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WO1997016547A1 - VECTEURS D'EXPRESSION ADENOVIRAUX ANTISENS DU K-ras ET LEUR APPLICATION EN THERAPIE ANTICANCEREUSE - Google Patents

VECTEURS D'EXPRESSION ADENOVIRAUX ANTISENS DU K-ras ET LEUR APPLICATION EN THERAPIE ANTICANCEREUSE Download PDF

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Publication number
WO1997016547A1
WO1997016547A1 PCT/US1996/017979 US9617979W WO9716547A1 WO 1997016547 A1 WO1997016547 A1 WO 1997016547A1 US 9617979 W US9617979 W US 9617979W WO 9716547 A1 WO9716547 A1 WO 9716547A1
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Prior art keywords
ras
cell
expression vector
polynucleotide
antisense
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PCT/US1996/017979
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English (en)
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WO1997016547A9 (fr
Inventor
Jack A. Roth
Ramon Alemany
Wei-Wei Zhang
Tapas Mukhopadhyay
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The Board Of Regents, The University Of Texas System
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Priority to AU76748/96A priority Critical patent/AU7674896A/en
Publication of WO1997016547A1 publication Critical patent/WO1997016547A1/fr
Publication of WO1997016547A9 publication Critical patent/WO1997016547A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 tumor biology.
  • the invention relates to a polynucleotide encoding an antisense construct that targets a known oncogene.
  • the invention relates to a polynucleotide encoding an antisense construct that targets a known oncogene.
  • invention relates to adenovirus expression vectors encoding an antisense K-ras and their use in inhibiting cancer.
  • Mutations of the ras gene family are found in more than 30% of human carcinomas, especially those of
  • mutated ras genes permanently transduce a strong mitogenic signal to stimulate cell proliferation. Therefore, blocking mutated ras has a clear antitumor potential, and different strategies have been used to achieve this objective.
  • the neoplastic phenotype associated with mutated ras genes has been reversed by antibodies to p21, by fragments of natural p21 ligands (e.g., NF1 and c-Raf-1), and by dominant negative ras mutants.
  • K-ras mutations may arise prior to invasion and can easily be detected in sputum samples. The presence of this mutation correlates with a poor clinical outcome.
  • Initial studies have shown that K-ras expression in tumor cell lines can be inhibited by transfection of a plasmid construct that expresses a K-ras antisense RNA. This K-ras construct was then inserted into a retroviral vector and similar results were achieved following infection of tumor cells and in an orthotopic nude mouse model. Mukhopadhyay et al. (1991); Georges et al.
  • retroviral system is not without its limitations.
  • vector-borne genotoxicity is associated with integration.
  • Retroviruses also are unstable, require specific
  • the present invention addresses the need for
  • compositions and, in particular, use in the treatment of cancer.
  • the present invention encompasses adenovirus
  • expression vectors that comprise a promoter functional in eukaryotic cells and a polynucleotide encoding a K-ras antisense construct, the polynucleotide being under transcriptional control of the promoter and positioned such that the transcript produced is antisense.
  • the adenovirus lacks at least a portion of the El region.
  • the adenoviral expression vectors further comprise a
  • constructs further comprise a selectable marker.
  • the polynucleotide is derived from the genome. In other embodiments, the polynucleotide is a cDNA or synthetically generated polynucleotide. Still other embodiments include a combination of cDNA and genomic DNA, for example, in a mini-gene construct. Further embodiments include
  • fragments of K-ras that correspond to introns and/or splice junctions are fragments of K-ras that correspond to introns and/or splice junctions.
  • the present invention also includes pharmaceutical compositions comprising an expression vector with a promoter functional in eukaryotic cells and a
  • polynucleotide encoding a K-ras antisense transcript along with a pharmaceutically acceptable buffer, solvent or diluent.
  • the expression vector and pharmaceutically acceptable buffer, solvent or diluent are supplied in a kit.
  • the present invention further comprises a method for inhibiting K-ras function in a cell.
  • This method comprises contacting such a cell with an expression vector as described above, wherein the polynucleotide is positioned in an antisense orientation with respect to the promoter.
  • the cell is a transformed cell and the
  • the cell is a lung, pancreas or colon cancer cell.
