EP0975750A2 - Proteines modifiees de suppression des retinoblastomes - Google Patents

Proteines modifiees de suppression des retinoblastomes

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Publication number
EP0975750A2
EP0975750A2 EP98908570A EP98908570A EP0975750A2 EP 0975750 A2 EP0975750 A2 EP 0975750A2 EP 98908570 A EP98908570 A EP 98908570A EP 98908570 A EP98908570 A EP 98908570A EP 0975750 A2 EP0975750 A2 EP 0975750A2
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EP
European Patent Office
Prior art keywords
amino acid
cell
tumor suppressor
dna segment
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP98908570A
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German (de)
English (en)
Inventor
Hong-Ji Xu
Shi-Xue Hu
William F. Benedict
Yunli Zhou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baylor College of Medicine
University of Nebraska
University of Texas System
Original Assignee
Baylor College of Medicine
University of Nebraska
University of Texas System
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Application filed by Baylor College of Medicine, University of Nebraska, University of Texas System filed Critical Baylor College of Medicine
Publication of EP0975750A2 publication Critical patent/EP0975750A2/fr
Ceased legal-status Critical Current

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4736—Retinoblastoma protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates generally to the field of molecular and cellular biology. More particularly, it concerns modifications of the retinoblastoma tumor suppressor. The present invention further relates to the use of the instant modified retinoblastoma tumor suppressors in situations where providing a tumor suppressor or normal cell growth suppressor is indicated.
  • Cancers and tumors are the second most prevalent cause of death in the United States, causing approximately 450,000 deaths per year. One in three Americans will develop cancer, and one in five will die of cancer (Scientific American Medicine, part 12, I, 1, section dated 1987). While substantial progress has been made in identifying some of the likely environmental and hereditary causes of cancer, the statistics for the cancer death rate indicates a need for substantial improvement in the therapy for cancer and related diseases and disorders.
  • cancer genes A number of genes have been implicated in the etiology of cancer. These genes have been identified in connection with hereditary forms of cancer, and in a large number of well- studied tumor cells. Study of cancer genes has helped provide some understanding of the process of tumorigenesis. While a great deal more remains to be learned about cancer genes, the presently known cancer genes serve as useful models for understanding tumorigenesis. Cancer genes are broadly classified into "oncogenes" which, when activated, promote tumorigenesis, and “tumor suppressor genes” which, when damaged, fail to suppress tumorigenesis. While these classifications provide a useful method for conceptualizing tumorigenesis, it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
  • the oncogenes are somatic cell genes that are mutated from their wild-type alleles (the art refers to these wild-type alleles as protooncogenes) into forms which are able to induce tumorigenesis under certain conditions.
  • the oncogenes ras and myc are considered as models for understanding oncogenic processes in general.
  • the ras oncogene is believed to encode a cytoplasmic protein
  • the myc oncogene is believed to encode a nuclear protein.
  • the collaborative model of oncogene tumorigenesis must be qualified by the observation that a cell expressing the ras oncogene that is surrounded by normal cells does not undergo full transformation. However, if most of the surrounding cells are also r ⁇ s-expressing, then the ras oncogene alone is sufficient to induce tumorigenesis in a r ⁇ s-expressing cell. This observation validates the multiple hit theory of tumorigenesis because a change in the tissue environment of the cell hosting the oncogene may be considered a second hit.
  • events that collaborate with the activation of an oncogene such as ras or myc may include the inactivation of a negative regulatory factor or factors, i.e., a tumor suppressor protein (Weinberg, 1989; Goodrich et al, 1992a).
  • a negative regulatory factor or factors i.e., a tumor suppressor protein
  • Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation.
  • the gene coding for a tumor suppressor protein is mutated or deleted, the resulting mutant protein or the complete lack of a tumor suppressor protein may fail to correctly regulate cellular proliferation. This can lead to abnormal cellular proliferation, particularly if there is already existing damage to the cellular regulatory mechanism.
  • the lack of control of cellular proliferation has been linked to the development of a wide variety of human cancers (Weinberg, 1991). A number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes.
  • tumor suppressor genes and candidate tumor suppressor genes include, but are not limited to, the retinoblastoma (RB) gene (Friend et al, 1986; Fung et al, 1987; Lee et al, 1987a), the wild-type p53 gene (Finlay et al, 1989; Baker et al, 1990), the deleted in colon carcinoma (DCC) gene (Fearon et al, 1990a; 1990b), the neurofibromatosis type 1 (NF-1) gene (Wallace et al, 1990; Viskochil et al, 1990; Cawthon et al, 1990), the Wilms tumor (WT-1) gene (Call et al, 1990; Gessler et al, 1990; Pritchard- Jones et al, 1990), the von Hippel-Lindau (NHL) disease tumor suppressor gene (Duan et al, 1995), the Maspin (Zou et al, 1994), Brush- 1 (Schott et al
  • the first tumor suppressor gene identified was the retinoblastoma (RB) gene, which causes the hereditary retinoblastoma (Knudson, 1971; Murphree and Benedict, 1984; Knudson, 1985).
  • the retinoblastoma (RB) gene which was cloned in the middle 1980s, is one of the best studied tumor suppressor genes.
  • the RB gene has been shown to be missing or defective in a majority of retinoblastomas, sarcomas of the soft tissues and bones, and in approximately 20 to 40 percent of breast, lung, prostate and bladder carcinomas (Lee et al, WO 90/05180; Bookstein et al., 1991; Benedict et al, 1990).
  • the most direct proof that the cloned RB gene is indeed a tumor suppressor gene is the observed recovery of tumor suppression function in RB-minus tumor cells from the introduction of a cloned intact copy of the RB gene.
  • a number of reports have indicated that replacement of the normal RB gene in RB-defective tumor cells from disparate types of human cancers could suppress their tumorigenic activity in nude mice (Huang et al, 1988; Goodrich and Lee, 1993; Zhou et al, 1994b).
  • the tumor cell lines studied were derived from widely disparate types of human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung.
  • the RB cDNA open reading frame sequence (McGee et al, 1989) contains a second in-frame AUG codon located in exon 3, at nucleotides 355-357.
  • the protein initiated from this second AUG codon lacks the N-terminal 112 amino acid residues of the full-length RB protein,
  • _ 94 were RB . Even more striking was that the pRB expression also significantly reduced colony formation of two RB + (with normal RB alleles) tumor cell lines examined, namely the fibrosarcoma cell line, HT1080, and the cervical carcinoma cell line, HeLa (Xu et al, 1994b), no while no such effects were observed when an additional pRB -coding gene(s) was introduced by transfection using plasmid vectors (Fung et al, 1993) or by microcell fusion (Anderson et al,
  • the modified retinoblastoma tumor suppressors of the present invention overcome the shortcomings of those described in the art, providing a broad spectrum tumor suppressor with surprising beneficial effects.
  • the present invention provides broad-spectrum modified retinoblastoma tumor suppressor proteins that are suprisingly at least as effective, and in most cases more effective, than the corresponding wild-type retinoblastoma tumor suppressor proteins in inhibiting cell growth.
  • the invention provides retinoblastoma tumor suppressor proteins that have a modified N-terminal region.
  • the invention further provides methods of making and using the modified retinoblastoma tumor suppressor proteins, particularly in circumstances wherein cell growth inhibition is desired.
  • the present invention provides methods for treating diseases, as exemplified by, but not limited to cancer, that are characterized by abnormal cellular proliferation.
  • a broad-spectrum tumor suppressor gene is a genetic sequence coding for a protein that, when inserted into and expressed in an abnormally proliferating host cell, e.g., a tumor cell, suppresses abnormal proliferation of that cell irrespective of the cause of the abnormal proliferation.
  • the invention provides an isolated DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB or pRB 56 , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification.
  • pRB 4 and pRB 56 refer to retinoblastoma proteins that have a molecular weight of 94 kDa and 56 kDa, respectively.
  • the pRB and pRB retinoblastoma proteins are fragments of the full length wild-type retinoblastoma protein that have 112 and 379 contiguous amino acids deleted from the N-terminus, respectively.
  • N-terminal or “N-terminal region”, as used herein, will be understood to refer to the region of a protein corresponding to as much as the first approximately 40% of the amino acid sequence. Thus, these terms will be understood to include up to about the first 5%, the first 10%, the first 15%, the first 20%, the first 25%, the first 30% or the first 35% of the amino acid sequence of a protein. However, these values are only approximations, and therefore will be understood to include intermediate values, such as 2%, 3%, 6%, 7%, 11%, 13%, 17%, 18%, 22%, 26%, 33%, 37%, 38%, 41%, 42% and the like.
  • modified refers to deletions and/or mutations of the wild-type protein sequence. In certain embodiments, it may also refer to insertion of a heterologous amino acid or amino acids into the wild-type protein sequence. In yet other aspects, the term may refer to post-translational alteration of the wild-type amino acid sequence.
  • the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region that comprises a first sequence region from which at least one amino acid has been deleted.
  • the deletion may produce a modified retinoblastoma tumor suppressor protein with a biological activity equal to, or in certain embodiments, greater than the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein.
  • the gene encodes a modified retinoblastoma tumor suppressor protein wherein at least two amino acids have been deleted from the first sequence region. In other embodiments of the invention at least about five amino acids, at least about ten amino acids, at least about 25 amino acids, at least about 50 amino acids, at least about 75 amino acids or at least about 100 amino acids have been deleted from the first sequence region.
  • the gene encodes a modified retinoblastoma tumor suppressor protein wherein at least about 150 amino acids, at least about 200 amino acids, at lest about 250 amino acids, at least about 300 amino acids or at least about 370 amino acids have been deleted from the first sequence region.
  • intermediate sized deletions are also provided, exemplified by, but not limited to, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
  • the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region that comprises at least a first sequence region located between about amino acid 1 and about amino acid 50 from which at least one amino acid has been deleted. It will be understood that "between about amino acid 1 and about amino acid 50" includes amino acid 1 and amino acid 50, and it is thus so with other deletions described herein. Amino acid 1 is the N-terminal amino acid, and the numbers increase toward the C-terminus.
  • the first sequence region is located between about amino acid 51 and about amino acid 100, between about amino acid 101 and about amino acid 150, between about amino acid 151 and about amino acid 200, between about amino acid 201 and about amino acid 250 or between about amino acid 251 and about amino acid 300.
  • the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 100, between about amino acid 51 and about amino acid 150, between about amino acid 101 and about amino acid 200, between about amino acid
  • the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 150.
  • the first sequence region is located between about amino acid 51 and about amino acid 200, between about amino acid 101 and about amino acid 250 or between about amino acid 151 and about amino acid 300.
  • the gene encodes a modified retinoblastoma tumor suppressor protein wherein the first sequence region is located between about amino acid 1 and about amino acid 200, between about amino acid 51 and about amino acid 250, between about amino acid 101 and about amino acid 300, between about amino acid 1 and about amino acid 250, between about amino acid 51 and about amino acid 300, between about amino acid 1 and about amino acid 300 or between about amino acid 1 and about amino acid 370.
  • the modified retinoblastoma tumor suppressor protein is a modified retinoblastoma protein wherein about amino acid 2 through about amino acid 34 have been deleted from the first sequence region.
  • the location of these particular amino acids is in reference to the human wild-type retinoblastoma protein, but will be understood to correspond to analogous regions of homologous retinoblastoma proteins.
  • about amino acid 2 through about amino acid 55 have been deleted from the first sequence region.
  • about amino acid 2 through about amino acid 78 have been deleted from the first sequence region.
  • amino acid 2 through about amino acid 97 have been deleted from the first sequence region.
  • about amino acid 2 through about amino acid 148 have been deleted from the first sequence region.
  • the modified retinoblastoma tumor suppressor protein is a modified retinoblastoma protein wherein about amino acid 31 through about amino acid 107 have been deleted from the first sequence region. In another embodiment of the invention about amino acid 77 through about amino acid 107 have been deleted from the first sequence region. In a further embodiment of the invention about amino acid 111 through about amino acid 181 have been deleted from the first sequence region. In yet another embodiment of the invention about amino acid 111 through about amino acid 241 have been deleted from the first sequence region. In still another embodiment of the invention about amino acid 181 through about amino acid 241 have been deleted from the first sequence region. In a particular embodiment of the invention about amino acid 242 through about amino acid 300 have been deleted from the first sequence region.
  • the N-terminal region of the modified retinoblastoma tumor suppressor protein further comprises at least a second sequence region from which at least one amino acid has been deleted.
  • about amino acid 2 through about amino acid 34, and about amino acid 76 through about amino acid 112 have been deleted.
  • about amino acid 2 through about amino acid 55, and about amino acid 76 through about amino acid 112 have been deleted.
  • Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification wherein the gene encodes a modified retinoblastoma tumor suppressor protein comprising at least a first N-terminal mutation, and wherein the modified retinoblastoma tumor suppressor protein has an increased biological activity in comparison to the biological activity of the corresponding wild type retinoblastoma tumor suppressor protein.
  • the gene encodes a modified retinoblastoma protein comprising a mutation at position 111.
  • the modified retinoblastoma protein comprises glycine at position 111 in place of aspartic acid.
  • the modified retinoblastoma tumor suppressor protein comprises at least a second N-terminal mutation.
  • the gene encodes a modified retinoblastoma protein comprising a mutation at position 111 and a mutation at position 112.
  • the modified retinoblastoma protein comprises glycine at position 111 in place of aspartic acid, and aspartic acid at position 112 in place of glutamic acid.
  • the gene encodes a modified retinoblastoma tumor suppressor protein comprising an N-terminal region from which at least one amino acid has been deleted, and which contains at least one amino acid mutation.
  • the gene encodes a modified retinoblastoma tumor suppressor protein that comprises a contiguous amino acid sequence from at least about position 370 to about position 928 of SEQ ID NO:2.
  • the gene encodes a modified retinoblastoma tumor suppressor protein that comprises a contiguous amino acid sequence from at least about position 3 to about position 928 of SEQ ID NO:2.
  • a contiguous amino acid sequence will be understood to be a contiguous amino acid sequence of at least about 8, about 10, about 12, about 15, about 20, about 25, about 50 or about 100 amino acids and so on up to the full length amino acid sequence.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:29.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2691 of SEQ ID NO:28.
  • a contiguous nucleic acid sequence will be understood to be a contiguous nucleic acid sequence of at least about 8, about 10, about 12, about 15, about 17, about 20, about 25, about 50 or about 100 nucleotides and so on up to the full length nucleotide sequence.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:31.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2628 of SEQ ID NO:30.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:33.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2559 of SEQ ID NO:32. In a further embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:35. In yet another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2502 of SEQ ID NO:34. In still another embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:37. In a particular embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2349 of SEQ ID NO:36. In an additional embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:39.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2559 of SEQ ID NO:38.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:41.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2697 of SEQ ID NO:40.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:43.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2583 of SEQ ID NO:42.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:45.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2397 of SEQ ID NO:44.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:47. In another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2613 of SEQ ID NO:46. In a further embodiment of the invention the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:49. In yet another embodiment of the invention the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2619 of SEQ ID NO:48.
