WO1996021014A2 - Production and administration of high titer recombinant retroviruses - Google Patents
Production and administration of high titer recombinant retroviruses Download PDFInfo
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- WO1996021014A2 WO1996021014A2 PCT/US1995/016852 US9516852W WO9621014A2 WO 1996021014 A2 WO1996021014 A2 WO 1996021014A2 US 9516852 W US9516852 W US 9516852W WO 9621014 A2 WO9621014 A2 WO 9621014A2
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Definitions
- the present invention relates generally to .recombinant retroviruses, and more specifically, to high titer recombinant retroviral particle preparations suitable for a variety of applications.
- recombinant retroviral gene delivery methods have been most extensively utilized, in part due to: (1) the efficient entry of genetic material (the vector genome) into cells; (2) an active, efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific control of gene expression; (5) a general lack of pre-existing host immunity; and (6) substantial knowledge and clinical experience which has been gained with such vectors.
- retroviruses are diploid positive-strand R ⁇ A viruses that replicate through an integrated D ⁇ A intermediate.
- the retroviral genome upon infection by the R ⁇ A virus, the retroviral genome is reverse-transcribed into D ⁇ A by a virally encoded reverse transcriptase that is carried as a protein in each retrovirus.
- the viral D ⁇ A is then integrated pseudo- randomly into the host cell genome of the infecting cell, forming a "provirus" which is inherited by daughter cells.
- Wild-type retroviral genomes (and their proviral copies) contain three genes
- gag, pol and env genes which are preceded by a packaging signal ( ⁇ ), and two long terminal repeat (LTR) sequences which flank both ends.
- ⁇ packaging signal
- LTR long terminal repeat
- the gag gene encodes the internal structural (nucleocapsid) proteins.
- the pol gene codes for the RNA-depende DNA polymerase which reverse transcribes the RNA genome
- the env gene encodes t retroviral envelope glycoproteins.
- the 5' and 3' LTRs contain c.-. -acting elements necessa to promote transcription and polyadenylation of retroviral RNA.
- Adjacent to the 5 1 LTR are sequences necessary for reverse transcription the genome (the tRNA primer binding site) and for efficient e ⁇ capsidation of retroviral RN into particles (the ⁇ sequence). Removal of the packaging signal prevents encapsidati (packaging of retroviral RNA into infectious virions) of genomic RNA, although t resulting mutant can still direct synthesis of all proteins encoded in the viral genome.
- Recombinant retroviruses and various uses thereof have been described numerous references including, for example, Mann et al. (Cell 33.153, 1983), Cane a Mulligan (Proc. Nat'l. Acad. Sci.
- a foreign gene of interest m be inco ⁇ orated into the retrovirus in place of the normal retroviral RNA.
- the foreign gene is also introduced into the cell, a may then be integrated into the host's cellular DNA as if it were the retrovirus itse Expression of this foreign gene within the host results in expression of the foreign protein the host cell.
- Such methods have included, for example chemical treatment with 10 carbon tetrachloride in mineral oil (Kaleko et al., Human Gene Therapy 2:21-32, 1991 Alternatively, others have suggested excising large portions of the liver (e.g., 70% Rettinger et al., PNAS 91: 1460-1464, 1994; 70% in Moscioni et al., Surgery 7/3:304-31 1993; ) in order to stimulate the rapid division of hepatocytes and thereby increase t infection of such cells.
- retroviruses of avian, murine, feline, and simi origin are all inactivated and lysed by normal human serum. (Welsh et al., Nature 257:61 614, 1975; Welsh et al., Virology 74:432-440, 1976; Banapour et al., Virology 752:268- 271, 1986; and Cooper et al., Immunology of the Complement System. Pub., American Press, Inc., pp.
- the present invention provides recombinant retrovirus compositions which are capable of surviving inactivation in human serum.
- the present invention provides high titer recombinant retrovirus compositions which allow delivery of therapeutics or palliatives by routes not previously deemed possible, and without the need to induce replication of cells by chemical treatment or by excision of a target organ such as the liver. The present invention provides these, as well as other related advantages.
- the present invention provides high titer compositions comprising recombinant retroviruses; as well as methods for utilizing these compositions.
- methods are provided for obtaining measurable levels of a protein, nucleic acid molecule, or enzymatic product in a bodily fluid or cells of a human, comprising the step of administering to a human a recombinant retroviral preparation having a titer on HT1080 cells of greater than 10-> cfu/ml, wherein the recombinant retroviral preparation is capable of directing the expression of a protein, nucleic acid molecule, or enzyme which generates an enzymatic product, such that measurable levels of the protein, nucleic acid molecule, or enzymatic product may be obtained in the bodily fluid or cells of said human.
- the titer may be greater than 10 ⁇ cfu/ml, 10 ⁇ cfu/ml, 10 8 cfu ml, 10 9 cfu/ml, 10 10 cfu/ml, or 10 1 1 cfu ml.
- methods for obtaining measurable levels of a protein, nucleic acid molecule, or enzymatic product in a bodily fluid or cells of a human, comprising the steps of administering to a human a recombinant retroviral preparation having a titer in human serum and on HT1080 cells equivalent to its' titer in heat-inactivated serum and on HT1080 cells, wherein the recombinant retroviral preparation is capable of directing the expression of a protein, nucleic acid molecule, or enzyme which generates an enzymatic product, such that measurable levels of the protein, nucleic acid molecule, or enzymatic product may be obtained in the bodily fluid or cells of said human.
- measurable levels a protein, nucleic acid molecule, or enzymatic product refers to a statisically significant lev of detection over background, utilizing any suitable technique (e.g., antibody-mediate detection of a protein, PCR analysis for the presence of a nucleic acid molecule, visualization of enzymatic products).
- 'equivalent" titers are deemed to be those which are substantially the same withi a given assay, generally, within about three-fold of each other.
- the recombinant retrovirus i administered to a site such as the cerebral spinal fluid, bone marrow, joints, arteri endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selecte from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selecte from the group consisting of tumors and interstitial spaces.
- th recombinant retrovirus may be administered intraocularly, intranasally, sublinually, orall topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracraniall intramuscularly, or subcutaneously.
- the protein is a vir antigen obtained from a virus such as influenza virus, respiratory syncytial virus, HPV, HB HCV, EBV, HTV, HSV, FeLV, FIV, Hantavirus, HTLV I, HTLV II and CMV.
- the protein is a cytokine such as E -l, IL-2, IL-3, EL-4, IL-5, IL-6, HL-7, IL- IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, ⁇ -IFN, G-CSF and GM-CSF, or a recept for any of these cytokines.
- the nucleic acid molecule may be an antisens sequence, a non-coding non-heterologous sense sequence, and a ribozyme sequence.
- the protein is a toxin.
- Figure 1 is a schematic illustration of p31N2R5(+).
- Figure 2 is a schematic illustration of pN2R3(-).
- Figure 3 is a schematic illustration of p31N25 ⁇ (+).
- Figure 4 is a schematic illustration of pN2R3(+).
- Figure 5 is a schematic illustration of pN2R5(-).
- Figure 6 is a schematic illustration of p31N25 ⁇ (+). - 5 -
- Figure 7 is a schematic illustration of pTK ⁇ A.
- Figure 8 is a schematic illustration of pPrTK ⁇ A.
- Figure 9 is a schematic illustration of pTK-1 and pTK-3.
- Figure 10 is a bar graph which illustrates the effect of Ganciclovir on CT26, CT26 bgal and CT26TK Neo cells.
- Figure 11 is a graph which illustrates the effect of tumor volume over time in a Ganciclovir dose study of mice injected with CT26TK Neo.
- Figure 12 is a series of four photographs of mice, illustrating the effect of different dose regimens of Ganciclovir on intraperitoneal tumor growth.
- Figure 13 is a series of four photographs of mice, illustrating the effect of different dose regimens of Ganciclovir on subcutaneous tumor growth.
- Figure 14 is a graph illustrating the effect of Ganciclovir in CT26 versus CT26TK Neo cells.
- Figure 15 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovirus lyophilized in a formulation buffer containing mannitol.
- Figure 16 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovirus lyophilized in a formulation buffer containing lactose.
- Figure 17 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovirus lyophilized in a formulation buffer containing trehalose.
- Figures 18A-18D are representative graphs comparing stability of liquid non- lyophilized recombinant retrovinis stored at -80°C versus lyophilized formulated recombinant retrovirus stored at -20°C, using various saccharides.
- Figure 19 is a bar graph which depicts the results of a reverse transcriptase assay on samples sliced from a gel. Slice 1 is from the lowest part of the gel, and slice 8 from the highest.
- Figure 20 depicts a 8% to 25% gradient polyacrylamide gel.
- Lane 1 is DA/ ⁇ gal S-500 purified;
- Lane 2 is DA/ ⁇ gal crude supernatant;
- Lane 3 is HIV-IT crude supernatant;
- lane 4 is DAC 6A3 crude supernatant;
- Lane 5 is HBc/SA2 crude supernatant; and
- Lane 5 is molecular weight markers.
- Figure 21 is a schematic illustration of pDHF811.
- Figures 22A-D are graphs which indicate the total quantity of lactate in low seed and high seed cultures ( Figures 22 A and 22B, respectively) and the level of lactate production per day in low seed and high seed cultures ( Figures 22C and 22D, respectively).
- Figure 23 is a bar graph which depicts the titer of the cell line 2X- ⁇ -gal un different initial seeding conditions.
- Vector construct refers to a nucleic acid construct which carries, and wit certain embodiments, is capable of directing the expression of a nucleic acid molecule interest.
- the retroviral vector must include at least one transcriptional promoter/enhancer locus defining element(s), or other elements which control gene expression by other mea such as alternate splicing, nuclear RNA export, post-translational modification of messengerg or post-transcriptional modification of protein.
- Such vector constructs must also included packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negati strand primer binding sites appropriate to the retrovirus used (if these are not already pres in the retroviral vector).
- the recombinant retroviral vector may also include signal which directs polyadenylation, selectable markers such as Neomycin resistance, T hygromycin resistance, phleomycin resistinacne histidinol resistance, or DHFR, as well one or more restriction sites and a translation termination sequence.
- such vectors typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin second strand DNA synthesis, and a 3' LTR or a portion thereof.
- Retrovirus refers to a retrovirus which carries least one gene of interest.
- the retrovi ⁇ is may also contain a selectable marker.
- T recombinant retrovirus is capable of reverse transcribing its genetic material into DNA a inco ⁇ orating this genetic material into a host cell's DNA upon infection.
- the present invention provides high titer recombin retroviral preparations which are suitable for administration to humans. Such preparatio provide the unexpected result of providing efficatious gene therapy for a variety of disea (and by a variety of routes) that were previously not amenable for gene therapy.
- the present invention provides recombinant retroviruses which are capable of survivi inactivation in human serum, thereby allowing more efficient gene transfer over prolong periods of time.
- the present invention provides recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest.
- retroviral gene delivery vehicles may be utilized within the context of the present invention, including for example those described in EP 0,415,731; WO 90/07936;
- Retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
- Preferred retroviruses for the preparation or construction of retroviral gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma Virus.
- Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 79:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR- 590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998), and Moloney Murine Leukemia Virus (ATCC No. VR-190).
- retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC";
- retroviral gene delivery vehicles Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vehicles given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:488, 1985).
- portions of the retroviral gene delivery vehicles may be derived from different retroviruses.
- retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
- recombinant retroviruses may be made by introducing a vector construct as discussed above, into a cell (termed a "packaging cell") which contains those elements necerney for production of infectious recombinant retrovirus which are lacking in the vector construct.
- a wide variety o retrovector constructs may be utilized within the present invention in order to prepar recombinant retroviruses.
- retrovector constructs comprising a 5' LTR, a tRNA binding site, a packagin signal, one or more heterologous sequences, an origin of second strand DNA synthesis and 3' LTR, wherein the vector construct lacks gag/pol or env coding sequences.
- LTRs Lon Terminal Repeats
- U5 Lon Terminal Repeats
- R and U3 Lon Terminal Repeats
- U5 Lon Terminal Repeats
- U5 Lon Terminal Repeats
- U3 Lon Terminal Repeats
- LTRs may be readily identified in the provirus due to their precise duplication either end of the genome.
- a 5' LTR should be understood to include a 5 promoter element and sufficient LTR sequence to allow reverse transcription and integratio of the DNA form of the vector.
- the 3' LTR should be understood to include polyadenylation signal, and sufficient LTR sequence to allow reverse transcription an integration of the DNA form of the vector.
- the tRNA binding site and origin of second strand DNA synthesis are als important for a retrovirus to be biologically active, and may be readily identified by one o skill in the art.
- retroviral tRNA binds to a tRNA binding site by Watson-Cric base pairing, and is carried with the retrovirus genome into a viral particle.
- the tRNA i then utilized as a primer for DNA synthesis by reverse transcriptase.
- the tRNA binding sit may be readily identified based upon its location just downstream from the 5' LT
- the origin of second strand DNA synthesis is, as its name implies, important for th second strand DNA synthesis of a retrovirus. This region, which is also referred to as th poly-purine tract, is located just upstream of the 3' LTR.
- th poly-purine tract is located just upstream of the 3' LTR.
- certain preferred retrovector constructs which are provided herein als comprise a packaging signal, as well as one or more nucleic acid molecules (e.g. heterologous sequences), each of which is discussed in more detail below.
- retrovector constructs are provided whic lack both gag/pol and env coding sequences.
- the phrase "lacks gag/pol o env coding sequences" should be understood to mean that the retrovector does not contain least 20, preferably at least 15, more preferably at least 10, and most preferably at least consecutive nucleotides which are found in gag/pol or env genes, and in particular, withi gag/pol or env expression cassettes that are used to construct packaging cell lines for th retrovector construct.
- Packaging cell lines suitable for use with the above-described retrovector constructs may b readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see als WO 92/05266), and utilized to create producer cell lines (also termed vector cell lines or M VCLs") for the production of recombinant vector particles.
- packaging cell lines are made from human (e.g., HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that are capable of surviving inactivation in human serum.
- One representative assay that may be utilized in order to determine the whether recombinant retroviruses are inactivated in human serum is set forth in more detail below in Example 11.
- packaging cell lines that produce greater than recombinant retroviral particles at titers greater than 10 ⁇ or 10? cfu/ml (in crude supernatant) may readily be obtained.
- titers are generally obtained from titer assays on HT1080 cells, which produce a three-fold lower titer than titers obtained on murine 3T3 cells.
- nucleic acid molecules may be carried and/or expressed by the recombinant retroviruses of the present invention.
- the nucleic acid molecules which are described herein do not occur naturally in the recombinant retrovirus that carries it, and provides some desirable benefit, typically an ability to fight a disease, or other pathogenic agent or condition.
- pathogenic agent refers to a cell that is responsible for a disease state. Representative examples of pathogenic agents include tumor cells, autoreactive immune cells, hormone secreting cells, cells which lack a function that they would normally have, cells that have an additional inappropriate gene expression which does not normally occur in that cell type, and cells infected with bacteria, viruses, or other intracellular parasites.
- pathogenic agent may also refer to a cell that over-expresses or inappropriately expresses a recombinant retrovirus (e.g., in the wrong cell type), or that has become tumorigenic due to inappropriate insertion into a host cell's genome.
- nucleic acid molecules which may be carried and/or expressed by the recombinant retroviruses of the present invention include genes and other nucleic acid molecules which encode a substance, as well as biologically active nucleic acid molecules such as inactivating sequences that inco ⁇ orate into a specified intracellular nucleic acid molecule and inactivate that molecule.
- a nucleic acid molecule is considered to be biologically active when the molecule itself provides the desired benefit without requiring the expression of a substance.
- the biologically active nucleic acid molecule may be an inactivating sequence that inco ⁇ orates into a specified intracellular nucleic acid molecule and inactivates that molecule, or the molecule may be a tRNA, rRNA or mRNA that has configuration that provides a binding capability.
- Substances which may be encoded by the nucleic acid molecules describe herein include proteins (e.g., antibodies including single chain molecules immunostimulatory molecules (such as antigens) immunosuppressive molecules, blockin agents, palliatives (such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and oth material capable of inhibiting a function of a pathogenic agent) cytokines, variou polypeptides or peptide hormones, their agonists or antagonists, where these hormones ca be derived from tissues such as the pituitary, hypothalamus, kidney, endothelial cells, live pancreas, bone, hemopoetic marrow, and adrenal.
- proteins e.g., antibodies including single chain molecules immunostimulatory molecules (such as antigens) immunosuppressive molecules, blockin agents, palliatives (such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and oth material capable of
- Such polypeptides can be used fo induction of growth, regression of tissue, suppression of immune responses, apoptosis, gen expression, blocking receptor-ligand interaction, immune responses and can be treatment fo certain anemias, diabetes, infections, high blood pressure, abnormal blood chemistry o chemistries (e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevate LDL with lowered HDL), levels of Alzheimer associated amaloid protein, bon erosion/calcium deposition, and controlling levels of various metabolites such as steroi hormones, purines, and pyrimidines.
- abnormal blood chemistry o chemistries e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevate LDL with lowered HDL
- levels of Alzheimer associated amaloid protein bon erosion/calcium deposition
- various metabolites such as steroi hormones, purines, and pyrimidines.
- the palliative when "capable of inhibiting a function" is utilized within th context of the present invention, it should be understood that the palliative either directl inhibits the function or indirectly does so, for example, by converting an agent present in th cells from one which would not normally inhibit a function of the pathogenic agent to on which does.
- functions for viral diseases include adso ⁇ tion, replicatio gene expression, assembly, and exit of the virus from infected cells.
- suc functions for cancerous diseases include cell replication, susceptibility to external signal (e.g., contact inhibition), and lack of production of anti-oncogene proteins.
- suc functions for cardiovascular disease include inappropriate growth or accumulation material in blood vessels, high blood pressure, undesirable blood levels of factors such a cholesterol or low density lipoprotein that predispose to disease, localized hypoxia, an inappropriately high and tissue-damaging levels of free radicals.
- Examples of such function for neurological conditions include pain, lack of dopamine production, inability to replac damaged cells, deficiencies in motor control of physical activity, inappropriately low levels various peptide hormones derived from neurological tissue such as the pituitary hypothalamus, accumulation of Alzheimer's Disease associated amyloid plaque protein, an inability to regenerate damaged nerve junctions.
- Examples of such functions for autoimmun or inlammatory disease include inappropriate production of cytokines and lymphokine inappropriate production and existence of autoimmune antibodies and cellular immun responses, inappropriate disruption of tissues by proteases and collagenases, inhibition of th normal action of proteases, lack of production of factors normally supplied by destroyed cells, and excessive or aberrant regrowth of tissues under autoimmune attack.
- the methods are provided for administration of a recombinant retrovirus which directs the expression of a palliative.
- the palliative may be a DNA molecule, an RNA molecule, or some combination of the two.
- palliatives that act directly to inhibit the growth of cells include toxins such as ricin (Lamb et al., Eur. J. Biochem. 148:265-210, 1985), abrin (Wood et al., Eur. J. Biochem. 198:123-132, 1991; Evensen et al., J. of Biol Chem. 266:6848-6852, 1991; Collins et al., J. of Biol. Chem. 265:8665-8669, 1990; Chen et al., Fed of Eur. Biochem Soc. 309:115-118, 1992), diphtheria toxin (Tweten et al., J. Biol Chem.
- the recombinant retrovirus carries a gene specifying a product which is not in itself toxic, but when processed or modified by a protein, such as a protease specific to a viral or other pathogen, is converted into a toxic form.
- the recombinant retrovirus could carry a gene encoding a proprotein chain, which becomes toxic upon processing by the HTV protease.
- a synthetic inactive proprotein form of the toxic ricin or diphtheria A chains could be cleaved to the active form by arranging for the HTV virally encoded protease to recognize and cleave off an appropriate "pro" element.
- the recombinant retrovirus directs the expression of a substance capable of activating an otherwise inactive precursor into an active inhibitor of a pathogenic agent, or a conditional toxic palliative, which are palliatives that are toxic for the cell expressing the pathogenic condition.
- a wide variety of inactive precursors may be converted into active inhibitors of a pathogenic agent.
- antiviral nucleoside analogues such as AZT or ddl are metabolized by cellular mechanisms to the nucleotide triphosphate form in order to specifically inhibit retroviral reverse transcriptase, and thus viral replication - 12 -
- Recombinant retroviruse which direct the expression of a gene product (e.g., a protein) such as He ⁇ es Simplex Viru Thymidine Kinase (HSVTK) or Varicella Zoster Virus Thymidine Kinase (VZVTK) whic assists in metabolizing antiviral nucleoside analogues to their active form are therefore usefu in activating nucleoside analogue precursors (e.g., AZT or ddC) into their active form.
- a gene product e.g., a protein
- HSVTK He ⁇ es Simplex Viru Thymidine Kinase
- VZVTK Varicella Zoster Virus Thymidine Kinase
- AZ or ddl therapy will thereby be more effective, allowing lower doses, less generalized toxicity and higher potency against productive infection.
- Additional nucleoside analogues whos nucleotide triphosphate forms show selectivity for retroviral reverse transcriptase but, as result of the substrate specificity of cellular nucleoside and nucleotide kinases are no phosphorylated, will be made more efficacious.
