WO2014121005A1 - Retroviral vector packaging cell lines and methods of purifying and producing retroviral particles - Google Patents
Retroviral vector packaging cell lines and methods of purifying and producing retroviral particles Download PDFInfo
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Definitions
- nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows:
- Retroviral vector particles are useful for any of a variety of gene transfer applications (e.g., gene therapy).
- obstacles to the effective production of retroviral vector particles exist.
- the production of retroviral vector particles using packaging cell lines may produce a retroviral vector particle product that is contaminated with other cell components that could, for example, be toxic to a patient.
- Retroviral particles produced from murine packaging cells may not efficiently infect human target cells and may be subject to inactivation by the human immune system.
- Murine packaging cell genomes also may contain endogeneous, inducible replication competent retroviruses that can disadvantageously result in replication competent retroviruses upon recombination with introduced retroviral packaging genes.
- An embodiment of the invention provides a method of purifying retroviral vector particles, the method comprising: (a) (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1 D8, V5, or influenza hemagglutinin (HA) epitope tag; (b) culturing the cells of (a) to produce epitope-tagged retroviral vector particles; (c) producing a first supernatant comprising the epitope-tagged retroviral vector particles and cells; (d) centrifuging the first supernatant to produce a second
- Another embodiment of the invention provides a method of producing retroviral vector particles, the method comprising: (a) (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; (b) transfecting the cells with a vector encoding a gene of interest; (c) inserting the gene of interest into a cell genome by Cre recombinase-mediated cassette exchange (RMCE); (d) culturing the cells of (c) to produce retroviral vector particles by the cells; (e) producing a first supernatant comprising the retroviral vector particles and cells; (f) centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and (g) separating the second supernatant including the retroviral vector particles from the
- Still another embodiment of the invention provides retroviral packaging cells comprising HT1080, 293 (also known as HEK cells) or LNCaP cells expressing Gag, Pol, and Env proteins.
- Retroviral packaging cells comprising a CI 1D8, V5, or HA epitope tag on a cell surface protein, wherein the cells are human or mouse cells.
- Still another embodiment of the invention provides retroviral packaging cells comprising two lox sites flanking a target site for a gene of interest.
- Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising transfecting HT1080, 293 or LNCaP cells with one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins.
- Still another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag.
- Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells.
- Still another embodiment of the invention provides methods of treating a disease in a mammal in need thereof, the method comprising administering to the mammal the retroviral vector particles purified or produced by any of the inventive methods described herein, wherein the gene of interest encodes a therapeutic gene suitable for treating the disease.
- Another embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising transfecting target cells with the retroviral vector particles purified or produced according to any of the inventive methods described herein and administering the transfected target cells to the mammal in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
- Another embodiment of the invention provides a retroviral vector particle comprising Gag, Pol, and Env proteins, and a retroviral vector comprising a therapeutic gene, wherein the Env protein comprises a CI 1 D8, V5, or HA epitope tag.
- Figure 1 is a schematic showing the insertion of an epitope tag between a signal peptide and a surface subunit (SU) of the envelope protein to produce a pre-protein that undergoes signal peptide cleavage to produce an envelope protein having an epitope tag positioned on the amino terminus.
- SU surface subunit
- retroviral vector particles may be effectively purified using a CI 1D8, V5, or HA epitope tag.
- an embodiment of the invention provides a method of purifying retroviral vector particles comprising (a) (i) introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or influenza hemagglutinin (HA) epitope tag; (b) culturing the cells of (a) to produce epitope-tagged retroviral vector particles; (c) producing a first supernatant comprising the epitope-tagged retroviral vector particles and cells; (a) introducing a nucleo
- the retroviral vector particle may be any suitable retroviral vector particle known in the art.
- the retroviral vector particle may be chosen based on any of a variety of factors including, for example, the particular type of retroviral vector to be enveloped and/or the type of cell and/or the type of tissue to be targeted for gene therapy (also referred to herein as a "target cell” or "target tissue").
- the retroviral vector particles are gammaretroviral vector particles.
- the retroviral vector particles are lentiviral particles. Lentiviral particles may provide several advantages including, for example, the ability to provide long-term and stable gene expression and the ability to infect non-dividing target cells, such as, for example, neurons.
- the retroviral vector particles may be derived from any suitable species of retrovirus.
- the retroviral vector particles may be derived from Gibbon Ape Leukemia Virus (GALV), murine leukemia virus (MLV), wooly monkey virus (WMV), koala retrovirus (KoRV), spleen leukosis virus (SNV), feline leukemia virus (FeLV), or RD1 14.
- GALV retroviral vector particles may provide numerous advantages including, for example, the ability to infect a broad range of target cells and non-toxicity (or reduced toxicity) to target cells, including, for example, neurons, microglia, hematopietic stem cells, astrocytes, T cells, and B cells.
- retroviral vector particle and “retroviral particle” refer to particles that contain a viral envelope and have the ability to infect cells but are not replication competent. In contrast, viruses are replication competent.
- the method comprises (i) introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag;.
- Introducing a nucleotide sequence into cells may be carried out in any suitable manner known in the art. See, for example, Green et al.
- nucleotide sequence into cells may include, but are not limited to, transfection and targeted genome editing using, for example, zinc finger nucleases, CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs) (see, e.g., Joung et al., Nat. Rev. Mol. Cell Biol, 14(1): 49-55 (2013)).
- TALENs transcription activator-like effector nucleases
- Introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag advantageously provides a cell that is capable of producing retroviral vector particles (also referred to as a "packaging cell").
- the packaging cell may provide stable (i.e., constitutive) or transient expression of retroviral particles.
- the packaging cell is a mouse cell that provides stable expression of retroviral particles.
- the packaging cell is a human cell that provides transient expression of retroviral particles.
- a nucleotide sequence encoding an epitope tag is introduced into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein.
- the cells into which a nucleotide sequence is introduced already express Gag, Pol, and Env proteins and may be, for example, any viral packaging cell.
- Exemplary viral packaging cells include, but are not limited to, PG13 packaging cells and STAR packaging cell lines for lentiviral vectors (Ikeda et al., Nat. Biotechnol., 21 : 569-572 (2003)).
- the viral packaging cell is a PG13 packaging cell or STAR GALV.
- the method may comprise introducing a nucleotide sequence encoding an epitope tag into the cells using targeted genome editing such as, for example, TALENs to obtain expression of an epitope-tagged Env protein.
- one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins are introduced into cells, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag.
- the cells into which the one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins are introduced do not already express Gag, Pol, and Env proteins and may not be a packaging cell prior to the introduction of the one or more nucleotide sequences.
- the cell may be a human cell that is not a packaging cell such as, for example, HT1080, 293 or LNCaP cells, or a mouse cell that is not a packaging cell such as, for example, NIH3T3 cells.
- the cells may become a packaging cell after introduction of the one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into the cell.
- Nucleic acid as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
- a vector comprising any of the nucleic acids described herein may be introduced into the cells.
- the term "vector” refers to a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
- the vectors are not naturally-occurring as a whole.
- the vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
- the vectors can comprise naturally-occurring, non-naturally-occurring intemucleotide linkages, or both types of linkages.
- the non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector.
- the vector may be any suitable vector including, for example, commercially available vectors.
- a vector that is constructed so that the genes within the inverted repeats (IR) of the plasmid are positioned so that the genes are not disrupted during integration of all or a portion of the vector into the cell genome.
- IR inverted repeats
- Such vectors are commercially available and may include, for example, the PIGGYBAC plasmid available from System Bioscience (Mountain View, CA).
- the vectors described herein are recombinant expression vectors.
- the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
- the replication can be in vitro replication or in vivo replication.
- the nucleic acids described herein may also be recombinant.
- vectors and nucleic acids described herein can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra, and Ausubel et al., supra.
- Constructs of vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
- Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, cyto megalovirus and the like.
- the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA based.
- regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA based.
- the nucleic acid(s) may encode any suitable retroviral Gag, Pol, and Env proteins known in the art that are suitable for the particular type of retroviral vector particle chosen and/or the type of retroviral vector to be enveloped.
- the Gag, Pol, and/or Env protein(s) may be wild-type or enhanced, and may be derived from any suitable retrovirus species such as, for example, those described herein with respect to other aspects of the invention.
- the nucleic acid(s) encode a wild-type GALV Env protein comprising both DRISAVKIL (SEQ ID NO: 1) and
- the nucleic acid(s) encode substituted Gag, Pol, and/or Env proteins.
- the nucleic acid(s) may encode a substituted GALV Env protein that includes three amino acid substitutions with respect to the wild-type GALV protein.
- the nucleic acid(s) may encode a substituted GALV Env protein comprising both DRISAVQAL (SEQ ID NO: 3) and VLTQKALENE (SEQ ID NO: 4).
- the substituted GALV Env protein encoded by such nucleic acid(s) may facilitate the ability of the GALV retroviral vector particle to envelope a lentiviral vector as described in Merten et al., J.
- Retroviral vector particles expressing the substituted Env protein comprising both DRISAVQAL (SEQ ID NO: 3) and VLTQKALENE (SEQ ID NO: 4) may, advantageously, envelope lentiviral vectors.
- the terms "Env” and "Env protein” refer to both wild-type and substituted Env proteins unless specified otherwise.
- the cells into which a nucleic acid has been introduced express an Env protein that further comprises a CI 1D8, V5, or HA epitope tag.
- the Env protein further comprises (i) a CI 1D8 epitope (also referred to as a "CI 1D8 tag” or “CI 1D8 epitope tag”), (ii) a V5 epitope (also referred to as a "V5 tag” or “V5 epitope tag”), or (iii) an HA epitope (also referred to as an "HA tag” or "HA epitope tag”) which specifically binds to a CI 1 D8 antibody, V5 antibody, or HA antibody, respectively .
- the CI 1D8, V5, or HA epitope tag may comprise any suitable amino acid sequence that specifically binds to a CI 1D8, V5, or HA antibody, respectively.
- HA and V5 epitope tags may provide numerous advantages such as, for example, a higher titer yield and easier purification.
- the nucleic acid(s) may encode a CI 1D8 epitope comprising QVMTITPPQAMGPNLVLP (SEQ ID NO: 6) or a functional fragment of SEQ ID NO: 6.
- the functional fragment of SEQ ID NO: 6 may comprise less than the entire SEQ ID NO: 6 and at least 5 contiguous amino acid residues of SEQ ID NO: 6 such that the functional fragment specifically binds to a CI 1D8 antibody.
- the functional fragment comprises from about 5 to about 17 contiguous amino acid residues of SEQ ID NO: 6, preferably from about 6 to about 16 contiguous amino acid residues of SEQ ID NO: 6, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 6.
- the functional fragment comprises MGPNL (SEQ ID NO: 5).
- Exemplary CI 1D8 epitopes useful for the invention include, but are not limited to, SEQ ID NOs: 5-14.
- the nucleic acid(s) may encode a V5 epitope comprising GKPIPNPLLGLDST (SEQ ID NO: 15) or a functional fragment of SEQ ID NO: 15.
- the functional fragment of SEQ ID NO: 15 may comprise less than the entire SEQ ID NO: 15 and at least 5 contiguous amino acid residues of SEQ ID NO: 15 such that the functional fragment specifically binds to a V5 antibody.
- the functional fragment comprises from about 5 to about 14 contiguous amino acid residues of SEQ ID NO: 15, preferably from about 6 to about 12 contiguous amino acid residues of SEQ ID NO: 15, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 15.
- the nucleic acid(s) may encode an HA epitope comprising YPYDVPDYA (SEQ ID NO: 16) or a functional fragment of SEQ ID NO: 16.
- the functional fragment of SEQ ID NO: 16 may comprise less than the entire SEQ ID NO: 16 and at least 5 contiguous amino acid residues of SEQ ID NO: 6 such that the functional fragment specifically binds to a HA antibody.
- the functional fragment comprises from about 5 to about 9 contiguous amino acid residues of SEQ ID NO: 16, preferably from about 6 to about 8 contiguous amino acid residues of SEQ ID NO: 16, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 16.
- the epitope tag may comprise one or more copies of the any of the epitope tag amino acid sequences described herein.
- the epitope tag can comprise 1 , 2, 3, 4, 5, or more, copies of the epitope tag amino acid sequence.
- the epitope tag may comprise YPYDVPDYAYPYDVPDYA (SEQ ID NO: 17),
- introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag or an epitope-tagged Env protein, wherein the epitope tag is a CD1 1D8, V5, or HA epitope tag provides numerous advantages.
- the CI 1D8, V5, or HA epitope tag may allow the retroviral vector particles to be purified and/or detected using CI 1D8, V5, or HA antibodies, respectively.
- the CI 1D8, V5, or HA epitope tag may allow the retroviral vector particles to be purified and/or detected using CI 1D8, V5, or HA antibodies, respectively.
- the CI 1D8, V5, or HA epitope tag may allow the retroviral vector particles to be purified and/or detected using CI 1D8, V5, or HA antibodies, respectively.
- the CI 1D8, V5, or HA epitope tag may allow the retroviral vector particles to be purified and/or detected using CI 1D8, V5, or HA antibodies
- the nucleic acid(s) vector(s) may encode an Env protein having the epitope tag positioned at any suitable position in the Env protein amino acid sequence.
- the epitope tag is positioned in the Env protein such that the epitope tag is recognized by the appropriate antibody.
- the epitope tag may be inserted into a proline rich region (PRR) of the Env protein.
- PRR proline rich region
- the epitope tag may be inserted into the GALV Env protein at GALV envelope protein residue 219 or 264 as described in Farrell et al, J. Virol., 76: 4267-74 (2002).
- the epitope tag may be positioned at the amino terminus of the Env protein.
- the epitope tag may be inserted between a signal peptide (such as, for example, SEQ ID NO: 20) and a surface (SU) subunit of the envelope protein (such as, for example, SEQ ID NO: 21) to produce a preprotein that includes the epitope tag positioned at the carboxyl terminus of the signal peptide and at the amino terminus of the Env protein.
