Cell line for recombinant protein and / or viral vector production

Abstract

To provide a method for producing a human embryonic kidney (HEK) cell line that does not express functional endogenous dihydrofolate reductase (DHFR) and does not express functional endogenous glutamine synthetase (GS).SOLUTION: The present invention provides a method comprising the steps of: (a) mutating or knocking out the endogenous DHFR gene and the GS gene in a HEK cell line; (b) stably or transiently transfecting the HEK cell line with an AAV vector genome comprising a first heterologous nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence and a first selectable marker; and (c) stably or transiently transfecting the HEK cell line with a second heterologous nucleic acid sequence and a second selectable marker.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] [Background Art]

[0002] Glutamine synthetase (GS) is an enzyme in the synthesis of the amino acid L-glutamine. Therefore, GS-negative cell lines are auxotrophic for L-glutamine. GS has been reported as a selectable marker gene in CHO cells based on recombinant protein expression systems (Wurm et al. (2004) Nature Biotechnology 22: 1393-1398). An expression cassette containing the GS gene can be selected using the GS inhibitor methionine sulfoximine when the cassette is introduced into a GS-negative CHO strain.

[0002]

[0003] Dihydrofolate reductase (DHFR, 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase) is an enzyme in both eukaryotic and prokaryotic cells and catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate (an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, glycine, and methyl compounds). DHFR-deficient cells grow only in a medium supplemented with certain factors involved in folate metabolism or when DHFR is provided to the cells, for example, as a transgene.

[0003] [Summary of the Invention]

[0004] Cells and cell lines capable of producing therapeutic proteins, antibodies, vectors, and viral vectors, such as lentiviral vectors and adeno-associated virus (AAV) vectors, are disclosed herein. The cells and / or cell lines can have mutations or deletions such that the expression or function of DHFR and / or GS is substantially reduced or eliminated in one or both of the endogenous dihydrofolate reductase (DHFR- / -) or glutamine synthetase (GS- / -) genes.

[0004]

[0005] Reduction can be achieved, for example, by knockout of a single allele of the DHFR and / or GS gene. Reduction can be achieved by mutations (e.g., substitutions or deletions) in the DHFR and / or GS gene that reduce the function or activity of the corresponding protein. Elimination can be achieved by knockout of both alleles of the DHFR and / or GS gene.

[0005]

[0006] In certain embodiments, the cells and / or cell lines of the invention are based on or derived from human embryonic kidney (HEK) cells or cell lines such as HEK293. Human embryonic kidney (HEK) cells or cell lines such as HEK293 have mutations or deletions in one or both of the endogenous dihydrofolate reductase (DHFR- / -) or glutamine synthetase (GS- / -) genes such that the expression and / or function of the DHFR and / or GS protein is substantially reduced or eliminated, as disclosed herein.

[0006]

[0007] In certain embodiments, the cells and / or cell lines of the invention are based on or derived from human adenocarcinoma alveolar basal epithelial cells or cell lines. Human A459 cells or cell lines have mutations or deletions in one or both of the endogenous dihydrofolate reductase (DHFR- / -) or glutamine synthetase (GS- / -) genes such that the expression and / or function of the DHFR and / or GS protein is substantially reduced or eliminated, as disclosed herein.

[0007]

[0008] In certain embodiments, the cells and / or cell lines of the invention are based on or derived from African green monkey kidneys. Vero cells or cell lines have mutations or deletions in one or both of the endogenous dihydrofolate reductase (DHFR- / -) or glutamine synthetase (GS- / -) genes such that the expression and / or function of the DHFR and / or GS protein is substantially reduced or eliminated, as disclosed herein.

[0008]

[0009] Cell lines can be selected (cloned) from individual cells. The clone can be expanded and, in a forward manner, HEK cells having a mutation or deletion such that the expression and / or function of DHFR and / or GS is substantially reduced or eliminated in one or both of the endogenous dihydrofolate reductase (DHFR- / -) or glutamine synthetase (GS- / -) genes, such as HEK293, human A459 cells, and / or Vero cells, can be provided as a stable cell line.

[0009]

[0010] In some aspects herein, human embryonic kidney (HEK) cells, human A459 cells, and Vero cells that do not express a functional endogenous dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS) are disclosed. In one aspect herein, a human embryonic kidney (HEK) cell line, a human A459 cell line, and a Vero cell line that do not express a functional endogenous dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS) are presented.

[0010]

[0011] In some embodiments, HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines are stably or transiently transfected with a first heterologous nucleic acid sequence and optionally stably or transiently transfected with a second heterologous nucleic acid sequence. In some embodiments, HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines are stably or transiently transfected with a first heterologous nucleic acid sequence and a first selectable marker and optionally stably or transiently transfected with a second heterologous nucleic acid sequence and a second selectable marker. In certain embodiments, the first heterologous nucleic acid sequence encodes a therapeutic protein or polynucleotide sequence, and in certain embodiments, the second heterologous nucleic acid sequence encodes a therapeutic protein or polynucleotide sequence. The therapeutic protein or polynucleotide sequence encoded by the first heterologous nucleic acid sequence and the optional therapeutic protein or polynucleotide sequence encoded by the second heterologous nucleic acid sequence may be the same or different. In some embodiments, the first and / or second selectable marker does not confer resistance to an antibiotic. In certain embodiments, the first and / or second selectable marker provides a means for amplifying the first and / or second heterologous nucleic acid sequence. In some aspects, the first and / or second selectable marker comprises a nucleic acid encoding a protein having DHFR function. In some aspects, the first and / or second selectable marker comprises a nucleic acid encoding a protein having GS function. In some embodiments, the first selectable marker comprises a nucleic acid encoding a protein having DHFR function and the second selectable marker comprises a nucleic acid encoding a protein having GS function.

[0011]

[0012] In some embodiments of the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein, the first heterologous nucleic acid sequence comprises a first vector, and in some embodiments, an optional second heterologous nucleic acid sequence comprises a second vector. The first vector and the optional second vector may be the same or different. In some embodiments, the first vector and the optional second vector each comprise a selectable marker comprising a nucleic acid encoding a protein having DHFR function or a nucleic acid encoding a protein having GS function. In certain embodiments, the first vector comprises a first viral vector and the optional second vector comprises a second viral vector. In some embodiments, the first and / or second viral vectors comprise an AAV vector genome. In some embodiments where the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines comprise the first and second viral vectors, each of the viral vectors comprises an AAV vector genome or a portion thereof. In certain embodiments, the AAV vector genome comprises one or two AAV ITRs flanking the 5' and / or 3' end of the heterologous nucleic acid sequence.

[0012]

[0013] In one aspect, the copy number of the heterologous nucleic acid sequence and / or vector and / or viral vector and / or AAV vector genome in HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines is between 10 and 5000 copies per cell. In some embodiments, the copy number of the heterologous nucleic acid sequence and / or vector and / or viral vector and / or AAV vector genome in HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines is 1 to 5 copies per cell, 5 to 10 copies per cell, 10 to 50 copies / cell, 50 to 100 copies per cell, 100 to 250 copies per cell, 250 to 500 copies per cell, 500 to 1,000 copies per cell, 1,000 to 2,000 copies per cell, or between about 2,000, 3,000, 4,000 or 5,000 copies per cell or more. In one embodiment, the copy number of the AAV vector genome in HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines is at least 1,000 copies per cell, and the yield of rAAV vector particles is at least 1×10 8 vg / ml, at least 1×10 9 vg / ml, at least 1×10 10 vg / ml, at least 1×10 11 vg / ml or at least 2×10 11 vg / ml. In some embodiments, the copy number appears to be stable over many passages, e.g., at least 5, 10, 15, 20, 30, 40, 50, or more passages, and the production of the AAV vector is stable and consistent, e.g., within about 10 to 30% of the amount produced from any fewer cell passages.