  • the gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO: 51.
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2790 of SEQ ID NO:50.
  • the invention thus provides a gene encodes a modified retinoblastoma protein comprising a contiguous amino acid sequence of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:
  • the gene comprises a contiguous nucleic acid sequence from between position 7 and position 2691 of
  • SEQ ID NO:28 from between position 7 and position 2628 of SEQ ID NO:30, from between position 7 and position 2559 of SEQ ID NO:32, from between position 7 and position 2502 of
  • SEQ ID NO:34 from between position 7 and position 2349 of SEQ ID NO:36, from between position 7 and position 2559 of SEQ ID NO:38, from between position 7 and position 2697 of
  • SEQ ID NO:40 from between position 7 and position 2583 of SEQ ID NO:42, from between position 7 and position 2397 of SEQ ID NO:44, from between position 7 and position 2613 of SEQ ID NO:46, from between position 7 and position 2619 of SEQ ID NO:48 or from between position 7 and position 2790 of SEQ ID NO:50.
  • Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB or pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, where the DNA segment is operationally positioned under the control of a promoter. In one embodiment of the invention this DNA segment is operationally positioned under the control of a recombinant promoter. In another embodiment of the invention the DNA segment is further defined as a recombinant vector. In a particular aspect of the present invention, the recombinant vector is an adenoviral vector. In another aspect, the recombinant vector is a retro viral vector.
  • the DNA segment is further defined as a component of a tetracycline responsive expression system.
  • the DNA segment is operatively positioned downstream of a promoter comprising a tetracycline operator nucleic acid sequence; the tetracycline responsive expression system further comprising a second sequence region comprising an isolated gene encoding a fusion protein comprising a transcriptional transactivation domain operatively attached to a tetracycline repressor protein, the second sequence region operatively positioned downstream of a minimal promoter.
  • the tetracycline responsive expression system is comprised within an adenoviral vector.
  • the adenoviral vector is comprised within a recombinant adenovirus.
  • the invention also provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, which is comprised within a host cell.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is a human cell.
  • the host cell is a tumor cell.
  • the host cell is comprised within an animal. In a particular embodiment of the invention the animal is a human subject.
  • Another embodiment of the invention provides a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB 94 , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, which is dispersed in a pharmaceutically acceptable excipient.
  • Yet another embodiment of the invention provides an isolated DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification, wherein the modified retinoblastoma tumor suppressor protein is characterized as: comprising an
  • the modified retinoblastoma tumor suppressor protein has a biological activity at least about equivalent to the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein; or comprising an N-terminal region that comprises a first sequence region comprising at least one mutation, and wherein the modified retinoblastoma tumor suppressor protein has an increased biological activity in comparison to the biological activity of the corresponding wild-type retinoblastoma tumor suppressor protein.
  • the DNA segments as described above are contemplated for use in expressing a modified retinoblastoma tumor suppressor protein, for example in a host cell.
  • the DNA segments are contemplated for use in inhibiting cellular proliferation, or in the preparation of a medicament for inhibiting cellular proliferation or treating cancer, for example in a human patient.
  • the use of the instant DNA segments in the preparation of a modified retinoblastoma tumor suppressor protein, in inhibiting cellular proliferation, and in the preparation of a medicament for inhibiting cellular proliferation or treating cancer is provided.
  • the medicament is intended for administration to a human patient, or formulated for parenteral administration.
  • the invention further provides a modified retinoblastoma tumor suppressor protein other than pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification.
  • the invention also provides a recombinant host cell comprising a DNA segment comprising an isolated gene encoding a modified retinoblastoma tumor suppressor protein other than pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification.
  • the host cell is a prokaryotic host cell.
  • the host cell is E. coli.
  • the host cell is a eukaryotic host cell.
  • the host cell is a tumor cell.
  • the DNA segment is introduced into the cell by means of a recombinant vector.
  • the invention further provides a method of inhibiting cellular proliferation, comprising contacting a cell with an effective inhibitory amount of a first modified retinoblastoma tumor suppressor protein other than pRB , the modified retinoblastoma tumor suppressor protein comprising an N-terminal modification.
  • the first modified retinoblastoma tumor suppressor protein comprises a modified retinoblastoma protein from which amino acids 111 through 241 have been deleted.
  • the first modified retinoblastoma tumor suppressor protein comprises a modified retinoblastoma protein that comprises a mutation at position 111 and position 112.
  • the first modified retinoblastoma tumor suppressor protein is prepared by expressing a DNA segment encoding the modified retinoblastoma tumor suppressor protein in a recombinant host cell and collecting the modified retinoblastoma tumor suppressor protein expressed by the cell.
  • the cell is contacted with the first modified retinoblastoma tumor suppressor protein by providing to the cell a DNA segment that expresses the first modified retinoblastoma tumor suppressor protein in the cell.
  • the cell is provided with a tetracycline responsive expression vector system that expresses the first modified retinoblastoma tumor suppressor protein in the cell.
  • the vector system is an adenoviral vector system.
  • Another aspect of the invention provides a method of inhibiting cellular proliferation, comprising contacting a tumor cell with an effective inhibitory amount of a first modified retinoblastoma tumor suppressor protein other than pRB , the protein comprising an N-terminal modification.
  • the cell is located within an animal and the first modified retinoblastoma tumor suppressor protein, or a gene encoding the modified retinoblastoma tumor suppressor protein, is administered to the animal in a pharmaceutically acceptable vehicle.
  • the term "gene” is defined as an isolated DNA segment that includes the coding region of the protein, or a portion thereof. Thus the term “gene” includes genomic DNA, cDNA or RNA encoding the protein.
  • the animal is a human subject.
  • the cell is further contacted with a second tumor suppressor protein.
  • the cell is contacted with a modified retinoblastoma protein and a wild-type retinoblastoma, p53 or other tumor suppressor protein.
  • the invention further provides a method of inhibiting cellular proliferation, comprising contacting a cell with a retinoblastoma protein and a p53 protein in a combined amount effective to inhibit cellular proliferation in the cell.
  • the invention also provides a method of treating cancer, comprising administering to an animal with cancer a pharmaceutically acceptable composition comprising a biologically effective inhibitory amount of a first modified retinoblastoma tumor suppressor protein, other than pRB , that comprises an N-terminal modification.
  • cancer or “tumor” are clinically descriptive terms which encompass a myriad of diseases characterized by cells that exhibit unchecked and abnormal cellular proliferation.
  • tissue when applied to tissue, generally refers to any abnormal tissue growth, i.e., excessive and abnormal cellular proliferation.
  • a tumor may be "benign” and unable to spread from its original focus, or “malignant” and capable of spreading beyond its anatomical site to other areas throughout the hostbody.
  • cancer is an older term which is generally used to describe a malignant tumor or the disease state arising therefrom.
  • the art refers to an abnormal growth as a neoplasm, and to a malignant abnormal growth as a malignant neoplasm.
  • abnormal cellular proliferation is the result of a failure of one or more of the mechanisms controlling cell growth and division.
  • the mechanisms controlling cell growth and division include the genetic and tissue- mediated regulation of cell growth, mitosis and differentiation. These mechanisms are thought to act at the cell nucleus, the cell cytoplasm, the cell membrane and the tissue-specific environment of each cell. The process of transformation of a cell from a normal state to a condition of excessive or abnormal cellular proliferation is called tumorigenesis.
  • tumorigenesis is usually a multistep progression from a normal cellular state to, in some instances, a full malignancy. It is therefore believed that multiple "hits" upon the cell regulatory mechanisms are required for full malignancy to develop. Thus, in most instances, it is believed that there is no single cause of excessive proliferation, but that these disorders are the end result of a series of cumulative events.
  • a malignant tumor or cancer capable of unchecked and rapid spread throughout the body is the most feared and usually the deadliest type of tumor, even so-called benign tumors or growths can cause significant morbidity and mortality by their inappropriate growth.
  • a benign tumor can cause significant damage and disfigurement by inappropriate growth in cosmetically sensitive areas, or by exerting pressure on central or peripheral nervous tissue, blood vessels and other critical anatomical structures.
  • FIG. 1 Relative activities of the modified hCMV promoters.
  • the 5637 bladder carcinoma cells (lanes 1-5) and Saos2 osteocarcinoma cells (lanes 6-10) were transfected with reporter plasmids in which CAT gene expression was driven by the various modified (mhCMVp3, lanes 2 and 7; mhCMVp2, lanes 3 and 8; mhCMVpl, lanes 4 and 9) or full-length hCMV promoters (lanes 5 and 10).
  • the % CAT activity is shown on the vertical axis.
  • the CAT activity of the cells transfected with the plasmid carrying the full-length hCMV promoter (lanes 5 and 10) is defined as 100 percent.
  • FIG. 2 Expression of tTA from the modified mCMVp-t7 i cassette has no squelching effects on the 5637 cell growth.
  • a method of staining cells with crystal violet followed by measuring OD 550 was used for quantification of relative cell numbers (OD 550 shown on vertical axis; Gillies et al, 1986). Shown is the growth parent cells with (A) and without (D) tetracycline, and the mCMVp-t7 ⁇ 4 transfected cells with ( ⁇ ) and without (O) tetracycline. Days after transfection are shown on the horizontal axis. FIG. 3A, FIG. 3B and FIG. 3C.
  • FIG. 3A Representative long-term clone from the ⁇ -reconstituted osteosarcoma cell line (Saos-2, clone 11).
  • FIG. 3B Representative long-term clone from the i?5-reconstituted breast carcinoma cell line (MDA-MB-468, clone 19-4).
  • FIG. 3C Representative long-term clone from the i?5-reconstituted bladder carcinoma cell line (5637, clone 34-6). The cells were grown in the presence of 0.5 ⁇ g/ml of Tc (D) versus absence of Tc (O).
  • FIG. 4A, FIG. 4B and FIG. 4C The effects of tetracycline-regulatable pRB expression on soft agar colony formation.
  • FIG. 4A Percent colony formation (vertical axis) for three independent Saos2 osteosarcoma cell line clones (RBI 10 C14, lane 2; RBI 10 Oi l, lane 3; RBI 10 C113, lane 4) and the Saos2 parent strain (lane 1).
  • FIG. 4B Percent colony formation (vertical axis) for two independent MDA-MB-468 breast carcinoma cell line clones (Rbl lO C119-4, lane 2; RM 10O20-1, lane 3) and the MDA-MB-468 parent strain (lane 1).
  • FIG. 4C Percent colony formation (vertical axis) for two independent 5637 bladder carcinoma cell line clones (Rbl lO C134-6, lane 2; Rbl lO C136-9, lane 3) and the 5637 parent strain (lane 1). Soft agar colony formation of tumor cells with tetracycline-regulatable pRB expression was completely abrogated by induction of pRB in tetracycline-free medium. Colony formation is shown in the presence (open bar) and the absence (hatched bar) of tetracycline.
  • FIG. 5 Time course analysis of the pRB and pRB expression in representative, Tc- regulatable Saos-2 cell clones in Tc-free media and its effects on DNA synthesis, using a
  • Tumor Suppressor Proteins 1. Retinoblastoma Based upon study of the isolated RB cDNA clone, the predicted RB gene product has 928 amino acids and an expected molecular weight of 106 kDa (Lee et al, 1987a; 1987b). The natural factor corresponding to the predicted RB gene expression product has been identified as a nuclear phosphoprotein having an apparent relative molecular mass (M r ) of between 105 and 114 kDa (Lee et al, 1987b; Xu et al, 1989b; Yokota et al, 1988; Whyte et al, 1988). The literature
  • RR generally refers to the protein encoded by the RB gene as pi 10 .
  • normal human cells show an RB protein pattern consisting of a lower sharp band with an Mr of 110 kD and a broader, more variable region above this band with an M r ranging from 110 kD to 116 kD.
  • the 110 kD band is the underphosphorylated RB protein, whereas the broader region represents the phosphorylated RB protein.
  • the heterogeneity of the molecular mass results from a varying degree of phosphorylation (Xu et al. , 1989b).
  • the RB protein shows cyclical changes in phosphorylation during the cell cycle. Most RB protein is unphosphorylated during GI phase, but most (perhaps all) RB molecules are phosphorylated in S and G2 phases (Xu et al, 1989b; DeCaprio et al, 1989; Buchkovich et al, 1989; Chen et al, 1989; Mihara et al, 1989).
  • the established components of the pRB pathway include the E2F transcription factors, which are involved in transcriptional control of numerous cellular genes responsible for the advances of cells through the cell cycle (Nevins, 1992; La Thangue, 1994).
  • the pRB also interacts with certain GI phase cyclins (Koff et al, 1992; Resnitzky and Reed, 1995; Geng et al, 1996). Therefore, the RB gene apparently plays a key role in cell growth regulation being involved in the major decisions during the GI phase of the cell cycle which govern cell proliferation, quiescence and differentiation (Weinberg, 1995). Furthermore, only the underphosphorylated RB protein binds to SV40 large T antigen.
  • the phosphorylation status of pRB undergoes a readily distinguishable alteration at a time close to and perhaps contemporaneous with the R point transition of the cell cycle (Weinberg, 1995).
  • middle GI phase the only pRB species detected is an underphosphorylated form.
  • the majority of pRB synthesized after middle GI phase is hyperphosphorylated.
  • pRB hyperphosphorylation occurs in late GI, preceding the Gl/S boundary (Xu et al, 1991a; Mittnacht et al, 1994).
  • pRB maintains this hyperphosphorylated status throughout the remainder of the cell cycle, becoming dephosphorylated only upon evolution from M/early GI (Ludlow et al, 1990; Xu et al, 1991a; Mittnacht et al, 1994).
  • the underphosphorylated form of pRB is able to form complexes with the transcription factor E2Fs or directly interact with the E2F site, and switches the E2F site from a positive to negative element in transcriptional control.
  • the E2F site is present in the promoters of diverse cellular genes that are responsible for the advances of cells through the cell cycle, including c-myc, B-myb, cdc2, dihydrofolate reductase, thymidine kinase, and the RB as well as the E2F-1 gene itself (Chellappan et al, 1991; Nevins, 1992; Weintraub et al, 1992; La Thangue, 1994; Shan et al, 1994; Sardet et al, 1995; Shan et al, 1996). Since hyperphosphorylated pRB appears to have lost the ability to interact with E2Fs, the inhibitory function of pRB on cell growth can be abrogated by hyperphosphorylation.