- the preprotein may undergo cleavage of the signal peptide to produce a mature envelope protein including the epitope tag positioned at the amino terminus of the Env protein.
- An epitope tag positioned at the amino terminus of the Env protein may provide numerous advantages such as, for example, a higher titer yield and easier purification using, for example, an antibody-conjugated column as compared to an epitope tag that is located in the Env protein at a position other than the amino terminus.
- the cells into which one or more nucleic acids are introduced are mammalian cells.
- the cells may be derived from any suitable mammal.
- the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal may be from the order
- Artiodactyla including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
- the mammal may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
- a preferred mammal is the mouse.
- the cells are human and the human cells are HT1080, 293 or LNCaP cells.
- HT1080 is a human fibrosarcoma cell line
- 293 is a human embryonic kidney cell line
- LNCaP is a human carcinoma cell line.
- the cells are mouse cells.
- the mouse cells may be PG13 packaging cells, which are derived from NIH3T3 mouse cells.
- the method may further comprise transfecting the cells with a vector encoding a gene of interest.
- Transfecting cells with a vector encoding a gene of interest may be carried out in any suitable manner known in the art that will enable the cell to produce retroviral vector particles capable of infecting target cells with the gene of interest.
- the gene of interest may be any suitable gene useful for any of a variety of applications.
- the gene is a therapeutic gene.
- the therapeutic gene can encode any beneficial protein or polypeptide that replaces missing or defective protein required by the target cell or subject into which the vector is transferred, or can encode a cytotoxic polypeptide that can be directed, e.g., to cancer target cells or other target cells whose death would be beneficial to the subject.
- the therapeutic gene can also encode antisense RNAs that can bind to, and thereby inactivate, mRNAs made by the subject that encode harmful proteins.
- the therapeutic gene can also encode ribozymes that can effect the sequence-specific inhibition of gene expression by the cleavage of mRNAs.
- antisense polynucleotides can be produced from a therapeutic gene that includes an expression cassette that contains a sequence that promotes cell-type specific expression (Wirak et al., EMBO 10:289 (1991)).
- an expression cassette that contains a sequence that promotes cell-type specific expression (Wirak et al., EMBO 10:289 (1991)).
- RNA and DNA see Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).
- Other examples of therapeutic genes which can be administered to a target cell or subject as part of the retroviral vectors described herein can include, but are not limited to, the following: nucleic acids encoding secretory and nonsecretory proteins and nucleic acids encoding therapeutic agents.
- TNFs tumor necrosis factors
- interferons such as interferon-a, interferon- ⁇ , and interferon- ⁇ , interleukins (IL), such as IL- 1 , IL- ⁇ ⁇ , and ILs-2 through -14
- GM- CSF granulocyte-macrophage colony-stimulating factor
- adenosine deaminase cellular growth factors, such as lymphokines; soluble CD4; Factor VIII; Factor IX; T-cell receptors; Low-Density Lipoprotein (LDL) receptor; ApoE; ApoC; alpha- 1 antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosis transmembrane receptor (CFTR); insulin; Fc receptors for antigen binding domains of antibodies, such as immunoglobulins; anti TNFs, antigen binding domains of antibodies, such as immunoglobulins; antigen binding domains of antibodies, such as immunoglobul
- therapeutic genes that may be employed include, but are not limited to, was, gp91-phox, il2yc, and ada.
- the therapeutic gene is chosen considering several factors, including the cell and/or tissue to be targeted for therapy.
- the target cell is a blood cell
- particularly useful therapeutic genes to use are those which allow the blood cells to exert a therapeutic effect, such as a gene encoding a clotting factor for use in treatment of hemophilia.
- Another target cell is the lung airway cell, which can be used to administer nucleic acids, such as those coding for the cystic fibrosis transmembrane receptor, which could provide a gene therapeutic treatment for cystic fibrosis.
- target cells include muscle cells where useful therapeutic genes, such as those encoding cytokines and growth factors, can be transduced and the protein the nucleic acid encodes can be expressed and secreted to exert its effects on other cells, tissues and organs, such as the liver.
- cancer cells corresponding or derived from lung, muscle, brain and other tissues can be target tissues.
- Additional target cells for gene therapy may include, but are not limited to, immune cells (e.g., T cells and B cells), stem cells (e.g., embryonic stem cells and/or hematopoietic stem cells) and cells of the central nervous system (e.g., neurons, astrocytes, and microglia).
- the therapeutic gene can encode more than one gene product, limited only by the size of vector that can be packaged by the caspid of the particular retroviral vector particle chosen.
- the method comprises inserting the gene of interest into a (packaging) cell genome by Cre recombinase-mediated cassette exchange (RMCE).
- Inserting the gene of interest into the packaging cell genome by Cre RMCE can, advantageously, direct the insertion of the gene of interest into any suitable specific target site in the packaging cell genome.
- the target site chosen may, for example, be a transcriptionally active site that facilitates optimal transcription of the gene of interest.
- the target site chosen may advantageously avoid inserting of the gene of interest at an undesirable site such as, for example, a site that disrupts the expression of an endogenous gene the normal expression of which is necessary for the healthy function of the cell and/or a site that causes uncontrolled cell division (e.g., by activating an oncogene).
- the packaging cells are human cells
- the target site is in a human chromosome 19-specific adeno-associated virus (AAV) integration site.
- AAV integration site may be any suitable AAV integration site.
- the AAV integration site is AAVS 1.
- the method further comprises transfecting the cells with a lox insertion vector encoding lox sites, a first marker gene and a defective second marker gene.
- the first marker gene can be any suitable marker gene which allows for selection of transfected cells.
- Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
- Suitable marker genes include, for instance, LacZ, green fluorescent protein (GFP), luciferase, puromycin, blasticidin, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
- the first marker gene is an antibiotic resistance gene.
- the lox insertion vector may include an appropriate promoter oriented to promote expression of the first marker gene.
- the promoter may comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the first marker gene.
- the selection of promoters e.g., strong, weak, and inducible, is within the ordinary skill of the artisan.
- the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
- the promoter can be a non-viral promoter or a viral promoter, e.g., a retroviral promoter, a GALV promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, chicken beta-actin promoter with CMV enhancer (CAG) promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.
- a viral promoter e.g., a retroviral promoter, a GALV promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, chicken beta-actin promoter with CMV enhancer (CAG) promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.
- a retroviral promoter e.g., a retroviral promoter, a GALV promoter, a cytomegalovirus (CMV) promoter, an SV40
- exemplary promoters include, but are not limited to, a PGK (phosphoglycerate kinase 1) promoter, a glial fibrillary acidic protein (GFAP) (promoter), a neuronal-specific nuclear protein (NeuN) promoter, and an elongation factor-1 alpha (EFl ) promoter.
- PGK phosphoglycerate kinase 1
- GFAP glial fibrillary acidic protein
- NeN neuronal-specific nuclear protein
- EFl elongation factor-1 alpha
- the lox insertion vector may comprise two lox sites flanking the combination of the promoter and the first marker gene.
- the lox sites may be any suitable lox sites.
- the lox sites are loxP and lox2722.
- loxp is oriented 5' of the combination of the promoter and the first marker gene and lox2722 is oriented 3' of the combination of the promoter and the first marker gene.
- the loxp is oriented 3' of the combination of the promoter and the first marker gene and lox2722 is oriented 5' of the combination of the promoter and the first marker gene.
- the defective second marker gene may be any suitable marker gene such as, for example, any of those described above which has been mutated so that it is not expressed until the mutation is corrected upon Cre-mediated recombination.
- the mutation may be any suitable mutation.
- the defective second marker gene is mutated to lack an initiation codon.
- the defective second marker gene encodes a marker that is different from that which is encoded by the first marker gene (e.g., resistance to a different antibiotic).
- the defective second marker gene may be positioned outside of the lox-flanked combination of promoter and first marker gene.
- the lox insertion vector may comprise, from the 5' to the 3' end, a lox site, a promoter, a first marker gene, a lox site, and the defective second marker gene.
- the lox insertion vector may comprise, from the 5' to the 3' end, the defective second marker gene, a lox site, a promoter, a first marker gene, a lox site, and the defective second marker gene.
- the lox insertion vector may further comprise nucleotide sequences that are homologous to a left and right arm of the target site (e.g., the AAV integration site) flanking the combination of the promoter, marker gene, lox sites, and defective second marker gene.
- the nucleotide sequences homologous to left and right arms of the AAV integration sites are known in the art and may be any suitable size that provides for insertion of the lox sites, promoter, first marker gene, and defective second marker gene in the target site.
- the nucleotide sequences that are homologous to a left and right arm of the target site may be from about 3 to about 4 kb.
- Transfecting the cells with a lox insertion vector encoding a marker gene flanked by lox sites and a defective second marker gene results in the integration of the lox sites, the promoter, the first marker gene, and the defective second marker gene into the target site of the genome of the packaging cell.
- the lox sites, the promoter, the first marker gene, and the defective second marker gene are integrated into the target site of the genome of the packaging cell in the same orientation as described for the lox insertion vector.
- the method may further comprise selecting transfected cells using the marker encoded by the first marker gene.
- transfecting the cells with a vector encoding a gene of interest comprises transfecting the cells with one or more vectors encoding Cre recombinase and the gene of interest.
- the vector encoding the gene of interest may comprise the gene of interest flanked by lox sites oriented in a position to facilitate integration of the gene of interest into the packaging cell genome by Cre RMCE using the lox sites already inserted into the genome as described above.
- the lox sites in the vector encoding the gene of interest may be any suitable lox sites and are preferably the same lox sites already inserted into the genome.
- the vector encoding the gene of interest may further encode an internal ribosome entry site (IRES), a 5' long terminal repeat (LTR), and a 3' LTR, and/or an appropriate promoter oriented to promote expression of the gene of interest and the second marker gene.
- the vector encoding the gene of interest is a self-inactivating (SIN) vector such that integration of components into the cell genome does not include integration of a viral promoter into the cell genome.
- SIN vectors may lack a viral promoter at both the 5' and 3' ends upon integration into the cell genome.
- the promoter for the gene of interest may be as described herein with respect to other aspects of the invention.
- the promoter is a 5' long tenninal repeat (LTR) packaging site ( ⁇ ) promoter (e.g., PGK).
- the vector encoding the gene of interest may further encode a component that corrects the mutation that prevents the defective second marker gene from being expressed.
- the vector encoding the gene of interest may further include an initiation codon.
- the component correcting the mutation may be oriented in the vector encoding the gene of interest such that the component is inserted into the defective second marker gene in the genome of the cell to correct the mutation and allow for expression of the formerly defective second marker gene.
- the lox sites, gene of interest and its promoter, the IRES, and the component that corrects a mutation may be oriented in the vector in such a manner so as to facilitate their integration into the genome of the packaging cell at the target site.
- the vector encoding the gene of interest may comprise, from the 5' to the 3' end, loxp, a 5' LTR packaging site ( ⁇ ) promoter, gene of interest, 3' LTR, IRES, the component that corrects a mutation in the second marker gene, and lox2722.
- Cre RMCE exchanges the first marker gene with the gene of interest so that the first marker gene and its promoter are removed from the cell genome and replaced with the gene of interest, its promoter, IRES, and the component that corrects the mutation in the defective second marker gene.
- the packaging cells may contain a genome comprising the gene of interest, its promoter, the IRES, and the second, corrected marker gene.
- the gene of interest, its promoter, the IRES, and the second marker gene may be located at the target site and oriented in such as way as to allow the cell to produce retroviral vector particles that envelope a retroviral vector containing the gene of interest that can be integrated into a target cell upon infection of the target cell with the retroviral vector particles.
- the cell genome may comprise, from the 5' to the 3' end, loxp, a 5' LTR packaging site ( ⁇ ) promoter, gene of interest, 3'LTR, IRES, the corrected, second marker gene, and lox2722.
- viral promoters are not integrated into the cell genome.
- the method may further comprise selecting for cells encoding the gene of interest using the marker encoded by the corrected, second marker gene.
- Inserting the gene of interest into a cell genome by Cre RMCE may provide many advantages. For example, because the first marker gene is replaced with the gene of interest in the genome of the packaging cell, the first marker gene does not have to be incorporated into the genome bearing the therapeutic gene. The removal of the first marker gene from the genome of the packaging cell may, advantageously, provide more room for a larger therapeutic gene. For example, the inventive methods may provide space of up to about 8 kb for a therapeutic gene as opposed to only about 5 kb of space that would remain for a therapeutic gene if the first marker gene remained in the packaging cell genome.
- the method comprises culturing the cells to produce epitope-tagged retroviral vector particles.
- the cells may be cultured in any suitable manner known in the art that causes the cells to produce retroviral vector particles.
- the epitope tag may be as described herein with respect to other aspects of the invention.
- the method comprises producing a first supernatant comprising the epitope- tagged retroviral vector particles and cells.
- the first supernatant may also comprise aggregates of cells and small particles and/or debris.
- the small particles and/or debris may include particles and debris that are smaller than cells such as, for example, cellular components (e.g., membranes and/or vesicles).
- the first supernatant may be produced in any suitable manner known in the art.
- the method comprises centrifuging the first supernatant to produce a second supernatant comprising the epitope-tagged retroviral vector particles and small particles and/or debris, e.g., cellular components.
- Centrifuging the first supernatant may be carried out in any suitable manner known in the art and at any suitable spin speed. In an embodiment of the invention, centrifuging the first supernatant may be carried out at a spin speed of about 3 to about 4,000 g. Centrifuging the first supernatant may further comprise forming a pellet comprising the cells described above.
- the method comprises separating the second supernatant from the cells described above. Separating the second supernatant from the cells may be earned out in any suitable manner known in the art. In an embodiment of the invention, separating the second supernatant from the cells may comprise physically separating the second supernatant, including the cellular components and the epitope-tagged retroviral vector particles, from cells present in a pellet formed after centrifuging. The second supernatant that is separated from the cells contains no cells or fewer cells as compared to that contained in the first supernatant.