  • Another embodiment of the invention is a method of treating a mammal with cancer. This method comprises administering to an animal a pharmaceutical composition comprising an expression vector having a promoter
  • the mammal is a human.
  • administering is via intratumoral instillation.
  • the cancer is lung cancer.
  • FIG. 1 Adenoviral Vector Construction.
  • a 2 kB genomic fragment containing exons 2 and 3 and intron 2 of the K-ras protooncogene was cloned between the CMV promoter and the SV40 polyadenylation signal in sense and antisense orientations.
  • These expression constructs were inserted into the polylinker site of pXCJL.1, which contains the left arm of Adenovirus type 5 (Ad5) with the exception of an E1 deletion.
  • Ad5 Adenovirus type 5
  • FIG. 2 Growth Curve of Transduced H460a Cell In Vitro. At the indicated days following initial infection (MOI of 100 pfu/cell, day 0), cells were incubated with [ 3 H] thymidine for 4 h and harvested, and the incorporated radioactivity was counted (cpm). The plot represents combined data from three studies. Similar curves were obtained by cell counting (P ⁇ .001) by analysis of
  • the present invention involves the use of adenoviral expression vectors in the reversal of the transformed state of certain tumor cells.
  • the adenovirus genome provides an advantageous framework in which to insert a therapeutic gene, in this instance, an antisense polynucleotide for a K-ras antisense construct.
  • Preferred forms of the virus are replication defective and can only be grown on special, helper cell lines that provide the missing replicative functions in trans.
  • Such an engineered adenovirus can be propagated in vitro to high titers for use in treating cancer cells.
  • antisense constructs containing introns bind to "sense" intron regions found on the RNA transcript of the gene, and affect proper RNA processing. Thus, subsequent translation of protein-coding RNA's into their corresponding proteins is inhibited or prevented.
  • the use of antisense introns may prove advantageous, in certain situations, because genetic diversity in
  • non-coding regions may be higher than in coding regions.
  • intron is intended to refer to gene regions that are transcribed into RNA molecules, but processed out of the RNA before it is translated into a protein.
  • exon regions are those which are translated into protein.
  • a "distinct" intron region is intended to refer to an intron region that is sufficiently different from an intron region of another gene such that cross hybridization would not occur under physiologic conditions.
  • the intracellular concentration of monovalent cation is approximately 160 mM (10 mM Na + ; 150 mM K + ). The intracellular
  • concentration of divalent cation is approximately 20 mM (18 mM Mg + ; 2 mM Ca ++ ).
  • the intracellular protein concentration which would serve to decrease the volume of hybridization and, therefore, increase the effective concentration of nucleic acid species, is 150 mg/ml. Constructs can be tested in vi tro under conditions that mimic these in vivo conditions. Typically, where one intron exhibits sequence homology of no more than 20% with respect to a second intron, one would not expect hybridization to occur between antisense and sense introns under physiologic conditions.
  • K-ras antisense polynucleotide is intended to refer to molecules complementary to the RNA of K-ras or the DNA corresponding thereto.
  • “Complementary” polynucleotides are those which are capable of base-pairing according to the standard
  • the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C cytosine
  • A:T thymine
  • A:U uracil
  • Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • polynucleotides leads to triple-helix formation
  • targeting RNA will lead to double-helix formation.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vi tro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is
  • the most effective antisense constructs will include regions complementary to intron/exon splice junctions.
  • a preferred antisense constructs will include regions complementary to intron/exon splice junctions.
  • antisense sequences mean polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example,
  • sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen positions.
  • sequences which are "completely complementary” will be sequences which are entirely complementary throughout their entire length and have no base mismatches.
  • Other sequences with lower degrees of homology also are contemplated.
  • an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., a ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • polynucleotides may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In other embodiments, however, the polynucleotides may be complementary DNA (cDNA).
  • cDNA is DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region (s) for the corresponding protein.
  • the antisense polynucleotide may be produced synthetically.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used.
  • polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • K-ras The DNA and protein sequences for K-ras are provided below. It is contemplated that natural variants of K-ras exist that have different sequences than those disclosed herein. Thus, the present invention is not limited to use of the provided polynucleotide sequence for K-ras but, rather, includes use of any naturally-occurring variants. Depending on the particular sequence of such variants, they may provide additional advantages in terms of target selectivity, i . e . , avoid unwanted antisense inhibition of K-ras-related transcripts. The present invention also encompasses chemically synthesized mutants of these sequences.
