WO2000053775A9 - Prevention and treatment of viral infections - Google Patents
Prevention and treatment of viral infectionsInfo
- Publication number
- WO2000053775A9 WO2000053775A9 PCT/US2000/006333 US0006333W WO0053775A9 WO 2000053775 A9 WO2000053775 A9 WO 2000053775A9 US 0006333 W US0006333 W US 0006333W WO 0053775 A9 WO0053775 A9 WO 0053775A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- virus
- rna
- toxin
- viras
- Prior art date
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Classifications
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
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- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- 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
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- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8263—Ablation; Apoptosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Definitions
- This invention pertains to the prevention and treatment of viral infections by making cells, plants, and animals non-permissive for viral infections.
- Vaccines can stimulate both cellular and humoral immune responses to viruses.
- the type of immunity predominating in a particular situation depends on the antigen used in a vaccine, any adjuvants administered with the antigen, and the route by which the vaccine is administered. Different vaccines elicit humoral (antibody) protection, cell-mediated immunity, or sometimes both.
- Antibodies are generally more effective against viral surface antigens.
- Cell-mediated immunity can be effective against both surface viral antigens and core viral antigens. Core antigens tend to be more highly conserved, so immune system recognition of core antigens tends to provide greater cross-protection against related viruses.
- Antisense technology uses gene sequences complementary to the message transcribed from the targeted gene to inhibit the translation of that message, thereby inhibiting the expression of the targeted gene. This relatively new technology is currently being investigated to block replication of cancerous cells, to inhibit the functioning of virus-specific genes, and to study normal gene function. Some practical applications of antisense nucleic acid sequences to inhibit the expression of essential genes of viruses to prevent and treat virus infections appear promising. See Wagner, R.W. et al., "Antisense technology and prospects for therapy of viral infections and cancer, " Mol. Med. Today, vol. 1, pp. 31-38 (1997); Kilkuskie, R.E. et al.,
- Methods to transform cells include inhalation or injection of free DNA, shooting DNA-coated particles into cells by gene-gun technology, introduction of DNA as complexes with carrier polymers, electroporation, incorporation of nucleic acids in liposomes, utilization of receptor- mediated endocytosis, or by using one of the many different types of viral vectors. See Nakanishi, M., "Gene introduction into animal tissues," Crit. Rev. Ther. Drug Carrier Syst., vol. 12, pp. 263-310 (1995); Robbins, P.D. et al., "Viral vectors for gene therapy," Pharmacol. Ther., vol. 80, pp.
- a preferred vector for transforming a cell's genome is the transposon-based vector disclosed in Cooper, United States patent no. 5,719,055.
- the vector may be introduced, e.g., via electroporation or lipofection, using protocols known in the art.
- the modification may occur in the germ line, in somatic cells only, or in both somatic cells and germ cells. Only transformations of germ cells will be inherited by subsequent generations. In humans, genetic modification of the germ line is generally considered off-limits today due to ethical concerns. However, various experimental treatments have been tested involving modification of somatic cells in humans (such as hematopoietic stem cells) to correct genetic defects.
- nucleic acid vaccines have been introduced into cells to produce antigens to stimulate a specific immune response against target viruses. A great deal more could be accomplished if additional genetic-based techniques were available to provide protection against viral infection without compromising other functions of uninfected cells and uninfected organisms.
- IE promoter to drive the expression of the lacZ gene to yield ⁇ -galactosidase, which is readily detected in live cells by its ability to convert exogenously-supplied IPTG to a dark blue reaction product.
- This use of the IE promoter to drive production of ⁇ -galactosidase is a common research tool; such a construct has been cloned into cultured cells for use as an early signal of infection by herpesviruses. When cloned into cells, the marker is activated following infection with a herpesvirus, because the herpesvirus cannot replicate without activating this promoter. In this manner, infected cells can readily be identified by counting the colored cells. See Sandri-Goldin, R.M.
- toxin Transcription of the toxin in the HIV retroviral systems has been reported to be "leaky,” i.e., toxin can be expressed at low levels in the absence of the inducer. See, e.g., J. Ragheb et al., "Inhibition of human immunodeficiency virus type 1 by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha2, or diphtheria toxin compared with inhibition by transdominant Rev," Rev. Hum. Gene. Ther., vol. 10, pp. 103-112 (1999). This
- leaky expression may be typical of retroviral systems.
- This invention presents a completely new approach to preventing, treating, and curing viral infections.
- This approach is based upon designing genetic codes for toxins so that the toxins can only be expressed in cells that are infected by a virus.
- These "genetic drugs” can be introduced into cells in a variety of ways, either to prevent or to cure viral infections by killing virus-infected cells.
- This method can be used to kill cells infected by many different kinds of viruses. These novel techniques may be used both to prevent the establishment of viral infections and to treat existing viral infections.
- the method can also be used to kill cancer cells that express viruses, whether or not the viruses actually cause the cancer.
- the common theme of the three different methods of implementing the invention is to encrypt the genetic code for a toxin in such a way that an effective amount of the toxin is expressed within, and only within, virus-infected cells, leading to the death of only the infected cells and thereby terminating the infection.
- Methods to express toxins only within virus- infected cells include: (I) the control of toxin expression using virus-specific gene regulatory mechanisms; (II) the control of toxin expression using antisense codes for toxins that can be translated only within virus-infected cells; or (III) the use of negative DNA codes for toxins that can only be translated in virus-infected cells. Infection by many viruses of prokaryotes and eukaryotes may be treated or prevented by one or more of these three methods.
- the host cell will be killed prior to the formation of mature or infectious virions, thereby terminating the infectious cycle of the virus. If the encrypted code for toxin production is introduced to a cell that is already producing viruses, then the infected cell will be killed, thereby terminating the production of viruses.
- the sequences encoding the toxins will be in different coding forms depending on the type of virus.
- the toxin is coded in the conventional manner, with the expression of the toxin dependent upon the virus-specific regulation of gene expression.
- the toxins are encoded either in the form of an antisense message, or in a form that will be transcribed as an antisense message within the infected cell, and converted to positive messenger RNA only in the presence of an infecting RNA virus.
- negative DNA of the toxin construct may be converted into positive DNA using a virus-specific 3' -OH terminal sequence as a primer for DNA polymerase.
- Method I is designed to impart transgenic cells, tissues, or organisms with the ability to prevent infection by DNA viruses. Most DNA viruses rely upon virus-specific gene regulatory mechanisms that use promoters and inducers to regulate viral gene expression.
- virus-specific regulatory information that is identical to or functionally homologous with at least one viral nucleic acid regulatory region is used in a mechanism to kill cells promptly following infection by a virus.
- a gene encoding a toxin is placed under the control of a virus- specific regulatory sequence. In the absence of viral infection, these virus-specific regulatory sequences are not activated, and the toxin gene is not expressed. When viral infection occurs, the presence of a virus-specific inducer leads to expression of the toxin gene. The expressed toxin kills the infected cell, thereby terminating the infectious cycle of the virus.
- Method II is designed for RNA viruses other than retroviruses. Method II may be used to treat infections by RNA viruses in transgenic or non-transgenic organisms. Method II may also be used to impart transgenic cells, tissues or organisms with the ability to prevent infection by RNA viruses. Method II may be used against any virus whose replication is based on RNA, i.e., any virus wherein an RNA template is used to replicate RNA, including negative-stranded RNA viruses, positive-stranded RNA viruses, and double-stranded RNA viruses (such as reoviruses).
- an otherwise non-functional antisense code or negative message for a toxin is transcribed by a virus-specific enzyme, RNA-dependent RNA polymerase, resulting in a functional, positive message that can be translated to a toxin by the cell's ribosomes.
- All RNA viruses (other than retroviruses) rely upon RNA-dependent RNA polymerase to convert negative or antisense RNA into positive or messenger RNA, the form that can be translated into peptides or proteins.
