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• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• Retroviruses are a diverse taxonomic group of viruses that infect a wide range of mammalian and non- mammalian hosts including humans, horses, cats, mice and chickens. These infections range from asymptomatic to lethal . Some of the more devastating clinical results of retrovirus infection are tumor formation, leukemia, immunodeficiency and neurologic disfunction. Retroviruses have single-stranded (ss) ribonucleic acid genomes (RNA) . After infection of a host cell, the enzyme reverse transcriptase (RT) , encoded by one of the retroviral pol genes, converts the ssRNA genome into double-stranded (ds) deoxyribonucleic acid (DNA) .
• ss single-stranded
• ds double-stranded
• DNA deoxyribonucleic acid
• the protease gene (prt) is necessary for the generation of a functional RT enzyme as prt cleaves the polyprotein gag-RT precursor.
• a third retroviral enzyme called integrase (int) facilitates integration of the dsDNA into the host cell genome.
• a host- encoded RNA polymerase transcribes genes encoded by the integrated retroviral sequences.
• mRNA viral messenger RNA' s
• mRNA viral messenger RNA' s
• infectious retroviral particles are then assembled. Each new particle contains two copies of the viral RNA.
• the fidelity of the packaging of viral RNA is achieved by virtue of a particular sequence within the retrovirus genome, designated .
• Retroviral vectors are recombinant retroviruses that can be utilized for the introduction of genes into eukaryotic cells.
• all known retroviral vectors carry endogenous reverse transcriptase and int, thereby facilitating incorporation of the retroviral vector DNA into the host genome. This permits exogenous genes that have been recombined into the retroviral vector DNA to be inserted into the host's genetic material.. If the integration is stable and compatible with the host, the exogenous gene is expressed.
• retrovirus vectors allow the targeting of cells capable of infection by a particular retrovirus, the question of selective killing of RIC's remains.
• a retroviral vector that can infect and integrate into the genome of a non-infected host cell presents an unacceptable risk in and of itself.
• one object of the present invention is to provide a retroviral vector that (i) is capable of infecting target cells, (ii) does not integrate into the genome of, or otherwise replicate in, non-infected host cells and (iii) can either kill infected cells or inhibit the replication of retroviruses therein.
• Another object of the present invention is to provide a packaging cell line that (i) will support the replication of the retroviral vector described above but (ii) does not confer on that virus the ability to integrate or replicate in non-infected host cells.
• Yet another object of the present invention is to provide a method of treatment for an animal infected with a retrovirus using an integration-incompetent vector, produced in a replication-supporting cell line.
• a retroviral vector is provided that lacks all sequences except the LTR, the sequence, a selectable marker and one or more genes capable of effecting either (i) the inhibition of replication of retroviruses in a RIC or (ii) the selective killing of a RIC.
• a retroviral vector is provided, where inhibition of retroviral replication is the result of an anti-sense RNA expressed by the retroviral vector.
• a retroviral vector is provided, wherein selective killing is the result of a toxin expressed by the retroviral vector.
• a retroviral vector where selective killing is the result of a surface-expressed, non-retroviral, antigenic protein expressed by the retroviral vector.
• a retroviral vector is provided, where selective killing is the result of the interaction between a gene expressed by the retroviral vector and an agent administered to the patient.
• a packaging cell line contains a retroviral construct which lacks the sequence and one or more of the RT, int and prt genes but contains all the structural genes necessary for assembly of virions.
• a method of therapy wherein a therapeutic, integration-incompetent retroviral vector is used to treat subjects suffering from a retroviral infection where the vector is only active in cells infected the retrovirus.
• the therapeutic retroviral vectors of the present invention exhibit normal host range functions, at least at the level of attachment and internalization.
• the present vectors do not integrate into the genome of cells unless those cells have already been infected with a disease-causing retrovirus.
• therapeutic vectors of the present invention also carry genes that facilitate either (i) the killing of RIC's or (ii) the inhibition of the replication of RIC's.
• the key to the selective treatment of RIC's using these therapeutic vectors is the reliance of the vectors on the presence of reverse transcriptase, integrase and protease in the cells which they infect. This reliance results from the absence of at least one of a functional RT, ⁇ nt, or prt coding sequences in both the vector and the cell line in which the vector is grown and packaged.