- the method comprises contacting a stationary phase comprising anantibody with the second supernatant and binding the epitope-tagged retroviral vector particles to the antibody, wherein the antibody is a CI 1D8, V5, or HA antibody.
- the stationary phase may be any stationary phase known in the art that is suitable for separation by affinity
- the stationary phase may be beads (e.g., metallic and/or polymeric beads). Suitable exemplary stationary phases that are commercially available include SEPHAROSE beads (Sigma-Aldrich, St. Louis, MO).
- the stationary phase is cross-linked.
- the stationary phase may be beads cross-linked with protein G or protein A.
- the stationary phase may comprise a CI 1D8, V5, or HA antibody conjugated to the stationary phase.
- Suitable CI 1D8, V5, and HA antibodies are known in the art and are commercially available (e.g., Santa Cruz Biotechnology, Inc., Santa Cruz, CA; Thermo Fisher Scientific, Waltham, MA; and Novus Biologicals, Littleton, CO).
- the antibody chosen will be one that specifically binds to and recognizes the particular epitope tag employed.
- the antibody when the epitope tag is a CI 1D8 epitope tag, the antibody is a CI 1 D8 antibody, when the epitope tag is a V5 epitope tag, the antibody is a V5 antibody, and when the epitope tag is an HA epitope tag, the antibody is an HA antibody.
- Contacting the stationary phase with the second supernatant may be carried out in any suitable manner known in the art and may comprise physically contacting the stationary phase with the second supernatant, including the cellular components and the epitope-tagged retroviral vector particles, such that the epitope-tagged retroviral vector particles in the second supernatant bind to the antibody.
- the method comprises separating the cellular components from the epitope- tagged retroviral vector particles. Separating the cellular components from the epitope- tagged retroviral vector particles may be carried out in any suitable manner and may include physically separating the cellular components from the epitope-tagged retroviral vector particles. Separating cellular components from the epitope-tagged retroviral vector particles may be carried out by binding the epitope-tagged retroviral vector particles to the antibody- conjugated stationary phase while not binding the cellular components to the antibody- conjugated stationary phase. In an embodiment, components in the second supernatant that lack an epitope (e.g., cellular components) will not bind to the antibody.
- cellular components e.g., cellular components
- the cellular components may be removed from the stationary phase (and the epitope-tagged retroviral vector particles bound to the stationary phase) in any suitable manner, e.g., by washing the stationary phase with a suitable buffer (e.g., PBS).
- the method may comprise removing the components that lack an epitope (e.g., cellular components) and which are not bound to the antibody from the epitope-tagged retroviral vector particles.
- the method comprises eluting the epitope-tagged retroviral vector particles from the stationary phase to produce purified epitope-tagged retroviral vector particles. Eluting the epitope-tagged retroviral vector particles from the stationary phase may be carried out in any suitable manner. In an embodiment, eluting the epitope-tagged retroviral vector particles from the stationary phase may be carried out by contacting the stationary phase with buffer containing any of the CI 1D8, V5, or HA epitopes described herein. The epitope chosen will be one that specifically binds to and recognizes the particular antibody employed.
- the epitope when the antibody is a CI 1D8 antibody, the epitope is a CI 1D8 epitope, when the antibody is a V5 antibody, the epitope is a V5 epitope, and when the antibody is an HA antibody, the epitope is an HA epitope.
- the elution buffer may comprise any suitable amount of peptide (epitope), for example, about 60 ⁇ to about 100 ⁇ of peptide.
- the method may comprise displacing the epitope-tagged retroviral vector particles with the epitopes and binding the epitopes to the antibody on the stationary phase to produce an eluent comprising purified epitope-tagged retroviral vector particles.
- the eluent comprising purified epitope-tagged retroviral vector particles contains no cellular components or fewer cellular components as compared to that contained in the second supernatant.
- purified means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity.
- the purity can be at least about 50%, can be greater than 60%, 70% or 80%, or can be 100%.
- Purifying the epitope-tagged retroviral vector particles may provide many advantages. For example, purifying the epitope-tagged retroviral vector particles may reduce and/or remove cellular components and debris that may be toxic to target cells, target tissues, and/or to a patient. In this regard, the inventive methods may advantageously provide a purified retroviral vector particle product that is safer for delivery to target cells, target tissues, and/or to patients. For example, the inventive methods may provide purified retroviral vector particles that can be more safely administered to the central nervous system because it may reduce or avoid the administration of cellular components and/or debris that can cause inflammation in the central nervous system.
- inventive methods of purifying retroviral particles using an epitope tag may, also, advantageously reduce the time, expense, and labor associated with other methods of purifying retroviral particles that do not use epitope tagged retroviral particles (see, e.g., Biffi et al., Science, 341 : 1233158 (2013) (e.g., Supplemental Figure S3)).
- the method may further comprise
- concentrating the purified epitope-tagged retroviral vector particles may be carried out in any suitable manner known in the art.
- concentrating the purified epitope-tagged retroviral vector particles is carried out by passing the purified epitope-tagged retroviral vector particles (e.g., the eluent) through a filter having any suitable molecular weight cut-off that retains the epitope-tagged retroviral vector particles.
- the molecular weight cut-off may be about 100,000 or more.
- Exemplary filters useful for the inventive methods include AMICON filters (available from Millipore, Billerica, MA).
- Another embodiment of the invention provides a method of producing retroviral vector particles, the method comprising: (a) (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; (b) transfecting the cells with a vector encoding a gene of interest; (c) inserting the gene of interest into a cell genome by Cre RMCE; (d) culturing the cells of (c) to produce retroviral vector particles by the cells; (e) producing a first supernatant comprising the retroviral vector particles and cells; (f) centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and (g) separating the second supernatant including the retroviral vector particles from the cells of (e).
- the method comprises (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins;.
- Cells that express Gag, Pol, and Env proteins may be packaging cells as described herein with respect to other aspects of the invention.
- Introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins to obtain expression of Gag, Pol, and Env proteins may be carried out as described herein with respect to other aspects of the invention.
- the nucleic acid(s) encode an Env protein that does not comprise a CI 1D8, V5, or HA epitope. In an embodiment of the invention, the nucleic acid(s) encode an Env protein, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
- the method comprises transfecting the cells with a vector encoding a gene of interest; inserting the gene of interest into a cell genome by Cre RMCE; culturing the cells to produce retroviral vector particles by the cells; producing a first supernatant comprising the retroviral vector particles and cells; centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and separating the second supernatant including the retroviral vector particles from the cells.
- the method further comprises purifying the retroviral vector particles from the cellular components.
- Purifying the retroviral vector particles from the cellular components may be carried out by any suitable method.
- the nucleic acid(s) encode an Env protein, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag
- purifying the retroviral vector particles from the cellular components may be earned out as described herein with respect to other aspects of the invention.
- the method further comprises concentrating the retroviral vector particles. Concentrating the retroviral vector particles may be carried out as described herein with respect to other aspects of the invention. Retroviral Packaging Cells
- Another embodiment of the invention provides retroviral packaging cells comprising HT1080, 293 or LNCaP cells expressing Gag, Pol, and Env proteins.
- the HT1080, 293 or LNCaP cells may be transfected with one or more nucleic acid(s)encoding Gag, Pol, and Env proteins as described herein with respect to other aspects of the invention.
- Human packaging cells for producing retroviral vector particles provide many advantages. For example, retroviruses produced from human packaging cells may more efficiently infect human target cells as compared to retroviruses produced from non-human packaging cells, e.g., murine packaging cells. Retroviruses produced from human packaging cells may also be less likely to be inactivated by the human immune system as compared to retroviruses produced from non-human packaging cells, e.g., murine packaging cells, or may avoid inactivation by the human immune system.
- human packaging cells advantageously do not include endogeneous, inducible replication competent retroviruses (such as those present in, e.g., murine packaging cells) that can disadvantageously result in replication competent retroviruses upon recombination with introduced retroviral packaging genes.
- the Env protein expressed by the HT1080, 293 or LNCaP cell does not comprise a CI 1D8, V5, or HA epitope tag.
- the Env protein expressed by the HT1080, 293 or LNCaP cell comprises a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
- Another embodiment of the invention comprises retroviral packaging cells comprising a CI 1D8, V5, or HA epitope tag on a cell surface protein, wherein the cells are human or mouse cells.
- the CI 1 D8, V5, or HA epitope tag may comprise any of the CI 1D8, V5, or HA epitopes described herein with respect to other aspects of the invention, respectively.
- Any suitable cell surface protein may comprise the CI 1D8, V5, or HA epitope tag.
- the Gag, Pol, or Env may comprise the CI 1 D8, V5, or HA epitope tag.
- Env comprises the CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
- Mouse packaging cells for producing retroviral vector particles also provide many advantages. It has been observed that some human cells expressing epitope-tagged GALV may express APOBEC3, which may mediate hypermutation of retroviral vectors, and after passage, may result in reduced vector titers. Hypermutation of retroviral vectors may be avoided or reduced in mouse packaging cells. Accordingly, retroviruses produced from mouse packaging cells may provide increased vector titers as compared to human packaging cells. 293 cells advantageously have no APOBEC3 activity.
- any of the retroviral packaging cells described herein may further comprise two lox sites flanking a target site for a gene of interest.
- the two lox sites, the target site, and the gene of interest may be as described herein with respect to other aspects of the invention and may be positioned in the cell genome as described herein with respect to other aspects of the invention.
- another embodiment of the invention provides retroviral packaging cells comprising two lox sites flanking a target site for a gene of interest.
- Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising transfecting HT1080, 293 or LNCaP cells with one or more vectors encoding Gag, Pol, and Env proteins.
- Transfecting HT1080, 293 or LNCaP cells with one or more vectors encoding Gag, Pol, and Env proteins may be carried out as described herein with respect to other aspects of the invention.
- the method comprises transfecting HT1080, 293 or LNCaP cells with a vector encoding an Env protein that does not comprise a CI 1D8, V5, or HA epitope tag.
- the method comprises transfecting HT1080, 293 or LNCaP cells with a vector encoding a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
- Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag.
- the cells may be from any mammal described herein such as, for example, human or mouse.
- the human cells may be any suitable human cells.
- the mouse cells may be any suitable mouse cells.
- the human cells are HT1080, 293 or LNCaP cells.
- the mouse cells are PG13 cells.
- the method comprises introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag or an epitope-tagged Env protein, wherein the epitope tag is a CI 1 D8, V5, of HA epitope tag
- the method may further comprise purifying the cells using a CI 1D8, V5, or HA antibody. Purifying the cells using a CI 1 D8, V5, or HA antibody may be carried out as described herein with respect to other aspects of the invention.
- the methods of producing retroviral packaging cells may further comprise cloning two lox sites flanking a target site for a gene of interest into a genome of the cells. Cloning two lox sites flanking a target site for a gene of interest into a genome of the cells may be carried out as described herein with respect to other aspects of the invention.
- an embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells.
- Cells that express Gag, Pol, and Env proteins may be packaging cells as described herein with respect to other aspects of the invention.
- Introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells may be carried out as described herein with respect to other aspects of the invention.
- the lox sites may be positioned in a human chromosome 19-specific AAV integration site, as described herein with respect to other aspects of the invention.
- the methods of producing retroviral packaging cells may further comprise transfecting the cells with a vector encoding a gene of interest. Transfecting the cells with a vector encoding a gene of interest may be carried out as described herein with respect to other aspects of the invention.
- the method comprises inserting the gene of interest into the cell genome by Cre RMCE. Inserting the gene of interest into the cell genome by Cre RCME may be carried out as described herein with respect to other aspects of the invention.
- the retroviral vector particles produced or purified by the inventive methods or produced by the inventive retroviral packaging cells may be useful for a variety of applications.
- the retroviral vector particles containing a retroviral vector encoding a therapeutic gene may be useful for delivering the therapeutic gene to a target cell or tissue as part of gene therapy to treat or prevent any of a variety of diseases.
- an embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising administering to the mammal the retroviral vector particles purified or produced according to any of the inventive methods described herein in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
- Another embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising transfecting target cells with the retroviral vector particles purified or produced according to any of the inventive methods described herein and administering the transfected target cells to the mammal in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
- the mammal may be any mammal.
- the mammal may be any of the mammals described herein with respect to other aspects of the invention.
- the mammal is a human.
- the retroviral vector particles can be administered, as described herein, with a Multiplicity of Infection (MOl) of 10.
- MOl is the ratio of infectious virus particles to the number of cells being infected.
- an MOl of 0.1 results in the average inoculation of 1 virus particle for every 10 cells.
- the general theory behind MOl is to introduce one infectious virus particle to every target (host) cell that is present in the culture. However, more than one virus may infect the same cell which leaves a percentage of cells uninfected. This occurrence can be reduced by using a higher MOl to ensure that every cell is infected.
- the provided retroviral vector particles can therefore be administered to cells, as described herein, with a MOl of about 1 to about 100, such as for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100.
- inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.
- the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.
- the method comprises transfecting target cells with the retroviral vector particles carrying the gene of interest.
- the target cells may be any of the target cells described herein with respect to other aspects of the invention.
- the target cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage. Most preferably the target cells can include, for instance, muscle, lung, and brain cells (for example, microglial cells), and the like.
- the target cell may be a microglial cell.
- the microglial cells may be targeted for gene therapy by, for example, infecting bone marrow- derived hematopoetic stem cells ex vivo. Bone marrow-derived stem cells may migrate into the central nervous system and develop into microglial cells following brain conditioning. This conditioning may ablate brain-resident myeloid precursors, allowing turnover of microglia following hematopoietic stem cell transplantation (Biffi et al., Science,
- the target cell can be a cancer cell.
- the target cell of the present can be a tumor cell.
- the cancer can be any cancer, including but not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma
- Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- the route of administration in accordance with the present invention is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intramuscular, intrarterial, subcutaneous, intralesional routes, by aerosol or intranasal routes, or by sustained release systems as noted below.