- Those RNA viruses with an antisense RNA genome also carry this enzyme within the virion.
- RNA viruses that use either double- stranded RNA or single-stranded (i.e., messenger) RNA as their genomes
- the RNA-dependent RNA polymerase is encoded in the viral genome.
- the RNA-dependent RNA polymerase either must be expressed by the host cell, or it must have been previously packaged in the virion, in order for the viral genome to replicate.
- RNA-dependent RNA polymerase is not present in a cell, so the antisense code for the toxin is not translated.
- the antisense toxin message is converted into positive sense RNA, or messenger RNA, by the RNA-dependent RNA polymerase.
- the positive or messenger RNA is then translated to yield the toxin, thus killing the infected cell prior to formation of viral progeny, and thereby terminating the infectious cycle.
- Method III targets primarily viruses such as retroviruses and parvoviruses that use a virus-specific nucleic acid sequence as a primer to initiate the copying of a single-strand viral genome. Method III is used to treat existing viral infections. The primer is needed because
- DNA-dependent DNA polymerase requires a primer with an available 3' -OH to function, as does the retroviral polymerase (reverse transcriptase).
- toxins are encoded in negative single-stranded DNA flanked in the 3' direction by one or more regions complementary to the 3' end of the nucleic acid of the targeted viruses.
- a virus-specific primer e.g., the 3' terminal end of the viral nucleic acid
- the positive strand is then transcribed to form the functional RNA message of the toxin, which is in turn translated into toxin, which kills only the infected cells.
- Method III is intended for the treatment of virus-infected cells, rather than for the creation of transgenic resistant cells or organisms.
- the negative strand toxin coding sequence with a site to hybridize with the 3' -OH region of the virus nucleic acid and other complementary sequences necessary for transcription and translation, can be produced in large amounts for therapeutic purposes through means known in the art, for example by using single- stranded PCR. See, e.g., F. M. Ausubel et al. (Eds), Current Protocols in Molecular Biology, vol. 2, Chapter 15 ("The Polymerase Chain Reaction") John Wiley and Sons (Wiley
- Method I is designed to impart transgenic cells, tissues, or organisms with the ability to prevent infection by DNA viruses.
- Method I uses a gene that encodes a toxin, and that is placed under the regulatory control of a virus-specific promoter. Activation of the virus- specific promoter depends upon the presence of a virus-specific inducer.
- Productive DNA virus infections of host cells typically require the activation of at least one virus-specific promoter by a virus-specific inducer.
- this invention uses virus-specific sequences to directly activate or allow the expression of a toxin to kill a virus-infected cell.
- a virus-specific promoter is linked to a sequence encoding a toxin as a mechanism to kill virus-infected cells.
- This genetic construct is introduced into host cell genomes (or otherwise stably maintained in the cell, e.g., as an episome), where it remains inactive unless viral infection occurs. Following viral infection, the virus-specific promoter is activated by the virus-specific inducer, the toxin is produced, and the infected cell is killed before the virus can replicate. Viral infection may be halted long before the conventional immune system even becomes "aware" of the presence of an infection, and before any symptoms of disease are exhibited. Method I can be particularly useful for preventing infections by viruses that are responsible for persistent or chronic infection.
- This toxin construct can be introduced into the germ lines of plants and non-human animals to become a permanent part of the genome, or into progenitor cells of tissues (e.g., hematopoietic stem cells) in humans to provide longer lasting protection.
- a DNA viral promoter is linked to a sequence encoding a toxin as follows.
- Either an immediate-early promoter or an early promoter from Herpes Simplex I (HSV-I) is used as the regulatory sequence.
- HSV-I Herpes Simplex I
- a functional subunit of the promoter for viral protein ICP4 (an immediate early promoter, also called IE175) from GenBank accession number X06461, bases 843-1202 (D. McGeoch et al. (1986); G. Byrne et al. (1989); U.S. Patent 5,221,778) is linked to DNA encoding a toxic peptide, e.g.
- this construct is delivered to a cell or to an animal genome, where it would be idle in the absence of viral infection.
- HSV-I herpes virus
- the ICP4 promoter is induced to begin expressing the toxic peptide at the same time the infecting virus attempts to initiate the expression of wild-type ICP4 protein.
- Expression of the toxin causes cell death very early in the virus replication cycle, preventing the formation of mature virions.
- herpes simplex I herpes simplex II
- varicella zoster virus pseudorabies
- bovine herpes virus bovine herpes virus
- equine herpes virus Marek's disease.
- the promoters used are not normally present in the host, but instead are promoters specific to viruses. These virus-specific promoters are activated only in response to infection of a cell by a virus using the same inducer/promoter mechanism of gene regulation, or a mechanism with sufficient homology to cross-react (a fairly common circumstance among viral regulatory sequences, as discussed further below). A gene product of the virus is required to induce or activate the promoter to allow transcription of the code.
- Virus-specific promoters often have very strong activity. In native viruses they function to induce the production of large amounts of gene product as part of the replication cycle of the virus. The efficiency of many virus-specific promoters has made such promoters common choices in cloning/expression technologies where large amounts of expressed product are desired.
- One commonly used virus promoter is the very late bacculovirus promoter, which is responsible for the production of the inclusion bodies or polyhedra that accumulate in the form of a protein crystal surrounding the newly produced mature bacculovirus. See V. Lucklow, "Bacculovirus systems for the expression of human gene products," Curr. Opin. Biotech. , vol. 4, pp. 564-572 (1993); L.
- lytic peptides Genes encoding both natural and designed lytic peptides have been cloned into the bacculovirus genome under the control of the late bacculovirus promoter, and have been expressed to produce lytic peptides (Hellers et al., 1991; Choi, 1996).
- the bacculovirus system is an excellent example of the use of virus-specific regulation to drive the expression of genes, including those encoding toxins such as lytic peptides.
- the very late regulatory function used in this expression system is activated after many infectious virions have been formed, providing additional modified viruses to continue the process. Early expression of toxic compounds would terminate the infectious cycle and limit the yield of the desired expressed products.
- bacculoviruses are best known as insect viruses and as laboratory tools, they are also major pathogens of cultured shrimp (Loh, P.C. et al. 1997).
- Literature searches were conducted to identify virus-specific promoter sequences from representatives from different virus families. The results are included in Table II (below), which also includes RNA replication promoters useful in Method II. The promoter sequences reported in Table II should suffice to cover many viruses of medical, veterinary, and agricultural significance due to sequence homology, as discussed below. The different viruses may be classified into "treatment groups" accordingly.
- the promoter will be included if a sufficiently large number of bases upstream of the transcription initiation site are ligated to a sequence encoding the toxin.
- the fact that "extra” bases may also be included in addition to the promoter is acceptable, even if those "extra” bases might have the effect of down-regulating the promoter under certain circumstances. Even where such down-regulation may exist, the toxin gene will still be expressed when the corresponding early native viral gene is expressed, as both will be under the control of the same regulatory elements.
- upstream bases needed to encompass a particular promoter may readily be determined in a particular case, and for the reasons just given, the precise number of bases is not crucial. In general, sequences of 500, 1000, or 1500 bases upstream from the transcription initiation site should suffice in most cases. Alternatives 2-4
- virus-specific promoters Existing published information on virus-specific promoters is of three types. (1) For some commonly-studied viruses many of the promoter sequences have been reported, and may be used directly in the practice of the present invention. (2) For some viruses, available data indicate that expression of viral proteins is regulated by virus-specific promoters, but the sequences are currently unknown. (3) For many viruses, available data are too limited to provide much of a guide, except by analogy and inference based on related viruses whose promoter functions are known. For the second and third categories, techniques such as the following illustrative alternatives may be used to isolate and clone appropriate regulatory regions and link them to toxin genes:
- Viral DNA is randomly digested with a restriction enzyme recognizing 4 bp sites.
