserted gene. The packaging cell line provides the proteins necessary for particle assembly (Markowitz et al., J. Virol. 62:1120

[1988] ).

[0067] In some preferred embodiments, the retroviral vectors are pseudotyped, for example, utilizing the G protein of VSV as a membrane-associated protein. Unlike retroviral envelope proteins that bind to specific cell surface protein receptors to allow entry into cells, the VSV G protein interacts with phospholipid components of the plasma membrane (Mastromarino et al., J. Gen. Virol. 68:2359

[1977] ). Because VSV entry into cells does not depend on the presence of specific protein receptors, VSV has an extremely broad host range. Retroviral vectors pseudotyped with the VSV G protein have the altered host range characteristics of VSV (i.e., they can infect almost any species of vertebrate, invertebrate, and insect cells). Importantly, VSV G pseudotyped retroviral vectors can be concentrated more than 2000-fold by ultracentrifugation without significant loss of infectivity (Burns et al. Proc. Natl. Acad. Sci. USA 90:8033

[1993] ).

[0068] The present invention is not limited to the use of the VSV G protein, as the viral G protein is used as a heterologous membrane-associated protein within the viral particle (see, e.g., U.S. Patent No. 5,512,421, incorporated herein by reference). The G proteins of viruses in the genus Vesiculovirus other than VSV, such as Piry and Chandipura viruses, which are highly homologous to the VSV G protein and, like the VSV G protein, contain a covalently bound palmitic acid (Brun et al. Intervirol. 38:274

[1995] and Masters et al., Virol. 171:285 (1990)). Thus, the G proteins of the Piry and Chandipura genera can be used in place of the VSV G protein for pseudotyping of virus particles. Furthermore, the VSV G proteins of viruses in the genus Lyssavirus, such as rabies and Mokola viruses, show a high degree of conservation (amino acid sequence and functional conservation) with the VSV G protein. For example, the Mokola virus G protein has been shown to function in a manner similar to the VSV G protein (i.e., mediating membrane fusion) and therefore can be used in place of the VSV G protein for pseudotyping of virus particles (Mebatsion et al., 2002). al., J.Virol.69:1444

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

[0069] In some preferred embodiments, the vector is a lentiviral vector. Lentiviruses (e.g., Equine infectious anemia virus, Caprine arthritis-encephalitis virus, Human immunodeficiency virus) are a subfamily of retroviruses that can integrate into non-dividing cells. Lentiviral genomes and proviral DNA contain three genes found in all retroviruses, namely gag, pol, and env, which are flanked by two LTR sequences. The gag gene encodes internal structural proteins (e.g., matrix, capsid, 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 transcription and polyadenylation of viral RNA. Additional genes in the lentiviral genome include the vif, vpr, tat, rev, vpu, nef, and vpx genes.

[0070] Various lentiviral vectors and packaging cell lines are known in the art and find use in the present invention (see, for example, U.S. Patent Nos. 5,994,136 and 6,013,516, both of which are incorporated herein by reference). In addition, the VSV G protein has also been used to pseudotype retroviral vectors based on human immunodeficiency virus (HIV) (Naldini et al., Science 272:263

[1996] ). Thus, the VSV G protein can be used to generate various pseudotyped retroviral vectors, which are not limited to vectors based on MoMLV. Lentiviral vectors can also be modified as described above to contain various regulatory sequences (e.g., signal peptide sequences, RNA export factors, and IRES). After generation of the lentiviral vector, the lentiviral vector can be used to transfect host cells as described above for retroviral vectors.

[0071] In some preferred embodiments, the vector is an adeno-associated virus (AAV) vector. AAV is a human DNA parvovirus that belongs to the Dependovirus genus. The AAV genome is composed of a linear, single-stranded DNA molecule containing approximately 4680 bases. The genome contains inverted terminal repeats (ITRs) at each end that function in cis as origins of DNA replication and as viral packaging signals. The internal non-repetitive portion of the genome contains two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for viral proteins involved in virion replication and packaging. At least four families of viral proteins are expressed in the Rep and cap regions of the AAV rep region. The AAV genome is synthesized from Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3 (for detailed descriptions of the AAV genome, see, e.g., Muzyczka, Current Topics Microbiol. Immunol. 158:97-129

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

[1994] ).

[0072] To produce reproductive infection, AAV requires co-infection with an unrelated helper virus, such as adenovirus, herpes virus, or smallpox. In the absence of such co-infection, AAV establishes a latent state by inserting its genome into the chromosome of the host cell. Subsequent infection with helper virus rescues the integrated copy, which can then replicate to produce infectious viral progeny. Unlike non-pseudotyped retroviruses, AAV has a wide host range and can replicate in any species of cell, as long as it is co-infected with a helper virus that also reproduces in that species. Thus, for example, human AAV replicates in canine cells co-infected with canine adenovirus. Moreover, unlike retroviruses, AAV is not associated with any human or animal disease, and upon integration, it does not appear to change the biological properties of the host cell, but can integrate into undivided cells. It has also been recently discovered that AAV is capable of site-specific integration into the host cell genome.

