WO2001051630A1 - Compositions antisens et methodes afferentes - Google Patents

Compositions antisens et methodes afferentes Download PDF

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Publication number
WO2001051630A1
WO2001051630A1 PCT/US2001/000754 US0100754W WO0151630A1 WO 2001051630 A1 WO2001051630 A1 WO 2001051630A1 US 0100754 W US0100754 W US 0100754W WO 0151630 A1 WO0151630 A1 WO 0151630A1
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nucleic acid
antisense
antisense nucleic
cell
sequence
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PCT/US2001/000754
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English (en)
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Christopher M. Kearney
Mary K. Wood
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Baylor University
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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
    • 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
    • C12N15/1131—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 viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates generally to the fields of biochemistry and cell biology. More particularly, it concerns antisense compositions and methods.
  • antisense originally referred to an oligonucleotide sequence designed to complement a pre-mRNA or mRNA, both of which are “sense” molecules, so that the ultimate translation of that RNA would be inhibited. Theoretically, when the two molecules were in close proximity, they would hybridize to each other and the target RNA would therefore no longer be accessible to translation initiation factors and ribosomes.
  • antisense now includes oligonucleotides ("ON”; short RNA molecules) and oligodeoxynucleotides (“ODN”; short, single stranded DNA molecules) that bind double stranded DNA or areas of double stranded secondary structure in RNA to form triplexes. Indeed, the current definition of antisense covers the entire range of ON/ODN/nucleic acid interaction.
  • antisense has been used in two main areas of research: loss-of- function studies and therapeutic use.
  • loss-of-function studies a gene or its mRNA is "turned off by complementary antisense and the resulting phenotype noted.
  • RNA and DNA antisense have been used to both control and study the functions of many diverse genes in a variety of prokaryotic and eukaryotic organisms (Green et al. 1986; Mol et al. 1990; Murray 1992).
  • Technology involving the development of antisense therapeutics is also showing rapid growth.
  • antisense therapeutics disease- associated genes or their RNA transcripts are targeted by antisense molecules designed to inhibit their expression (Brysch and Schlingensiepen 1994; Crooke 1998; Lavrovsky et al.
  • Some of the primary targets for therapeutic antisense design have been proto-oncogenes, such as c-myc, N- yc, c- myh, c-fos, N-r-xs * , c-H-ras, BCL-2, c-raf- ⁇ , cdc-2 and c-mos (Fabbro et al. 1998) and some of the DNA and RNA viruses (Cohen 1991; Cowsert et al. 1993; Field 1998).
  • antisense therapeutic agents have also been tested or are currently in clinical trials against such diverse conditions as rheumatoid arthritis (Nietfeld et al.
  • Antiviral antisense drugs have the potential to be quite specific for their viral targets without interfering with other nontarget nucleic acids within the cell. Theoretically, therefore, these drugs should be less cytotoxic.
  • the design of multiple antisense molecules targeting various sequences within a single virus would permit rapid drug rotation in response to the development of resistance by that virus.
  • antiviral antisense "cocktails" may be necessary when targeting viral populations having a high degree of genetic variability (Bull et al. 1998).
  • Bull et al. also suggest that the solution to this problem may involve simply targeting viral sequences that are relatively unchanging within that viral population.
  • Effective antisense design is still routinely a trial-and-error process with complementarity to the nucleic acid target being only one of the design considerations.
  • Other factors that have been reported as affecting the effectiveness of an antisense design include nonspecific interactions of the antisense with nontarget nucleic acids and nucleic acid-binding proteins, as well as the sequence context of antisense :target hybridization. This last factor includes not only the actual linear base sequence, but also the secondary and tertiary structures formed by both the antisense and the target.
  • antisense drug design is a more rational process than most traditional drug design protocols.
  • ODNs have been the most frequent form of antisense molecule used because of the greater intracellular stability of antisense DNA as compared to antisense RNA.
  • Most ODNs in use today are relatively short (12-25 nucleotides) and are chemically modified to increase their resistance to intracellular nucleases and to increase their affinity for the target molecules (Uhlmann and Peyman 1990). Unfortunately, these chemical modifications are often accompanied by an increase in cytotoxity, which limits the use of such ODNs in vivo.
  • ODNs the phosphorothioate ODNs
  • cytotoxicity a group of ODNs
  • a high level of intracellular stability a high level of intracellular stability
  • one of the oxygen atoms of the phosphate group normally found in the nucleic acid backbone is replaced with a sulfur atom (Cohen 1993).
  • sulfur increases the nuclease resistance of this ODN, the intracellular half-life is still only 12 to 24 hours (Crooke et al. 1995).
  • 9teen phosphorothioate antisense drugs had been allowed into clinical trials (Dean et ?/. 1996).
  • RNAs with varying degrees of success are produced with varying degrees of success (Weiss et al. 1999).
  • One way of producing this kind of antisense is by reverse transcribing a complementary DNA (cDNA) from a target mRNA and then cloning this cDNA into an expression vector in an antisense orientation.
  • the cloned nucleotide sequence is therefore reversed as compared with the normal sequence and RNA transcripts produced from this cloned sequence are able to hybridize with the target mRNA.
  • the expression vector is then either transfected or injected into the target cells. These vectors are transient, but remain within the cell long enough for antisense RNA transcription to occur.
  • Such expression vectors are quite convenient for rapidly screening a variety of antisense molecules to see which ones will affect translation of the target mRNAs.
  • antisense in plants has generally been geared more toward regulating and/or investigating plant gene expression rather than toward controlling viral infections. This was due primarily to the early development of relatively effective viral control and prevention measures.
  • Crop viruses have been partially controlled by crop management practices such as crop rotation, insect/nematode/fungal vector control, the planting of virus-free seed, and the development of virus-resistant plant varieties by selective breeding.
  • crop management practices such as crop rotation, insect/nematode/fungal vector control, the planting of virus-free seed, and the development of virus-resistant plant varieties by selective breeding.
  • these control measures are limited in their ability to prevent virus infections.
  • Another virus control measure is cross protection.
  • Cross protection is a process whereby a strain of the plant virus that produces only mild symptoms in the plant is inoculated onto the host plant. This mild virus infection protects the plant from infection by a more virulent strain of the virus (Fulton 1986 from PN). Cross protection does work although crop yields are often slightly reduced.
  • Tobamovirus in a "sense" orientation so that a normal TMN coat protein mR ⁇ A was now expressed in the transgenic tobacco plant (Abel et al. 1986). Tobacco is normally very susceptible to TMN infection, but the new transgenic plant was not. In addition, these plants were also resistant to certain of the Tobamoviruses closely related to TMN ( ⁇ ejidat and Beachy 1990). Since that time, coat protein-mediated resistance (CPMP) has been used successfully against a variety of plant viruses. Other viral genes, such as replicase and movement protein genes, have also been used as host plant transgenes but with varying degrees of success (Beachy 1997).
  • CPMP coat protein-mediated resistance
  • the antisense and target RNAs are not predicted to be linear at all. Indeed, secondary structures seem to be required for initial binding between the two RNAs (Hjalt and Wagner 1995; Simons 1993). A high degree of complementarity, however, is important.
  • Antisense length also seems to be important. Specificity for a particular target RNA requires an antisense length of at least 11 to 15 nucleotides (Helene and Toulme 1990). Below that length, the antisense tends to bind nonspecifically to a variety of targets. As antisense length increases, the affinity of the antisense for its target also potentially increases because of the increased number of hydrogen bonds possible between the two molecules (Mirabelli and Crooke 1993). There are dangers though in increasing antisense length ⁇ the antisense RNA has more opportunity to partially bind to nontarget RNAs (therefore decreasing its specificity for the target) and it may develop a higher degree of secondary structure (Ecker 1993) which may affect its ability to stably bind to the target.