- the digest is timed to yield nucleotide fragments in the 1000 bp range.
- the digested viral genome can be size-fractionated on a sucrose gradient; then 0.5 mL fractions are harvested and analyzed by agarose gel electrophoresis.
- the fractions yielding the majority of products in the 1000 bp range are purified by dialysis to remove the sucrose, and the DNA is then concentrated by standard procedures known in the art.
- the restriction enzyme used is Sau3A I (a 4-bp cutter).
- the fragments are cloned into a plasmid vector containing a sequence coding the desired toxin.
- the fragments are cloned upstream of the toxin sequence into a BamH I site; BamH I has a 6 bp recognition sequence, but the 4 bp overhanging sequence generated is the same as that of Sau3A I, allowing sticky-end ligation to occur.
- the new plasmid construct is then transformed into E. coli for propagation; the DNA is harvested and used to transform appropriate cells in 96-well plates. Each well may be challenged with the virus and screened for cell lysis or death. Non-transformed cells infected with the same virus are used as controls. Cells expressing the toxin upon viral infection should lyse (or die) more quickly than cells that lyse due to the normal viral infection cycle.
- the plasmid containing the viral promoter may be identified. By careful timing and observation, this system allows the determination of viral promoters used early and late in the viral cycle. Confirmation can be obtained, for example, by assay with antibodies against the toxin to confirm expression of the toxin and to determine the timing of that expression. This approach may be used generally to clone any promoter from any virus. For details of the protocols used, see F. M. Ausubel et al. (Eds), Current Protocols in Molecular Biology, vols. 1-3, John Wiley and Sons (Wiley Interscience) (1999); and T. Maniatis et al. (Eds.), Molecular Cloning: A Laboratory Manual, vols. 1-3, Cold Spring Harbor Laboratory Press (2nd ed. 1989).
- Alternative 3 This alternative is similar to Alternative 2 above, except that a reporter gene is initially used in lieu of the toxin gene.
- the reporter gene could, for example, be the lacZ gene or another gene encoding ⁇ -galactosidase, which causes the development of a blue color in the presence of IPTG.
- Clones expressing the reporter gene only in the presence of viral infection are presumptively under the control of promoters responsive to a virus-specific inducer. The use of a reporter molecule can make initial screening easier. The clones identified as having a virus-specific promoter can then be used in practicing the invention.
- the reporter gene in the plasmid is replaced by a toxin gene (for example, the reporter gene is removed from the vector with an appropriate restriction enzyme, and a sequence encoding the toxin is ligated into the same location).
- the resulting construct may then be used in the present invention.
- Alternative 4 This alternative is similar to Alternative 3 above, except that the reporter gene is directly and randomly inserted into the viral genome rather than into random fragments of that genome. Infected cells expressing the reporter gene are then candidates for appropriate virus-specific promoters. A second round of screening (as described for
- the DNA virus to be used in the initial proof-of-concept experiments is the he ⁇ esvirus, both because of the importance of the he ⁇ esvirus family in causing diverse diseases in humans and animals, including cancers, and because a great deal is known about he ⁇ esvirus-specific regulatory mechanisms (Roizman, B., 1996; Roizman, B., and Sears, A.S., 1996).
- a preferred vector for transforming a cell's genome is the transposon-based vector disclosed in Cooper, United States patent no. 5,719,055.
- the he ⁇ esvirus-specific immediate early (IE) promoter and promoters may be used to drive the expression of foreign genes.
- Glorioso et al. (1995) reported using the he ⁇ esvirus IE promoter to drive expression of the lacZ gene to yield ⁇ -galactosidase, which is readily detected in live cells by its ability to convert exogenously-supplied IPTG to a dark blue reaction product.
- the ⁇ -galactosidase marker gene is expressed only following infection with a he ⁇ esvirus, when viral replication will activate the IE promoter. In this manner infected cells can readily be identified by counting the colored cells.
- NS1 and NS2 are downstream of a promoter (TATA box) at location mp4.
- TATA box a promoter
- NS1 a large, nonstructural protein
- NS1 from the human parvovirus B19 has been reported to activate the p6 promoter, which in turn controls the transcription of all other B19 genes.
- Mice minute virus (MMV) NS1 trans- activates the p4 and p6 promoters.
- HI NS1 trans-activates the p38 promoter.
- the MMV p38 promoter can be regulated in trans by either or both of the NS proteins.
- the NS2 nonstructural protein appears to regulate gene expression in a manner that depends on the cell type. See X. Li et al., "The parvovirus HI NS2 protein affects viral gene expression through sequences in the 3' untranslated region," Virology, vol. 194, pp. 10 ff (1993).
- a sequence coding a toxin e.g. phor21
- NS1 e.g., promoter p38 or p6, along with appropriate stop codon and polyadenylation signals.
- the promoter /toxin construct is cloned into a delivery plasmid and used to transfect cells of an animal. Following infection of a transformed cell by a parvovirus, the protein NS1 is expressed early in the replication cycle. NS1, in turn, activates the promoter controlling expression of the toxin. The toxin is expressed, and the infected cell is destroyed.
- RNA complementary to a sequence encoding a toxin is introduced into a cell, where it remains untranslated.
- a strand of negative, or antisense, RNA cannot be translated, so production of the toxin does not occur.
- the virus provides the RNA-dependent RNA polymerase necessary to convert the complementary toxin message to the translatable positive RNA, as the RNA polymerase performs its normal function of converting viral negative RNA into positive RNA. Translation of the message for the toxin results in death of the cell prior to formation of infectious progeny.
- Antisense messages can either be introduced into cells in the RNA form (in which case it would be preferred to cap the ends of the RNA to inhibit digestion by exonucleases), or they can be transcribed as antisense RNA intracellularly from complementary DNA sequences that are introduced into the cells or that are inco ⁇ orated into the genome. Genetic information encoding the negative RNA message can be part of the genome under regulatory control such as low level constitutive production, or it can be transcribed in response to specific inducers such as stress inducers or interferons. Alternatively, that information could be encoded in a plasmid, or it could be introduced directly as linear or circular DNA or RNA.
- the 3'-terminal end of a rotavirus such as porcine rotavirus strain OSU, comprising at least the terminal 26 nucleotides beginning with CC-3', is used as the promoter for RNA replication.
- a rotavirus such as porcine rotavirus strain OSU
- the promoter is linked, i.e. ligated, to a (-) strand sequence encoding a toxin, including appropriate stop codon and polyadenylation sequences.
- This promoter /toxin construct sits dormant in the cell until infection by a rotavirus (or related virus) occurs.
- Viral proteins encoding the RNA replicase recognize the promoter and begin transcribing the sequence to the (+) strand mRNA as a consequence of the viral replication process.
- the (+) strand mRNA is then translated into an active toxin peptide, e.g., hecate or phor21 (a highly lytic, designed peptide), resulting in cell death before mature infections virions are formed.
- the complete RNA sequence of this prototype example targeted against OSU rotavirus is listed below as SEQ. ID NO. 3.
- RNA virus When an RNA virus enters a susceptible host cell, the RNA-dependent RNA polymerase packaged with the virus, or encoded in the genome of the virus, converts negative RNA to positive RNA, an essential step to allow translation of the viral genes. If a negative RNA code for a toxin is also present, the same enzyme will convert the toxin code of negative RNA into positive or messenger RNA, which can then be translated to produce toxin, resulting in death of only the infected cell. Table I lists some of the more important viruses from the class of single-stranded negative-sense RNA viruses.