- the HSVTK gene may be expresse under the control of a constitutive macrophage or T-cell-specific promoter, and introduce into macrophage or T-cells.
- Constitutive expression of HSVTK results in more effectiv metabolism of nucleotide analogues such as AZT or ddl to their biologically activ nucleotide triphosphate form, and thereby provides greater efficacy, delivery of lower doses less generalized toxicity, and higher potency against productive infection.
- nucleoside analogues whose nucleotide triphosphate forms show selectivity for retrovira reverse transcriptase but, as a result of the substrate specificity of cellular nucleoside an nucleotide kinases are not phosphorylated, may also be utilized within the context of th present invention.
- recombinant retroviruses ar provided which direct the expression of a substance that activates another compound wit little or no cytotoxicity into a toxic product in the presence of a pathogenic agent, thereb effecting localized therapy to the pathogenic agent.
- expression of the gen product from the recombinant retrovirus is limited to situations wherein an entity associate with the pathogenic agent, such as an intracellular signal identifying the pathogenic state, i present, thereby avoiding destruction of nonpathogenic cells.
- This cell-type specificity ma also be conferred at the level of infection, by targeting the recombinant retrovirus to cell having or being susceptible to the pathogenic condition.
- recombinant retroviruses ar provided which direct the expression of a gene product(s) that activates a compound wit little or no cytotoxicity into a toxic product.
- a wide variety of gene products whic either directly or indirectly activate a compound with little or no cytotoxicity into a toxi product may be utilized within the context of the present invention.
- Representative example of such gene products include HSVTK and VZVTK which selectively monophosphorylat certain purine arabinosides and substituted pyrimidine compounds, converting them t cytotoxic or cytostatic metabolites.
- gangicylovir acyclovir or any of their analogues (e.g., FIAC, DHPG) to HSVTK, phosphorylates the drug into its corresponding active nucleotide triphosphate form.
- analogues e.g., FIAC, DHPG
- the recombinant retrovirus directs the expression of the he ⁇ es simplex virus thymidine kinase ("HSVTK”) gene downstream, and under the transcriptional control of an HTV promoter (which is known to be transcriptionally silent except when activated by HIV tat protein).
- HSVTK simplex virus thymidine kinase
- expression of the tat gene product in human cells infected with HTV and carrying the recombinant retrovirus causes increased production of HSVTK.
- the cells are then exposed to a drug such as ganciclovir, acyclovir or its analogues (FIAC, DHPG).
- these drugs are known to be phosphorylated by HSVTK (but not by cellular thymidine kinase) to their corresponding active nucleotide triphosphate forms.
- Acyclovir triphosphates inhibit cellular polymerases in general, leading to the specific destruction of cells expressing HSVTK in transgenic mice (see Borrelli et al., Proc. Natl Acad Sci. USA 85:1512, 1988).
- Those cells containing the recombinant retrovirus and expressing HIV tat protein are selectively killed by the presence of a specific dose of these drugs.
- expression of a conditionally lethal HSVTK gene may be made even more --TV-specific by including cis-acting elements in the transcript ("CRS/CAR"), which require an additional HTV gene product, rev, for optimal activity (Rosen et al., Proc. Natl Acad Sci. USA 55:2071, 1988). More generally, cis elements present in mRNAs have been shown in some cases to regulate mRNA stability or translatability. Sequences of this type (i.e., post-transcriptional regulation of gene expression) may be used for event- or tissue-specific regulation of vector gene expression. In addition, multimerization of these sequences (i.e., rev-responsive "CRS/CAR” or tat- responsive "TAR" elements for HTV) may be utilized in order to generate even greater specificity.
- recombinant retroviruses may be generated which carry a gene for phosphorylation, phosphoribosylation, ribosylation, or other metabolism of a purine- or pyrimidine-based drug.
- genes may have no equivalent in mammalian cells, and might come from organisms such as a virus, bacterium, fungus, or protozoan.
- Representative examples include: E. coli guanine phosphoribosyl transferase ("gpt”) gene product, which converts thioxanthine into thioxanthine monophosphate (see Besnard et al., Mol Cell. Biol.
- alkaline phosphatase which will convert inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds
- fungal e.g., Fusarium oxysporum
- bacterial cytosine deaminase which will convert 5-fluorocytosine to the toxic compound 5-fluorouracil
- carboxypeptidase G2 which will cleave the glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard
- Penicillin- V amidase which will conver phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compound
- Conditionally lethal gene products of this type have application to many presently know purine- or pyrimidine-based anticancer drugs, which often require intracellular ribosylation
- conditionally lethal gen product could also metabolize a nontoxic drug, which is not a purine or pyrimidine analogu to a cytotoxic form (see Searle et al., Brit. J. Cancer 53:377-384, 1986).
- the target pathogen is a mammalian virus recombinant retroviruses
- vectors may be constructed to take advantage of the fact tha mammalian viruses in general tend to have "immediate early" genes, which are necessary fo subsequent transcriptional activation of other viral promoter elements.
- Gene products of thi nature are excellent candidates for intracellular signals (or "identifying agents") of vir infection.
- conditionally lethal genes transcribed from transcriptional promote elements that are responsive to such viral "immediate early" gene products could specificall kill cells infected with any particular virus.
- the human ⁇ and ⁇ interfero promoter elements are transcriptionally activated in response to infection by a wide variety o nonrelated viruses
- the introduction of vectors expressing a conditionally lethal gene produc like HSVTK, for example, from these viral-responsive elements (VREs) could result in th destruction of cells infected with a variety of different viruses.
- recombinant retroviruses ar provided that produce substances such as inhibitor palliatives, that inhibit viral assembly.
- the recombinant retrovirus codes for defective gag, pol, env or other vir particle proteins or peptides which inhibit in a dominant fashion the assembly of vir particles. Such inhibition occurs because the interaction of normal subunits of the vir particle is disturbed by interaction with the defective subunits.
- inhibitory palliatives are to expres inhibitory genes, such as viral inhibitory genes, in conjunction with the expression of gene which increase the probability of infection of the resistant cell by the virus in question. Th result is a nonproductive "dead-end" event which would compete for productive infectio events.
- a recombinant retrovirus may be administered tha inhibits HTV replication (by expressing anti-sense tat, etc., as described above) and als overexpress proteins required for infection, such as CD4.
- a relatively sma number of vector-infected HTV-resistant cells act as a "sink” or "magnet” for multipl nonproductive fusion events with free virus or virally infected cells.
- recombinant retroviruses ar provided for the expression substances such as inhibiting peptides or proteins specific fo viral protease. Viral protease cleaves the viral gag and gag/pol proteins into a number o smaller peptides. Failure of this cleavage in all cases leads to complete inhibition of production of infectious retroviral particles.
- the HTV protease is known to be an aspartyl protease, and these are known to be inhibited by peptides made from amino acids from protein or analogues. Recombinant retroviruses that inhibit HTV will express one or multiple fused copies of such peptide inhibitors.
- recombinant retroviruses that express a palliative, wherein the palliative has a membrane anchor and acts as an anti-tumor agent(s).
- a palliative may be constructed, for example, as an anti-tumor agent - membrane anchor fusion protein.
- the membrane anchor aspect of the fusion protein may be selected from a variety of sequences, including, for example, the transmembrane domain of well known molecules.
- membrane anchor sequences are regions of a protein that bind the protein to a membrane.
- transmembrane regions that span the lipid bilayer of the cell membrane, and interact with the hydrophobic center region (proteins containing such regions are referred to integral membrane proteins), and (2) domains which interact with an integral membrane protein or with the polar surface of the membrane (such proteins are referred to as peripheral, or extrinsic, proteins).
- Membrane anchors for use within the present invention may contain transmembrane domains which span the membrane one or more times.
- the membrane binding region spans the membrane once
- the transmembrane domain of rhodopsin spans the membrane seven times
- that of the photosynthetic reaction center of Rhodopseudomonas viridis spans the membrane eleven times (see Ross et al., J. Biol. Chem. 257:4152, 1982; Garbers, Pharmac. Ther. 50:337-345, 1991; Engelman et al., Proc. Natl Acad Sci.
- Membrane anchors for use in the present invention can also include, for example, phosphoinositol anchors. Regardless of the number of times the protein crosses the membrane, the membrane spanning regions typically have a similar structure. More specifically, the 20 to 25 amino-acid residue portion of the domain that is located inside the membrane generally consists almost entirely of hydrophobic residues (see Eisenberg et al., Ann. Rev. Biochem. 53:595-623, 1984).
- membrane spanning region of glycophorin 28 of the 34 residues in the membrane spanning region of glycophorin are hydrophobic (see Ross et al., supra; Tomita et al., Biochemistry 77:4756-4770, 1978).
- the membrane spanning regions typically have an alpha helical structure, as determined by X-ray diffraction, crystallography and cross-linking studies (see Eisenberg et d ⁇ .,supra; Heijne and Manoil, supra).
- the location of thes transmembrane helices within a given sequence can often be predicted based o hydrophobicity plots. Stryer et al., Biochemistry, 3rd. ed. 304, 1988.
- Particularly preferre membrane anchors for use within the present invention include naturally occurring cellul proteins (that are non-immunogenic) which have been demonstrated to function a membrane signal anchors (such as glycophorin).
- a DNA sequence i provided which encodes a membrane anchor - gamma interferon fusion protein.
- this fusion protein may be constructed by genetically fusing the sequence whic encodes the membrane anchor of the gamma-chain of the Fc receptor, to a sequence whic encodes gamma-interferon.
- recombinant retroviruses which have therapeutic effect by encoding one or more ribozymes (RNA enzymes) (Haseloff an Gerlach, Nature 334:585, 1989) which will cleave, and hence inactivate, RNA molecule corresponding to a pathogenic function.
- ribozymes function by recognizing a specifi sequence in the target RNA and this sequence is normally 12 to 17 bp, this allows specifi recognition of a particular RNA sequence corresponding to a pathogenic state, such as HI tat, and toxicity is specific to such pathogenic state. Additional specificity may be achieve in some cases by making this a conditional toxic palliative, as discussed above.
- recombinant retroviruses comprising biologically active nucleic acid molecule that is an antisense sequence (an antisense sequenc may also be encoded by a nucleic acid sequence and then produced within a host cell vi transcription).
- the antisense sequence is selected from the grou consisting of sequences which encode influenza virus, HIV, HSV, HPV, CMV, and HB
- the antisense sequence may also be an antisense RNA complementary to RNA sequence necessary for pathogenicity.
- the biologically active nucleic acid molecule ma be a sense RNA (or DNA) complementary to RNA sequences necessary for pathogenicity.
- the biologically active nucleic acid molecule may be a antisense sequence.
- antisense sequences are designed to bind to RNA transcript and thereby prevent cellular synthesis of a particular protein, or prevent use of that RN sequence by the cell.
- Representative examples of such sequences include antisense thymidin kinase, antisense dihydrofolate reductase (Maher and Dolnick, Arch. Biochem.
- antisense RNA may be utilized as an anti-tumor agent in order to induce a potent Class I restricted response.
- high levels of specific antisense sequences are believed to induce the increased expression of interferons (including gamma-interferon), due to the formation of large quantities of double- stranded RNA.
- the increased expression of gamma interferon boosts the expression of MHC Class I antigens.
- Preferred antisense sequences for use in this regard include actin RNA, myosin RNA, and histone RNA.
- Antisense RNA which forms a mismatch with actin RNA is particularly preferred.
- the substances of the invention include a surface protein that is itself therapeutically beneficial. For example, in the particular case of HIV, expression of the human CD4 protein specifically in HIV-infected cells may be beneficial in two ways:
- CD4/HTV env complex has been implicated as a cause of cell death, additional expression of CD4 (in the presence of excess HI V-env present in HIV-infected cells) leads to more rapid cell death and thus inhibits viral dissemination.
- This may be particularly applicable to monocytes and macrophages, which act as a reservoir for virus production as a result of their relative refractility to HTV-induced cytotoxicity (which, in turn, is apparently due to the relative lack of CD4 on their cell surfaces).
- Still further aspects of the present invention relate to recombinant retroviruses capable of immunostimulation.
- the ability to recognize and defend against foreign pathogens is essential to the function of the immune system.
- the immune system must be capable of distinguishing "self* from "nonself (i.e., foreign), so that the defensive mechanisms of the host are directed toward invading entities instead of against host tissues.
- Cytolytic T lymphocytes are typically induced, or stimulated, by the display of a cell surface recognition structure, such as a processed, pathogen-specific peptide, in conjunction with a MHC class I or class II cell surface protein.
- Diseases suitable to treatment include viral infections such as influenza virus, respiratory syncytial virus, HPV, HBV, HCV, EBV, HTV, HSV, FeLV, FTV, Hantavirus, HTLV I, HTLV II and CMV, cancers such as melanomas, renal carcinoma, breast cancer, ovarian cancer and other cancers, and heart disease.
- viral infections such as influenza virus, respiratory syncytial virus, HPV, HBV, HCV, EBV, HTV, HSV, FeLV, FTV, Hantavirus, HTLV I, HTLV II and CMV
- cancers such as melanomas, renal carcinoma, breast cancer, ovarian cancer and other cancers, and heart disease.
- the invention provides methods for stimulating a specifi immune response and inhibiting viral spread by using recombinant retroviruses that direct th expression of an antigen or modified form thereof in susceptible target cells, wherein th antigen is capable of either (1) initiating an immune response to the viral antigen or (2) preventing the viral spread by occupying cellular receptors required for viral interactions.
- Expression of the protein may be transient or stable with time.
- the recombinant retrovirus is preferably designed to express a modified form of the antigen which will stimulate an immune response and whic has reduced pathogenicity relative to the native antigen.
- an immune response is achieve when cells present antigens in the correct manner, i.e., in the context of the MHC class and/or II molecules along with accessory molecules such as CD3, ICAM-1, ICAM-2, LFA-1, or analogs thereof (e.g., Altmann et al., Nature 335:512, 1989).
- An immune response can also be achieved by transferring to an appropriate immune cell (such as a T lymphocyte) (a) the gene for the specific T-cell receptor that recognizes the antigen of interest (in the context of an appropriate MHC molecule i necessary), (b) the gene for an immunoglobulin which recognizes the antigen of interest, or (c) the gene for a hybrid of the two which provides a CTL response in the absence of the MHC context.
- an appropriate immune cell such as a T lymphocyte
- a) the gene for the specific T-cell receptor that recognizes the antigen of interest in the context of an appropriate MHC molecule i necessary
- the antigen generated from a recombinant retrovirus may be i a form which will elicit either or both an HLA class I- or class H-restricted immune response.
- the antigen is preferably selected from gp 160, gp 120, and gp 41, which have been modified to reduce their pathogenicity.
- the selected antigen is modified to reduce the possibility of syncytia, to avoid expression of epitopes leading to a disease enhancing immune response, to remov immunodominant, but haplotype-specific epitopes or to present several haplotype-specifi epitopes, and allow a response capable of eliminating cells infected with most or all strains o HTV.
- the haplotype-specific epitopes can be further selected to promote the stimulation o an immune response within an animal which is cross-reactive against other strains of HIV.
- Antigens from other HTV genes or combinations of genes, such as gag, pol, rev, vif, nef, prot, gag/pol, gag prot, etc., may also provide protection in particular cases.
- HIV is only one example. This approach should be effective against man virally linked diseases or cancers where a characteristic antigen (which does not need to be membrane protein) is expressed, such as in HPV and cervical carcinoma, HTLV-I-induce leukemias, prostate-specific antigen (PSA) and prostate cancer, mutated p53 and colon carcinoma and melanoma, melanoma specific antigens (MAGEs), and melanoma, mucin and breast cancer.
- a characteristic antigen which does not need to be membrane protein
- substances which are carried and/or expressed by the recombinant retroviruses of the present invention may also include "immunomodulatory factors," many of which are set forth above.
- Immunomodulatory factors refer to factors that, when manufactured by one or more of the cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence of the factor.
- the factor may also be expressed from a non- recombinant retrovirus derived gene, but the expression is driven or controlled by the recombinant retrovirus.
- the quality or potency of a response may be measured by a variety of assays known to one of skill in the art including, for example, in vitro assays which measure cellular proliferation (e.g., -- i H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure -**Cr release) (see, Warner et al., AIDS Res. and Human Retroviruses 7:645- 655, 1991).
- Immunomodulatory factors may be active both in vivo and ex vivo. Representative examples of such factors include cytokines, such as IL-1, IL-2 (Karupiah et al., J. Immunology 744:290-298, 1990; Weber et al., J.
- Immunomodulatory factors may also be agonists, antagonists, or ligands for these molecules For example soluble forms of receptors can often behave as antagonists for these types o factors, as can mutated forms of the factors themselves.
- an immunomodulatory factor cited above is a member of th B7 family of molecules (e.g., B7.1-.3 costimulatory factor). Briefly, activation of the ful functional activity of T cells requires two signals. One signal is provided by interaction o the antigen-specific T cell receptor with peptides which are bound to majo histocompatibility complex (MHC) molecules, and the second signal, referred to a costimulation, is delivered to the T cell by antigen presenting cells.
- MHC majo histocompatibility complex
- the second signal i required for interleukin-2 (EL-2) production by T cells appears to involve interaction o the B7.1-.3 molecule on antigen-presenting cells with CD28 and CTLA-4 receptors o T lymphocytes (Linsley et al., J. Exp. Med, 773:721-730, 1991a and J. Exp. Med., 774:561 570, 1991).
- B7.1-.3 may be introduced into tumo cells in order to cause costimulation of CD8 + T cells, such that the CD ⁇ +T cells produc enough IL-2 to expand and become fully activated.
- CD8 + T cells can kill tumor cell that are not expressing B7 because costimulation is no longer required for further CT function.
- Vectors that express both the costimulatory B7.1-.3 factor, and, for example, a immunogenic HBV core protein may be made utilizing methods which are described herein. Cells transduced with these vectors will become more effective antigen presenting cells. Th HBV core-specific CTL response will be augmented from the fully activated CD8 + T cell vi the costimulatory ligand B7.1-.3.
- immunomodulatory factor may be based upon known therapeutic effects of the factor, or, experimentally determined.
- a known therapeutic effector in chronic hepatitis B infections is alpha interferon. This has been found to be efficacious in compensating patient's immunological deficit, and thereby assisting recovery from the disease.
- a suitable immunomodulatory factor may be experimentally determined. Briefly, blood samples are first taken from patients with a hepatic disease. Peripheral bloo lymphocytes (PBLs) are restimulated in vitro with autologous or HLA matched cells (e.g.
- EBV transformed cells that have been transduced with a recombinant retrovirus whic directs the expression of an immunogenic portion of a hepatitis antigen and th immunomodulatory factor.
- These stimulated PBLs are then used as effectors in a CTL assa with the HLA matched transduced cells as targets.
- the immunomodulatory factor gamma interferon is particularly preferred.
- the present invention also includes recombinant retroviruses which encode immunogenic portions of desired antigens including, for example, viral, bacterial or parasite antigens.
- desired antigens including, for example, viral, bacterial or parasite antigens.
- various immunogenic portions of the HBV S antigens may be combined in order to present an immune response when administered by one of the recombinant retroviruses described herein.
- particular combinations of antigens may be preferred for administration in particular geographic regions.
- epitopes that are found in all human hepatitis B virus S samples are defined as determinant "a".
- the immunological variability is due to single nucleotide substitutions in two areas of the hepatitis B virus S antigen open reading frame resulting in the following amino acid changes: (1) exchange of lysine- 122 to arginine in the hepatitis B virus S antigen open reading frame causes a subtype shift from d to y, and (2) exchange of arginine- 160 to lysine causes the shift from subtype r to w. In black Africa, subtype ayw is predominant, whereas in the U.S. and northern Europe the subtype adw2 is more abundant (Molecular Biology of the Hepatitis B Virus, McLachlan (ed.), CRC Press, 1991).
- a vector for administration which is appropriate to the particular hepatitis B virus subtype which is prevalent in the geographical region of administration.
- Subtypes of a particular region may be determined by two-dimensional double immunodifiusion or, preferably, by sequencing the S antigen open reading frame of HBV virus isolated from individuals within that region.
- HBV pol also presented by HBV are pol ("HBV pol"), ORF 5, and ORF 6 antigens.
- HBV pol the polymerase open reading frame of HBV encodes reverse transcriptase activity found in virions and core-like particles in infected liver tissue.
- the polymerase protein consists of at least two domains: the amino terminal domain encodes the protein that primes reverse transcription, and the carboxyl terminal domain which encodes reverse transcriptase and RNase H activity.
- Immunogenic portions of HBV pol may be administered to a warm ⁇ blooded animal by introducing into the animal a recombinant retrovirus which expresses the antigen of interest in order to generate an immune response within the animal.
- HBV antigens such as ORF 5 and ORF 6, (Miller et al., Hepatology 9:322-321, 1989), may be expressed utilizing recombinant retroviruses as described herein.
- at least one immunogenic portion of a hepatitis B antigen can be inco ⁇ orated into a recombinant retrovirus.
- the immunogenic portion(s) which are inco ⁇ orated into the recombinant retrovirus may be of varying length, although it is generally preferred that the portions be at least 9 amino acids long, and may include the entire antigen. Immunogenicity of a particular sequence is often difficult to predict, although T cell epitopes may be predicted utilizing the HLA A2.1 motif described by Falk et al.