- the retroviral vector particles and/or the transfected target cells may be administered continuously by infusion or by bolus injection.
- An effective amount of retroviral vector particles and/or the transfected target cells to be employed therapeutically will depend, for example, upon the therapeutic and treatment objectives, the route of administration, the age, condition, and body mass of the patient undergoing treatment or therapy, and auxiliary or adjuvant therapies being provided to the patient. Accordingly, it will be necessary and routine for the practitioner to titer the dosage and modify the route of administration, as required, to obtain the optimal therapeutic effect.
- a typical daily dosage might range from about l xl O 4 retroviral vector particles and/or the transfected target cells /dose to about lxl O 9 retroviral vector particles and/or the transfected target cells /dose or more, preferably from about 1x10 to about 1x10 retroviral vector particles and/or the transfected target cells /dose, depending on the above-mentioned factors.
- the clinician will administer retroviral vector particles and/or the transfected target cells until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
- the retroviral vector particles used in the context of the present invention can, themselves, be linked to a detectable label.
- a detectable label allows for the presence of, or the amount of the viral titer to be determined.
- the detectable label is the C 1 1 D8, V5, or HA epitope tag.
- Alternative methods of vector delivery such as convection may enhance distribution of the retroviral vector comprising the therapeutic gene and, thus, more widespread tumor killing than the simple intratumoral injection.
- an alternative method for efficient and widespread delivery of macromolecules and particles to tumors is convection-enhanced infusion, which is used to supplement simple diffusion and to improve vector distribution by bulk flow inside and outside the tumor.
- Stereotactic injection and subsequent infusion by maintaining a positive pressure gradient is able to improve the distribution of large molecules in animal models (Lieberman D.M., et al., J. Neurosurg. 82: 1021-1029 (1985)).
- this convection- enhanced infusion technique was able to mediate delivery of virus particles to tumors with an approximate volume of 100 mm 3 , and also beyond the tumor borders into the surrounding brain tissue (Nilaver et al., Proc. Natl. Acad. Sci. USA 92: 9829-9833 (1995)).
- Intravascular methods of vector application make use of a natural and ubiquitously distributed network of arteries, veins and capillaries, which is present in every normal tissue and is even denser in malignant tumors.
- Intravascular applications such as intra-arterial injection of virus vectors, are capable of delivering a vector to the largest proportion of tumor cells and surrounding tissues without afflicting mechanical injury to normal brain tissue or having other toxic consequences (Spear et al., J. Neurovirol. 4: 133-147 (1998); Muldoon et al., "Delivery of therapeutic genes to brain and intracerebral tumors; in Chiocca E.A., and Breakefield X.O. (eds.), "Gene Therapy for Neurological Disorders and Brain Tumors," Boston: Humana Press, pp 128-139 (1997)).
- the inventive methods of purifying and/or producing retroviral vector particles may, advantageously, provide a retroviral vector particle comprising Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag, and a retroviral vector comprising a therapeutic gene.
- another embodiment of the invention provides a retroviral vector particle comprising Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag, and a retroviral vector comprising a therapeutic gene.
- the retroviral vector particles, Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1 D8, V5, or HA epitope tag, and therapeutic gene may be as described herein with respect to other aspects of the invention.
- the retroviral vector particles may be useful for any of a variety of applications, including but not limited to the methods of treatment described herein with respect to other aspects of the invention.
- This example demonstrates a method of making a retroviral packaging cell.
- HT1080 cells are transfected with a first vector encoding Gag, Pol, and a puromycin resistance gene. The transfected cells are selected with puromycin. The selected cells are transfected with a second vector encoding a CI 1D8 epitope-tagged Env protein and a hygromycin resistance gene. The cells are selected with hygromycin. The hygromycin- selected cells are ready to be transfected with a vector encoding a gene of interest.
- This example demonstrates a method of transfecting a packaging cell with a vector encoding a gene of interest.
- a vector comprising, from the 5' to the 3' end, loxp, a pgk (phosphoglycerate kinase 1) promoter, a blasticidin resistance gene, lox2722, and a neomycin resistance gene lacking an initation codon is introduced into the genome of the HT1080 packaging cell described in Example 1 between the AAVS1 right arm and AAVS1 left arm genome sites. The cells are selected with blasticidin.
- a Cre expression plasmid and a plasmid containing a vector genome comprising, from the 5' to the 3' end, loxp, a 5' LTR packaging site ( ⁇ ) promoter (e.g., PGK), an IL2yc gene, a 3' LTR, a 628 bp IRES (internal ribosome entry site), and an ATG codon sequence are transfected into the cells that were selected with blasticidin.
- ⁇ 5' LTR packaging site
- IL2yc gene e.g., PGK
- a 3' LTR e.g., PGK
- IRES internal ribosome entry site
- the cells After Cre mediated recombination, the cells contain a genome with the IL2yc gene and an intact ATG-containing G418 resistance gene. From the 5' to the 3' end, the packaging cell genome contains: loxp, a 5' LTR packaging site ( ⁇ ) promoter (e.g., PGK), an IL2yc gene, a 3' LTR, a 628 bp IRES, and the neomycin resistance gene including an initiation codon oriented so that the neomycin resistance gene can be expressed.
- ⁇ 5' LTR packaging site
- the cells are selected with G418.
- the cells produce retroviral vector particles comprising a CI 1 D8 epitope-tagged Env protein and a vector comprising the IL2yc gene.
- This example demonstrates a method of purifying a retroviral vector particle.
- the cells described in Example 2 produce a first supernatant comprising retroviral vector particles comprising a CI 1 D8 epitope-tagged Env protein and a vector comprising the IL2yc gene.
- the first supernatant also contains cells.
- the first supernatant is placed in a tube and centrifuged at a speed of 2,000 g to produce a second supernatant and a pellet comprising the cells.
- the second supernatant comprises the CI 1D8 epitope-tagged retroviral vector particles and cellular components.
- CI 1D8 monoclonal antibody is cross-linked to SEPHAROSE beads, an affinity column is prepared including the cross-linked beads, and the column is equilibrated as described in Grant et al., J. Immunol., 131 (6): 3042-48 (1983).
- the second supernatant is removed from the centrifugation tube and applied to a column including the cross-linked SEPHAROSE beads.
- the column is washed with phosphate buffered saline (PBS) buffer to remove the cellular components.
- PBS phosphate buffered saline
- the retroviral vector particles are eluted from the column in PBS buffer containing 80 ⁇ MGPNL (SEQ ID NO: 5) peptide.
- the retroviral vector particles are concentrated by passing the eluted retroviral vector particles through an
- the virus fraction is measured using an ELISA plate reader with CI 1D8 antibodies.
- the particles are assessed for infectivity in 70 ⁇ peptide eluate.
- This example demonstrates the titer of retroviral particle obtained after purifying the retroviral particles using an epitope tag.
- a nucleic acid comprising a nucleotide sequence encoding a V5 epitope
- GKPIPNPLLGLDST (SEQ ID NO: 15), HA epitope YPYDVPDYA (SEQ ID NO: 16), or CI 1D8 epitope QVMTITPPQAMGPNLVLP (SEQ ID NO: 6) is introduced into the gene encoding GALV Env protein in PG13 cells using targeted genome editing using TALENs.
- the cells produce retroviral particles including the V5, HA, or CI 1 D8 epitope positioned at the amino terminus of the Env protein as shown in Figure 1.
- the retroviral particles are purified as described in Example 3 using V5, HA, or CI 1D8 antibodies.
- the titer of retroviral particles is measured and compared to that obtained with PG 13 cells that produce wild-type GALV retroviral particles (i.e., retroviral particles that lack an epitope tag).
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Abstract
The invention provides methods of purifying retroviral vector particles using a C11D8, V5, or influenza hemagglutinin (HA) epitope tag. The invention also provides methods of producing retroviral vector particles comprising inserting a gene of interest into the cell genome by Cre recombinase-mediated cassette exchange (RMCE). Retroviral packaging cells, methods of producing retroviral packaging cells, methods of treatment, and retroviral vector particles are also provided.
Description
RETROVIRAL VECTOR PACKAGING CELL LINES AND METHODS OF PURIFYING AND PRODUCING RETROVIRAL PARTICLES
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/759,516, filed February 1 , 2013, which is incorporated herein by reference in its entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a computer-readable
nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 4,887 Byte ASCII (Text) file named "715813_ ST25.txt," dated January 29, 2014.
BACKGROUND OF THE INVENTION
[0003] Retroviral vector particles are useful for any of a variety of gene transfer applications (e.g., gene therapy). However, obstacles to the effective production of retroviral vector particles exist. For example, the production of retroviral vector particles using packaging cell lines may produce a retroviral vector particle product that is contaminated with other cell components that could, for example, be toxic to a patient. Retroviral particles produced from murine packaging cells may not efficiently infect human target cells and may be subject to inactivation by the human immune system. Murine packaging cell genomes also may contain endogeneous, inducible replication competent retroviruses that can disadvantageously result in replication competent retroviruses upon recombination with introduced retroviral packaging genes.
[0004] Accordingly, there is a need for improved retroviral packaging cells and methods of producing retroviral vector particles.
BRIEF SUMMARY OF THE INVENTION
[0005] An embodiment of the invention provides a method of purifying retroviral vector particles, the method comprising: (a) (i) introducing a nucleic acid comprising a nucleotide
sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1 D8, V5, or influenza hemagglutinin (HA) epitope tag; (b) culturing the cells of (a) to produce epitope-tagged retroviral vector particles; (c) producing a first supernatant comprising the epitope-tagged retroviral vector particles and cells; (d) centrifuging the first supernatant to produce a second supernatant comprising the epitope-tagged retroviral vector particles and cellular components; (e) separating the second supernatant from the cells of (c); (f) contacting a stationary phase comprising an antibody with the second supernatant and binding the epitope-tagged retroviral vector particles to the antibody, wherein the antibody is a CI 1D8, V5, or HA antibody; (g) separating the cellular components from the epitope- tagged retroviral vector particles; and (h) eluting the epitope-tagged retroviral vector particles from the stationary phase to produce purified epitope-tagged retroviral vector particles.
[0006] Another embodiment of the invention provides a method of producing retroviral vector particles, the method comprising: (a) (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; (b) transfecting the cells with a vector encoding a gene of interest; (c) inserting the gene of interest into a cell genome by Cre recombinase-mediated cassette exchange (RMCE); (d) culturing the cells of (c) to produce retroviral vector particles by the cells; (e) producing a first supernatant comprising the retroviral vector particles and cells; (f) centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and (g) separating the second supernatant including the retroviral vector particles from the cells of (e).
[0007] Still another embodiment of the invention provides retroviral packaging cells comprising HT1080, 293 (also known as HEK cells) or LNCaP cells expressing Gag, Pol, and Env proteins.
[0008] Another embodiment of the invention provides Retroviral packaging cells comprising a CI 1D8, V5, or HA epitope tag on a cell surface protein, wherein the cells are human or mouse cells.
[0009] Still another embodiment of the invention provides retroviral packaging cells comprising two lox sites flanking a target site for a gene of interest.
[0010] Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising transfecting HT1080, 293 or LNCaP cells with one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins.
[0011] Still another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag.
[0012] Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells.
[0013] Still another embodiment of the invention provides methods of treating a disease in a mammal in need thereof, the method comprising administering to the mammal the retroviral vector particles purified or produced by any of the inventive methods described herein, wherein the gene of interest encodes a therapeutic gene suitable for treating the disease.
[0014] Another embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising transfecting target cells with the retroviral vector particles purified or produced according to any of the inventive methods described herein and administering the transfected target cells to the mammal in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
[0015] Another embodiment of the invention provides a retroviral vector particle comprising Gag, Pol, and Env proteins, and a retroviral vector comprising a therapeutic gene, wherein the Env protein comprises a CI 1 D8, V5, or HA epitope tag.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0016] Figure 1 is a schematic showing the insertion of an epitope tag between a signal peptide and a surface subunit (SU) of the envelope protein to produce a pre-protein that undergoes signal peptide cleavage to produce an envelope protein having an epitope tag positioned on the amino terminus.
DETAILED DESCRIPTION OF THE INVENTION
Methods of Purifying Retroviral Vector Particles
[0017] It has been discovered that retroviral vector particles may be effectively purified using a CI 1D8, V5, or HA epitope tag. Accordingly, an embodiment of the invention provides a method of purifying retroviral vector particles comprising (a) (i) introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or influenza hemagglutinin (HA) epitope tag; (b) culturing the cells of (a) to produce epitope-tagged retroviral vector particles; (c) producing a first supernatant comprising the epitope-tagged retroviral vector particles and cells; (d) centrifuging the first supernatant to produce a second supernatant comprising the epitope-tagged retroviral vector particles and cellular components; (e) separating the second supernatant from the cells of (c); (f) contacting a stationary phase comprising an antibody with the second supernatant and binding the epitope-tagged retroviral vector particles to the antibody, wherein the antibody is a CI 1D8, V5, or HA antibody; (g) separating the cellular components from the epitope-tagged retroviral vector particles; and (h) eluting the epitope-tagged retroviral vector particles from the stationary phase to produce purified epitope-tagged retroviral vector particles.
[0018] The retroviral vector particle may be any suitable retroviral vector particle known in the art. The retroviral vector particle may be chosen based on any of a variety of factors including, for example, the particular type of retroviral vector to be enveloped and/or the type of cell and/or the type of tissue to be targeted for gene therapy (also referred to herein as a "target cell" or "target tissue"). In an embodiment of the invention, the retroviral vector particles are gammaretroviral vector particles. In another embodiment of the invention, the retroviral vector particles are lentiviral particles. Lentiviral particles may provide several
advantages including, for example, the ability to provide long-term and stable gene expression and the ability to infect non-dividing target cells, such as, for example, neurons. The retroviral vector particles may be derived from any suitable species of retrovirus. For example, the retroviral vector particles may be derived from Gibbon Ape Leukemia Virus (GALV), murine leukemia virus (MLV), wooly monkey virus (WMV), koala retrovirus (KoRV), spleen leukosis virus (SNV), feline leukemia virus (FeLV), or RD1 14. GALV retroviral vector particles may provide numerous advantages including, for example, the ability to infect a broad range of target cells and non-toxicity (or reduced toxicity) to target cells, including, for example, neurons, microglia, hematopietic stem cells, astrocytes, T cells, and B cells.