[0073] In view of the above characteristics, several recombinant AAV vectors have been developed for gene transfer (e.g., U.S. Pat. Nos. 5,173,414, 5,139,941, WO 2013 / 023361, and WO 2013 / 023361). 92 / 01070 and WO 93 / 03769, both 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. al., J. Exp. Med. 179:1867-1875

[1994] ).

[0074] Recombinant AAV virions can be produced in suitable host cells transfected with both AAV helper plasmids and AAV vectors. AAV helper plasmids generally contain AAV rep and cap coding regions, but lack AAV ITRs. Thus, the helper plasmid cannot replicate or package itself. AAV vectors generally contain a selected gene of interest bounded by AAV ITRs that confer viral replication and packaging functionality. Both the helper plasmid and the AAV vector carrying the selected gene are introduced into suitable host cells by transient transfection. The transfected cells are then infected with a helper virus, such as adenovirus. Such a helper virus transactivates the AAV promoter present on the helper plasmid, which induces the transcription and translation of the AAV rep and cap regions. Recombinant AAV virions carrying the selected gene can be prepared and purified. Once the AAV vectors are generated, they can be used to transfect host cells at a desired multiplicity of infection (see, e.g., U.S. Patent No. 5,843,742, incorporated herein by reference) to produce high copy numbers of host cells. As will be appreciated by those skilled in the art, AAV vectors can also be modified as described above to contain various regulatory sequences (e.g., signal peptide sequences, RNA export factors, and IRES).

[0075] In some preferred embodiments, the vector is a transposon-based vector. Transposons are mobile genetic elements that can move or transpose from one location in a genome to another. Transposition within a genome is controlled by a transposase enzyme encoded by the transposon. Tn5 (see, e.g., de la Cruz et al., J. Bact. 175: 6932-38

[1993] ), Tn7 (see, e.g., Craig, Curr. Topics Microbiol. Immunol. 204: Many examples of transposons are known in the art, including, but not limited to, Tn10 (see, e.g., Morisato and Kleckner, Cell 51:101-111 (1987)). The ability of transposons to integrate into genomes has been exploited to create transposon-based 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-based vectors are introduced into host cells by methods known in the art (e.g., electroporation, lipofection, or microinjection). Thus, the ratio of transposon vector to host cell can be adjusted to provide a desired multiplicity of infection to generate high copy number host cells of the invention.

[0076] A transposon vector suitable for use in the present invention generally comprises a nucleic acid encoding a protein of interest sandwiched between two transposon insertion sequences. Some vectors also comprise a nucleic acid sequence encoding a transposase enzyme. In these vectors, one of the insertion sequences is located between the transposase enzyme and the nucleic acid encoding the protein of interest, so that the insertion sequence is not integrated into the genome of the host cell during recombination. Alternatively, the transposase enzyme may be provided by a suitable method (e.g., lipofection or microinjection). As will be understood 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 factors, and IRES).

[0077] In some preferred embodiments, the vector comprises a selection marker. Suitable selection markers include, but are not limited to, glutamine synthetase (GS), dihydrofolate reductase (DHFR), and the like. 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 which are incorporated herein by reference. In some particularly preferred embodiments, the selection marker is GS, and has a sequence that shares at least 80%, 90%, 95%, 99%, or 100% identity with the sequence of positions 1789 to 2910 of SEQ ID NO:2. In some preferred embodiments, the selection marker utilized is compatible with a host cell line that is defective in the production of the enzyme encoded by the 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 cells expressing the protein with resistance to an antibiotic. Suitable antibiotic resistance markers include genes that confer resistance to neomycin (neomycin resistance gene (neo)), hygromycin (hygromycin B phosphotransferase gene), puromycin (puromycin N-acetyl-transferase), etc.

[0078] In some embodiments, the nucleic acid sequence encoding the selection marker is operably linked to a promoter. In some particularly preferred embodiments, the promoter sequence is altered, for example by mutation, to reduce promoter activity compared to the unaltered version of the promoter. These promoters can be described as weak promoters in that the expression of the gene linked to the promoter occurs at low levels, as opposed to high levels. Genes controlled by strong promoters produce more mRNA, and therefore more protein product, than genes controlled by weak promoters. Thus, the present invention preferably utilizes promoters for altered selection markers, which produce less mRNA than the corresponding unaltered promoter.

[0079] In some preferred embodiments, the promoter operably linked to the selection marker is a long terminal repeat (LTR) from a retrovirus or lentivirus. In some particularly preferred embodiments, the promoter is an LTR that is altered by removing either all or part of the U3 region of the LTR. In some embodiments, the promoter operably linked 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 to SEQ ID NO: 3, and the SIN LTR most preferably has a deletion in the U3 region that reduces the promoter activity compared to the promoter activity when the deletion is not present in the U3 region.