- peptides may be synthesized and used as targets in an in vitro cytotoxic assay.
- Other assays may also be utilized, including, for example, ELISA which detects the presence of antibodies against the newly introduced vector, as well as assays which test for T helper cells, such as gamma-interferon assays, IL-2 production assays, and proliferation assays.
- At least one immunogenic portion of a hepatitis C antigen can be inco ⁇ orated into a recombinant retrovirus.
- Preferred immunogenic portion(s) of hepatitis C may be found in the C and NS3-NS4 regions since these regions are the most conserved among various types of hepatitis C virus (Houghton et al., Hepatology 74:381-388, 1991).
- Particularly preferred immunogenic portions may be determined by a variety of methods. For example, as noted above for the hepatitis B virus, identification of immunogenic portions of the polypeptide may be predicted based upon amino acid sequence.
- CTL epitopes for the HLA A2.1 haplotype may be predicted utilizing the HLA A2.1 motif described by Falk et al. (Nature 351:290, 1991). From this analysis, peptides are synthesized and used as targets in an in vitro cytotoxic assay.
- methods for destroying hepatitis B carcinoma cells comprising the step of administering to a warm ⁇ blooded animal a recombinant retrovirus which directs the expression of an immunogenic portion of antigen X, such that an immune response is generated.
- Sequences which encode the HBxAg may readily be obtained by one of skill in the art given the disclosure provided herein.
- a 642 bp Neo I-Taq I is recovered from ATCC 45020, and inserted into recombinant retroviruses as described above for other hepatitis B antigens.
- the X antigen is a known transactivator which may function in a manner similar to other potential oncogenes (e.g., EIA).
- EIA potential oncogenes
- Various methods may be utilized to render the X antigen non- tumorigenic including, for example, by truncation, point mutation, addition of premature stop codons, or phosphorylation site alteration.
- the sequence or gene o interest which encodes the X antigen is truncated.
- Truncation may produce a variety of fragments, although it is generally preferable to retain greater than or equal to 50% of the encoding gene sequence. In addition, it is necessary that any truncation leave intact some of the immunogenic sequences of the gene product.
- multiple translational termination codons may be introduced into the gene. Insertion of termination codons prematurely terminates protein expression, thus preventing expression of the transforming portion of the protein.
- the X gene or modified versions thereof may be tested for tumorigenicity in a variety of ways.
- Representative assays include tumor formation in nude mice, colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice.
- methods for destroying hepatitis C carcinoma cells comprising the step of administering to a warm ⁇ blooded animal a recombinant retrovirus which directs the expression of an immunogenic portion of a hepatitis C antigen.
- Preferred immunogenic portion(s) of a hepatitis C antigen may be found in the polypeptide which contains the Core antigen and the NS1-NS5 regions (Choo et al., Proc. Natl Acad Sci. USA 55:2451-2455, 1991).
- Particularly preferred immunogenic portions may be determined by a variety of methods. For example, as noted above preferred immunogenic portions may be predicted based upon amino acid sequence.
- CTL epitopes for the HLA A2.1 haplotype may be predicted utilizing the HLA A2.1 motif described by Falk et al. (Nature 357:290, 1991).
- Another method that may also be utilized to predict immunogenic portions is to determine which portion has the property of CTL induction in mice utilizing retroviruses (see, Warner et al., AIDS Res. and Human Retroviruses 7:645-655, 1991).
- CTL induction in mice may be utilized to predict cellular immunogenicity in humans.
- Preferred immunogenic portions may also be deduced by determining which fragments of the polypeptide antigen or peptides are capable of inducing lysis by autologous patient lymphocytes of target cells (e.g., autologous EBV-transformed lymphocytes) expressing the fragments after vector transduction of the corresponding genes.
- Preferred immunogenic portions may also be selected in the following manner. Briefly, blood samples from a patient with a target disease, such as HCV, are analyzed with antibodies to individual HCV polypeptide regions (e.g., HCV core, El, E2/SNI and NS2-NS5 regions), in order to determine which antigenic fragments are present in the patient's serum.
- antigens are useful as immunogenic portions within the context of the present invention (Hayata et al., Hepatology 73:1022-1028, 1991; Davis et al., N. Eng. Med 327:1501-1506, 1989).
- Additional immunogenic portions of a chosen antigen may be obtained by truncating the coding sequence.
- a chosen antigen such as those from th hepatitis B or C virus
- the following sites may be truncated: Bst UI, Ssp I, Ppu Ml, and Msp (Valenzuela et al., Nature 250:815-19, 1979; Valenzuela et al., Animal Virus Genetics ICN/UCLA Symp. Mol. Cell Biol, 1980, B. ⁇ . Fields and R. Jaenisch (eds.), pp. 57-70, Ne York: Academic). Further methods for determining suitable immunogenic portions as wel as methods are also described below in the context of hepatitis C.
- immunogeni portions for inco ⁇ oration into recombinant retroviruses include HBeAg, HBcAg, an HBsAgs.
- more than one immunogenic portion may be inco ⁇ orated into the recombinant retrovirus.
- a recombinant retrovirus may be prepared which directs the co-expressio of both an immunogenic portion of the hepatitis B antigen, as well as an immunogeni portion of the hepatitis C polypeptide.
- Such constructs may be administered in order t prevent or treat acute and chronic hepatitis infections of either type B or C.
- a recombinant retrovirus may be prepared which directs the co expression of both an immunogenic portion of the hepatitis B X antigen, as well as a immunogenic portion of the hepatitis C polypeptide.
- Such a construct may similarly b administered in order to treat hepatocellular carcinoma that is associated with either hepatiti B or C.
- a vector may also be utilized a a prophylactic treatment for the disease. Immunogenic portions may also be selected by other methods.
- the HLA A2.1/Kb transgenic mouse has been shown to be useful as a model for human T cell recognition of viral antigens.
- th murine T-cell receptor repertoire recognizes the same antigenic determinants recognized b human T-cells.
- the CTL response generated in the HLA A2.1/K transgenic mouse is directed toward virtually the same epitope as those recognized by huma CTLs of the HLA A2.1 haplotype (VitieUo et al., J. Exp. Med 773:1007-1015, 1991 Vitiello et al., Abstract of Molecular Biology of Hepatitis B Virus Symposia, 1992).
- Immunogenic proteins of the present invention may also be manipulated by variety of methods known in the art, in order to render them more immunogenic
- Representative examples of such methods include: adding amino acid sequences tha correspond to T helper epitopes; promoting cellular uptake by adding hydrophobic residues by forming paniculate structures; or any combination of these (see generally, Hart, op. cit. Milich et al., Proc. Natl. Acad Sci. USA 55:1610-1614, 1988; Willis, Nature 340:323-324, 1989; Griffiths et al., J. Virol. 65:450-456, 1991).
- the present invention also includes compositions and methods for treating, as well as vaccines for preventing, various feline diseases, including for example feline leukemia vims ("FeLV”) and feline immunodeficiency virus (“FTV”) infections. These viruses are discussed more fully in PCT application number US 93/09070.
- feline leukemia virus is a retrovirus of the oncornavirus subfamily. FeLV is presently believed to exist in three subgroups - A, B or C - which are differentiated by their envelope antigens gp70 and pl5E. FeLV is also comprised of a number of core antigens, including pi 5, pl2, p27, and plO, which are highly conserved for all subgroups of FeLV (see Geering et al., Vir. 36:678-680, 1968; Hardy et al., JAVMA 755:1060-1069, 1971; Hardy et al., Science 766:1019-1021, 1969).
- the recombinant retrovirus directs the expression of at least one portion of a feline leukemia virus antigen selected from the group consisting of p ⁇ 5gag, p ⁇ 2gag, p21gag, plOgag, pl4pol, pSQpol, p46pol, gp70env, and p ⁇ 5env.
- a feline leukemia virus antigen selected from the group consisting of p ⁇ 5gag, p ⁇ 2gag, p21gag, plOgag, pl4pol, pSQpol, p46pol, gp70env, and p ⁇ 5env.
- the recombinant retrovirus directs the expression of gp85em>. Sequences which encode these antigens may be readily obtained given the disclosure provided herein (see Donahue et al., J. Vir. 62(3): 722-731, 1988; Stewart et al., J. Vir.
- Feline immunodeficiency virus has been classified as a retrovirus of the lentivirus subfamily, based upon the magnesium requirement for reverse transcriptase (RT) and the mo ⁇ hology of viral particles (see Pederesen et al., Science 235:190-193, 1987).
- feline immunodeficiency virus is mo ⁇ hologically and antigenically distinct from other feline retroviruses, including feline leukemia virus, type C oncorna virus (RD-114), and feline syncytium-forming virus (FeSFV) (see Yamamoto et al., "Efficacy of experimental FIV vaccines, (Abstract), First International Conference of Feline Immunodeficiency Virus researchers, University of California, Davis, CA, Sep. 4-7, 1991).
- feline leukemia virus type C oncorna virus
- FeSFV feline syncytium-forming virus
- the recombinant retrovirus directs the expression of at least one immunogenic portion of an feline immunodeficiency virus antigen selected from the group consisting of pl5gag, p24gag, plOgag, p ⁇ 3pol, p62pol, p ⁇ 5pol and p36pol.
- the recombinant retrovirus directs the expression of gp68env, gp27ewv and rev.
- "rev” is understood to refer to the antigen corresponding to the rev open reading frame (see, Phillips et al., First International Conference, supra).
- ras * a non-tumorigenic gene
- the ras* gene is an attractive target because it is causally linked to th neoplastic phenotype, and indeed may be necessary for the induction and maintenance o tumorigenesis in a wide variety of distinct cancers, such as pancreatic carcinoma, colo carcinoma and lung adenocarcinoma.
- ras* genes are found in pre-neoplasti tumors, and therefore immune intervention therapy may be applied prior to detection of malignant tumor.
- Normal ras genes are non-tumorigenic and ubiquitous in all mammals. The are highly conserved in evolution and appear to play an important role in maintenance of th cell cycle and normal growth properties.
- the normal ras protein is a G-protein which bind GTP and has GTPase activity, and is involved in transmitting signals from the external milie to the inside of the cell, thereby allowing a cell to respond to its environment.
- Ras * genes o the other hand alter the normal growth regulation of neoplastic cells by uncoupling cellula behavior from the environment, thus leading to the uncontrolled proliferation of neoplasti cells.
- ras gene Mutation of the ras gene is believed to be an early event in carcinogenesis (Kuma et al., "Activation of ras Oncogenes Preceding the Onset of Neoplasia," Science 245:1101 1104, 1990), which, if treated early, may prevent tumorigenesis.
- Ras* genes occur in a wide variety of cancers, including for exampl pancreatic, colon, and lung adenocarcinomas.
- the spectrum of mutation occurring in the ras* genes found in a variety of cancers is quite limited. These mutation alter the GTPase activity of the ras protein by converting the normal on/off switch to constitutive ON position.
- Tumorigenic mutations in ras occur primarily (in vivo) in only codons: 12, 13 and 61. Codon 12 mutations are the most prevalent in both human an animal tumors.
- p53 p53
- p53 is a nuclear phosphoprotein which was originally discovered in extracts of transforme cells, and thus was initially classified as an oncogene (Linzer and Levine, Cell 77:43-5 1979; Lane and Crawford, Nature 275:261-263, 1979). It was later discovered that th original p53 cDNA clones were mutant forms of p53 (Hinds et al., J. Virol. 63:739-74 1989). It now appears that p53 is a tumor suppressor gene, which negatively regulates th cell cycle, and that mutation of this gene may lead to tumor formation. Of colon carcinom that have been studied, 75%-80% show a loss of both p53 alleles, one through deletion, an the other through point mutation. Similar mutations are found in lung cancer, and in brain and breast tumors.
- p53 mutations e.g., p53**, p53* 2 , etc.
- the majority of p53 mutations are clustered between amino-acid residues 130 to 290 (see Levine et al., Nature 357:453-456, 1991; see also the following references which describe specific mutations in more detail: Baker et al., Science 244:217-221, 1989; Nigro et al., Nature 342:705-708, 1989 (p53 mutations cluster at four "hot spots" which coincide with the four highly conserved regions of the genes and these mutations are observed in human brain, breast, lung and colon tumors); Vogelstein, Nature 345:681-682, 1990; Takahashi et al., Science 246:491-494, 1989; Iggo et al., Lancet 335:675-679, 1990; James et al., Proc.
- Certain alterations of the p53 gene may be due to certain specific toxins.
- Bressac et al. (Nature 350:429-431, 1991) describes specific G to T mutations in codon 249, in patients affected with hepatocellular carcinoma.
- One suggested causative agent of this mutation is aflatoxin Bi, a liver carcinogen which is known to be a food contaminant in Africa.
- retinoblastoma is a childhood eye cancer associated with the loss of a gene locus designated Rb, which is located in chromosome band 13ql4. A gene from this region has been cloned which produces a nuclear phosphoprotein of about 110 kd (Friend et al., Nature 323:643, 1986; Lee et al., Science 235:1394, 1987; and Fung et al., Science 236:1657, 1987).
- Rb is believed to be a negative regulator of cellular proliferation, and has a role in transcriptional control and cell-cycle regulation. Rb binds to at least seven proteins found in the nucleus, and in particular, appears to be involved with a cellular transcription factor which has been designated both E2F (Bagchi et al., Cell 62:659-669, 1990) and DRTF (Shivji and La Thangue, Mol. Cell. Biol. 77:1686-1695, 1991). Rb is believed to restrict cellular growth by sequestering a variety of nuclear proteins involved in cellular proliferation. Deletions within the Rb gene have been detected which evidence that the Rb gene may be responsible for tumorigenicity.
- deletions include, for example, a deletion in exon 21 in a prostate cancer and bladder cancer cell line (Bookstein et al., Science 247:712-715, 1990; Horowitz et al., Science 243:931, 1989), a deletion of exon 16 in a small-cell carcinoma of the lung (Shew et al., Cell Growth and Diff. 7:17, 1990), and a deletion between exons 21 and 27 (Shew et al., Proc. Natl. Acad. Sci. USA 87:6, 1990). Deletion of these exons results in the production of a protein containing a novel coding sequence at the junction of the deleted exons. This novel protein coding sequence may be used as a marker of tumorigenic cells, and an immune response directed against this novel coding region may eliminate tumorigenic cells containing the Rb exon deletion.
- recombinant retroviruses are provided which direct the expression of an altered gene which causes Wilms' tumor.
- Wilms' tumor is typically found in children younger than 16 years of age. One child in 10,000 will develop this tumor, which comprises approximately 5% of childhood cancers. The tumor usually presents itself as a large abdominal mass which is surrounded by a fibrous pseudocapsule. Approximately 7% of the tumors are multifocal in one kidney, and 5.4% are involved with both kidneys.
- the Wilms' tumor gene has been localized to chromosome l lpl3, and a cDNA clone (wtl) has been isolated that is characteristic of a tumor suppressor gene (Call et al., Cell 60:509, 1990; Gessler et al., Nature 343:744, 1990; Rose et al., Cell 60:495, 1990; and Haber et al., Cell 67:1257, 1990).
- the wtl gene encodes a protein which contains four zinc fingers and a glutamine and proline rich amino terminus. Such structures are believed to be associated with transcriptional and regulatory functions.
- Mutations of the Wilms 1 tumor gene include the insertion of lysine, threonine, and serine between the third and forth zinc fingers.
- a wtl protein which contains such insertions does not bind to the EGR-1 site.
- a second alternative mutation results in the insertion of about 17 amino acids in the region immediately NH2-terminal to the zinc finger domain (Madden et al., Science 253:1550-1553, 1991; Call et al., Cell 60:509, 1990; Gessler et al., Nature 343:744, 1990; Rose et al., Cell 60:495, 1990; Haber et al., Cell 67:1257, 1990; and Buckler et al., Mol. Cell. Biol. 77:1707, 1991).
- mucins are large molecular weight glycoproteins which contain approximately 50% carbohydrate.
- Polymo ⁇ hic epithelial mucin (PEM) is a tumor-associated mucin (Girling et al., Int. J. Cancer 43:1072-1076, 1989) which is found in the serum of cancer patients.
- PEM polymo ⁇ hic epithelial mucin
- the full-length cDNA sequence has been identified (Gendler et al., J. Biol. Chem. 265(25): 15286-15293, 1990; Lan et al., J. Biol. Chem.
- recombinant retroviruses which direct the expression of an altered DCC (deleted in colorectal carcinomas) gene.
- DCC deficiency C-cell-adhesion molecule
- NCAM neural cell-adhesion molecule
- contactin contactin
- the DCC gene is expressed in normal colonic mucosa, but its expression is reduced or absent in the majority of colorectal carcinomas (Solomon, Nature 343:412-414, 1990). This loss of expression has been associated in some cases with somatic mutations of the DCC gene.
- a contiguous stretch of DNA comprising 370 kb has been cloned which encodes an approximately 750 amino acid protein (Fearon et al., "Identification of a Chromosome 18q Gene That Is Altered in Colorectal Cancers," Science 247:49-56, 1990).
- recombinant retroviruses are provided which direct the expression of MCC (mutated in colorectal cancer) or APC. Both MCC and APC have been identified as tumor suppressor genes (Kinzler et al., Science 257:1366-1370, 1991) which undergo mutation in familial adenomatous polyposis - 30 -
- FAP familial adenomatous polyposis gene
- recombinant retroviruses which direct the expression of an altered receptor which is functionally locked or stuck in an "ON” or “OFF” mode.
- many cellular receptors are involved in cell growth by monitoring the external environment and signaling the cell to respond appropriately. If either the monitoring or signaling mechanisms fail, the cell will no longer respond to the external environment and may exhibit uncontrolled growth.
- Many different receptors or receptor-like structures may function as altered cellular components, including, for example, neu and mutated or altered forms of the thyroid hormone receptor, the PDGF receptor, the insulin receptor, the Interleukin receptors (e.g., IL-1, -2, -3, etc. receptors), or the CSF receptors, such as the G-CSF, GM-CSF, or M-CSF receptors.
- neu also referred to as the Human Epidermal Growth Factor Receptor "HER” or the Epidermal Growth Factor ⁇ GF” receptor
- HER Human Epidermal Growth Factor Receptor
- ⁇ GF Epidermal Growth Factor ⁇ GF
- This clone encodes a protein that has extracellular, transmembrane, and intracellular domains (Schechter, Nature 372:513, 1984; Coussens et al., Science 230:1132, 1985) and thus is believed to encode the neu receptor.
- rat neu gene isolated from chemically induced neuroglioblastoma cells indicate that it contains a single mutation at position 664 from valine to glutamic acid (Bargmann et al., EMBOJ. 7:2043, 1988).
- baby rats which were treated with N-ethyl-N-nitrosourea developed malignant tumors of the nervous system. All 47 trigeminal schwannomas and 12 neurinomas which developed carried a T to A transversion at position 664 of the neu gene (Nik-tin et al., Proc. Natl. Acad Sci USA 55:9939-9943, 1991).
- chromosome 3p21-p25 has been associated with small-cell lung carcinomas (Leduc et al., Am. J. Hum. Genet. 44:282-
- Alterations in receptors as described above result in the production of protein(s) (or receptors) containing novel coding sequencers).
- the novel protein(s) encoded by these sequencers) may be used as a marker of tumorigenic cells, and an immune response directed against these novel coding region(s) may be utilized to destroy tumorigenic cells containing the altered sequence(s) or gene(s).
- the altered cellular component is associated with making the cell tumorigenic, then, it is necessary to make the altered cellular component non-tumorigenic.
- the sequence or gene of interest which encodes the altered cellular component is truncated in order to render the gene product non-tumorigenic.
- the gene encoding the altered cellular component may be truncated to a variety of sizes, although it is preferable to retain as much as possible of the altered cellular component.
- any truncation leave intact at least some of the immunogenic sequences of the altered cellular component.
- multiple translational termination codons may be introduced into the gene which encodes the altered cellular component, downstream of the immunogenic region. Insertion of termination codons will prematurely terminate protein expression, thus preventing expression of the transforming portion of the protein.
- the ras * gene is truncated in order to render the ras protein non-tumorigenic.
- the carboxy-terminal amino acids of ras * functionally allow the protein to attach to the cell membrane. Truncation of these sequences renders the altered cellular component non-tumorigenic.
- the ras* gene is truncated in the purine ring formation, for example around the sequence which encodes amino acid number 110.
- the ras* gene sequence may be truncated such that as little as about 20 amino acids (including the altered amino acid(s) are encoded by the recombinant retrovirus, although preferably, as many amino acids as possible should be expressed (while maintaining non-tumorigenicity).
- the p53 protein is modified by truncation in order to render the cellular component non-tumorigenic.
- p53* is truncated to a sequenc which encodes amino acids 100 to 300, thereby including all four major "hot spots.”
- altered cellular components which are oncogenic may also be truncate in order to render them non-tumorigenic.
- both neu and bcr/abl may b truncated in order to render them non-tumorigenic.
- Non-tumorigenicity may be confirme by assaying the truncated altered cellular component as described above.
- altered cellular component is onl associated with non-tumorigenic cells in general, and is not. required or essential for makin the cell tumorigenic, then it is not necessary to render the cellular component non tumorigenic.
- altered cellular components which are no tumorigenic include Rb , ubiquitin , and mucin .