  • pRB is an R point guardian.
  • pRB exerts most of its growth inhibitory effects in the first two thirds of the GI phase. A cell that has progressed through early and middle GI encounters the R point gate. Should conditions be ready for advance into the remainder of the cell cycle, pRB will undergo phosphorylation and functional inactivation, causing it to open the gate and to permit the cell to proceed into late GI. Cells that lack normal pRB function for various reasons will proceed freely into late GI.
  • pRB Without pRB, the upstream components of the cell cycle clock that regulate pRB phosphorylation, such as cyclin D, cyclin E and their corresponding cyclin-dependent kinases (CDKs) (Kato et al, 1993; Ewen et al, 1993) lose much of their influences in the decision of the cell to pass through the R point gate.
  • CDKs cyclin-dependent kinases
  • pRB allows the cell cycle clock to control the expression of numerous genes that mediate advance of the cell through a critical phase of its growth cycle being involved in the major decisions concurrent with the R point transition. Functional loss of pRB deprives the cell of this clock and thus of an important mechanism for braking cell proliferation.
  • RB gene Various mutations of the RB gene are known, and these are generally inactive. Mutations in RB are seen in virtually all cases of retinoblastoma; additionally, the RB gene products could potentially be inactivated by hyperphosphorylation, and by viral oncoprotein-like cellular protein binding. Although the RB gene was initially named because deletions or mutations within the gene caused the rare childhood ocular tumor, retinoblastoma, loss of pRB function is not only causally related to the retinoblastoma, but is also linked to the progression of many common human cancers. Additionally, there is growing evidence suggesting that the RB protein status is potentially a prognostic marker in urothelial carcinoma, non-small cell lung carcinoma, and perhaps also in some other types of human neoplasms (Xu, 1995).
  • the most direct proof that the cloned RB gene is indeed a tumor suppressor gene comes from introduction of a cloned intact copy of the gene into cancer cells with observed tumor suppression function.
  • a number of reports have indicated that replacement of the normal RB gene in RB-defective tumor cells from disparate types of human cancers could suppress their tumorigenic activity in nude mice (Huang et al, 1988; Goodrich and Lee, 1993; Zhou et al, 1994b).
  • the tumor cell lines studied were derived from widely disparate types of human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung (Table 2).
  • RB may also play a role in elicitation of immunogenicity of tumor cells (Lu et al, 1994; Lu et al, 1996), anti-angiogenesis (Dawson et al, 1995) and suppression of tumor invasiveness (Li et al, 1996), which make the emerging RB gene therapy even more attractive.
  • preclinical studies have recently demonstrated that treatment of established human xenograft tumors in nude mice by recombinant adenovirus vectors expressing either wild-type or an N-terminal truncated retinoblastoma protein resulted in regression of the treated tumors (Xu et al, 1996).
  • the RB cDNA open reading frame sequence (McGee et al, 1989) contains a second in-frame AUG codon located in exon 3, at nucleotides 355-357. The deduced second AUG
  • RR codon-initiated RB protein would be 98 kD, or 12 kD smaller than the pi 10 protein. It has been proposed that the lower molecular weight bands are the underphosphorylated (98 kD) and phosphorylated (98-104 kD) RB protein translated from the second AUG codon of the RB mRNA (Xu et al, 1989b), and this was later shown conclusively (Xu et al, U.S. Patent
  • This protein is referred to as the p94 protein.
  • RR pi 10 coding gene did not change the neoplastic phenotype of such tumor lines (Huang et al, 1988).
  • Somatic cell mutations of the p53 gene are said to be the most frequently mutated gene in human cancer (Weinberg, 1991).
  • the normal or wild-type p53 gene is a negative regulator of cell growth, which, when damaged, favors cell transformation (Weinberg, 1991).
  • the p53 expression product is found in the nucleus, where it may act in parallel
  • RR both p53 and pi 10 proteins are targeted for binding or destruction by the oncoproteins of SV40, adenovirus and human papillomavirus.
  • Tumor cell lines deleted for p53 have been successfully treated with wild-type p53 vector to reduce tumorigenicity (Baker et al, 1990).
  • the introduction of either p53 or RB 11 into cells that have not undergone lesions at these loci does not affect cell proliferation (Marshall, 1991; Baker et al, 1990; Huang et al, 1988).
  • Such experiments suggest that sensitivity of cells to the suppression of their growth by a tumor suppressor gene is dependent on the genetic alterations that have taken place in the cells.
  • Neurofibromatosis type 1 or von Recklinghausen neurofibromatosis results from the inheritance of a predisposing mutant allele or from alleles created through new germline mutations (Marshall, 1991).
  • the neurofibromatosis type 1 gene referred to as the NFl gene, is a relatively large locus exhibiting a mutation rate of around 10 . Defects in the NFl gene result in a spectrum of clinical syndromes ranging from cafe-au-lait spots to neurofibromas of the skin and peripheral nerves to Schwannomas and neurofibrosarcomas.
  • the NFl gene encodes a protein of about 2485 amino acids that shares structural similarity with three proteins that interact with the products of the ras protooncogene (Weinberg, 1991).
  • the NFl amino acid sequence shows sequence homology to the catalytic domain of ras GAP, a GTPase- activating protein for p21 ras (Marshall, 1991).
  • NFl in cell cycle regulation
  • it is a suppressor of oncogenically activated p21 ras in yeast (Marshall, 1991 citing Ballester et al, 1990).
  • other possible pathways for NFl interaction are suggested by the available data (Marshall, 1991; Weinberg, 1991).
  • no attempts to treat NFl cells with a wild-type NFl gene have been undertaken due to the size and complexity of the NFl locus. Therefore, it would be highly desirable to have a broad-spectrum tumor suppressor gene able to treat NFl and any other type of cancer or tumor.
  • the multiple steps in the tumorigenesis of colon cancer are readily monitored during development by colonoscopy.
  • the combination of colonoscopy with the biopsy of the involved tissue has uncovered a number of degenerative genetic pathways leading to the result of a malignant tumor.
  • One well studied pathway begins with large polyps in which 60% of the cells carry a mutated, activated allele of K-ras. A majority of these tumors then proceed to the inactivation-mutation of the gene referred to as the deleted in colon carcinoma (DCC) gene, followed by the inactivation of the p53 tumor suppressor gene.
  • DCC deleted in colon carcinoma
  • the DCC gene is a more than approximately one million base pair gene coding for a 190- kD transmembrane phosphoprotein which is hypothesized to be a receptor (Weinberg, 1991), the loss of which allows the affected cell a growth advantage. It has also been noted that the DCC has partial sequence homology to the neural cell adhesion molecule (Marshall, 1991) which might suggest a role for the DCC protogene in regulating cell to cell interactions.
  • the large size and complexity of the DCC gene, together with the complexity of the K-ras, p53 and possibly other genes involved in colon cancer tumorigenesis demonstrates a need for a broad-spectrum tumor suppressor gene and methods of treating colon carcinoma cells which do not depend upon manipulation of the DCC gene or on the identification of other specific damaged genes in colon carcinoma cells.
  • Tumor Suppressor Proteins examples include, but are not limited to; the Wilms tumor (WT-1) gene (Call et al, 1990; Gessler et al, 1990; Pritchard-Jones et al, 1990), the von Hippel-Lindau (VHL) disease tumor suppressor gene (Duan et al, 1995), the Maspin (Zou et al, 1994), Brush- 1 (Schott et al, 1994) and BRCA 1 genes (Miki et al, 1994; Futreal et al, 1994) for breast cancer, and the multiple tumor suppressor (MTS) or pi 6 gene (Serrano et al, 1993; Kamb et al, 1994).
  • WT-1 Wilms tumor
  • VHL von Hippel-Lindau
  • MLS multiple tumor suppressor
  • the tumor suppressor genes may be stably integrated into the genome of the cell.
  • the genes may be stably maintained in the cell as a separate, episomal segment of DNA.
  • Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance or replication independent of or in synchronization with the host cell cycle. How the tumor suppressor gene is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.
  • adenovirus vectors and particularly tetracycline-controlled adenovirus vectors. These vectors may be employed to deliver and express a wide variety of genes, including, but not limited to, tumor suppressor genes such as the retinoblastoma and p53 genes, in addition to cytokine genes such as tumor necrosis factor , the interferon gene family and the interleukin gene family.
  • tumor suppressor genes such as the retinoblastoma and p53 genes
  • cytokine genes such as tumor necrosis factor , the interferon gene family and the interleukin gene family.
  • a preferred method for delivery of the expression constructs involves the use of an adenovirus expression vector.
  • adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
  • "Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct in host cells with complementary packaging functions and (b) to ultimately express a heterologous gene of interest that has been cloned therein.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • adenovirus a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences (Grunhaus and Horwitz, 1992).
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because wild-type adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5 '-tripartite leader
  • recombinant adenovirus is generated from homologous recombination between a shuttle vector and a master plasmid which contains the backbone of the adenovirus genome. Due to the possible recombination between the backbone of the adenovirus genome, and the cellular DNA of the helper cells which contain the missing portion of the viral genome, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
  • adenovirus generation and propagation of most adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (El A and E1B; Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of most adenovirus vectors is at least 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the preferred helper cell line is 293.
  • Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25%) of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
  • AdPK adenovirus mediated gene delivery to multiple cell types has been found to be much less efficient compared to epithelial derived cells.
  • a new adenovirus, AdPK has been constructed to overcome this inefficiency (Wickham et al, 1996).
  • AdPK contains a heparin- binding domain that targets the virus to heparan-containing cellular receptors, which are broadly expressed in many cell types. Therefore, AdPK delivers genes to multiple cell types at higher efficiencies than unmodified adenovirus, thus improving gene transfer efficiency and expanding the tissues amenable to efficient adenovirus mediated gene therapy.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect (Brough et al. , 1996).
  • Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 to 10 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No severe side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1991; Rich et al, 1993).
  • trachea instillation Rosenfeld et al, 1991; 1992
  • muscle injection Rogot et al, 1993
  • peripheral intravenous injections Herz and Gerard, 1993
  • stereotactic inoculation into the brain Le Gal La Salle et al, 1993.
  • Recombinant adenovirus and adeno-associated virus can both infect and transduce non-dividing human primary cells.
  • Adeno-associated virus is also an attractive system for use in construction of vectors for delivery of and expression of tumor suppressor genes as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo.
  • AAV has a broad host range for infectivity (Tratschin et al, 1984; Laughlin et al, 1986; Lebkowski et al, 1988; McLaughlin et al, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each incorporated herein by reference.
  • AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al, 1994; Lebkowski et al, 1988; Samulski et al, 1989; Yoder et al, 1994; Zhou et al, 1994a; Hermonat and Muzyczka, 1984; Tratschin et al, 1985; McLaughlin et al, 1988) and genes involved in human diseases (Flotte et al, 1992; Luo et al, 1994; Ohi et al, 1990; Walsh et al, 1994; Wei et al, 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.
  • AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992).
  • another virus either adenovirus or a member of the herpes virus family
  • helper virus the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al, 1990; Samulski et al, 1991).
  • rAAV is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994).
  • recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al. , 1988; Samulski et al, 1989; each inco ⁇ orated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al, 1991; incorporated herein by reference).
  • the cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function.
  • rAAV virus stocks made in such fashion are contaminated with adenovirus which must be inactivated by heat shock or physically separated from the rAAV particles (for example, by cesium chloride density centrifugation).
  • adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes could be used (Yang et al, 1994; Clark et al, 1995). Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al, 1995).
  • the retroviruses are a group of single- stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retroviral vectors can limit their use for stable gene transfer in eukaryotic cells.
  • a murine leukemia virus-derived vector has been developed in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus (Burns et al, 1993).
  • These vectors can be concentrated to extremely high titers (10 colony forming units/ml), and can infect cells that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. These vectors may facilitate gene therapy model studies and other gene transfer studies that require direct delivery of vectors in vivo.
  • Baculovirus expression vectors are useful tools for the production of proteins for a variety of applications (Summers and Smith, 1987; O'Reilly et al, 1992; also U.S. Patent Nos., 4,745,051 (Smith and Summers), 4,879,236 (Smith and Summers), 5,077,214 (Guarino and Jarvis), 5,155,037 (Summers), 5,162,222, (Guarino and Jarvis), 5,169,784 (Summers and Oker- Blom) and 5,278,050 (Summers), each incorporated herein by reference).
  • the inventors contemplate the construction of baculoviral expression vectors wherein gene expression is regulated by tetracycline. These vectors might be particularly useful, for example, where the desired protein is toxic to the insect cells. In these instances, production of the protein can be turned off until the cells have reached a very high density, thereby still allowing for the production of large quantities of the desired protein.
  • Baculovirus expression vectors are recombinant insect vectors in which the coding region of a particular gene of interest is placed behind a promoter in place of a nonessential baculoviral gene.
  • the classic approach used to isolate a recombinant baculovirus expression vector is to construct a plasmid in which the foreign gene of interest is positioned downstream of the polyhedrin promoter. Then, via homologous recombination, that plasmid can be used to transfer the new gene into the viral genome in place of the wild-type polyhedrin gene (Summers and Smith, 1987; O'Reilly et al, 1992).
  • the resulting recombinant virus can infect cultured lepidopteran insect cells or larvae and express the foreign gene under the control of the polyhedrin promoter, which is strong and provides very high levels of transcription during the very late phase of infection.
  • the strength of the polyhedrin promoter is an advantage of the use of recombinant baculoviruses as expression vectors because it usually leads to the synthesis of large amounts of the foreign gene product during infection.
  • viral vectors may be employed for construction of expression vectors in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and
  • Sindbis virus and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;
  • the expression constructs to be delivered are housed within an infective virus that has also been engineered to express a specific binding ligand.
  • the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
  • a novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • tumor suppressor genes of the present invention are contemplated.
  • the expression construct In order to effect expression of a gene construct, the expression construct must be delivered into a cell. As described herein, a preferred mechanism for delivery is via viral infection, where the expression construct is encapsidated in an infectious viral particle. However, several non- viral methods for the transfer of expression constructs into eukaryotic and prokaryotic cells also are contemplated by the present invention. In one embodiment of the present invention, the expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL).
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987). Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non- histone chromosomal proteins (HMG-1) (Kato et al, 1991).
  • HMG-1 nuclear non- histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • the expression construct is introduced into the cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
  • Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al, 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al, 1986) in this manner.
  • the expression construct is introduced to the cells using calcium phosphate precipitation.
  • Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
  • mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990).