[0080] It will be understood that if the vector is a retroviral vector, the vector is constructed such that the SIN LTR is the 3'LTR of the retroviral vector sequence. Due to the fact that the U3 deletion is copied to the 5' and 3'LTR during reverse transcription, the integrated SIN vector contains only the LTR with the U3 deletion. A map of an exemplary retroviral vector of the present invention is shown in FIG. 2. As can be seen, the vector contains a SIN 3'LTR. When reverse transcribed, the hCMV-MOMuSV shown in the vector map is replaced by the SIN 3'LTR shown in the vector map, so that when the vector is integrated into the host cell chromosome, the SIN LTR operably binds to and drives expression of the GS cDNA shown.

[0081] In certain preferred embodiments of the invention, the nucleic acid sequence encoding the protein of interest within the expression vector is operably linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Promoters useful in the present invention include the LTR or SV40 promoter, E. coli lac or trp, phage lambda P 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, as well as other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0082] As noted above, an exemplary map of a vector of the invention is shown in Figure 3, which corresponds to SEQ ID NO: 2. In some preferred embodiments, the vector comprises the following elements in 5' to 3' order: -5'LTR (exemplified by hCMV-MoMuSV LTR) -Retroviral packaging region - a nucleic acid encoding a selectable marker (exemplified by GS cDNA) - internal promoters (exemplified by the simian CMV (sCMV) promoter) - a nucleic acid sequence encoding a protein of interest operably linked to an internal promoter (exemplified by the "Anyway" sequence) -WPRE sequence -SIN 3′ LTR.

[0083] In some embodiments, the vector is a plasmid that includes 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., a 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.

[0084] In some embodiments, the vector comprises a single polyA signal sequence downstream of the nucleic acid encoding the protein of interest. For example, in some embodiments, the vector comprises the following elements in 5' to 3' order: promoter (e.g., SIN)-selectable marker-stop codon-promoter (e.g., CMV)-protein of interest-stop codon-polyA.

[0085] In some embodiments, the invention provides host cells and host cell cultures, where the host cells express a protein of interest from the vectors described above. In a preferred embodiment, the host cells are mammalian host cells. Several mammalian host cells are known in the art. Generally, as described in more detail below, these host cells are capable of growing and surviving when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and growth factors. Typically, the cells are capable of expressing and secreting large amounts of a particular protein of interest into the medium. Examples of suitable mammalian host cells include Chinese hamster ovary cells (CHO-K1, ATCC CCl-61), bovine mammary epithelial cells (ATCC CRL 10274; bovine mammary epithelial cells), monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cells (293 cells or 293 cells subcloned for growth in suspension culture; see, e.g., Graham et al., J. Gen Virol., 36:59 1977), baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251

[1980] ), monkey kidney (CV1 ATCC CCL 70), African green monkey kidney (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 10), and the like. CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065), mouse mammary carcinoma (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 (amniotic cell production of CEVEC) cells, and human hepatoma lines (Hep G2).

[0086] In some particularly preferred embodiments, the host cells are modified to become, or are naturally deficient in, an enzymatic activity that is necessary for the growth or survival of the cells in the presence of a selection agent and that is provided by a selection marker. For example, Chinese Hamster Ovary (CHO) cells are modified to be GS deficient. In some preferred embodiments, where the vector contains a GS selection marker, the host cell line is GS deficient. In some particularly preferred embodiments, the GS deficient host cell line is CHOZN® GS, available from Merck KGaA. - / - In other embodiments, where the selectable marker is, for example, DHFR, the cell line may preferably be deficient in DHFR activity (i.e., - Suitable DHFR- cell lines include, but are not limited to, CHO-DG44 and its derivatives.

[0087] The vectors can be introduced into the host cells by any suitable means. In some preferred embodiments, vectors (e.g., retroviral vectors) encoding the protein of interest have been generated and can be used to transfect or transduce the host cells. Preferably, the host cells are transfected or transduced with the integrating vector at a multiplicity of infection sufficient to result in integration of at least one, and preferably at least two or more, retroviral vectors. In some embodiments, a multiplicity of infection of 10 to 1,000,000 can be utilized, such that the genome of the infected host cell contains 2 to 1000 copies of the integrating vector, preferably 5 to 1000 copies of the integrating vector, and most preferably 20 to 500 copies of the integrating vector. In other embodiments, a multiplicity of infection of 10 to 10,000 is utilized. When non-pseudotyped retroviral vectors are used for infection, the host cells are incubated with medium from retroviral producer cells containing the desired titer (i.e., colony forming units·CFU) of infectious vector. When using pseudotyped retroviral vectors, the vector is concentrated to an appropriate titer by ultracentrifugation before being added to host cell culture.Alternatively, the concentrated vector can be diluted in a medium suitable for the cell type.Furthermore, when it is desired for the host cell to express two or more proteins of interest, the host cell can be transfected with multiple vectors, each of which contains the nucleic acid encoding a different protein of interest.