- th altered cellular component must also be immunogenic. Immunogenicity of a particula sequence is often difficult to predict, although T cell epitopes often possess an immunogeni amphipathic alpha-helix component. In general, however, it is preferable to determin immunogenicity in an assay.
- Representative assays include an ELISA which detects th presence of antibodies against the newly introduced vector, as well as assays which test for helper cells such as gamma-interferon assays, IL-2 production assays, and proliferatio assays.
- a particularly preferred method for determining immunogenicity is the CTL assay.
- a sequence encoding at least one anti-tumor agent has been obtained, i is preferable to ensure that the sequence encodes a non-tumorigenic protein.
- Various assay are known and may easily be accomplished which assess the tumorigenicity of a particula cellular component. Representative assays include tumor formation in nude mice or rats colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice
- tumor formation in nud mice or rats is a particularly important and sensitive method for determining th tumorigenicity of an anti-tumor agent.
- Nude mice lack a functional cellular immune syste
- recombinan retrovirus is delivered to syngeneic murine cells, followed by administration into nude mice The mice are visually examined for a period of 2 to 8 weeks after administration in order t determine tumor growth. The mice may also be sacrificed and autopsied in order t determine whether tumors are present.
- Tumorigenicity testing of cell lines considered for production o biological drugs Abnormal Cells, New Products and Risk, Hopps and Petricciani (eds.), Tissue Culture Association, 1985; and Levenbook et al., J. Biol. Std 73:135-141, 1985). Tumorigenicity may also be assessed by visualizing colony formation in soft agar (MacPherson and Montagnier, Vir. 23:291-294, 1964). Briefly, one property of normal non- tumorigenic cells is "contact inhibition" (i.e., cells will stop proliferating when they touch neighboring cells). If cells are plated in a semi-solid agar support medium, normal cells rapidly become contact inhibited and stop proliferating, whereas tumorigenic cells will continue to proliferate and form colonies in soft agar.
- Transgenic animals such as transgenic mice, may also be utilized to assess the tumorigenicity of an anti-tumor agent (e.g., Stewart et al., Cell 35:627-637, 1984; Quaife et al., Cell 45:1023-1034, 1987; and Koike et al., Proc. Natl. Acad Sci. USA 56:5615-5619, 1989).
- an anti-tumor agent e.g., Stewart et al., Cell 35:627-637, 1984; Quaife et al., Cell 45:1023-1034, 1987; and Koike et al., Proc. Natl. Acad Sci. USA 56:5615-5619, 1989.
- the gene of interest may be expressed in all tissues of the animal. This unregulated expression of the transgene may serve as a model for the tumorigenic potential of the newly introduced gene.
- the present invention also provides recombinant retroviruses capable of immune down-regulation.
- specific down-regulation of inappropriate or unwanted immune responses such as in autoimmune or pseudo-autoimmune diseases such as chronic hepatitis, diabetes, rheumatoid arthritis, graft vs. host disease and Alzheimer's, or in transplants of heterologous tissue such as bone marrow, can be engineered using immune- suppressive viral gene products, or active portion thereof, which suppress surface expression of transplantation (MHC) antigen.
- MHC transplantation
- an "active portion" of a gene product is that fragment of the gene product which must be retained for biological activity.
- Such fragments or active domains can be readily identified by systematically removing nucleotide sequences from the protein sequence, transforming target cells with the resulting recombinant retrovirus, and determining MHC class I presentation on the surface of cells using FACS analysis or other immunological assays, such as a CTL assay. These fragments are particularly useful when the size of the sequence encoding the entire protein exceeds the capacity of the viral carrier.
- the active domain of the MHC antigen presentation inhibitor protein can be enzymatically digested and the active portion purified by biochemical methods.
- a monoclonal antibody that blocks the active portion of the protein can be used to isolate and purify the active portion of the cleaved protein (Harlow et al., Antibodies: A Laboratory Manual, Cold Springs Harbor, 1988).
- the suppression is effected by specifically inhibitin the activation of display of processed peptides in the context of self MHC molecules alon with accessory molecules such as CD8, intercellular adhesion molecule -1 (ICAM-1), ICAM 2, ICAM-3, leukocyte functional antigen-1 (LFA-1) (Altmann et al., Nature 335:521, 1989) the B7.1-.3 molecule (Freeman et al., J. Immunol.
- Components of the antigen presentation pathway include th 45 Kd MHC class I heavy chain, ⁇ 2-microglobulin, processing enzymes such as proteases accessory molecules, chaperones such as calnexin (Gaczynska, et al, Nature, 365: 264-282 1993), and transporter proteins such as PSF1, TAP1 and TAP 2 (Driscoll, et al, Nature 365: 262-263, 1993).
- recombinant retroviruses which direc the expression of a gene product or an active portion of a gene product capable of binding 2-microglobulin.
- transport of MHC class I molecules to the cell surface for antige presentation requires association with ⁇ 2-microglobulin.
- proteins that bind ⁇ 2 microglobulin and inhibit its association with MHC class I indirectly inhibit MHC class antigen presentation.
- Suitable proteins include the H301 gene product.
- the H30 gene obtained from the human cytomegalovirus (CMV) encodes a glycoprotein wit sequence homology to the ⁇ 2-microglobulin binding site on the heavy chain of the MH class I molecule (Browne et al., Nature 347:110, 1990).
- H301 binds ⁇ 2-microglobulin thereby preventing the maturation of MHC class I molecules, and renders transformed cell unrecognizable by cytotoxic T-cells, thus evading MHC class I restricted immun surveillance.
- recombinant retroviruses are provided whic direct the expression of a protein or active portion of a protein that binds to newl synthesized MHC class I molecules intracellularly. This binding prevents migration of th MHC class I molecule from the endoplasmic reticulum, resulting in the inhibition of termin glycosylation. This blocks transport of these molecules to the cell surface and prevents cel recognition and lysis by CTL.
- one of the products of the E3 gene may be use to inhibit transport of MHC class I molecules to the surface of the transformed cell.
- E3 encodes a 19 kD transmembrane glycoprotein, E3/19K, transcribed from th E3 region of the adenovirus 2 genome.
- tissu cells are transformed with a recombinant retrovirus containing the E3/19K sequence, whic upon expression produces the E3/19K protein.
- the E3/19K protein inhibits the surface expression of MHC class I surface molecules, and cells transformed by the recombinant retrovirus evade an immune response. Consequently, donor cells can be transplanted with reduced risk of graft rejection and may require only a minimal immunosuppressive regimen for the transplant patient. This allows an acceptable donor-recipient chimeric state to exist with fewer complications.
- Similar treatments may be used to treat the range of so-called autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis or chronic hepatitis B infection.
- Another alternative method of immunosuppression involves the use of antisense message, ribozyme, or other gene expression inhibitor specific for T-cell clones which are autoreactive in nature. These block the expression of the T-cell receptor of particular unwanted clones responsible for an autoimmune response.
- the anti-sense, ribozyme, or other gene may be introduced using a viral vector delivery system.
- a recombinant retrovirus that expresses a protein or an active portion thereof suspected of being capable of inhibiting MHC class I antigen presentation is transformed into a tester cell line, such as BO
- the tester cell lines with and without the sequence encoding the candidate protein are compared to stimulators and/or targets in the CTL assay. A decrease in cell lysis corresponding to the transformed tester cell indicates that the candidate protein is capable of inhibiting MHC presentation.
- This blocking action may occur intracellularly, on the cell membrane, or extracellularly.
- the blocking action of a viral or, in particular, a recombinant retrovirus carrying a gene for a blocking agent can be mediated either from inside a susceptible cell or by secreting a version of the blocking protein to locally block the pathogenic interaction.
- the two agents of interaction are the gp 120/gp 41 envelope protein and the CD4 receptor molecule.
- an appropriate blocker would be a recombinant retrovirus expressing either an HIV env analogue that blocks HI entry without causing pathogenic effects, or a CD4 receptor analogue.
- the CD4 analogu would be secreted and would function to protect neighboring cells, while the gp 120/gp 41 i secreted or produced only intracellularly so as to protect only the vector-containing cell.
- I may be advantageous to add human immunoglobulin heavy chains or other components t CD4 in order to enhance stability or complement lysis. Delivery of a recombinant retroviru encoding such a hybrid-soluble CD4 to a host results in a continuous supply of a stabl hybrid molecule.
- HTV env Vector particles leading to expression of HTV env may also be constructed. I will be evident to one skilled in the art which portions are capable of blocking viru adso ⁇ tion without overt pathogenic side effects (Willey et al., J. Virol 62:139, 1988; Fishe et al., Science 233:655, 1986).
- Another aspect of the invention involves the delivery of suppressor gene which, when deleted, mutated or not expressed in a cell type, lead to tumorigenesis in tha cell type.
- Reintroduction of the deleted gene by means of a viral vector leads to regressio of the tumor phenotype in these cells. Since malignancy can be considered to be an inhibitio of cellular terminal differentiation compared with cell growth, the delivery and expression o gene products which lead to differentiation of a tumor should also, in general, lead t regression.
- Sequences which encode the above-described nucleic acid molecules may b obtained from a variety of sources.
- plasmids which contain sequences tha encode altered cellular products may be obtained from a depository such as the America Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such a Advanced Biotechnologies (Columbia, Maryland).
- ATCC America Type Culture Collection
- ATCC No. 41000 containing a to T mutation in the 12th codon of ras
- ATCC No. 41049 containing a G to mutation in the 12th codon
- nucleic acid molecules that encode the above-described substances, a well as other nucleic acid molecules that are advantageous for use within the presen invention may be readily obtained from a variety of sources, including for exampl depositories such as the American Type Culture Collection (ATCC, Rockville, Maryland), o from commercial sources such as British Bio-Technology Limited (Cowley, Oxfor England).
- Representative examples include BBG 12 (containing the GM-CSF gene codin for the mature protein of 127 amino acids), BBG 6 (which contains sequences encodin gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATC No. 20663 (which contains sequences encoding alpha interferon), ATCC Nos.
- 31902, 3190 and 39517 which contains sequences encoding beta interferon
- ATCC No 67024 whilec contains a sequence which encodes Interleukin-lb
- ATCC Nos. 39405, 39452, 39516, 39626 and 39673 which contains sequences encoding Interleukin-2
- ATCC Nos. 59399, 59398, and 67326 which contain sequences encoding Interleukin-3
- ATCC No. 57592 which contains sequences encoding Interleukin-4
- ATCC Nos. 59394 and 59395 which contain sequences encoding Interleukin-5
- ATCC No. 67153 which contains sequences encoding Interleukin-6).
- ATCC No. 45020 contains the total genomic DNA of hepatitis B (extracted from purified Dane particles) (see Figure 3 of Blum et al., 77G 5(5): 154-158, 1989) in the Bam HI site of pBR322 (Moriarty et al., Proc. Natl Acad Sci. USA 75:2606-2610, 1981). (Note that correctable errors occur in the sequence of ATCC No. 45020.)
- cDNA sequences for use with the present invention may be obtained from cells which express or contain the sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159. See also PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed ), Stockton Press, 1989) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
- a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
- Nucleic acid molecules which are carried and/or expressed by the recombinant retroviruses described herein may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., APB DNA synthesizer model 392 (Foster City, California).
- Retroviral vectors are known to preferentially infect dividing cells. (See Miller, et al., (1990) Molec. Cell Biol. 10:4239.) There are variety of techniques that may be used to increase the number of dividing cells in target tissues and thereby enhance the efficiency of target cell infection by the retroviral vectors of the invention. For example, growth factors may be used to stimulate target tissues to enter the portion of the cell cycle in which retrovector integration can take place.
- Such growth factors and their target tissues include, but are not limited to, the following: Protein S and Gas6 acting on nervous system cells and smooth muscle cells; thrombin acting on smooth muscle cells, gastrointestinal epithelium, Uver fat storage cells, dental pulp cells, fibroblasts, endothelial cells, mesangial cells, and astrocytes; coagulation Factor Xa acting on smooth muscle cells; nerve growth factor acting on nervous system cells; CSF-1 acting on placenta and endometrium; IGF-1 acting on kidney, bone, skin, adipose tissue, airway smooth muscle cells, gastrointestinal epithelium, neural tissue, muscle, and follicular cells; insulin acting on gastrointestinal epithelium, skin, and adipose tissue; KGF acting on urothelium, mammary epithelium, skin, liver, and gastrointestinal epithelium; TGF acting on gastrointestinal epithelium, dental pulp, neural tissue, fibroblasts, connective tissue, inner ear sensory epithelium, colon
- Growth factors can also be used in combination, particularly but not limited to mixtures consisting of one or several of EGF, IGF, PDGF, FGF, or KGF.
- multiple growth factors known to stimulate cell division of particular target tissue can used in corn-nation to increase the proportion of dividing cells in the tissue.
- the actions of the above and other growth factors can also be potentiated wit substances including but not limited to dextan sulfate, heparin, and other sulfate glycos-trninoglycans, FBP, leukotrienes, prostaglandins, oleic acid, HGF activators, androgens, estrogens, ethanol, PF4, and TGF beta antagonists.
- Treatment with growth factors or the other substances described above can occur by adminsitering the substances in vivo or can also be used in other treatment modalities such as ex vivo treatment.
- Liver is an attractive organ for gene therapy because it is easily accessible via the circulation and is the source of a varitey of proteins involved in genetic disorders, including factor VTU.
- Gene therapy targeting the liver using the retroviral vectors of the invention can be performed with or without pretreatment to incude benign hype ⁇ lasia of the liver.
- Pretreatment to induce benign liver hype ⁇ lasia can be effected, for example, by treatment with hepatic growth factor (hGF) and/or transforming growth factor alpha.
- hGF hepatic growth factor
- transforming growth factor alpha See eg. Liu, et al. (1994) Hepatology 19:1521.
- liver cell growth can also be stimulated by nutritional minipulation.
- a variety of different nutritional periods can be used. For instance, a period of protein deprivation followed by consumption of a high protein meal can be used to stimulate DNA synthesis and cell division. (See Mead, et al. (1990) Cancer Res. 50:7023-7030.)
- cyclooxidase inhibitors such as indomethacin
- indomethacin is known to increase DNA synthesis and cell clearance in duodenal and jejunal mucusa (see Uribe, et al. (1992) Dig. Dis. Sci. 37:403-408).
- pretreatment with indomethacin or other non-steroidal anti inflammatory drugs can be used to increase cell proliferation in the gastric mucosa.
- prostaglandins in particular, prostraglandin E2 can be used after introduction of the gene therapy vehicle in order to increase the retention of the transduced intestinal cells.
- the present invention provides high titer recombinant retroviral preparation suitable for administration to humans.
- cell culture methods as described below are provided in order to enable the production of high titer recombinant retroviruses.
- a wide variety of methods may be utilized, including for example, the use of fermenters or bioreactors, roller bottles, cell hotels or cell factories, and hollow fiber culture.
- cells are preferably grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J. at concentrations ranging from 3 to 15 g/L microcarrier. Suitable media, and growth conditions are described by way of a representative illustration in Example 17.
- suitable conditions include those described above for bioreactors, with the exception that microcarrier beads are not utilized.
- cells are grown in 850 cm 2 roller bottles ("FALCON" Corning, Corning New York) containing DMEM media, along with 15%-20% FBS.
- the bottles are sealed to avoid contaminatior-, although "open” bottles may also be utilized under appropriate conditions (e.g., 5% CO2).
- the roller bottles are incubated at a temperature of 37°C, with a rotation speed of 0.5 ⁇ m/minute.
- Cell factories may also be utilized for the large scale cell culture and production of recombinant retroviruses.
- cell factories also termed 'bell hotels'
- cell factories typically contain 2, 10, or 40 trays, are molded from virgin polystyrene, treated to provide a Nuclon D surface, and assembled by sonic welding one to another.
- these factories have two port tubes which allow access to the chambers for adding reagents or removing culture fluid.
- a 10-layer factory provides 6000cm 2 of surface area for growing cells, roughly the equivalent of 27 T-225 flasks.
- Cell factories are available from a variety o manufactureres, including for example Nunc.
- Most cell types are capable of producing high titer vector into the media for 3-6 days, allowing for multiple harvests. Each cell type is tested to determine the optimal harvest time after seeding the culture and optimal number of harvest days.
- Cells are typically initially grown in DMEM supplemented with 2-20% FBS in roller bottles until the required number of cells for seeding a cell factory is obtained. Cells are then seeded into the factories and 2 liters of culture supernatant containing vector is harvested from each day for four days. Fresh DMEM/FBS is used to replenish the cultures.
- hollow fiber culture methods are provided for the production of recombinant retroviruses.
- high titer retroviral production using hollow fiber cultures is based on increasing viral concentration as the cells are being cultured to a high density in a reduced volume of media.
- Cells are fed nutrients and waste products are diluted using a larger volume of fresh media which circulates through the lumen of numerous capillary fibers.
- the cells are cultured on the exterior spaces of the capillary fibers in a bioreactor chamber where cell waste products are exchanged for nutrients by diffusion through 30,000 Dalton pores in the capillary fibers.
- Retroviruses which are produced from the cell lines are too large to pass through the 30,000 Dalton pore membrane, and thus concentrate in the hollow fiber bioreactor along side of the cells.
- the volume of media being cultured on the cell side is approximately 10 to 100 fold lower then volumes required for equivalent cell densities cultured in tissue culture dishes or flasks. This decrease fold in volume inversely correlates with the fold induction of titer when hollow fiber retroviral titers are compared to tissue culture dishes or flasks.
- This 10-100 fold induction in titer is seen when an individual retroviral producer cell line is amiable to hollow fiber growth conditions. To achieve maximum cell density, the individual cells must be able to grow in very close proximity and on top of each other.
- the harvest procedure in its original design, is a procedure which uses syringes to evacuate and replace culture sups to harvest the produced vector.
- This syringe procedure has an associated high risk of possible contamination, which requires a significant number manual connections and disconnection's of media flow paths.
- a convenient procedure has been devised which will reduce the risk of contaminating the cultures, increase the daily volumes which can harvested and reduce the time required to handle the culture system.
- the harvesting procedure is now performed using a batch peristaltic pump drive to deliver precise volumes of fresh media to replace equal volumes of harvest material which is then delivered through thin line tubing into a collection bottle stored at 4°C.
- the pump batch sequence is activated by a timer which can be set at specific time points and the pump can be adjusted to harvest any set volume of harvest material, twenty-four hours a day.
- the collected supernatant can then be frozen, pooled with earlier harvests, or processed as described elsewhere.
- This collection procedure can be used for any hollow fiber system including the Cellco (Rockville, Maryland), Unisyn (Tustin, CA), or Cellex (Coon Rapids, MN) systems including ceramic matrix high density culture systems.
- the present invention provides methods for concentrating and purifying recombinant retroviruses, in order to increase the purity of therapeutic preparation, as well as to increase the titer of recombinant retrovirus that may be given.
- a wide variety of methods may be utilized for increasing viral concentration and purity, including for example, precipitation of recombinant retroviruses with ammonium sulfate, polyethylene glycol ("PEG”) concentration, concentration by centrifugation (either with or without gradients such as PERCOLL, or "cushions” such as sucrose, use of concentration filters (e.g., Amicon filtration), and 2-phase separations.
- concentration filters e.g., Amicon filtration
- recombinant retroviruses may be concentrated b centrifugation, and more particularly, low speed centrifugation.
- low spee centrifugation allows concentration of recombinant retroviruses, while avoiding th difficulties associated with pelleting that accompanies high speed centrifugation (e.g., vir destruction or inactivation).
- Particularly preferred methods for concentrating viruses by lo speed centrifugation are described below in more detail in Example 15.
- recombinant retroviruses may b concentrated by an aqueous two-phase separation method.
- polymeric aqueous tw phase systems may be prepared by dissolving two different non-compatible polymers i water.
- Many pairs of water-soluble polymers may be utilized in the construction of suc two-phase systems, including for example polyethylene glycol (“PEG”) or methylcellulos and dextran or dextran sulfate (see Walter and Johansson, Anal. Biochem.
- an aqueous two-phase system may be established suitable fo purifying recombinant retroviruses. Utilizing such procedures, approximately 1.4 liters crude research grade supernatant may be reduced to a 10 mL volume, while recoverin approximately 50% of the total starting retrovirus.
- recombinant retroviruse may be prepared either from roller bottles, cell factories, or bioreactors prior t concentration.
- daily harvests of recombinant retroviruses from producer cells i preferred, followed by addition of fresh media.
- Removed media containing the recombina retrovirus may be frozen at -70°C, or more preferably, stored at 2°C to 8°C in large poole batches prior to processing.
- the recombinant retrovirus pool i first clarified through a 0.8 um filter (1.2um glass fiber pre-filter, 0.8um cellulose acetat connected in series with a 0.65 um filter (Sartorious).
- This filter arrangement provide approximately 2 square feet of filter, and allows processing of about 15-20 liters of poole material before clogging.
- a singl 0.65um cartridge (2 sq. ft.) normally suffices for volumes up to 40 liters.
- a 5 sq. ft. filter may be required.
- the filter is rinsed with buffer (150 mM NaCl, 25 mM Tris, pH 7.2-7.5). This step has allowed recoveries of recombinant retrovirus ranging from 80% to 120%.
- recombinant retroviruses are concentrated by tangential flow ultrafiltration utilizing Filtron units and Sigma Screen cassettes with a 300,000 mw cut off.