  • the expression construct is delivered into the cell using DEAE- dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Further embodiments of the present invention include the introduction of the expression construct by direct microinjection or sonication loading.
  • Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985), and LTK" fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et ⁇ /., 1987).
  • the expression construct is introduced into the cell using adenovirus assisted transfection.
  • Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al, 1992; Curiel, 1994).
  • receptor-mediated delivery vehicles that may be employed to deliver the construct to the target cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In view of the cell type-specific distribution of various receptors, this delivery method adds a degree of specificity to the present invention. Specific delivery in the context of another mammalian cell type is described by Wu and Wu (1993; inco ⁇ orated herein by reference).
  • Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a DNA-binding agent. Others comprise a cell receptor-specific ligand to which the DNA construct to be delivered has been operatively attached.
  • Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et al, 1994; Myers, EPO 0273085), which establishes the operability of the technique.
  • the ligand will be chosen to correspond to a receptor specifically expressed on the neuroendocrine target cell population.
  • the DNA delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome.
  • the nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally inco ⁇ orated into the liposome membrane.
  • the liposome will thus specifically bind to the receptors of the target cell and deliver the contents to the cell.
  • Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
  • EGF epidermal growth factor
  • the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • Nicolau et al (1987) employed lactosyl-ceramide, a galactose-terminal asialoganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into the target cells in a similar manner.
  • the present invention also provides recombinant candidate screening and selection methods which are based upon whole cell assays and which, preferably, employ a reporter gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional.
  • reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture.
  • a genetic marker is provided which is detectable by standard genetic analysis techniques, such as DNA or RNA amplification by PCRTM or hybridization using fluorometric, radioisotopic or spectrophotometric probes.
  • Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by their activity, as will be known to those skilled in the art.
  • Contemplated for use in the present invention is green fluorescent protein (GFP) as a marker for transgene expression (Chalfie et al, 1994). The use of GFP does not need exogenously added substrates, only irradiation by near UV or blue light, and thus has significant potential for use in monitoring gene expression in living cells.
  • GFP green fluorescent protein
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • Other marker genes within this class are well known to those of skill in the art, and are suitable for use in the present invention.
  • reporter genes which confer detectable characteristics on a host cell are those which encode polypeptides, generally enzymes, which render their transformants resistant against toxins.
  • Examples of this class of reporter genes are the neo gene (Colberre-Garapin et al, 1981) which protects host cells against toxic levels of the antibiotic G418, the gene conferring streptomycin resistance (U. S. Patent 4,430,434), the gene conferring hygromycin B resistance (Santerre et al, 1984; U. S.
  • Patents 4,727,028, 4,960,704 and 4,559,302) a gene encoding dihydrofolate reductase, which confers resistance to methotrexate (Alt et al, 1978), the enzyme HPRT, along with many others well known in the art (Kaufman, 1990).
  • tumor suppressor proteins exemplified by the retinoblastoma protein
  • the present invention contemplates the use of tumor suppressor proteins, exemplified by the retinoblastoma protein, which contain modifications within the N-terminal region which confer equal or greater tumor suppression activity on the resultant protein
  • alteration of the unmodified C-terminal portion of the protein such that biological activity is maintained also falls within the scope of the present invention.
  • modification and changes may be made in the structure of, for example, the retinoblastoma protein, and still obtain a molecule having like or otherwise desirable characteristics.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of tumor suppression activity. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. Equally, the same considerations may be employed to create a protein or polypeptide with countervailing ⁇ e.g., antagonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of tumor suppressor proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.
  • Conservative substitutions well known in the art include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, inco ⁇ orated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular tumor suppressor or cytokine protein.
  • site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically.
  • This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence a ⁇ angement.
  • a genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones inco ⁇ orating the mutagenic oligonucleotide.
  • PCRTM with commercially available thermostable enzymes such as Taq polymerase may be used to inco ⁇ orate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector.
  • thermostable enzymes such as Taq polymerase
  • the PCRTM-mediated mutagenesis procedures of Tomic et al. (1990) and Upender et al (1995) provide two examples of such protocols.
  • thermostable ligase in addition to a thermostable polymerase may also be used to inco ⁇ orate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector.
  • the mutagenesis procedure described by Michael (1994) provides an example of one such protocol.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
  • oligonucleotide directed mutagenesis procedure is intended to refer to a process that involves the template-dependent extension of a primer molecule.
  • template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Patent 4,237,224, specifically inco ⁇ orated herein by reference in its entirety.
  • compositions of the proteins, nucleic acids, including vectors such as tetracycline-regulated vectors, recombinant viruses and cells in a form appropriate for the intended application.
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the therapeutic agent dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium, and preferably encapsulated.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti- cancer agents, can also be inco ⁇ orated into the compositions.
  • Solutions of the active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent growth of microorganisms.
  • Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.
  • unit dose refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • compositions of the present invention will often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intratumoral, peritumoral or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intratumoral, peritumoral or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains a second agent(s) as active ingredients will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the active compounds may be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the particular methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, peritumoral and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th
  • other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
  • the expression vectors and delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • the injection can be general, regional, local or direct injection, for example, of a tumor. Also contemplated is injection of a resected tumor bed, and continuous perfusion via catheter. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the vectors of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified.
  • a typical compositions for such pu ⁇ oses comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as theyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well known parameters.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the route is topical, the form may be a cream, ointment, salve or spray.
  • unit dose refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
  • the methods of the present invention may be combined with any other methods generally employed in the treatment of the particular disease or disorder that the patient exhibits.
  • the methods of the present invention may be used in combination with classical approaches, such as surgery, radiotherapy and the like. So long as a particular therapeutic approach is not known to be detrimental in itself, or counteracts the effectiveness of the tumor suppressor therapy, its combination with the present invention is contemplated.
  • one or more agents are used in combination with cytokine gene therapy and/or tumor suppressor gene therapy, there is no requirement for the combined results to be additive of the effects observed when each treatment is conducted separately, although this is evidently desirable, and there is no particular requirement for the combined treatment to exhibit synergistic effects, although this is certainly possible and advantageous.
  • any surgical intervention may be practiced in combination with the present invention.
  • any mechanism for inducing DNA damage locally within tumor cells is contemplated, such as ⁇ -irradiation, X-rays, UV-irradiation, microwaves and even electronic emissions and the like.
  • the directed delivery of radioisotopes to tumor cells is also contemplated, and this may be used in connection with a targeting antibody or other targeting means.
  • Cytokine therapy also has proven to be an effective partner for combined therapeutic regimens. Various cytokines may be employed in such combined approaches.
  • cytokines examples include IL-l ⁇ IL-l ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF- ⁇ , GM-CSF, M-CSF, G-CSF, TNF ⁇ , TNF ⁇ , LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ .
  • Cytokines are administered according to standard regimens, consistent with clinical indications such as the condition of the patient and relative toxicity of the cytokine. Below is an exemplary, but in no way limiting, table of cytokine genes contemplated for use in certain embodiments of the present invention.
  • compositions of the present invention can have an effective amount of an engineered virus or cell for therapeutic administration in combination with an effective amount of a compound (second agent) that is a chemotherapeutic agent as exemplified below.
  • a compound that is a chemotherapeutic agent as exemplified below.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • chemotherapeutic agents may be used in combination with the therapeutic genes of the present invention. These can be, for example, agents that directly cross-link DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • compositions of the present invention in combination with the administration of a chemotherapeutic agent, one would simply administer to an animal at least a first modified retinoblastoma tumor suppressor as disclosed herein in combination with the chemotherapeutic agent in a manner effective to result in their combined anti-tumor actions within the animal. These agents would therefore be provided in an amount effective and for a period of time effective to result in their combined presence and their combined actions in the tumor environment.
  • the modified retinoblastoma tumor suppressor and chemotherapeutic agents may be administered to the animal simultaneously, either in a single composition or as two distinct compositions using different administration routes.
  • the modified retinoblastoma tumor suppressor treatment may precede or follow the chemotherapeutic agent treatment by intervals ranging from minutes to weeks.
  • the chemotherapeutic factor and modified retinoblastoma tumor suppressor are applied separately to the animal, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the chemotherapeutic agent and modified retinoblastoma tumor suppressor composition would still be able to exert an advantageously combined effect on the tumor.
  • both agents are delivered in a combined amount effective to inhibit its growth, irrespective of the times for administration.
  • chemotherapeutic agents are intended to be of use in the combined treatment methods disclosed herein.
  • Chemotherapeutic agents contemplated as exemplary include, e.g., etoposide (VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
  • chemotherapeutic agents will be generally around those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics.
  • agents such as cisplatin, and other DNA alkylating may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Agents that directly cross-link nucleic acids, specifically DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination.
  • Agents such as cisplatin, and other DNA alkylating agents may be used.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m at 21 day intervals for adriamycin, to 35-50 mg/m for etoposide intravenously or double the intravenous dose orally.
  • Agents that disrupt the synthesis and fidelity of polynucleotide precursors may also be used. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
  • 5-FU 5-fluorouracil
  • Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree, Taxus brevifolia. It binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Taxol is currently being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. Maximal doses are 30 mg/m per day for 5 days or 210 to 250 mg m given once every 3 weeks. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
  • chemotherapeutic agents that are useful in connection with combined therapy are listed in Table 4. Each of the agents listed therein are exemplary and by no means limiting. The skilled artisan is directed to "Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Chlorambucil macroglobulinemia Hodgkin's disease, non- Hodgkin's lymphomas
  • Alkyl Sulfonates Busulfan Chronic granulocytic leukemia
  • BCNU Carmustine
  • Nitrosoureas Lo ustine (CCNU) primary brain tumors, small-cell lung
  • Semustine (methyl-CCNU) Primary brain tumors, stomach, colon Table 4 (Continued)
  • streptozotocin carcinoid dacarbazine (DTIC; Malignant melanoma, Hodgkin's disease, soft-
  • Cytarabine cytosi ⁇ e Acute granulocytic and acute lymphocytic continued arabinoside
  • Cytarabine cytosi ⁇ e Acute granulocytic and acute lymphocytic continued arabinoside
  • leukemias Mercaptopurine Acute lymphocytic, acute granulocytic and
  • VLB Vinblastine
  • Vinca Alkaloids Acute lymphocytic leukemia, neuroblastoma,
  • Etoposide Hodgkin's disease, non-Hodgkin's lymphomas, Tertiposide acute granulocytic leukemia, Kaposi's sarcoma Table 4 (Continued)
  • Doxorubicin acute leukemias breast, genitourinary, thyroid, lung, stomach, neuroblastoma Testis, head and neck, skin, esophagus, lung and
  • Bleomycin genitourinary tract Hodgkin's disease, non- Hodgkin's lymphomas
  • Interferon alfa Modifiers non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma, chronic granulocytic leukemia
  • Adrenocortical Mitotane ( ⁇ ,/?'-DDD) Adrenal cortex Table 4 (Continued)
  • purification and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • the term "purified protein or peptide " as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally- obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "- fold purification number".
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater -fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or a most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • Gel chromatography is unsu ⁇ assed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adso ⁇ tion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N- acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography is immunoaffinity chromatography.
  • the ability to produce biologically active polypeptides is increasingly important to the pharmaceutical industry.
  • the present invention discloses compositions and methods for the efficient regulated expression of, for example, tumor suppressor genes in cells, allowing for the production of these proteins in vitro from previously refractory cell types.
  • Mammalian cultures have advantages over cultures derived from the less advanced lifeforms in their ability to post-translationally process complex protein structures such as disulfide- dependent folding and glycosylation. Indeed, mammalian cell culture is now the preferred source of a number of important proteins for use in human and animal medicine, especially those which are relatively large, complex or glycosylated.
  • aspects of the present invention take advantage of the biochemical and cellular capacities of mammalian cells as well as of recently available bioreactor technology.
  • Growing cells according to the present invention in a bioreactor allows for large scale production and secretion of complex, fully biologically-active polypeptides into the growth media.
  • the purification strategy can be greatly simplified, thus lowering production cost.
  • Anchorage-dependent and non-anchorage-dependent cultures Animal and human cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage- dependent cells requiring attachment to a solid substrate for their propagation ⁇ i.e., a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • Large scale suspension culture based on microbial (bacterial and yeast) fermentation technology has clear advantages for the manufacturing of mammalian cell products. The processes are relatively straightforward to operate and scale up. Homogeneous conditions can be provided in the reactor which allows for precise monitoring and control of temperature, dissolved oxygen, and pH, and ensure that representative samples of the culture can be taken.
  • suspension cultured cells cannot always be used in the production of biologicals. Suspension cultures are still considered to have tumorigenic potential and thus their use as substrates for production put limits on the use of the resulting products in human and veterinary applications (Petricciani, 1985; Larsson and Litwin, 1987). Viruses propagated in suspension cultures as opposed to anchorage-dependent cultures can sometimes cause rapid changes in viral markers, leading to reduced immunogenicity (Bruemann, 1980). Finally, sometimes even recombinant cell lines can secrete considerably higher amounts of products when propagated as anchorage-dependent cultures as compared with the same cell line in suspension (Nilsson and Mosbach, 1987). For these reasons, different types of anchorage- dependent cells are used extensively in the production of different biological products.
  • the current invention includes cells which are anchorage-dependent of nature.
  • Anchorage-dependent cells when grown in suspension, will attach to each other and grow in clumps, eventually suffocating cells in the inner core of each clump as they reach a size that leaves the core cells unsustainable by the culture conditions. Therefore, an efficient means of large-scale culture of anchorage-dependent cells is also provided in order to effectively take advantage of the cells' capacity to secrete heterologous proteins.
  • Instrumentation and controls for bioreactors have been adapted, along with the design of the fermentors, from related microbial applications. However, acknowledging the increased demand for contamination control in the slower growing mammalian cultures, improved aseptic designs have been implemented, improving dependability of these reactors. Instrumentation and controls include agitation, temperature, dissolved oxygen, and pH controls. More advanced probes and autoanalyzers for on-line and off-line measurements of turbidity (a function of particles present), capacitance (a function of viable cells present), glucose/lactate, carbonate/bicarbonate and carbon dioxide are also available. Maximum cell densities obtainable in suspension cultures are relatively low at about 2-4 x 10 6 cells/ml of medium (which is less than 1 mg dry cell weight per ml), well below the numbers achieved in microbial fermentation.
  • the stirred reactor design has successfully been used on a scale of 8000 liter capacity for the production of interferon (Phillips et al, 1985; Mizrahi, 1983).
  • Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1 :1 to 3:1.
  • the culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
  • the main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1.
  • the airlift reactor scales up relatively readily, has good mass transfer of gasses and generates relatively low shear forces.