  • Affinity is determined not only by antisense length but also by the GC versus AU content of the duplex.
  • a high GC content allows the antisense to bind strongly to nontarget RNAs even if there is a partial mismatch between the two molecules, whereas an antisense RNA with high AU content may not bind strongly even to its target sequence. Brysch (1994) therefore suggests that the best specificity and affinity is found in antisense RNAs containing an even distribution of AUs and GCs.
  • Ecker et al. (1992) have developed some optimization rules for antisense design. They include the following: (1) avoid targeting long stems but instead target single stranded areas, short stems, and bulges; (2) avoid antisense designs that contain extremely stable secondary or tertiary structures; (3) choose antisense lengths that allow all bases to bind the target. Again, the ribosome's ability to unwind double stranded structures within its template RNA must be considered. In competition studies done by Lawson et al (1989), eIF-4F was able to remove oligonucleotides complementary to the first 15 nucleotides of capped mRNA. Therefore, it might be more efficient to design antisense that prevents the initial binding of the ribosomal subunits rather than to attempt to design antisense with a high enough affinity for the target that the ribosome could not remove it.
  • the present invention concerns novel antisense compositions that overcome many of these problems and are more efficient at inhibitng expression of targetted genes.
  • This invention is based on antisense nucleic acid compositions comprising sequences complementary to a first region in a target nucleic acid and to a second region in a target nucleic acid.
  • the antisense sequences are designed to hybridize to complementary nucleic acid target regions in a target nucleic acid, and inhibit translation, processing, transport, or binding by proteins or riboproteins.
  • the sequences of the antisence nucleic acid compositions may be contiguous or noncontiguous.
  • the first and second regions of the target nucleic acid are not contiguous.
  • the invention encompasses antisense nucleic acid compositions and methods for their use.
  • the invention provides an antisence nucleic acid comprising sequences complementary to a first region in a target nucleic acid and a second region in the target nucleic acid.
  • the invention provides an antisence nucleic acid comprising a first sequence complementary to a first region in a target nucleic acid and a second sequence complementary to a second region in the target nucleic acid.
  • the invention comprises method of contacting a target nucleic acid with the antisence nucleic acids described above.
  • one or both of the first or second sequences of the antisense nucleic acid may be complementary to regions of the target nucleic acid that comprise, for example, a 5' non-translated region, an AUG, a translation initiation factor binding sequence, a ribosome subunit binding sequence, a Shine Dalgarno sequence, a 3' non-translated sequence, a poly-addition site, a 3' mRNA cleavage site, a coding region, or an intron, intron branch, intron/exon junction, or a splice sequence.
  • regions of the target nucleic acid that comprise, for example, a 5' non-translated region, an AUG, a translation initiation factor binding sequence, a ribosome subunit binding sequence, a Shine Dalgarno sequence, a 3' non-translated sequence, a poly-addition site, a 3' mRNA cleavage site, a coding region, or an intron, intron branch, intron/
  • the target nucleic acid encodes, for example, one or more oncogenes, angiogenic genes, tumor suppressors, inducers of apoptosis, enzymes, transcription factor regulators, cell cycle regulators, viral sequences and bacterial sequences.
  • the antisense nucleic acid may be about 20, 25, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000, or about 4000 bases or longer.
  • the antisense nucleic acid may be comprised in an expression vector.
  • the target nucleic acid is mRNA. In other embodiments the invention encompasses a method of inhibiting transcription of mRNA. In other embodiments, the invention includes methods of treating a disease state that is associated with expression of a selected gene product, wherein the target nucleic acid is an mRNA encoding the selected gene product.
  • the target nucleic acid is in a cell.
  • the type of such cell may be, for example, an animal cell, a plant cell, and a human cell.
  • the invention provides a method for protecting a cell from pathogen attack comprising contacting a nucleic acid of said pathogen with an antisense nucleic acid comprising sequences complementary to two regions in a target nucleic acid, wherein the target nucleic acid is a pathogen nucleic acid.
  • the pathogen is a virus.
  • the cell may be a plant or animal cell and the target nucleic acid may be mRNA.
  • the protecting comprises inhibiting viral replication.
  • the antisense nucleic acid may be comprised in an expression vector.
  • the first sequence and the second sequence may be linked by a linker.
  • the linker may comprise, for example, a nucleic acid segment, or non-nucleic acid such as an organic or inorganic chemical group.
  • the invention provides a method for screening for genes of a given function using the loop-inducing antisense format as a more predictably effective means to inhibit gene expression.
  • the invention comprises a pharmaceutical composition comprising an antisense nucleic acid comprising sequences complementary to two regions in a target nucleic acid, wherein the regions are not contiguous in the target nucleic acid.
  • the pharmaceutical composition is dispersed in a pharmacologically acceptable buffer, diluent or excipient.
  • the antisense nucleic acid may be comprised in an expression vector, may further be of any type contemplated by the invention.
  • the invention provides a cell comprising an antisense nucleic acid in accordance with the invention.
  • the cell may comprise a eukaryotic cell, including an animal, human or plant cell.
  • Another embodiment of the invention includes a plant or animal comprising such a cell.
  • FIG. 4A and FIG. 4B Construction of pPZP-AL plasmids.
  • FIG. 5 TFP transgene R ⁇ A expression levels. All plants gave a positive value except nontransgenic plants, which had density values of zero.
  • FIG. 6 TF ⁇ transgene R ⁇ A expression levels. All plants gave a positive value except nontransgenic plants, which had density values of zero.
  • FIG. 7 Development of systemic TMN infection in TFP plants. All plants shown in FIG. 5 and FIG. 6 were initially screened for resistance to TMN. The most resistant plants were selected for further screening, as seen here and in subsequent figures.
  • FIG. 8 Development of systemic TMN infection in TF ⁇ plants.
  • FIG. 9 Development of systemic TMN infection in TQP plants.
  • FIG. 10 Development of systemic TMN infection in TQ ⁇ plants.
  • FIG. 11 Development of systemic TMN infection in T 0 plants.
  • FIG. 12. Time of first appearance of lesions. Bars from left to right are for the following plants in each series: (GFP (55); GF ⁇ (31);GQP (44); GQ ⁇ (24);Gzero (7)). Numbers in () indicate the number of individual plants used to get the average value.
  • FIG. 13. Total lesion number for transgenic Nicotiana tabacum cv. Xanthi NN. Bars from left to right are for the following plants in each series: (GFP (41); GFN (44); GQP (72); GQN (10). Numbers in () indicate the number of individual plants used to get the average value.
  • FIG. 14 Average lesion diameters for GFP plants
  • FIG. 15 Average lesion diameters for transgenic Nicotiana tabacum cv. Xanthi NN.
  • the present invention overcomes deficiencies in the prior art by describing antisense nucleic acids compositions comprising a first and a second domain.
  • the domains are designed to hybridize to complementary nucleic acid target domains in a target RNA, and inhibit translation, processing, transport, or binding by proteins or riboproteins.
  • the first and the second target domain may be contiguous or noncontiguous in the target RNA.
  • Target domains include, for example, AUG, 5' non- translated sequences, translation initiation factor binding sites, ribosome subunit binding sites, Shine Dalgarno sequence, 3' nontranslated sequences, poly-A tail, 3' cleavage sites, coding regions, introns, intron branch sites, intron/exon junctions, or splice sequences.
  • Target RNA may be viral, bacterial, animal, human, and plant.
  • Methods for using these antisense compositions include inhibiting translation of an RNA and treating diseases in a cell, an animal, a plant or a human. Therapeutic antisense compositions are also described.