[0088] In each case, the host cells are exposed to the medium containing the infectious retroviral vector for a period of time sufficient to allow infection and subsequent integration of the vector. In general, the amount of medium used to overlay the cells should be kept as small as possible to support the maximum amount of integration events per cell. As a general guideline, the number of colony forming units (cfu) per milliliter should be around 10, depending on the number of integration events desired. 5 ~10 7 It should be cfu / mL.

[0089] In some embodiments, after transfection or transduction, the cells are propagated, then trypsinized and re-plated. Individual colonies are then selected to provide clonally selected cell lines. In yet further embodiments, the clonally selected cell lines are screened by Southern blotting or PCR assay to confirm that the desired number of integration events have occurred. It is also contemplated that clonal selection allows for the identification of cell lines that produce superior proteins. In other embodiments, the cells are not clonally selected after transfection.

[0090] In some embodiments, the host cells are transfected with vectors encoding different proteins of interest. The vectors encoding the different proteins of interest can be used to transfect cells simultaneously (e.g., the host cells are exposed to a solution containing vectors encoding the different proteins of interest) or the transfections can be sequential (e.g., the host cells are first transfected with a vector encoding a first protein of interest, a period of time is allowed to pass, and then the host cells are transfected with a vector encoding a second protein of interest). In some preferred embodiments, the host cells are transfected with an integrating vector encoding a first protein of interest, high-expressing cell lines containing multiple integrated copies of the integrating vector are selected (e.g., clonal selection), and the selected cell lines are transfected with an integrating vector encoding a second protein of interest. This process can be repeated to introduce multiple proteins of interest. In some embodiments, the infection multiplicity can be manipulated (e.g., increased or decreased) to increase or decrease expression of the protein of interest. Similarly, different promoters can be utilized to vary expression of the protein of interest. It is contemplated that these transfection methods can be used to construct host cell lines that contain entire exogenous metabolic pathways or to provide host cells with increased capacity to process proteins (e.g., the host cell can provide enzymes required for post-translational modifications).

[0091] In yet another embodiment, cell lines are transfected sequentially with vectors encoding the same gene. In some preferred embodiments, host cells are transfected with an integrated vector encoding a protein of interest (e.g., at an MOI of about 10-1,000,000, preferably 100-10,000), cell lines containing one or more integrated copies of the integrated vector or high concentrations of the desired protein are selected (e.g., clonal selection), and the selected cell lines are retransfected with the vector (e.g., at an MOI of about 10-1,000,000, preferably 100-10,000). In some embodiments, cell lines containing at least two integrated copies of the vector are identified and selected. This process may be repeated multiple times until a desired level of protein expression is obtained, and may be repeated to introduce vectors encoding multiple proteins of interest. Unexpectedly, sequential transfection of the same gene results in increased protein production from the resulting cells rather than merely addition.

[0092] The present invention contemplates various sequential transfection procedures. In some embodiments utilizing retroviral vectors, sequential transduction procedures are provided. In preferred embodiments, sequential transduction is performed on a pool of cells. In these embodiments, an initial pool of host cells is contacted with the retroviral vector, preferably at a multiplicity of infection ranging from about 0.5 to about 1000 vectors / host cell. The cells are then cultured in an appropriate medium (e.g., with a selection agent) for several days. An aliquot of the cells is then taken, the number of integrated vectors is determined, and frozen for potential future use. The remaining cells are then contacted again, preferably at a multiplicity of infection ranging from about 0.5 to about 1000 vectors / host cell. The process is repeated until cells with the desired number of integrated vectors are obtained. For example, the process can be repeated up to 10-20 times, or more. In some embodiments, cells can be clonally selected if desired after any particular transduction step, but by utilizing a pool of cells in the absence of transduction, the time to reach the desired integrated vector copy number is reduced.

[0093] In some embodiments of the present invention, after transformation of a suitable host strain and growth of the host strain to an appropriate cell density in a medium, the protein of interest is secreted during the culture of the host cells. In some preferred embodiments utilizing an amplifiable marker, it is contemplated that the culture medium of the transduced host cells in the medium contains an inhibitor of the gene. Suitable inhibitors include, but are not limited to, methotrexate for inhibition of DHFR, and methionine sulfoximine (Msx) or phosphinothricin for inhibition of GS. It is contemplated that when the concentration of these inhibitors is increased in the cell culture system, cells with a high copy number of the amplifiable marker (and thus the gene or genes of interest) or containing a high-producing insert are selected.

[0094] Thus, the host cells containing the above-mentioned vectors are cultured according to methods known in the art. Suitable culture conditions for mammalian cells are well known in the art (see, for example, J. Immunol. Methods (1983) 56:221-234

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

[1992] ).