• therapeutic vectors of the present invention infect cells that do not contain active reverse transcriptase, integrase or protease, the vector RNA rapidly will be degraded.
• the retroviral vector RNA will be converted to DNA.
• the DNA copy then integrates into the host chromosome, resulting in "superinfection" of the host cell. After integration, genes encoded by the retroviral vector will be expressed.
• the present invention also provides mechanisms for killing infected cells or inhibiting their replication.
• the mechanisms can be direct, i.e., caused by vector-based products, or they can be indirect, i . e . , caused by the interaction of some other factors with vector-based products.
• An example of a direct mechanism is where the vector carries a gene encoding a toxin. Expression of the toxin after integration of the vector would kill the host cell .
• Another example of the direct mechanism would be the expression of an anti-sense RNA from sequences carried by the vector. The antisense RNA would be designed to hybridize to transcripts of one of the essential genes expressed by the disease-causing retrovirus, thereby blocking translation.
• An indirect method might involve the expression of a surface bound, heterologous, antigenic protein encoded by the vector. If sufficient levels of circulating antibodies are not produced in response to the antigen or are not already present due to prior immunization, antibodies or antibody-conjugates specific for this antigen that effect cell killing are then administered to the patient.
• the antigenic protein might also elicit a cellular response capable of killing RIC's.
• the killing might be effected by the interaction of a therapeutic gene product and a pharmacologic agent administered to the patient which interacts with the therapeutic gene.
• Retroviral vectors according to the present invention generally contain a long terminal repeat (LTR) , packaging i ⁇ ) sequence, a selectable marker and one or more therapeutic genes under the control of appropriate promoters. Preferably, all structural genes are removed since this eliminates the possibility of recombination with proviral sequences.
• LTR long terminal repeat
• the LTR, and any other regulatory sequences should be compatible with trans- acting factors provided by the host cell.
• the reverse transcriptase, protease and integrase of the disease-causing virus must be compatible with signals contained in the vector. Thus, selection of the starting material from which the therapeutic vector is generated depends, to a large measure, on the nature of the disease-causing retrovirus of the disease to be treated.
• Selectable marker genes are often drug resistance genes or genes that produce color or light reactions upon appropriate treatment.
• Favored drug markers are hygromycin-B-phosphotransferase, aminoglycoside phosphotransferase and xanthine-guanine phosphoribosyltransferase igpt) .
• a favored color marker is ⁇ -galactosidase.
• a favored fluorescent marker is luciferase.
• Therapeutic genes include, but are not limited to xenogenic major histocompatibility complex proteins, toxins, immunogenic membrane-bound proteins and appropriate antisense constructs.
• xenogenic MHC molecules these could include class I and II antigens from rat, mouse, horse, pig or other species.
• Appropriate toxins include ricin A chain, Pseudomonas exotoxin and pokeweed antiviral protein.
• Immunogenic membrane bound proteins include any surface bound protein from a species other than the host for which the corresponding antibody is abundantly available.
• an immunogenic membrane bound protein could be derived from a pathogen for which an individual already possesses antibodies or for which the individual could be vaccinated.
• Antisense constructs are derived from any viral gene that is essential to viral replication, assembly or expression (e.g., tar, ⁇ ) .
• genes which render target cells susceptible to drugs such as thymidine kinase ( tk) , also are contemplated.
• Retroviral sequences are cloned into standard vectors such as pBR322 and its derivatives, the pUC plasmid series or any of a variety of commercially available vectors. Ligation is accomplished by directional (dual site) or non-directional cloning (blunt-end or single site) .
• linker sequences at particular restriction enzyme sites in a given region of DNA. This is accomplished by cleaving a single site within a target sequence and ligating a linker into that site. The introduction of even a few base pairs may result in frame-shift leading to premature termination of a polypeptide encoded by the target sequence. Alternatively, the region "downstream" of the linker insertion may still code for a polypeptide sequence, but that polypeptide may be completely different than the original polypeptide sequence. Finally, if the insertion consists of a sequence that is of the length 3n, where n is 1, and no "stop" codons are introduced, the only change will be the addition of n number of aminp acids at the insertion site. Insertional mutagenesis can also be applied to non-coding regions but with less predictable results.