  • antisense technology including antisense transgene construction, viral and non-viral delivery and expression vectors, and antisense oligonucleotides. Descriptions of examples are given for target domains and target genes as well as methods for administering antisense compositions and disease treatment.
  • Antisense Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary is intended to refer to polynucleotides that are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and either adenine paired with thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine et al.
  • Antisense construct 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 RNAs may be employed to inhibit gene transcription, translation, or both within a host cell, either in vitro 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 contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means 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 complementary nucleotides at thirteen or fourteen positions. Naturally, sequences that are completely complementary will be sequences that are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology are also contemplated. For example, an antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • ribozyme e.g., ribozyme; see below
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized 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.
  • A. Antisense Transgenes within certain embodiments expression vectors used in therapeutic applications. Expression requires that appropriate signals be provided in the vectors, which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells are also defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells are also defined.
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • expression construct is meant to include any type of genetic construct containing a polynucleotide coding for a gene product in which part or all of the polynucleotide encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the polynucleotide encoding a gene of interest.
  • the polynucleotide encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the polynucleotide to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of D ⁇ A, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for
  • R ⁇ A 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 SN40 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 elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a polynucleotide sequence of interest is not believed to be important, so long as it is capable of directing the expression of 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 (CMN) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CPN human cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
  • Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • a transgene or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it may be desirable to prohibit or reduce expression of one or more of the transgenes.
  • transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes.
  • inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.
  • the Ecdysone-Inducible Mammalian Expression System (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mR ⁇ A transcripts are attained.
  • both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid.
  • Engineering of this type of system into the gene transfer vector of interest would therefore be useful.
  • Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
  • expression of the transgene could be activated with ecdysone or muristeron A.
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-OnTM system gene expression is turned on in the presence of doxycycline
  • Tet-OffTM system gene expression is turned on in the absence of doxycycline.
  • the gene of interest is cloned into a plasmid behind a promoter that has tetracycline- responsive elements present in it.
  • a second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-OffTM system, of the NP16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • the tetracycline-controlled transactivator which is composed, in the Tet-OffTM system, of the NP16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription.
  • Tet-OffTM system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
  • An inducible system particularly useful with the present invention is a radiation-inducible system.
  • Ionizing radiation-inducible promoters include a CArG domain of an Egr-1 promoter, a los promoter, a c-jun promoter, and a T ⁇ F- ⁇ promoter, which can be operatively linked to a protein expression region.
  • U.S. Patent 5,612,318, dealing with induction of expression from these promoters, is specifically inco ⁇ orated by reference.
  • CMN immediate early promoter if often used to provide strong transcriptional activation.
  • Modified versions of the CMN promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLN or MMTN are often used.
  • viral promoters that may be used depending on the desired effect include SN40, RSN LTR, HIN-1 and HIN-2 LTR, adenovirus promoters such as from the El A, E2A, or MLP region, AAN LTR, cauliflower mosaic virus, HSN-TK, and avian sarcoma virus.
  • tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non- targeted tissues.
  • promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein (hK2) may be used to target gene expression in the prostate.
  • promoters as those that are hormone or cytokine regulatable.
  • promoters that are hormone regulatable include MMTN, MT-1, ecdysone and RuBisco.
  • Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones are expected to be useful in the present invention.
  • Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen (Kageyama et al, 1987), c-fos, T ⁇ F-alpha, C- reactive protein (Arcone et al, 1988), haptoglobin (Oliviero et al, 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al, 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al, 1988), angiotensinogen (Ron et al, 1991), fibrinogen, c-jun (inducible by phorbol esters, T ⁇ F-alpha, UN radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothione
  • Tumor specific promoters such as osteocalcin, hypoxia-responsive element
  • HRE HRE
  • MAGE-4 MAGE-4
  • CEA alpha-fetoprotein
  • GRP78/BiP tyrosinase
  • Other promoters that could be used according to the present invention include Lac-regulatable, chemotherapy inducible (e.g. MDR), and heat (hyperthermia) inducible promoters, radiation-inducible (e.g., EGR (Joki et al, 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acid promoters, UI snRNA (Bartlett et al, 1996), MC-1, PGK, ⁇ -actin and ⁇ -globin. Many other promoters that may be useful are listed in Walther and Stein (1996).
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers 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 otlier 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 frequently overlapping and contiguous, often seeming to have a very similar modular organization.
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • 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 such as human growth hormone and SN40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • retrovirus the adenoviral infection of 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 humans.
  • 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 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 EIB) 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 shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, 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 the current 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 (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 D3 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 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 El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
  • MOI multiplicities of infection
  • 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., Nero 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 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • 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.
  • 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 ⁇ 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 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, 1990; Rich et al, 1993).
  • 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 retro viral 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 polynucleotide 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).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or tlirough the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Narmus et al, 1981).
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
  • Lentiviruses can also be used as vectors in the present application.
  • lentiviruses present the opportunity to transduce nondividing cells and potentially achieve regulated expression.
  • the development of lentiviral vectors requires the design of transfer vectors to ferry the transgene with efficient encapsidation of the transgene R ⁇ A and with full expression capability, and of a packaging vector to provide packaging machinery in trans but without helper virus production.
  • a knowledge of packaging signal is required-the signal to be included in the transfer vector but excluded from the packaging vector.
  • Exemplary human lentiviruses are human immunodeficiency virus type 1 and type 2 (HIN-1 and HIN-2).
  • HIN-2 is likely better suited for gene transfer than HIN-1 as it is less pathogenic and thus safer during design and production; its desirable nuclear import and undesirable cell-cycle arrest functions are segregated on two separate genes (Arya et al., 1998; Blo er et ., 1997).
  • AAN utilizes a linear, single-stranded D ⁇ A of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (NP), designated NP-1, NP-2 and NP-3. The second, the rep gene, encodes four non-structural proteins ( ⁇ S). One or more of these rep gene products is responsible for transactivating AAN transcription.
  • the three promoters in AAN are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
  • the splice site derived from map units 42-46, is the same for each transcript.
  • the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
  • AAN is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAN requires "helping" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAN replication. Low level expression of AAN rep proteins is believed to hold AAN structural expression in check, and helper virus infection is thought to remove this block.
  • the terminal repeats of an AAN vector can be obtained by restriction endonuciease digestion of AAN or a plasmid such as psub201, which contains a modified AAN genome (Samulski et al 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAN.
  • the ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAN ITRs which is required to allow function, i.e. stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
  • AAN-based vectors have proven to be safe and effective vehicle for gene delivery in vitro, and these vectors are now being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo.
  • wide variations in AAN transduction efficiency in different cells and tissues in vitro as well as in vivo has been repeatedly observed (Ponnazhagan et al, 1997b; 1997c; 1997d; 1997d) et al (Carter and Flotte, 1996 ; Chatterjee et al, 1995; Ferrari et al, 1996; Fisher et al, 1996; Flotte et al, 1993; Goodman et al, 1994; Kaplitt et al, 1994; 1996, Kessler et al, 1996; Koeberl et al, 1997; Mizukami et al , 1996; Xiao et al , 1996).
  • AAN-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1996; Flotte et al, 1993).
  • the prospects for treatment of muscular dystrophy by AAN- mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor IX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart appear promising since AAN-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al. , 1996; Flotte et al. , 1993; Kaplitt et al. , 1994; 1996; Koeberl et al. , 1997; McCown et al, 1996; Ping et al, 1996; Xiao et al, 1996).
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAN) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990). With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences.
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al (1984) successfully injected polyomavirus DNA in the form of calcium phosphate 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 intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • 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.
  • 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, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.
