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WO1996035455A1 - Therapie genique par transduction de cellules epitheliales orales - Google Patents

Therapie genique par transduction de cellules epitheliales orales Download PDF

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Publication number
WO1996035455A1
WO1996035455A1 PCT/US1996/006648 US9606648W WO9635455A1 WO 1996035455 A1 WO1996035455 A1 WO 1996035455A1 US 9606648 W US9606648 W US 9606648W WO 9635455 A1 WO9635455 A1 WO 9635455A1
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vector
oral
agent
cells
host
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PCT/US1996/006648
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English (en)
Inventor
Bruce Trapnell
Edward J. Shillitoe
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Genetic Therapy, Inc.
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Application filed by Genetic Therapy, Inc. filed Critical Genetic Therapy, Inc.
Priority to AU57389/96A priority Critical patent/AU5738996A/en
Publication of WO1996035455A1 publication Critical patent/WO1996035455A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/70—Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites

Definitions

  • This invention relates to gene therapy for the treatment of diseases or disorders. More particularly, this invention relates to the treatment of diseases or disorders through transduction of oral epithelial cells with viral vectors including a polynucleotide encoding a therapeutic agent. BACKGROUND OF THE INVENTION
  • Oral cancer cells that carry papillomavirus may- have their malignant or potentially malignant phenotype modified by the use of antisense molecules that are directed to the transforming genes of papillomaviruses (Steele, et al . , Cancer Res.. Vol. 52, pgs.
  • the malignant phenotype of oral cancer cells may be suppressed by transduction with a wild-type p53 gene (Liu, et al . . Cancer Res .. Vol. 54, pgs. 3662-3667 (1994) ; Clayman, et al . , Cancer Res.. Vol. 55, pgs. 1-6 (1995)) .
  • Other approaches include the transfer of a toxic phenotype gene to the oral mucosa (O'Malley, et al . , Arch. Otolarvncrol . -Head and Neck Surer., Vol. 119, pgs. 1191-1197
  • adenovirus including a negative selective marker such as, for example, the Herpes Simplex Virus thymidine kinase gene
  • a negative selective marker such as, for example, the Herpes Simplex Virus thymidine kinase gene
  • optimal delivery systems should be developed for the transfer of these genetic constructs to the oral mucosa and to oral cancers.
  • Transduction of epithelial cells can result in long-term expression of the transduced gene.
  • Basal oral epithelial cells may be transduced in vi tro with retroviruses and grown to a differentiated state.
  • Adenoviruses also may be effective vectors for transduction of oral epithelial cells because they can transduce a variety of tissues effectively.
  • Trapnell Adv. Dru ⁇ Deliver y Rev.. Vol. 12, pgs. 185-189 (1993); Trapnell, et al . , Current Opinion Biotech, in press (1994)
  • Adenoviruses have been used to transduce skin cells i vivo with long-term expression of the transgene. (Setoguchi, et al . , J. Invest. Dermatol.. Vol. 102, pgs. 415-421 (1994)) . SUMMARY OF THE INVENTION
  • the present invention is directed to the treatment or prevention of a disease or disorder in a host by delivery of a therapeutic agent to oral epithelial cells of a host.
  • a therapeutic agent may be delivered to a host by transducing oral epithelial cells of the host in vivo by administering to the host systemically (such as through a branch of the external carotid artery) a viral vector which includes a polynucleotide encoding the therapeutic agent.
  • a viral vector which includes a polynucleotide encoding the therapeutic agent.
  • Such method may be employed, for example, in the treatment of tumors of the oral epithelium.