- bioreactor material containing 12% to 16% FBS
- 4 to 5 liters of material may be concentrated per cassette.
- 5-6 liters of material may be concentrated per cassette.
- cell factories containing 10% FBS 8 to 9 liters of material may be concentrated per cassette.
- Utilizing a pressure differential of 2 psi between filtrate (8 psi) and retentate (10 psi), up to 80 liters of material may be concentrated to a volume of less than 500 mL in under two hours. This process also provides a yield of about 80%.
- DNAse is added to a concentration of 50 U/mL, and recirculated at a lower pump speed with the filtrate line closed for 30 minutes. If retroviruses have been trapped within a gel layer formed during the ultrafiltration, this step will break down trapped retrovirus, and improve recovery.
- Discontinuous diafiltration is then accomplished by addition of 4 liters of additional buffer, and utilizing the same cross differential pressure set forth above. Generally, recovery after this step is approximately 70%. Concentrated material is then subjected to column chromatography on a
- Tangential flow filtration may once again be utilized in order to further reduce the volume to under 200 mL.
- the concentrated material is sterilized by filtration through a 0.2um Millipore filter (PVDF, or Sterivex).
- methods are provided for quantitating retroviral particles utilizing non-denaturing gels (e.g., 4-15% gradient polyacrylamide gels), along with methods for estimating or quantitating the resultant products such as, for example, staining with coomassie blue or silver stain, followed by densitometry scanning.
- non-denaturing gels e.g., 4-15% gradient polyacrylamide gels
- methods for estimating or quantitating the resultant products such as, for example, staining with coomassie blue or silver stain, followed by densitometry scanning.
- methods while not capable of discriminating between viable and non-viable vector particles, are advantageous because they are relatively simple and quick.
- assays are provided for titering recombinant retrovirus in a sample. Typically, such assays may be based upon presence of a selectable marker, or formation of blue colonies.
- recombinant retroviruses which do not include a gene coding for a selectable marker. Therefore, antibody and PCR assays, the latter of which is described below, may be employed in order to determine retrovirus titer.
- various primers are required. Such primers can readily be designed by those skilled in the art and will depend on the retroviral vector backbone employed and the components thereof, the particular region(s) desired to be amplified, etc. Representative examples of particular primer pairs include those specific for LTR sequences, packaging signal sequences or other regions of the retroviral backbone, include primers specific for the nucleic acid molecule (i.e., non-heterologous sequence) of interest.
- a PCR titering assay is performed by growing a known number of cells, transduced with a recombinant retrovirus on 6- ell plates for at least 16 hr. before harvest. One well per plate is sacrificed for counting. Cells from the other wells are lysed and their contents isolated. DNA is prepared using a QUIAmp DNA isolation kit (QUIAgen, Inc.,Chatsworth, CA). DNAs are resuspended in 5 x 10 cell equivalents/ ⁇ L per sample. To calculate titer, a standard curve is generated using DNA isolated from 5 x
- Negative Control ( ⁇ L) 0 12.5 25 37.5 45 47.5 50 0
- ⁇ L total volume are then initiated by adding 45.0 ⁇ L of a reaction mix containing the following components per tube to be tested: 24.5 ⁇ L water, 5 ⁇ L 10X reaction PCR buffer, 4 ⁇ L of 25 mM MgCb, 4 ⁇ L dNTPs (containing 2.5 mM of each of dATP, dGTP, dCTP, and dTTP), 5 ⁇ L of primer mix (100 ng or each primer), 0.25 ⁇ L TaqStart monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 ⁇ L TaqStart buffer (Clontech Labs, Inc.), and 0.25 ⁇ L AmpliTaq DNA polymerase (Perkin-Elmer, Inc., Norwalk, CN).
- thermocycler Just prior to aliquoting the reaction mix to the reaction tubes, 1 ⁇ L of ⁇ - 32 P dCTP (250 ⁇ Ci; 3000 C/mmol, 10 mCi/mL, Amersham Co ⁇ ., Arlington Heights, IL) is added into the reaction mix. After aliquoting 45.0 ⁇ L the reaction mix into each of the reaction tubes, the tubes are capped and placed into a thermocycler. The particular denaturation, annealing, elongation times and temperatures, and number of thermocycles will vary depending on size and nucleotide composition of the primer pair used. 20 - 25 amplification thermocycles are then performed.
- the scanning results are then downloaded and plotted on a log scale as cpm (ordinate) versus percent positive control DNA (abscissa).
- Titers infectious units mL
- Titers are calculated by multiplying the number of cells from which DNA was isolated by the percentage (converted to decimal form) determined from the standard curve based on the detected radioactivity, divided by the volume of recombinant retrovirus used to transduce the cells.
- other methods of detection such as colorimetric methods, may also be employed to label the amplified products.
- recombinant retrovirus which has been purified or concentrated as described above may be preserved by first adding a sufficient amount of a formulation buffer to the media containing the recombinant retrovirus, in order to form an aqueous suspension.
- the formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water.
- the aqueous solution may also contain one or more amino acids.
- the recombinant retrovirus can also be preserved in a purified form. More specifically, prior to the addition of the formulation buffer, the crude recombinant retrovirus described above may be clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Co ⁇ ., Nortborough, MA). Within one embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the recombinant retrovirus in a more desirable buffered solution. The diafiltrate is then passed over a Sephadex S-500 gel column and a purified recombinant retrovirus is eluted.
- a cross flow concentrating system Frtron Technology Co ⁇ ., Nortborough, MA
- the formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water.
- the aqueous solution may also contain one or more amino acids.
- the crude recombinant retrovirus can also be purified by ion exchange column chromatography. This method is described in more detail in U.S. Patent Application Serial No. 08/093,436.
- the crude recombinant retrovirus is clarified by passing it through a filter, and the filtrate loaded onto a column containing a highly sulfonated cellulose matrix.
- the recombinant retrovirus is eluted from the column in purified form by using a high salt buffer.
- the high salt buffer is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column.
- a sufficient amount of formulation buffer is then added, as discussed above, to the purified recombinant retrovirus and the aqueous suspension is either dried immediately or stored, preferably at -70°C.
- the aqueous suspension in crude or purified form can be dried by lyophilization or evaporation at ambient temperature.
- lyophilization involves the steps of cooling the aqueous suspension below the glass transition temperature or below the eutectic point temperature of the aqueous suspension, and removing water from the cooled suspension by sublimation to form a lyophilized retrovirus. Briefly, aliquots of the formulated recombinant retrovirus are placed into an Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo 12K). A multistep freeze drying procedure as described by Phillips et al.
- water is removed from the aqueous suspension at ambient temperature by evaporation.
- water is removed through spray drying (EP 520,748).
- spray drying Within the spray drying process, the aqueous suspension is delivered into a flow of preheated gas, usually air, whereupon water rapidly evaporates from droplets of the suspension.
- Spray drying apparatus are available from a number of manufacturers (e.g., Drytec, Ltd., Tonbridge, England; Lab-Plant, Ltd., Huddersfield, England). Once dehydrated, the recombinant retrovirus is stable and may be stored at -20°C to 25°C.
- the resulting moisture content of the dried or lyophilized retrovirus may be determined through use of a Karl-Fischer apparatus (EM Science AquastarTM V1B volumetric titrator, Cherry Hill, NJ), or through a gravimetric method.
- the aqueous solutions used for formulation are composed of a saccharide, high molecular weight structural additive, a buffering component, and water.
- the solution may also include one or more amino acids.
- the combination of these components act to preserve the activity of the recombinant retrovirus upon freezing and lyophilization, or drying through evaporation.
- a preferred saccharide is lactose
- other saccharides may be used, such as sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose or galactose.
- combinations of saccharides can be used, for example, lactose and mannitol, or sucrose and mannitol.
- a particularly preferred concentration of lactose is 3%-4% by weight.
- the concentration of the saccharide ranges from 1% to 12% by weight.
- the high molecular weight structural additive aids in preventing viral aggregation during freezing and provides structural support in the lyophilized or dried state.
- structural additives are considered to be of "high molecular weight" if they are greater than 5000 m.w.
- a preferred high molecular weight structural additive is human serum albumin.
- other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone.
- a particularly preferred concentration of human serum albumin is 0.1% by weight.
- the concentration of the high molecular weight structural additive ranges from 0.1% to 10% by weight.
- amino acids function to further preserve viral infectivity upon cooling and thawing of the aqueous suspension.
- amino acids function to further preserve viral infectivity during sublimation of the cooled aqueous suspension and while in the lyophilized state.
- a preferred amino acid is arginine, but other amino acids such as lysine, ornithine, serine, glycine, glutamine, asparagine, glutamic acid or aspartic acid can also be used.
- a particularly preferred arginine concentration is 0.1% by weight.
- the amino acid concentration ranges from 0.1% to 10% by weight.
- the buffering component acts to buffer the solution by maintaining a relatively constant pH.
- buffers may be used, depending on the pH range desired, preferably between 7.0 and 7.8. Suitable buffers include phosphate buffer and citrate buffer.
- a particularly preferred pH of the recombinant retrovirus formulation is 7.4, and a preferred buffer is tromethamine.
- the aqueous solution contain a neutral salt which is used to adjust the final formulated recombinant retrovirus to an appropriate iso- osmotic salt concentration.
- Suitable neutral salts include sodium chloride, potassium chloride or magnesium chloride.
- a preferred salt is sodium chloride.
- Aqueous solutions containing the desired concentration of the components described above may be prepared as concentrated stock solutions.
- a particularly preferred method of preserving recombinant retroviruses in a lyophilized state for subsequent reconstitution comprises the steps of (a) combining an infectious recombinant retrovirus with an aqueous solution to form an aqueous suspension, the aqueous suspension including 4% by weight of lactose, 0.1% by weight of human serum albumin, 0.03% or less by weight of NaCl, 0.1% by weight of arginine, and an amount of tromethamine buffer effective to provide a pH of the aqueous suspension of approximately 7.4, thereby stabilizing the infectious recombinant retrovirus; (b) cooling the suspension to a temperature of from -40°C to -45°C to form a frozen suspension; and (c) removing water from the frozen suspension by sublimation to form a lyophilized composition having less than 2% water by weight of the lyophilized composition, the composition being capable of infecting mammalian cells upon reconstitution. It is preferred that the recombinant retrovirus be replication defective and
- mannitol and lactose lyophilized recombinant retrovirus formulations were assayed for preservation of viral activity under various storage temperatures as a function of time.
- Figure 3 illustrates the results of assays of trehalose recombinant retrovirus formulations for preservation of viral activity under various storage temperatures as a function of time.
- Figure 4 depicts a comparison of the viral infectivity of frozen formulated recombinant retrovirus (-80°C) as a liquid and the viral infectivity of lyophilized recombinant retrovirus stored at -20°C.
- Mannitol formulations may lose considerable activity upon lyophilization (5-6 fold), but appear to remain stable subsequent to the lyophilization event. Although not preferable, such a loss is acceptable assuming sufficient amounts of retrovirus are present in the aqueous solution.
- the lyophilized or dehydrated retroviruses of the subject invention may be reconstituted using a variety of substances, but are preferably reconstituted using water. In certain instances, dilute salt solutions which bring the final formulation to isotonicity may also be used. In addition, it may be advantageous to use aqueous solutions containing components known to enhance the activity of the reconstituted retrovirus. Such components include cytokines, such as IL-2, polycations, such as protamine sulfate, or other components which enhance the transduction efficiency of the reconstituted retrovirus. Lyophilized or dehydrated recombinant retrovirus may be reconstituted with any convenient volume of water or the reconstituting agents noted above that allow substantial, and preferably total solubilization of the lyophilized or dehydrated sample.
- high titer recombinant retroviral particles of the present invention may be administered to a wide variety of locations including, for example, into sites such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces.
- the recombinant retrovirus may be administered intraocularly, intranasally, sublinually, orally, topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracranially, intramuscularly, or subcutaneously.
- Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel.
- compositions of the present invention include the following: Oral administration is easy and convenient, economical (no sterility required), safe (over dosage can be treated in most cases), and permits controlled release of the active ingredient of the composition (the recombinant retrovirus). Conversely, there may be local irritation such as nausea, vomiting or diarrhea, erratic abso ⁇ tion for poorly soluble drugs, and the recombinant retrovirus will be subject to "first pass effect" by hepatic metabolism and gastric acid and enzymatic degradation. Further, there can be slow onset of action, efficient plasma levels may not be reached, a patient's cooperation is required, and food can affect abso ⁇ tion.
- Preferred embodiments of the present invention include the oral administration of recombinant retroviruses that express genes encoding erythropoietin, insulin, GM-CSF cytokines, various polypeptides or peptide hormones, their agonists or antagonists, where these hormones can be derived from tissues such as the pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hemopoetic marrow, and adrenal.
- Such polypeptides can be used for induction of growth, regression of tissue, suppression of immune responses, apoptosis, gene expression, blocking receptor-ligand interaction, immune responses and can be treatment for certain anemias, diabetes, infections, high blood pressure, abnormal blood chemistry or chemistries (e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevated LDL with lowered HDL), levels of Alzheimer associated amaloid protein, bone erosion/calcium deposition, and controlling levels of various metabolites such as steroid hormones, purines, and pyrimidines.
- the recombinant retroviruses are first lyophilized, then filled into capsules and administered.
- Buccal/sublingual administration is a convenient method of administration that provides rapid onset of action of the active component(s) of the composition, and avoids first pass metabolism. Thus, there is no gastric acid or enzymatic degradation, and the abso ⁇ tion of recombinant retroviruses is feasible. There is high bioavailability, and virtually immediate cessation of treatment is possible. Conversely, such administration is limited to relatively low dosages (typically about 10-15 mg), and there can be no simultaneous eating, drinking or swallowing.
- Preferred embodiments of the present invention include the buccal/sublingual administration of recombinant retroviruses that contain genes encoding self and/or foreign MHC, or immune modulators, for the treatment of oral cancer; the treatment of Sjogren's syndrome via the buccal/sublingual administration of such recombinant retroviruses that contain IgA or IgE antisense genes; and, the treatment of gingivitis and periodontitis via the buccal/sublingual administration of IgG or cytokine antisense genes.
- Rectal administration provides a negligible first pass metabolism effect (there is a good blood/lymph vessel supply, and absorbed materials drain directly into the inferior vena cava), and the method is suitable of children, patients with emesis, and the unconscious.
- the method avoids gastric acid and enzymatic degradation, and the ionization of a composition will not change because the rectal fluid has no buffer capacity (pH 6.8; charged compositions absorb best). Conversely, there may be slow, poor or erratic abso ⁇ tion, irritation, degradation by bacterial flora, and there is a small abso ⁇ tion surface (about 0.05m 2 ). Further, lipidophilic and water soluble compounds are preferred for abso ⁇ tion by the rectal mucosa, and abso ⁇ tion enhancers (e.g., salts, EDTA, NSAID) may be necessary.
- Preferred embodiments of the present invention include the rectal administration of recombinant retroviruses that contain genes encoding colon cancer antigens, self and/or foreign MHC, or immune modulators. - 51 -
- Nasal administration avoids first pass metabolism, and gastric acid and enzymatic degradation, and is convenient.
- nasal administration is useful for recombinant retrovirus administration wherein the recombinant retrovirus is capable of expressing a polypeptide with properties as described herein. Conversely, such administration can cause local irritation, and abso ⁇ tion can be dependent upon the state of the nasal mucosa.
- Pulmonary administration also avoids first pass metabolism, and gastric acid and enzymatic degradation, and is convenient. Further, pulmonary administration permits localized actions that minimize systemic side effects and the dosage required for effectiveness, and there can be rapid onset of action and self-medication. Conversely, at times only a small portion of the administered composition reaches the brochioli/alveoli, there can be local irritation, and overdosing is possible. Further, patient cooperation and understanding is preferred, and the propellant for dosing may have toxic effects.
- Preferred embodiments of the present invention include the pulmonary administration of recombinant retroviruses that express genes encoding IgA or IgE for the treatment of conditions such as asthma, hay fever, allergic alveolitis or fibrosing alveolitis, the CFTR gene for the treatment of cystic fibrosis, and protease and collagenase inhibitors such as ⁇ -1-antitrypsin for the treatment of emphysema.
- Ophthalmic administration provides local action, and permit prolonged action where the administration is via inserts.
- Preferred embodiments of the present invention include the ophthalmic administration of recombinant retroviruses that express genes encoding IgA or IgE for the treatment of hay fever conjunctivitis or vernal and atomic conjunctivitis; and ophthalmic administration of recombinant retroviruses that contain genes encoding melanoma specific antigens (such as high molecular weight-melanoma associated antigen), self and/or foreign MHC, or immune modulators.
- Transdermal administration permits rapid cessation of treatment and prolonged action leading to good compliance. Further, local treatment is possible, and avoids first pass metabolism, and gastric acid and enzymatic degradation. Conversely, such administration may cause local irritation, is particularly susceptible to tolerance development, and is typically not preferred for highly potent compositions.
- Preferred embodiments of the present invention include the transdermal administration of recombinant retroviruses that express genes encoding IgA or IgE for the treatment of conditions such as atopic dermatitis and other skin allergies; and transdermal administration of recombinant retroviruses encoding genes encoding melanoma specific antigens (such as high molecular weight-melanoma associated antigen), self and or foreign MHC, or immune modulators.
- Vaginal administration provides local treatment and one preferred route for hormonal administration. Further, such administration avoids first pass metabolism, and gastric acid and enzymatic degradation, and is preferred for administration of compositions wherein the recombinant retroviruses express peptides.
- Preferred embodiments of the present invention include the vaginal administration of recombinant retroviruses that express genes encoding self and or foreign MHC, or immune modulators.
- Other preferred embodiments include the vaginal administration of genes encoding the components of sperm such as histone, flagellin, etc., to promote the production of sperm-specific antibodies and thereby prevent pregnancy. This effect may be reversed, and/or pregnancy in some women may be enhanced, by delivering recombinant retroviruses vectors encoding immunoglobulin antisense genes, which genes interfere with the production of sperm-specific antibodies.
- Intravesical administration permits local treatment for urogenital problems, avoiding systemic side effects and avoiding first pass metabolism, and gastric acid and enzymatic degradation. Conversely, the method requires urethral catheterization and requires a highly skilled staff.
- Preferred embodiments of the present invention include intravesical administration of recombinant retrovirus encoding antitumor genes such as a prodrug activation gene such thymidine kinase or various immunomodulatory molecules such as cytokines.
- Endoscopic retrograde cystopancreatography (goes through the mouth; does not require piercing of the skin) takes advantage of extended gastroscopy, and permits selective access to the biliary tract and the pancreatic duct. Conversely, the method requires a highly skilled staff, and is unpleasant for the patient.
- Many of the routes of administration described herein e.g., into the CSF, into bone marrow, into joints, intravenous, intra-arterial, intracranial intramuscular, subcutaneous, into various organs, intra-tumor, into the interstitial spaces, intra-peritoneal, intralymphatic, or into a capillary bed) may be accomplished simply by direct administration using a needle, catheter or related device.
- one or more dosages may be administered directly in the indicated manner: into the cerebral spinal fluid at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; into bone marrow at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; into joint(s) at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; intravenously at dosages greater than or equal to 10 , 10 , 10 or 10 cfu; intra-arterially at dosages greater than or equal to 10 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; intra-cranially at dosages greater than or equal to 10 9 , 10 or 10 cfu; intra-muscularly at dosages
- Recombinant retrovirus may be delivered to the target from outside of the body (as an outpatient procedure) or as a surgical procedure, where the vector is administered as part of a procedure with other pu ⁇ oses, or as a procedure designed expressly to administer the vector.
- Other routes and methods for administration include the non-pareneral routes as well as administration via multiple sites.
- the N2R5 construct is mutated by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression.
- This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites.
- the Pst I-Pst I mutated fragment is purified from the SK + plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-mutated 200 bp fragment.
- the plasmid pUC31 is derived from pUC19 (Stratagene, La Jolla, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Neo I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is designated pUC31 N2R5gM.
- a 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK + resulting in a construct designated N2R3".
- a 1.0 Kb Cla I-Hind III fragment is purified from this construct.
- the Cla I-Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector (Kriegler etal, Cell 35:483, 1984; St. Louis etal, PNAS 55:3150- 3154,1988), comprising a SV40 early promoter driving expression of the neomycin (neo) phosphotransferase gene, is cloned into the SK + plasmid.
- This construct is designated SK SW -neo A 1.3 Kb Cla I-BstB I gene fragment is purified from the SK + SV2-weo plasmid.
- KT-3B or KT-1 vectors are constructed by a three part ligation in which the Xho I-Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I- Hind HI fragment are inserted into the Xho I-Hind m site of pUC31/N2R5gM plasmid. This gives a vector designated as having the KT-1 backbone.
- the 1.3 Kb Cla I-BstB I neo gene fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation to yield a vector designated as having the KT-3B backbone.
- EXAMPLE 2 ORAL ADMINIS TRA ⁇ ON OF RECOMBINANT RETROVIRUSES EXPRESSING FACTOR v ⁇ i
- a retroviral backbone e.g., KT-1, lacking a selectable marker gene is employed.
- a gene encoding full length factor VHI can be obtained from a variety of sources.