[0019] As used herein, the terms "retroviral vector particle" and "retroviral particle" refer to particles that contain a viral envelope and have the ability to infect cells but are not replication competent. In contrast, viruses are replication competent.
[0020] In an embodiment, the method comprises (i) introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag;. Introducing a nucleotide sequence into cells may be carried out in any suitable manner known in the art. See, for example, Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, New York (2012) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY (2007). Exemplary methods of introducing a nucleotide sequence into cells may include, but are not limited to, transfection and targeted genome editing using, for example, zinc finger nucleases, CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs) (see, e.g., Joung et al., Nat. Rev. Mol. Cell Biol, 14(1): 49-55 (2013)).
[0021] Introducing a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or introducing one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag advantageously provides a cell that is capable of producing retroviral vector particles (also referred to as a "packaging cell"). The packaging cell may provide stable (i.e., constitutive) or transient expression of retroviral
particles. In an embodiment of the invention, the packaging cell is a mouse cell that provides stable expression of retroviral particles. In an embodiment of the invention, the packaging cell is a human cell that provides transient expression of retroviral particles.
[0022] In an embodiment, a nucleotide sequence encoding an epitope tag is introduced into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein. In this regard, the cells into which a nucleotide sequence is introduced already express Gag, Pol, and Env proteins and may be, for example, any viral packaging cell.
Exemplary viral packaging cells include, but are not limited to, PG13 packaging cells and STAR packaging cell lines for lentiviral vectors (Ikeda et al., Nat. Biotechnol., 21 : 569-572 (2003)). Preferably, the viral packaging cell is a PG13 packaging cell or STAR GALV. In an embodiment, when the cells into which a nucleotide sequence is introduced already express Gag, Pol, and Env proteins, the method may comprise introducing a nucleotide sequence encoding an epitope tag into the cells using targeted genome editing such as, for example, TALENs to obtain expression of an epitope-tagged Env protein.
[0023] In an embodiment, one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins are introduced into cells, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag. In this regard, the cells into which the one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins are introduced do not already express Gag, Pol, and Env proteins and may not be a packaging cell prior to the introduction of the one or more nucleotide sequences. For example, the cell may be a human cell that is not a packaging cell such as, for example, HT1080, 293 or LNCaP cells, or a mouse cell that is not a packaging cell such as, for example, NIH3T3 cells. The cells may become a packaging cell after introduction of the one or more nucleotide sequences encoding Gag, Pol, and epitope-tagged Env proteins into the cell.
[0024] "Nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
[0025] In an embodiment of the invention, a vector comprising any of the nucleic acids described herein may be introduced into the cells. For puiposes herein, the term "vector"
refers to a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. In an embodiment of the invention, the vectors are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The vectors can comprise naturally-occurring, non-naturally-occurring intemucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector. The vector may be any suitable vector including, for example, commercially available vectors. In some embodiments, it may be advantageous to employ a vector that is constructed so that the genes within the inverted repeats (IR) of the plasmid are positioned so that the genes are not disrupted during integration of all or a portion of the vector into the cell genome. Such vectors are commercially available and may include, for example, the PIGGYBAC plasmid available from System Bioscience (Mountain View, CA).
[0026] In an embodiment, the vectors described herein are recombinant expression vectors. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. The nucleic acids described herein may also be recombinant.
[0027] The vectors and nucleic acids described herein can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra, and Ausubel et al., supra. Constructs of vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, cyto megalovirus and the like.
[0028] Desirably, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g.,
bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA based.
[0029] The nucleic acid(s) may encode any suitable retroviral Gag, Pol, and Env proteins known in the art that are suitable for the particular type of retroviral vector particle chosen and/or the type of retroviral vector to be enveloped. In an embodiment of the invention, the Gag, Pol, and/or Env protein(s) may be wild-type or enhanced, and may be derived from any suitable retrovirus species such as, for example, those described herein with respect to other aspects of the invention. In an embodiment of the invention, the nucleic acid(s) encode a wild-type GALV Env protein comprising both DRISAVKIL (SEQ ID NO: 1) and
VLRQKALENE (SEQ ID NO: 2). In another embodiment, the nucleic acid(s) encode substituted Gag, Pol, and/or Env proteins. For example, the nucleic acid(s) may encode a substituted GALV Env protein that includes three amino acid substitutions with respect to the wild-type GALV protein. In this regard, the nucleic acid(s) may encode a substituted GALV Env protein comprising both DRISAVQAL (SEQ ID NO: 3) and VLTQKALENE (SEQ ID NO: 4). The substituted GALV Env protein encoded by such nucleic acid(s) may facilitate the ability of the GALV retroviral vector particle to envelope a lentiviral vector as described in Merten et al., J. Virol, 79: 834-40 (2005) and Christodoulopoulos et al, J. Virol, 75: 4129-38 (2001). Retroviral vector particles expressing the substituted Env protein comprising both DRISAVQAL (SEQ ID NO: 3) and VLTQKALENE (SEQ ID NO: 4) may, advantageously, envelope lentiviral vectors. As used herein, the terms "Env" and "Env protein" refer to both wild-type and substituted Env proteins unless specified otherwise.
[0030] In an embodiment, the cells into which a nucleic acid has been introduced express an Env protein that further comprises a CI 1D8, V5, or HA epitope tag. In this regard, the Env protein further comprises (i) a CI 1D8 epitope (also referred to as a "CI 1D8 tag" or "CI 1D8 epitope tag"), (ii) a V5 epitope (also referred to as a "V5 tag" or "V5 epitope tag"), or (iii) an HA epitope (also referred to as an "HA tag" or "HA epitope tag") which specifically binds to a CI 1 D8 antibody, V5 antibody, or HA antibody, respectively . The CI 1D8, V5, or HA epitope tag may comprise any suitable amino acid sequence that specifically binds to a CI 1D8, V5, or HA antibody, respectively. HA and V5 epitope tags may provide numerous advantages such as, for example, a higher titer yield and easier purification.
[0031] In an embodiment, the nucleic acid(s) may encode a CI 1D8 epitope comprising QVMTITPPQAMGPNLVLP (SEQ ID NO: 6) or a functional fragment of SEQ ID NO: 6.
The functional fragment of SEQ ID NO: 6 may comprise less than the entire SEQ ID NO: 6 and at least 5 contiguous amino acid residues of SEQ ID NO: 6 such that the functional fragment specifically binds to a CI 1D8 antibody. In an embodiment of the invention, the functional fragment comprises from about 5 to about 17 contiguous amino acid residues of SEQ ID NO: 6, preferably from about 6 to about 16 contiguous amino acid residues of SEQ ID NO: 6, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 6. In an embodiment of the invention, the functional fragment comprises MGPNL (SEQ ID NO: 5). Exemplary CI 1D8 epitopes useful for the invention include, but are not limited to, SEQ ID NOs: 5-14.
[0032] In an embodiment, the nucleic acid(s) may encode a V5 epitope comprising GKPIPNPLLGLDST (SEQ ID NO: 15) or a functional fragment of SEQ ID NO: 15. The functional fragment of SEQ ID NO: 15 may comprise less than the entire SEQ ID NO: 15 and at least 5 contiguous amino acid residues of SEQ ID NO: 15 such that the functional fragment specifically binds to a V5 antibody. In an embodiment of the invention, the functional fragment comprises from about 5 to about 14 contiguous amino acid residues of SEQ ID NO: 15, preferably from about 6 to about 12 contiguous amino acid residues of SEQ ID NO: 15, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 15.
[0033] In an embodiment, the nucleic acid(s) may encode an HA epitope comprising YPYDVPDYA (SEQ ID NO: 16) or a functional fragment of SEQ ID NO: 16. The functional fragment of SEQ ID NO: 16 may comprise less than the entire SEQ ID NO: 16 and at least 5 contiguous amino acid residues of SEQ ID NO: 6 such that the functional fragment specifically binds to a HA antibody. In an embodiment of the invention, the functional fragment comprises from about 5 to about 9 contiguous amino acid residues of SEQ ID NO: 16, preferably from about 6 to about 8 contiguous amino acid residues of SEQ ID NO: 16, more preferably at least about 7 contiguous amino acid residues of SEQ ID NO: 16.
[0034] In an embodiment, the epitope tag may comprise one or more copies of the any of the epitope tag amino acid sequences described herein. For instance, the epitope tag can comprise 1 , 2, 3, 4, 5, or more, copies of the epitope tag amino acid sequence. In this regard, the epitope tag may comprise YPYDVPDYAYPYDVPDYA (SEQ ID NO: 17),
GKPIPNPLLGLDSTGKPIPNPLLGLDST (SEQ ID NO: 1 8), or MGPNLMGPNL (SEQ ID NO: 19).
[0035] Introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag or an epitope-tagged Env protein, wherein the epitope tag is a CD1 1D8, V5, or HA epitope tag provides numerous advantages. For example, the CI 1D8, V5, or HA epitope tag may allow the retroviral vector particles to be purified and/or detected using CI 1D8, V5, or HA antibodies, respectively. Additionally, the CI 1D8, V5, or HA epitope tag
advantageously may not reduce or eliminate the ability of the retroviral vector particle to bind to or infect a target cell.
[0036] The nucleic acid(s) vector(s) may encode an Env protein having the epitope tag positioned at any suitable position in the Env protein amino acid sequence. In an
embodiment of the invention, the epitope tag is positioned in the Env protein such that the epitope tag is recognized by the appropriate antibody. In an embodiment of the invention, the epitope tag may be inserted into a proline rich region (PRR) of the Env protein. For example, the epitope tag may be inserted into the GALV Env protein at GALV envelope protein residue 219 or 264 as described in Farrell et al, J. Virol., 76: 4267-74 (2002). In an embodiment of the invention, the epitope tag may be positioned at the amino terminus of the Env protein. For example, as shown in Figure 1 , the epitope tag may be inserted between a signal peptide (such as, for example, SEQ ID NO: 20) and a surface (SU) subunit of the envelope protein (such as, for example, SEQ ID NO: 21) to produce a preprotein that includes the epitope tag positioned at the carboxyl terminus of the signal peptide and at the amino terminus of the Env protein. The preprotein may undergo cleavage of the signal peptide to produce a mature envelope protein including the epitope tag positioned at the amino terminus of the Env protein. An epitope tag positioned at the amino terminus of the Env protein may provide numerous advantages such as, for example, a higher titer yield and easier purification using, for example, an antibody-conjugated column as compared to an epitope tag that is located in the Env protein at a position other than the amino terminus.
[0037] In an embodiment, the cells into which one or more nucleic acids are introduced are mammalian cells. In this regard, the cells may be derived from any suitable mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal may be from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammal may be of the order Primates, Ceboids, or Simoids
(monkeys) or of the order Anthropoids (humans and apes). A preferred mammal is the mouse.
[0038] In an embodiment of the invention, the cells are human and the human cells are HT1080, 293 or LNCaP cells. HT1080 is a human fibrosarcoma cell line, 293 is a human embryonic kidney cell line and LNCaP is a human carcinoma cell line. In another embodiment of the invention, the cells are mouse cells. For example, the mouse cells may be PG13 packaging cells, which are derived from NIH3T3 mouse cells.
[0039] The method may further comprise transfecting the cells with a vector encoding a gene of interest. Transfecting cells with a vector encoding a gene of interest may be carried out in any suitable manner known in the art that will enable the cell to produce retroviral vector particles capable of infecting target cells with the gene of interest. The gene of interest may be any suitable gene useful for any of a variety of applications.
[0040] In an embodiment, the gene is a therapeutic gene. The therapeutic gene can encode any beneficial protein or polypeptide that replaces missing or defective protein required by the target cell or subject into which the vector is transferred, or can encode a cytotoxic polypeptide that can be directed, e.g., to cancer target cells or other target cells whose death would be beneficial to the subject. The therapeutic gene can also encode antisense RNAs that can bind to, and thereby inactivate, mRNAs made by the subject that encode harmful proteins. The therapeutic gene can also encode ribozymes that can effect the sequence-specific inhibition of gene expression by the cleavage of mRNAs. In one aspect, antisense polynucleotides can be produced from a therapeutic gene that includes an expression cassette that contains a sequence that promotes cell-type specific expression (Wirak et al., EMBO 10:289 (1991)). For general methods relating to antisense
polynucleotides, see Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). Other examples of therapeutic genes which can be administered to a target cell or subject as part of the retroviral vectors described herein can include, but are not limited to, the following: nucleic acids encoding secretory and nonsecretory proteins and nucleic acids encoding therapeutic agents.