[0013]

[0014] In some embodiments, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines presented herein further comprise the rep and / or cap sequences of AAV. In some embodiments, the rep and / or cap sequences of AAV are provided by a plasmid transiently or stably transfected into HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines. In some embodiments, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines presented herein further comprise the helper function sequences of AAV.

[0014]

[0015] In one embodiment, the HEK cells or HEK cell line presented herein is HEK293.

[0015]

[0016] In some embodiments, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines presented herein are in a culture or growth medium or in a medium suitable for long-term storage. In some embodiments, the culture medium or growth medium contains methotrexate (MTX) and / or methionine sulfoximine (MSX).

[0016]

[0017] In one aspect, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines presented herein produce rAAV vector particles that package one or more heterologous nucleic acid sequences (e.g., the first and / or second heterologous nucleic acids described herein). In some embodiments, the rAAV vector particles are produced in a greater amount than the amount produced by HEK293 cells transiently transfected with an AAV vector genome having a heterologous nucleic acid sequence that expresses a functional endogenous DHFR and / or GS. In certain embodiments, the produced AAV vector particles contain an empty capsid of rAAV and / or a lesser amount (e.g., at least 1%, less than at least 10%, or less than at least one-half) of rAAV empty capsids and / or a lesser amount (e.g., at least 1%, less than at least 10%, or less than at least one-half) of rAAV particles that package contaminating DNA produced by HEK293 cells transiently transfected with an rAAV vector genome that expresses a functional endogenous DHFR and / or GS and has a heterologous nucleic acid sequence.

[0017]

[0018] In one aspect, the heterologous nucleic acid sequence encodes one or more therapeutic proteins. In one aspect, the heterologous nucleic acid sequence encodes one or more inhibitory factors. In some embodiments, the heterologous nucleic acid sequence comprises one or more inhibitory nucleic acid sequences. In some embodiments, the heterologous nucleic acid sequence encodes a therapeutic protein and / or comprises an inhibitory nucleic acid sequence. In certain embodiments, the therapeutic protein comprises a blood coagulation factor. In certain embodiments, the therapeutic protein comprises an immunoglobulin sequence (e.g., the amino acid sequence of an immunoglobulin). In some embodiments, the inhibitory nucleic acid sequence comprises small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA.

[0018]

[0019] In one aspect, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein are stably transfected with a first heterologous nucleic acid sequence. In some embodiments, the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein are stably transfected with a first and / or a second heterologous nucleic acid sequence.

[0019]

[0020] In some aspects, virus or rAAV vector particles isolated and / or purified from the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein are presented herein.

[0020]

[0021] In some aspects, therapeutic proteins isolated and / or purified from the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein are presented herein.

[0021]

[0022] In some aspects, methods of manufacturing a therapeutic protein, viral vector, and / or rAAV vector particle, comprising culturing the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein under conditions that allow for the production and / or secretion of the therapeutic protein, viral vector, or rAAV vector particle described herein, and isolating or purifying the therapeutic protein, viral vector, or rAAV vector particle from the cell culture, culture medium, or cell culture and culture medium (e.g., the cell culture, culture medium, or cell culture and culture medium comprising the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines) are presented herein.

[0022]

[0023] In some aspects, a method for producing rAAV vector particles includes culturing the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines described herein under conditions that allow for the production and / or secretion of rAAV vector particles, and isolating or purifying rAAV vector particles from the cell culture, culture medium, or both the cell culture and the culture medium, wherein the HEK cells or cell lines, human A459 cells or cell lines, and / or Vero cells or cell lines have at least 1,000 copies of the AAV vector genome per cell, and the rAAV vector particle yield is at least 1×10 8 vg / ml, or at least 1×10 9 vg / ml, or at least 1×10 10 vg / ml, or at least 1×10 11 vg / ml or at least 2×10 11 vg / ml, as presented herein.

[0023]

[0024] In some embodiments, the first and / or second heterologous nucleic acid sequences described herein encode gene products selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase.

[0024]

[0025] In one aspect, the HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines, or rAAV vector particles described herein, encode a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase, and the methods described herein that include the first and / or second heterologous nucleic acid sequences are presented herein.

[0025]

[0026] In one aspect, provided herein is a method described herein, wherein HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines, or a first and / or a second heterologous nucleic acid sequence encode a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 - IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules.

[0026]

[0027] In one aspect, provided herein is a method described herein, wherein HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines, or rAAV vector particles contain a first and / or a second heterologous nucleic acid sequence encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 - IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules.

[0027]

[0028] In one aspect, the HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines, or the first and / or second heterologous nucleic acid sequences described herein, that encode a protein useful for the correction of inborn errors selected from the group consisting of carbamoyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionine beta-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence, are presented herein by the methods described herein.

[0028]

[0029] In one aspect, the HEK cells or cell lines, human A459 cells or cell lines and / or Vero cells or cell lines, or rAAV vector particles described herein, or a method described herein that includes first and / or second heterologous nucleic acid sequences encoding a protein useful for correcting a congenital error selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionine beta-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence is presented herein.

[0029]

[0030] In some embodiments, a method of producing a human embryonic kidney (HEK) cell line, a human A459 cell line, and a Vero cell line that do not express a functional endogenous dihydrofolate reductase (DHFR), the method comprising mutating or knocking out the endogenous DHFR gene is presented herein.

[0030]

[0031] In some embodiments, a method of producing a human embryonic kidney (HEK) cell line, a human A459 cell line, and a Vero cell line that do not express a functional endogenous glutamine synthetase (GS), the method comprising mutating or knocking out the endogenous GS gene is presented herein.

[0031]

[0032] In some embodiments, provided herein is a method for producing human embryonic kidney (HEK) cell lines, human A459 cell lines, and Vero cell lines that do not express functional endogenous dihydrofolate reductase (DHFR) and glutamine synthetase (GS), the method comprising the step of mutating or knocking out the endogenous DHFR gene and GS gene.

Brief Description of the Drawings

[0032]

Figure 1

Figure 2

[0033] [Modes for Carrying Out the Invention]

[0035] Clones of HEK cells such as HEK293 cells, in which such genes have been modified or knocked out, are the first clones of human cells having a DHFR− / − and / or GS− / − genomic background. These cells and cell lines can be used to produce many different recombinant biological materials, such as recombinant proteins (e.g., antibodies such as monoclonal antibodies) and viral vectors. The recombinant proteins and viral vectors produced can be used for the treatment of diseases. In certain applications, the viral vectors produced (e.g., lentiviral or AAV) can be used for the application of knock-in (e.g., introducing an abnormal or missing functional protein) gene therapy. In certain applications, the viral vectors produced (e.g., lentiviral or AAV) can be used for the application of knock-out (e.g., introducing an inhibitory sequence such as antisense that targets an endogenous protein with abnormal or undesirable expression or function, e.g., a mutant protein that causes or is associated with a medical condition or disease) gene therapy.

[0034]

[0036] The present invention provides a benefit for the production by a gene amplification system in a well-characterized human cell line of biological agents, such as proteins, and other biological materials including recombinant proteins, antibodies, AAV, lentivirus, and other viral vectors including other viruses. An additional benefit relates to the production of proteins that require the human intracellular environment for folding, modification (post-translation), and function.

[0035]

[0037] The present invention creates a new production system using well-characterized human cells or cell lines. The parental clones selected to establish rAAV-producing cell lines are engineered from HEK (e.g., HEK293) cells, human A459 cells or Vero cells in which the DHFR and / or GS genes are substantially reduced or eliminated, such as a single or double knockout of the DHFR and / or GS genes. Human HEK (e.g., HEK293), human A459 and / or Vero cells or cell lines in which the DHFR and / or GS genes are substantially reduced or eliminated, e.g., the single or double knockout of the DHFR and / or GS genes of the present invention, allows post-translational modification of biological products closer to their native modification in humans and will therefore improve the safety and biological activity of biological products.