• genes or genetic control regions may easily be introduced into a vector.
• Selectable markers like hygromycin, tk, neomycin and gpt or therapeutic genes such as MHC molecules, toxins or antisense constructs are examples of genes whose expression which might be useful in the context of this invention.
• all the genes should be next to one another and under the control of the same promoter.
• the therapeutic gene should be located upstream of the marker gene to ensure that all selection-resistant clones contain sequences from both ends of the inserted segment
• Packaging cell lines are mammalian cells that carry "proviruses" integrated into their chromosomes. The proviral sequences, however, are defective in their ability to be packaged and, thus, produce empty virions. These cell lines are used to support the replication of retroviral vectors that lack certain genes necessary for the assembly of the viral capsid.
• Retroviral sequences are genetically-manipulated rendering them packaging-incompetent. These sequences are then transfected into host cells capable of supporting retroviral replication.
• Popular cells for this purpose include the various fibroblasts, H9 cells, MOLT-3 cells, MT4 cells and others. Transfection can be accomplished by a variety of means including electroporation, calcium phosphate coprecipitation, lipofection or protoplast fusion. Some of the transfected sequences will integrate into the host cell DNA and will be maintained in a stable fashion.
• a selectable marker is usually covalently linked to the retroviral sequences used for transfection. Such markers are discussed above.
• proviruses contained in packaging cells in addition to lacking ⁇ sequence, also lack the sequence for at least one of a functional reverse transcriptase, a functional protease and a functional integrase.
• a retroviral vector lacking one or more of these enzymes the retrovirus produced will be incapable of integrating into the host genome of a normal cell it infects subsequently. Should this same retroviral vector infect a host cell already infected by an integration competent retrovirus, the latter will provide enzymes necessary for the integration of the former and superinfection will occur.
• Type I packaging cell lines contain a helper provirus wherein the packaging sequence
• Type II packaging cell lines contain a helper provirus wherein the packaging sequence is deleted and the protease gene is defective. Thus, type II cell produce virions that do not contain proviral RNA and functional aspartate protease.
• TYPE III CELLS. Type III packaging cell lines contain a helper provirus wherein the packaging sequence is deleted and the integrase gene is defective. Thus, type III cell produce virions that do not contain proviral RNA and functional integrase .
• a suitable retroviral provirus can be constructed using virtually any retroviral genome .
• genomic human immunodeficiency virus type 1 (HIV-1) DNA can be obtained from American Type Culture Collection (ATCC #53069-pBT-l) . See U.S. Patent 4,784,941.
• Deletion of, the ⁇ sequence can be achieved by either oligo-directed mutagenesis, restriction digestion + Ba231 treatment, or other standard techniques
• Type I cells In order to inactivate the gene encoding reverse transcriptase, part or all of the RT gene can be deleted, usually by restriction digestion and religation of the RT sequences.
• a selectable marker replaces the deleted RT sequences. The presence of the marker activity indicates that the RT sequences have not been "rescued" by recombination with wild-type sequences.
• Type II cells The cells are constructed in a manner similar to that described for TYPE I cells with the exception that the protease gene is subjected to inactivation/deletion.
• Type III cells In these cells, the integrase gene is subjected to mutagenesis rather than either the RT or protease genes. Again, it is preferable to insert a marker gene in the integrase coding region that acts as a selectable surrogate for the i ⁇ t " phenotype. CULTIVATION AND HARVESTING OF THERAPEUTIC RETROVIRAL VECTORS Once a therapeutic vector has been generated, along with an appropriate packaging cell line, the vector DNA is transfected into the packaging cells by any of the methods discussed above in the section dealing with packaging cell lines.
• the transfected cells i.e., the producing cells, are cultured and the packaged retroviral vectors are isolated as concentrated culture supernatant and stored at -70°C. These stocks are stable for several years.
• the marker genes in either or both the packaging cells and the vector are drug resistance/selection genes, the transfected cells can be subjected to single or dual antibiotic selection.
• a gene encoding a toxin may be included in the vector.
• the toxin must be under the control of a promoter that is not active in the packaging or producing cells but is active in the target cells.