  • Figure 1 is a schematic of the construction of plasmid pHR
  • Figure 2 is a schematic of the construction of an adenoviral vector including an ITR, an encapsidation signal, a Rous Sarcoma Virus promoter, and an adenoviral tripartite leader (TPL) sequence;
  • Figure 3 is a schematic of the construction of pAvS6;
  • Figure 4 is a map of plasmid pAvS6;
  • Figure 5 is a map of plasmid pAvS6-nLacZ;
  • Figure 6 is a schematic of the construction of AvlLacZ4;
  • Figure 7A is a photograph of a microscope slide of oral sub-mucous connective tissue upon abrasion of the oral epithelium with the flat surface of a scalpel, followed by transduction of the oral sub-mucous connective tissue cells with adenovirus AvlLacZ4;
  • Figure 7B is a photograph of a microscope slide of connective tissue adjacent to a salivary duct following topical application of adenovirus AvlLacZ4 to the oral mucous membrane;
  • Figure 7C is a photograph of a microscope slide of adipocytes transduced with AvlLacZ4 along a needle track following subcutaneous injection of the adenovirus;
  • Figures 8A, 8B, and 8C are photographs of sections of the cheek pouch, buccal mucosa, and tongue, respectively, of a hamster after such tissues were transduced with AvlLacZ4 through intracardiac injection of the adenovirus;
  • Figures 9A, 9B, and 9C are histological sections of the tissues shown in Figures 8A, 8B, and 8C, respectively;
  • Figure 10A is a photograph of a microscope slide of a monolayer of uninfected Tul38 cells, magnified 50 times;
  • Figure 10B is a photograph of a microscope slide of a monolayer of Tul38 cells exposed to AvlLacZ4 at a multiplicity of infection (MOD of 50, magnified 50 times;
  • Figure IOC is a photograph of a microscope slide of a monolayer of Tul38 cells exposed to AvlLacZ4 at a multiplicity of infection (MOD of 100, magnified 50 times;
  • Figure 10D is a photograph of a microscope slide of a monolayer of Tul38 cells exposed twice to AvlLacZ4 at a multiplicity of infection of 100, magnified 50 times;
  • Figure 11 is a photograph of a microscope slide of a section of a raft culture of Tul38 cells exposed to AvlLacZ4, magnified 400 times;
  • Figure 12 is a photograph of a microscope slide of a section of an established subcutaneous solid tumor of human Tul38 cells in a nude mouse wherein AvlLacZ4 was injected into the tumor, magnified 125 times.
  • a process for treating or preventing a disease or disorder of a host by delivery of a therapeutic agent to oral epithelial cells of the host comprises transducing oral epithelial cells of a host in vivo by administering to the host systemically a viral vector including a polynucleotide encoding a therapeutic agent to produce in said oral epithelial cells said therapeutic agent.
  • a viral vector including a polynucleotide encoding a therapeutic agent to produce in said oral epithelial cells said therapeutic agent.
  • therapeutic is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.
  • oral epithelial cells includes, but is not limited to, normal oral epithelial cells of the mouth (including the floor of the mouth) , lips, tongue, gums, palate, and cheek, as well as pre-cancerous cells and tumor cells of the oral epithelium.
  • polynucleotide as used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single- and double-stranded DNA, as well as single- and double-stranded RNA. ' The term also includes modified polynucleotides such as methylated or capped polynucleotides. Applicants have found that, although topical application of viral vectors to oral epithelial cells has been unsuccessful in transducing oral epithelial cells, one may transduce oral epithelial cells successfully with viral vectors by administering such vectors systemically.
  • the method of the present invention is employed in treating pre-cancerous conditions of the oral epithelium.
  • the viral vector includes a polynucleotide encoding an agent which prevents the transformation of normal oral epithelial cells into malignant cells, thereby preventing the formation of cancerous tumors; and/or inhibits, prevents, or destroys the growth of pre- cancerous lesions.
  • Such agents include, but are not limited to, tumor suppressor proteins, such as, for example, p53 protein, Rb protein, taxol, and vinblastine; antisense oligonucleotides to papillomaviruses (and in particular antisense oligonucleotides directed to the transforming genes of papillomaviruses); and ribozymes.
  • tumor suppressor proteins such as, for example, p53 protein, Rb protein, taxol, and vinblastine
  • antisense oligonucleotides to papillomaviruses and in particular antisense oligonucleotides directed to the transforming genes of papillomaviruses
  • ribozymes include, but are not limited to, tumor suppressor proteins, such as, for example, p53 protein, Rb protein, taxol, and vinblastine.
  • the method of the present invention may be employed to treat the pre-cancerous lesions of the mouth or cheek such as leukoplakia, dysplasia, and erythroplasia.
  • the cells of the lesion are transduced with a viral vector, which is administered to a host systemically, and which includes a polynucleotide encoding a tumor suppressor protein, such as p53 protein.
  • a tumor suppressor protein such as p53 protein.
  • the agent is an antisense polynucleotide to a papillomavirus.
  • Papillomaviruses are associated with the transformation of normal cells into malignant phenotypes.
  • antisense polynucleotides include, but are not limited to, antisense polynucleotides to the E6 and E7 genes of human papillomavirus 18. Examples of such antisense polynucleotides are described further in Steele, et al., Cancer Research. Vol. 53, pgs. 2330-2337 (May 15, 1993) and in Steele, et al. , Cancer Research. Vol. 53, pgs. 4706-4711 (September 1, 1992) .
  • the agent is a ribozyme.
  • ribozymes which may be employed include, but are not limited to, ribozymes which cleave transcripts encoding papillomavirus proteins.