[0036]

[0038] Eliminating (e.g., knocking out) one or both of the DHFR and GS genes creates one or two selectable markers for HEK293 cells, human A459 cells and / or Vero cells. The HEK cells and cell lines, human A459 cells and cell lines and / or Vero cells and cell lines of the invention in which a selectable marker for DHFR is stably integrated can be selected by culturing the cells in a culture medium. Moreover, when a heterologous nucleic acid sequence different from the selectable marker for DHFR (e.g., two separate plasmids) or as a single polynucleotide sequence (e.g., in the same plasmid such as an AAV vector plasmid) is introduced into one cell, both can be integrated. Thus, if the DHFR transgene adds or contains a heterologous nucleic acid sequence (e.g., encoding a protein or nucleic acid of interest), cells expressing both DHFR and the protein or nucleic acid of interest can be selected.

[0037]

[0039] The HEK cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention in which a selectable marker of GS is stably integrated can be selected by culturing the cells in a culture medium. When a heterologous nucleic acid sequence different from the selectable marker of GS (for example, two separate plasmids) or as a single polynucleotide sequence (for example, in the same plasmid such as an AAV vector plasmid) is introduced into one cell, both can be integrated. Therefore, when the GS transgene adds or contains a heterologous nucleic acid sequence (for example, encoding a protein or nucleic acid of interest), cells expressing both GS and the protein or nucleic acid of interest can be selected.

[0038]

[0040] In response to inhibitors such as methotrexate (MTX), the copy number of the DHFR gene, and thus the heterologous nucleic acid sequence integrated adjacent to the DHFR gene, can be amplified in the HEK cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention. In response to inhibitors such as methionine sulfoximine (MS), the copy number of the GS gene, and thus the heterologous nucleic acid sequence integrated adjacent to the GS gene, can be amplified in the HEK cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention.

[0039]

[0041] Therefore, the sequence encoding the protein or nucleic acid of interest integrated adjacent to or together with the exogenous DHFR and / or GS can be amplified by gradually exposing the cells to increasing concentrations of MTX and / or MS, resulting in increased expression of the encoded protein or nucleic acid of interest.

[0040]

[0042] The research disclosed in this specification shows that HEK293 clones with high copy numbers of the rAAV genome were obtained. In certain examples, the rAAV genome encodes copies of the rAAV genome that reached levels exceeding a thousand copies, significantly higher than the most stable cell line reported for therapeutic human FIX. The high copy number of the rAAV genome resulted in high rAAV-hFix production. The high copy number appears to be stable over many passages, for example, at least 5, 10, 15, 20, 30, 40, 50, or more passages.

[0041]

[0043] In addition to high rAAV production, the quality of rAAV preparations produced from cell lines that produce HEK293 rAAV vectors was also evaluated. High copies of the rAAV genome in stable clones of HEK293 appear to be able to further reduce DNA impurities packaged in rAAV particles, thereby reducing the ratio of empty particles to vectors with packaged genomes.

[0042]

[0044] In addition to the high copy number of the rAAV genome in HEK293 clones that produce stably, the HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the present invention also enable gene amplification through the induction of MTX and / or MSX, which will further amplify the rAAV genome in HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines. The amplification increases the number of rAAV genomes in a forward manner, thereby increasing rAAV production by the engineered HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines.

[0043]

[0045] The HEK cells or cell lines of the invention, human A459 cells and cell lines, and / or Vero cells and cell lines, e.g., the knock-in clones of HEK cells (single DFHR- / - or GS- / -, or double DFHR- / - / GS- / - of HEK293), can be used to produce rAAV vectors of any AAV serotype. For example, the HEK cells or cell lines of the invention, human A459 cells or cell lines, and / or Vero cells or cell lines, e.g., the knock-in clone of rAAV-hFix DFHR- / - / GS- / - of HEK293 of a vector of a virus (e.g., AAV), can be used to produce rAAV-hFix vectors of any AAV serotype, and the vector production can be used for the treatment of hemophilia B by gene therapy.

[0044]

[0046] Certain non-limiting methods for using the HEK cells or cell lines of the present invention to produce vectors of viruses (e.g., AAV) are described in U.S. Patent Application Publication No. 2013 / 0072548 (U.S. Patent Application No. 13 / 561,753); U.S. Patent Application Publication No. 2014 / 0349403 (U.S. Patent Application No. 14 / 364,623); and U.S. Patent Application Publication No. 2014 / 0323556 (U.S. Patent Application No. 14 / 216,778).

[0045]

[0047] "Selectable marker" refers to a polynucleotide or gene that, when introduced under appropriate selective culture conditions and expressed by cells, enables the selection of cells expressing the selectable marker. The selectable marker can be DHFR and / or GS, in particular, a protein having the function or activity of DHFR and / or GS, e.g., a polynucleotide or gene encoding a DHFR and / or GS protein expressed by or in the HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention.

[0046]

[0048] All mammalian and non-mammalian forms of the DHFR protein and GS protein, as well as the encoding nucleic acids, are clearly included. Suitable DHFR and GS proteins, and thus genes known to those skilled in the art, can be used. The DHFR and GS proteins can be derived from any species as long as they retain at least partial function or activity in the HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention. The DHFR and / or GS proteins include naturally occurring polymorphs.

[0047]

[0049] DHFR and / or GS may be wild-type DHFR and / or GS or their functional variants or derivatives. The terms "variant" or "derivative" refer to DHFR and / or GS proteins having one or more amino acid sequence exchanges (e.g., deletions, substitutions, or additions) with respect to the amino acid sequences of the respective DHFR and / or GS proteins, fusion proteins containing the DHFR and / or GS proteins, or their functional fragments (this term may also refer to nucleic acid sequences encoding such proteins). Variants include DHFR and / or GS proteins that retain at least partial function or activity of the DHFR and / or GS proteins.

[0048]

[0050] Variants and derivatives of the DHFR and / or GS proteins that are modified to provide additional structure and / or function, as well as the aforementioned functional fragments that still have at least one function of the DHFR and / or GS proteins. For example, the DHFR and / or GS proteins can also be used as selectable markers that are more or less sensitive to antifolate drugs such as MTX, or more or less sensitive to MS, compared to the wild-type DHFR or GS proteins respectively, and / or the DHFR or GS proteins endogenously expressed by HEK (e.g., HEK293) cells, human A459 cells, and / or Vero cells.

[0049]

[0051] For example, the DHFR protein is used as a selectable marker that is more sensitive than the endogenous DHFR enzyme expressed in the HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines, and / or Vero cells and cell lines of the invention to DHFR inhibitors such as MTX. Such DHFR provides, in a forward direction, a means for the selectable marker of DHFR and for the strong amplification of heterologous nucleic acid / vector sequences in a forward direction.

[0050]

[0052] The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or integration of nucleic acids. Vectors can be used for genetic manipulation (i.e., "cloning the vector") to introduce / move polynucleotides into cells and to transcribe or translate the inserted polynucleotides in cells. An "expression vector" is a vector that contains a gene or nucleic acid sequence having regulatory regions necessary for expression in a host cell. Vector nucleic acid sequences generally contain at least an origin of replication for propagation in cells and optionally additional elements such as heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), optionally selectable markers (e.g., DHFR, GS, etc.), polyadenylation signals.

[0051]

[0053] Viral vectors are derived from or based on one or more nucleic acid elements containing the genome of a virus. Certain viral vectors include lentiviruses, pseudotyped lentiviruses, and parvovirus vectors, such as adeno-associated virus (AAV) vectors. Parvoviruses, including AAV, can enter cells and introduce nucleic acid / gene material, such that the nucleic acid / gene material can be stably maintained in the cells, and thus are useful as gene therapy vectors. In addition, these viruses can introduce nucleic acid / gene material into specific sites, such as specific sites on chromosome 19. Since AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenicity or disease.