- One such source is the plasmid pCIS-F8 (see EP 0 260 148), which contains a full length factor Vm cDNA whose expression is under the control of a CMV major immediate- early (CMV MIE) promoter and enhancer.
- the factor VDI cDNA contains approximately 80 bp of 5' untranslated sequence from the factor VHI gene and a 3' untranslated region of about 500 bp.
- a CMV intron sequence, or "cis" element is spanning about 280 bp, comprises a splice donor site from the CMV major immediate-early promoter about 140 bp upstream of a splice acceptor from an immunoglobulin gene.
- a plasmid, designated pJW-2 encoding a retroviral vector for expressing full length factor VQI is constructed using the KT-1 backbone from pKT-1. Briefly, in order to facilitate directional cloning of the factor VDI cDNA insert into pKT-1, the unique Xho I site is converted to a Not I site by site directed mutagenesis. The resultant plasmid vector is then opened with Not I and Cla I. pCIS-F8 is digested to completion with Cla I and Eag I, for which there are two sites, to release the fragment encoding full length factor VHI. This fragment is then ligated into the Not I/Cla I restricted vector to generate a plasmid designated pJW-2.
- a plasmid vector encoding a truncation of about 80% (approximately 370 bp) of the 3' untranslated region of the factor VHI cDNA, designated pND-5, is constructed in a pKT-1 vector as follows: As described for pJW-2, the pKT-1 vector employed has its Xho I restriction site replaced by that for Not I.
- the factor VDI insert is generated by digesting pCIS-F8 with Cla I and Xba I, the latter enzyme cutting 5' of the factor VHI stop codon. The approximately 7 kb fragment containing all but the 3' coding region of the factor VTH gene is then purified.
- pCIS-F8 is also digested with Xba I and Pst I to release a 121 bp fragment containing the gene's termination codon. This fragment is also purified and then ligated in a three way ligation with the larger fragment encoding the rest of the factor VIII gene and Cla I/Pst I restricted BLUESCRIPT® KS + plasmid (Stratagene, supra) to produce a plasmid designated pND-2.
- the unique Sma I site in pND-2 is then changed to a Cla I site by ligating Cla I linkers (New England Biolabs, Beverly, MA) under dilute conditions to the blunt ends created by a Sma I digest. After recircularization and ligation, plasmids containing two Cla I sites are identified and designated pND-3.
- the factor VDI sequence in pND-3 bounded by Cla I sites and containing the full length gene with a truncation of much of the 3' untranslated region, is cloned as follows into a plasmid backbone derived from a Not I/Cla I digest of pKT-1 (a pKT-1 derivative by cutting at the Xho I site, blunting with Klenow, and inserting a Not I linker (New England Biolabs)), which yields a 5.2 kb Not I/Cla I fragment.
- pCIS-F8 is cleaved with Eag I and Eco RV and the resulting fragment of about 4.2 kb, encoding the 5' portion of the full length factor Vm gene, is isolated.
- pND-3 is digested with Eco RV and Cla I and a 3.1 kb fragment is isolated. The two fragments containing portions of the factor VHI gene are then ligated into the Not I/Cla I digested vector backbone to produce a plasmid designated pND- 5.
- the precursor DNA for the B-deleted FV--Q is obtained from Miles Laboratory.
- This expression vector is designated p25D and has the exact backbone as pCISF ⁇ above.
- the Hpa I site at the 3' of the FVHI8 cDNA in p25D is modified to Cla-I by oligolinkers.
- An Ace I to Cla I fragment is clipped out from the modified p25D plasmid. This fragment spans the B-domain deletion and includes the entire 3' two-thirds of the cDNA.
- An Ace I to Cla I fragment is removed from the RetrovectorTM JW-2 above, and replaced with the modified B-domain deleted fragment just described. This is designated B- del-1.
- L33env, and BCenv express HIV-1 WBenv, Warner et al, AIDS Res. and Human Retrovirus 7:645, 1991
- transduced with the KT-ND5 vector, carrying the amphotropic or VSVG envelope protein are examined for the expression of factor VHI.
- Non-transduced cells are also analyzed for factor Vm expression and compared with KT-ND5 transduced cells to determine the effect of transduction on protein expression.
- Murine cell lines L33-KT-ND5, L33e ⁇ v-KT-ND5, L33ewv, L33, BC10ME, BC10ME-KT-ND5, BCenv, and BCe/_v-KT-ND5, are tested for expression of the KT-ND5 molecule.
- Cells are grown to subconfluent density and the supernatant is removed following centrifugation at 200 xg. The samples are diluted and assayed by the COATEST® Factor Vm assay (KabiVitrum Diagnostica, Molndal, Sweden).
- the assay is performed as follows: 100 ⁇ l of culture media sample is mixed with 200 ⁇ l of working buffer provided in the kit. The mixture is incubated at 37°C for 4 - 5 min., after which 100 ⁇ L of a 0.025 M CaCl 2 stock solution is added, followed by a 5 min. 37°C incubation. 200 ⁇ L of the chromogenic reagent (20 mg S-2222, 335 ⁇ g synthetic thrombin inhibitor, 1-2581, in 10 mL) is then mixed in. After a 5 min. incubation at 37°C, 100 ⁇ L of 20% acetic acid or 2% citric acid is added to stop the reaction.
- Absorbance is then measured against a blank comprising 50 mM Tris, pH 7.3, and 0.2% bovine serum albumin (BSA).
- BSA bovine serum albumin
- a standard curve based on dilutions of normal human plasma 1.0 IU factor Vm/mL is used and the assays should be performed in plastic tubes. Serum levels of factor Vm in non-hemophilic patients are in the range of 200 ng/mL.
- Non-transduced cells are analyzed to compare with KT-ND5 transduced cells and determine the effect that transduction has on expression.
- the packaging cell line, HX (WO92/05266), are seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish on day 1 with Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS). On day 2, the media is replaced with 5.0 ml fresh media 4 hours prior to transfection.
- DMEM Dulbecco's Modified Eagle Medium
- FBS fetal bovine serum
- a standard calcium phosphate-DNA co-precipitation is performed by mixing 40.0 ⁇ l 2.5 M CaC-2, 10 ⁇ g plasmid DNA, and deionized H 2 O to a total volume of 400 ⁇ l.
- 29 2 3 cells (WO 92/05266) ( these are 293 cells expressing gag and pol) are transfected with the vector DNA and the plasmid pMLP-VSVG ( or other VSVG encoding plasmids) to yield VSVG psuedotyped vector particles that are harvested and stored as described above.
- DA amphotropic cell line derived from a D17 cell line ATCC No. 183, WO 92/05266
- cells are seeded at 5.0 x 10 5 cells/10 cm tissue culture dish in 10 ml DMEM and 10% FBS, 4 ⁇ g ml polybrene (Sigma, St. Louis, MO) on day 1.
- FBS 4 ⁇ g ml polybrene
- 3.0 ml, 1.0 ml and 0.2 ml of the freshly collected virus-containing HX media is added to the cells.
- the cells are incubated with the virus overnight at 37°C.
- the media is removed and 1.0 ml DMEM, 10% FBS with 800 ⁇ g/ml G418 is added to the plate.
- a G418 resistant pool is generated over a period of a week.
- the pool of cells is dilution cloned by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/ml and adding 0.1 ml to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37°C, 10% CO 2 . Twenty-four clones are selected and expanded up to 24 well plates, 6 well plates then 10 cm plates at which time the clones are assayed for expression and the supernatants are collected and assayed for viral titer.
- the titer of the individual clones is determined by infection of HT 1080 cells, (ATCC No. CCL 121). On day 1, 5.0 x 10 5 HT1080 cells are plated on each well of a 6 well microtiter plate in 3.0 ml DMEM, 10% FBS and 4 ⁇ g ml polybrene. On day 2, the supernatant from each clone is serially diluted 10 fold and used to infect the HT1080 cells in
- cell lines are derived that produce greater than or equal to 10 6 cfu/ml in culture.
- the packaging cell line HX is transduced with vector generated from the DA vector producing cell line in the same manner as described for transduction of the DA cells from HX supernatant.
- Transduction of the DA or HX cells with vectors lacking a neo selectable marker was performed as described above. However, instead of adding G418 to the cells on day 3, the cells are cloned by limiting dilution. Titer is analyzed as described above.
- DA cells are seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% irradiated (2.5 megarads minimum) FBS. On day 2, the media is replaced with 5.0 ml fresh media 4 hours prior to transfection.
- a standard calcium phosphate-DNA coprecipitation is performed by mixing 60 ⁇ l 2.0 M CaC- 2 , 10 ⁇ g MLP-G plasmid, 10 ⁇ g KT-ND5 retroviral vector plasmid, and deionized water to a volume of 400 ⁇ l.
- DA cells are seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish in 10 ml DMEM and 10% FBS, 4 mg ml polybrene (Sigma, St. Louis, MO) on day 1.
- 10 ml DMEM 10% FBS, 4 mg ml polybrene (Sigma, St. Louis, MO)
- FBS 4 mg ml polybrene
- 2.0 ml 2.0 ml, 1.0 ml or 0.5 ml of the freshly collected and filtered G-pseudotyped virus containing supernatant is added to the cells.
- the cells are incubated with the virus overnight at 37°C.
- the medium is removed and 10 ml DMEM, 10% irradiated FBS with 800 ⁇ g/ml G418 is added to the plate. Only cells that have been transduced with the vector and contain the neo selectable marker will survive.
- a G418 resistant pool is generated over the period of 1-2 weeks.
- the pool is tested for expression and then dilution cloned by removing the cells from the plate, counting the cell suspension, diluting the cell suspension down to 10 cells/ml and adding 0.1 ml to each well (1 cell/well) of a 96-well plate. Cells are incubated for 2 weeks at 37°C, 10% CO2 Twenty-four clones are selected and expanded up to 24-well plates, then 6- well plates, and finally 10 cm plates, at which time the clones are assayed for expression and the supernatants are collected and assayed for viral titer as described above.
- the extended S + L assay determines whether replication competent, infectious virus is present in the supernatant of the cell line of interest.
- the assay is based on the empirical observation that infectious retroviruses generate foci on the indicator cell line MiCli (ATCC No. CCL 64.1).
- the MiCli cell line is derived from the MvlLu mink cell line (ATCC No. CCL 64) by transduction with Murine Sarcoma Virus (MSV). It is a non- producer, non-transformed, revertant clone containing a replication defective murine sarcoma provirus, S + , but not a replication competent murine leukemia provirus, L ⁇ .
- Infection of MiCli cells with replication competent retrovirus "activates" the MSV genome to trigger "transformation" which results in foci formation.
- Supernatant is removed from the cell line to be tested for presence of replication competent retrovirus and passed through a 0.45 ⁇ filter to remove any cells.
- MvlLu cells are seeded at 1.0 x 10 5 cells per well (one well per sample to be tested) of a 6 well plate in 2 ml DMEM, 10% FBS and 8 ⁇ g ml polybrene.
- MvlLu cells are plated in the same manner for positive and negative controls on separate 6 well plates. The cells are incubated overnight at 37°C, 10% CO 2 .
- 1.0 ml of test supernatant is added to the MvlLu cells.
- the negative control plates are incubated with 1.0 ml of media.
- the positive control consists of three dilutions (200 focus forming units (flu), 20 fiu and 2 fiu each in 1.0 ml media) of MA virus (referred to as pAM in Miller et al, Molec. and Cell Biol. 5:431, 1985) which is added to the cells in the positive control wells.
- MA virus referred to as pAM in Miller et al, Molec. and Cell Biol. 5:431, 1985
- the cells are incubated overnight.
- the media is aspirated and 3.0 ml of fresh DMEM and 10% FBS is added to the cells.
- the cells are allowed to grow to confluency and are split 1:10 on day 6 and day 10, amplifying any replication competent retrovirus.
- the media on the MvlLu cells is aspirated and 2.0 ml DMEM and 10% FBS is added to the cells.
- the Midi cells are seeded at 1.0 x 10 5 cells per well in 2.0 ml DMEM, 10% FBS and 8 ⁇ g/ml polybrene.
- the supernatant from the MvlLu cells is transferred to the corresponding well of the MiCli cells and incubated overnight at 37°C, 10% CO2.
- the media is aspirated and 3.0 ml of fresh DMEM and 10% FBS is added to the cells.
- the cells are examined for focus formation (appearing as clustered, refractile cells that overgrow the monolayer and remain attached) on the monolayer of cells.
- the test article is determined to be contaminated with replication competent retrovirus if foci appear on the MiCl. cells. Using these procedures, it can be shown that the HBV core producer cell lines are not contaminated with replication competent retroviruses.
- Mus dunni As an alternate method to test for the presence of RCR in a vector-producing cell line, producer cells are cocultivated with an equivalent number of Mus dunni (NIH NIAID Bethesda, MD) cells. Small scale cocultivations are performed by mixing of 5.0 x 10 s Mus dunni cells with 5.0 x 10 s producer cells and seeding the mixture into 10 cm plates (10 ml standard culture media/plate, 4 ⁇ g ml polybrene) at day 0. Every 3-4 days the cultures are split at a 1 : 10 ratio and 5.0 x 10 5 Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide maximum amplifcation of RCR.
- Mus dunni NIH NIAID Bethesda, MD
- culture supernatants are harvested, passed through a 0.45 ⁇ cellulose-acetate filter, and tested in the MdH marker rescue assay.
- Large scale cocultivations are performed by seeding a mixture of 1.0 x 10* Mus dunni cells and 1.0 x 10 8 producer cells into a total of twenty T-150 flasks (30 ml standard culture media/flask, 4 ⁇ g/ml polybrene). Cultures are split at a ratio of 1:10 on days 3, 6, and 13 and at a ratio of 1:20 on day 9.
- the final supernatants are harvested, filtered and a portion of each is tested in the MdH marker rescue assay.
- the MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral vector encoding the hygromycin B resistance gene (Palmer et al, PNAS 84: 1055-1059, 1987).
- the retroviral vector can be rescued from MdH cells upon infection of the cells with RCR.
- One ml of test sample is added to a well of a 6-well plate containing 10 5 MdH cells in 2 ml standard culture medium (DMEM with 10% FBS, 1% 200 mM L-glutamine, 1% non-essential amino acids) containing 4 ⁇ g/ml polybrene. Media is replaced after 24 hours with standard culture medium without polybrene.
- DMEM standard culture medium
- HT1080 cells are set up at 2 x 10 4 cells per well in six well tissue culture plates containing 2 mis standard growth media (DME + 10% FBS ).
- ND-5 FvTH retroviral vector particles from a confluent vector producing cell line are harvested as a HX-ND-5 clone. They are filtered through .45 ⁇ m syringe filters prior to testing the supernatants. (Alternatively the filtered media supernatants may be frozen at 80 in aliquots for later use.) Polybrene is added to each well such that the final concentration is 8 ⁇ g per ml. Thirty minutes later, either diluted or undiluted retroviral vector supernatant is added to duplicate wells.
- Typical volumes and dilutions are 0.5 ml per well and four or more 1:3 serial dilutions in growth media.
- two wells are transduced with the same volume of growth media only.
- the wells are refeed with 2mls of fresh media and the cells allowed to reach confluence, which may typically be about day four or five.
- the cells are again refeed with one ml per well fresh growth media. Twenty four hours later the media is harvested and filtered as above.
- the expression of vector transduced human cells for FVH1 is detected by the Coatest R assay as described above in Example 2B1. Activity is assayed relative to supernatant from the control wells by counting the cells per well from the two control wells and normalizing FVD3 expression data per lx 10 6 cells per 24 hours.
- the intestinal epithelium is an attractive site for gene delivery due to its rapidly proliferating tissue mass and the known location of stem cells in the crypts of Lieberkuhn.
- the deep location of the stem cells in the crypts and the protective role of the mucus gel layer makes the retrovirus relatively inaccessible to the tissue cells.
- the accessibility of the retroviral vector to these stem cells can be improved in animal models by the in vivo mucus removal method of Sandberg, J., et al.,( Human Gene Therapy 5:3232- 329, 1994).
- ESH 4 25 ⁇ g/ml in 1.0 M NaHCO 3 /0.5 M NaCl, pH 9.0
- ESH 4 is coupled to the ELISA plates overnight at 4°C, washed with 0.1% Tween 20 in PBS, and blocked with 1% BSA in PBS.
- the samples are applied in 0.05 M Tris-HCI/1 M NaCl/2% BSA, pH 7.5, over 4 hr at room temperature, the plates are washed, and ESH 8 (2.5 ⁇ g ml in 0.05 M Tris-HCI/1 M NaCl/2% BSA, pH 7.5,) which has been biotinylated with N- hydroxysuccinimidobiotin (Pierce, Rockford, IL.) is added for 2 hr at room temperature.
- the color reaction is performed with peroxidase-conjugated streptavidin (Boehringer Mannheim, Indianapolis, IN.) and o- phenylenediamine dihydrochloride as substrate.
- the human factor VHI:c standard from the National Institute for Biological Standards and Control, Hertfordshire, U.K.
- normal rat plasma are used as references.
- Lyophilized recombinant retrovirus containing the gene for Factor VIII expression is formulated into an enteric coated tablet or gel capsule according to known methods in the art. These are described in the following patents: US 4,853,230, EP 225, 189, AU 9,224,296, AU 9,230,801, and WO 92144,52.
- the capsule is administered orally to be targeted to the jejunum.
- expression of Factor VIII is measured in the plasma and blood by the Coatest R Factor Vm assay as described in Example 2B1.
- pUC31 is a modification of pUC19 (Stratagene, San Diego, CA) carrying additional restriction sites (Xho I, Bgl ⁇ , BssH H, and Neo I) between the Eco RI and Sac I sites of the polylinker.
- Plasmid N2R3+/- is thereby created from ligation of the SK plasmid with the 1.0 Kb 3' LTR fragment.
- the plasmids p31N2R5+/- and p31N2R3+/- are constructed from the ligation of pUC31 with the 2.8 Kb 5' LTR and packaging signal (Y) or the 1.0 Kb 3' LTR fragment, respectively.
- N2 refers to the vector source
- R refers to the fact that the fragment is an Eco RI fragment
- 5 and 3 refer to 5' or 3' LTRs
- + or - refers to the orientation of the insert (see Figures 1-6 for examples of LTR subclones).
- a 1.2 Kb Cla lEco RI 5' LTR and W fragment from N2 is subcloned into the same sites of an SK vector. This vector is designated pN2CR5.
- the 5' LTR containing a 6 bp deletion of the splice donor sequence (Yee et al, Cold Spring Harbor, Quantitative Biology, 51:1021, 1986) is subcloned as a 1.8 Kb Eco RI fragment into pUC31.
- This vector is designated p31N25D[+], Figure 6.
- HSV-1 thymidine kinase gene The coding region and transcriptional termination signals of HSV-1 thymidine kinase gene (HSVTK) are isolated as a 1.8 Kb Bgl H Pvu II fragment from plasmid 322TK (3.5 kb Bam HI fragment of HSV-1 (McKnight et al) cloned into Bam HI of pBR322 (ATCC No. 31344)) and cloned into Bgl ⁇ /Sma I-digested pUC31. This construct is designated pUCTK. For constructs which require deletion of the terminator signals, pUCTK is digested with Sma I and Bam HI and the 0.3 Kb fragment containing the (A) n signal is removed.
- the remaining coding sequences and sticky-end Bam HI overhang are reconstituted with a double-stranded oligonucleotide made from the following oligomers: 5'GAGAGATGGGGGAGGCTAACTGAG3'(SEQUENCEID.NO.1)
- pTKD A Figure 7.
- pPrTKDA Figure 8
- HSVTK promoter and coding sequence lacking an (A) signal
- pTKD A is linearized with Bgl II treated with alkaline phosphatase, and gel purified.
- a 0.8 Kg fragment contained the HSVTK transcriptional promoter is isolated as a Bam HI/Bgl II fragment from p322TK. 3. Products from (1) and (2) are ligated, transformed into bacteria, and positive clones are screened for the proper orientation of the promoter region. A resultant clone is designated pPrTKDA ( Figure 8).
- the retroviral provectors pTK-1 and pTK-3 are constructed essentially as described below.
- HSVTK coding sequences lacking transcriptional termination sequences are isolated as a 1.2 Kb Xho I/Bam HI fragment from pTKDA ( Figure 2).
- pTK-3 is constructed by linearizing TK-1 with Bam HI, filling in the 5' overhang and blunt-end ligating a 5'-filled Cla I/Cla I fragment containing the bacterial lac UV5 promoter, SV40 early promoter, plus Tn5 neo r gene obtained from pAFVXM retroviral vector (Krieger et al, Cell 39:483, 1984; St. Louis et al, PNAS 85:3150, 1988).
- Kanamycin-resistant clones are isolated and individual clones are screened for the proper orientation by restriction enzyme analysis ( Figure 9).
- constructs were used to generate infectious recombinant vector particles in conjunction with a packaging cell line, such as DA as described above.
- CT26 cells colon tumor 26, Brattain, Baylor College of Medicine, Houston, TX
- CT26 cells are transduced with G-pseudotyped TK-3 vector. Twenty-four hours after adding the viral supernatant, the CT26 cells are placed under G-418 selection (450 Ig/ml). After 10 days incubation, a G-418 selected pool is obtained and designated CT26TK Neo.