[0041] Further examples of therapeutic agents that may be encoded by the therapeutic gene may include, but are not limited to, tumor necrosis factors (TNFs), such as TNF-a; interferons, such as interferon-a, interferon-β, and interferon-γ, interleukins (IL), such as IL- 1 , IL-Ι β, and ILs-2 through -14; granulocyte-macrophage colony-stimulating factor (GM- CSF); adenosine deaminase; cellular growth factors, such as lymphokines; soluble CD4;
Factor VIII; Factor IX; T-cell receptors; Low-Density Lipoprotein (LDL) receptor; ApoE; ApoC; alpha- 1 antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosis transmembrane receptor (CFTR); insulin; Fc receptors for antigen binding domains of antibodies, such as immunoglobulins; anti-human immunodeficiency virus (HIV) decoy tar elements; and antisense sequences which inhibit viral replication, such as antisense sequences which inhibit replication of hepatitis B or hepatitis non-A, non-B virus. Further exemplary therapeutic genes that may be employed include, but are not limited to, was, gp91-phox, il2yc, and ada. The therapeutic gene is chosen considering several factors, including the cell and/or tissue to be targeted for therapy. Where the target cell is a blood cell, for example, particularly useful therapeutic genes to use are those which allow the blood cells to exert a therapeutic effect, such as a gene encoding a clotting factor for use in treatment of hemophilia. Another target cell is the lung airway cell, which can be used to administer nucleic acids, such as those coding for the cystic fibrosis transmembrane receptor, which could provide a gene therapeutic treatment for cystic fibrosis. Other target cells include muscle cells where useful therapeutic genes, such as those encoding cytokines and growth factors, can be transduced and the protein the nucleic acid encodes can be expressed and secreted to exert its effects on other cells, tissues and organs, such as the liver. In addition, cancer cells corresponding or derived from lung, muscle, brain and other tissues can be target tissues. Additional target cells for gene therapy may include, but are not limited to, immune cells (e.g., T cells and B cells), stem cells (e.g., embryonic stem cells and/or hematopoietic stem cells) and cells of the central nervous system (e.g., neurons, astrocytes, and microglia). Furthermore, the therapeutic gene can encode more than one gene product, limited only by the size of vector that can be packaged by the caspid of the particular retroviral vector particle chosen.
[0042] In an embodiment of the invention, the method comprises inserting the gene of interest into a (packaging) cell genome by Cre recombinase-mediated cassette exchange (RMCE). Inserting the gene of interest into the packaging cell genome by Cre RMCE can, advantageously, direct the insertion of the gene of interest into any suitable specific target site in the packaging cell genome. The target site chosen may, for example, be a transcriptionally active site that facilitates optimal transcription of the gene of interest. Alternatively or, preferably, additionally, the target site chosen may advantageously avoid inserting of the gene of interest at an undesirable site such as, for example, a site that disrupts the expression of an endogenous gene the normal expression of which is necessary for the healthy function of the cell and/or a site that causes uncontrolled cell division (e.g., by activating an
oncogene). When the packaging cells are human cells, preferably the target site is in a human chromosome 19-specific adeno-associated virus (AAV) integration site. The AAV integration site may be any suitable AAV integration site. Preferably, the AAV integration site is AAVS 1.
[0043] In an embodiment, the method further comprises transfecting the cells with a lox insertion vector encoding lox sites, a first marker gene and a defective second marker gene.
[0044] The first marker gene can be any suitable marker gene which allows for selection of transfected cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes include, for instance, LacZ, green fluorescent protein (GFP), luciferase, puromycin, blasticidin, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. In an embodiment of the invention, the first marker gene is an antibiotic resistance gene.
[0045] The lox insertion vector may include an appropriate promoter oriented to promote expression of the first marker gene. The promoter may comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the first marker gene. The selection of promoters, e.g., strong, weak, and inducible, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a retroviral promoter, a GALV promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, chicken beta-actin promoter with CMV enhancer (CAG) promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus. Other exemplary promoters include, but are not limited to, a PGK (phosphoglycerate kinase 1) promoter, a glial fibrillary acidic protein (GFAP) (promoter), a neuronal-specific nuclear protein (NeuN) promoter, and an elongation factor-1 alpha (EFl ) promoter.
[0046] The lox insertion vector may comprise two lox sites flanking the combination of the promoter and the first marker gene. The lox sites may be any suitable lox sites.
Preferably, the lox sites are loxP and lox2722. In an embodiment of the invention, loxp is oriented 5' of the combination of the promoter and the first marker gene and lox2722 is oriented 3' of the combination of the promoter and the first marker gene. Alternatively, the loxp is oriented 3' of the combination of the promoter and the first marker gene and lox2722 is oriented 5' of the combination of the promoter and the first marker gene.
[0047] The defective second marker gene may be any suitable marker gene such as, for example, any of those described above which has been mutated so that it is not expressed until the mutation is corrected upon Cre-mediated recombination. The mutation may be any suitable mutation. In an embodiment, the defective second marker gene is mutated to lack an initiation codon. Preferably, the defective second marker gene encodes a marker that is different from that which is encoded by the first marker gene (e.g., resistance to a different antibiotic).
[0048] The defective second marker gene may be positioned outside of the lox-flanked combination of promoter and first marker gene. For example, the lox insertion vector may comprise, from the 5' to the 3' end, a lox site, a promoter, a first marker gene, a lox site, and the defective second marker gene. Alternatively, the lox insertion vector may comprise, from the 5' to the 3' end, the defective second marker gene, a lox site, a promoter, a first marker gene, a lox site, and the defective second marker gene.
[0049] The lox insertion vector may further comprise nucleotide sequences that are homologous to a left and right arm of the target site (e.g., the AAV integration site) flanking the combination of the promoter, marker gene, lox sites, and defective second marker gene. The nucleotide sequences homologous to left and right arms of the AAV integration sites are known in the art and may be any suitable size that provides for insertion of the lox sites, promoter, first marker gene, and defective second marker gene in the target site. For example, the nucleotide sequences that are homologous to a left and right arm of the target site may be from about 3 to about 4 kb.
[0050] Transfecting the cells with a lox insertion vector encoding a marker gene flanked by lox sites and a defective second marker gene results in the integration of the lox sites, the promoter, the first marker gene, and the defective second marker gene into the target site of the genome of the packaging cell. In an embodiment, the lox sites, the promoter, the first marker gene, and the defective second marker gene are integrated into the target site of the genome of the packaging cell in the same orientation as described for the lox insertion vector. The method may further comprise selecting transfected cells using the marker encoded by the first marker gene.
[0051] In an embodiment of the invention, transfecting the cells with a vector encoding a gene of interest comprises transfecting the cells with one or more vectors encoding Cre recombinase and the gene of interest. The vector encoding the gene of interest may comprise the gene of interest flanked by lox sites oriented in a position to facilitate integration of the
gene of interest into the packaging cell genome by Cre RMCE using the lox sites already inserted into the genome as described above. The lox sites in the vector encoding the gene of interest may be any suitable lox sites and are preferably the same lox sites already inserted into the genome. The vector encoding the gene of interest may further encode an internal ribosome entry site (IRES), a 5' long terminal repeat (LTR), and a 3' LTR, and/or an appropriate promoter oriented to promote expression of the gene of interest and the second marker gene. In a preferred embodiment of the invention, the vector encoding the gene of interest is a self-inactivating (SIN) vector such that integration of components into the cell genome does not include integration of a viral promoter into the cell genome. SIN vectors may lack a viral promoter at both the 5' and 3' ends upon integration into the cell genome. The promoter for the gene of interest may be as described herein with respect to other aspects of the invention. In an embodiment of the invention, the promoter is a 5' long tenninal repeat (LTR) packaging site (ψ) promoter (e.g., PGK).
[0052] The vector encoding the gene of interest may further encode a component that corrects the mutation that prevents the defective second marker gene from being expressed. For example, when the mutation is the lack of an initiation codon, the vector encoding the gene of interest may further include an initiation codon. The component correcting the mutation may be oriented in the vector encoding the gene of interest such that the component is inserted into the defective second marker gene in the genome of the cell to correct the mutation and allow for expression of the formerly defective second marker gene.
[0053] The lox sites, gene of interest and its promoter, the IRES, and the component that corrects a mutation may be oriented in the vector in such a manner so as to facilitate their integration into the genome of the packaging cell at the target site. For example, the vector encoding the gene of interest may comprise, from the 5' to the 3' end, loxp, a 5' LTR packaging site (ψ) promoter, gene of interest, 3' LTR, IRES, the component that corrects a mutation in the second marker gene, and lox2722.
[0054] Transfecting the cells with a vector encoding Cre recombinase results in the activation of Cre RMCE. Cre RMCE exchanges the first marker gene with the gene of interest so that the first marker gene and its promoter are removed from the cell genome and replaced with the gene of interest, its promoter, IRES, and the component that corrects the mutation in the defective second marker gene. Upon completion of Cre RMCE, the packaging cells may contain a genome comprising the gene of interest, its promoter, the IRES, and the second, corrected marker gene. The gene of interest, its promoter, the IRES,
and the second marker gene may be located at the target site and oriented in such as way as to allow the cell to produce retroviral vector particles that envelope a retroviral vector containing the gene of interest that can be integrated into a target cell upon infection of the target cell with the retroviral vector particles. For example, upon completion of Cre RMCE, the cell genome may comprise, from the 5' to the 3' end, loxp, a 5' LTR packaging site (ψ) promoter, gene of interest, 3'LTR, IRES, the corrected, second marker gene, and lox2722. In an embodiment of the invention, upon completion of Cre RMCE, viral promoters are not integrated into the cell genome. The method may further comprise selecting for cells encoding the gene of interest using the marker encoded by the corrected, second marker gene.
[0055] Inserting the gene of interest into a cell genome by Cre RMCE may provide many advantages. For example, because the first marker gene is replaced with the gene of interest in the genome of the packaging cell, the first marker gene does not have to be incorporated into the genome bearing the therapeutic gene. The removal of the first marker gene from the genome of the packaging cell may, advantageously, provide more room for a larger therapeutic gene. For example, the inventive methods may provide space of up to about 8 kb for a therapeutic gene as opposed to only about 5 kb of space that would remain for a therapeutic gene if the first marker gene remained in the packaging cell genome.
[0056] The method comprises culturing the cells to produce epitope-tagged retroviral vector particles. The cells may be cultured in any suitable manner known in the art that causes the cells to produce retroviral vector particles. The epitope tag may be as described herein with respect to other aspects of the invention.
[0057] The method comprises producing a first supernatant comprising the epitope- tagged retroviral vector particles and cells. In an embodiment of the invention, the first supernatant may also comprise aggregates of cells and small particles and/or debris. The small particles and/or debris may include particles and debris that are smaller than cells such as, for example, cellular components (e.g., membranes and/or vesicles). The first supernatant may be produced in any suitable manner known in the art.
[0058] The method comprises centrifuging the first supernatant to produce a second supernatant comprising the epitope-tagged retroviral vector particles and small particles and/or debris, e.g., cellular components. Centrifuging the first supernatant may be carried out in any suitable manner known in the art and at any suitable spin speed. In an embodiment of the invention, centrifuging the first supernatant may be carried out at a spin speed of about 3
to about 4,000 g. Centrifuging the first supernatant may further comprise forming a pellet comprising the cells described above.
[0059] The method comprises separating the second supernatant from the cells described above. Separating the second supernatant from the cells may be earned out in any suitable manner known in the art. In an embodiment of the invention, separating the second supernatant from the cells may comprise physically separating the second supernatant, including the cellular components and the epitope-tagged retroviral vector particles, from cells present in a pellet formed after centrifuging. The second supernatant that is separated from the cells contains no cells or fewer cells as compared to that contained in the first supernatant.
[0060] The method comprises contacting a stationary phase comprising anantibody with the second supernatant and binding the epitope-tagged retroviral vector particles to the antibody, wherein the antibody is a CI 1D8, V5, or HA antibody. The stationary phase may be any stationary phase known in the art that is suitable for separation by affinity
chromatography. For example, the stationary phase may be beads (e.g., metallic and/or polymeric beads). Suitable exemplary stationary phases that are commercially available include SEPHAROSE beads (Sigma-Aldrich, St. Louis, MO). In an embodiment of the invention, the stationary phase is cross-linked. In this regard, the stationary phase may be beads cross-linked with protein G or protein A.
[0061] The stationary phase may comprise a CI 1D8, V5, or HA antibody conjugated to the stationary phase. Suitable CI 1D8, V5, and HA antibodies are known in the art and are commercially available (e.g., Santa Cruz Biotechnology, Inc., Santa Cruz, CA; Thermo Fisher Scientific, Waltham, MA; and Novus Biologicals, Littleton, CO). The antibody chosen will be one that specifically binds to and recognizes the particular epitope tag employed. For example, when the epitope tag is a CI 1D8 epitope tag, the antibody is a CI 1 D8 antibody, when the epitope tag is a V5 epitope tag, the antibody is a V5 antibody, and when the epitope tag is an HA epitope tag, the antibody is an HA antibody. Contacting the stationary phase with the second supernatant may be carried out in any suitable manner known in the art and may comprise physically contacting the stationary phase with the second supernatant, including the cellular components and the epitope-tagged retroviral vector particles, such that the epitope-tagged retroviral vector particles in the second supernatant bind to the antibody.
[0062] The method comprises separating the cellular components from the epitope- tagged retroviral vector particles. Separating the cellular components from the epitope- tagged retroviral vector particles may be carried out in any suitable manner and may include physically separating the cellular components from the epitope-tagged retroviral vector particles. Separating cellular components from the epitope-tagged retroviral vector particles may be carried out by binding the epitope-tagged retroviral vector particles to the antibody- conjugated stationary phase while not binding the cellular components to the antibody- conjugated stationary phase. In an embodiment, components in the second supernatant that lack an epitope (e.g., cellular components) will not bind to the antibody. The cellular components may be removed from the stationary phase (and the epitope-tagged retroviral vector particles bound to the stationary phase) in any suitable manner, e.g., by washing the stationary phase with a suitable buffer (e.g., PBS). The method may comprise removing the components that lack an epitope (e.g., cellular components) and which are not bound to the antibody from the epitope-tagged retroviral vector particles.
[0063] The method comprises eluting the epitope-tagged retroviral vector particles from the stationary phase to produce purified epitope-tagged retroviral vector particles. Eluting the epitope-tagged retroviral vector particles from the stationary phase may be carried out in any suitable manner. In an embodiment, eluting the epitope-tagged retroviral vector particles from the stationary phase may be carried out by contacting the stationary phase with buffer containing any of the CI 1D8, V5, or HA epitopes described herein. The epitope chosen will be one that specifically binds to and recognizes the particular antibody employed. For example, when the antibody is a CI 1D8 antibody, the epitope is a CI 1D8 epitope, when the antibody is a V5 antibody, the epitope is a V5 epitope, and when the antibody is an HA antibody, the epitope is an HA epitope. The elution buffer may comprise any suitable amount of peptide (epitope), for example, about 60 μΜ to about 100 μΜ of peptide. The method may comprise displacing the epitope-tagged retroviral vector particles with the epitopes and binding the epitopes to the antibody on the stationary phase to produce an eluent comprising purified epitope-tagged retroviral vector particles. The eluent comprising purified epitope-tagged retroviral vector particles contains no cellular components or fewer cellular components as compared to that contained in the second supernatant.