  • sequences that have between about 50% and about 75%, or between about 76% and about 99% of nucleotides that are identical to the nucleotides disclosed herein will be preferred.
  • Sequences that are within the scope of "a K-ras antisense polynucleotide” are those that are capable of base-pairing with a polynucleotide segment containing the complement of the K-ras sequences
  • K-ras antisense sequences may be full length genomic or cDNA copies, or large fragments thereof, they also may be shorter
  • oligonucleotides Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of base-pairing. Both binding affinity and sequence specificity of an
  • oligonucleotide to its complementary target increases with increasing length. It is contemplated that
  • oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs will be used. While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo
  • oligonucleotide to its complementary target increases with increasing length. It is contemplated that
  • oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used.
  • antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines.
  • Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al ., 1993).
  • ribozyme is refers to an RNA-based enzyme capable of targeting and cleaving particular base sequences in K-ras DNA and RNA. Ribozymes can either be targeted directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes may be used and applied in much the same way as described for antisense polynucleotide. Ribozyme sequences also may be modified in much the same way as described for antisense polynucleotide. For example, one could incorporate non-Watson-Crick bases, or make mixed RNA/DNA
  • oligonucleotides or modify the phosphodiester backbone.
  • the nucleotide and amino acid sequences of K-ras are as follows: The following sequence includes a genomic fragment of K-ras from base 67 to base 1961. This genomic
  • fragment includes exon 2.
  • the exon begins at base 618 and ends at base 796.
  • the underlined sequences are examples of oligonucleotide primer hybridization
  • adenoviral expression vector is meant to include those constructs containing adenovirus sequences sufficient to (i) support packaging of the construct and ( ii ) to express an
  • antisense polynucleotide that has been cloned therein.
  • expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • double-stranded DNA virus allows substitution of a large piece of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992).
  • retrovirus the infection of adenoviral DNA into host cells does not result in chromosomal integration because 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 can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in the human.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair (bp) inverted terminal repeats (ITR), which are cis elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal 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 E1 region (E1A 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
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible
  • wild-type adenovirus may be generated from this process.
  • adenovirus can package approximately 105% of the adenovirus genome
  • the maximum capacity of the current adenovirus vector is under 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 and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1 deleted virus is incomplete. For example, leakage of viral gene
  • 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 propagation of adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 L
  • Sterlin, Stone, UK (5 g/l) 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 1-4 h. The medium is then replaced with 50 ml of fresh medium and shaking started. 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.
  • adenovirus vector be replication defective, or at least
  • 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 method of 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 E1 region.
  • the position of insertion of the K-ras construct within the adenovirus sequences is not critical to the present invention.
  • the polynucleotide encoding a K-ras antisense transcription unit also may be inserted in lieu of the deleted E3 region in E3
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 11 plaque-forming unit (PFU)/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
  • 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., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation
  • the polynucleotide encoding the K-ras polynucleotide typically is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location in relation to the polynucleotide to control RNA polymerase initiation and expression of the polynucleotide.
  • promoter will be used here to refer to a group of transcriptional control modules that are
  • promoters including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter
  • the particular promoter that is employed to control the expression of a K-ras polynucleotide is not believed to be critical, so long as it is capable of expressing the polynucleotide in the targeted cell.
  • a human cell it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the K-ras polynucleotide.
  • CMV cytomegalovirus
  • polynucleotides is contemplated as well, provided that the levels of expression are sufficient to produce a growth inhibitory effect.
  • a promoter with well-known properties, the level and pattern of expression of a polynucleotide following transfection can be optimized. For example, selection of a promoter which is active in specific cells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin (liver tumors), CC10 (lung tumor) and
  • prostate-specific antigen prostate tumor
  • K-ras antisense polynucleotides selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the antisense
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of K-ras antisense constructs. This list is not intended to be exhaustive of all the possible elements involved in the promotion of K-ras antisense expression but, merely, to be exemplary thereof.