  • examples of such ribozymes are ribozymes RzllO and Rz558, which cleave the human papillomavirus 16 (HPV-16) genome immediately 3' to nucleotides 110 and 558, respectively, of the viral genomic DNA, and are described further in Lu, et al., Cancer Gene Therapy. Vol. 1, No. 4, pgs. 267-277 (1994) .
  • Ribozymes RzllO and Rz558 cleave the transcripts encoding the HPV-16 E6 and E7 open reading frames in proximity to the translational start sites.
  • the method of the present invention may be employed in treating tumors, and in particular cancerous tumors, of the oral epithelium.
  • tumors may be treated by transducing tumor cells of the oral epithelium by administering systemically to a host (preferably a human patient) a viral vector including a polynucleotide encoding an agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor.
  • Polynucleotides encoding an agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor include, but are not limited to, genes encoding tumor suppressor proteins such as antisense polynucleotides that are directed to the transforming genes of papillomaviruses and ribozymes as hereinabove described; antisense polynucleotides directed to oncogenes, ribozymes which cleave oncogenes or transcripts thereof; and negative selective markers or "suicide" genes.
  • tumor suppressor proteins such as antisense polynucleotides that are directed to the transforming genes of papillomaviruses and ribozymes as hereinabove described
  • antisense polynucleotides directed to oncogenes, ribozymes which cleave oncogenes or transcripts thereof include negative selective markers or "suicide" genes.
  • the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the tumor is a tumor suppressor protein, such as, for example, p53 protein, Rb protein, taxol, or vinblastine.
  • a tumor suppressor protein such as, for example, p53 protein, Rb protein, taxol, or vinblastine.
  • the agent is an antisense polynucleotide to a papillomavirus, whereby one may inhibit, prevent, or destroy the growth of tumor cells containing papillomavirus by transducing such tumor cells with antisense polynucleotides to papillomavirus such as those hereinabove described.
  • the method of the present invention may be employed to treat laryngeal papillomas which have spread to the oral epithelium, wherein the laryngeal papilloma cells are transduced with a viral vector including an antisense polynucleotide to a papillomavirus.
  • the method of the present invention may be employed to treat nasopharyngeal cancers which have spread to the oral epithelium.
  • Such cancer cells may be infected with the Epstein-Barr Virus (EBV) .
  • EBV Epstein-Barr Virus
  • the agent which is capable of providing for the inhibition, prevention, or destruction of the oral tumor upon expression of such agent is a negative selective marker; i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents, or destroys the growth of the oral tumor.
  • an interaction agent is administered to the host. The interaction agent interacts with the negative selective marker in order to prevent, inhibit, or destroy the growth of the oral tumor.
  • Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase; and cytosine deaminase.
  • thymidine kinase such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase
  • cytosine deaminase include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the interaction or chemotherapeutic agent preferably is a nucleoside analogue, for example, one selected from the group consisting of ganciclovir, acyclovir, and l-2-deoxy-2-fluoro- ⁇ -D-arabinofuranosil-5-iodouracil (FIAU) .
  • Such interaction agents are utilized efficiently by the viral thymidine kinases as substrates, to produce a substance which is lethal to the oral tumor cells expressing the viral thymidine kinases, thereby resulting in inhibition of the growth of or the destruction of the oral tumor.
  • the negative selective marker is cytosine deaminase.
  • cytosine deaminase is the negative selective marker
  • a preferred interaction agent is 5- fluorocytosine. Cytosine deaminase converts 5-fluorocytosine to 5-fluorouracil, which is highly cytotoxic.
  • the oral tumor cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5-fluorouracil to inhibit the growth of and/or destroy the oral tumor.
  • the interaction agent is administered in an amount effective to inhibit, prevent, or destroy the growth of the tumor cells.
  • the interaction agent may be administered in an amount from about 5 mg to about 15 mg/kg of body weight, preferably about 10 mg/kg, depending on overall toxicity to a patien .
  • the viral vectors including a polynucleotide encoding a negative selective marker are administered systemically as hereinabove described to a host, or administered directly to the tumor, a "bystander effect" may result, i.e., tumor cells of the oral epithelium which were not originally transduced with the negative selective marker may be killed upon administration of the interaction agent.
  • the transformed tumor cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non- transformed tumor cells, which then become susceptible to the action of the interaction agent. It also is possible that one or both of the negative selective marker and the interaction agent are communicated between tumor cells.