- Table I was compiled from Murphy, F.A., 1996 and from “Classification and Nomenclature of Viruses,” Francki, Knudson, and Brown (Eds.) 1991. Each of these viruses, like all non-retro RNA viruses, requires RNA-dependent RNA polymerase in order to replicate. Table I demonstrates the broad potential of Method II to kill cells infected by diverse single-stranded negative-sense viruses. (Note: "single-stranded” means that the nucleic acid polymers carried in the virion are not, in general, base-paired to complementary nucleic acid polymers. Some viruses have fragmented genomes, multiple nucleic acid polymers. Such fragmented genomes are considered “single-stranded” if they are not, in general, base-paired to complementary nucleic acid polymers.)
- Arenaviridae Junin virus (Argentine hemorrhagic Humans fever virus) Lassa fever virus Humans
- the infecting genome is either in the form of positive-sense single-stranded RNA or double-stranded RNA.
- copies of positive RNA must be generated from negative RNA templates using virus-specific RNA-dependent RNA polymerase, prior to packaging the genome as part of the maturation process. Examples of other single-stranded and double-stranded RNA viruses using this enzyme are given in Francki, Knudson, and Brown (Eds.) 1991; Mu ⁇ hy, F.A., 1996; and Fields, Virology, 1996.
- RNA toxin message present in the same cell will also be converted to positive RNA, which will then be translated to yield the cell-killing toxin. This chain of events will occur only in a virus-infected cell. Therefore, a cell infected by any non-retro RNA virus is killed by the construct of Method II. The negative toxin message is otherwise benign.
- RNA promoter or RNA-dependent RNA polymerase recognition site
- RNA promoter or recognition sequences tend to be highly conserved within genera and families of viruses, allowing broad protection with just a few different RNA promoters.
- different virus-specific promoters may be linked to toxin genes; the promoters could be linked in tandem to drive the expression of a single sequence encoding a toxin; or various promoter and toxin coding sequences could be linked in alternate fashion along the same nucleic acid polymer or on different nucleic acid polymers.
- RNA promoters examples include specific RNA promoters listed below in Table II, the references cited in Table II, and in the following references: Adkins, S. et al., "Subgenomic RNA promoters dictate the mode of recognition by bromoviral RNA-dependent RNA polymerases," Virology, vol. 252, pp. 1-8 (Dec. 1998); Brown, D. et al., "Template recognition by an RNA-dependent RNA polymerase: identification and characterization of two RNA binding sites on Q beta replicase," Biochemistry, vol. 34, pp. 14765-14774 (1995); Chapman, M.R.
- mRNA amplification system by viral replicase in transgenic plants FEBS Lett., vol. 336, pp. 171-174 (1993); Mushegian, A.R. et al, "Genetic elements of plant viruses as tools for genetic engineering,” Microbiol. Rev., vol. 59, pp. 548-578 (1995); O'Reilly, E.K. et al., "Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure," Virology, vol. 252, pp. 287-303
- negative or antisense genetic constructs are known to be useful to interrupt gene function in a variety of applications, including inhibition of viruses or cancers, and to determine the function of genes and gene products, they have been widely studied, and much is known about their manufacture and delivery.
- the same general techniques may be used to manufacture and introduce negative RNA toxin constructs into cells in accordance with the present invention. See, e.g., Guo and Kemphues, 1995; Montgomery and Fire, 1998; Tabara et al, 1998; US Pat. No. 5831069; US Pat. No. 5759829; US Pat. No. 5811537; US Pat. No. 5691317; US Pat. No. 5734039; US Pat. No. 5242906; US Pat. No. 5316930; Wagner, R.W. et al, "Antisense technology and prospects for therapy of viral infections and cancer," Mol.
- antisense oligonucleotides are not used to inhibit gene function, but are instead used to encrypt the genetic codes of toxins in such a way that the toxins are expressed only in cells infected by non-retro RNA viruses. I.e. , the negative message functions to produce a gene product, while the conventional use of antisense technology has been to inhibit production of a gene product.
- the complementary sequence of the toxin message in Method II could, as one example, be contained as a complementary code within an otherwise functional (-H)RNA strand coding another polypeptide; which could be converted to a functional message for the toxin when the virus-specific RNA-dependent RNA polymerase synthesizes the complementary polymer.
- This variation could be useful if the efficiency of the RNA-dependent RNA polymerase is much greater for the composite (+) RNA than for the toxin-encoding (-)RNA alone.
- Method II has been described with reference to RNA viruses, it will also work with DNA viruses that encode an RNA-dependent RNA polymerase, for example, the Hepatitis C virus, a hepadnavirus.
- an "indirect” or “conditional” toxin is a compound whose toxic effect depends on the presence of a second factor, typically an externally-administered pharmaceutical.
- a second factor typically an externally-administered pharmaceutical.
- virus thymidine kinase is itself non-toxic, but it generates a lethal toxin in the presence of a compound such as ganciclovir or acyclovir.
- the use of a conditional toxin such as thymidine kinase in Method II or Method III could allow the fine-tuning of the treatment protocol.
- Ganciclovir and acyclovir are already approved for other uses in humans, and are considered to be safe.
- Negative strand RNA viruses depend on RNA-dependent RNA polymerase (Rd-Rp) to replicate the viral genome to a (+) RNA strand before translation to yield viral proteins can occur.
- Rd-Rp RNA-dependent RNA polymerase
- Many Rd-Rp recognition sites/promoters are on the 3 '-end of the genome, with a critical base or multiple base sequence at that end of the genome, such as CC-3' in rotaviruses, that serves as an essential signal for replication of the viral genome. See Wentz, M.J. et al., "Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro," - rc ⁇ . Virol.
- RNA viruses such as the Flaviviridae (which includes viruses of humans and other animals).
- the Rd-Rp recognizes short templates on the 3 '-end to initiate positive strand RNA synthesis.
- the 21 nucleotides on the 3 '-end contain the sequence that is recognized to initiate RNA synthesis.
- the precise mechanisms of the nucleotide sequence/RNA polymerase interaction are not yet known, it is known that the recognition sequences needed for polymerase activity are contained in this 21 nucleotide sequence. See Kao, C.C.
- RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element, " J. Gen. Virol, vol. 78, pp. 3045-3049 (1997).
- a DNA plasmid vector will be constructed with a constitutive promoter controlling the transcription of a sequence that, when transcribed as mRNA, will have the first 30 bp from the 3 '-end of the influenza virus genome linked to (-) strand mRNA for toxin. In the absence of Rd-Rp, the (-) mRNA will persist in the cytoplasm for only a short time before being degraded.
- the Rd-Rp from the virus binds to the 30 bp site, the (+) strand mRNA is synthesized, and the cell's normal ribosomal machinery translates the encoded toxic peptide, killing the cell prior to the formation of mature virions.
- Method III includes a negative DNA sequence that is complementary to a sequence encoding a toxin, where the sequence also contains, in the 3' direction from the negative DNA toxin code, one or more sequences complementary to the 3'- terminal portion of a single-stranded DNA virus or of a retrovirus that uses single-stranded DNA as part of its infectious cycle.
- the negative-sense DNA may be delivered to cells via means known in the art, for example, via liposomes or via a modified negative-DNA virus delivery vector. This embodiment mimics the replication of negative-sense DNA viral genomes, in which a 3'-OH is provided by hybridization, to allow replication of the negative viral genome to form positive DNA.
- a toxin is encoded in (-)DNA, a functional message cannot be produced unless the (-)DNA is first converted into ( + )DNA.
- a primer with a free 3' -OH must hybridize to the (-)DNA toxin code before DNA polymerase will begin producing the corresponding (+)DNA.
- This primer can be provided by the 3 '-terminal portion of the virus genetic code itself, with a complementary sequence in the (-)DNA located in the 3'- direction from the toxin code.