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and
  • promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of viral promoters, cellular
  • promoters/enhancers and inducible promoters/enhancers that could be used in the K-ras antisense polynucleotide expression vector (Table 2 and Table 3). Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a K-ras construct. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible
  • Eukaryotic cells can support cytoplasmic transcription from certain bacteriophage promoters if the appropriate bacteriophage polymerase is provided, either as part of the delivery complex or as an additional genetic expression vector.
  • the delivery of an expression vector in a cell may be identified in vi tro or in vivo by including a marker in the expression vector.
  • the marker would result in an identifiable change to the transfected cell permitting easy identification of expression.
  • a drug selection marker aids in cloning and in the selection of transformants.
  • enzymes such as herpes simplex virus thymidine kinase (tk)
  • CAT chloramphenicol acetyltransferase
  • Immunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed along with the polynucleotide encoding K-ras antisense. Further examples of selectable markers are well known to one of skill in the art.
  • polyadenylation signal The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. The inventors have employed the SV40 polyadenylation signal in that it was convenient and known to function well in the target cells employed. Also contemplated as an element of the expression construct is a terminator.
  • the expression vector In order to effect expression of antisense K-ras constructs, the expression vector must be delivered into a cell. As described above, the preferred mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious adenovirus particle.
  • lipofectamine-DNA complexes lipofectamine-DNA complexes, cell sonication (Fechheimer et al . , 1987), gene bombardment using high velocity microprojectiles (Yang et al . , 1990), polycations
  • the adenoviral expression vector may simply consist of naked recombinant vector. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
  • Dubensky et al . (1984) successfully injected polyomavirus DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct
  • DNA encoding an antisense K-ras construct may also be transferred in a similar manner in vivo .
  • Another embodiment of the invention for transferring a naked DNA expression vector 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.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ.
  • DNA encoding a K-ras antisense construct may be delivered via this method.
  • the expression vector 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
  • lipofectamine-DNA complexes are also contemplated.
  • 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
  • HVJ hemagglutinating virus
  • liposome-encapsulated DNA (Kaneda et al . , 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression vectors have been successfully employed in transfer and expression of a polynucleotide in vi tro and in vivo, then they are applicable for the present invention.
  • a bacteriophage promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacteriophage
  • Receptor-mediated gene targeting vehicles generally consist of two
  • a cell receptor-specific ligand a cell receptor-specific ligand and a cell receptor-specific ligand
  • DNA-binding agent DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al . , 1993).
  • ASOR asialoorosomucoid
  • transferrin transferrin
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand for example, Nicolau et al . (1987) employed lactosyl-ceramide, a
  • an adenoviral expression vector also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems, with or without liposomes.
  • epidermal growth factor EGF
  • Mannose can be used to target the mannose receptor on liver cells.
  • CD5 CD5
  • CD22 lymphoma
  • CD25 T-cell leukemia
  • MAA melanoma
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a polynucleotide into the cells, in vi tro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues. Anderson et al., U.S. Patent
  • HS-tK herpes simplex-thymidine kinase
  • compositions of the present invention To kill cells, such as malignant or metastatic cells, using the methods and compositions of the present invention, one would generally contact a "target" cell with an expression vector and at least one DNA damaging agent. These compositions would be provided in a
  • This process may involve contacting the cells with the expression vector and the DNA damaging agent(s) or factor(s) at the same time.
  • the K-ras treatment may precede or follow the DNA damaging agent treatment by intervals ranging from minutes to weeks.
  • the DNA damaging factor and K-ras expression vector are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the DNA damaging agent and expression vector would still be able to exert an advantageously combined effect on the cell.
  • DNA damaging agents or factors are defined herein as any chemical compound or treatment method that induces DNA damage when applied to a cell.
  • agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • chemotherapeutic agents function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein.
  • Chemotherapeutic agents contemplated to be of use include, e . g. ,
  • adriamycin 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • DNA damaging agents whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • the use of cisplatin in combination with a K-ras antisense expression vector is particularly
  • a DNA damaging agent in addition to the expression vector. This may be achieved by irradiating the localized tumor site with DNA damaging radiation such as X-rays, UV-light, ⁇ -rays or even microwaves.
  • the tumor cells may be contacted with the DNA damaging agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a DNA damaging compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more
  • a DNA damaging compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more
  • the DNA damaging agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a K-ras expression vector, as described above.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 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 damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
  • 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 2 at 21 day intervals for adriamycin, to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally.