[0052]

[0054] The term "recombinant" as a modified gene of a vector, e.g., of a recombinant virus, such as a lentiviral or parvovirus (e.g., rAAV) vector, and as a modified gene of sequences such as recombinant polynucleotides and polypeptides, generally means that the composition has been engineered (i.e., altered) in a manner that does not occur naturally. A specific example of a recombinant vector, such as an AAV vector, would be when a polynucleotide that is not normally present in the genome of a wild-type virus (e.g., AAV) is inserted into the genome of the virus. Examples of recombinant vectors would be when a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into the vector, with or without the 5', 3' and / or intron regions normally associated with the gene within the genome of a virus (e.g., AAV). Although the term "recombinant" is not necessarily used herein with respect to vectors, such as viral and AAV vectors, and sequences such as polynucleotides, recombinant forms containing polynucleotides are clearly included notwithstanding any such omission.

[0053]

[0055] A "vector" or "rAAV vector" of a recombinant virus is derived from the wild-type genome of a virus (e.g., AAV) by using a molecular method that removes the wild-type genome from the virus and replaces it with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for AAV, the inverted terminal repeat (ITR) sequences of the AAV genome are retained in the rAAV vector. A "recombinant" viral vector (e.g., rAAV) is clearly distinguishable from the genome of a virus (e.g., AAV) because all or part of the viral genome has been replaced with a non-native sequence, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, with respect to the nucleic acid of the virus (e.g., AAV) genome. Therefore, the incorporation of a non-native sequence defines the viral vector (e.g., AAV) as a "recombinant" vector and, in the case of AAV, can be referred to as an "rAAV vector".

[0054]

[0056] The sequences of recombinant vectors (e.g., lentiviral, parvovirus-, AAV) can be packaged and are herein referred to as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. When the recombinant vector sequence is capsid-formed or packaged in AAV particles, the particles can also be referred to as "rAAV". Such rAAV particles contain proteins that capsid-form or package the vector genome. Specific examples include viral envelope proteins and, in the case of AAV, capsid proteins.

[0055]

[0057] The vector “genome” refers to the portion of a recombinant plasmid sequence that is ultimately packaged or capsid - formed to form viral (e.g., rAAV) particles. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non - vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” and the plasmid backbone is important for the processes necessary for plasmid cloning and amplification, propagation, and recombinant virus production, but is not itself packaged or capsid - formed into viral (e.g., rAAV) particles. Thus, the vector “genome” refers to the nucleic acid that is packaged or capsid - formed by a virus (e.g., rAAV).

[0056]

[0058] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined on the basis of the lack of cross - reactivity between antibodies when comparing one AAV to another. The difference in cross - reactivity is usually due to differences in the sequence / epitopes of the capsid proteins (e.g., due to differences in the AAV serotype in the VP1, VP2, and / or VP3 sequences).

[0057]

[0059] Based on the conventional definition, a serotype means that the virus of interest has been tested for neutralizing activity against sera specific for all existing and characterized serotypes and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants arise, there may or may not be a serological difference from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, for mutant viruses with modified capsid sequences, serological tests for neutralizing activity must be further performed to determine whether they are of another serotype according to the conventional definition of serotype. Thus, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype and are not serologically distinct.

[0058]

[0060] Recombinant vectors (e.g., rAAV) include any viral strain or serotype. As non-limiting examples, the plasmid of a recombinant vector (e.g., AAV) or the genome or particle (capsid) of a vector (e.g., AAV) can be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, etc. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other. As non-limiting examples, the plasmid of a recombinant vector (e.g., rAAV) based on the genome of one serotype or the genome or particle (capsid) of a vector (e.g., AAV) can be identical to one or more of the capsid proteins that package the vector. In addition, the plasmid of a recombinant vector (e.g., AAV) or the genome of a vector (e.g., AAV) can be based on an AAV (e.g., AAV2) serotype genome that is distinct from one or more of the capsid proteins that package the vector genome, in which case at least one of the three capsid proteins can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or variants thereof. AAV vectors therefore contain gene / protein sequences identical to those characteristic of a particular serotype, as well as mixed serotypes.

[0059]

[0061] In various exemplary embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to the capsid protein of one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In various exemplary embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to the ITR of one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

[0060]

[0062] Recombinant vectors (e.g., rAAV), such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, among others, as well as variants, hybrids, and chimeric sequences, can be constructed using recombinant techniques known to those of skill in the art to contain one or more heterologous polynucleotide sequences (transgenes) adjacent to the ITR sequences of one or more functional AAVs. Such vectors are partially or completely deleted for, e.g., the rep and / or cap genes, but have one or more of the wild-type AAV genes that retain at least one functional adjacent ITR sequence necessary for rescuing, replicating, and packaging the recombinant vector in rAAV vector particles. The rAAV vector genome thus contains, in cis form, the sequences necessary for replication and packaging (e.g., functional ITR sequences).

[0061]

[0063] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and all forms of oligonucleotides. Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0062]

[0064] A "heterologous" nucleic acid sequence refers to a polynucleotide inserted into a vector (e.g., AAV) for the purpose of moving / delivering the polynucleotide into a cell, mediated by the vector. A heterologous nucleic acid sequence is typically distinct from the nucleic acid of the vector (e.g., AAV), i.e., it is non-native with respect to the nucleic acid of the virus (e.g., AAV). Once moved / delivered into a cell, the heterologous nucleic acid sequence contained within the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous polynucleotide contained within the vector that has been moved / delivered into a cell may not be expressed. The term "heterologous" is not limited to use herein in reference to nucleic acid sequences and polynucleotides, but references to a nucleic acid sequence or polynucleotide are intended to include heterologous nucleic acid sequences and polynucleotides, even in the absence of a "heterologous" transgene, notwithstanding the omission.

[0063]

[0065] The "polypeptides", "proteins" and "peptides" encoded by a "nucleic acid sequence" include, as a result, the naturally occurring proteins, as well as functional partial sequences, modified forms or sequence variants, provided that the modified forms or variants retain to some extent the functionality of the native full-length protein, including the native full-length sequence. Such polypeptides, proteins and peptides encoded by a nucleic acid sequence can be identical to the endogenous proteins that are defective, or insufficiently or incompletely expressed, in the mammal to be treated, but they need not be.

[0064]

[0066] The term "transgene" is used herein for convenience to refer to a nucleic acid (e.g., heterologous) that is intended for or has been introduced into a cell or organism. A transgene can include any nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence.

[0065]

[0067] In a cell having a transgene, the transgene has been introduced / moved by transfection or transduction of the cell, e.g., by a plasmid or vector such as AAV. The terms "transduction" and "transfect" refer to the introduction of a molecule such as a nucleic acid into a cell (e.g., HEK293) or host organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, the introduced nucleic acid can be stably maintained in that cell or organism and further passed on to or inherited by the progeny cells or organisms of the recipient cell or organism.

[0066]

[0068] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell means a cell in which there has been a genetic change after the incorporation of a foreign molecule, such as a nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced" cell is a cell into which a foreign nucleic acid has been introduced or its progeny. The cell can be propagated (cultured), and the introduced protein can be expressed by the cell, or the nucleic acid can be transcribed, or a vector such as rAAV can be produced. For use and methods of gene therapy, transduced cells can be present in a subject.

[0067]

[0069] With respect to a cell, the term "stable" or "stably integrated" as used herein means that a nucleic acid sequence such as a selectable marker or a heterologous nucleic acid sequence, or a plasmid or vector, is inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.), or is maintained extrachromosomally in a recipient cell or host organism and remains in the chromosome or is maintained extrachromosomally for a period of time. In the case of cultured cells, a nucleic acid sequence such as a selectable marker or a heterologous nucleic acid sequence, or a plasmid or vector inserted into a chromosome can be maintained over multiple cell passages.