  • cancers which may be found in the oral epithelium and which may be treated in accordance with the method of the present invention include, but are not limited to, squamous cell carcinomas of the mouth and oral cavity, including the floor of the mouth, tongue, cheek, gums, or palate; adenocarcinoma of the oral cavity; lip cancers; Kaposi's sarcoma; and laryngeal papillomas and nasopharyngeal cancers as hereinabove described, which may have spread to the oral epithelium.
  • the viral vector is administered systemically to an animal host.
  • animal hosts include mammalian hosts, including human and non-human primate hosts.
  • the viral vector may be administered intravascularly to the host at a point in close proximity to the oral epithelial cells . The localization of the
  • SUBST ⁇ TJTE SHEET (RULE 26) intravascular administration is preferred in that such localization provides for improved transduction of cells in the area where required.
  • the vector is delivered to oral epithelial cells by administering such vector to a branch of the external carotid artery which supplies blood to oral epithelial cells.
  • Branches of the external carotid artery to which the vector may be administered include, but are not limited to, the lingual artery; the ascending pharyngeal artery; the facial artery; and the maxillary artery.
  • a catheter (along with other devices, such as a guide wire, if needed) is directed into the carotid artery, then into the external carotid, and then into the desired branch of the external carotid artery.
  • the catheter is directed to an appropriate point in the desired branch of the external carotid artery, and the vector is administered into the artery through the catheter for transduction of oral epithelial cells.
  • Viral vectors which may be transduced into the oral epithelial cells include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated virus vectors and Herpes Virus vectors.
  • the viral vector is an adenoviral vector.
  • the adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome (Shenk, et al . , Curr. Top. Microbiol. Immunol.. 111(3) : 1-39 (1984)) .
  • the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
  • the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; and at least one DNA sequence encoding a therapeutic agent .
  • the vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.
  • the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which the viral particles are produced.
  • This temperature sensitive mutant is described in Ensinger, et al . , J. Virology, 10:328-339 (1972), Van der Vliet, et al . , J . Virology. 15:348-354 (1975), and Friefeld, et al . , Virology. 124:380-389 (1983) .
  • Such a vector in a preferred embodiment, is constructed first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter) ; a multiple cloning site; a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.
  • the vector also may contain a tripartite leader sequence.
  • the DNA segment corresponding to the adenoviral genome serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome.
  • the plasmid may also include a selectable marker and an origin of replication.
  • the origin of replication may be a bacterial origin of replication. Representative examples of such shuttle plasmids include pAvS6, shown in Figure 4.
  • the DNA encoding the therapeutic agent then may be inserted into the multiple cloning site. One may amplify the expression of the DNA encoding the therapeutic agent by adding to the
  • SUBSTTTUTE SHEET (RULE 26) plasmid increased copies of the DNA encoding the therapeutic agent .
  • This construct is then used to produce an adenoviral vector.
  • Homologous recombination is effected with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted.
  • Such homologous recombination may be effected through co- transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells (ATCC No. CRL 1573) , by CaP0 4 precipitation.
  • a recombinant adenoviral vector is formed that includes DNA sequences derived from the shuttle plasmid between the NotI site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.
  • the homologous recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome.
  • a vector which includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela enhancer sequence; a promoter; the DNA sequence which encodes a therapeutic agent; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR.
  • the vector also may include a tripartite leader sequence.
  • This vector may then be transfected into a helper cell line, such as the 293 helper cell line, which will include the Ela and Elb DNA sequences, which are necessary for viral replication, and to generate infectious adenoviral particles.
  • the adenoviral vector particles are administered systemically to a host such that the adenoviral vector particles transduce oral epithelial cells in a host by methods such as those hereinabove described.
  • the adenoviral vector particles which are administered systemically and
  • SUBS ⁇ TUTESHEET(RULE26) preferably intravascularly, then transduce oral epithelial cells.
  • the adenoviral particles are administered in an amount effective to produce a therapeutic effect in a host.
  • the adenoviral particles are administered in an amount of at least IO 4 plaque forming units (pfu) , and in general such amount does not exceed about IO 13 pfu, and preferably is from about IO 7 pfu to about 10" pfu.
  • the exact dosage of adenoviral vector particles to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient to be treated, and the nature and extent of the disease or disorder to be treated.
  • the adenoviral particles may be administered as part of a preparation having a titer of adenoviral particles of at least IO 6 pfu/ml, and in general not exceeding 10 12 pfu/ml.
  • the adenoviral particles may be administered in combination with a pharmaceutically acceptable carrier in a volume up to 10 ml.
  • the adenoviral vector particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient, such as, for example, a liquid carrier such as a saline solution, protamine sulfate (Elkins-Sinn, Inc., Cherry Hill, N.J.) , water, aqueous buffers such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, MO) .