• the vector RNA is converted to DNA and integrates into the chromosome of the target cell, the toxin is transcribed and translated by target cell synthetic enzymes.
• the toxin selected for its toxicity inside the cell, kills the target. In so doing, the production of the disease-causing retrovirus is eliminated.
• the vector DNA encodes a surface- bound, antigenic protein not normally expressed in the patient.
• This antigen may elicit a protective humoral or cellular immune response from the patient that results in destruction of antigen-expressing cells.
• the antigen may be one for which the patient has already acquired immunologic sensitivity.
• an antibody or antibody-conjugate may be provided passively to the patient which effects cell killing. This approach may be preferable where multiple rounds of superinfection are desired.
• Another version of toxin action involves the expression of a gene that interacts with a pharmaceutical substance, resulting in the formation of a toxic compound. By administering the pharmaceutical substance before or after infection with the therapeutic vector, one can regulate the number of rounds of replication of the therapeutic vector.
• the therapeutic vector encodes a gene which is the complement to a gene essential for the replication of the disease-causing retrovirus.
• the essential gene and the antisense gene are transcribed, they hybridize to each other, thereby preventing translation of the essential gene's message. Without the essential product, replication of the disease-causing retrovirus is blocked. Similarly, a defective essential gene can be employed to thwart replication.
• the therapeutic vector encodes a non-functional form of some gene essential for replication of the disease-causing retrovirus.
• this non-functional protein When this non-functional protein is generated, it supplants use of the normal protein. To the extent that this use can overwhelm use of the normal protein in the replication cycle, the production of infectious disease-causing retrovirus is reduced. In addition, it may be possible to attack cellular processes which do not adversely affect the cell but specifically inhibit viral replication.
• the target for such treatment would be cellular enzymes that are not required for the maintenance of cells but are required at some point in the viral replication cycle.
• a therapeutic vector may be deployed in a clinical setting.
• One approach is to culture packaging and producer cells in vi tro and purify large quantities of the therapeutic vector.
• the vector would then be administered in one or more doses to the patient via an intravenous or subcutaneous route.
• An alternative approach involves the use of a patient's own cells in an ex vivo/ in vivo context. Briefly, cells amenable to in vi tro culture are isolated from an patient and transfected, as discussed above, to generate packaging and producer cell lines. The producer cells would then be reintroduced into the patient and would produce therapeutic vector for the period during which the cells remained viable. In this way, the total amount of therapeutic virus provided, as well as the period during which the patient is exposed to the vector, is increased.
• An even more aggressive regimen involves the same general strategy as set out in the previous paragraph, with the exception that the patient's cells are immortalized prior to their introduction back into the patient. Because the producing cells are rendered immortal, their longevity in vivo is greatly enhanced, further increasing the total dose and exposure of the patient to the therapeutic vector.
• tk gene is part of the therapeutic vector and renders retrovirally-infected cells susceptible to gangcyclovir treatment. This cycle can be repeated until all retrovirally-infected cells have been eliminated.
• Another variation of the foregoing strategy involves the use of a separate therapeutic gene under the control of a cell specific promoter.
• producer cells would be rendered immortal and tk + , but the therapeutic vector would not encode the tk gene product nor would the therapeutic product be expressed in the producer cells.
• tumor cells make an ideal target for conversion into packaging and producer cells.
• Tumor cells transfected with a therapeutic vector construct already are immortalized and need only been rendered tk + before use in vivo .
• gangcyclovir killing of the transformed tumor cells in vivo also may render non-transformed tumor cells susceptible to the drug therapy by virtue of the "bystander effect.” See Culver et al . , Science 256:1550- 1552 (1992) .
• Example 1 Construction of a packaging cell line.
• Vectors containing the genomic sequences of the appropriate retroviruses can be obtained from the American Type Culture Collection, Rockville, MD.
• the retroviral packaging sequence ⁇ approximately 350 bp near the 5' -end of genomic retroviral DNA, are deleted by standard techniques. Similar deletions are described in the literature. See for example, Mann and Baltimore, J. Virol . 54:401-407 (1985) ; Mann et al . , Cell 33:153-159 (1983) .