  • a batch process is a closed system in which a typical growth profile is seen. A lag phase is followed by exponential, stationary and decline phases. In such a system, the environment is continuously changing as nutrients are depleted and metabolites accumulate. This makes analysis of factors influencing cell growth and productivity, and hence optimization of the process, a complex task. Productivity of a batch process may be increased by controlled feeding of key nutrients to prolong the growth cycle. Such a fed-batch process is still a closed system because cells, products and waste products are not removed.
  • perfusion of fresh medium through the culture can be achieved by retaining the cells with a fine mesh spin filter and spinning to prevent clogging.
  • Spin filter cultures can produce cell densities of approximately 5 x 10 cells/ml.
  • a true open system and the most basic perfusion process is the chemostat in which there is an inflow of medium and an outflow of cells and products.
  • Culture medium is fed to the reactor at a predetermined and constant rate which maintains the dilution rate of the culture at a value less than the maximum specific growth rate of the cells (to prevent washout of the cell mass from the reactor).
  • Culture fluid containing cells, cell products and byproducts is removed at the same rate.
  • Non-perfused attachment systems Traditionally, anchorage-dependent cell cultures are propagated on the bottom of small glass or plastic vessels.
  • the restricted surface-to- volume ratio offered by classical and traditional techniques, suitable for the laboratory scale, has created a bottleneck in the production of cells and cell products on a large scale.
  • a number of techniques have been proposed: the roller bottle system, the stack plates propagator, the spiral film bottles, the hollow fiber system, the packed bed, the plate exchanger system, and the membrane tubing reel.
  • roller bottle Being little more than a large, differently shaped T-flask, simplicity of the system makes it very dependable and, hence, attractive.
  • Fully automated robots are available that can handle thousands of roller bottles per day, thus eliminating the risk of contamination and inconsistency associated with the otherwise required intense human handling. With frequent media changes, roller bottle cultures can achieve cell densities of close to 0.5 x 10 ⁇ cells/cm 2 (corresponding to 10 cells/bottle or 10 cells/ml of culture media).
  • microcarrier cultures offer a high surface-to- volume ratio (variable by changing the carrier concentration) which leads to high cell density yields and a potential for obtaining highly concentrated cell products.
  • Cell yields are up to 1-2 x 10 cells/ml when cultures are propagated in a perfused reactor mode.
  • cells can be propagated in one unit process vessels instead of using many small low-productivity vessels ⁇ i.e., flasks or dishes). This results in far better utilization and a considerable saving of culture medium.
  • propagation in a single reactor leads to reduction in need for facility space and in the number of handling steps required per cell, thus reducing labor cost and risk of contamination.
  • the well-mixed and homogeneous microcarrier suspension culture makes it possible to monitor and control environmental conditions ⁇ e.g., pH, pO 2 , and concentration of medium components), thus leading to more reproducible cell propagation and product recovery.
  • environmental conditions e.g., pH, pO 2 , and concentration of medium components
  • microcarriers settle out of suspension easily, use of a fed- batch process or harvesting of cells can be done relatively easily.
  • the mode of the anchorage-dependent culture propagation on the microcarriers makes it possible to use this system for other cellular manipulations, such as cell transfer without the use of proteolytic enzymes, cocultivation of cells, transplantation into animals, and perfusion of the culture using decanters, columns, fluidized beds, or hollow fibers for microcarrier retainment.
  • microcarrier cultures are relatively easily scaled up using conventional equipment used for cultivation of microbial and animal cells in suspension.
  • microencapsulation One method which has shown to be particularly useful for culturing mammalian cells is microencapsulation.
  • the mammalian cells are retained inside a semipermeable hydrogel membrane.
  • a porous membrane is formed around the cells permitting the exchange of nutrients, gases, and metabolic products with the bulk medium surrounding the capsule.
  • Several methods have been developed that are gentle, rapid and non-toxic and where the resulting membrane is sufficiently porous and strong to sustain the growing cell mass throughout the term of the culture. These methods are all based on soluble alginate gelled by droplet contact with a calcium-containing solution. Lim (U.S.
  • Patent 4,321,883 describes cells concentrated in an approximately 1% solution of sodium alginate which are forced through a small orifice, forming droplets, and breaking free into an approximately 1% calcium chloride solution. The droplets are then cast in a layer of polyamino acid that ionically bonds to the surface alginate. Finally the alginate is reliquefied by treating the droplet in a chelating agent to remove the calcium ions. Other methods use cells in a calcium solution to be dropped into a alginate solution, thus creating a hollow alginate sphere. A similar approach involves cells in a chitosan solution dropped into alginate, also creating hollow spheres.
  • Microencapsulated cells are easily propagated in stirred tank reactors and, with beads sizes in the range of 150-1500 mm in diameter, are easily retained in a perfused reactor using a fine-meshed screen.
  • the ratio of capsule volume to total media volume can kept from as dense as 1 :2 to 1 : 10.
  • intracapsular cell densities of up to 10 the effective cell density in the culture is 1-5 x 10 7 .
  • the advantages of microencapsulation over other processes include the protection from the deleterious effects of shear stresses which occur from sparging and agitation, the ability to easily retain beads for the pu ⁇ ose of using perfused systems, scale up is relatively straightforward and the ability to use the beads for implantation.
  • Perfusion refers to continuous flow at a steady rate, through or over a population of cells (of a physiological nutrient solution). It implies the retention of the cells within the culture unit as opposed to continuous-flow culture which washes the cells out with the withdrawn media ⁇ e.g., chemostat).
  • the idea of perfusion has been known since the beginning of the century, and has been applied to keep small pieces of tissue viable for extended microscopic observation. The technique was initiated to mimic the cells milieu in vivo where cells are continuously supplied with blood, lymph, or other body fluids. Without perfusion, cells in culture go through alternating phases of being fed and starved, thus limiting full expression of their growth and metabolic potential.
  • perfused culture is to grow cells at high densities ⁇ i.e., 0.1-5 x 10 cells/ml).
  • the medium In order to increase densities beyond 2-4 x 10 cells/ml (or 2 x 10 cells/cm ), the medium has to be constantly replaced with a fresh supply in order to make up for nutritional deficiencies and to remove toxic products.
  • Perfusion allows for a far better control of the culture environment (pH, pO 2 , nutrient levels, etc.) and is a means of significantly increasing the utilization of the surface area within a culture for cell attachment.
  • Microcarrier and microencapsulated cultures are readily adapted to perfused reactors but, as noted above, these culture methods lack the capacity to meet the demand for cell densities above 10 cells/ml. Such densities will provide for the advantage of high product titer in the medium (facilitating downstream processing), a smaller culture system (lowering facility needs), and a better medium utilization (yielding savings in serum and other expensive additives). Supporting cells at high density requires efficient perfusion techniques to prevent the development of non-homogeneity.
  • the cells of the present invention may, irrespective of the culture method chosen, be used in protein production and as cells for in vitro cellular assays and screens as part of drug development protocols. J. Kits
  • kits All the essential materials and reagents required for the various aspects of the present invention may be assembled together in a kit.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the instant compositions may be formulated into a single or separate pharmaceutically acceptable syringeable composition.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.
  • kits of the invention may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • the kits of the invention may also include an instruction sheet defining administration of the gene therapy and/or the chemotherapeutic drug.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • instructions for use of the kit components is typically included.
  • PCRTM primers were designed and synthesized according to the sequences of RB cDNA.
  • the sense primers were determined by the RB cDNA sequences downstream of the deleted N-terminal sequence. All primers contain a Hwdlll restriction site (underlined) at the 5'-end and the consensus Kozak cassette (GCCGCC) followed by an ATG (italics).
  • GCCGCC consensus Kozak cassette
  • the anti-sense primer 5'-GTCCAAGAGAATTCATAAAAGG-3' overlaps with the EcoRI site (underlined) at the nucleotide +900 of the RB cDNA
  • the anti-sense primer was paired with each sense primer described above to amplify various modified 5'-RB cDNA fragments using plasmid F7 as template (which contains the full-length RB cDNA).
  • pCMVRBd 31-107 a deletion of amino acids 31 to 107 of the wild type RB protein
  • pCMVRBd 77 a deletion of amino acids 31 to 107 of the wild type RB protein
  • pCMVRBm 111 ⁇ 2 a mutation of amino acid 111 of the wild type RB protein from aspartic acid to glycine and a mutation of amino acid 112 from glutamic acid to aspartic acid
  • pCMVRBd 111-181 a deletion of amino acids 111 to 181 of the wild type RB protein
  • pCMVRBd ⁇ ⁇ -24] a deletion of amino acids 111 to 241 of the wild type RB protein
  • pCMVRBd 181 . 241 a deletion of amino acids 181 to 241 of the wild type RB protein
  • pCMVRBd 242 a deletion of amino acids 242 to 300 of the wild type RB protein.
  • an RB cDNA fragment from nucleotide position +325 to +910 was amplified from the plasmid F7 by PCRTM using the primers 5'-GCGCCTGAGGACCTAGATGAGATGTCGTTC-3' (SEQ ID NO: 19) and OMRbAS300 (SEQ ID NO: 13).
  • This RB cDNA fragment was digested with Bsu36l (underlined) and EcoRI (from OMRbAS300), and inserted into plasmid pCMVRB 110 digested with the same enzymes, to replace the original RB cDNA fragment from nucleotides +91 to +900.
  • the nucleic acid sequence of pRB ⁇ 31-107 is S ⁇ Q ID NO:38, and the corresponding amino acid sequence is S ⁇ Q ID NO:39.
  • an RB cDNA fragment (nucleotides +328 to +910) was amplified from the plasmid F7 by PCRTM using the oligonucleotides 5'-GCGGTTAACCCTAGATGAGATGTCGTTCACT-3' (SEQ ID NO:20) and OMRbAS300 (SEQ ID NO: 13), followed by digestion with Hpal (underlined) and EcoRI.
  • the amplified, digested fragment was inserted into plasmid pCMVRB digested with the same enzymes, to replace the RB cDNA fragment from nucleotides +230 to +900.
  • the nucleic acid sequence of pRB ⁇ 77-107 is S ⁇ Q ID NO:40, and the corresponding amino acid sequence is S ⁇ Q ID NO:41.
  • pCMVRBm ⁇ l/m For the construction of pCMVRBm ⁇ l/m , two pairs of primers were used to change nucleotide A (position +332 of the wild-type RB cDNA) to G, in order to change the codon for aspartic acid (GAT) to glycine (GGT), thus creating a new restriction enzyme site, Avrll, and nucleotide G (position +336 of the wild-type RB cDNA) to T, in order to change the codon for glutamic acid (GAG) to aspartic acid (GAT).
  • the first pair of primers are
  • OMRbS332 S ⁇ Q ID NO:23; the mutated bases are in bold
  • OMRbAS300 S ⁇ Q ID
  • H d III and Avrll (underlined), and those amplified with OMRbS332 and OMRbAS300 were digested with Avrll and EcoRI. These fragments were ligated together into plasmid pCMVRB 110 digested with H dIII and EcoRI to replace the corresponding wild-type RB cDNA sequences.
  • the nucleic acid sequence of pRBml l l/112 is S ⁇ Q ID NO: 50, and the corresponding amino acid sequence is S ⁇ Q ID NO:51.
  • pCMVRBd ⁇ n-241 For the construction of pCMVRBd ⁇ n-241 , a 5' RB cDNA fragment containing nucleotides +1 to +331 was obtained by digestion of pCMVRBm H 1 with HmdIII and Avrll. The 3' RB cDNA fragment beginning from nucleotide +722 was isolated from the same plasmid digested with Pvu ⁇ l and BamHI. Then the two DNA fragments (in-frame) were ligated into pCMV-G digested with HmdIII and BamHI. The nucleic acid sequence of pRB ⁇ l 11-241 is SEQ ID NO:44, and the corresponding amino acid sequence is SEQ ID NO:45.
  • pCMVRBd 181 . 241 a 5'-RB cDNA fragment containing nucleotides +1 to +538 was amplified from plasmid F7 by PCRTM with primers OMRBS1 (SEQ ID NO:21) and 5'-CCCGATATCAACTGCTGGGTTGTGTCAAATA-3' (SEQ ID NO:25) using plasmid F7 as a template.
  • the obtained RB cDNA fragment was cut with HmdIII and EcoRV (underlined), and inserted into pCMVRB 110 to replace the original 5' RB cDNA fragment between the HmdIII and Pvull sites.
  • the nucleic acid sequence of pRB ⁇ 181-241 is S ⁇ Q ID NO:46, and the corresponding amino acid sequence is S ⁇ Q ID NO:47.
  • primers OMRBS1 S ⁇ Q ID NO:21
  • 5'- CCCGAATTCGTTTTATATGGTTCTTTGAGCAA-3* S ⁇ Q ID NO:26
  • the amplified product was digested with HmdIII and EcoRI (underlined), and inserted into pCMVRB digested with the same enzymes to replace the original 5' RB cDNA sequences from nucleotides +1 to +900.
  • the nucleic acid sequence of pRB ⁇ 242-300 is S ⁇ Q ID NO:48
  • the corresponding amino acid sequence is S ⁇ Q ID NO:49.
  • Tumor cells were seeded onto coverslips in medium containing tetracycline and transfected with plasmids expressing pRB , pRB or other mutant RB proteins.
  • the cells were incubated with 1 ml of fresh medium containing 10 ⁇ Ci [ 3 H]-methyl thymidine (Amersham, Arlington Heights, IL) for 2 hours at 37°C, then fixed and immunochemically stained for expression of RB protein as described previously (Xu et al., 1991a; 1991b). Stained slides were subsequently coated with a thin layer of gelatin and dried at 37°C overnight.
  • the slides were then overlaid with autoradiographic emulsion (Type NTB2, Eastman Kodak, Rochester, NY) and exposed for 2 days. After development, slides were examined under a light microscope. Twenty-four hours after transfection, cells were processed for immunocytochemical staining of RB protein and [ H]- thymidine inco ⁇ oration assay as described above.
  • autoradiographic emulsion Type NTB2, Eastman Kodak, Rochester, NY
  • the pRB mutants with any deletions between amino acid 55 and 181 significantly inhibit DNA synthesis after being introduced into the tumor cells.
  • cells transfected with pRBs containing deletions only between amino acid 181 and 241 showed weaker inhibition of DNA synthesis than those transfected with plasmids expressing pRBs carrying deletions between amino acid 55 and 181, although these were still more effective than cells transfected with the full-length pRB expression plasmid.
  • modifications that combine certain of the above deletions for example a deletion between amino acid 1 and amino acid 241, would be expected to have similar significant DNA synthesis inhibitory activity.
  • a pRB carrying a point mutation at amino acid position 111 converting aspartic acid to glycine significantly suppressed DNA synthesis, further suggesting that this region is vital for regulating pRB function.