[0064] The term "purified" as used herein means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For
example, the purity can be at least about 50%, can be greater than 60%, 70% or 80%, or can be 100%.
[0065] Purifying the epitope-tagged retroviral vector particles may provide many advantages. For example, purifying the epitope-tagged retroviral vector particles may reduce and/or remove cellular components and debris that may be toxic to target cells, target tissues, and/or to a patient. In this regard, the inventive methods may advantageously provide a purified retroviral vector particle product that is safer for delivery to target cells, target tissues, and/or to patients. For example, the inventive methods may provide purified retroviral vector particles that can be more safely administered to the central nervous system because it may reduce or avoid the administration of cellular components and/or debris that can cause inflammation in the central nervous system. The inventive methods of purifying retroviral particles using an epitope tag may, also, advantageously reduce the time, expense, and labor associated with other methods of purifying retroviral particles that do not use epitope tagged retroviral particles (see, e.g., Biffi et al., Science, 341 : 1233158 (2013) (e.g., Supplemental Figure S3)).
[0066] In an embodiment of the invention, the method may further comprise
concentrating the purified epitope-tagged retroviral vector particles. Concentrating the purified epitope-tagged retroviral vector particles may be carried out in any suitable manner known in the art. In an embodiment, concentrating the purified epitope-tagged retroviral vector particles is carried out by passing the purified epitope-tagged retroviral vector particles (e.g., the eluent) through a filter having any suitable molecular weight cut-off that retains the epitope-tagged retroviral vector particles. For example, the molecular weight cut-off may be about 100,000 or more. Exemplary filters useful for the inventive methods include AMICON filters (available from Millipore, Billerica, MA).
Methods of Producing Retroviral Vector Particles
[0067] Another embodiment of the invention provides a method of producing retroviral vector particles, the method comprising: (a) (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; (b) transfecting the cells with a vector encoding a gene of interest; (c) inserting the gene of interest into a cell genome by Cre RMCE; (d) culturing the cells of (c) to produce retroviral vector particles by the cells; (e) producing a first supernatant comprising the
retroviral vector particles and cells; (f) centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and (g) separating the second supernatant including the retroviral vector particles from the cells of (e).
[0068] The method comprises (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins;. Cells that express Gag, Pol, and Env proteins may be packaging cells as described herein with respect to other aspects of the invention. Introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins to obtain expression of Gag, Pol, and Env proteins may be carried out as described herein with respect to other aspects of the invention. In an embodiment of the invention, the nucleic acid(s) encode an Env protein that does not comprise a CI 1D8, V5, or HA epitope. In an embodiment of the invention, the nucleic acid(s) encode an Env protein, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
[0069] The method comprises transfecting the cells with a vector encoding a gene of interest; inserting the gene of interest into a cell genome by Cre RMCE; culturing the cells to produce retroviral vector particles by the cells; producing a first supernatant comprising the retroviral vector particles and cells; centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and separating the second supernatant including the retroviral vector particles from the cells. These steps may be carried out as described herein with respect to other aspects of the invention.
[0070] In an embodiment, the method further comprises purifying the retroviral vector particles from the cellular components. Purifying the retroviral vector particles from the cellular components may be carried out by any suitable method. In an embodiment in which the nucleic acid(s) encode an Env protein, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag, purifying the retroviral vector particles from the cellular components may be earned out as described herein with respect to other aspects of the invention.
[0071] In an embodiment, the method further comprises concentrating the retroviral vector particles. Concentrating the retroviral vector particles may be carried out as described herein with respect to other aspects of the invention.
Retroviral Packaging Cells
[0072] Another embodiment of the invention provides retroviral packaging cells comprising HT1080, 293 or LNCaP cells expressing Gag, Pol, and Env proteins. The HT1080, 293 or LNCaP cells may be transfected with one or more nucleic acid(s)encoding Gag, Pol, and Env proteins as described herein with respect to other aspects of the invention.
[0073] Human packaging cells for producing retroviral vector particles provide many advantages. For example, retroviruses produced from human packaging cells may more efficiently infect human target cells as compared to retroviruses produced from non-human packaging cells, e.g., murine packaging cells. Retroviruses produced from human packaging cells may also be less likely to be inactivated by the human immune system as compared to retroviruses produced from non-human packaging cells, e.g., murine packaging cells, or may avoid inactivation by the human immune system. Moreover, human packaging cells advantageously do not include endogeneous, inducible replication competent retroviruses (such as those present in, e.g., murine packaging cells) that can disadvantageously result in replication competent retroviruses upon recombination with introduced retroviral packaging genes.
[0074] In an embodiment of the invention, the Env protein expressed by the HT1080, 293 or LNCaP cell does not comprise a CI 1D8, V5, or HA epitope tag. Preferably, the Env protein expressed by the HT1080, 293 or LNCaP cell comprises a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
[0075] Another embodiment of the invention comprises retroviral packaging cells comprising a CI 1D8, V5, or HA epitope tag on a cell surface protein, wherein the cells are human or mouse cells. The CI 1 D8, V5, or HA epitope tag may comprise any of the CI 1D8, V5, or HA epitopes described herein with respect to other aspects of the invention, respectively. Any suitable cell surface protein may comprise the CI 1D8, V5, or HA epitope tag. For example, the Gag, Pol, or Env may comprise the CI 1 D8, V5, or HA epitope tag. Preferably, Env comprises the CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
[0076] Mouse packaging cells for producing retroviral vector particles also provide many advantages. It has been observed that some human cells expressing epitope-tagged GALV may express APOBEC3, which may mediate hypermutation of retroviral vectors, and after passage, may result in reduced vector titers. Hypermutation of retroviral vectors may be avoided or reduced in mouse packaging cells. Accordingly, retroviruses produced from
mouse packaging cells may provide increased vector titers as compared to human packaging cells. 293 cells advantageously have no APOBEC3 activity.
[0077] Any of the retroviral packaging cells described herein may further comprise two lox sites flanking a target site for a gene of interest. The two lox sites, the target site, and the gene of interest may be as described herein with respect to other aspects of the invention and may be positioned in the cell genome as described herein with respect to other aspects of the invention. Accordingly, another embodiment of the invention provides retroviral packaging cells comprising two lox sites flanking a target site for a gene of interest.
Methods of Producing Retroviral Packaging Cells
[0078] Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising transfecting HT1080, 293 or LNCaP cells with one or more vectors encoding Gag, Pol, and Env proteins. Transfecting HT1080, 293 or LNCaP cells with one or more vectors encoding Gag, Pol, and Env proteins may be carried out as described herein with respect to other aspects of the invention.
[0079] In an embodiment of the invention, the method comprises transfecting HT1080, 293 or LNCaP cells with a vector encoding an Env protein that does not comprise a CI 1D8, V5, or HA epitope tag. Preferably, the method comprises transfecting HT1080, 293 or LNCaP cells with a vector encoding a CI 1D8, V5, or HA epitope tag as described herein with respect to other aspects of the invention.
[0080] Another embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag. The cells may be from any mammal described herein such as, for example, human or mouse. The human cells may be any suitable human cells. The mouse cells may be any suitable mouse cells. In an embodiment of the invention, the human cells are HT1080, 293 or LNCaP cells. In an embodiment of the invention, the mouse cells are PG13 cells. Introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or introducing one or more nucleic acids comprising a nucleotide
sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag may be carried out as described herein with respect to other aspects of the invention.
[0081] In those embodiments in which the method comprises introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag or an epitope-tagged Env protein, wherein the epitope tag is a CI 1 D8, V5, of HA epitope tag , the method may further comprise purifying the cells using a CI 1D8, V5, or HA antibody. Purifying the cells using a CI 1 D8, V5, or HA antibody may be carried out as described herein with respect to other aspects of the invention.
[0082] The methods of producing retroviral packaging cells may further comprise cloning two lox sites flanking a target site for a gene of interest into a genome of the cells. Cloning two lox sites flanking a target site for a gene of interest into a genome of the cells may be carried out as described herein with respect to other aspects of the invention.
[0083] In this regard, an embodiment of the invention provides a method of producing retroviral packaging cells, the method comprising (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells. Cells that express Gag, Pol, and Env proteins may be packaging cells as described herein with respect to other aspects of the invention. Introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins and cloning two lox sites flanking a target site for a gene of interest into a genome of the cells may be carried out as described herein with respect to other aspects of the invention. In an embodiment of the invention, the lox sites may be positioned in a human chromosome 19-specific AAV integration site, as described herein with respect to other aspects of the invention.
[0084] In an embodiment of the invention, the methods of producing retroviral packaging cells may further comprise transfecting the cells with a vector encoding a gene of interest. Transfecting the cells with a vector encoding a gene of interest may be carried out as described herein with respect to other aspects of the invention. In an embodiment of the invention, the method comprises inserting the gene of interest into the cell genome by Cre RMCE. Inserting the gene of interest into the cell genome by Cre RCME may be carried out as described herein with respect to other aspects of the invention.
Methods of Treatment
[0085] The retroviral vector particles produced or purified by the inventive methods or produced by the inventive retroviral packaging cells may be useful for a variety of applications. For example, the retroviral vector particles containing a retroviral vector encoding a therapeutic gene may be useful for delivering the therapeutic gene to a target cell or tissue as part of gene therapy to treat or prevent any of a variety of diseases. In this regard, an embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising administering to the mammal the retroviral vector particles purified or produced according to any of the inventive methods described herein in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
[0086] Another embodiment of the invention provides a method of treating or preventing a disease in a mammal in need thereof, the method comprising transfecting target cells with the retroviral vector particles purified or produced according to any of the inventive methods described herein and administering the transfected target cells to the mammal in an amount effective to treat or prevent the disease in the mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating or preventing the disease.
[0087] The mammal may be any mammal. For example, the mammal may be any of the mammals described herein with respect to other aspects of the invention. Preferably, the mammal is a human.
[0088] The retroviral vector particles can be administered, as described herein, with a Multiplicity of Infection (MOl) of 10. The MOl is the ratio of infectious virus particles to the number of cells being infected. Thus, an MOl of 0.1 results in the average inoculation of 1 virus particle for every 10 cells. The general theory behind MOl is to introduce one infectious virus particle to every target (host) cell that is present in the culture. However, more than one virus may infect the same cell which leaves a percentage of cells uninfected. This occurrence can be reduced by using a higher MOl to ensure that every cell is infected. The provided retroviral vector particles can therefore be administered to cells, as described herein, with a MOl of about 1 to about 100, such as for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100.
[0089] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes
as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.
Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.
[0090] In an embodiment, the method comprises transfecting target cells with the retroviral vector particles carrying the gene of interest. The target cells may be any of the target cells described herein with respect to other aspects of the invention. The target cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage. Most preferably the target cells can include, for instance, muscle, lung, and brain cells (for example, microglial cells), and the like.
[0091] In an embodiment of the invention, the target cell may be a microglial cell. The microglial cells may be targeted for gene therapy by, for example, infecting bone marrow- derived hematopoetic stem cells ex vivo. Bone marrow-derived stem cells may migrate into the central nervous system and develop into microglial cells following brain conditioning. This conditioning may ablate brain-resident myeloid precursors, allowing turnover of microglia following hematopoietic stem cell transplantation (Biffi et al., Science,
341 :1233158 (2013) and Capotondo et al, PNAS, 109: 15018-15023 (2012)).
[0092] In addition, the target cell can be a cancer cell. For example, in an embodiment, the target cell of the present can be a tumor cell. With respect to the inventive methods, the cancer can be any cancer, including but not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer.
[0093] The retroviral vector particles and/or the transfected target cells to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes, prior to, or following reconstitution.
[0094] Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration in accordance with the present invention is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intramuscular, intrarterial, subcutaneous, intralesional routes, by aerosol or intranasal routes, or by sustained release systems as noted below. The retroviral vector particles and/or the transfected target cells may be administered continuously by infusion or by bolus injection.
[0095] An effective amount of retroviral vector particles and/or the transfected target cells to be employed therapeutically will depend, for example, upon the therapeutic and treatment objectives, the route of administration, the age, condition, and body mass of the patient undergoing treatment or therapy, and auxiliary or adjuvant therapies being provided to the patient. Accordingly, it will be necessary and routine for the practitioner to titer the dosage and modify the route of administration, as required, to obtain the optimal therapeutic effect. A typical daily dosage might range from about l xl O4 retroviral vector particles and/or the transfected target cells /dose to about lxl O9 retroviral vector particles and/or the transfected target cells /dose or more, preferably from about 1x10 to about 1x10 retroviral vector particles and/or the transfected target cells /dose, depending on the above-mentioned factors. Typically, the clinician will administer retroviral vector particles and/or the transfected target cells until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
[0096] The retroviral vector particles used in the context of the present invention can, themselves, be linked to a detectable label. Such a detectable label allows for the presence of, or the amount of the viral titer to be determined. Preferably, the detectable label is the C 1 1 D8, V5, or HA epitope tag.
[0097] Alternative methods of vector delivery such as convection may enhance distribution of the retroviral vector comprising the therapeutic gene and, thus, more widespread tumor killing than the simple intratumoral injection. For example, an alternative method for efficient and widespread delivery of macromolecules and particles to tumors is convection-enhanced infusion, which is used to supplement simple diffusion and to improve
vector distribution by bulk flow inside and outside the tumor. Stereotactic injection and subsequent infusion by maintaining a positive pressure gradient is able to improve the distribution of large molecules in animal models (Lieberman D.M., et al., J. Neurosurg. 82: 1021-1029 (1985)). When applied, for example, to rat brain tumors, this convection- enhanced infusion technique was able to mediate delivery of virus particles to tumors with an approximate volume of 100 mm3, and also beyond the tumor borders into the surrounding brain tissue (Nilaver et al., Proc. Natl. Acad. Sci. USA 92: 9829-9833 (1995)).