- Such terminal portions could include, for example, the RU region of the retrovirus genome (Coffin. J.M., 1996), or the terminal portion of a parvovirus genome (Berns, K.I., 1996). Only in a cell infected by a target virus will the essential primer be present (the 3 '-terminal portion of the viral genetic code itself). Hybridization with the 3'- portion of the virus DNA allows complementary synthesis of the (-)DNA code to form the
- ( + )DNA toxin code ( + )DNA toxin code.
- the (-I-)DNA is synthesized to form a double helix with the complementary (-)DNA
- the ( + )mRNA for the toxin is transcribed and translated into toxin, thus terminating infection by killing the host cell.
- Method III may be used against ss (-)DNA viruses, ss (+)DNA viruses, and retroviruses.
- the complete (-)DNA sequence of a prototype example targeted against canine parvovirus is listed below as SEQ. ID NO. 4.
- the SEQ. ID No. 5 (for Method I) construct will be modified by replacing the he ⁇ esvirus promoter with the commercially available cytomegalovirus (CMV) promoter, using standard techniques such as those found in references such as Ausubel et al. (1999).
- CMV cytomegalovirus
- the CMV promoter is a universal promoter for mammalian cells, resulting in high levels of constitutive transcription.
- the 3' -OH end of the virus will anneal in position to initiate replication from the negative strand that encodes the toxin construct, to yield positive DNA.
- the host cell DNA-dependent DNA polymerase will then initiate the steps leading to transcription and translation of the active toxin.
- the negative DNA will be produced by single strand PCR using the positive strand of the construct as the template, according to the techniques described in Ausubel et al. (1999).
- the next experimental step will be in vivo testing, creating transgenic plants and animals that are non-permissive for specific viral infections.
- mouse models for various human and animal diseases will be tested by introducing a he ⁇ esvirus-protective construct into the germ lines of mice.
- he ⁇ esvirus resistance will be introduced into chickens, which are susceptible to Marek's disease, caused by a type of he ⁇ esvirus. Furthermore, because Marek's disease results in tumors, the utility of this invention in preventing virus-associated cancers will also be demonstrated.
- bacteria and yeasts of economic significance and in plants and animals of agricultural significance or of significance as companion animals, including major crops such as wheat, rice, corn, barley, potatoes, soya, sweet potatoes, yams, and casava; mammals such as cows, pigs, horses, sheep, goats, dogs, and cats; insects such as bees; and other animals such as fish (e.g. , catfish, tilapia, salmon), insects (e.g., honeybees, silkworms), crustaceans (e.g., shrimp, crabs, lobsters, crawfish, prawns), and birds (e.g. , chickens, turkeys, ostriches, and parrots).
- major crops such as wheat, rice, corn, barley, potatoes, soya, sweet potatoes, yams, and casava
- mammals such as cows, pigs, horses, sheep, goats, dogs, and cats
- insects such as bees
- other animals such as fish (e.g. ,
- This invention is based on regulatory features and enzyme functions of viruses that are less likely to "drift" or to mutate in response to selective pressure than are those features of viruses that have been targeted by previous drug treatments or by vaccinations.
- the regulatory sequences of viruses tend to be highly conserved, as are the RNA polymerase function and the polymerase recognition sequence.
- Sub-optimal levels of treatment are not likely to create selective pressures favoring treatment-resistant mutations, as has often occurred with other drugs such as chemical inhibitors, antibodies, or antibiotics.
- the inducer and the promoter both would have to develop compensatory mutations simultaneously in order to retain function and to evade the effect of the novel constructs.
- Simultaneous compensatory mutations in each of two factors are far less likely to occur than either would be alone. Furthermore, even if such a mutational event did occur, it would be relatively easy to identify the alteration, for example by sequencing the mutated promoter, and then to synthesize a new construct based on the mutated promoter, in accordance with the present invention. By contrast, it is extremely difficult, time-consuming, and expensive to identify the reason why a conventional drug has become ineffective and to redesign the drug accordingly.
- Method II if the recognition sequence used by the RNA-dependent RNA polymerase mutated, the enzyme would also have to mutate simultaneously in order to recognize the new sequence.
- viruses The most variable characteristics of viruses are surface proteins that can be recognized by humoral and cellular immune mechanisms of the host. These regions can differ widely between genetically related viruses, and can even be diverse within populations of viruses originally derived from the same clone.
- the extensive diversity of surface proteins presents substantial obstacles to the successful development of vaccines, and also limits the ability of drugs to inhibit the functions of these ever-changing targets.
- viral core proteins tend to be more stable genetically, and are conserved between related viruses to a much greater extent.
- Core proteins show some promise for vaccine development, because cellular processing can present core antigens on cell surfaces where they may be recognized by cell-mediated immune mechanisms.
- a substantial drawback to this approach is that mature, cell-free viruses cannot be inactivated by immune mechanisms directed against core antigens; and mature viruses are often released prior to the destruction of the infected cell by immune mechanisms.
- Virus-specific regulatory sequences that are never expressed as proteins are essential to the cycle of replication for many viruses. Because the regulatory sequences are not expressed, they are not attacked by the immune system or other defense mechanisms of host cells. Thus there has been no selective pressure for viruses to develop diversity in these sequences. Viral regulatory sequences tend to be highly conserved, both in sequence and function. See M. Martin et al, "Identification of a transactivating function mapping to the putative immediate- early locus of human he ⁇ esvirus 6," J. Virology, vol. 65, pp. 5381-5390 (1991). Regulatory regions are fairly resistant to mutation.
- RNA viruses the RNA-dependent RNA polymerase, used in Method II of the present invention, could not readily mutate to lose its function without ending the ability of the virus to replicate.
- vimses do mutate in response to selective pressure from traditional antisense inhibition (Bull, J.J. et al. 1998), the virus could not readily eliminate the RNA-dependent RNA polymerase function without losing its ability to reproduce.
- Viral mutations are less likely to interfere with this new strategy of encrypting the genetic codes of toxins than for other methods of viral inhibition, including vaccinations, drug treatments, and conventional antisense inhibition of viral genes.
- a single construct will often provide protection against different species of viruses, because regulatory functions are typically conserved within groups of viruses.
- the conservation of viral-specific functions, including gene regulation and RNA-dependent RNA polymerase functions, ensures that many constructs designed in accordance with the present invention will be effective against many different species of viruses.
- this conservation of virus-specific sequences and functions enhances the utility of the prevention and treatment strategies of this invention, as viral infections can be prevented or treated even where the identity of a particular virus is unknown.
- this invention can be used to prevent or treat infections caused by so-called “emerging” viruses, viruses that might be used as biological weapons, and "hidden” viruses contained in congeneric transplant tissues and organs, or in xenotransplants, even though the exact species of the virus may not be known.
- This invention may be applied to prokaryotic or eukaryotic cells, including germ cells and somatic cells of plants and animals.
- Applications include introduction of a construct in accordance with the present invention into the germ lines of agriculturally significant plants and animals or companion animals to produce virus-resistant breeds; or introduction into somatic cells of humans, other animals, or plants to prevent or treat viral infection.
- hematopoietic stem cells of a patient could be transformed with a construct in accordance with the present invention and then transfused back into the patient. The patient will then have a "reservoir" of non-permissive cells.
- the construct could be introduced in vivo or ex vivo into a patient's cells, for example via liposomes or other carriers containing transformation vectors known in the art, for example the high-efficiency transformation vector of Cooper, United States patent no. 5,719,055, to introduce the construct into both infected and uninfected cells of the patients.
- Active replication of a virus would trigger the toxin and result in the death of the cell prior to release of mature virions, preventing (or at least reducing) further spread of the virus.
- DNA construct without any of the flanking sequences necessary to promote inco ⁇ oration into a chromosome may be introduced into cells by a high efficiency vector such as a liposome.
- somatic cells will include the introduction of constructs in accordance with the present invention into subdermal or mucosal tissue to generate virus- resistant skin cells or mucosal cells.