[0068]

[0070] "Expression control element" refers to a nucleic acid sequence that affects the expression of an operably linked nucleic acid. Control elements include expression control elements such as promoters and enhancers described herein. A vector sequence including an rAAV vector can include one or more "expression control elements". Typically, such elements are included to facilitate transcription of an appropriate heterologous polynucleotide and, if appropriate, translation (e.g., promoter, enhancer, splicing signals for introns, maintenance of the correct reading frame of a gene that allows in-frame translation of the mRNA, and stop codons, etc.). Such elements typically act in cis form and are referred to as "cis-acting" elements, but can also act in trans form.

[0069]

[0071] Expression control can be affected at levels such as transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the nucleic acid being transcribed. Expression control elements can also be placed at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be adjacent to the transcribed sequence or at a distance (e.g., 1 - 10, 10 - 25, 25 - 50, 50 - 100, 100 - 500, or more nucleotides from the polynucleotide), even at a considerable distance. Nevertheless, due to the length limitation of a certain vector, e.g., the rAAV vector, expression control elements are typically within 1 - 1000 nucleotides from the nucleic acid being transcribed.

[0070]

[0072] Functionally, the expression of operably linked nucleic acids can be at least partially controlled by an element (e.g., a promoter), such that the element regulates the transcription of the nucleic acid and, where appropriate, the translation of the transcript. A specific example of an expression control element is a promoter, which is typically located 5' to the sequence being transcribed. A promoter typically increases the amount expressed from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0071]

[0073] As used herein, "enhancer" can refer to a sequence located adjacent to a nucleic acid sequence such as a selectable marker or a heterologous nucleic acid sequence. Enhancer elements are typically located upstream of promoter elements, but can also function and be located downstream or within the sequence. Therefore, enhancer elements can be located within 100 base pairs, 200 base pairs, or 300 or more base pairs of, either upstream or downstream of, e.g., a selectable marker and / or a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Enhancer elements typically increase the expression of an operably linked nucleic acid above that produced by a promoter element.

[0072]

[0074] The term "operably linked" means that the regulatory sequences necessary for the expression of a nucleic acid sequence are placed in an appropriate position relative to the sequence such that they have an effect on the expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in expression vectors, such as rAAV vectors.

[0073]

[0075] In an example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element is adapted to regulate the expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked are arranged in such a relationship (cis or trans) that at least one of the two DNAs can have a physiological effect on the other sequence.

[0074]

[0076] Thus, additional elements for a vector include, but are not limited to, expression control elements (e.g., promoters / enhancers), transcriptional termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) that are present adjacent to sequences such as the AAV ITR sequences, or one or more copies of an intron.

[0075]

[0077] Additional elements include, for example, polynucleotide sequences of fillers or stuffers for improving packaging and reducing the presence of contaminating nucleic acids. AAV vectors typically accept DNA inserts having a size range generally of about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, a stuffer or filler is included to adjust the length to near or at the normal size of the viral genomic sequence that is acceptable for packaging of the vector into rAAV particles. In various embodiments, the nucleic acid sequence of the filler / stuffer is a non-translated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 Kb, the polynucleotide sequence of the filler or stuffer has a length having an overall length between about 3.0 and 5.5 Kb, or between about 4.0 and 5.0 Kb, or between about 4.3 and 4.8 Kb when combined with the sequence (e.g., inserted into a vector).

[0076]

[0078] A "therapeutic protein" is, in one embodiment, a peptide or protein that can alleviate or reduce symptoms resulting from an insufficient amount, absence, or defect of a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can provide a benefit to a subject, for example, by correcting a genetic defect, correcting a (expression or functional) deficiency of a gene, and the like.

[0077]

[0079] Non-limiting examples of heterologous nucleic acids encoding gene products useful according to the present invention (e.g., therapeutic proteins) include "congestion" or blood coagulation disorders such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, coagulation factors VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor deficiencies, combined FV / FVIII deficiencies, thalassemia, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; anemia, trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation abnormalities, bleeding associated with disseminated intravascular coagulation (DIC); overcoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, low molecular weight antithrombotic agents (i.e., FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency, including but not limited to those that can be used in the treatment of diseases or disorders.

[0078]

[0080] Nucleic acid molecules, vectors, such as cloning vectors, expression vectors (e.g., vector genomes) and plasmids, can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information enables the preparation of nucleic acid molecules by various means. For example, the nucleic acid encoding factor IX (FIX) can be produced by various standard cloning, recombinant DNA techniques, cell expression or in vitro translation and chemical synthesis techniques. The purity of the polynucleotide can be determined by sequencing, gel electrophoresis, etc. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes that detect homologous nucleotide sequences; (2) antibody screening using, for example, expression libraries to detect polypeptides with shared structural features; (3) polymerase chain reaction (PCR) in genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtracted nucleic acid libraries.

[0079]

[0081] As disclosed herein, a method for producing a recombinant viral vector, such as an rAAV vector according to the present invention, is to express a heterologous nucleic acid encoding a therapeutic protein or polynucleotide in HEK (e.g., HEK293) cells or cell lines of the invention, human A459 cells or cell lines and / or Vero cells or cell lines. The cells or cell lines provide helper functions for packaging a viral (e.g., AAV) vector and produce rAAV under appropriate culture conditions. Accordingly, the present invention provides HEK (e.g., HEK293) cells and cell lines, human A459 cells and cell lines and / or Vero cells and cell lines for producing recombinant viral vectors such as rAAV vectors, and a method for manufacturing recombinant viral vectors such as rAAV vectors. The HEK (e.g., HEK293) cells or cell lines of the present invention, human A459 cells or cell lines and / or Vero cells or cell lines include the expression of a heterologous nucleic acid and simultaneously provide helper functions for packaging a viral (e.g., AAV) vector. The method includes the expression of a heterologous nucleic acid in HEK (e.g., HEK293) cells or cell lines of the invention, human A459 cells or cell lines and / or Vero cells or cell lines that provide helper functions for packaging a viral (e.g., AAV) vector.

[0080]

[0082] As also disclosed herein, a method for producing a recombinant protein according to the present invention is to express a nucleic acid encoding such a protein in HEK (e.g., HEK293) cells or cell lines of the invention, human A459 cells or cell lines and / or Vero cells or cell lines. Accordingly, the present invention also provides cells for producing a recombinant protein and a method for producing a recombinant protein. The HEK (e.g., HEK293) cells or cell lines of the present invention, human A459 cells or cell lines and / or Vero cells or cell lines include the expression of a nucleic acid encoding a recombinant protein. The method includes the expression of a nucleic acid encoding a recombinant protein in HEK (e.g., HEK293) cells or cell lines of the invention, human A459 cells or cell lines and / or Vero cells or cell lines.

[0081]

[0083] When used with respect to a modified gene of a composition, the term "isolated" means that the composition is made by human hand or is separated from its in vivo environment, either completely or at least in part, from what occurs in nature. Generally, an isolated composition is substantially free of one or more materials that are normally associated therewith, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0082]

[0084] With respect to proteins, the terms "isolated protein" or "isolated and purified protein" are sometimes used herein. This term primarily refers to a protein produced by the expression of a nucleic acid molecule. Alternatively, this term may refer to a protein that is sufficiently separated from other proteins that are naturally associated therewith and thus exists in a "substantially pure" form as a result.

[0083]

[0085] The term "isolated" does not exclude combinations made by human hand, such as recombinant vector sequences, or virus particles that package or capsid a vector genome (e.g., rAAV) and pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms of a composition, such as hybrid / chimeric, multimer / oligomer, modified (e.g., phosphorylated, glycosylated, lipid-modified) or derivatized forms, or forms expressed in a host cell made by human hand.

[0084]

[0086] When referring to a particular nucleotide sequence or amino acid sequence, the phrase "consisting essentially of" means a sequence having the properties of the given sequence. For example, when used with respect to an amino acid sequence, the phrase includes molecular modifications that do not affect the sequence itself and its essential and novel properties.