  • (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the
  • K-ras expression vectors to patients with K-ras-linked cancers will be a very efficient method for delivering a therapeutically effective gene to counteract the clinical disease.
  • the chemo- or radiotherapy may be directed to a particular, affected region of the
  • 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-1 ⁇ IL-1 ⁇ , 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
  • tumor-related gene conceivably can be targeted in this manner, for example, p53, p21, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, other ras molecules, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. It also may be desirable to combine anti-sense K-ras therapy with an antibody-based gene therapy treatment involving the use of a
  • compositions of the present invention comprise an effective amount of the expression vector, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients also can be incorporated into the compositions.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as
  • 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
  • the expression vectors and delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of therapeutic 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 will be by orthotopic, intradermal subcutaneous,
  • compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a
  • the composition may contain 10 mg, 25 mg, 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 ethyloleate.
  • 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 the pharmaceutical composition 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.
  • an effective amount of the therapeutic agent is determined based on the intended goal, for example (i) inhibition of tumor cell proliferation or (ii)
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, 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. In certain embodiments, it may be desireable to provide a continuous supply of therapeutic compositions to the patient. For intravenous or intraarterial routes, this is accomplished by drip system. For topical
  • delayed release formulations could be used that provided limited but constant amounts of the therapeutic agent over and extended period of time.
  • continuous perfusion of the region of interest may be preferred. This could be accomplished by catheterization, post-operatively in some cases, followed by continuous administration of the therapeutic agent.
  • the time period for perfusion would be selected by the clinician for the particular patient and situation, but times could range from about 1-2 h, to 2-6 h, to about 6-10 h, to about 10-24 h, to about 1-2 days, to about 1-2 weeks or longer.
  • kits This generally will comprise selected adenoviral expression vectors. Also included may be various media for replication of the expression vectors and host cells for such replication. Such kits will comprise distinct containers for each individual reagent.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the expression vector may be formulated into a
  • the container means may itself be an inhalent, syringe, pipette, eye dropper, or other such like
  • kits 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.
  • the components of the kit 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
  • kits of the present invention also will be understood.
  • kits of the invention 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.
  • 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 inhalent, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • 293 cells (293S, human embryonic kidney cells) at passage thirty-one, grown in minimal essential medium with nonessential amino acids and 10% horse serum, were used for cotransfections.
  • a selected population of 293 cells with faster growing properties (293F) was grown in DMEM 4 with 10% FBS and used for virus amplification.
  • the human NSCLC cell line H460a was maintained in RPMI medium with 5% fetal bovine serum (FBS). This cell line was derived from a
  • recombinant adenovirus, subconfluent cell monolayers were first incubated with the virus in a minimal amount of complete medium (1 ml/60-mm plate, 37°C in CO 2 incubator, 1 h rocking plates every 10 min to avoid drying).
  • I-Sal I genomic fragment from the K-ras protooncogene containing exons 2 (176 bp) and 3 (130 bp) with flanking intron sequences and complete intron 2 (1.7 kB) was obtained from the plasmid Apr1-neo-Kras (Mukhopadhyay et al., 1991). After blunting the ends with the Klenow, the fragment was cloned between the CMV promoter and SV40 poly A signal in both sense (S) and antisense (AS) orientations.
  • S sense
  • AS antisense
  • AdCMV-pA empty vectors. Viruses were subsequently plaque-isolated on 293S cells and amplified in 293F by standard procedures (Zhang et al., 1994;
  • the viruses were purified by two CsCl gradients (a step gradient of 1.5-1.35-1.25 g/ml, 150,000g 1 h and a continuous gradient of 1.35 g/ml, 150,000g 16 h). After dialysis, stocks were kept at -80°C in a solution containing 10 mM Tris-HCl, pH 7.5; 1 mM MgCl 2 ; and 10% glycerol. Titers of purified viruses were determined by plaque assays (Graham and Prevec, 1991).
  • [ 3 H] thymidine uptake assays cells grown at 50-60% confluence in 60-mm plates were infected for 24 h, trypsinized, counted and seeded in triplicate 96-well plates at 1 ⁇ 10 3 cells/well. At the specified day, 10 ⁇ l of a 1:10 dilution of [ 3 H] thymidine (5 Ci/mmol, Amersham) in DMEM with 3% FBS was added to each well and incubated for 4 h. Then cells were washed and harvested to filters for radioactivity counting. Direct cell number assays were performed as described elsewhere (Zhang et al., 1994).