  • the modified retinoblastoma genes and proteins described above have a number of practical utilities, including, but not limited to, gene therapy. For these aspects, expression systems are needed. While systems such as those described above are appropriate for certain embodiments, they have certain shortcomings in relation to gene therapy using cytotoxic constructs.
  • the original tetracycline-responsive gene expression system of Gossen and Bujard (1992) is an attractive system, but has certain drawbacks, such as squelching effects on cell growth (Gill and Ptashne, 1988). To overcome these and other drawbacks, the inventors have improved the tetracycline-responsive gene expression system.
  • the original tetracycline repressor/operator-based regulatory system consists of two plasmids, pUHD15-l and pUHC13-3 (U. S. Patent 5,464,758, inco ⁇ orated in its entirety herein by reference; Gossen and Bujard 1992).
  • pUHC13-3 is a tetracycline (Tc; tet) sensitive expression vector containing a hybrid minimal human CMV promoter, in which tet operator sequences had been inserted upstream of the TATA box.
  • pUHD15-l contains sequences encoding a tetracycline responsive transactivator (tTA), with expression driven by a wild-type CMV promoter.
  • the inventors found that efficiently reversible transgene expression was observed in many tumor cell lines studied.
  • attempts to isolate long-term clones expressing the reporter gene in a tetracycline-responsive manner were unsuccessful. This was most likely caused by the high intracellular levels of the tTA transactivator, whose expression was driven by the strong CMV promoter/enhancer sequence in the plasmid pUHD15-l.
  • the tTA transactivator contains the VP-16 activating domain, which is known to have squelching effects on cell growth (Gill and Ptashne, 1988).
  • the tTA expression cassette was first modified by replacing the strong CMVp enhancer (Boshart et al, 1985) in the original pUHD15-l plasmid with a pair of 19 bp imperfect direct repeat sequence (a portion of the CMVp enhancer; SEQ ID NO:5).
  • the modification of the hCMV promoter/enhancer was done by removal of a portion of the 5' enhancer sequences from the hCMV promoter.
  • oligonucleotide primers were designed based on the published sequence of the hCMV promoter (Boshart et al, 1985). A Xhol and an EcoRI restriction enzyme site (underlined) was added to the 5' end of each sense and the anti-sense oligo, respectively.
  • the sense oligos are: 5'-CCGCTCGAGCAATGGGCGTGATAGCGG-3' (OMCMVsl; S ⁇ Q ID NO:6); 5'-CCGCTCGAGCACCAAAATCAACGGGA-3' (OMCMVs2; S ⁇ Q ID NO:7) and 5'- CCGCTCGAGC AACTCCGCCCCATTGAC-3 ' (OMCMVs3; S ⁇ Q ID NO:8), respectively, and they shared the same anti-sense primer, 5'-TAGACATATGAATTCGCGGCC-3' (OMCMVas; SEQ ID NO:9).
  • PCRTM amplification with primer pairs of OMCMVsl + OMCMVas; OMCMVs2 + OMCMVas and OMCMVs3 + OMCMVas, generated three shorter versions of CMV promoter with lengths of 282bp (namely mhCMVpl), 203bp (mhCMVp2) and 168bp (mhCMVp3) respectively.
  • the purified shortened CMV promoter/enhancer fragments were double digested with Xhol and EcoRI, and inserted into pUHD15-l to replace the original hCMV promoter. This produced three new tTA expressing plasmids, namely pmCMV 1 -tTA, pmCMV2-tTA and pmCMV3-tTA.
  • CAT chloramphenicol acetyltransferase
  • the CAT expression plasmids were introduced into three cell lines, the tumor cell lines 5637 and Saos2, and the embryonal kidney cell line 293, via the Lipofectin method (Life Technologies, Gaithersburg, MD). Forty-eight hours after transfection, cell lysates were prepared and CAT activity was measured by a CAT FLASH assay kit from Stratagene (Stratagene, La Jolla, CA).
  • FIG. 1 is a graphical representation of the CAT activity in the 5637 and Saos-2 cell lines. The more enhancer sequences that were deleted, the weaker was the promoter that remained. The order of promoter activity from strongest to weakest is hCMV, mhCMVpl, mhCMVp2 and mhCMVp3. The activity of mhCMVpl is 17.7% of the full-length hCMV promoter, while the mhCMVp3 activity is only 3.3% of the hCMV promoter in 5637 cells (FIG. 1).
  • mhCMVpl (S ⁇ Q ID NO:5) was chosen for the modified tetracycline regulatable gene expression system.
  • mhCMVpl showed optimal tetracycline-controlled transactivator (tTA) expression with no squelching effects on host cell growth (FIG. 2), an important characteristic for potential use in human gene therapy.
  • a single plasmid vector named EC1214A was constructed.
  • This plasmid contains: 1) the modified tetracycline-responsive transactivator (tTA) expression cassette to eliminate the squelching effects of tTA on host cell growth; 2) the tTA-dependent promoter from plasmid pUHC13-3; 3) a generic intron sequence; 4) a multiple cloning site downstream of the promoter and intron; and 5) a neo ⁇ - expression cassette to allow G418 selection. Expression in this system is regulated by tetracycline, or a tetracycline analog.
  • a "tetracycline analog” will be understood to be any one of a number of compounds that are closely related to tetracycline, and which bind to the tet repressor with at least an affinity (K. of at least 10 6 /M, preferably with a K a of 10 9 /M, and more preferably with a K a of 10 ⁇ /M.
  • affinity K. of at least 10 6 /M, preferably with a K a of 10 9 /M, and more preferably with a K a of 10 ⁇ /M.
  • Exemplary, but in no way limiting, of such tetracycline analogs are those disclosed by Hlavka and Boothe (1985), Mitschef (1978), the Noyee Development Co ⁇ oration (1969), Evans (1968) and Dowling (1955), each of which is inco ⁇ orated herein in its entirety.
  • Plasmid pMLSIS.CAT (Choi et al., 1991) contains an generic intron sequence which consists of a portion of the 5 '-untranslated leader from the adeno virus-major-late region, which contains part of the first exon of the tripartite and the first intervening sequence, as well as a synthetic splice donor/acceptor sequence derived from an IgG variable region.
  • telomere sequence was then replaced by a HSV thymidine kinase (TK) gene polyadenylation signal sequence to generate a plasmid, named pCMV*-G-TKpA.
  • TK thymidine kinase
  • Plasmid pRc/CMV (Invitrogen, San Diego, CA) was double digested with restriction enzymes Nrul and Xbal. The 5' overhang from the Xbal digest was filled in by Klenow fragment of DNA polymerase (Life Technologies, Gaithersburg, MD), and the blunt-ended insert was ligated to a DNA fragment containing mhCMVl-tTA obtained from plasmid pmCMVl-tTA (Example 2). The new plasmid was named pmCMVl-tTA.neo.
  • a DNA fragment containing the tTA-dependent promoter, the generic intron and the TK polyadenylation signal was isolated from plasmid pCMV*-G-TKpA, and inserted into the Bgl ⁇ l site of plasmid pmCMVl-tTA.neo to produce a vector named EC1214A, which carries both the tTA expression cassette and the tTA-dependent promoter as well as a selection marker, the neomycin resistance gene.
  • the original tetracycline repressor/operator-based tet-on system also consists of two plasmids, pUHD17-lneo (or pUHD172-lneo) and pUHC13-3 (Gossen et al, 1995).
  • pUHC13-3 is a tetracycline sensitive expression vector containing a hybrid minimal human CMV promoter, in which tet operator sequences had been inserted upstream of the TATA box.
  • pUHD17-lneo or pUHD172-lneo contains sequences encoding a reverse tetracycline responsive transactivator
  • rtTA rtTA
  • rtTA transactivator contains the VP-16 activating domain, which is known to have squelching effects on cell growth (Gill and Ptashne, 1988).
  • the rtTA expression cassette was first modified by replacing the strong CMVp enhancer (Boshart et al. , 1985) in the pUHD17-lneo or pUHD172-lneo plasmid with a pair of 19 bp imperfect direct repeat sequence (SEQ ID NO:5).
  • the modification of the hCMV promoter/enhancer was done by removal of a portion of the 5' enhancer sequences from the hCMV promoter (Example 2).
  • the new rtTA expressing plasmid was named pmCMVl-rtTA.
  • a single plasmid vector named EC1214B was constructed using pmCMVl-rtTA.
  • This plasmid contains: 1) the modified reverse tetracycline-responsive transactivator (rtTA) expression cassette to eliminate the squelching effects of rtTA on host cell growth; 2) the rtTA-dependent promoter from plasmid pUHC13-3; 3) a generic intron sequence; 4) a multiple cloning site downstream of the promoter and intron; and 5) a neo ⁇ - expression cassette to allow G418 selection.
  • the construction was performed as outlined in Example 3.
  • plasmid F7 (Takahashi et al, 1991) or p4.95BT (Friend et al., 1987), containing the full-length RB gene cDNA, was digested with the restriction enzymes ⁇ 4cyl at nucleotide -322 and Seal at +3230 (the A of the second in-frame ATG start codon was designated nucleotide +19).
  • the 5' overhangs generated by the Acyl digest were treated with E. coli DNA polymerase I in the presence of all four dNTPs to generate blunt ends.
  • BamHI linkers were ligated onto the fragment, and the fragment was then digested with BamHI to remove excess linkers and generate BamHI ends (Maniatis et al, 1989; Ausubel et al, 1992).
  • the resultant RB cDNA fragment of 3552 bp was inserted into the unique BamHI site of EC1214A to generate pCMV*-tTA-RB 110 .
  • terminal truncated pRB are in a suboptimal context for initiation of translation in higher eukaryotes. For example, there is an out-of-frame AUG codon at the nucleotide -5 position (the
  • a of the ATG start codon for the pRB cDNA is designated nucleotide +1), and the leading
  • the modified 5'-RB cDNA fragment was obtained by PCRTM using plasmid F7 carrying the full-length RB 110 cDNA as the template.
  • the sense primer used for the PCRTM reaction (5'- CCCAAGCTTGCCGCCATGTCGTTCACTTTTAC-3'; SEQ ID NO:12) contained a HmdIII restriction site (underlined) and a Kozak cassette (italics; Kozak, 1987).
  • the PCRTM product was digested with HmdIII and EcoRI, then ligated with a DNA fragment containing the 3'-RB cDNA fragment between EcoRI (position +900) and BamHI (+3548) isolated from plasmid F7.
  • the entire RB 94 cDNA fragment was inserted into the HmdIII and BamHI sites of ⁇ C1214A to produce the inducible pRB expression plasmid, pCMV*-tTA-RB 94 .
  • the modified, single-plasmid tetracycline-responsive mammalian gene expression system has been used to obtain various stable tumor cell lines in which expression of the wild-type or the N-terminal truncated retinoblastoma (RB) tumor suppressor gene, or the p53 tumor suppressor gene can be reversibly turned on and off without detectable leakage.
  • RB retinoblastoma
  • a breast carcinoma cell line, MDA-468 (HTB132) was obtained from ATCC and cultured in Leibovitz's L-15 (Life Technologies, Gaithersburg, MD) with 10% FBS (Life Technologies, Gaithersburg, MD).
  • An osteosarcoma cell line, Saos2 was cultured in medium McCoy's 5A (Life Technologies, Gaithersburg, MD) with 15% FBS (Zhou et al, 1994b).
  • a bladder carcinoma cell line, 5637 (HTB9) obtained from ATCC was cultured with RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FBS. All cell culture media were supplemented with 0.5% penicillin/streptomycin. Saos2 and 5367 cells were incubated at 37°C in a 5% CO 2 incubator, while MDA-468 cells were cultured at 37°C without CO 2 .
  • Tumor cells were transfected with the pRB 11 and pRB expression plasmids, pCMV*- tTA-RB U0 and pCMV*-tTA-RB 94 via the Lipofectin method according to the manufacturer's instruction manual (Life Technologies, Gaithersburg, MD). During transfection and the subsequent procedures except where specified, 0.5 ⁇ g/ml of tetracycline (Sigma, St. Louis, MO) was added to the transfection and culture media. Forty-eight hours after transfection, G418 (Life Technologies, Gaithersburg, MD.) was added to the culture media at a concentration of 300 ⁇ g/1. Two to three weeks later, single colonies were isolated by cloning rings.
  • tetracycline Sigma, St. Louis, MO
  • Tumor cells were seeded into 60-mm culture dishes or onto sterile coverslips at concentrations that would reach about 40% confluent next day. Twenty hours later, proper amount of plasmid DNA was mixed with Lipofectin reagent in Opti-MEM medium according to the manufacture's instruction manual (Life Technologies, Gaithersburg, MD). Cells were overlaid with the DNA-Lipofectin complex and incubated in a CO 2 incubator at 37°C overnight. Next day, fresh medium was added to replace the DNA-Lipofectin. Twenty-four or forty-eight hours later, cells were fixed for immunochemical staining or lysed for preparation of cell lysates.
  • Immunocytochemical staining was performed as described previously (Xu et al., 1989a).
  • cells grown on coverslips were fixed in 45% (vol/vol) acetone/ 10% (wt / vol) formaldehyde/0.1 M phosphate buffer for 5 min. After being washed six times with phosphate-buffered saline, cells were blocked with 1% non-fat milk/1.5% goat serum or horse serum in phosphate buffer for 4 hours at room temperature.
  • the RB-WL-1 anti-RB antibody or Canji's monoclonal anti-RB antibody (QED, San Diego, CA) was diluted to 2 ⁇ g/ml or 0.5 ⁇ g/ml respectively in the same solution plus 0.02% Triton X-100, and was incubated with the cell overnight. After being washed, the coverslips were processed for immunostaining with the avidin biotinylated peroxidase complex (ABC) method according to the technical manual (Vector Laboratories, Burlingame, CA).
  • ABSC avidin biotinylated peroxidase complex
  • Cell lysate was prepared as previously described (Xu et al, 1991a; 1991b). Briefly, cultured cells in 60 mm dishes were lysed with 0.6 ml of ice-cold lysis buffer containing 100 mM NaCl, 0.2% NP-40, 0.2% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl (pH8.0) with 50 ⁇ g/ml aprotinin and 1 mM PMSF. The cell lysate was passed through 21 gauge needle several times and clarified by centrifugation.