  • 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 are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Nicolau et al, (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.
  • 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).
  • 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. Since these expression constructs have been successfully employed in transfer and expression of polynucleotides in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a polynucleotide encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a 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, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and Wu, 1990
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a polynucleotide encoding a particular gene 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 may be used as the receptor for mediated delivery of a polynucleotide encoding a gene in many tumor cells that exhibit upregulation of EGF receptor.
  • Mannose can be used to target the mannose receptor on liver cells.
  • antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
  • 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 vitro, 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.
  • AS-ODNs Antisense oligodeoxynucleotides
  • mRNA messenger RNA
  • oligonucleotides or “ODNs” or “polynucleotides” or “oligos” or
  • oligomers or “n-mers" of the present invention are preferably deoxyoligonucleotides (i.e. DNAs), or derivatives thereof; ribo-oligonucleotides (i.e.
  • RNAs or derivatives thereof
  • PNAs peptide nucleic acids
  • the oligonucleotides may also comprise phosphorothioate antisense oligonucleotides.
  • substantially complementary when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to the sequence, and thus will specifically bind to a portion of an mRNA. As such, typically the sequences will be highly complementary to the mRNA "target" sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e.
  • oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch.
  • highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
  • Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or '% exact-match') to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds.
  • the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%o, and even 100% exact match complementary to the target mRNA to which the designed oligonucleotide specifically binds.
  • Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • "DNA sequence” refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct.
  • the DNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • sequences are preferably provided in the form of an open reading frame uninterrupted by internal nonfranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
  • RNA sequence refers to an RNA polymer, in the form of a separate fragment or as a component of a larger RNA construct, such as a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the RNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector, or alternatively, by chemically synthesizing the RNA molecule completely or partially in vitro.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of specifically binding to an mRNA molecule and acting as an antisense construct to alter, reduce, or inhibit the transcription of the message into polypeptide, and thus, ultimately affect the concentration, amount, or activity of the final gene product in situ, in vitro, or in vivo.
  • the targeting of antisense oligonucleotides to bind mRNA is one mechanism to shut down protein synthesis.
  • the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U. S. Patent 5,739,119 and U. S. Patent 5,759,829, each specifically incorporated herein by reference in its entirety).
  • antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E- selectin, STK-1, striatal GABA A receptor and human EGF (Jaskulski et al, 1988; Vasanthakumar and Ahmed, 1989; Peris et al, 1998; U. S. Patent 5,801,154; U. S. Patent 5,789,573; U. S. Patent 5,718,709 and U. S. Patent 5,610,288, each specifically incorporated herein by reference in its entirety).
  • Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U. S. Patent 5,747,470; U. S. Patent 5,591,317 and U. S. Patent 5,783,683, each specifically inco ⁇ orated herein by reference in its entirety).
  • the oligonucleotide compounds of the invention bind to the messenger RNA coding for human ACE thereby inhibiting expression of the protein.
  • the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura).
  • compounds that are antisense to the DNA or mRNA sense strand are compounds which have a nucleoside sequence complementary to the sense strand. Table 1 shows the four possible sense strand nucleosides and their complements present in an antisense compound. TABLE 1
  • the present invention broadly includes oligonucleotide compounds which are capable of binding to the sense mRNA.
  • the invention includes compounds which are not strictly antisense: the compounds may have some non-complementary bases provided such compound have sufficient binding affinity for mRNA to inhibit expression.
  • antisense compounds of the present invention may also differ from native
  • C 1-4 alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon-atoms.
  • Antisense compounds also may be substituted at the 3' and/or 5' ends by a substituted acridine derivative.
  • substituted acridine means any acridine derivative capable of intercalating nucleotide strands such as DNA.
  • Preferred substituted acridines are 2-methoxy-6-chloro-9-pentylaminoacridine, N-(6-chloro-2-methoxyacridinyl) -O-methoxydiisopropylaminophosphinyl- 3-aminopropanol, and N-(6-chloro-2-methoxyacridinyl)-
  • P(0) (0) -substituted acridine means a phosphate covalently linked to a substitute acridine.
  • the oligonucleotide antisense compounds have at least 9 to about 35 or so nucleotides in length.
  • nucleotides includes nucleotides in which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridines.
  • Preferred oligonucleotide antisense compounds have at least 9 to about 25 nucleotides, while more preferred compounds have at least 9 to about 15 or so nucleotides.
  • Compounds having fewer than 9 nucleotides are less desirable because they generally have less specificity and compounds having greater than about 35 nucleotides are less desirable because their physical size and charge will attenuate the crossing of the lipophilic cell membrane. Thus, they are less likely to enter cells.
  • the reaction scheme involves lH-tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates which are reacted with sulfur in 2,6-lutidine to give phosphate compounds.
  • Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylamine/tetra-hydrofuran, room temperature, 1 h). The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared. Such compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 h and then are further deprotected by heating at about 50°C overnight to yield compounds.
  • Compounds in which at least one X is oxygen are prepared by substituting I -H 2 0 for the sulfur in 2,6-lutidine.
  • Antisense oligonucleotide compounds in which at least X is CH 3 or other C 1-4 alkyl are prepared by the following published procedure: Agarwal and Riftina, 1979.
  • the reaction sequence is conducted on a solid support.
  • the reaction procedure involves phosphorylation of the 3'-hydroxyl group of a 5'-protected nucleoside using methylphosphonoditriazolide as the phosphorylating reagent followed by benzene sulfonyl-catalyzed coupling of the methylphosphonates to yield the methyl phosphonate oligonucleotide.
  • Methylphosphonoditriazolide is prepared in situ from equimolar quantities of methylphosphonodichloridate, triethylamine, and triazole. Benzene sulfonyl tetrazole also was prepared in situ from pyridine, benzene sulfonic acid and triethylamine. Repeating this reaction sequence followed by cleavage from the support and deprotection yield antisense compounds.
  • Antisense compounds in which R is P(0) (O)-substituted acridine also are prepared by the following published procedures: Asseline and Thuong, 1989; Stein et ah, 1988.
  • nucleoside phosphoramidite-bearing acridine derivative which then is reacted with 2, 2'-dithiodiethanol attached to a support. The elongation chain then is carried out of an automatic solid-phase DNA synthesized as described above.
  • published procedures also include synthesis of nucleoside phosphoramidite-bearing acridine derivatives by reacting substituted 9-(3-hydroxypropyl) amino acridines with N- ethyldiisopropylamine followed by N,N-diisopropylmethylphosphonanidic chloride.
  • antisense compounds in which R is P(0) (0)- substituted acridine are prepared by an extra round of synthesis using the acridinyl phosphoramidites in acetonitrile.
  • Highly preferred target regions of the mRNA are those which are at or near the AUG translation initiation codon, and those sequences which were substantially complementary to 5' regions of the mRNA.
  • Nucleotides at either end derived by the same process are also envisioned by this invention.
  • the effective length of the ODN is from 9-25 nucleotides.
  • the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense ODN, in which the phosphate substituents are replaced by phosphorothioates.
  • one or both ends of the oligonucleotide may be substituted by one or more acridine derivatives which intercalates nucleotide strands of DNA.
  • Target domains in the target nucleic acid are defined as being "non-contiguous" in the primary RNA sequence. Non-contiguous target domains are where the first domain and second domain are separated by at least 1 nucleic acid base.