[0095] The host cell culture of the present invention is prepared in a medium suitable for the particular cells being cultured. Commercially available media such as ActiPro medium (Hyclone), ExCell Advanced Fed Batch medium (SAFC), Ham's F10 (Sigma, St. Louis, MO), Minimal Essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are exemplary nutrient solutions. Suitable media are also described in U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 5,122,469, 4,560,655, and WO 90 / 03430 and WO 87 / 00195, the disclosures of which are incorporated herein by reference. Any of these media may be supplemented as necessary with serum, hormones and / or other growth factors (insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin / gentamicin), trace elements (usually defined as inorganic compounds present in the micromolar range at final concentrations), lipids (linoleic acid or other fatty acids) and their suitable carriers, and glucose, or an equivalent energy source. In some preferred embodiments where a selection marker such as GS is utilized, for example, the medium lacks glutamine. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art.

[0096] The present invention also contemplates the use of various culture systems (e.g., petri dishes, 96-well plates, roller bottles, and bioreactors) for the transfected host cells. For example, the transfected host cells can be cultured in a perfusion system. Perfusion culture refers to providing a continuous flow of medium through the culture medium that maintains a high cell density. The cells are suspended and do not require a solid support for growth. In general, fresh nutrients must be continuously supplied, along with the removal of toxic metabolites, and ideally, the selective removal of dead cells. Filtration, entrapment, and microencapsulation methods are all suitable for refreshing the culture environment at a sufficient rate.

[0097] As another example, in some embodiments, fed-batch culture procedures can be used. In preferred fed-batch cultures, mammalian host cells and medium are initially fed to the culture vessel, additional culture nutrients are fed to the culture medium continuously or in discrete increments during the culture, and cells and / or products are periodically harvested or not harvested before the end of the culture. Fed-batch cultures can include, for example, semi-continuous fed-batch cultures, in which all the culture medium (including cells and medium) is periodically removed and replaced with fresh medium. Fed-batch cultures are distinguished from simple batch cultures, in which all components for cell culture (including cells and all culture nutrients) are fed to the culture vessel at the beginning of the culture process. Fed-batch cultures can also be distinguished from perfusion cultures, insofar as the supernatant is not removed from the culture vessel during the process (in perfusion cultures, the cells are kept in the culture medium, for example, by filtration, encapsulation, anchoring to microcarriers, and medium is continuously or intermittently introduced and removed from the culture vessel). In some particularly preferred embodiments, the batch culture is carried out in roller bottles.

[0098] Furthermore, the cells of the culture can be propagated according to any scheme or routine that may be suitable for the particular host cell and the particular production plan contemplated. Thus, the present invention contemplates single-step or multi-step culture procedures. In a single-step culture, the host cells are seeded into a culture environment and the process of the present invention is used during one production phase of the cell culture. Alternatively, a multi-step culture is envisioned. In a multi-step culture, the cells can be cultured in several steps or stages. For example, the cells can be grown in a first step or growth phase culture. In this stage, the cells, optionally removed from storage, are seeded into a medium suitable to promote growth and high viability. The cells can be maintained in the growth phase for a suitable period of time by adding fresh medium to the host cell culture.

[0099] Fed-batch or continuous cell culture conditions are devised to improve the growth of mammalian cells in the growth phase of the cell culture. In the growth phase, the cells are grown under conditions and for a period of time maximized for growth. The culture conditions, such as temperature, pH, dissolved oxygen (dO2), etc., used for a particular host will be apparent to one of skill in the art. Generally, the pH is adjusted to a level of about 6.5-7.5 using either an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH). The suitable temperature range for culturing mammalian cells, such as CHO cells, is about 30°C-38°C, and the suitable dO2 is 5-90% air saturation.

[0100] Following the polypeptide production phase, the polypeptide of interest is recovered from the culture medium using techniques well established in the art. The protein of interest is preferably recovered from the culture medium as a secreted polypeptide (e.g., secretion of the protein of interest is induced by a signal peptide sequence), although the protein of interest may also be recovered from a host cell lysate. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The polypeptide is then purified from contaminating soluble proteins and polypeptides. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, and protein A sepharose columns to remove contaminants such as IgG. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may also be useful to inhibit proteolysis during purification. Additionally, the protein of interest can be fused in frame to a marker sequence that allows purification of the protein of interest. Non-limiting examples of marker sequences include a hexahistidine tag (which may be provided by a vector, preferably the pQE-9 vector), and a hemagglutinin (HA) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (see, e.g., Wilson et al. (See, e.g., W. et al., Cell, 37:767

[1984] .) One of skill in the art will recognize that purification methods suitable for a subject polypeptide may require modification to account for changes in the characteristics of the polypeptide upon expression in recombinant cells.