- Transient transfection e.g., with plasmids that do not integrate into the genome, may be used to cure viral infections, or progenitor cells may be permanently transformed.
- a toxin should have the following characteristics:
- the toxin should be capable of being readily produced either under the regulatory control mechanisms of a virus- specific promoter; or from messenger RNA after conversion from antisense RNA; or from negative single-stranded DNA after conversion to positive DNA using viral nucleic acid as a primer, for example under the control of a constitutive promoter in the last case.
- a suitable toxin may be one of the many toxic peptides known in the art.
- the toxin should be capable of killing an infected cell prior to release of mature virions or capable of killing persistently infected cells.
- the toxin should not kill uninfected cells, whether or not they contain the construct, and whether or not an uninfected cell is near an infected cell that is killed by expression of the construct.
- toxins from plants, animals, and bacteria satisfying these criteria.
- there are many bacterial toxins that use an A/B subunit motif in which the A subunit is toxic once it enters a cell but has no ability to cross cell membranes unassisted, and in which the B subunit (or multi-subunit complex) binds to cells but has no toxicity on its own.
- the A subunit even when injected systemically, is non-toxic. See, e.g., Balfanz et al, 1996; Middlebrook and Dorland, 1984. Nucleic acids coding for the A or active subunit could be used in this invention because the A subunit will already be inside the cell when it is produced, so it will not be necessary to include sequences coding for the B or cell-binding component.
- the A subunit will kill the cell in which it is expressed, but will not damage other cells when released by cell lysis because the A subunit could not gain access to the interior of other cells. Examples include the A subunit of cholera toxin, which destroys ion balance, and the A subunit of diphtheria toxin, which terminates protein synthesis.
- toxins comprise a single peptide chain having separate domains, where one domain functions to enable entry into the cell and a second domain is toxic.
- Such a multidomain peptide toxin could be truncated, using genetic engineering to produce a construct that only codes for the toxin domain.
- truncated toxin that has been used in other systems to kill artificially targeted cells is the truncated form of exotoxin A from Pseudomonas aeruginosa (Brinkman et al., 1993, Pastan and FitzGerald, 1991, and Wels et al., 1995)
- the commonly used ricin toxin from plants also uses this same type of A/B subunit motif.
- Lee, H.P. et al. "Immunotoxin Therapy for Cancer," JAMA, vol. 269, pp. 78-81 (1993).
- catalytic toxins such as diphtheria toxin A polypeptide have been successfully used (in another context) to selectively kill cell lineages in transgenic mice without evidence of non-specific "leakiness.”
- Catalytic toxins such as diphtheria toxin A polypeptide have been successfully used (in another context) to selectively kill cell lineages in transgenic mice without evidence of non-specific "leakiness.”
- Leakiness of transcription is not expected to be a major problem using viral promoters other than those from retroviruses.
- other classes of "non-catalytic" peptide toxins may be more appropriate for general use, because a small degree of "leakiness" in transcription would not be lethal.
- the class of peptides called "lytic peptides,” or “antimicrobial amphipathic peptides,” is preferred. These peptides are relatively small, generally containing 20 to 50 amino acids (or even fewer), and are capable of forming an amphipathic alpha helix in a hydrophobic environment, wherein at least part of one face is predominantly hydrophobic and at least part of the other face is predominately hydrophilic and is positively charged at physiological pH.
- Such structures can be predicted by applying the amino acid sequence to the Edmundson helical wheel (Schiffer and Edmundson, 1967).
- lytic peptides are widely distributed in nature and vary significantly in toxicity. They can also be designed to possess different levels of lytic activity. Many of these toxins are inactivated by serum factors, and cause systemic tissue damage only when present in high concentrations. Typically, when applied to cells in culture, a few micrograms per mL are required to kill the cultured cells. The level of toxicity of lytic peptides is determined by the amino acid composition and sequence. Different peptides can have widely differing levels of toxicity. In addition, relatively few molecules should be needed to kill a cell if the cell produces the molecules internally. A further discussion of lytic peptides suitable for use in this invention appears below.
- lytic peptides Due to the role played by lytic peptides in nature, especially to protect against bacterial infections, the production of lytic peptides by various cloning technologies has been widely investigated and published. A consistent finding of workers who have attempted to express lytic peptides by cloning is that the lytic peptides kill the cells expressing the cloned lytic peptide gene, thereby seriously reducing product yields. In nature most lytic peptides are produced with signal sequences directing the product to be stored in membrane-bound vesicles, to be secreted on mucosal surfaces.
- the killing of the cell producing the cloned lytic peptide has been viewed as an unwanted consequence, resulting in failure of the experiment.
- the killing of cells that produce the cloned lytic peptide does not represent a failed experiment, but rather is the desired response to viral infection.
- lytic peptides may be used in the treatment of viral infections in accordance with the present invention.
- a virus-specific promoter would be linked to a sequence encoding such an indigenous lytic peptide, but devoid of the signal sequence normally used with the peptide.
- the only new genetic element introduced into the cell is a promoter of a target virus. Promoters are neither transcribed nor translated, so no foreign peptide or protein is ever expressed in the course of protecting the cell from viral infection.
- a difficulty in proving causality in humans is that the viruses often associated with and expressed from tumors can also be found in non-cancerous cell types, as well as in individuals without symptoms of cancer.
- the present invention to treat virus-associated cancers, it does not matter whether the virus actually induced the cancer or is just growing there: if the virus uses virus-specific mechanisms to induce virus-specific genes, then the virus-infected cells of the cancer can be destroyed by the present invention.
- novel methods of the present invention have the potential to alleviate many of the problems of curing persistent viral infections.
- RNA cancer-causing or cancer-associated viruses appear to be closely related to one another genetically. Therefore it is likely that a single construct or a small number of different constructs will suffice to treat a wide variety of RNA virus-based cancers.
- the use of the present invention is non-toxic to any cell that does not contain the virus-specific regulatory sequences or enzymes exploited by the present invention. Additionally, it is not necessary — although it is certainly possible— to selectively target the toxin to the cancer cells; either way, the toxin would be expressed only in infected cells containing the virus-specific inducers.
- the modification of non-human germ lines to prevent viral infections has potentially broad (though sometimes indirect) implications for public health. See Gubler, 1998.
- the invention will, for example, be applied to modifying insect or arachnid vectors of viral diseases.
- the state of the art in manipulating insect genomes is quite advanced. Previous attempts to modify mosquitoes to be non-permissive for human pathogenic viruses have successfully reduced virus loads in the vector (Powers, 1996); and in a few cases have eliminated the ability of the vector to transmit the virus, albeit through the use of complex methods of double infections that are not likely to be applicable in the field.
- the genetic additions introduced by the present invention are relatively small, and are not even expressed unless a mosquito (for example) is infected by the target virus.
- mosquito-borne viruses that may be controlled through the present invention include the viruses responsible for yellow fever and dengue fever. See, e.g., K. Olson et al, "Genetically engineered resistance to Dengue-2 virus transmission of mosquitoes," Science, vol. 272, pp. 884-886 (1996).
- influenza virus is known to infect domesticated animals, particularly pigs, ducks, and chickens; and to recombine or mutate in those animals to make new strains that are infectious in humans.
- the development and spread of new influenza viruses could be reduced by making pigs, ducks, and chickens non- permissive to the influenza virus in accordance with Method II.
- the present invention could reduce the application of insecticides on crops.
- the damage to crops following predation by insects is largely due not to the direct effect of feeding by the insects, but to the effects of viral pathogens carried by the insects. If a crop plant were made non-permissive to viral infection in accordance with the present invention, then the need to control at least some insect pests would be reduced, thereby reducing the use of pesticides on the crop.
- a plasmid vector was designed to force inco ⁇ oration of the desired transgene into a recipient chromosome.