  • VDF Immobilon polyvinylidene difluoride membranes
  • a crystal violet staining method was used to measure the cell growth changes in the presence or absence of tetracycline (Gillies et al, 1986). Briefly, cells were seeded into 24-well plates in duplicate. In one set of the plates, cells were grown in medium containing 0.5 ⁇ g/ml tetracycline, while in duplicate plates, the same cells were cultured in non-tetracycline media. At each time point, cells were fixed with 1% glutaraldehyde in PBS and stained using 0.5% of crystal violet. After cells at all desired time points were collected, the crystal violet dye was extracted from the stained cells by incubating cells with Sorenson's solution containing 0.9% trisodium citrate, 0.02 N chloric acid and 45% ethanol (vol/vol). The extracted dyes were diluted properly with the Sorenson's solution and optical absorbencies at ⁇ 550 were measured. Growth curves were obtained by plotting the OD 550 against the time.
  • the tumorigenicity test has been described previously (Takahashi et al, 1991). Two groups of athymus nude mice were set up for each cell clone to be tested. One group of mice were given regular water, while the other group was given water containing 5 mg/ml of tetracycline. A total of 5 ⁇ l0 cells from each RB - or RB -reconstituted clone were injected subcutaneously in 0.2 ml of phosphate buffered saline into the right flank of nude mice. RB- negative parental controls including Saos2, 5637 and MDA-468 cells were injected at the identical concentration into the left flank of the same mice. Tumors were scored 4 weeks after injection.
  • Tumor cells were seeded onto coverslips and transfected with plasmids expressing pRB , pRB or other mutant RB proteins. Twenty-four hours after transfection, cells were processed for immunocytochemical staining of RB protein and [ H] -thymidine inco ⁇ oration assay as described in Xu et al. (1991b; 1991c).
  • the tumor cell lines studied were derived from widely disparate types of human cancers such as the retinoblastoma, osteosarcoma, carcinomas of the bladder, prostate, breast and lung (Goodrich and Lee, 1993; Xu, 1996; Xu, 1995 for review). Although it has been well documented that correction of the RB gene defect alone in tumor cells carrying multiple genetic alterations was sufficient to revert their malignant phenotype, it was more puzzling than it appeared at first sight (Klein, 1990).
  • RB-defective tumor cell lines were used to establish long-term inducible RB expression clones. They were the osteosarcoma cell line, Saos2, the bladder cancer cell line,
  • MDA-468 as recipient cells was that they are the RB-defective tumor cells most in use for RB- replacement studies.
  • the tumor cells were transfected with the inducible RB expression plasmid, pCMV*-tTA-RB 110 and the pRB 94 expression plasmid, pCMV*-tTA-RB 94 in the presence of tetracycline. After selection in 400 ⁇ g/ml of G418 for approximately 2 to 4 weeks, well separated single colonies were isolated and maintained in tetracycline containing media. A small portion of the isolated clones were cultured separately in the absence of tetracycline (Tc) for 24 to 48 hours and stained with an anti-RB antibody, RB-WL-1. Tight control of pRB protein expression in the stable clones of Tc-responsive / ⁇ -reconstituted 5637 bladder carcinoma and MDA-MB-468 breast carcinoma cells is seen.
  • Tc tetracycline
  • the RB-reconstituted 5637 cells grown in the presence of 0.5 ⁇ g/ml of Tc in the culture medium are RB" by immunocytochemical staining, while after removal of Tc, the pRB expression was turned on in the RB-reconstituted 5637 cells as shown by RB + immunocytochemical staining.
  • the MDA-MB-468 breast carcinoma tumor cells were also RB" by immunocytochemical staining in the presence of 0.5 ⁇ g/ml of Tc in culture medium, whereas after removal of Tc, the pRB expression was turned on in the RB-reconstituted MDA-MB-468 breast carcinoma cells as shown by RB + immunocytochemical staining.
  • tetracycline is an inhibitor, rather than an inducer, in this tetracycline-responsive expression system.
  • the minimal concentration of tetracycline required to shut off RB expression was also tested. It was found that as little as 0.1 ⁇ g/ml of tetracycline can inhibit RB expression to non- detectable level by immunostaining, indicating that the tetracycline-regulated expression system is very sensitive to tetracycline.
  • the Saos2 and 5637 clones also failed to synthesize DNA, which were followed by noticeable mo ⁇ hological changes and finally, by cell death.
  • the cellular mo ⁇ hology was markedly altered after pRB expression was induced in Tc-free medium, including cell enlargement, flattening, and lower nucleocytoplasmic ratio than cycling Gl/S cells.
  • changes in mo ⁇ hology and growth rate after either transient or stable RB-replacement with a non-regulatable system have not been well documented in the literature (Goodrich et al, 1992b; Takahashi et al, 1991 ; Zhou et al, 1994b).
  • the phenotypes of the established Tc-regulatable RB + tumor lines in Tc-free medium were quite similar to those documented previously for RB plasmid-transfected (or RB retrovirus vector-infected) tumor cell mass cultures (Huang et al, 1988; Templeton et al, 1991; Qin et al, 1992). All tumor cell clones under permissive condition for pRB expression were unable to form colonies in soft agar (FIG. 4A, FIG. 4B and FIG. 4C), and were non-tumorigenic in nude mice.
  • SA- ⁇ -gal (pH 6 activity) independent of senescence or age.
  • SA- ⁇ -gal is not a universal marker of replicative senescence, which is not su ⁇ rising.
  • the SA- ⁇ -gal provides a simple assay allowing the further characterization the RB-mediated tumor cell growth cessation.
  • the majority (>99.9%) of young (early passage) human WI-38 fibroblasts are SA- ⁇ -gal negative.
  • the senescent (at population doubling level greater than 52) WI-38 cells were strongly SA- ⁇ -gal positive. All tetracycline-responsive tumor cell clones examined so far were SA- ⁇ -gal negative in the presence of tetracycline (RB”), and were SA- ⁇ -gal positive in tetracycline-free medium
  • the tumor cells with both wild-type p53 and pRB expression displayed more intense SA- ⁇ -gal positive staining as compared to tumor cells only expressing pRB .
  • the results imply that the mechanisms for tumor suppression by pRB and p53 were different from each other, but expression of pRB and p53 together had synergistic effects on RB-mediated tumor cell senescence.
  • pRB-mediated replicative senescence was tumor-specific.
  • the young WI-38 fibroblasts at early passage infected with recombinant adenovirus vector, AdCMVpRBl 10 at multiplicity of infection (MOI) of 100 remained SA- ⁇ -gal negative, and they resumed a normal growth pattern about one week post-infection. Therefore pRB is a relatively safe reagents for anticancer gene therapy.
  • the emerging RB gene therapy also may be beneficial in treating post-surgery residue tumors, superficial cancers, or premalignancies, as well as non-malignant, hype ⁇ roliferative disorders in certain circumstances (Chang et al, 1995; Xu et al, 1996).
  • pRB may also play a role in inhibition of angiogenesis and in elicitation of immunogenicity of tumor cells.
  • CM serum-free conditioned media
  • the class II proteins present peptides derived from proteolytically processed antigens to CD4 + T lymphocytes as part of the immune response. Therefore, pRB likely has a role in mediating tumor immunogenicity as well.
  • telomerase activity which was presumably essential for an extended proliferative life-span of neoplastic cells, was repressed in the tumor cell lines after induction of pRB (but not p53) expression.
  • pRB plays a critical role in the intrinsic cellular senescence program. From a practical standpoint, findings imply that cytostatic gene therapy using RB (or RB and p53 together) may result in differential elimination of tumor cells through cellular senescence and crisis. At the same time the replicative lifespan of normal cells in vivo may not be affected. This could provide a potential basis for designing tumor-specific tumor suppressor gene therapy and anti-telomerase gene therapy.
  • TSR tumor suppressor resistance
  • MDR multiple drug resistance
  • pRB 94 exerted su ⁇ risingly more potent cell growth suppression as compared to the full-length pRB protein in a diversity of tumor cell lines examined, including those having a normal endogenous RB gene.
  • Tumor cells transfected with the pRB -expressing plasmids displayed multiple mo ⁇ hological changes frequently associated with cellular senescence. They failed to enter S phase and rapidly died (Xu et al, 1994b; Resnitzky and Reed, 1995).
  • 94 cells may account for the enhanced tumor cell growth suppression by pRB .
  • pRB another truncated version of pRB, named pRB , beginning at amino acid 379, has also been reported as a more potent inhibitor of cell cycle progression compared to the full-length pRB (Wills et al, 1995).
  • the modified system is threefold: 1) it is suitable for establishing long-term stable cell lines with inducible gene expression because of lower constitutive expression of the tTA peptide; 2) the system is now contained within a single plasmid so that only one round of transfection and selection is required; and 3) of importance, the single-plasmid tetracycline-responsive mammalian gene expression system is readily convertible to tetracycline-controlled viral vectors (Examples 7-12 below).
  • the desired cDNA fragment of a gene of interest is first inserted into the single-plasmid tetracycline-regulatable plasmid vector, EC1214A (Example 3) or EC1214B (Example 4).
  • the tetracycline-responsive foreign gene expression cassette and the modified tTA (or rtTA) expression cassette from the corresponding EC1214A or EC1214B plasmid vectors are then recovered using standard methods in the art for DNA manipulation (Maniatis et al, 1989;
  • Ad5dl309 genome and E1/E3 deletion mutation (Microbix Biosystems, Inc.) into 293 cells using the LIPOFECTIN reagent (GIBCO/BRL Life Technologies).
  • the co-transfection of 293 cells is performed in the presence (for tet-off system) or absence (for tet-on system) of 0.5 ⁇ g/ml of tetracycline.
  • a fragment containing a gene of interest is first inserted into the single- plasmid tetracycline-regulatable plasmid vector, EC1214A or EC1214B.
  • the tetracycline- responsive foreign gene expression cassette and the modified tTA (or rtTA) expression cassette from the corresponding EC1214A or EC1214B plasmid vectors are then recovered and inserted, respectively, into the shuttle plasmid, p ⁇ ElsplA and the master adenovirus plasmid, pBHGl l.
  • the resultant recombinant shuttle plasmids and the recombinant master adenovirus plasmid are co-transfected into 293 cells.
  • Co-transfection of 293 cells with the recombinant shuttle plasmid and the recombinant master adenovirus plasmid produce infectious virions by in vivo recombination, in which the minigene cassette expressing the gene of interest and the modified tTA (or rtTA) expression cassette are replaced the ⁇ E1 region or ⁇ E1 and ⁇ E3 regions of the Ad5dl309 genome, respectively.
  • Presence of recombinant adenoviruses in the transfected 293 cells is initially identified by cytopathic effect (CPE).
  • CPE cytopathic effect
  • Recombinant viruses are then isolated by screening adenovirus plaques from 293 cell monolayers after infection with the virus supernatants, and further characterized by restriction enzyme digestion mapping, PCRTM, or by expression of the gene of interest in virus-infected host cells in a tetracycline-regulatable manner.
  • the recombinant adenoviruses containing the desired foreign gene as well as the modified tTA (or rtTA) expression cassettes are subjected to at least three rounds of plaque purification.
  • High-titer stocks of the tetracycline-controlled recombinant adenoviruses are prepared by methods modified from Graham and Prevec, (1991).
  • the concentrated viral suspension is desalted by gel filtration through Sephadex G50 to generate a final purified virus stock about 10 plaque-forming units (pfu) per ml in PBS.
  • a replication-deficient adenovirus vectors expressing N-terminal truncated pRB ⁇ 4 protein has been used in in vivo animal studies of human cancer gene therapy (Xu et al, 1996).
  • the ratio of viral particles to plaque-forming units of the AdCMVpRB94 virus supernatants increased dramatically with passage, making it difficult for large-scale preparation of high-titer stocks of the AdCMVpRB94 virus for human cancer gene therapy clinical trials. This was probably caused by the super cell growth suppression effects of pRB94 protein on the 293 virus-producing cell line.
  • the modified tetracycline-responsive mammalian gene expression system has been used in a similar manner as described above to generate a tetracycline-controlled pRB ⁇ -containing adenovirus vector, AdVtTA.RB94, which is designed for delivery of high-dose pRB 94 gene therapy.
  • the entire tetracycline regulation cassette can be inserted into the El region of the adenovirus genome, or the RB 4 expression cassette can be inserted into the El region of the adenovirus genome, while the transcriptional transactivation fusion protein expression cassette is inserted into the E3 region of the adenovirus genome.
  • Over-expression of pRB in tumor cells will cause tumor cell-specific senescence and cell death.
  • the pRB 94 cDNA has a modified optimal initiator context sequence. Expression of the pRB94 protein in transduced human tumor cells by AdVtTA.RB94 can be reversibly turned off and on.
  • the novel AdVtTA.RB94 recombinant adenovirus vector can be propagated efficiently in 293 cells with increased yield and quality.
  • the tumor cells with both wild-type p53 and pRB expression displayed more intense SA- ⁇ -gal positive staining as compared to tumor cells only expressing pRB .
  • the results imply that the mechanisms for tumor suppression by pRB and p53 were different from each other, but expression of pRB and p53 together had synergistic effects on RB-mediated tumor cell senescence.
  • Insertion of both the modified tetracycline-responsive transactivator (tTA) expression cassette and the tTA-dependent pRB expression cassette into the El region of the Ad5 genome facilitates construction of an adenovirus vector simultaneously expressing two tumor suppressor genes, named AdVtTA.RB110/p53.
  • AdVtTA.RB110/p53 the smaller p53 expression cassette is inserted into the E3 region of the 34 kb master plasmid, pBHGl l, through ligation reaction. Since attempts to replace both RB and p53 genes in the same cell have never been successful (Wang et al, 1993), the inventors reasoned that adenovirus vectors simultaneously expressing the two tumor suppressor genes should be built in the regulatable gene expression system.
  • the kat retrovirus production system produces high titer retrovirus supernatant capable of transducing efficiently hematopoietic cell types refractory to conventional retrovirus transduction (Finer et al, 1994).
  • the kat retrovirus plasmid vector with a hybrid LTR with will be combined with EC1214A (Example 3) to generate a retrovirus with Tc-regulatable expression. Since some success using standard retroviral vectors have been reported in the literature, the Tc-controlled retroviral vector may work better than the Tc-controlled adenoviral vector for transduction of certain cell types, such as hematopoietic stem cells.
  • EXAMPLE 11 Therapeutic Administration of Modified RB Constructs A. Treatment of Human Bladder Cancers in vivo.
  • the human bladder cancer represents an ideal model for practicing tumor suppressor gene therapy of solid tumors by infusing the instant modified RB protein expression retroviral vectors into the bladder.
  • the original experimental model of human bladder cancer was established by Jones and colleagues (Ahlering et al, 1987). It has been shown that human bladder tumor cells of RT4 cell line established from a superficial papillary tumor, which usually does not metastasize, produced tumors only locally when injected by a 22-gauge catheter into the bladder of female nude mice. In contrast, the EJ bladder carcinoma cells which were originally isolated from a more aggressive human bladder cancer produced invasive tumors in the nude mouse bladders which metastasized to the lung spontaneously. Therefore, this model can be used for treatment of experimental bladder cancer by in vivo gene transfer with retroviral vectors.