  • a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein.
  • the viral vector is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the vector is generally a replication incompetent retrovirus particle.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env) , are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.
  • the retroviral vector may be one of a series of vectors based on the N2 vector containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed.
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
  • the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al., Biotechnicrues. 7:980-990, 1989) .
  • Packaging-defective helper viruses for production of retroviral vectors are known in the art and examples thereof are described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) .
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989 ) .
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 08/340,805, filed November 11, 1994, and in PCT Application No. WO91/10728, published July 25, 1991, and incorporated herein by reference in their entireties.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechni ⁇ ues. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters) .
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell line.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , PA12, T19-14X, VT- 19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines, as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) .
  • the vector containing the nucleic acid sequence encoding the therapeutic agent may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the producer cells generate retroviral vector particles, which are administered to a host systemically, preferably intravascularly, whereby such retroviral vector particles transduce oral epithelial cells.
  • the vectors then produce the therapeutic agent in the oral epithelial cells.
  • the retroviral vector particles are administered to the host in an amount effective to produce a therapeutic effect in the host.
  • the retroviral vector particles are administered in an amount of at least IO 5 colony forming units (cfu), and in general not exceeding 10 g cfu, and preferably about IO 8 cfu.
  • the exact dosage of retroviral vector particles is dependent upon the factors hereinabove mentioned with respect to the adenoviral particles.
  • the retroviral vector particles are administered as part of a preparation having a titer of retroviral vector particles of at least IO 5 cfu/ml and in general not exceeding IO 9 cfu/ml.
  • the retroviral vector particles may be administered in combination with a pharmaceutically acceptable carrier, such as those hereinabove described with respect to the adenoviral vector particles, in a volume up to 10 ml.
  • Example 1 A. Adenoviral Gene Transfer Vector
  • the adenoviral vector used in this example was a replication deficient Ela/Elb " , E3 _ deletion mutant expressing a nuclear-targeted ⁇ -galactosidase gene under the control of the RSV-LTR promoter.
  • Such vector hereinafter is referred to as AvlLacZ4.
  • the vector was produced by recombination of the expression cassette with a cotransfected adenoviral genome in 293 cells for complementation of the El defect, thereby allowing virion production. Viral supernatants were harvested by 3 freeze-thaw cycles, followed by purification by ultracentrifugation through two cesium chloride gradients.
  • AvlLacZ4 is constructed from the adenoviral shuttle vector pAvS6.
  • the construction of AvlLacZ4 is described in detail as follows: (i) Construction of pAvS6
  • the adenoviral construction shuttle plasmid pAvS6 was constructed in several steps using standard cloning techniques including polymerase chain reaction based cloning techniques. First, the 2913 bp Bglll, Hindlll fragment was removed from Ad-dl327 and inserted as a blunt fragment into the Xhol site of pBluescript II KS- (Stratagene, La Jolla, CA) ( Figure 1) .
  • Ad-dl327 (Thimmappaya, et al . , Cell. Vol.
  • pg. 543 (1983) is identical to adenovirus 5 except that an Xbal fragment including bases 28591 to 30474 (or map units 78.5 to 84.7) of the adenovirus 5 genome, and which is located in the E3 region, has been deleted.
  • the orientation of this fragment was such that the Bglll site was nearest the T7 RNA polymerase site of pBluescript II KS " and the Hindlll site was nearest the T3 RNA polymerase site of pBluescript II KS " .
  • This plasmid was designated pHR. ( Figure 1) .
  • the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification ( Figure 2) .
  • the ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260] ) were amplified (amplification 1) together from Ad-dl327 using primers containing NotI or AscI restriction sites.
  • the Rous Sarcoma Virus LTR promoter was amplified (amplification 2) from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San Diego, CA) using primers containing an AscI site and an Sfil site. DNA products from amplifications 1 and 2 were joined using the "overlap" PCR method (amplification 3) with only the NotI primer and the Sfil primer. Complementarity between the AscI containing end of each initial DNA amplification product from reactions 1 and 2 allowed joining of these two pieces during amplification.
  • the TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad- dl327 [identical to similar sequences from Ad5, Genbank accession #M73260] ) from cDNA made from mRNA isolated from 293 cells infected for 16 hrs. with Ad-dl327 using primers containing Sfil and Xbal sites respectively. DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and Xbal primers, thus creating the complete gene block.
  • the ITR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and Xbal and inserted into the NotI, Xbal cleaved pHR plasmid.
  • This plasmid was designated pAvS6A and the orientation was such that the NotI site of the fragment was next to the T7 RNA polymerase site ( Figure 3) .