[0101] experiment The present invention provides a unique method of combining retroviral-type transduction (herein referred to as GPEx® technology) in combination with a glutamine synthase (GS) knockout CHO cell line system that unexpectedly improved the ability of cells to produce high titers and specific productivity of a protein of interest. The GS knockout cell line used in these studies was the CHOZN cell line available from MilliporeSigma. The GPEx® technology was performed in a similar manner to that previously performed in regular CHO cells. However, in the viral expression vector, the GS gene used for selection in the GS knockout cell line was driven from a very weak promoter derived from a SIN (self-inactivating) LTR present in one version of the GPEx expression vector we used at Catalent. The combination of the vector, the regular GPEx cell line production process, and the GS knockout CHO cell line resulted in up to 7-fold higher production levels in unmodified CHO cell lines than the conventional GPEx process.

[0102] Test 1: Two pooled cell lines were generated from two different viral vectors. Both lines were generated using the normal GPEx transduction process 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 was that one vector would have a full-length Moloney Murine Leukemia Virus (MMLV) LTR driving expression of the GS gene after insertion into the cell line, and the other vector would have an MMLV SIN LTR driving expression of GS. The genetic constructs used to generate the retrovector particles are shown in Figures 1 and 2. The sequences for the genetic constructs are shown in Figures 3 and 4.

[0103] Development of CHOZN cell lines Retrovector Generation: The expression constructs outlined above were introduced into a HEK293 cell line that constitutively produces the MLV gag, pro, and pol proteins. An envelope containing expression plasmid was also co-transfected with each of the gene constructs. Co-transfection resulted in the generation of high titer replication-incompetent retrovectors that were concentrated by ultracentrifugation and used for cell transduction (1, 2). Transduction of CHO cells with retrovectors generated using the above constructs results in a 3'LTR sequence that is duplicated at the 5' end of the sequence that replaces the hCMV-MoMuSV 5'LTR and subsequently controls expression of the GS gene in pooled cell lines.

[0104] Retrovector transduction into CHOZN cells: Pooled cell lines containing retrovector inserts from each of the above constructs were generated by transducing the CHOZN Chinese Hamster Ovary parent cell line with retrovectors made from gene constructs developed to express the Anyway protein. Single cycle transduction was performed to generate pooled cell lines for each of the two constructs. Upon completion of cell transduction, cell lines were placed in glutamine-free medium to perform GS selection.

[0105] Fed-batch production of Anyway from two pooled cell populations: After transduction, the pooled cell lines were scaled up in duplicate in 250 mL shake flasks in a fed-batch study for productivity. Each shake flask was seeded with 300,000 viable cells per mL in a 50 mL working volume of ExCell Advanced Fed Batch Medium (SAFC) and incubated in a humidified (70-80%) shaking incubator at 110 rpm, 5% CO2, and 37°C. During production, cultures were fed every other day starting on day 3 with a feeding supplement. Glucose was monitored daily and replenished if the concentration was below 4 g / L. Cultures were terminated when viability was below 50%.

[0106] result The results are shown in Table 1 and Figure 5. These results are surprising. At similar average gene copy numbers in the two pools, there was a large titer difference between the two pooled cell lines. Therefore, even at slightly higher average gene copy numbers, wild-type or full-length LTR gene constructs resulted in significantly lower titers, which also led to significantly lower cell-specific productivity per gene insert. Selection or competitive advantage of high-expressing cells containing SIN versions of gene constructs occurs.

[0107] [Table 1]

[0108] Test 2: Based on previous results, we designed an experiment to compare how the traditional GPEx technology is implemented with how the technology is implemented in exactly the same way (but in combination with the CHOZN GS knockout cell line and using the SIN-GS vector described above). The processes were as identical as possible with only two main differences. The first process, which is traditional GPEx, uses the GPEx® Chinese Hamster Ovary (GCHO) parent cell line, and the new version uses the CHOZN cell line. The second difference is that the genetic constructs used to make the retrovectors were different. For traditional GPEx, the construct does not contain the GS gene, and for the new version, the construct contains the GS gene. All other elements of the genetic constructs were similar. Each of the constructs again expresses the Anyway protein. After the pooled cell lines were completed for each method, the production of the cell lines was compared in a fed-batch analysis.

[0109] Development of GCHO and CHOZN cell lines: Retrovector generation: The expression constructs were introduced into a HEK293 cell line that constitutively produces MLV gag, pro, and pol proteins. An envelope containing expression plasmid was also co-transfected with each of the gene constructs. Co-transfection resulted in the generation of replication-incompetent high-titer retrovectors, which were concentrated by ultracentrifugation and used for cell transduction (1, 2).

[0110] Retrovector transduction into CHOZN cells: Either the CHOZN Chinese hamster ovary parental cell line or the GCHO Chinese hamster ovary parental cell line was transduced with retrovectors made from either the GS-containing or non-GS-containing genetic constructs, both of which were developed to express the Anyway protein, to generate pooled cell lines containing retrovector inserts from each of the constructs. Three cycles of transduction were performed to generate pooled cell lines for each of the two constructs in the corresponding cell line. Additional cycles typically increase the number of inserts, and subsequently the gene copy number, in the cell line. Upon completion of each cycle of transduction, the CHOZN-based cell pools were placed in glutamine-free medium for GS selection. Conventional GPEx cells were not selected as per usual procedures.