[0085]

[0087] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0086]

[0088] All patent applications, publications, patents, and other references cited herein, as well as GenBank citations and ATCC citations, are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0087]

[0089] All features disclosed in this specification may be combined in any combination. Each feature disclosed herein may also be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless otherwise explicitly stated, the disclosed features (e.g., nucleic acid sequences, vectors, viral vectors, rAAV vectors, etc.) are examples of classes of equivalent or similar features.

[0088]

[0090] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "a nucleic acid sequence" or "a selectable marker" include a plurality of such nucleic acid sequences and selectable markers, and references to "a vector" include a plurality of such vectors, such as rAAV vectors.

[0089]

[0091] All numerical values or ranges of numbers used in this specification, unless otherwise clearly indicated in the context, include integers within such ranges and fractions or integers of values within such ranges. Thus, by way of example, a reference to an identity of 80% or more includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0090]

[0092] A reference to an integer with "more than" or "less than" includes any number greater than or less than the number mentioned, respectively. Thus, for example, a reference to less than 100 includes 99, 98, 97, etc., all the way down to the number 1; less than 10 includes 9, 8, 7, etc., all the way down to the number 1.

[0091]

[0093] All numerical values or ranges used in this specification are inclusive. Further, all numerical values or ranges, unless otherwise clearly indicated in the context, include fractions and integers of values within such ranges, as well as fractions of integers within such ranges. Thus, by way of example, a reference to a range of numbers such as 1 to 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Therefore, a reference to the range of 1 to 50 includes up to and including 50, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc. (and so on).

[0092]

[0094] References to a series of ranges include ranges that combine the boundary values of different ranges within that series of ranges. Thus, by way of example, references to a series of ranges such as 1 - 10, 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60, 60 - 75, 75 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 400, 400 - 500, 500 - 750, 750 - 1,000, 1,000 - 1,500, 1,500 - 2,000, 2,000 - 2,500, 2,500 - 3,000, 3,000 - 3,500, 3,500 - 4,000, 4,000 - 4,500, 4,500 - 5,000, 5,500 - 6,000, 6,000 - 7,000, 7,000 - 8,000, or 8,000 - 9,000 include ranges such as 10 - 50, 50 - 100, 100 - 1,000, 1,000 - 3,000, 2,000 - 4,000, etc.

[0093]

[0095] The present invention is generally disclosed herein using affirmative language, and numerous embodiments and aspects are described. The present invention also specifically includes embodiments in which certain subject matters such as substances or materials, method steps and conditions, protocols, or procedures are excluded in whole or in part. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Thus, with respect to what is not included in the present invention, even if the present invention is not generally expressed herein, nevertheless, aspects that are not expressly excluded by the present invention are disclosed herein.

[0094]

[0096] Some embodiments of the present invention are described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention, and adapt the present invention to various uses and conditions. Therefore, the following examples are intended to be illustrative but not intended to limit the scope of the claimed present invention.

Examples

[0095] Example 1 This example describes the production of the HEK cells and cell lines of the invention, and subsequent transfer of the viral genome and production of the viral (AAV) vector.

[0096]

[0097] The HEK cells and cell lines of the invention can be produced by knocking out the endogenous DHFR gene and / or GS gene of the cells in various ways. For example, one non-limiting method involves zinc finger nucleases (ZFNs) for targeted cleavage and gene inactivation (see, for example, U.S. Patent Application Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; 2008 / 0015164; and International Publication No. 07 / 014275). ZFNs provide the ability to define the location of double-strand DNA breaks (DSBs) at selected genomic positions. Removal of this site-specific DSB is carried out by the cell's own DNA repair machinery through homologous recombination repair processes or by non-homologous end joining (NHEJ) when donor DNA is provided. See, for example, Urnov et al. (2005) Nature 435:646-651 (2005); Moehle et al. (2007) Proc Natl Acad Sci USA 104:3055-3060 (2007); Bibikova et al. (2001) Mol Cell Biol 21:289-297; Bibikova et al. (2003) Science 300:764; Porteus et al. (2005) Nature Biotechnology 23:967-973; Lombardo et al. (2007) Nature Biotechnology 25:1298-1306; Perez et al. (2008) Nature Biotechnology 26:808-816; Bibikova et al. (2002) Genetics 161:1169-1175; Lloyd et al. (2005) Proc Natl Acad Sci USA 102:2232-2237; Morton et al. (2006) Proc Natl Acad Sci USA 103:16370-16375.

[0097]

[0098] CRISPR / Cas9 editing is another method that can be used to produce HEK, human A459 and / or Vero cells, cell lines and cell clones of the invention with reduced expression of endogenous DHFR and / or GS, or with knockout of the endogenous DHFR gene and / or GS gene. The CRISPR / Cas9 system for modification / deletion of the targeted gene has been described in detail (see, for example, Qi LS et al. Cell. 152(5), 1173-1183 (2013); Cong L. et al. Science. 339(6121), 819-823 (2013); Hsu PD et al. Cell. 157(6), 1262-1278 (2014); Hsu PD et al. Nat Biotechnol. 31(9), 827-832 (2013); and Doudna JA, Charpentier E. Science 346(6213), 1258096 (2014)).

[0098]

[0099] Constructs of rAAV-hFix related to either the DHFR gene expression cassette or the GS expression cassette were constructed and transfected into the HEK293 DHFR- / - / GS- / - cell line. Stable clones were isolated using MTX or MSX as selection markers. Clones containing a high copy number of the rAAV-hFiX genome and producing more rAAV vectors were maintained for further characterization. Some of the isolated cell clones showed stable and high AAV production (Figure 2).

[0099] Example 2 This example illustrates certain non-limiting features of the HEK cells and cell lines of the invention.

[0100]

[0100] For example, generally,[[]] A. Clones of stable producers. B. Manufactured cell clones of human mammals with knocked-out DHFR and / or GS genes use the DHFR or GS gene (or both) as a selection marker to enable the selection of stable clones expressing the gene of interest, and the clones can create any gene of interest or viral vector. C. More stable production of suitable human cell lines for producing biological products with gene amplification functions that provide high productivity. D. The DHFR and / or GS-negative genomic background of the created cell line enables gene amplification of the gene of interest, thus leading to high specific productivity. E. A genomic background without contamination, no need to introduce antibiotic markers to select positive producer clones, and thus more stable clones for producing recombinant proteins, viral vectors such as rAAV vectors. F. Significantly reduced labor costs and material costs.

[0101]

[0101] For example, for rAAV production, A. Improved rAAV yield B. Ease of scale-up for large-scale production / mass production of rAAV. C. A more stable production system compared to production systems involving helper viruses, such as production systems using adenovirus as a helper or baculovirus-based production systems. D. Producing rAAV vectors with a low amount of empty capsids by reducing empty particles and thus reducing DNA impurities packaged in the rAAV vector. E. High copy number of the rAAV genome achieved for a certain cell clone, the identified high rAAVhFix producer clone. F. Double knock-in of DHFR / GS using the rAAVhFix genome enables substantial amplification of the rAAVhFix genome, thus leading to high rAAV production.

[0102]

[0102] For example, with respect to protein production, A. HEK, human A459 and / or Vero cells and cell lines can fold and modify biological products closer to their native state when produced in humans, and thus, the production of products from HEK, human A459 and / or Vero cells and cell lines is more stable and stronger. B. HEK (e.g., HEK293) and A549 cells are human cell lines and not of another non-human species, so biological products produced from HEK (e.g., clones of the created HEK293DHFR- / - / GS- / - cells), and / or human A459 allow for post-translational modifications closer to their native products in the human body.

[0103] [Related Applications]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 315,480, filed March 30, 2016. The entire contents of the foregoing application are hereby incorporated by reference herein, including the text, tables, sequence listings, and drawings in their entirety.