  • the recombinant, replication-deficient adenoviral vector AvlLac Z4, which expresses a nuclear-targetable B- galactosidase enzyme was constructed in two steps. First, a transcriptional unit consisting of DNA encoding amino acids 1 through 4 of the SV40 T-antigen fqllowed by DNA encoding amino acids 127 through 147 of the SV40 T-antigen (containing the nuclear targeting peptide Pro-Lys-Lys-Lys-Arg-Lys-Val) , followed by DNA encoding amino acids 6 through 1021 of E. coli B-galactosidase, was constructed using routine cloning and PCR techniques and placed into the EcoRV site of pAvS6 to yield pAvS6-nlacZ ( Figure 5) .
  • AvlLacZ4 The infectious, replication-deficient, AvlLacZ4 was assembled in 293 cells by homologous recombination. To accomplish this, plasmid pAvS6-nLacZ was linearized by cleavage with Kpnl. Genomic adenoviral DNA was isolated from purified Ad-dl327 viruses by Hirt extraction, cleaved with Clal, and the large (approximately 35 kb) fragment was isolated by agarose gel electrophoresis and purified. The Clal fragment was used as the backbone for all first
  • SUBST ⁇ UTE SHEET (RULE 26) generation adenoviral vectors and the vectors derived from it are known as Avl.
  • IMEM-2 IMEM plus 2% FBS, 2mM glutamine (Bio Whittaker 046764)
  • IMEM-10 Improved minimal essential medium (Eagle's) with 2x glutamine plus 10% vol./vol. fetal bovine serum) plus 2mM supplemental glutamine (Bio Whittaker 08063A) and incubated at 37°C for approximately three days until the cytopathic effect, a rounded appearance and "grapelike" clusters, was observed.
  • Cells and supernatant were collected and designated as CVL-A.
  • AvlLacZ4 vector (a schematic of the construction of which is shown in Figure 6) was released by three cycles of freezing and thawing of the CVL-A. Then, a 60 mm dish of 293 cells was infected with 0.5 ml of the CVL-A plus 3 ml of IMEM-10 and incubated for approximately three days as above. Cells and supernatant from this infection then were processed by three freeze/thaw cycles in the same manner.
  • AvlLacZ4 also is described in Yei, et al . , Human Gene Therapy. Vol. 5, pgs. 731-744 (1994); Trapnell, Advanced Dru g Delivery Reviews ' . Vol. 12, pgs. 185-199 (1993), and Smith, et
  • the resultant viral stock was titered by plaque assay on 293 cells using a standard protocol involving a 1.5 hour adsorption period in DMEM/2% FBS, followed by washout and agar overlay of the cell monolayer. (Graham, et al . , Virology. Vol. 52, pgs. 456-467 (1973)) .
  • the absence of wild-type virus was checked by polymerase chain reaction assays of the stock using primers amplifying a 337 bp fragment of the El gene. The stock was negative for wild- type adenovirus using this assay.
  • the virus stock then were frozen at -80°C and stored until used.
  • the virus stock had a titer of 1.5x10" pfu/ml.
  • ⁇ -D-galactosidase activity in tissues was detected by histochemical staining, using the method of MacGregor, et al . , Methods in Molecular Biology, Vol. 7: Gene Expression in vivo, Murray, et al . , eds., Humana Press Inc. (Clifton, N.J.) (1989) .
  • Tissues or cell monolayers were divided into two portions. One was snap frozen in liquid nitrogen and sectioned on a cryostat. Staining then was performed on sections on a microscope slide.
  • the other portion was fixed in glutaraldehyde/ paraformaldehyde and the entire tissue was stained before being embedded and sectioned. With raft cultures, only fixation was used. For all specimens, a neutral red counterstain was applied to the sections, and over 200 cells in a representative field were examined. The transduction frequency was expressed as a percentage of cells that were blue.
  • mice For topical application of adenovirus to the oral mucosa of mice, the mice (C3H female mice, purchased at 6 weeks of age from Harlan Sprague-Dawley, Indianapolis, Indiana) were anesthetized with a mixture of ketamine and aceprozamine and divided into groups of three animals.
  • the AvlLacZ4 adenovirus was diluted to 1 x IO 9 pfu/ l and applied to the oral mucosa in a volume of 0.1 ml.
  • the virus was placed directly in the mouth for one minute.
  • the surface cells of the buccal mucosa first were removed by careful scraping with the flat edge of a scalpel blade.
  • DMSO dimethyl sulfoxide
  • a fifth group received a sub-mucosal or sub-cutaneous injection of 0.1 ml of virus at 1 x IO 8 pfu/ml with a 28 gauge needle.