  • the first domain and second domain are separated by at least 2 nucleic acid bases, or the first domain and second domain are separated by at least 3 nucleic acid bases, or the first domain and second domain are separated by at least 4 nucleic acid bases, or the first domain and second domain are separated by at least 5 nucleic acid bases, or the first domain and second domain are separated by at least 6 nucleic acid bases, or the first domain and second domain are separated by at least 7 nucleic acid bases, or the first domain and second domain are separated by at least 8 nucleic acid bases, or the first domain and second domain are separated by at least 9 nucleic acid bases, or the first domain and second domain are separated by at least 10 nucleic acid bases, or the first domain and second domain are separated by at least 11 nucleic acid bases, or the first domain and second domain are separated by at least 12 nucleic acid bases, or the first domain and second domain are separated by at least 13 nucleic acid bases, or the first domain and second domain are separated by at least 14 nucleic acid bases, or the first domain and second domain are
  • 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 contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used.
  • Other domains include AUG, 5' non-translated, Translation Initiation Factor, Ribosome Subunit Binding, Snine Dalgarno, 3' nonfranslated, Poly A, 3' Cleavage site, Coding Region, Intron, etc.
  • the site of attachment of the antisense construct may be less concerned with the functionality of that site than with the specific RNA secondary structure of the site. The induction of loop formation and sequestration of the 5' cap is thought to be most important.
  • RNA genomes consisting of one or more single stranded RNAs, are positive sense, meaning that they act as messenger RNAs (mRNAs) for the translation of viral proteins after the virus successfully infects a host cell.
  • mRNAs messenger RNAs
  • the RNA genomes are surrounded by a protein coat called the capsid, which is specific for that virus and, especially in the animal viruses, the capsid may be surrounded by a lipid- rich envelope.
  • Table 2 lists some of the positive sense single stranded viruses known to infect vertebrates, plants, and bacteria, along with their taxonomic classification. Many of the plant viruses are not currently classified by family, but instead, are given a group classification.
  • Pestivirus Bovine diarrhea virus hog cholera virus Hepatitis virus Hepatitis virus C C
  • viruses have a variety of replication and translation strategies (Levy et al. 1994; Matthews 1991), but, in general, they undergo the following infection process.
  • the virion (a single, intact virus particle) attaches to the outside of the host cell and the viral genomic RNA enters the cell either as naked RNA or as RNA complexed with other viral components.
  • TNN tobacco mosaic virus
  • the entire virion enters the cell.
  • viral proteins Once inside the host cell, viral proteins are translated and the genomic R ⁇ A is replicated.
  • One of the first viral proteins translated is generally the viral replicase, which is then responsible for replication of the viral genome.
  • progeny virions are assembled within the host cell and released from the cell to begin another infection cycle.
  • RNA virus replication details the general scheme of positive sense single stranded RNA virus replication.
  • the viral replicase attaches to the 3' end of the positive sense genomic RNA and begins transcribing a negative sense RNA strand. It is possible that several replicases will be able to follow one another on the positive sense template strand.
  • the negative sense strands act as templates for synthesis of new positive sense strands.
  • the new positive sense strands either act as new templates for additional negative strand synthesis or are translated into one or more viral proteins. Each virus species has its own particular translation strategy.
  • RNA is double stranded and may represent a newly synthesized negative strand completely complexed with its positive sense template (see FIG. IB).
  • the Rl form is partially single stranded and partially double stranded. It may represent the situation depicted in FIG. 1A or lC.
  • TMN's genomic R ⁇ A is an attractive target for antisense design.
  • nt nucleotides 1-192 and nt 379-581, respectively, of the positive sense TMN genomic R ⁇ A
  • SEQ ID ⁇ O:l positive sense TMN genomic R ⁇ A
  • this loop would prevent ribosomal recognition of the viral cap and/or the translation regulatory sequences within the omega sequence so that translation of the viral replicase would be inhibited.
  • the loop structure might also possibly prevent transcription of the full-length negative sense intermediate necessary for successful viral replication. Inhibition of translation or replication may also occur via an endonuclease-sponsored degradation of the RNA, with the formation of the loop being a better inducer of endonuciease attack that normal antisense binding.
  • the 3' end of the negative sense TMN strand is the exact complement of the 5' end of the positive sense strand.
  • a second set of two antisense sequences, complementary to the first antisense pair and thus complementary to the 3' end of the negative sense TMN R ⁇ A strand were fused to form a 395-nucleotide antisense molecule theoretically capable of causing loop formation at the 3' end of the negative sense TMN R ⁇ A.
  • the negative sense strand serves as a template for replication of new genomic R ⁇ As and all subgenomic R ⁇ As produced by TMN, so this second antisense molecule might be able to inhibit TMN replication.
  • Gene therapy has become an increasingly viable endeavor in the past decade for the mere reason that genetic defects responsible for numerous genetic diseases have been identified.
  • genes include cytokines, hormones, transporters, enzymes and receptors. Examples include the genes responsible for cystic fibrosis (CF), surfactant protein B deficiency and alpha- 1-antitrypsin deficiency.
  • CF cystic fibrosis
  • antisense oncogene constructs, tumor suppressor genes, inducers of apoptosis, repair genes and toxins have been identified as potential therapeutics in various cancers.
  • Oncogenes that are targets for antisense constructs are ras, myc, neu, raf, erb, src, fins, jun, trk, ret, hst, gsp, bcl-2 and abl.
  • Targets for this embodiment will include angiogenic genes such as NEGFs and angiopoeiteins as well as the oncogenes (e.g., ras, myc, neu, raf, erb, src, fins, jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and abl.
  • angiogenic genes such as NEGFs and angiopoeiteins
  • the oncogenes e.g., ras, myc, neu, raf, erb, src, fins, jun, trk,
  • Tumor suppressors that may be employed according to the present invention include p21, pl5, BRCA1, BRCA2, IRF-1, PTE ⁇ , RB, APC, DCC, ⁇ F-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, NHL, FCC, p53, CDK's and MCC.
  • Inducers of apoptosis such as Bax, Bak, Bcl-X s , Bad, Bim, Bik, Bid, Harakiri, Ad EIB, Bad and ICE-CED3 proteases, similarly could find use according to the present invention.
  • Such enzymes include for example, human copper zinc superoxide dismutase (U.S. Patent No. 5,196,335), cytosine deaminase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-1 -phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, -L-iduronidase, glucose-6- phosphate dehydrogenase, ⁇ -glucuronidase, HSV thymidine kinase and human thymidine kinase and extracellular proteins such as collagenase and matrix metalloprotease .
  • human copper zinc superoxide dismutase U.S. Patent No. 5,196,335
  • cytosine deaminase cytosine deaminase
  • transcription factors for example C/EBP ⁇ , h B, NfkB and Par-4.
  • Cell cycle regulators provide possible advantages, when combined with other genes. Such cell cycle regulators include for example, p27, pl6, p21, p57, pl8, p73, pl9, pl5, E2F-1, E2F-2, E2F-3, pl07, pl30 and E2F-4.
  • Other cell cycle regulators include anti-angiogenic proteins, such as soluble Fltl (dominant negative soluble NEGF receptor), soluble Wnt receptors, soluble Tie2/Tek receptor, soluble hemopexin domain of matrix metalloprotease 2 and soluble receptors of other angiogenic cytokines (e.g., NEGFR1/KDR, NEGFR3/Flt4, both NEGF receptors).
  • soluble Fltl dominant negative soluble NEGF receptor
  • Wnt receptors soluble Wnt receptors
  • Tie2/Tek receptor soluble Tie2/Tek receptor
  • soluble hemopexin domain of matrix metalloprotease 2 soluble hemopexin domain of matrix metalloprot
  • compositions of the present invention are prepared as pharmaceutical compositions, i.e. in a form appropriate for in vivo applications. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • the compositions may be administered via any suitable route, including parenterally or by injection.
  • 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 also can 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 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 carrier can 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.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the antisense constructs 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.
  • the preferred 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.