[0111] result As expected, the copy number increased with repeated cycles of transduction in both methods, however, significantly higher gene copy numbers were observed in each of the novel GPEx cell pools compared to the conventional GPEx, as shown in Table 2.

[0112] [Table 2]

[0113] Fed-batch production of Anyway from two pooled cell populations (three cycles of transduction): After transduction, the pooled cell lines were scaled up in duplicate in 250 mL shake flasks in a fed-batch study for productivity. Each shake flask was seeded with 300,000 viable cells per mL in a working volume of 50 mL ActiPro medium (HyClone) and incubated in a humidified (70-80%) shaking incubator at 120 rpm, 5% CO2, and a temperature of 37°C (34°C from day 6). Cultures were fed six times during the production run using two different feeding supplements. Glucose was monitored and replenished if the concentration was below 4 g / L. Cultures were terminated when viability was below 50%.

[0114] The results are shown in Table 3 and Figure 6. Again, a very unexpected result was observed. The optimized conventional GPEx clone cell line producing Anyway product expressed at up to 1.8g / L, and the new GPEx pool exceeded that expression by more than 2-fold before any clone selection. The new GPEx pool has both high copy number and high production per gene insert, as seen in experiment #1. Combining these two, a striking difference in cell-specific productivity was obtained with almost 3-fold higher pg / cell / day for the new GPEx pool. The viable overall cell density was also higher in the new GPEx pool compared to the conventional pool, again driving the substantial titer difference observed.

[0115] [Table 3]

[0116] Tests 3 and 4: The above data is for Fc fusions made from a single gene. Experiments 3 and 4 utilize transduction of separate heavy and light chain vectors (two light chain transductions for both, and three heavy chain transductions) with two different antibodies, "Peelaway" and "Yourway", using both GPEx® technology and a novel process using the SIN LTR to drive GS expression.

[0117] Retrovector generation: The expression constructs were introduced into a HEK293 cell line that constitutively produces MLV gag, pro, and pol proteins. An envelope containing expression plasmid was also co-transfected with each of the gene constructs. Co-transfection resulted in the generation of replication-incompetent high-titer retrovectors, which were concentrated by ultracentrifugation and used for cell transduction (1, 2).

[0118] Retrovector transduction into CHOZN cells: Pooled cell lines containing retrovector inserts from each of the constructs were generated by transducing either the CHOZN Chinese Hamster Ovary parent cell line or the GCHO Chinese Hamster Ovary parent cell line with retrovectors made from either the GS-containing or non-GS-containing heavy and light chain gene constructs developed to express the two different antibodies. Two cycles of light chain transduction and three cycles of heavy chain transduction were performed to generate pooled cell lines for each of the four constructs in each of the corresponding antibody cell lines. Additional cycles typically increase the number of inserts, and subsequently the gene copy number, in the cell lines. Upon completion of each cycle of transduction, the CHOZN-based cell pools were placed in glutamine-free medium for GS selection. Conventional GPEx cells were not selected as per normal procedures.

[0119] Production of Peelaway and Yourway in fed-batch culture from pooled population cell lines (2 cycles of light chain transduction and 3 cycles of heavy chain transduction): After transduction, the pooled cell lines were scaled up in duplicate in 250 mL shake flasks in a fed-batch study for productivity. Each shake flask was seeded with 300,000 viable cells per mL in a working volume of 50 mL ActiPro medium (HyClone) and incubated in a humidified (70-80%) shaking incubator at 120 rpm, 5% CO2, and a temperature of 37°C (34°C from day 6). Cultures were fed six times during the production run using two different feeding supplements. Glucose was monitored and replenished if the concentration was below 4 g / L. Cultures were terminated when viability was below 50%.

[0120] result The results are shown in Table 4 and Figure 5. A significant titer advantage was observed with the new vector and process.

[0121] [Table 4]

[0122] [Table 5]

[0123] Additional copy number analysis was performed on the four Yourway cell pools. The cell pool generated after one light chain transduction and two heavy chain transduction cycles was compared to the cell pool generated after two light chain transduction cycles and three heavy chain transduction cycles. Each of these two pooled cell lines was either set to continue growth in glutamine-containing medium or placed in medium lacking glutamine. Gene copy numbers relative to the total number of genes, number of heavy chain genes, and number of light chain genes were calculated for each of the four pooled cell lines.

[0124] [Table 6]

[0125] These data indicate that although each of the cells in these pools has multiple copies of the GS gene driven from the SIN LTR, there is significant selection occurring for those with more copies, and based on the data above, for those copies that are expressed at higher concentrations.