  • a control group of mice had no exposure to virus.
  • Each experiment was performed at least twice, and some were repeated with administration of virus at 1 x 10 10 pfu/ml. The mice were evaluated for transgene expression at 72 hours after treatment.
  • hamster oral mucosa For topical administration to hamster oral mucosa, three hamsters (LSH hamsters purchased at 6 weeks of age from Harlan Sprague-Dawley, Indianapolis, Indiana) were anesthetized and the interior of the cheek pouches were rinsed with saline. The pouch on one side was exposed to DMSO for 5 minutes while the other side remained untreated. The adenovirus was diluted to 1 x 10 10 pfu/ml. Cotton swabs were soaked in 0.1 ml of virus and were placed in each cheek pouch. The swabs remained in place for several hours, until the hamsters awoke and removed them. A control group was not exposed to adenovirus .
  • the oral mucosa of the mice did not show ⁇ -galactosidase activity following surface application of any dose of adenovirus. Drying, scraping, or use of DMSO did not result in epithelial transduction, although scraping of the surface frequently with the flat surface of a scalpel blade prior to application of virus frequently led to transduction of sub- mucosal connective tissue (Figure 7A) and to transduction of connective tissue adjacent to a salivary duct following topical application to oral mucous membrane (Figure 7B) . Sub-mucosal injection produced scattered staining of fibroblasts, adipocytes, or muscle cells, frequently along the needle track ( Figure 7C) , but not of the overlying epithelial cells.
  • Example 3 Ex vivo administration of adenovirus Three mice were killed and slices of tongue and buccal mucosa were removed and incubated at 37°C in a petri dish. Some slices were exposed to adenovirus in a volume of 0.1 ml at a concentration of 2 x 10 10 pfu/ml for 1 hour, while control slices were not exposed to virus. All tissues then were incubated in culture medium for a further 72 to 96 hours.
  • Oral mucosa was removed from two human patients undergoing surgical treatment of oral malignancies.
  • the tissues then were processed in the same way as the mouse tissue, using adenovirus at 1 x 10 10 pfu/ml.
  • the specimens then were incubated for a further 24 hours in culture medium.
  • Ex vivo exposure to the adenovirus produced some ⁇ - galactosidase activity in the exposed cut surface of connective tissues. No activity was seen in the oral epithelium in either the mouse or human samples.
  • Example 4 Systemic administration of adenovirus Two hamsters were anesthetized, and 0.1 ml of adenovirus at 1 x 10" pfu/ml was injected into the heart. A control hamster was not infected. After 72 hours, the animals were killed and tissues were removed and processed.
  • the Tu 138 cell line was established from a moderately differentiated gingivo-labial squamous cell carcinoma, and is described in Liu, et al . , Cancer Research, Vol. 54, pgs. 3662-3667 (1994) .
  • the Tu 138 cell line is cytokeratin positive and tumorigenic in nude mice.
  • the cells were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) with penicillin and streptomycin.
  • FBS fetal bovine serum
  • the medium then was removed from confluent monolayers of confluent Tu 138 cells and replaced with suspensions of adenovirus at lxlO 8 pfu/ml or at 2xl0 8 pfu/ml in DMEM/F12 medium with 2% FBS.
  • the cells were incubated at 37°C for 1 hour with agitation every 15 minutes.
  • Medium with 10% FBS then was added and the cells were incubated further at 37°C.
  • FIG. 10A is a photograph of a slide of a monolayer of Tu 138 cells that was not infected with adenovirus.
  • Figure 10B is a photograph of a slide of a monolayer of Tu 138 cells exposed to lxlO 8 pfu/ml of AvlLacZ4, which corresponds to a multiplicity of infection (MOD of 50.
  • Figure IOC is a photograph of a slide of a monolayer of Tu 138 cells exposed to 2xl0 8 pfu/ml of AvlLacZ4, which corresponds to a multiplicity of infection (MOD of 100.
  • Figure 10D is a photograph of a slide of a monolayer of Tu 138 cells exposed to 2xl0 8 pfu/ml of AvlLacZ4, repeated one time.
  • Figure 10B With a single application of virus at lxlO 8 pfu/ml, about 30% of the cells were transduced (Figure 10B) , and 60% of cells were transduced when the virus concentration was increased to 2xl0 8 pfu/ml, which represented a multiplicity of infection (MOD of 100.
  • the proportion of transduced cells was increased to 100% by applying a second infection of 2xl0 8 pfu/ml. ( Figure 10D) .