- the stable inco ⁇ oration of a transgene relied on homologous recombination of a transgene into the recipient chromosome, which occurs at a very low frequency in most systems.
- a plasmid was constructed to contain a mini-transposon.
- a "mini-transposon” is one in which the transposon 's insertion sequences have been shortened to prevent unwanted homologous recombination, and in which the transposase has been removed from between the transposon insertion sequences to an upstream position under control of an inducible promoter.
- the result is two-fold: 1) expression of the transposase can be controlled, and 2) once the transposon carrying the desired gene has been delivered to a recipient chromosome, the remainder of the plasmid is destroyed, which prevents future transposition events from occurring, since the transposase is lost with the rest of the plasmid.
- the first gene transformed using this transposon system was that encoding the lytic peptide cecropin B under control of an acute phase promoter, both originating from the giant silk moth Hyalophora cecropia.
- the plasmid carrying the transposon system was named pCep90 (carrying the native cecropin B gene plus 2 kilobases of moth DNA flanking the gene on either side, for a total of 5.9 kbp of insert between the insertion sequences).
- a streamlined version of pCep90 was also prepared, named pPC6, modified to include only 1.8 kbp of the cecropin B gene between the insertion sequences. (The 2 kbp flanking each side of the cecropin B gene were removed).
- the ICP4 (or Vmw 175) gene is an immediate-early gene isolated from He ⁇ es Simplex I (HSV I), as sequenced and described by M. Murchie et al, "DNA sequence analysis of an immediate-early gene region of the he ⁇ es simplex virus type 1 genome (map coordinates 0.950 to 0.978)," J. Gen. Virol, vol. 62 (Pt 1), pp. 1-15 (1982) Byrne et al.
- mice (1989) linked the ICP4 promoter to a gene encoding chloramphenicol acetyltransferase (CAT) and a simian virus polyadenylation signal to create transgenic mice capable of expressing the CAT protein in the presence of he ⁇ es viral proteins responsible for turning on immediate early proteins in an HSV
- CAT chloramphenicol acetyltransferase
- the vector containing this ICP4/CAT/PolyA was designated pIE.
- the pIE plasmid contained 360 bp of the ICP4 promoter.
- plasmids pICP/Phor21/-e ⁇ and pICP/Phor21/ ⁇ c The base plasmid for both was pPC6 (described above), modified as follows: 1) To make the intermediate plasmid pBTnNeo, the cecropin B promoter and gene were removed from between the insertion sequences of pPC6 and replaced with a gene encoding neomycin/kanamycin resistance and a multiple cloning site.
- the neomycin gene allowed selection for eukaryotic cells containing the transposon in the presence of the antibiotic neomycin or its analog, G418, as well as selection for cells containing the transposon in prokaryotic cells using the antibiotic kanamycin.
- the multiple cloning site allowed easy cloning of a desired gene (in this case, ICP4/Phor21) between the insertion sequences. The result was a transposon carrying a selectable marker and the gene encoding antiviral activity.
- the intermediate plasmid pBTnLac carried the same transposon as described above, but the cecropin B gene and promoter were replaced with a gene encoding the a fragment of the ⁇ -galactosidase gene, commonly referred to as lacZ.
- lacZ a gene encoding the a fragment of the ⁇ -galactosidase gene, commonly referred to as lacZ.
- the lacZ gene carries a multiple cloning site to allow easy insertion of a desired gene of interest (in this case, ICP4/Phor21) and easy selection of plasmids containing the desired gene using blue/white color screening.
- PCR primers to amplify the ICP4 promoter from pIE. These primers had restriction sites added to the ends — Spel on the 5' end of the promoter and Hind III on the 3' end. A sequence encoding Phor21 was synthesized and then amplified with PCR primers containing restriction sites - Hind III on the
- the poly A termination sequence was amplified from cecropin B with PCR primers containing Kpn I on the 5' end and Spe I on the 3' end. Digestion with the appropriate restriction enzymes and subsequent ligation using T4 DNA ligase (New England Biolabs, Beverly, MA) insured correct 5 '-3' orientation of the ICP4 promoter to Phor21 and correct 5 '-3' orientation of Phor21 to the poly A. Restriction digestion of the Spe I sites from the ligated product allowed sticky end ligation into the Spe I site in either pBTnNeo or pBTnlac.
- the sequence for the completed construct is SEQ. ID NO. 5. (All procedures described in the preparation of the plasmids, e.g., restriction digests, ligation etc., were conducted using either the standard protocols found in F. M. Ausubel et al. (Eds) (1999), or following the manufacturer's suggested protocols.)
- Each cell type described in Tables III - V will be challenged with HSV 1 and observed for increased cytopathic effect. It is expected that cells expressing Phor 21 under control of the viral promoter will lyse more quickly than will control cells when a high virus titer is used, i.e., a titer sufficiently high to infect essentially all cells in the culture.
- a high virus titer i.e., a titer sufficiently high to infect essentially all cells in the culture.
- a low virus titer e.g., 1 viral particle per 10 cells
- cells containing ICP/Phor21 that become infected with virus will die and will be replaced by dividing uninfected cells, while all control cells will be killed by the spread of infectious HSV 1 particles.
- the low virus titer condition approximates the results expected in vivo. Preliminary in vitro results for the anti-herpes construct.
- neomycin (GeneticaTM G418) was added to the 60% confluent monolayers to generate selective pressure. After eight days, cells were transferred from the wells to 25 cm 2
- the He ⁇ es virus inoculum was obtained by infecting a 162 cm 2 Falcon flask of confluent normal Vero cells with a 1:40 dilution of He ⁇ es Simplex Virus 1 infected tissue culture fluid, obtained from the American Type Culture Collection, accession number VR-733.
- the tissue culture fluid was harvested, the cells were removed by centrifugation, and the infected supernatant was frozen in 1 mL aliquots.
- PCR analysis of the Vero cells confirmed that the construct was present in the putatively transgenic Vero cells, but not in the control (untransformed) Vero cells.
- DNA was extracted from: (a) Vero cells that had been transfected with Superfect and pICP4/Phor21/-e ⁇ , and (b) control Vero cells receiving no treatment. Approximately 3.4 ⁇ g of DNA was obtained from each group of cells, of which " 0.07 ⁇ g was used for PCR reactions with primers specific to the 5' end of ICP4 and the 3' polyA end of the ICP4/Phor21 gene.
- mice Twenty mice total will be used in the initial experiment: 10 males and 10 females. Five males and five females will be used in the control group, and five each in the treatment group.
- the pICP/Phor21/ ⁇ c vector (SEQ. ID NO. 5) will be complexed to
- mice will receive Superfect only. The mice will be held for ten days to allow any uninco ⁇ orated vector to be cleared from the bloodstream. On day 10, a 50-100 ⁇ l blood sample will be drawn from each mouse, and DNA from the blood will be extracted using a Qiagen Blood Kit for DNA extraction. Each mouse's fur will be numbered with a black marker, so that each DNA sample may be associated with a specific mouse.
- PCR will be conducted on each sample using primers specific to ICP4 on the 5' end and polyA on the3' end. PCR protocols will be as described in Ausubel et al. (1999). Each PCR sample will be electrophoresed on a 1 % agarose gel with a 1 kilobase ladder as a reference marker, stained with 0.5 mg/mL ethidium bromide, and visualized on a U.V. light source. Male and female mice positive for ICP4/phor21 will be paired for mating. DNA from blood samples from all F, mice resulting from these crosses will be extracted, and PCR conducted as described above.
- mice positive for ICP4/Phor21 by PCR will be challenged with the same virulent isolate of he ⁇ es used in the in vitro experiments, at a viral load sufficient to cause disease in normal mice; an equal number of controls will be treated in the same manner. Expected in vivo results. Mice containing the ICP4/Phor21 construct will rid themselves of the virulent he ⁇ es virus without becoming viremic, while the control mice will die from the challenge. Chickens. In vivo transformation and challenge of chickens will be generally similar to that described above for mice. Sexually immature chickens will be lipofected using the same SEQ. ID NO. 5 DNA/Superfect ratios and the same amount of DNA per gram body weight.