  • infected cells were trypsinized, mixed with 0.35% agarose and plated over a base layer of 0.7% agarose as described elsewhere (Zhang et al., 1993). Colonies were counted 10 days later.
  • a 2 kB fragment was inserted downstream of a strong promoter. This fragment was chosen because it has been shown to block p21 protein expression in other systems without affecting the expression of the other proteins of the ras family (Zhang et al., 1993).
  • the steps used to construct the virus are parallel to those used to generate the adenoviral vector Ad5CMV-p53 (Zhang et al., 1994).
  • the fragment is inserted in an expression cassette.
  • this cassette is inserted into the E1-deleted region of the Ad5 left arm.
  • this construct is cotransfected with a
  • FIG. 1 shows these steps schematically.
  • the structure of the virus so produced was confirmed by restriction analysis.
  • the Xba I sites at the end of exon 3 in the K-ras fragment and in front of the CMV promoter allows clear distinction between the sense and the antisense constructs.
  • Example 3 Expression of Antisense K-ras RNA in Infected Cells
  • the first step in assessing the effect of AdKrasAS is to define an appropriate range of dose and toxicity. It was assumed that the more antisense RNA present in the cell, the stronger the growth inhibitory-effects would be, with a limit imposed by the toxic effects of large doses of viral proteins.
  • H460a cells were infected with an adenovirus expressing the ⁇ -gal gene (Ad5CMV-LacZ; Zhang et al., 1994), at an increasing multiplicity of infection (MOI).
  • AdKrasAS affects the pattern of K-ras mRNA expression.
  • Protein production was analyzed by Western blot using a monoclonal antibody specific for the p21 protein. Three days after infection with AdKrasAS at an MOI of 100 pfu/cell (65% of cells transduced), the level of p21 protein was less than half (30%) of that found in
  • AdKrasS-infected cells Another approach used to study the growth-inhibitory effect of AdKrasAS was to test the colony-forming ability of transduced cells. Plates with H460a cells infected with AdKrasAS consistently (three studies) showed about ten-fold fewer colonies; most cells remained as single cells (number of colonies, 121 ⁇ 24), as compared with uninfected cells (1304 ⁇ 182), AdKrasS-infected cells
  • AdKrasAS markedly decreased the capacity of human lung cancer cells to achieve anchorage-independent growth.
  • Bos "ras oncogenes in human cancer: A review,” Cancer Res., 49:2682, 1989. Boshart et al., "A very strong enhancer is located
  • Oligodeoxyribonucleotides Complementary mRNA of the Human c-Harvey-ras Oncogene on Cell Proliferation," J. Cancer Res. Clin. Oncol., 116 (Suppl. Part
  • Graham and van der Eb "A new technique for the assay of infectivity of human adenovirus 5 DNA", Virology, 52:456-467, 1973. Graham and Prevec, "Manipulation of adenovirus vectors," In: E.J. Murray (ed.), Methods in Molecular Biology, Vol. 7: Gene transfer and expression protocols, Clifton, N. J.: The Humana Press, 1991. Graham et al., "Characteristics of a human cell line
  • Hermonat and Muzycska "Use of adenoassociated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells," Proc. Natl. Acad. Sci. USA, 81:6466-6470, 1984.
  • Kaneda et al. "Increased expression of DNA cointroduced with nuclear protein in adult rat liver," Science,
  • Racher et al. Biotechnology Techniques, 9:169-174, 1995.
  • Ragot et al. "Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdxmice," Nature, 361:647-650, 1993.
  • Renan "Cancer genes: current status, future prospects, and applicants in radiotherapy/oncology,” Radiother. Oncol., 19:197-218, 1990.