  • Example 6 Transduction of raft cultures of Tu 138 cells in vi tro Raft cultures of Tu 138 cells were established as described by Kopan, et al . , J. Cell. Biol., Vol. 109, pgs. 295-307 (1989) .
  • Fibroblast-seeded collagen gels were prepared at a concentration of 5xl0 4 cells/ml in 24-well plates. The collagen-fibroblast mixture was allowed to gel at 37°C for approximately 30 minutes, and then was incubated in complete medium overnight. The medium then was aspirated and Tu 138 cells were overlaid at 1x10° cells/ml and incubated to confluency.
  • the gels then were raised to the air-liquid interface by placing a collagen-coated nylon disc on top of a stainless steel mesh platform and then placing the gel onto the nylon disc. Medium then was added to the level of the platform. A 0.05 ml drop of virus suspension at 1.5xl0 10 pfu/ml was placed on the center of the raft and was not allowed to contact the submerged media. The rafts were exposed for 1 hour and then washed with media, and incubated for a further 24 hours at 37°C.
  • Raft cultures of Tu 138 cells showed a close resemblance to stratified epithelium of the oral cavity.
  • virus at a concentration of 1.5xl0 10 pfu/ml was pipetted onto the raft culture, the diffuse superficial layers expressed the ⁇ - galactosidase gene with transduction evident up to four cells deep. ( Figure 11) . No expression was found in the fibroblasts of the supporting base.
  • Example 7 In vivo transduction of tumor cells in nude mice
  • six nude female mice at 4 to 6 weeks of age obtained from Harlan Sprague-Dawley, Indianapolis, Indiana
  • lxlO 7 Tu 138 cells then were injected subcutaneously into the flanks of the nude mice.
  • Tumor modules then were injected directly using a total volume of 0.1 ml of virus suspension at a concentration of lxlO 10 pfu/ml.
  • 48 hours after the injection of the adenovirus AvlLacZ4 the mice were killed and the tumors were removed.
  • the squamous carcinomas that were induced in the nude mice by the injection of the Tu 138 cells were observed to grow subcutaneously with a pseudocapsule.
  • Intratumoral injections of 0.1 ml of adenovirus AvlLacZ4 at lxlO 10 pfu/ml produced diffuse expression of /3-galactosidase activity in about 30% of the cells within the tumor. This was seen at sites that were several cell layers distant to the injection site ( Figure 12) , but ⁇ -galactosidase activity did not extend beyond the pseudocapsule.

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Abstract

L'invention concerne un procédé pour traiter ou prévenir une maladie ou un trouble chez un hôte. Ce procédé consiste à administrer un agent thérapeutique aux cellules épithéliales orales, ce qui assure la transduction in vivo de ces cellules par l'administration systémique, à l'hôte, d'un vecteur viral comprenant un polynucléotide codant un agent thérapeutique pour produire cet agent dans les cellules épithéliales orales. Ce procédé peut être utilisé pour traiter les cellules épithéliales orales pré-cancéreuses ainsi que les tumeurs cancéreuses de l'épithélium oral.
PCT/US1996/006648 1995-05-11 1996-05-10 Therapie genique par transduction de cellules epitheliales orales WO1996035455A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005835A1 (fr) * 1993-08-26 1995-03-02 Baylor College Of Medicine Therapie genique pour tumeurs solides, papillomes et verrues
US5457189A (en) * 1989-12-04 1995-10-10 Isis Pharmaceuticals Antisense oligonucleotide inhibition of papillomavirus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457189A (en) * 1989-12-04 1995-10-10 Isis Pharmaceuticals Antisense oligonucleotide inhibition of papillomavirus
WO1995005835A1 (fr) * 1993-08-26 1995-03-02 Baylor College Of Medicine Therapie genique pour tumeurs solides, papillomes et verrues

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 53, issued 15 May 1993, STEELE et al., "Effects of Human Papillomavirus Type 18-Specific Antisense Oligonucleotides on the Transformed Phenotype of Human Carcinoma Cell Lines", pages 2330-2337. *
CANCER RESEARCH, Volume 54, issued 15 July 1994, LIU et al., "Growth Suppression of Human Head and Neck Cancer Cells by the Introduction of a Wild-Type p53 Gene Via a Recombinant Adenovirus", pages 3662-3667. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Volume 15D, Abstract CD414, issued 1991, STEELE et al., "Prospective Gene Therapy for Oral Cancer", page 33. *
ORAL ONCOLOGY, EUROPEAN JOURNAL OF CANCER, Volume 30B, Number 03, issued 1994, SHILLITOE et al., "Gene Therapy - Its Potential in the Management of Oral Cancer", pages 145-154. *

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