- CT26 TK Neo cells were seeded into six 10 cm plates at a density of 2.5 X 10 per plate.
- each of two other cell types CT26 and CT26 beta-gal (this cell line was transduced with a retroviral vector carrying the reporter gene ⁇ -galactosidase from E. coli.), were also seeded into six 10 cm 2 plates as controls.
- Five plates of each cell type were treated twice per day for four consecutive days with medium containing ganciclovir concentrations of 100 ⁇ g/ml, 50 ⁇ g/ml, 25 ⁇ g/ml, 12.5 ⁇ g/ml and 6.25 ⁇ g/ml.
- One plate of each cell type was left untreated. Afterwards, the cells were removed from each dish using trypsin-EDTA, resuspended in DMEM with 10% FBS and counted.
- mice In order to test whether in vivo transduction of a murine tumor could be used to treat the disease, an experiment was performed to determine the optimal concentration of ganciclovir necessary to eliminate a tumor that was transduced and selected in vitro to assure 100% transduction. Twelve groups of 3 mice each are injected with 2 X 10 CT26TK Neo cells. Six groups of mice are injected with these cells intraperitoneally (I P ) and six groups of mice are injected subcutaneously (S.C.). Two other groups of 3 mice each are injected with 2 X 10 5 unmodified CT26 cells (as a control) either I.P. or S.C..
- mice in the 125 mg Kg, 250 mg Kg and 500 mg/Kg treated groups were dead due to the toxic effects of ganciclovir.
- Mice in the 15.63 mg/Kg, 31.25 mg/Kg and 62.5 mg/Kg treated groups were treated for an additional 7 days and were able to tolerate the treatment. Tumor measurements were made for 23 days Figure 11).
- CT26TK Neo grew only slightly slower than unmodified CT26 in the absence of ganciclovir.
- Complete tumor regression was seen in the groups of mice treated with the 62.5 mg/Kg regimen. Partial tumor regression was seen in the 31.25 mg Kg treated groups. Little or no effect was seen in the 15.63 mg/Kg treated groups as compared to the 2 untreated control groups.
- AY-27 cells are rat carcinoma cells which have been induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide ( Selman, S., et al, J Urol 736:141, 1986).
- the AY-27 cells are transduced with G-pseudotyped TK-3 vector. Twenty-four hours after adding the viral supernatant, the AY-27 cells are placed under G-418 selection (450 ⁇ g ml).
- AY-27TK Neo cells are seeded into six 10 cm 2 plates at a density of 2.5 X 10 per plate.
- AY-27 and AY-27beta-ga/ are also seeded into sbc 10 cm plates as controls.
- ganciclovir concentrations 100 ⁇ g/ml, 50 ⁇ g/ml, 25 ⁇ g/ml, 12.5 ⁇ g/ml and 6.25 ⁇ g/ml.
- One plate of each cell type is left untreated. Afterwards, the cells are removed from each dish using trypsin-EDTA, resuspended in DMEM with 10% FBS and counted. The data generated can be used to determine the concentration which has the most cytotoxic effect o the AY-27, AY-27 beta-gal, or AY-27 TK Neo cells.
- Each dose regimen consists of 2 daily AM and PM I.P. injections of ganciclovir. The experiment is summarized in Table B below.
- Rats are treated for 12 days, eliminating those that die due to the higher concentrations of ganciclovir. Tumor measurements are made for 20 days, assessing tumor regression in order to determine an optimal ganciclovir concentration.
- AY-27 rat bladder carcinoma cells are transplanted into the bladders of 20 male Fischer 344 rats ( Charles River Breeding Laboratories, Portage, MD).
- tumor-bearing rats weighing between 200 to 300 grams are anesthetized, their bladders are surgically exteriorized and evacuated of urine with a 27-gauge needle.
- Viral vector particles are instilled into the bladders of 5 rats at 10 6 to 10 10 in cfu 200 to 2,000 ⁇ l of formulation buffer.
- the addition of 4 to 8 ⁇ g/ml of polybrene increases the efficiency of transduction.
- the cystotomy is repaired with 7-zero nonabsorbable suture.
- the virus is allowed to incubate in the presence of the tumor cells for 0.5 to 1.0 hour by keeping the animal anesthetized and thereby preventing voiding.
- Ten control rats receive 500 ⁇ l of formulation buffer only.
- 200 to 2,000 ⁇ l of vector can be instilled directly into the bladder by catheterization through the urethra following urine evacuation and rinsing with saline.
- the rats are placed on twice daily (AM and PM) injections of I.P. ganciclovir at the previously determined optimum dose (e g 62.5 mg/Kg body weight) for 4 to 12 days. Finally, the rats receive a single daily dose of ganciclovir until the end of the experiment (1 to 10 weeks). Whole bladders are removed and tumor growth is measured.
- a urinary (Foley) catheter is inserted through the urethra into the bladder and secured in place.
- the bladder is evacuated of urine and washed with 100 to 500 mis of sterile saline.
- Recombinant retroviruses containing the gene for thymidine kinase expression are instilled through the catheter into the bladder at 10 5 to 10 11 cfu in 10 to 500 ml of formulation buffer preferably containing 4 to 8 ⁇ g/ml of polybrene, or other enhancing excipients.
- the viral particles are allowed to incubate for 0.25 to 12 hours prior to removal of the catheter.
- ganciclovir is administered at 1 to 5mg/Kg I. V.
- the vector can be readrninistered multiple times (2 to 20), followed by ganciclovir administration. Due to the frequency of granulocytopenia and thrombocytopenia in patients receiving ganciclovir, it is recommended that neutrophil and platelet counts be performed every two days during the dosing of the drug. Tumor regression is monitored by x-ray and/or biopsy and the treatment repeated if required.
- the KT-ND5 viral supernatant in formulation buffer with 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipient, is nebulized using a DeVilbiss #15 Atomizer (DeVilbiss Health Care Division, Somerset, PA ) designed to produce 0.3 to 0.5 ⁇ m particles (Rousculp, M. , Human Gene Therapy 3:411-411, 1992.)
- the aerosol produced by this nebulizer uses compressed air in a laminar flow hood. The mist is directed into a polypropylene tube, and the condensed vapor, as well as the control viral supernatant, is resterilized by 0.22 ⁇ m filtration. Retroviral particles pass through this filter without any significant loss of functional activity.
- Rats are anesthetized and the trachea is exposed by anterior midline incision.
- Recombinant retroviral particles expressing the factor VD3 gene product are diluted in 300 ⁇ l of formulation buffer, with or without 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipient, at 10 5 to 10 10 cfu/ml and instilled into the trachea though a small gauge needle.
- Control animals are administered 300 ⁇ l of formulation buffer only. The incision is sutured and the rats are allowed to recover. Two to fourteen days following viral instillation, blood is drawn from the tail vein and examined for factor VHI production as described in Example 2Hi. 2.
- the recombinant retrovirus is administered using a DeVilbiss #15 Atomizer (as described above) for 2 to 60 minutes. Two to fourteen days following administration of the recombinant retoviruses, expression of Factor Vm is measured in the blood and plasma by the Coatest R Factor ⁇ TH assay ( as described in Example 2B1) .
- Cottontail rabbit papillomavirus provides an animal model for the highly oncogenic human papillomavirus (HPV).
- Papillomas can be induced in the cottontail rabbit and virus infection leads to three different outcomes in the rabbit (Lin, Y. et el , J Virol 67:382, 1993). First, papillomas appear and persist for the lifetime of the rabbit; second, papillomas spontaneously regress 2 to 3 months after infection; and third, papillomas progress to carcinomas after 8 to 15 months.
- control animals are administered 25 to 100 ⁇ l of formulation buffer only.
- the rabbits are placed on twice daily (AM and PM) injections of I.P. ganciclovir at the previously determined optimum dose (e.g. 62.5 mg Kg body weight) for 4 to 12 days.
- the rabbits receive a single daily dose of ganciclovir until the end of the experiment (1 to 10 weeks). Papilloma regression is visually monitored for 2 to 14 days.
- the clinical cutaneous lesions that result from the human papillomavirus include common warts, filiform warts, plantar warts, and anogenital warts (reviewed in Cobb, M., et al J. Am. Aca Derm. 22:547, 1990).
- patients are divided into two groups.
- 100 to 500 ⁇ l of recombinant retrovirus particles at a concentration of 10 9 cfu/ml in a formulated ointment, preferably containing 4 to 8 ⁇ g/ml of polybrene or other enhancing excipients are applied to the warts using a transdermal delivery system (TDS).
- TDS transdermal delivery system
- TDS Transdermal delivery systems
- the major components of TDS are a controlled release device composed of polymers, the drug to be administered, excipients, and enhancers, and a fastening system to fix the device to the skin.
- a number of polymers have been described which include gelatin, gum arabic, paraffin waxes, and cellulose acetate phthalate (Sogibayasi, K., et al, J. Controlled Release, 29:177-185, 1994).
- the second group receives 100 to 500 ⁇ l of vector particle in formulation buffer only. The areas are covered and the viral particles are allowed to incubate for 0.25 to 12 hours prior to removal of the TDS.
- ganciclovir is administered at 1 to 5mg/Kg I. V., preferably at a constant rate over 1 hour) every 12 hours for 2 to 21 days.
- the vector can be readministered multiple times (2 to 20), followed by ganciclovir administration. Due to the frequency of granulocytopenia and thrombocytopenia in patients receiving ganciclovir, it is recommended that neutrophil and platelet counts be performed every two days during the dosing of the drug. The regression of the wart is visually monitored for 2 to 14 days.
- the opalescent viral band is removed and adjusted to 1.34 g/cm 3 with CsCl and further centrifuged in a Ti 50 rotor for 16-20 hours at 30,000 ⁇ m.
- the visible viral band in the middle of the gradient is removed and stored at 4°C until purification of adeno viral DNA.
- the adenovirus band is incubated with protease for 1 hour at 37°C to digest proteins. After centrifugation at 7,800 xg for 10 minutes at 4°C, the particles are solubilized in 5% SDS at room temperature for 30 minutes before being extracted with equal volumes of phenol. The upper aqueous phase is removed, re-extracted with phenol, extracted three times with ether, and dialyzed in Tris buffer for 24 hours. The viral Ad2 DNA is precipitated in ethanol, washed in ethanol, and resuspended in Tris-EDTA buffer (pH 8.1). Approximately 0.5 mg of viral Ad2 DNA is isolated from virus produced in 1.0 L of cells.
- the viral Ad2 DNA is digested with EcoR I and separated by electrophoresis on a 1% agarose gel.
- the resulting 2.7 Kb Ad2 EcoR I D fragments, located in the Ad2 coordinate region 75.9 to 83.4, containing the E3/19K gene are eluted by electrophoresis, phenol extracted, ethanol precipitated, and dissolved in Tris-EDTA (pH 8.1).
- the E3/19K gene is cloned into the EcoR I site of PUC1813.
- PUC1813 is prepared as essentially described by Kay et al (Nucleic Acids Research 75:2778, 1987) and Gray et al. (PNAS 50:5842, 1983).
- the E3/19K is retrieved by EcoR I digestion and the isolated fragment is cloned into the EcoR I site of phosphatase-treated pSP73 plasmid. This construct is designated SP-E3/19K.
- the orientation of the SP-E3/19K cDNA is verified by using appropriate restriction enzyme digestion and DNA sequencing.
- the 5' end of the cDNA is adjacent to the Xho I site of the pSP73 polylinker and the 3' end adjacent to the Cla I site.
- the Xho I-Cla I fragment containing the E3/19K cDNA in either sense or antisense orientation is retrieved from the SP-E3/19K construct and cloned into the Xho I-Cla I site of the KT-3BB retroviral. This construct is designated KT- 3B/E3/19K.
- Ad2 DNA E3/19K gene including the amino teiminal signal sequence, followed by the intraluminal domain and caiboxy terminal cytoplasmic tail which allow the
- E3/19K protein to embed itself in the endoplasmic reticulum (ER) is located between viral nucleotides 28,812 and 29,288. Isolation of the Ad2 E3/19K gene from the viral genomic
- DNA is accomplished by PCR amplification, with the primer pair shown below:
- the forward primer corresponds to the Ad2 nucleotide sequences 28,812 to 28,835. (SEQUENCE ID No. 3)
- the reverse primer corresponds to the Ad2 nucleotide sequences 29,241 to 29,213. (SEQUENCE ID No. 4)
- both primers contain a five nucleotide "buffer sequence" at their 5' ends for efficient enzyme digestion of the PCT amplicon products.
- This sequence in the forward primer is followed by the Xho I recognition site and by the Cla I recognition site in the reverse primer.
- the E3/19K gene is flanked by Xho I and Cla I recognition sites.
- Amplification of the E3/19K gene from Ad2 DNA is accomplished with the following PCR cycle protocol:
- the E3/19K gene from the SP-E3/19K construct is removed and isolated by 1% agarose/TBE gel electrophoresis.
- the Xho I-Cla I E3/19K fragment is then ligated into the KT-3B retroviral backbone.
- This construct is designated KT-3B/E3/19K. It is amplified by transforming E. coli , DH5 alpha bacterial strain (Bethesda Research Labs, Gaithersburg, Maryland) with the KT-3B/E3/19K construct. Specifically, the bacteria is transformed with 1-100 ng of ligation reaction mixture DNA. The transformed bacterial cells are plated on LB plates containing ampicillin.
- the plates are incubated overnight at 37°C, bacterial colonies are selected and DNA prepared from them.
- the DNA is digested with Xho I and Cla I.
- the expected endonuclease restriction cleavage fragment sizes for plasmids containing the E3/19K gene are 780 and 1,300 bp.
- 293 2-3 cells (a cell line derived from 293 cells ATCC No. CRL 1573, (WO 92/05266) 5.0 x 10 s cells are seeded at approximately 50% confluence on a 6 cm tissue culture dish. The following day, the media is replaced with 4 ml fresh media 4 hours prior to transfection.
- a standard calcium phosphate-DNA coprecipitation is performed by mixing 10.0 ⁇ g of KT-3B/E3/19K plasmid and 10.0 ⁇ g MLP G plasmid with a 2M CaC solution, adding a lx HEPES buffered saline solution, pH 6.9, and incubating for 15 minutes at room temperature.
- the calcium phosphate-DNA coprecipitate is transferred to the 293 2-3 cells, which are then incubated overnight at 37°C, 5% CO . The following morning, the cells are rinsed three times in lx PBS, pH 7.0. Fresh media is added to the cells, followed by overnight incubation at 37°C, 10% CO 2 . The following day, the media is collected off the cells and passed through a 0.45 ⁇ filter. This supernatant is used to transduce packaging and tumor cell lines. Transient vector supernatant for other vectors are generated in a similar fashion.
- Packaging cell line transduction is performed as described in Example 2C.
- the following adherent human and murine cell lines are seeded at 5 x 10 s cells 10 cm dish with 4 ⁇ g ml polybrene: HT 1080, Hela, and BC10ME.
- the following day 1.0 ml of filtered supernatant from the DA E3/19K pool is added to each of the cell culture plates.
- 800 ⁇ g/ml G418 is added to the media of all cell cultures. The cultures are maintained until selection is complete and sufficient cell numbers are generated to test for gene expression.
- the transduced cell lines are designated HT 1080-E3/19K, Hela- E3/19K and BC10ME-E3/19K, respectively.
- EBV transformed cell lines are transduced by co-cultivation with irradiated producer cell line, such as DA-E3/19K.
- irradiated producer line cells are plated at 5.0 x 10 5 cells/6 cm dish in growth media containing 4 ⁇ g/ml polybrene. After the cells have been allowed to attach for 2-24 hours, 10 6 suspension cells are added. After 2-3 days, the suspension cells are removed, pelleted by centrifugation, resuspended in growth media containing lmg/ml G418, and seeded in 10 wells of a round bottom 96 well plate. The cultures were expanded to 24 well plates, then to T-25 flasks.
- Radio-immuno precipitation assay lysates are made from selected cultures for analysis of E3/19K expression.
- RIP A lysates are prepared from confluent plates of cells. Specifically, the media is first aspirated off the cells. Depending upon the size of the culture plate containing the cells, a volume of 100 to 500 ml ice cold RIP A lysis buffer (10 mM Tris, pH 7.4; 1% Nonidet P40; 0.1% SDS; 150 mM NaCl) is added to the cells. Cells are removed from plates using a micropipet and the mixture is transferred to a microfuge tube. The tube is centrifuged for 5 minutes to precipitate cellular debris and the supernatant is transferred to another tube.
- the supernatants are electrophoresed on a 10% SDS-polyacrylamide gel and the protein bands are transferred to an Immobilon membrane in CAPS buffer (10 mM CAPS (Aldrich, Milwaukee, WI) pH 11.0; 10% methanol) at 10 to 60 volts for 2 to 18 hours.
- CAPS buffer 10 mM CAPS (Aldrich, Milwaukee, WI) pH 11.0; 10% methanol
- the membrane is transferred from the CAPS buffer to 5% Blotto (5% nonfat dry milk; 50 mM Tris, pH 7.4; 150 mM NaCl; 0.02% Na azide, and 0.05% Tween 20) and probed with a mouse monoclonal antibody to E3/19K (Severinsson et al, J. Cell. Biol. 707:540-547, 1985).
- Antibody binding to the membrane is detected by the use of 125 I-Protein A. 2. FACS Analysis of KT3B-E3/19k-Vector Transduced Cells to Demonstrate Decreased Levels of Class I Expression Compared to Non-Transduced Cells.
- Cell lines transduced with the KT3b-E3/19K-vector are examined for MHC class I molecule expression by FACS analysis. Non-transduced cells are also analyzed for MHC class I molecule expression and compared with E3/19K transduced cells to determine the effect of transduction on MHC class I molecule expression.
- Murine cell lines BC10ME, BC10ME-E3/19K, P815 (ATCC No. TIB 64), and P815-E3/19K, are tested for expression of the H-2D* 1 molecule on the cell surface.
- Cells grown to subconfluent density are removed from culture dishes by treatment with Versene and washed two times with cold (4°C) PBS plus 1% BSA and 0.02% Na-azide (wash buffer) by centrifugation at 200g. Two million cells are placed in microfuge tubes and pelleted in a microfuge at 200g before removing the supernatant.
- Cell pellets are resuspended with the H- 2D d -specific Mab 34-2-12s (50ml of a 1:100 dilution of purified antibody, ATCC No. HB 87) and incubated for 30 min at 4°C with occasional mixing.
- Antibody labeled cells are washed two times with 1 ml of wash buffer (4°C) prior to removing the supernatant.
- Cells are resuspended with a biotinylated goat anti-mouse kappa light chain Mab (50ml, of a 1 : 100 dilution of purified antibody) (Amersham, Arlington Height, IL) and incubated for 30 min at 4°C.
- Rats are anesthetized and one eye is instilled with 5 to 100 ⁇ l of recombinant retroviral particles at a concentration of 10 5 to 10 10 cfu ml in formulation buffer, with or without 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipient,.
- Five to one hundred ⁇ l of solution containing formulation buffer only is added to the other eye to be used as a control.
- the solution is allowed to incubate for 1 hour before each eye is rinsed 3 times with 100 ⁇ l of saline .
- Two to seven days following the treatment the rat is sacrificed, the cornea is removed, and homogenized in 2 ml ice cold RIP A lysis buffer. Expression of E3/19K is detected by Western blot analysis as described in Example 6E1. 2. Hvr ⁇ r ⁇ n Administration Pro ocol
- the cornea may be incubated for 1 hour in 1 ml of retroviral vector particles at a concentration of 10 5 to 10 10 cfu/ml in formulation buffer, with or without 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipient, just prior to surgical attachment.
- the progress of the transplant is monitored by visually observing tissue viability.
- the nasal route has been shown to be effective for the administration of a number of molecules due to the extensive network of capillaries located under the nasal mucosa. This facilitates effective systemic abso ⁇ tion and when the drug is administered with abso ⁇ tion promoters, abso ⁇ tion occurs rapidly with high bioavailability (review in
- One group of six Fischer-344 rats are used for nasal administration of the retroviral vector particles for Factor Vm.
- One to fifty ⁇ l of retroviral vector particles at 10 9 cfu/ml in formulation buffer, with or without 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipients are applied with a pipette inserted about 3 to 5 mm into each nostril.
- Another group is administered formulation buffer without vector in the same manner. Blood samples are collected from the jugular or tail vein 1 to 14 days later and assayed for factor Vm production as described in Example 2Hi.
- the metere-dose nebulizer can deliver a predetermined volume of the formulation to the nasal cavity.
- Two groups of patients are used in this study.
- One group of patients receives 100 to 500 ⁇ l of retroviral vector particles at 10 9 cfu ml in formulation buffer, with or without 4 to 8 ⁇ g/ml of polybrene or other transduction enhancing excipients, applied to each nostril via nasal spray or nasal drops.
- Another group receives formulation buffer only applied in the same manner. Blood samples are collected 1 to 14 days later and assayed for factor Vm production as described in Example 2B1.
- Vector pDHF828 containing the full-length human growth hormone gene is constructed essentially as follows. Briefly, plasmid pDHF811, was constructed by removing the Xhol- Clal fragment of the KT-1 retroviral vector described above, and inserting the following oligonucleotide linkers by ligation of the cohesive ends:
- the linkers were annealed at 65°C for 20 minutes, 42°C for 20 minutes, 37°C for 20 minutes, and room temperature for 2 hours.