[0104]

[0103] The present invention may be as follows. (1) Human embryonic kidney (HEK) cells that do not express functional endogenous dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS). (2) Human embryonic kidney (HEK) cell lines that do not express functional endogenous dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS). (3) The HEK cells or cell lines according to (1) or (2), stably or transiently transfected with a first heterologous nucleic acid sequence, and optionally, stably or transiently transfected with a second heterologous nucleic acid sequence. (4) The HEK cell or cell line according to (1) or (2), which is stably or transiently transfected with a first heterologous nucleic acid sequence and a first selectable marker, and optionally stably or transiently transfected with a second heterologous nucleic acid sequence and a second selectable marker. (5) The HEK cell or cell line according to (3) or (4), wherein the first heterologous nucleic acid sequence encodes a therapeutic protein or polynucleotide sequence, and the optional second heterologous nucleic acid sequence encodes a therapeutic protein or polynucleotide sequence. (6) The HEK cell or cell line according to (4), wherein the therapeutic protein or polynucleotide sequence encoded by the first heterologous nucleic acid sequence is the same as or different from the therapeutic protein or polynucleotide sequence encoded by the optional second heterologous nucleic acid sequence. (7) The HEK cell or cell line according to (4), wherein the first or second selectable marker does not confer resistance to an antibiotic. (8) The HEK cell or cell line according to (4), wherein the first or second selectable marker provides a means for amplifying the first and / or second heterologous nucleic acid sequences. (9) The HEK cell or cell line according to (4), wherein the first or second selectable marker comprises a nucleic acid encoding a protein having DHFR function. (10) The HEK cell or cell line according to (4), wherein the first or second selectable marker comprises a nucleic acid encoding a protein having GS function. (11) The HEK cell or cell line according to (4), wherein the first selectable marker comprises a nucleic acid encoding a protein having DHFR function, and the second selectable marker comprises a nucleic acid encoding a protein having GS function. (12) The HEK cell or cell line according to any one of (3) to (11), wherein the first heterologous nucleic acid sequence comprises a first vector, and the optional second heterologous nucleic acid sequence comprises a second vector. (13) The HEK cell or cell line according to (11) or (12), wherein the first vector and the optional second vector are the same or different. (14) The HEK cell or cell line according to (11) or (12), wherein the first vector and the optional second vector each comprise a selectable marker comprising a nucleic acid encoding a protein having DHFR function or a nucleic acid encoding a protein having GS function. (15) The HEK cell or cell line according to any one of (12) to (14), wherein the first vector comprises a first viral vector, and the optional second vector comprises a second viral vector. (16) The HEK cell or cell line according to any one of (12) to (14), wherein the first or second viral vector comprises an AAV vector genome. (17) The HEK cell or cell line according to any one of (12) to (14), comprising a first and a second viral vector each comprising an AAV vector genome. (18) The HEK cell or cell line according to (17), wherein the AAV vector genome comprises one or two AAV ITRs adjacent to the 5' and / or 3' ends of the heterologous nucleic acid sequence. (19) The copy number of the heterologous nucleic acid sequence and / or vector and / or viral vector and / or AAV vector genome in the HEK cells or cell line is 1 to 5 copies per cell, 5 to 10 copies per cell, 10 to 50 copies / cell, 50 to 100 copies per cell, 100 to 250 copies per cell, 250 to 500 copies per cell, 500 to 1,000 copies per cell, 1,000 to 2,000 copies per cell, or about 2,000, 3,000, 4,000 or 5,000 copies per cell or more, optionally, the copy number appears to be stable over many passages, for example, at least 5 or more, 10, 15 or more passages, the HEK cells or cell line according to any one of (4) to (18). (20) The copy number of the AAV vector genome in the HEK cells or cell line is at least 1,000 copies per cell, and the yield of the rAAV vector particles is from the roller bottle of the HEK cells or the HEK cell line, optionally at least 1×10 8 vg / ml, or at least 1×10 9 vg / ml, or at least 1×10 10 vg / ml, or at least 1×10 11 vg / ml or at least 2×10 11 vg / ml, the HEK cells or cell line according to any one of (16) to (19). (21) The HEK cells or cell line according to any one of (1) to (20), further comprising the rep and / or cap sequence of AAV. (22) The HEK cells or cell line according to (20), wherein the rep and / or cap sequence of the AAV is provided by a plasmid transiently or stably transfected into the HEK cells or cell line. (23) The HEK cells or cell line according to any one of (1) to (22), further comprising an AAV helper function sequence. (24) The HEK cells or cell line according to any one of (1) to (23), which is HEK293. (25) The HEK cells or cell line according to any one of (1) to (24) in a culture medium or growth medium, or in a medium suitable for long-term storage. (26) The HEK cells or cell line according to any one of (9) to (25) in a culture medium or growth medium containing methotrexate (MTX) and / or methionine sulfoximine (MSX). (27) The HEK cells or cell line according to any one of (6) to (26), which produce rAAV vector particles packaged with the heterologous nucleic acid sequence. (28) The HEK cells or cell line according to (27), which are produced in an amount exceeding the amount produced by HEK293 cells transiently transfected with an AAV vector genome having the heterologous nucleic acid sequence and expressing functional endogenous DHFR and / or GS. (29) The HEK cells or cell line according to (27), which contain an amount of empty AAV capsids of rAAV that is less than the amount of rAAV particles packaging contaminating DNA produced by HEK293 cells transiently transfected with an rAAV vector genome having the heterologous nucleic acid sequence and expressing functional endogenous DHFR and / or GS, and / or an amount of rAAV particles packaging less contaminating DNA than that. (30) The HEK cells or cell line according to any one of (3) to (29), wherein the heterologous nucleic acid sequence encodes a therapeutic protein or an inhibitory nucleic acid sequence. (31) The HEK cells or cell line according to (30), wherein the therapeutic protein contains a blood coagulation factor or an immunoglobulin sequence. (32) The HEK cell or cell line according to (30), wherein the inhibitory nucleic acid sequence comprises small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA. (33) The HEK cell or cell line according to any one of (3) to (32), which is stably transfected with the first heterologous nucleic acid sequence. (34) The HEK cell or cell line according to any one of (3) to (32), which is stably transfected with the first and the second heterologous nucleic acid sequences. (35) Viral particles or rAAV vector particles isolated or purified from the HEK cell or cell line according to any one of (15) to (34). (36) A therapeutic protein isolated or purified from the HEK cell or cell line according to any one of (5) to (34). (37) A method for producing a therapeutic protein, a viral vector or an rAAV vector particle, comprising culturing the HEK cell or cell line according to any one of (5) to (34) under conditions that enable production and / or secretion of the therapeutic protein, the viral vector or the rAAV vector particle, and isolating or purifying the therapeutic protein, the viral vector or the rAAV vector particle from the cell culture, the culture medium, or the cell culture and the culture medium. (38) A method for producing rAAV vector particles, comprising culturing the HEK cell or cell line according to any one of (23) to (25) under conditions that enable production and / or secretion of the rAAV vector particles, and isolating or purifying the rAAV vector particles from the cell culture, the culture medium, or the cell culture and the culture medium, wherein when the HEK cell or cell line has at least 1,000 copies of the AAV vector genome per cell, the yield of the rAAV vector particles is at least 2×10 of the HEK cell or the HEK cell line 11A method, which is a vg / roller bottle. (39) The first and / or second heterologous nucleic acid sequences encode gene products selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase, the HEK cells or cell line or method according to any one of (3) to (38). (40) The HEK cell or cell line or method according to any one of (23)-(29), (37) or (38), wherein the rAAV vector particles comprise the first and / or the second heterologous nucleic acid sequence encoding a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase. (41) The HEK cell or cell line or method according to any one of (3)-(38), wherein the first and / or second heterologous nucleic acid sequence encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules. (42) The HEK cell or cell line or method according to any one of (23)-(29), (37) or (38), wherein the rAAV vector particles contain the first and / or the second heterologous nucleic acid sequence encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukin (IL1-IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulin, humanized antibody, single-chain antibody, T cell receptor, chimeric T cell receptor, single-chain T cell receptor, class I and class II MHC molecules. (43) The HEK cell or cell line or method according to any one of (3)-(38), wherein the first and / or the second heterologous nucleic acid sequence encodes a protein useful for correcting congenital errors, selected from the group consisting of carbamoyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionine beta-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence. (44) The HEK cell or cell line or method according to any one of (23)-(29), (37) or (38), comprising the first and / or the second heterologous nucleic acid sequence encoding a protein useful for correcting inborn errors, wherein the rAAV vector particles are selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathionine beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence. (45) A method for producing a human embryonic kidney (HEK) cell line that does not express a functional endogenous dihydrofolate reductase (DHFR), the method comprising the step of mutating or knocking out the endogenous DHFR gene. (46) A method for producing a human embryonic kidney (HEK) cell line that does not express a functional endogenous glutamine synthetase (GS), the method comprising the step of mutating or knocking out the endogenous GS gene. (47) A method for producing a human embryonic kidney (HEK) cell line that does not express a functional endogenous dihydrofolate reductase (DHFR) and glutamine synthetase (GS), the method comprising the step of mutating or knocking out the endogenous DHFR gene and GS gene.