  • adenovirus vaccines II. Antibody response and protective effect against acute respiratory disease due to adenovirus type 7," J. Infect. Dis.,

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Abstract

La présente invention concerne diverses constructions génétiques trouvant une utilisation, tant in vitro que in vivo, dans le domaine de la biologie tumorale et de la thérapie anticancéreuse. L'invention concerne notamment des vecteurs d'expression adénoviraux contenant un acide nucléique de K-ras en position antisens par rapport aux régions dirigeant la régulation. Selon une réalisation, le vecteur d'expression adénoviral est un vecteur adénoviral présentant une déficience de réplication auquel manque la région E1 et contenant un acide nucléique de K-ras. L'invention concerne également des procédés permettant d'inhiber la prolifération des cellules cancéreuses.
PCT/US1996/017979 1995-10-31 1996-10-31 VECTEURS D'EXPRESSION ADENOVIRAUX ANTISENS DU K-ras ET LEUR APPLICATION EN THERAPIE ANTICANCEREUSE WO1997016547A1 (fr)

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WO1999042137A3 (fr) * 1998-02-19 1999-10-21 Peter Bromley Regulation de genes therapeutiques par un promoteur de stress en therapie genique, et compositions et procedes associes
WO2001002556A3 (fr) * 1999-06-30 2001-08-16 Max Delbrueck Centrum Agents pour le diagnostic, le pronostic et la therapie relatifs a des maladies malignes
WO2001070951A3 (fr) * 2000-03-23 2002-05-02 Max Delbrueck Centrum Moyen de diagnostic et de therapie de maladies virales
WO2003033692A3 (fr) * 2001-10-16 2003-09-18 Univ Muenchen Tech Utilisation du promoteur tardif e2 adenoviral
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WO2007035962A3 (fr) * 2005-09-23 2007-05-10 California Inst Of Techn Methode de blocage de gene
US10155930B2 (en) 2002-05-27 2018-12-18 Per Sonne Holm Use of adenovirus and nucleic acids coding therefor
US10300096B2 (en) 2003-11-14 2019-05-28 Per Sonne Holm Use of adenoviruses and nucleic acids that code for said viruses
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042137A3 (fr) * 1998-02-19 1999-10-21 Peter Bromley Regulation de genes therapeutiques par un promoteur de stress en therapie genique, et compositions et procedes associes
WO2001002556A3 (fr) * 1999-06-30 2001-08-16 Max Delbrueck Centrum Agents pour le diagnostic, le pronostic et la therapie relatifs a des maladies malignes
WO2001070951A3 (fr) * 2000-03-23 2002-05-02 Max Delbrueck Centrum Moyen de diagnostic et de therapie de maladies virales
US7329649B2 (en) * 2001-08-20 2008-02-12 The Trustees Of Columbia University In The City Of New York Combinatorial methods for inducing cancer cell death
EP1425399A4 (fr) * 2001-08-20 2006-09-13 Univ Columbia Techniques combinatoires d'induction de la mort de cellules cancereuses
KR101015772B1 (ko) * 2001-10-16 2011-02-16 테크니쉐 우니베르지테트 뮌헨 아데노바이러스 ｅ2 후기 프로모터의 용도
US7572633B2 (en) 2001-10-16 2009-08-11 Per Sonne Holm Use of the adenoviral E2 late promoter
WO2003033692A3 (fr) * 2001-10-16 2003-09-18 Univ Muenchen Tech Utilisation du promoteur tardif e2 adenoviral
US8921100B2 (en) * 2001-10-16 2014-12-30 Technische Universität München Use of the adenoviral E2 late promoter
US10155930B2 (en) 2002-05-27 2018-12-18 Per Sonne Holm Use of adenovirus and nucleic acids coding therefor
US10538744B2 (en) 2002-05-27 2020-01-21 Per Sonne Holm Use of adenovirus and nucleic acids coding therefor
US10731136B2 (en) 2002-05-27 2020-08-04 Per Sonne Holm Use of adenovirus and nucleic acids coding therefor
US11268073B2 (en) 2002-05-27 2022-03-08 Per Sonne Holm Use of adenovirus and nucleic acids coding therefor
US10300096B2 (en) 2003-11-14 2019-05-28 Per Sonne Holm Use of adenoviruses and nucleic acids that code for said viruses
WO2007035962A3 (fr) * 2005-09-23 2007-05-10 California Inst Of Techn Methode de blocage de gene
WO2022226291A1 (fr) * 2021-04-22 2022-10-27 Dana-Farber Cancer Institute, Inc. Compositions et méthodes pour traiter le cancer

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