• the Bglll/Clal fragment containing the R and U5 sequences of the LTR as well as the 5' untranslated region and part of the N-terminal coding region of the gag gene, is subcloned into the corresponding site of a staging vector, e . g. , pSP70
• Additional Bal31 treatment (1.5 U of Bal31 in a final volume of 50 ⁇ l of buffer containing 650 ⁇ g/ml DNA, 600 mM NaCl, 12 mM CaCl 2 , 12 mM MgCl 2 , 20 mM Tris-HCl at pH 8.0, 1 mM EDTA, at 30°C for 10 min) may be used until the whole ⁇ sequence is removed.
• Bal digests are repaired by T4 DNA polymerase (1-2 U in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol supplement with 170 ⁇ g/ml bovine serum albumin and dNTP's) at 37°C for 30 min, ligated (15 U of T4 DNA ligase in a final volume of 20 ⁇ l of buffer containing 50 mM Tris-HCl at pH 8.0, 10 mM MgCl 2 , 10 ⁇ g/ml BSA 20 mM dithiothreitol, 1 mM ATP) with linkers (1 ⁇ g) containing rare restriction site PpuMI to facilitate further modification when needed.
• T4 DNA polymerase 1-2 U in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol supplement with 170 ⁇ g/m
• the linkers are synthesized using an automated DNA oligonucleotide synthesizer (Applied Biosystems, Foster City, CA) . Deletion of the desired sequence is confirmed by dideoxy sequencing. The modified Bglll/Clal fragment with the ⁇ sequence deleted is reinserted at the corresponding site in the HIV DNA genome.
• the next step is to create a series of proviral constructs that are defective for additional retroviral functions.
• Three different proviruses, which give rise to three distinct packaging cell lines (Types I-III) are contemplated. Each of these cell lines, in addition to producing empty virions, also lacks a particular retroviral gene product.
• Type I Using the packaging-deficient retrovirus sequences, a second deletion is undertaken by a strategy similar to that described above.
• the reverse transcriptase gene (RT) with the pol region is destroyed by digestion with Kpnl and PvuII restriction enzymes. Excision of the intervening sequence results in loss of 3' coding regions for reverse transcriptase.
• additional Bal31 treatment from the .Kpnl site can be carried out as described above.
• the gene for the selectable marker neomycin is cloned into the site vacated by the reverse transcriptase.
• Type II Using the packaging-deficient retrovirus sequences, a second deletion is undertaken by a strategy similar to that described above.
• the prt gene which precedes the RT coding sequence, is eliminated by Bal31 treatment at the Dral site located 180 base pairs downstream of the translation initiation site of the prt gene.
• the deletion is extended to eliminate the upstream 180 nucleotides, including the translation initiation codon.
• Bal31 digests are repaired by DNA polymerase treatment and ligated with linkers containing the restriction sites for Pmel and Pad and a translation initiation site at the 3'-end of the linker. Sequencing is performed to confirm an in-frame fusion of the ATG site of the linker to the rest of the prt sequence as well as the RT sequence.
• the gene for the selectable marker neomycin is cloned into the restriction sites in the linker.
• Type III Using the packaging-deficient retrovirus, a second deletion is undertaken by a strategy similar to that described above. A 1095 base pair BcoRI fragment of HIV-1 containing the 3' half of the HIV-1 integrase gene is subcloned into pUC18 and deletions originating from the 5' BcoRI site generated by digestion with Bal31. Bal digests are repaired by T4 DNA polymerase and ligated with a linker containing the restriction sites for Pmel and Pad and the selectable marker neomycin. The modified BcoRI fragment is reinserted into the corresponding site of the HIV-1 genome.
• modified HIV genomic DNA is mixed with 5-10 million H9 cells in electroporation buffer (20 mM HEPES: 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HP0 4 , 6 mM dextrose, 2 mM ⁇ -mercaptoethanol - pH 7.5) in a final volume of 0.7 ml in a 0.4 cm electroporation cuvette (BIORAD, Hercules, CA) . Following electroporation, cells are distributed into 96-well microtiter plates and overlayed with fresh tissue culture media.
• electroporation buffer 20 mM HEPES: 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HP0 4 , 6 mM dextrose, 2 mM ⁇ -mercaptoethanol - pH 7.5
• electroporation buffer 20 mM HEPES: 137 mM NaCl, 5 mM KCl, 0.7 mM
• Selection media containing 0.1-0.5 mg/ml of hygromycin B (Calbiochem, San Diego CA) is added 24 to 48 h after electroporation.