Claims

1. 1. A method for producing a human embryonic kidney (HEK) cell line that does not express a functional endogenous dihydrofolate reductase (DHFR) and that does not express a functional endogenous glutamine synthetase (GS), comprising: (a) mutating or knocking out the endogenous DHFR and GS genes in the HEK cell line; (b) stably or transiently transfecting said HEK cell line with an AAV vector genome comprising a first heterologous nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence and a first selectable marker; A method comprising:

2. 2. The method of claim 1, further comprising the step of: (c) stably or transiently transfecting said HEK cell line with a second heterologous nucleic acid sequence and a second selectable marker.

3. (a) the second heterologous nucleic acid sequence encodes a therapeutic protein or polynucleotide sequence; (b) the first or second selectable marker does not provide resistance to an antibiotic; (c) the first or second selectable marker provides a means for amplifying the first and / or second heterologous nucleic acid sequence; (d) the first or second selectable marker comprises a nucleic acid encoding a protein having DHFR function; or 3. The method of claim 2, wherein (e) the first or second selectable marker comprises a nucleic acid encoding a protein having GS function.

4. The method of claim 2 or 3, wherein the second heterologous nucleic acid sequence encodes a therapeutic protein or an inhibitory nucleic acid sequence.

5. 3. The method of claim 2, wherein the first selectable marker comprises a nucleic acid encoding a protein having DHFR function and the second selectable marker comprises a nucleic acid encoding a protein having GS function.

6. The method of claim 2 , wherein the second heterologous nucleic acid sequence comprises a second vector.

7. (a) the second vector comprises a second viral vector; (b) the second viral vector comprises an AAV vector genome; or 7. The method of claim 6, wherein (c) the AAV vector genome further comprises one or two AAV ITRs flanking the 5' and / or 3' ends of the heterologous nucleic acid sequence.

8. 7. The method of claim 6, wherein the second vector comprises a selectable marker comprising a nucleic acid encoding a protein with DHFR function or a nucleic acid encoding a protein with GS function, respectively.

9. 9. The method of any one of claims 1 to 8, wherein the copy number of said heterologous nucleic acid sequence and / or vector and / or viral vector and / or AAV vector genome in said HEK cell or HEK cell line is 1-5 copies per cell, 5-10 copies per cell, 10-50 copies / cell, 50-100 copies per cell, 100-250 copies per cell, 250-500 copies per cell, 500-1,000 copies per cell, 1,000-2,000 copies per cell, or 2,000, 3,000, 4,000 or 5,000 copies per cell or more.

10. or the copy number appears to be stable over many passages, or the copy number of the AAV vector genome in the HEK cell line is at least 1,000 copies per cell, or the yield of the rAAV vector particles is at least 1×10 １１ vg / ml or at least 2 x 10 １１ The method according to any one of claims 1 to 9, wherein the concentration is in vg / ml.

11. The method of any one of claims 1 to 10, further comprising the step of transfecting said HEK cell line with AAV rep and / or cap sequences.

12. The method of any one of claims 1 to 11, further comprising the step of transfecting the HEK cell line with AAV helper function sequences.

13. 13. The method of any one of claims 2 to 12, wherein the therapeutic protein or inhibitory nucleic acid encoded by the first heterologous nucleic acid sequence and the therapeutic protein or polynucleotide sequence encoded by the second heterologous nucleic acid sequence are the same or different.

14. The method of any one of claims 2 to 12, wherein the inhibitory nucleic acid comprises a small or short hairpin (sh)RNA, a microRNA (miRNA), a small or short interfering (si)RNA, a trans-splicing RNA, or an antisense RNA.

15. (a) the first and / or second heterologous nucleic acid sequence is selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF), GFα), platelet derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase; (b) the first and / or second heterologous nucleic acid sequence encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, or (c) the first and / or second heterologous nucleic acid sequence is selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, encoding a protein useful for correcting an inborn error selected from the group consisting of isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence; The method according to any one of claims 2 to 14.

16. (a) the rAAV vector particle is capable of expressing any one of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet derived growth factor ( the first and / or the second heterologous nucleic acid sequence encoding a gene product selected from the group consisting of PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase; (b) the rAAV vector particle comprises the first and / or the second heterologous nucleic acid sequence encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, or (c) the rAAV vector particle is selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystatin beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-coA dehydrogenase, and the like. the first and / or second heterologous nucleic acid sequence encoding a protein useful for correcting an inborn error selected from the group consisting of hepatic phosphorylase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence; The method according to any one of claims 2 to 14.

17. 1. A method for producing rAAV vector particles, comprising: (a) obtaining a human embryonic kidney (HEK) cell line that does not express a functional endogenous dihydrofolate reductase (DHFR) and that does not express a functional glutamine synthase (GS); (b) subjecting the HEK cell line to (i) an AAV vector genome comprising a first heterologous nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence, said therapeutic protein being a peptide or protein that itself corrects a genetic defect or genetic deficiency, thereby alleviating or alleviating a symptom resulting from an insufficient amount, absence or deficiency of a protein in a cell or subject; or (ii) a first heterologous nucleic acid sequence and a first selectable marker, stably or transiently transfecting (c) culturing the HEK cell line under conditions that allow for the production and / or secretion of rAAV vector particles; and (d) isolating or purifying the rAAV vector particles from the cell culture, the culture medium, or the cell culture and the culture medium.

18. 18. The method of claim 17, wherein the HEK cell line is in culture or growth medium, or in a medium suitable for long-term storage.

19. (a) the HEK cell line is in a culture or growth medium comprising methotrexate (MTX) and / or methionine sulfoxamine (MSX); and / or 19. The method of claim 17 or claim 18, wherein (b) the HEK cell line produces rAAV vector particles packaged with the heterologous nucleic acid sequence.

20. (a) the rAAV vector particles are produced in an amount that exceeds that produced by HEK293 cells expressing functional endogenous DHFR and GS that have been transiently transfected with an AAV vector genome carrying the heterologous nucleic acid sequence; or (b) the AAV vector particles produced contain less rAAV empty capsids and / or less rAAV particles packaging contaminating DNA than the amount of AAV empty capsids and / or the amount of rAAV particles packaging contaminating DNA produced by HEK293 cells expressing functional endogenous DHFR and GS that have been transiently transfected with a rAAV vector genome having the heterologous nucleic acid sequence.

20. The method according to any one of claims 17 to 19.

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