[0126] Experiment 5 For this experiment, Yourway antibody products were used. The heavy chain gene constructs used to generate the cell lines were identical to those used in experiment 4. Except for containing the light chain coding sequence and lacking the GS gene, the light chain gene constructs were identical in all respects to the heavy chain gene constructs. One transduction was performed for each retrovector containing the light chain (-GS) and heavy chain (+GS) to generate pooled cell lines.

[0127] Retrovector generation: The expression constructs were introduced into a HEK293 cell line that constitutively produces MLV gag, pro, and pol proteins. An envelope containing expression plasmid was also co-transfected with each of the gene constructs. Co-transfection resulted in the generation of replication-incompetent high-titer retrovectors, which were concentrated by ultracentrifugation and used for cell transduction (1, 2).

[0128] Retrovector transduction into CHOZN cells: Pooled cell lines containing retrovector inserts from each of the constructs were generated by transducing the CHOZN Chinese Hamster Ovary parent cell line with retrovectors generated from the GS-free light chain gene construct and the GS-containing heavy chain construct. One cycle of light chain transduction and one cycle of heavy chain transduction were performed to generate the pooled cell line. Upon completion of both cycles of transduction, the CHOZN-based cell pool was split, with one half placed in glutamine-free medium and the other half continued to be supplemented with glutamine during cell expression and subsequent productivity evaluation.

[0129] Production of Yourway in fed-batch culture from pooled population cell lines (one light chain transduction and one heavy chain transduction): Pooled cell lines were scaled up in 250 mL shake flasks for productivity in fed-batch studies. Shake flasks were seeded with 300,000 viable cells per mL in a working volume of 50 mL 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 from day 6 onwards). Cultures were fed six times during the production run with two different feeding supplements. Glucose was monitored and replenished if the concentration fell below 4g / L. Glutamine in glutamine-supplemented medium was monitored daily and replenished if the concentration fell below 2mM. Cultures were terminated when viability was below 50%.

[0130] result The results are shown in Table 7. Selection against glutamine increased the copy number of the heavy chain gene in the cell pool. Somewhat surprisingly, the copy number of the light chain was also increased, although not to the same extent as the heavy chain. Total production of antibody was also significantly higher than in cultures without selection, reaching concentrations shown in Table 5, and only two transductions were performed instead of the five completed for this experiment.

[0131] [Table 7]

[0132] Test 6 Genetic constructs were designed and generated to test whether the techniques behave similarly using conventional cell transfection methods compared to retrovector transduction. An example of a conventional plasmid transfection construct generated is shown in Figure 8 (plasmid map) and Figure 9 (actual plasmid sequence, SEQ ID NO: 4).

[0133] 1.Bleck,GT 2005 An alternative method for the rapid generation of stable,high-expressing mammalian cell lines (A Technical Review).Bioprocessing J.Sep / Oct.pp 1-7. 2.Bleck, GT,2010.GPEx(R)A Flexible Method for the Rapid Generation of Stable,High Expressing,Antibody Producing Mammalian Cell Lines Chapter 4 In:Current Trends in Monoclonal Antibody Development and Manufacturing,Biotechnology: Pharmaceutical Aspects,Edited by: SJShire et al.(c)2010 American Association of Pharmaceutical Scientists,DOI 10.1007 / 978-0-387-76643-0_4.

[0134] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it will be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. [Brief description of the drawings]

[0135] [Figure 1] 1 is a map of the full-length MMLV construct. [Diagram 2] 1 is a map of the SIN MMLV LTR construct. [Figure 3-1] The sequence of the full-length MMLV construct (SEQ ID NO:1). [Figure 3-2] Figure 3-1 continued [Figure 3-3] Figure 3-2 (continued) [Figure 4-1] 2 is the sequence of the SIN MMLV LTR construct (SEQ ID NO:2). [Figure 4-2] Figure 4-1 continued [Figure 4-3] Figure 4-2 (continued) [Diagram 5] Pooled cell line titer comparison of full-length and SIN MMLV LTR constructs. [Figure 6] 13 is a comparison of the potency of pooled cell lines between processes using full-length MMLV constructs and SIN MMLV LTR constructs. [Figure 7] This is the SIN LTR sequence (SEQ ID NO:3). [Figure 8] 4 is a map of the proviral plasmid construct (SEQ ID NO:4). [Figure 9-1] The sequence of the proviral plasmid construct (SEQ ID NO:4). [Figure 9-2] Figure 9-1 continued [Figure 9-3] Figure 9-2 continued

Claims

[Claim 1] A vector for expressing a protein of interest, comprising a nucleic acid sequence encoding a selectable marker operably associated with a first promoter sequence that has been modified to reduce promoter activity compared to an unmodified, i.e., wild-type, version of the first promoter sequence, and a nucleic acid sequence encoding the protein of interest operably linked to a second promoter sequence.

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