  • 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 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 inco ⁇ orated into the compositions.
  • compositions of the present invention may be formulated 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 also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamme, histidine, procaine and the like.
  • 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 injectable solutions, drug release capsules and the like.
  • 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 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.
  • a unit dose could be dissolved in 1 ml of isotonic NaCI 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 Edition, pages 1035-1038 and 1570-1580).
  • 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.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the invention concerns transgenic plants and methods for the creation thereof.
  • Suitable methods for plant transformation for use with the current invention are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh et al, 1993), by desiccation inhibition- mediated DNA uptake (Potrykus et al, 1985), by electroporation (U.S. Patent No. 5,384,253, specifically inco ⁇ orated herein by reference in its entirety), by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Patent No. 5,302,523, specifically inco ⁇ orated herein by reference in its entirety; and U.S.
  • Patent No. 5,464,765 specifically inco ⁇ orated herein by reference in its entirety
  • Agrobacterium-med ted transformation U.S. Patent No. 5,591,616 and U.S. Patent No. 5,563,055; both specifically inco ⁇ orated herein by reference
  • DNA coated particles U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,877; and U.S. Patent No. 5,538,880; each specifically inco ⁇ orated herein by reference in its entirety
  • Krzyzek et al U.S. Patent No. 5,384,253, inco ⁇ orated herein by reference in its entirety
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells.
  • recipient cells are made more susceptible to transformation by mechanical wounding.
  • friable tissues such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly.
  • pectolyases pectolyases
  • mechanically wounding in a controlled manner.
  • some species which have been transformed by electroporation of intact cells include maize (U.S. Patent No.
  • One also may employ protoplasts for electroporation transformation of plants (Bates, 1994; Lazzeri, 1995).
  • protoplasts for electroporation transformation of plants
  • the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ. No. WO 9217598 (specifically inco ⁇ orated herein by reference).
  • Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al, 1991), maize (Bhattacharjee et al, 1997), wheat (He et al, 1994) and tomato (Tsukada, 1989).
  • a suitable method for delivering transforming DNA segments to plant cells in accordance with the invention is microprojectile bombardment (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94/09699; each of which is specifically inco ⁇ orated herein by reference in its entirety).
  • particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, platinum, and preferably, gold.
  • cells in suspension are concentrated on filters or solid culture medium.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
  • Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species.
  • species which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al, 1994; Hensgens et al, 1993), wheat (U.S. Patent No.
  • Agrob ⁇ cterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
  • Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al, (1985), Rogers et al, (1987) and U.S. Patent No. 5,563,055, specifically inco ⁇ orated herein by reference in its entirety.
  • Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is the preferable method for transformation of dicots, including Ar ⁇ bidopsis, tobacco, tomato, and potato. Indeed, while Agrobacterium-mediated transformation has been routinely used with dicotyledonous plants for a number of years, it has only recently become applicable to monocotyledonous plants. Advances in -4grob cter/ * wm-mediated transformation techniques have now made the technique applicable to nearly all monocotyledonous plants. For example, Agrobacterium- mediated transformation techniques have now been applied to rice (Hiei et ⁇ l., 1997; Zhang et ⁇ l., 1997; U.S. Patent No.
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et ⁇ l, 1985; Lorz et ⁇ l., 1985; Omirulleh et al, 1993; Fromm et al, 1986; Uchimiya et al, 1986; Callis et al, 1987; Marcotte et /., 1988).
  • Examples of the use of direct uptake transformation of cereal protoplasts include transformation of rice (Ghosh-Biswas et al, 1994), sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh et al, 1993).
  • Transgenic Nicotiana tabacum cv. Xanthi NN and Xanthi nn were the local lesion and systemic tobacco plant hosts, respectively, for TMV-U1.
  • the local lesion response is a natural resistance response which allows quantification of viral replicative success in the presence or absence of transgenes by measuring the size and numbers of lesions.
  • the systemic response represents natural nonresistance.
  • TMN strain UI was propagated in nontransgenic N. tabacum cv. Xanthi nn. Nirions were extracted and purified according to the protocol of Gooding and Hebert (1967).
  • Nucleotide position designations represent positions within TMV, numbered according to Goelet (1982). The cloning procedures used are as previously described (Ausubel, 1998) with minor modifications. All PCR primers used in transgene construction are characterized in Table 3. Plasmids were propagated in Escherichia coli strain DH5 (Life Technologies, Inc.; Grand Island, NY) except for pRK2013, which was propagated in E. coli strain MM294. Maxi- and minipreps of each plasmid were done according to the alkaline lysis method of Birnboim (1983) using a polyethylene glycol precipitation (Lis, 1980) or hand-packed Sepharose® (Pharmacia Biotech, Inc.; Piscataway, NJ) columns.
  • Clones were prepared for PCR screening using a boiling lysis method. Briefly, a small amount of each colony was dispersed in 50 ⁇ l of water and placed in a boiling water bath for 5 minutes. Each sample (1 ⁇ l) was then added to a PCR mix. PCR products and restriction digests were electrophoresed on agarose gels and the DNA was purified from excised gel bands using sodium iodide and silica powder (GeneCleanTM; BIO 101, Inc.; La Jolla, CA). Table 3
  • pT7TMV containing the cDNA copy of the TMV-U1 genomic RNA
  • primers T85 and T83 to produce a 209 bp product (AL83/85) representing nt 1-192 of TMN-U1.
  • a different amplification using primers T84 and T86 produced a 231 bp product (AL84/86) representing nt 379-581 of TMN-U1.
  • Purified D ⁇ A was then digested with either S ⁇ cl and EcoRI (AL83/85) or Kpnl and EcoRI (AL84/86).
  • Digestion products were ligated into pBluescript® SK- (pBSK) (Stratagene; La Jolla, CA) and cloned to produce pBSK-AL83/85 and pBSK- AL84/86, respectively.
  • the full-length fragment (AL83/86) was constructed by digesting the previously cloned plasmids with S ⁇ cl and EcoRI (pBSK-83/85) or Kpnl and EcoRI (pBSK-84/86). The restriction fragments were ligated together, and then ligated into pBSK which had been digested with Kpnl and S ⁇ cl.
  • pBSK-AL clones were amplified with primers carrying appropriate restriction enzyme sites (see Table 3) to allow insertion of AL fragments into a pRTL2 vector (Topfer et al. 1987) carrying the ⁇ -glucuronidase gene (GUS).
  • pRTL2-GUS was kindly provided by Pal Maliga. The GUS marker gene was removed during the digests done to prepare pRTL2-GUS for AL insertion.
  • lumbers in ( ) represent sizes of PCR products obtained with the indicated primers.
  • b PCR products were digested with the indicated restriction enzymes. Restriction fragments of the size indicated in ( ) were then ligated into the pRTL2 plasmid vector, giving the pRTL2-AL clones listed in column 4.
  • c pRTL2-AL clones were restricted with E to give the indicated fragment sizes in bp. These fragments were then ligated into a pPZP plasmid vector.
  • pRTL2-AL clones except for pRTL2-AL84/86 (+) and pRTL2-AL84/86 (-), were digested with Pstl and ligated into pPZPl l l (Hajdukiewicz et al. 1994) (kindly provided by Pal Maliga) to produce pPZP-AL83/86 (+), pPZP-AL83/86 (-), pPZP-AL83/85 (+), and pPZP-AL83/85 (-) (see Table 4).
  • pRTL2-AL84/86 (+) was digested with H dIII and the 4283 bp fragment containing the TMN sequence was ligated into pPZP-AL83/85 (+), producing pPZP-AL83/85-84/86 (4-).