  • RB + human bladder carcinoma cell line SCaBER (ATCC HTB3) will be injected directly into the bladders of female athymic (nu/nu) nude mice (6 to 8 weeks of age) by a catheter as initially reported by Jones and colleagues (Ahlering et al, 1987). Development and progression of the nude mouse bladder tumors will be monitored using a fiber-optical system to which a TV monitor is attached. The experimental tumors will subsequently be treated with retrovirus vectors expressing the modified RB proteins of the present invention.
  • Supernatants with high virus titers will be obtained from tissue culture media of selected clones expressing high level of human modified RB protein and confirmed as free of replication- competent virus prior to use.
  • NCI-H460 (ATCC HTB 177) cells which have normal pRB 1 expression will be injected into the right mainstream bronchus of athymic (nu/nu) nude mice (10 cells per mouse). Three days later the mice will be inoculated endobronchically with supernatant from the modified RB, or wild-type RB retrovirus producer cells daily for three consecutive days. Tumor formation suppression in the group of mice treated with the modified RB retrovirus supernatant, in contrast, to the group which is treated with wild-type RB retrovirus supernatant, will indicate that the modified RB-expressing retrovirus inhibits growth of RB + non-small cell lung carcinoma (NSCLC) cells, whereas the wild-type RB-expressing retrovirus does not.
  • NSCLC non-small cell lung carcinoma
  • Non-small cell lung cancer patients having an endobronchial tumor accessible to a bronchoscope, and also having a bronchial obstruction, will be initially selected for modified RB gene therapy. Treatment will be administered by bronchoscopy under topical or general anesthesia. To begin the procedure, as much gross tumor as possible will be resected endoscopically. A transbronchial aspiration needle (21G) will be passed through the biopsy channel of the bronchoscope. The residual tumor site will then be injected with the appropriate modified RB retroviral vector supernatant, modified RB adenovirus suspension or modified RB- expressing plasmid vector-liposome complexes at a volume of 5 ml to 10 ml.
  • 21G transbronchial aspiration needle
  • Protamine may be added to a concentration of 5 ⁇ g/ml.
  • the injections of therapeutic viral or plasmid supernatant comprising one or more of the vectors will be administered around and within the tumor or tumors and into the submucosa adjacent to the tumor.
  • the injections will be repeated daily for five consecutive days and monthly thereafter.
  • the treatment may be continued as long as there is no tumor progression.
  • After one year the patients will be evaluated to determine whether it is appropriate to continue therapy.
  • the patients will wear a surgical mask for 24 hours following injection of the viral supernatant. All medical personnel will wear masks routinely during bronchoscopy and injection of the viral supernatant. Anti -tussive will be prescribed as necessary.
  • target tumor or cancer cells will be treated by introducing the instant modified RB proteins into cells in need of such treatment by any known method.
  • liposomes are artificial membrane vesicles that have been extensively studied for their usefulness as delivery vehicles of drugs, proteins and plasmid vectors both in vitro or in vivo (Mannino et al, 1988).
  • Proteins such as erythrocyte anion transporter (Newton et al, 1988), superoxide dismutase and catalase (Tanswell et al, 1990), and UV-DNA repair enzyme (Ceccoli et al, 1989) have been encapsulated at high efficiency with liposome vesicles and delivered into mammalian cells in vitro or in vivo.
  • small-particle aerosols provide a method for the delivery of drugs for treatment of respiratory diseases. For example, it has been reported that drugs can be administered in small-particle aerosols by using liposomes as a vehicle. Administered via aerosols, the drugs are deposited rather uniformly on the surface of the nasopharynx, the tracheobronchial tree and in the pulmonary area (Knight et al., 1988).
  • the therapeutic modified RB proteins will be purified, for example, from recombinant baculovirus AcMNPV -modified RB infected insect cells by immunoaffinity chromatography or any other convenient source.
  • the modified RB protein will then be mixed with liposomes and inco ⁇ orated into the liposome vesicles at high efficiency.
  • the encapsulated modified RB will still be active. Since the aerosol delivery method is mild and well-tolerated by normal volunteers and patients, the modified RB-containing liposomes can be administered to treat patients suffering from lung cancers of any stage and/or to prevent lung cancers in high-risk population.
  • the modified RB protein-containing liposomes may administered by nasal inhalation or by a endotracheal tube via small-particle aerosols at a dose sufficient to suppress abnormal cell proliferation. Aerosolization treatments will be administered to a patient for 30 minutes, three times daily for two weeks, with repetition as needed. The modified RB protein will thereby be delivered throughout the respiratory tract and the pulmonary area. The treatment may be continued as long as necessary. After one year, the overall condition of the patient will be evaluated to determine if continued therapy is appropriate.
  • telomerase activity which is believed to be essential for an extended proliferative life-span of neoplastic cells, was abrogated or repressed in the tumor cell lines after induction of pRB (but not p53) expression. Strikingly, when returned to an non-permissive medium for pRB expression, the pRB-induced senescent tumor cells resumed DNA synthesis and attempted to divide. However, most cells died in the process, a phenomenon similar to postsenescent crisis of SV40 T-antigen-transformed human diploid fibroblasts in late passage.
  • the original multiple-plasmid tetracycline repressor/operator-based regulatory system was improved as described in detail above. All ⁇ -reconstituted tumor cell lines used in this Example were subjected to at least two rounds of subcloning following the initial plasmid transfection and are considered pure clones. The homogeneity of these clones was verified by pRB nuclear staining. In addition, a panel assay (Zhou et al, 1994) was used to ensure stable expression of the functional pRB under permissive conditions.
  • the ⁇ -reconstituted tumor cells were all RB " in the presence of 0.5 ⁇ g/ml of Tc in culture medium; while the great majority (>99%) of the cells became RB + at 24 hours after removal of Tc as shown by immunocytochemical staining.
  • the first peak (Ml) contains cells with diploid DNA in GO/Gl
  • the second peak (M3) with twice the Pi-fluorescence intensity contains tetraploid G2/M cells
  • the area between the two peaks (M2) represents the total number of cells in S phase (Nicoletti et al, 1991).
  • SA- ⁇ -gal assay The assay was performed essentially as previously described (Dimri et al, 1995).
  • the cells were fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 min and stained with 5-bromo-4-chloro-3-indolyl ⁇ -D-galactoside (X-Gal) at pH 6.0 for 6 hours.
  • the staining solution contained 1 mg/ml X-Gal, 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl 2 .
  • the methodology was modified from the original TRAP assay as described by Kim et al. (Kim et al, 1994).
  • -10 cells grown in a 100-mm Petri dish were harvested and resuspended in 200 ⁇ l of ice-cold lysis buffer for 30 min on ice, followed by centrifugation at 100,000 x g for 30 min at 4°C.
  • the supernatant was diluted to 0.5 ⁇ g protein/ ⁇ l, of which 2 ⁇ l was used for each TRAP assay.
  • telomerase reaction was carried out at 30°C for 30 min, which was followed by a 2-step PCRTM amplification with [ ⁇ - 32 P]-labeled TS primer (94°C, 30 s and 60°C, 30 s for 33 cycles).
  • the PCRTM -amplified telomerase extension products were subjected to electrophoresis on a 12.5% polyacrylamide gel.
  • tumor cell clones were established, in which expression of the wild-type pRB can be reversibly turned on and off without significant leakage.
  • the RB- reconstituted tumor cell clones were obtained, respectively, from the breast carcinoma cell line, MDA-MB-468, the osteosarcoma cell line Saos-2, and the bladder carcinoma cell line, 5637. These tumor cell lines were chosen as host cells since they were known to contain both RB and p53 gene mutations (Wang et al, 1993; Chen et al, 1990; Berry et al, 1996; Masuda et al, 1987).
  • pRB protein induced in the tumor cells reached the highest level about 24 hours after removal of tetracycline from the cell culture medium, and then became completely dephosphorylated within 24 to 40 hours.
  • the effects of induction of pRB expression on tumor cell growth were subsequently examined in representative clones by measuring growth curves and ( H) thymidine inco ⁇ oration (Xu et al, 1994b), and by flow cytometric analysis (Nicoletti et al, 1991). Cell growth and DNA synthesis of all the long-term tumor cell clones studied ceased 24 to 48 hours after pRB expression was induced (FIG. 3A, FIG. 3B and FIG. 3C). The great majority of the tumor cells were arrested at G0/G1 phase of the cell cycle.
  • the growth cessation of the tumor cells was irreversible by stimulation with a variety of mitogens, such as serum growth factors, phytohemagglutinin (PHA) and concanavalin A (Con A). This was determined by continuous flat growth curves as shown in FIG. 3A, FIG. 3B and FIG. 3C and failure of the tumor cells to inco ⁇ orate ( H) thymidine in response to mitogenic stimulation.
  • mitogens such as serum growth factors, phytohemagglutinin (PHA) and concanavalin A (Con A).
  • PHA phytohemagglutinin
  • Con A concanavalin A
  • SA- ⁇ -gal senescence-associated ⁇ -galactosidase
  • the Tc-responsive RB- reconstituted tumor cell clones were totally SA- ⁇ -gal negative in the presence of Tc (i.e., in RB " status), and the majority of the tumor cells became SA- ⁇ -gal positive after induction of pRB expression for four to five days in Tc-free medium.
  • the detection of this senescence-associated biomarker in the tumor cells was coincident with the irreversible growth cessation of the tumor cell populations (FIG. 3 A, FIG. 3B and FIG. 3C).
  • the intensity of the SA- ⁇ -gal staining of the induced RB tumor cells was variable depending on the tumor cell types.
  • telomerase Since telomerase has recently emerged as an attractive candidate for a regulator in cellular senescence (Linskens et al, 1995; Klingelhutz et al, 1996), the effects of pRB and p53 replacement on the telomerase activity of the host tumor cells were determined. In this connection, several long-term stable tumor cell clones with Tc-regulatable wild-type p53 expression from the osteosarcoma cell line, Saos-2 were established. A telomeric repeat amplification protocol (TRAP) assay as recently described (Kim et al, 1994) was used to measure telomerase activity in tumor cells before and after induction of pRB (or p53) expression.
  • TTP telomeric repeat amplification protocol
  • the i?5-reconstituted tumor cell clones from all three ft ⁇ //>55-defective tumor types examined were positive for telomerase activity, whereas the relative telomerase activity was ⁇ 15 to >100 times lower in the tumor cells after turning on the pRB expression as estimated by densitometry of the digitized image.
  • the telomerase activity was nearly non-detectable in the pRB-expressing MDA-MB-468 and Saos-2 tumor cells.
  • the pRB-induced tumor cell senescence was stringently dependent on the continued expression of the functional pRB.
  • the ⁇ -reconstituted MDA-MB-468, Saos-2, and 5637 tumor cells became senescent.
  • these tumor cells returned to an non-permissive medium for pRB expression, however, a large number of tumor cells were observed that lost cell-cell adherence, detached from the Petri dishes and died.
  • a combined method was employed involving pRB immunocytochemical staining and ( H) thymidine in situ labeling of the tumor cells.
  • telomere reexpression of functional pRB in i?5-defective tumor cells induced growth cessation concurrently with inhibition of telomerase activity.
  • the tumor cells irreversibly lost mitogen responsiveness, entering a viable Gl-arrested state. They also exhibited pRB-dependent SA- ⁇ -gal positivity (a senescence-associated biomarker) and resistance to apoptotic cell death.
  • SA- ⁇ -gal positivity a senescence-associated biomarker
  • replacement of either wild-type pRB or p53 in the RB7p53 nuU Saos-2 was able to block tumor cell growth at the population level, but only pRB induced inhibition of telomerase.
  • withdrawal of pRB in pRB-induced senescent tumor cells led to a crisis-like phenotype.
  • telomere activity is shown.
  • PCRTM polymerase chain reaction
  • the pRB -reconstituted Saos-2 clone when maintained in non-permissive conditions for pRB (or p53) expression, the pRB -reconstituted Saos-2 clone apparently had much lower telomerase activity than the p53 -reconstituted Saos-2 clone.
  • the difference implies that, even before switching-on of the pRB expression in Tc-free medium, there must be low baseline expression of pRB from the Tc-responsive promoter in Saos-2 cells (Gossen and Bujard, 1995).
  • the leakiness of pRB in pRB-reconstituted tumor cells under non-permissive conditions is below the immunodetection threshold for pRB protein (Xu et al, 1991b), but it might be sufficient to inhibit the most telomerase activity. Since the tumor cells lacking telomerase activity likely resume telomere decline, this would eventually trigger the intrinsic cellular senescence program if it remains intact in the tumor cells.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Ludlow et al Cell, 56:57-65, 1989. Ludlow et al, Cell, 60:387-396, 1990.
  • Nicolas and Rubinstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 494-513, 1988.
  • GAG AAA GTT TCA TCT GTG GAT GGA GTA TTG GGA GGT TAT ATT CAA
  • AAG 288 Glu Lys Val Ser Ser Val Asp Gly Val Leu Gly Gly Tyr lie Gin Lys 80 85 90
  • GCT AAA GCT GTG GGA CAG GGT TGT GTC GAA ATT GGA TCA CAG CGA TAC 1344 Ala Lys Ala Val Gly Gin Gly Cys Val Glu He Gly Ser Gin Arg Tyr 435 440 445

Abstract

Cette invention se rapporte à des protéines de suppression des rétinoblastomes, à large spectre d'efficacité, qui possèdent au minimum une activité biologique identique, et dans la plupart des cas une activité biologique supérieure, à celle de la protéine correspondante de suppression des rétinoblastomes du type sauvage. Les protéines de la présente invention possèdent une région N-terminale modifiée, ladite région comportant notamment des délétions et/ou des mutations. La présente invention se rapporte également à des procédés de fabrication et d'utilisation desdites protéines modifiées de suppression des rétinoblastomes, notamment dans les cas où l'on souhaite inhiber la croissance cellulaire. Cette invention se rapporte par conséquent à des procédés de traitement de maladies, et notamment, mais pas exclusivement, du cancer, de telles maladies se caractérisant par une prolifération cellulaire anormale.
EP98908570A 1997-02-20 1998-02-19 Proteines modifiees de suppression des retinoblastomes Ceased EP0975750A2 (fr)

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WO1995007708A2 (fr) * 1993-09-13 1995-03-23 The Regents Of The University Of California Utilisation therapeutique d'un produit genique contre la predisposition au retinoblastome

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