• An alternative method of introducing the provirus involves transfection by calcium phosphate precipitation. Prior to transfection, modified HIV genomic DNA, carried in an appropriate vector, is linearized and adjusted to a concentration of 40-60 ⁇ g/ml in 0.1 X TE (pH 8.0, 1 mM Tris-HCl, 0.1 mM EDTA) .
• Chromosomal DNA is prepared from cloned, drug resistant cell lines and subjected to southern blot analysis to confirm the presence of retroviral sequences.
• One of the hybridization positive clones is then selected on the basis of high level production of empty virions.
• Example 2 Construction of a therapeutic retrovirus.
• herpes simplex virus thymidine kinase (tk) gene can be inserted into the PvuII restriction site within the linker such that transcription is controlled by the HIV LTR.
• a hygromycin selection marker with an SV40 promoter, which is different from the one used in the packaging vector, is obtained from the vector pSVhyg
• the therapeutic vector should then contain, in its 5' to 3' orientation, the HIV LTR, a packaging sequence ⁇ , a promoterless tk gene, a hygromycin resistant gene driven by an SV40 promoter, and a downstream HIV LTR.
• Cells expressing the tk gene are sensitive to the cytotoxic effect of a gancylcovir.
• Other therapeutic genes or combinations of genes such as a xenogenic MHC molecule from heterologous source, an antisense construct, a toxin such as ricin A chain or some other surface marker for which specific antibodies are available, can be used in place of or in conjunction with the tk gene.
• Example 3 Cultivation of a therapeutic retrovirus in a packaging cell line.
• Packaging cells lines (Types I-III) are produced by transfecting H9 cells with one of the constructs as described in Example 1. Individual clones are isolated by further culture in cloning cylinders or by limiting dilution in microtiter plates in the presence of 0.5 mg/ml of hygromycin B for at least five days. Individual clones so selected are expanded into cell lines. Cell lines producing the highest titers of empty virions are identified by a standard ELISA assay.
• reaction solution 100 ⁇ l, containing 167 ⁇ g of ortho-phenylene-diamine (Sigma, St. Louis, MO), 0.025% hydrogen peroxide in PBS
• Color is allowed to develop in the dark for 30 min.
• the reaction is stopped by the addition of 50 ⁇ l of 4N HCl solution to each well before measuring absorbance at 490 nm in an automated ELISA reader (Bio-Tek Instruments, Winooski, VT) .
• the packaging cell line with the highest level of empty virion production is used for transfection.
• Transfected cells are resuspended in 20 ml of culture media (DMEM + 10%'FCS + 100 ⁇ g/ml gentamicin) and distributed into the wells of two microtiter plates. After incubating the cells at 37°C in the presence of 5% C0 2 for two days, fresh media containing 0.5 mg/ml of hygromycin B (Calbiochem, San Diego, CA) is added. Colonies usually emerge 2-3 weeks post-transfection.
• DMEM + 10%'FCS + 100 ⁇ g/ml gentamicin 100 ⁇ g/ml gentamicin
• Skin fibroblasts from an asymptomatic HIV- positive patient are obtained and transformed in vi tro by standard techniques to generate an immortalized cell line.
• the immortalized fibroblasts are transfected by the methods described above and converted into packaging and producer cells, of which those producing the highest level of viral particles are selected and expanded in culture.
• the HS-tJ retroviral vector-producing fibroblasts (10 7 cells) are injected into the donor patient subcutaneously and, after about one week, gangcyclovir (5 mg/kg) is given intravenously at a constant rate over one hour, twice daily, for one week.
• the patient receives a second injection of HS-t.k retroviral vector-producing fibroblasts (10 7 cells) and, one week later, gangcyclovir is given twice daily for one week.
• This two-week treatment cycle is repeated a third time and the effectiveness of the therapy is monitored at the end of each treatment cycle by measuring the presence of HIV in plasma or blood cells using PCR for HIV-1 mRNA.
• the patient will receive gangcyclovir (5 mg/kg) , once daily.

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