  • pPZP-AL83/85-84/86 (-) was constructed by digesting pRTL2-AL84/86 (-) with Hind ⁇ ll and ligating it into pPZP-AL83/85 (-). All pPZP-AL clones were grown on LB plating media containing 25 ⁇ g chloramphenicol per ml.
  • Triparental matings (Rogers et al. 1986) and plant transformations were performed according to the protocol of Svab et al. (1995). Triparental matings utilized A. tumefaciens strain LBA4404 (Ooms et al. 1982), E. coli strain MM294 (pRK2013), and each of the E. coli strain D ⁇ 5 (pPZP-AL) clones individually. pPZPl 11 containing no TMN sequence was also mobilized into A. tumefaciens to act as a control. Successfully transformed A. tumefaciens clones were identified by growth on minimal A medium containing 25 ⁇ g of chloramphenicol per ml and then were single-cell purified.
  • Leaf pieces (1 cm ) from Nicotiana tabacum cv. Xanthi ⁇ and Xanthi nn were individually cocultivated with A. tumefaciens strain LBA4404 (pPZP-AL) or (pPZPl l l) clones on Murashigi and Skoog (MS) medium containing 100 ⁇ g kanamycin sulfate per ml.
  • MS Murashigi and Skoog
  • each R ⁇ A sample (8 ⁇ l) was mixed with 2 ⁇ l of loading buffer (10 mM sodium phosphate, pH 7.0; 0.25% bromphenol blue; 50% glycerol) and 1 ⁇ l of 500 ng/ml ethidium bromide, and then incubated at 65°C for 15 minutes. Samples were then chilled on ice for two minutes and loaded onto a 1.2% agarose gel prepared with 10 mM sodium phosphate buffer, pH 7. R ⁇ ase-free conditions were maintained throughout the procedure.
  • loading buffer 10 mM sodium phosphate, pH 7.0; 0.25% bromphenol blue; 50% glycerol
  • Leaf pieces from each unique transgenic plant line were tissue cultured on MS medium containing kanamycin sulfate (100 ⁇ g/ml) using sterile techniques described in the protocol of Svab et al. (1995). Shoots were excised and transferred to MS medium containing kanamycin sulfate (100 ⁇ g/ml), NAA (0.1 ⁇ g/ml), and BAP (1 ⁇ g/ml). Rooted shoots were potted in soil and kept under fluorescent lights at 21°C.
  • TMN virions/ ⁇ l of FES buffer 100 mM glycine pH 9.2, 60 mM K HPO 4 , 1% sodium pyrophosphate, 1% diatomaceous earth, 1% Bentone MA (RHEOX, Inc.; Hightstown, ⁇ J)
  • FES buffer 100 mM glycine pH 9.2, 60 mM K HPO 4 , 1% sodium pyrophosphate, 1% diatomaceous earth, 1% Bentone MA (RHEOX, Inc.; Hightstown, ⁇ J)
  • Transgenic N. tabacum cv. Xanthi nn plants were monitored for 30 days in the greenhouse for the first appearance of systemic symptoms in the newest upper leaves: vein clearing and/or a mosaic pattern. The plant lines that showed resistance were tissue cultured again to obtain additional test clones.
  • Xanthi ⁇ clones from each line were manually inoculated with 50 ⁇ l of TMN per half leaf (TMN was diluted in FES buffer to give approximately 50 lesions per inoculation on nontransgenic N. tabacum cv. Xanthi ⁇ plants). Lesions on all four leaves (eight half leaves) were counted at 60 hours post inoculation and randomly chosen lesions on leaves two and four were measured at 90 hours post inoculation. The time of first appearance of lesions was also noted for each of the eight half leaves on each plant.
  • Ten sets of plants were independently transformed using Agrobacterium- mediated transformation with pPZP-TMN-Ul sequences or with pPZP vector sequences only were produced using either Nicotiana tabacum cv. Xanthi nn (systemic host; five sets) or Xanthi ⁇ (local lesion host; five sets) plant tissue. Each set of plants was then challenged with TMN-U1 and monitored for inhibition of TMN multiplication.
  • the full-length transgene, designated AL83/86 carried nt 1-192 and nt 379-581 of the TMN-U1 genome.
  • the first antisense domain (nts. 1-192) corresponds to the entire 5' nonfranslated region of tmv, the initiation aug (nt.
  • the second antisense domain corresponds to a portion of the tmv replicase coding region.
  • These two TMN sequences were fused (see FIG. 4A and FIG. 4B) and then were ligated into the pPZPl l l Agrobacterium binary vector in a sense (+) orientation to produce pPZP-AL83/86 (+) (FIG. 4A).
  • This same full-length transgene was also ligated into pPZPl l l in an antisense (-) orientation to produce pPZP-AL83/86 (-).
  • Both fusion transgenes were under the control of a dual 35S promoter from cauliflower mosaic virus (CaMN) and had a CaMN polyadenylation signal downstream.
  • TMN multiplication equally as well as the putative loop-inducing fusion transgene, two other pPZP-AL plasmids were constructed and used in a triparental mating and plant transformation.
  • Each of the arms of a full-length fusion transgene was independently ligated into a single pPZPl l l vector to produce pPZP-AL83/85- AL84/86 (in either sense or antisense orientation), each arm having its own dual 35S promoter and polyadenylation signal (see FIG. 4B).
  • T N. tabacum cv. Xanthi nn
  • G N. tabacum cv. Xanthi ⁇
  • F full-length fusion transgene
  • Q two arms of full-length transgene under separate CaMN promoters
  • P positive sense
  • ⁇ negative sense.
  • FIG. 5 and FIG. 6 show the TFP and TF ⁇ transgene expression levels for each unique plant line in the two categories. For both the TFP and the TF ⁇ plants, most transgene R ⁇ A expression levels appear to be below 100 pg of transgene R ⁇ A per 7.5 ⁇ g of total R ⁇ A.
  • Each plant was inoculated with TMN and monitored for 30 days to detect symptoms of systemic infection. At least five plant lines that exhibited the most delay in symptom development within each category were chosen for a larger scale repeat of the screening trials. Some categories such as TQP and Tzero had not gone through the initial TMN screening process, so several more lines from these categories were included in the large-scale TMN challenge trials. Table 6 lists the plant lines and the number of clones in each line that were rechallenged with TMN.
  • FIG. 7, FIG. 8, FIG. 9, FIG. 10, and FIG. 11 show the percentage of plants in each plant line of each category that exhibited symptoms of systemic infection at various days post inoculation. From the this data, it can be seen that tfn lines 8, 9, and 12, showed greater delay in symptom development over the 30 day monitoring period (FIG. 8) than any other plant line from the tq or tzero controls (FIGS. 9-11). These lines had a lesser proportion of plants infected at either the middle or the end of the 30-day monitoring period.
  • T 0 18 and 21 Two of the T 0 plant lines (T 0 18 and 21) had a slight delay in symptom development as compared with the TQP and TQN plant lines, but this delay was not as great as that seen in TFP21, TFN8, and TFN9, for example.
  • greenhouse temperatures ranged from 20-33 °C, with an average daylight temperature of around 30°C.
  • Leaf tissue from apical leaves was ground in FES buffer and inoculated onto Nicotiana tabacum cv. Xanthi NN plants. Lesions were counted 4 days later.
  • TFN transgene is better able to inhibit TMN replication and symptom development than the TQ ⁇ , TQP, and Tzero transgenes (FIGS. 8-11).
  • antisense with the two domains contiguous to each other on the same antisense mR ⁇ A molecule (TF ⁇ ) better inhibits TMN replication than the antisense with the two domains on separate molecules (TQ ⁇ ). Since TF ⁇ is expected to induce loop formation while TQ ⁇ is not, and since both transgenes contain the identical sequences, this indicates that loop-induction is important for inhibition of TMN replication.