[0098] Other methods of vector application include, for example, intravascular methods. Intravascular methods of vector application make use of a natural and ubiquitously distributed network of arteries, veins and capillaries, which is present in every normal tissue and is even denser in malignant tumors. Intravascular applications, such as intra-arterial injection of virus vectors, are capable of delivering a vector to the largest proportion of tumor cells and surrounding tissues without afflicting mechanical injury to normal brain tissue or having other toxic consequences (Spear et al., J. Neurovirol. 4: 133-147 (1998); Muldoon et al., "Delivery of therapeutic genes to brain and intracerebral tumors; in Chiocca E.A., and Breakefield X.O. (eds.), "Gene Therapy for Neurological Disorders and Brain Tumors," Boston: Humana Press, pp 128-139 (1997)).
Retroviral Vector Particles
[0099] The inventive methods of purifying and/or producing retroviral vector particles may, advantageously, provide a retroviral vector particle comprising Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag, and a retroviral vector comprising a therapeutic gene. In this regard, another embodiment of the invention provides a retroviral vector particle comprising Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag, and a retroviral vector comprising a therapeutic gene. The retroviral vector particles, Gag, Pol, and Env proteins, wherein the Env protein comprises a CI 1 D8, V5, or HA epitope tag, and therapeutic gene may be as described herein with respect to other aspects of the invention. The retroviral vector particles may be useful for any of a variety of applications, including but not limited to the methods of treatment described herein with respect to other aspects of the invention.
[0100] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLE 1
[0101] This example demonstrates a method of making a retroviral packaging cell.
[0102] HT1080 cells are transfected with a first vector encoding Gag, Pol, and a puromycin resistance gene. The transfected cells are selected with puromycin. The selected cells are transfected with a second vector encoding a CI 1D8 epitope-tagged Env protein and a hygromycin resistance gene. The cells are selected with hygromycin. The hygromycin- selected cells are ready to be transfected with a vector encoding a gene of interest.
EXAMPLE 2
[0103] This example demonstrates a method of transfecting a packaging cell with a vector encoding a gene of interest.
[0104] A vector comprising, from the 5' to the 3' end, loxp, a pgk (phosphoglycerate kinase 1) promoter, a blasticidin resistance gene, lox2722, and a neomycin resistance gene lacking an initation codon is introduced into the genome of the HT1080 packaging cell described in Example 1 between the AAVS1 right arm and AAVS1 left arm genome sites. The cells are selected with blasticidin.
[0105] A Cre expression plasmid and a plasmid containing a vector genome comprising, from the 5' to the 3' end, loxp, a 5' LTR packaging site (ψ) promoter (e.g., PGK), an IL2yc gene, a 3' LTR, a 628 bp IRES (internal ribosome entry site), and an ATG codon sequence are transfected into the cells that were selected with blasticidin.
[0106] After Cre mediated recombination, the cells contain a genome with the IL2yc gene and an intact ATG-containing G418 resistance gene. From the 5' to the 3' end, the packaging cell genome contains: loxp, a 5' LTR packaging site (ψ) promoter (e.g., PGK), an IL2yc gene, a 3' LTR, a 628 bp IRES, and the neomycin resistance gene including an initiation codon oriented so that the neomycin resistance gene can be expressed.
[0107] The cells are selected with G418. The cells produce retroviral vector particles comprising a CI 1 D8 epitope-tagged Env protein and a vector comprising the IL2yc gene.
EXAMPLE 3
[0108] This example demonstrates a method of purifying a retroviral vector particle.
[0109] The cells described in Example 2 produce a first supernatant comprising retroviral vector particles comprising a CI 1 D8 epitope-tagged Env protein and a vector comprising the
IL2yc gene. The first supernatant also contains cells. The first supernatant is placed in a tube and centrifuged at a speed of 2,000 g to produce a second supernatant and a pellet comprising the cells. The second supernatant comprises the CI 1D8 epitope-tagged retroviral vector particles and cellular components.
[0110] CI 1D8 monoclonal antibody is cross-linked to SEPHAROSE beads, an affinity column is prepared including the cross-linked beads, and the column is equilibrated as described in Grant et al., J. Immunol., 131 (6): 3042-48 (1983). The second supernatant is removed from the centrifugation tube and applied to a column including the cross-linked SEPHAROSE beads. The column is washed with phosphate buffered saline (PBS) buffer to remove the cellular components. The retroviral vector particles are eluted from the column in PBS buffer containing 80μΜ MGPNL (SEQ ID NO: 5) peptide. The retroviral vector particles are concentrated by passing the eluted retroviral vector particles through an
AMICON filter. The virus fraction is measured using an ELISA plate reader with CI 1D8 antibodies. The particles are assessed for infectivity in 70 μΜ peptide eluate.
[0111] No statistically significant difference in titer is obtained with GALV that has a V5 or HA epitope inserted at the amino terminus as compared to the wild type GALV. GALV that has the CI 1 D8 epitope (SEQ ID NO: 6) inserted at the amino terminus yields a statistically significant lower titer (4 logs) as compared to the wild type GALV.
EXAMPLE 4
[0112] This example demonstrates the titer of retroviral particle obtained after purifying the retroviral particles using an epitope tag.
[0113] A nucleic acid comprising a nucleotide sequence encoding a V5 epitope
GKPIPNPLLGLDST (SEQ ID NO: 15), HA epitope YPYDVPDYA (SEQ ID NO: 16), or CI 1D8 epitope QVMTITPPQAMGPNLVLP (SEQ ID NO: 6) is introduced into the gene encoding GALV Env protein in PG13 cells using targeted genome editing using TALENs. The cells produce retroviral particles including the V5, HA, or CI 1 D8 epitope positioned at the amino terminus of the Env protein as shown in Figure 1. The retroviral particles are purified as described in Example 3 using V5, HA, or CI 1D8 antibodies. The titer of retroviral particles is measured and compared to that obtained with PG 13 cells that produce wild-type GALV retroviral particles (i.e., retroviral particles that lack an epitope tag).
[0114] No statistically significant difference in titer is obtained with GALV that has a V5 or HA epitope inserted at the amino terminus as compared to the wild type GALV. GALV
that has the CI 1D8 epitope (SEQ ID NO: 6) after residue 219 has a 50% reduction in titer as compared to the wild type GALV. GALV that has the CI 1D8 epitope (SEQ ID NO: 6) inserted at the amino terminus yields a statistically significant lower titer (2 logs) as compared to the wild type GALV.
[0115] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0116] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly
contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0117] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as
specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Claims
CLAIM(S):
1. A method of purifying retroviral vector particles, the method comprising:
(a) (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or influenza hemagglutinin (HA) epitope tag; (b) culturing the cells of (a) to produce epitope- tagged retroviral vector particles;
(c) producing a first supernatant comprising the epitope-tagged retroviral vector particles and cells;
(d) centrifuging the first supernatant to produce a second supernatant comprising the epitope-tagged retroviral vector particles and cellular components;
(e) separating the second supernatant from the cells of (c);
(f) contacting a stationary phase comprising an antibody with the second supernatant and binding the epitope-tagged retroviral vector particles to the antibody, wherein the antibody is a CI 1D8, V5, or HA antibody;
(g) separating the cellular components from the epitope-tagged retroviral vector particles; and
(h) eluting the epitope-tagged retroviral vector particles from the stationary phase to produce purified epitope-tagged retroviral vector particles.
2. The method of claim 1, wherein the cells are mouse cells.
3. The method of claim 1 or 2, wherein the retroviral vector particles are Gibbon Ape Leukemia Virus (GALV) particles.
4. The method of any one of claims 1 -3, wherein (a) further comprises transfecting the cells with a vector encoding a gene of interest.
5. The method of claim 4, comprising inserting the gene of interest into a cell genome by Cre recombinase-mediated cassette exchange (RMCE).
6. The method of claim 5, wherein the vector comprises two lox sites flanking the gene of interest and the cells comprise a genome comprising two lox sites flanking a target site for the gene of interest.
7. The method of claim 6, wherein the two lox sites comprise loxP and lox 2722.
8. The method of any one of claims 1-7, wherein the cells are PG13 packaging cells.
9. The method of any one of claims 1-8, wherein the retroviral vector particles are gammaretroviral vector particles.
10. The method of any one of claims 1-8, wherein the retroviral vector particles are lentiviral particles.
11. A method of producing retroviral vector particles, the method comprising:
(a) (i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins;
(b) transfecting the cells with a vector encoding a gene of interest;
(c) inserting the gene of interest into a cell genome by Cre recombinase-mediated cassette exchange (RMCE);
(d) culturing the cells of (c) to produce retroviral vector particles by the cells;
(e) producing a first supernatant comprising the retroviral vector particles and cells;
(f) centrifuging the first supernatant to produce a second supernatant comprising the retroviral vector particles and cellular components; and
(g) separating the second supernatant including the retroviral vector particles from the cells of (e).
12. The method of claim 1 1 further comprising (h) purifying the retroviral vector particles from the cellular components.
13. The method of claim 1 1 or 12, wherein the vector comprises two lox sites flanking the gene of interest and the cells comprise a genome comprising two lox sites flanking a target site for the gene of interest.
14. The method of claim 13, wherein the two lox sites comprise loxP and lox 2722.
15. The method of any one of claims 11-14, wherein the cells are mouse cells.
16. The method of any one of claims 11-15, wherein the cells are PG13 packaging cells.
17. The method of any one of claims 11-16, wherein the retroviral vector particles are gammaretroviral vector particles.
18. The method of any one of claims 11 -16, wherein the retroviral vector particles are lentiviral particles.
19. The method of any one of claims 1 1-16, wherein the retroviral vector particles are GALV particles.
20. The method of any one of claims 11-19, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag.
21. The method of claim 20, further comprising
contacting a stationary phase comprising a CI 1D8, V5, or HA antibody with the second supernatant;
binding the CI 1 D8, V5, or HA epitope-tagged retroviral vector particles to the C1 1 D8, V5, or HA antibody;
separating the cellular components from the CI 1D8, V5, or HA epitope-tagged retroviral vector particles; and
eluting the CI 1 D8, V5, or HA epitope-tagged retroviral vector particles from the stationary phase.
22. Retroviral packaging cells comprising HT1080, 293 or LNCaP cells expressing Gag, Pol, and Env proteins.
23. The retroviral packaging cells of claim 22, wherein the Env protein further comprises a CI 1D8, V5, or HA epitope tag.
24. Retroviral packaging cells comprising a CI 1D8, V5, or HA epitope tag on a cell surface protein, wherein the cells are human or mouse cells.
25. The retroviral packaging cells of any one of claims 22-24, wherein the cells comprise a genome comprising two lox sites flanking a target site for a gene of interest.
26. Retroviral packaging cells comprising two lox sites flanking a target site for a gene of interest.
27. The retroviral packaging cells of claim 25 or 26, wherein the two lox sites comprise loxP and lox 2722.
28. The retroviral packaging cells of any one of claims 24-27, wherein the cells are PG13 packaging cells.
29. A method of producing retroviral packaging cells, the method comprising transfecting HT1080, 293 or LNCaP cells with one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins.
30. The method of claim 29, wherein the Env protein further comprises a CI 1D8, V5, or HA epitope tag.
31. A method of producing retroviral packaging cells, the method comprising (i) introducing a nucleic acid comprising a nucleotide sequence encoding an epitope tag into cells that express Gag, Pol, and Env proteins to obtain expression of an epitope-tagged Env protein, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence
encoding Gag, Pol, and epitope-tagged Env proteins into cells to obtain expression of Gag, Pol, and epitope-tagged Env, wherein the epitope tag is a CI 1D8, V5, or HA epitope tag.
32. The method of claim 30 or 31 , further comprising purifying the cells using a CI 1D8, V5, or HA antibody.
33. The method of any one of claims 29-32, further comprising cloning two lox sites flanking a target site for a gene of interest into a genome of the cells.
34. A method of producing retroviral packaging cells, the method comprising
(i) providing cells that express Gag, Pol, and Env proteins, or (ii) introducing one or more nucleic acids comprising a nucleotide sequence encoding Gag, Pol, and Env proteins into cells to obtain expression of Gag, Pol, and Env proteins; and
cloning two lox sites flanking a target site for a gene of interest into a genome of the cells.
35. The method of claim 33 or 34, wherein the two lox sites comprise loxP and lox
2722.
36. The method of any one of claims 33-35, wherein the cells are PG13 packaging cells.
37. The method of any one of claims 29-36, further comprising transfecting the cells with a vector encoding a gene of interest.
38. The method of claim 37, comprising inserting the gene of interest into the cell genome by Cre recombinase-mediated cassette exchange (RMCE).
39. The retroviral vector particles purified according to the method of any one of claims 4-8 or the retroviral vector particles produced according to the method of any one of claims 1 1-21 for use in treating a disease in a mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating the disease.
40. Target cells transfected with the retroviral vector particles purified according to the method of any one of claims 4-8 or the retroviral vector particles produced according to the method of any one of claims 1 1 -21 for use in treating a disease in a mammal, wherein the gene of interest encodes a therapeutic gene suitable for treating the disease.
41. A retroviral vector particle comprising Gag, Pol, and Env proteins, and a retroviral vector comprising a therapeutic gene, wherein the Env protein comprises a CI 1D8, V5, or HA epitope tag.
42. The retroviral vector particle of claim 41, wherein the retroviral vector particle is a gammaretroviral vector particle.
43. The retroviral vector particle of claim 41, wherein the retroviral vector particle is a lentiviral particle.
44. The retroviral vector particle of claim 41 , wherein the retroviral vector particle is a GALV particle.
45. The method of any of claims 1-10 and 21, wherein eluting the epitope-tagged retroviral particles comprises contacting the stationary phase with buffer comprising a C1 1D8, V5, or HA epitope.
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