- the concentrations of both oligonucleotides was 18mM and the salt concentration was 100 mM NaCl.
- 50ml of 1.8 mM annealed linker was digested with Clal overnight to generate Clal ends.
- 3nM of KT-1 Xhol - Clal fragment was mixed with 90nM of linker, and the - 80 -
- Plasmid chGH 800 containing the full length cDNA of the hGH gene (Martial, R.A. et al., Science 205:602, 1979) was digested with Hind m, blunt-ended with the Klenow fragment enzyme, and cloned into the Srfl site of pDHF ⁇ ll. The resultant plasmid was designated pDHF828.
- the pDHF828 plasmid was then introduced into the HX packaging cell, using standard procedures and assayed using the HGH Chemiluminescence Kit (HGH 100T) ( Nichols Institute, San Juan Capistrano, CA.), according to a preferred modification of the kit protocol.
- HGH 100T HGH Chemiluminescence Kit
- Test samples were centrifuged for 5' at top speed in a microfuge before using them in order to remove fibrin and other debris. All samples were measured in quadruplicate, including the standards.
- the incubations are performed in 12 xl7 polypropylene tubes that have been stored in the dark.
- HX/HGH retrovector producing cell lines were generated with titers of 4.8 x 10 cfu/ml. Introduction of the plasmid into DX packaging cells resulted
- Crude recombinant retrovirus is obtained from a Celligan bioreactor (New Brunswick, New Brunswick, NJ) containing DA cells transformed with the recombinant retrovirus bound to the beads of the bioreactor matrix.
- the cells release the recombinant retrovirus into the growth media that is passed over the cells in a continuous flow process.
- the media exiting the bioreactor is collected and passed initially through a 0.8 micron filter then through a 0.65 micron filter to clarify the crude recombinant retrovirus.
- the filtrate is concentrated utilizing a cross flow concentrating system (Filtron, Boston, MA). Approximately 50 Units of DNase (Intergen, New York, NY) per ml of concentrate is added to digest exogenous DNA.
- the digest is diafiltrated using the same cross flow system to 150 mM NaCl, 25 mM tromethamine, pH 7.2.
- the diafiltrate is loaded onto a Sephadex S-500 gel column (Pharmacia, Piscataway, NJ), equilibrated in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
- the purified recombinant retrovirus is eluted from the Sephadex S- 500 gel column in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
- the formulation buffer containing lactose was prepared at a 2X concentrated stock solution.
- the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2 mg/ml arginine, 10 mg/ml HSA, and 100 mg/ml lactose in a final volume of 100 mis at a pH 7.4.
- the purified recombinant retrovirus is formulated by adding one part 2X lactose formulation buffer to one part S-500 purified recombinant retrovirus.
- the formulated recombinant retrovirus can be stored at -70°C to -80°C or dried.
- the formulated retrovirus is lyophilized in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer (Edwards High Vacuum, Tonawanda, NY).
- a Supermodulyo 12K freeze dryer Edwards High Vacuum, Tonawanda, NY.
- the lyophilized recombinant retrovirus is reconstituted with 1.0 ml water.
- the infectivity of the reconstituted recombinant retrovirus is determined by a titer activity assay.
- the assay is conducted on HT 1080 fibroblasts or 3T3 mouse fibroblast cell line (ATCC No. CCL 163). Specifically, 1 x 10 5 cells are plated onto 6 cm plates and incubated overnight at 37°C, 10% CO 2 . Ten microliters of a dilution series of reconstituted recombinant retroviruses are added to the cells in the presence of 4 mg/mL polybrene (Sigma, St. Louis, MO) and incubated overnight at 37°C, 10% CO 2 .
- polybrene Sigma, St. Louis, MO
- cells are selected for neomycin resistance in G418 containing media and incubated for 5 days at 37°C, 10% CO2. Following initial selection, the cells are re-fed with fresh media containing G418 and incubated for 5-6 days. After final selection, the cells are stained with Commassie blue for colony detection. The titer of the sample is determined from the number of colonies, the dilution, and the volume used.
- Figure 15 demonstrates that storage in lyophilized form at -20°C to refrigerator temperatures retains similar viral activity as a recombinant retrovirus stored in liquid at -80 to -20°C permitting less stringent temperature control during storage.
- the recombinant retrovirus utilized in this example was purified as described in Example 9A.
- the formulation buffer containing mannitol was prepared as a 2X concentrated stock solution.
- the formulation buffer contains 25 mM tromethamine, 35 mM NaCl, 2 mg/ml arginine, 10 mg ml HSA and 80 mg/ml mannitol at a final volume of 100 mis at a pH 7.4.
- the purified recombinant retrovirus is formulated by adding one part mannitol formulation buffer to one part S-500 purified recombinant retrovirus.
- the formulated recombinant retrovirus can be stored at this stage at -70°C to -80°C or dried.
- the formulated retrovirus is dried in an Edwards Refrigerated Chamber
- formulated liquid product was stored at both - 80°C and at -20°C in cycling freezers.
- the viral infectivity of these samples were compared to the viral infectivity of lyophilized samples, Figure 16
- the lyophilized samples were stored at -20°C, refrigerator temperature and room temperature. Activity of the samples upon reconstitution are determined using the titer assay described in Example 9A.
- Figure 16 demonstrates that storage in lyophilized form at -20°C to refrigerator temperature retains significant viral activity as compared to recombinant retrovirus stored in liquid at -80°C or -20°C, permitting less stringent temperature control during storage.
- the recombinant retrovirus utilized in this example was purified as described in Example 9A.
- the formulation buffer containing trehalose was prepared as a 2X concentrated stock solution.
- the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2.0 mg/ml arginine, 10.0 mg/ml HSA and 100 mg/ml trehalose at a final volume of 100 mis at a pH 7.2.
- the purified recombinant retrovirus is formulated by adding one part trehalose formulation buffer to one part S-500 purified recombinant retrovirus.
- the formulated recombinant retrovirus can be stored at this stage at -70°C to -80°C or dried.
- the formulated retrovirus is dried in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer.
- the vials are stoppered under a vacuum following nitrogen gas bleeding to 700 mbar. Upon removal, vials are crimped with aluminum seals.
- Example 9A Activity of the samples upon reconstitution are determined using the titer assay as described in Example 9A.
- Figure 17 demonstrates that storage in lyophilized form at -20°C to refrigerator temperature retains similar viral activity as compared to recombinant retrovirus stored in liquid at -80°C to -20°C permitting less stringent temperature control during storage.
- Viral infectivity of liquid formulated recombinant retrovirus samples stored at -80°C was compared to viral infectivity of lyophilized formulated recombinant retrovirus stored at -20°C. Initially, a bulk of recombinant retrovirus was received and formulated in four different ways as shown below. The formulated recombinant retrovirus was then frozen in bulk for 1.5 months subsequent to being quick thawed and freeze dried. Positive controls were stored at -80°C for comparison with lyophilized samples which were stored at -20°C after freeze-drying. The formulations are listed below:
- the y-axis on each of the 4 graphs (A, B, C, D) represent the normalized titer.
- t 0
- a titer value was established for both the -80°C liquid sample and the -20°C lyophilized sample.
- the titer obtained was divided by the zero time point titer value and the % of original entered onto the graph.
- the data demonstrates that post-lyphilization activity is maintained in the lyophilized sample (stored at -20°C) relative to the liquid sample (stored at -80°C).
- the formulated lyophilized recombinant retrovirus was stored in a -20°C freezer (a frost-free cycling freezer). Comparison to the formulated liquid recombinant retrovirus stored at -80°C indicates the lyophilized form permits less stringent control of storage conditions.
- Crude and purified solutions of recombinant retrovirus particles may be separated on gradient polyacrylamide gels utilizing, for example, the PHASTGEL system (Pharmacia Biotech). Briefly, samples are place on 4-15% polyacrylamide gels without pretreatment and electrophoresed for 35 minutes at 250V. The gels are then removed and stained with coomassie blue in order to detect virus and other protein components. The gels are then scanned by laser densitometry in order to determine the content of virus and other components.
- Virus bands may be identified by their relative molecular weight and by reverse transcriptase activity (RT).
- the pu ⁇ ose of this assay is to quantify the activity of reverse transcriptase (RT), an enzyme exclusively associated with all retroviruses.
- RT reverse transcriptase
- the relative amount of retrovirus in a sample can be determined by measuring the activity of this enzyme in a given preparation.
- moloney murine leukemia virus reverse transcriptase (Pharmacia, Newark, NJ) is diluted to a concentration of 1 ⁇ g/ml by addition of lx Tris/EDTA buffer solution containing 10 mM Tris-HCl and ImM EDTA, pH 8.0.
- A. Packaging Cell Line Preparation and Transduction Four different packaging cell lines were used to package pCB ⁇ -gal into infectious virions. Two cell lines were derived from D17 dog cells ("D") (ATCC CCL 183), and two were derived from the human embryonic kidney cell line 293 ("2") (ATCC CRL 1573). In the case of both dog and human packaging cells, one cell line expressed the amphotropic envelope from the 4070A virus (i.e., DA and 2 A, respectively) and the other expressed the xenotropic envelope from the NZB9-1 virus (i.e., DX and 2X, respectively). All packaging cells were grown in DMEM media (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat inactivated fetal bovine serum (Hyclone, Salt Lake City, TJT).
- Each of the four packaging cell lines was transduced with a G-pseudotyped pCB ⁇ -gal (see Burns et al. (1993), Proc. Nat'l Acad Sci. USA, vol. 90, pp:8033-8037 for a pseudotyping procedure).
- the selected cell pools were dilution cloned (except for the amphotropic vector producing human cell line which was maintained as a non-clonal pool).
- Vector producing cell lines producing the highest titer on HT1080 cells (ATCC No. CCL 121) were selected. Titers were determined approximately 2 days after transduction by G418 colony forming units or by X-gal staining of the monolayers. (See Current Protocols in Molecular Biology, Ed. Ausubel et al., for more details on the titering assays.)
- Retrovirus containing supernatants were collected from each of the confluent monolayers of the individual amphotropic envelope (DA and 2A) or xenotropic envelope (DX or 2X) vector producing cell lines and filtered through a 0.45 ⁇ m filter prior to aliquoting and storage at -70°C.
- DA and 2A amphotropic envelope
- DX or 2X xenotropic envelope
- Serum inactivation titer assays were performed as follows: Serum was drawn from at least two different human volunteers, chimpanzees, baboons, and macaque monkeys. Approximately 20-70 mL of blood was collected from each donor into serum separating tubes (Becton Dickinson, Los Angeles, CA). Blood was allowed to clot for 20-30 minutes at room temperature, after which time the samples were centrifuged at 2000 x g for 10 minutes at 4°C. Serum was frozen in approximately 1.1 mL aliquots and stored at -80°C.
- HFB hollow fiber bioreactor
- the growth media should be what ever the cell line has been adapted to. All liquids in the HFB when originally shipped should be aspirated and replaced with the complete growth media.
- the cells When seeding the bioreactor, the cells should not have been split more than 48 hours earlier and should be in log growth phase at the time of harvest for the seeding of the HFB. The cells are harvested by trypsinazation and pelleted by centrifugation.
- the cell pellet is then resuspended in 4 mL of 25% pre-conditioned media and delivered to the extra-capillary space by syringe using the side syringe ports found on the HFB. After seeding the HFB, allow the cells to adhere for 20 to 30 minutes before starting the circulation pump. During this time replace the media used to condition the HFB with 100-200 mL using 25% pre-conditioned media.
- the circulation feed pump is initiated with the starting flow rate set at 25 mL/min. (setting 5 with 2 long pump pins). After 1 hour from the time of switching the pump on, a one mL sample of media is collected in order to record the initial levels of lactate and ammonia.
- the original Cellco supplied reservoir feeding cap is exchanged for the larger reservoir cap (Unisyn-vender part #240820) adapted for the Cellco system with the addition of tubing and male luer lock fittings.
- This reservoir cap will accommodate the large 2 Liter Corning bottles. (To avoid the exchange of the reservoir caps during a culture run, initiate the culture run with the larger reservoir cap which can also support smaller bottle sizes.)
- daily lactate readings are assayed and recorded, one can calculate the daily levels of lactate production of the culture in order to determine when the culture reaches maximum cell density when the rate of lactate decreases and levels off.
- Figure 22 is a representative graph of data generated over a two week period of the vector producer cell line 2X- ⁇ -GALi 7 - ⁇ -j..
- one HFB was seeded with 1.3 x 10 7 cells (to represent the low seed culture) the other seeded with 1.6 x 10 8 cells.
- the cell line (2x- ⁇ -GAL ⁇ . 14 ) was able to initiate a good hollow fiber run under low and high seeding conditions. Being able to incubate the HFB with fewer cells, is only convenient for reducing the effort required for generating the number of cells required to start a culture. However a low seed start also extends the time it takes to reach optimal cell densities which usually yield the highest titers.
- the cell line used adapted very well to hollow fiber cultures which eventually required daily media changes of 500 mL per day.
- the graphs indicate that the culture was no longer allowed to expand based on the plateauing of daily production of lactate. Another indicator of the health of the cell culture was the drop in peak titer production which also correlated with the daily exposure of high levels of lactate (See Figure 23). These findings would indicate that optimal titers can be correlated with the maximum cell densities and the relative health of the culture.
- ⁇ -gal titers for the above experiment were determined from frozen samples and were titered on 293 cells assayed 48 or 72 hours after transduction. The transduced cells were stained for ⁇ GAL activity and individual cells counted on a hemocytometer giving a titer based on the number of blue cells /mL (BCT/mL). As shown in Figure 23, optimum titers were obtained on day 7 of the high seed culture at 1.8 xlO 8 BCT/mL from a 72 hour blue cell titer on 293 cells.
- Previous flat stock cultures of 2X b-GAL ⁇ - 14 cultures have been titered using 48 hour blue cell titers on HT1080 cells and have been calculated to be 5 xlO 6 BCT/mL. If one uses the values obtained from 48 hour blue cell titers, the increase in titer by using hollow fiber systems is ten fold higher than crude supernatants obtained from tissue culture dishes or flasks. These maximum titers were reached prior to hitting the daily 20 mmol L toxic levels lactate which appeared to reduce the titer produced the following week.
- Crude supernatants can be harvested every 9 hours with out any loss of titer (See Figure 2). It is predicted that 3 harvests per day can be achieved with minimum loss of titers. In addition, continuous hollow fiber cultures can be maintained for several weeks. When titers were compared between the low and the high seed culture, there was little differences by day 11 between the two seed cultures averaging 4 x 10 7 BCT/mL.
- the bottom layer (approximately 20 mL) is carefully eluted, and the inte ⁇ hase (approximately 1 mL) is collected in a 15 mL conical
- the FALCON tube The inte ⁇ hase containing vector is diluted to 10 mL by addition of PBS, and incubated at 37°C in order to bring the solution to room temperature and destabilize the micelles.
- KC1 is added to a final concentration 0.4 M, and mixed well. The tube is then placed on ice for ten minutes, and spun for 2 minutes at 2,000 ⁇ ra in a bench-top centrifuge. The supernatant is removed and filtered through a 0.45 um syringe filter. The other half of the inte ⁇ hase containing vector is separated by S-500
- 1.4 liters of crude research grade supernatant containing recombinant retroviral particles may be reduced to a 10 mL volume, with approximately 50% (+/-20%) being recovered when KC1 separation is utilized as the final step.
- S-500 chromatography is utilized as the final step, only about 10% of the initial recombinant retroviral particles are recovered in a 14 mL.
- the vector-containing solution may be further subjected to concentration utilizing an MY- membrane Amicon filter, thereby reducing the volume from 10 to 14 mL, down to less than 1 mL.
- DX/ND7 ⁇ gal clone 87 an expression vector, was grown in cell factories.
- Cells were grown in DMEM supplemented with Fetal Bovine Serum in roller bottles until enough cells to seed 20 10-layer cell factories (NUNC) at a 1:3 dilution were obtained.
- NUNC 10-layer cell factories
- Each 10-layer cell factory is seeded with approximately 0.8 liters of cell medium.
- Cells were seeded into the cell factory by pouring media containing cells into the factory so that the suspensions evenly fill the 10 layers. The factory is then carefully tilted away from the port side to prevent the suspension from redistribution in the common tube. Finally, the cell factory is rotated into its final upright position. A hepavent filter is attached to each port. The factory was then placed in a CO 2 incubator.
- Verification of the vector was performed by transduction of HT 1080 cells. These cells were harvested 2 days later and stained for ⁇ -gal protein. The titer of the supernatant was determined to be 2 x 10 7 /ml.
- Producer cell lines DA/ ⁇ gal and HX DN-7 were cultured in a culture flask and a roller bottle, respectively, containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum plus ImM L-GHutamine, Sodium pyruvate, non- essential amino acids and antibiotics. Viral supernatant was harvested from the flask and roller bottle, and were filtered through a 0.45 um syringe filter. The filtered supernatants were stored either at 4°C (HX/ND7), or frozen at -70°C (DA ⁇ -gal).
- DMEM Dulbecco's Modified Eagle's Medium
- Virus Concentration Viral supernatant was aliquoted into 50 ml sterile OAKRUDGE screw cap tubes, and placed into an SS34 rotor for use in a Sorvall centrifuge. The tubes were spun for 1 hour at 16,000 rpm (25,000g-force) at 4 * C. Upon completion of the spin, the tubes were removed, the supernatant decanted and a small opaque pellet resuspended in the DMEM media described above.
- Concentrated virus was titered on HT1080 cells plated 24 hours earlier at a cell density of 2x10-5 cells per well in a six well plate + 4 ug/ml polybrene. Briefly, virus preps were diluted from 1/10 to 1/10,000 and 50 ul of each dilution was used to infect one well from the six well plate. Plates were incubated overnight at 37°C. Forty-eight hours later, cells were fixed and stained with X-gal. The results are set forth below in Table 1.
- the resultant titer was determined by assaying HT1080 target cells set up at a concentration of 1 x 10 cells per well 24 hours prior to transduction of the viral sample. Cells were transduced in the presence of 8 ug/ml polybrene and 2 mL growth media (DMEM plus 10% FBS) per well. As shown in Table 1 below, approximately one hundred percent of the virus was recovered utilizing this procedure (note that titers are in BCFU/ml).
- the media constituents for virus production are DMEM-high glucose (Irvine Scientific, Santa Ana, CA.) basal media supplemented with FBS (10 to 20%), Glutamine (8 to 15mM), glucose (4.5 to 6.5 g L), Nonessential amino acids (IX), RPMI 1640 amino acids ( 0.2 to 9.6X), 10 mM HEPES, RPMI 1640 Vitamins (0.2 to 5X).
- FBS 10 to 20%
- Glutamine 8 to 15mM
- glucose 4.5 to 6.5 g L
- Nonessential amino acids IX
- RPMI 1640 amino acids 0.2 to 9.6X
- 10 mM HEPES RPMI 1640 Vitamins
- pH pH (6.9 to 7.6)
- dissolved oxygen 'DO" 5 to 90%
- the culture is then continuously perfused with fresh media with concurrent continuous harvesting in an escalating perfusion rate of 0.5 to 2.5 volumes/day.
- Cell retention is the result of differential sedimentation of cell covered beads in a decanting column.
- the bioreactor is monitored for viable cells, titer, glucose, lactate, ammonia levels, and lack of contamination. Viable cells and titer range from 1 x 10
Abstract
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1998000541A2 (en) * | 1996-07-03 | 1998-01-08 | Chiron Corporation | Methods for administration of recombinant gene delivery vehicles for treatment of human disease |
WO1999011292A2 (en) * | 1997-09-02 | 1999-03-11 | Chiron Corporation | Compositions and methods for treating arthritis utilizing gene therapy |
US6114113A (en) * | 1997-08-11 | 2000-09-05 | Chiron Corporation | High efficiency genetic modification method |
WO2003082343A1 (en) * | 2002-03-29 | 2003-10-09 | Japan Science And Technology Agency | Drugs for treating liver diseases with the use of hollow protein nanoparticles |
WO2003082344A1 (en) * | 2002-03-29 | 2003-10-09 | Japan Science And Technology Agency | Remedies with the use of hollow protein nanoparticles presenting growth factor or the like |
US6818439B1 (en) | 1994-12-30 | 2004-11-16 | Chiron Corporation | Methods for administration of recombinant gene delivery vehicles for treatment of hemophilia and other disorders |
EP2339010A2 (en) | 2002-05-01 | 2011-06-29 | Gbp Ip, Llc | Lentiviral vector particles resistant to complement inactivation |
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WO1994029440A1 (en) * | 1993-06-04 | 1994-12-22 | The Regents Of The University Of California | Generation, concentration and efficient transfer of vsv-g pseudotyped retroviral vectors |
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1995
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WO1994029440A1 (en) * | 1993-06-04 | 1994-12-22 | The Regents Of The University Of California | Generation, concentration and efficient transfer of vsv-g pseudotyped retroviral vectors |
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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 612, 1990, pages 407--414, XP002008048 D. MARKOWITZ ET AL.: "Retroviral gene transfer using safe and efficient packaging cell line" * |
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WO1996021014A3 (en) | 1996-09-26 |
AU4608096A (en) | 1996-07-24 |
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