  • TFP transgenic line 21 appeared to delay symptoms during the monitoring period, but TFP line 30 did not prevent the accumulation of symptoms (Table 7).
  • TF ⁇ would be expected to inhibit translation predominantly (binds to 5' end of (+) TMN R ⁇ A), and hence TMN replication.
  • TFP would be expected to inhibit viral replication directly (binds to 3' end of (-) R ⁇ A).
  • the efficacy of the TF ⁇ lines was demonstrated by the inhibition of the final accumulation of TMN (Table 7) and by line TF ⁇ 9, which was completely resistant to TMV challenge in the Spring screening trials and showed unusual symptom delay and resistance in the fall final trials.
  • GQP, GQN, and Gzero were inoculated with TMV to determine if the transgene would inhibit either TMV's ability to initiate an infection or the cell-to-cell movement of TMV.
  • Two aspects of infection initiation were (1) the time of the first appearance of lesions on the inoculated leaf and (2) the number of lesions that actually develop.
  • the first four fully expanded leaves of each test plant were inoculated with TMN. Each leaf was numbered according to its position on the plant, with leaf 1 being the top-most leaf. Leaf position determines the number of local lesions that will develop for a particular TMN inoculum concentration and the sizes of those lesions (Matthews 1991). Therefore, the data for each leaf position were not comparable to data associated with leaves at other positions on the plant.
  • FIG. 12 shows the average time of the first appearance of lesions on each leaf for each plant category.
  • the Gzero plants showed a possible delay in local lesion appearance for every leaf position. All other plant categories showed no significant differences in times of first appearance of lesions.
  • the average number of lesions that developed at each leaf position for each plant category is represented in FIG. 13. There were no significant differences found among the four categories.
  • FIG. 14 shows the average lesion diameters for leaves 2 and 4 of ten GFP plant lines. Each bar represents the average lesion diameters (15 lesions per plant) for each of one to six plants inoculated in that particular line.
  • FIG. 15 shows the average lesion diameters in each category (except for
  • Transgenic tobacco plants containing test and control antisense sequences against tobacco mosaic virus (TMN) were screened for resistance to TMN. These plants were the rooted shoots from transformed callus (R0 generation) and were therefore heterozygotes. These plants were self-pollinated to produce Rl generation plants. The Rl generation plants are self-pollinated to produce an R2 generation. The Rl generation is then screened for resistance to TMN using methods described above.
  • a determination of which plants are heterozygous and which are homozygous for the transgenes is made by examining, by PCR using primers specific to the transgene, the proportions of R2 seedlings containing the transgene for each Rl specimen.
  • An R2 ratio of roughly 3:1 indicates that the Rl specimen from which the R2 is derived is a heterozygote, while an R2 population that was 100% positive for the transgene indicates that the parental Rl is homozygous for the transgene.
  • Heterozygotes were screened, but it is commonly found in such plant virus antisense studies that only the homozygotes show measurable levels of resistance. Though some resistant lines were found, especially with the TF ⁇ (potentially loop- inducing) antisense construct, increasing the resistance with all constructs will more clearly differentiate between different levels of resistance between different constructs.
  • the initial screening was performed in the spring and yielded good resistance levels, but control plants were not included. The complete screening, with control plant lines, was conducted in the summer, resulting in the lower levels of resistance reported above.
  • the original antisense constructs were inserted in pBluescript cloning vectors to produce the pBSK-AL plasmid constructs. These constructs were then subcloned to produce the pRTL2 and pPZP vectors for use in plant transformation. Table 4 of Example 1 above lists these plasmid constructs.
  • RNA transcripts from the antisense sequence will be used to produce RNA transcripts from the antisense sequence for testing inhibition of TMN replication in nontransgenic tobacco protoplasts.
  • the transcripts will either be produced in vitro and coinoculated with the TMN R ⁇ A by electroporation of the protoplasts, and/or the transcripts and TMN R ⁇ A will be produced in the protoplasts by electroporation of the protoplasts to introduce transient expression vectors expressing said sequences.
  • antisense oligonucletides similar to those described in Example 10, may also be tested.
  • Example 6 The experiment described in Example 6 above may also be conducted with antisense sequences against other plant viruses, using protoplasts co-inoculated with R ⁇ A of other plant viruses via the methods outlined above.
  • antisense oligonucletides similar to those described in Example 10, may also be tested.
  • R ⁇ A transcripts from antisense sequences against other plant viruses will inhibit replication of other plant viruses in the tobacco protoplasts.
  • antisense oligonucletides similar to those described in Example 10, may also be tested.
  • Examples 6, 7 and 8 Experiments similar to those outlined in Examples 6, 7 and 8 may be carried out using human or animal cell cultures, according to methods known to those of skill in the art in view of the specification. In this case, human or animal cellular or viral RNA are the potential targets. In addition, antisense oligonucletides, similar to those described in Example 10, may also be tested in human or animal cell cultures. These studies will provide information on the manner in which antisense constructs affect human or animal cell cultures, and prove the therapeutic use of such constructs.
  • antisense DNA oligonucleotides will be hybridized to test mRNA and the hybrid added to a rabbit reticulocyte or wheat germ in vitro translation assay. Potentially loop- inducing test sequences will be used as well as two antisense oligonucleotides binding to identical target sequences but not inducing a loop in the target mRNA, since they are unlinked (control antisense sequence). In this way, the test and control sequence design is analogous to the design in the examples above.
  • the two antisense sequences will be 10-30 bases long, with a linker sequence in the oligonucleotide of 0-10 bases (or possibly composed of a non-nucleic acid polymer).
  • the antisense oligonucleotides may consist of DNA or phosphorothioates or other modified nucleic acids.
  • Antisense RNA derived from in vitro transcription, may also be tested. In this case, each antisense RNA section will be 30-300 bases long.
  • Target mRNAs may be viral or cellular, from plants, animals, humans or bacteria, with the antisense sequences being complementary to the target mRNAs. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
  • 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/or methods and in the steps or in the sequence of steps of the method 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.
  • Ghosh-Biswas et al "Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts," J. Biotechnol, 32(1):1-10, 1994. Ghosh-Choudhury et al, EMBO J, 6:1733-1739, 1987.
  • ISIS 2302 ISIS-1 antisense oligonucleotide

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Abstract

Cette invention a trait à des compositions d'acide nucléique antisens comportant des séquences complémentaires d'une première et d'une seconde région d'un acide nucléique cible. Ces séquences antisens sont conçues pour s'hybrider aux régions cibles de l'acide nucléique dans un ARN cible et inhiber la traduction génétique, le traitement, le transport ou la fixation par des protéines ou des riboprotéines. La première et la seconde région cibles peuvent être contiguës ou non dans l'acide nucléique cible. Les régions cibles comportent, mais sans limitation à deux, AUG, des séquences non traduites en 5', des sites de fixation de facteur d'initiation de traduction, des sites de fixation de sous-unité ribosomique, une séquence de Shine-Dalgarno, des séquences non traduites en 3', une queue poly A, un site de restriction, une région codante, une ramification d'intron, une jonction intron/exon ou une séquence d'épissage. L'acide nucléique cible peut être de type viral, bactérien, animal, humain ou végétal. Les méthodes d'utilisation de ces compositions antisens visent à l'inhibition d'un ARN et au traitement de maladies cellulaires chez l'animal, le végétal ou l'homme. L'invention concerne également des compositions antisens pharmaceutiques.
PCT/US2001/000754 2000-01-07 2001-01-08 Compositions antisens et methodes afferentes WO2001051630A1 (fr)

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