- 10 birds will receive an intravenous injection through a wing vein, 10 birds will be injected in the intraperitoneal cavity, 10 birds will be injected directly in the gonads, and 10 will receive Superfect only, administered by intravenous injection.
- the birds will be held for 10 days to allow uninco ⁇ orated DNA to clear, and PCR will be conducted to identify birds carrying the transgene.
- the birds positive for the transgene will be allowed to breed, and all F s will be screened as in the mouse experiment.
- the F s positive for the transgene by PCR will be challenged with Marek's disease he ⁇ esvirus, as will an equal number of controls. Birds containing the anti-he ⁇ es construct will be protected from the disease while the control birds will become viremic and die.
- Marek's disease challenge will not only provide an in vivo demonstration of the efficacy of Method I in preventing viral infection generally, it will also demonstrate specifically its efficacy in preventing what has previously been a major disease problem in the poultry industry.
- each segment was amplified with primers containing restriction enzyme sites on the ends to allow ligation to Phor21 in the proper orientation.
- the resulting sequence of rybozyme:Phor21: hpoll is being ligated into pBTnNeo and pBTnLac as otherwise described above.
- An otherwise identical plasmid is also being constructed with Phor21 in the positive sense as a negative control.
- the plasmid bearing the neomycin gene will be used for cell culture experiments, e.g., human 293 cells (ATCC), and the plasmid bearing the lac gene will be used for animal experiments, e.g., mice and chickens.
- the same experimental design as described above will be used for each cell type and each animal, with similar results expected following challenge.
- the initial in vitro challenge of Vero cells will be conducted with influenza A virus (H1N1), ATCC accession number VR-825. Later in vitro and in vivo challenges will be conducted using strains of influenza virus that are virulent in the particular species.
- Tobacco plants will be made non-permissive for tobacco mosaic virus (TMV), a positive strand RNA virus.
- TMV tobacco mosaic virus
- the construct will use the messenger strand sequence for the viral RNA-dependent RNA polymerase recognition sequence, located at the 3' portion of the viral genome, linked to a negative sense coding sequence for a toxin.
- RNA-dependent RNA polymerase recognition sequence located at the 3' portion of the viral genome, linked to a negative sense coding sequence for a toxin.
- the DNA constructs used for cloning into plant cells will have constitutive promoters to continuously yield the desired forms of the transcribed RNA message in the plant cells.
- the message will comprise a virus polymerase recognition sequence and the negative sense sequence coding the toxin.
- the negative toxin message is converted to a positive message encoding the toxin by the virus RNA-dependent RNA polymerase, the positive mRNA form of the toxin code is translated to yield the toxin gene product, resulting in death of the infected cell prior to formation of mature virions.
- the starting material for obtaining the viral RNA-dependent RNA polymerase (RDRP) recognition region of the TMV genome will be cDNA of the TMV genome, cloned into the pBR322 plasmid vector, and grown in E. coli.
- This clone is available from the American Type Culture Collection (ATCC accession no. 45138). See W. Dawson et al, 1986, "cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts," Proc. Natl. Acad. Sci. USA. vol. 83, pp. 1832-1836 (1986).
- the pBR322 vector containing the TMV genome is grown in E. coli and purified using the MaxiprepTM kit from Qiagen (Chatsworth, CA), using the manufacturer's recommended protocols.
- RNA polymerase recognition sequence of the TMV genome is then amplified by PCR.
- RNA polymerase recognition sequence is then ligated to a toxin construct linked to a promoter, a ribosome binding sequence, and a polyadenylation sequence, according to the methods of F. M. Ausubel et al. (Eds) (1999).
- the toxin construct encodes the Phor21 peptide previously described.
- PCR primers are used with different restriction endonuclease sites on each end, so that just the selected strand will be amplified, again using the methods of F. M. Ausubel et al. (Eds) (1999).
- This modified A. tumefaciens is an efficient vector to generate transgenic plants in a wide variety of species.
- the desired genes are introduced into plants by plant cell culture, callus culture, leaf explants, or meristem cultures.
- the prepared constructs are introduced into leaf explants of tobacco plants using the modified Ti plasmid as described by J. Topping, "Tobacco Transformation,” pp. 365-372 in G. Foster et al. (Eds.), Plant Virology Protocols (1998).
- Transformed plants are selected by antibiotic resistance, grown, and tested by PCR for the presence of the construct. See D. Worrall, "PCR analysis of transgenic tobacco plants,” pp. 417-424 in G. Foster et al. (Eds.), Plant Virology Protocols (1998).
- the transgenic tobacco plants and non-transgenic controls will be experimentally infected with TMV. Evaluation of resistance will be determined both visually, and by methods described in "PART V. Evaluation of Resistance,” pp. 455-509 in G. Foster et al. (Eds.), Plant Virology Protocols (1998).
- the transgenic plants will be resistant to infection, while the control plants will become diseased following infection. Lytic Peptides Useful in the Present Invention.
- Lytic peptides are small, basic peptides. Native lytic peptides appear to be major components of the antimicrobial defense systems of a number of animal species, including those of insects, amphibians, and mammals. They typically comprise 23-39 amino acids, although they can be smaller. For example, the protegrins from porcine leukocytes have 16-18 amino acids, and fragments down to 12 amino acids show activity against bacteria. See X-D Qu et al, "Protegrin Structure and Activity against Neisseria gonorrhoea," Infection and
- Lytic peptides have the potential for forming amphipathic alpha-helices. See Boman et al, "Humoral immunity in Cecropia pupae,” Curr. Top. Microbiol. Immunol, vol. 94/95, pp. 75-91 (1981); Boman et al, “Cell-free immunity in insects,” Annu. Rev. Microbiol, vol. 41, pp. 103-126 (1987); Zasloff, "Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial DNA sequence of a precursor," Proc. Natl. Acad. Sci. USA, vol. 84, pp.
- Known amino acid sequences for lytic peptides may be modified to create new peptides that would also be expected to have lytic activity by substitutions of amino acid residues that preserve the amphipathic nature of the peptides (e.g., replacing a polar residue with another polar residue, or a non-polar residue with another non-polar residue, etc.); by substitutions that preserve the charge distribution (e.g., replacing an acidic residue with another acidic residue, or a basic residue with another basic residue, etc.); or by lengthening or shortening the amino acid sequence while preserving its amphipathic character or its charge distribution.
- substitutions of amino acid residues that preserve the amphipathic nature of the peptides e.g., replacing a polar residue with another polar residue, or a non-polar residue with another non-polar residue, etc.
- substitutions that preserve the charge distribution e.g., replacing an acidic residue with another acidic residue, or a basic residue with another basic residue, etc.
- Lytic peptides and their sequences are disclosed in Yamada et al, "Production of recombinant sarcotoxin LA in Bombyx mori cells," Biochem. J., vol. 272, pp. 633-666 (1990); Taniai et al,
- Families of naturally-occurring lytic peptides include the cecropins, the defensins, the sarcotoxins, the melittins, and the magainins.
- Boman and coworkers in Sweden performed the original work on the humoral defense system of Hyalophora cecropia, the giant silk moth, to protect itself from bacterial infection. See Hultmark et al, "Insect immunity. Purification of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia," Eur. J. Biochem., vol. 106, pp. 7-16 (1980); and Hultmark et al., "Insect immunity.
- cecropin D Isolation and structure of cecropin D. and four minor antibacterial components from cecropia pupae
- Infection in H. cecropia induces the synthesis of specialized proteins capable of disrupting bacterial cell membranes, resulting in lysis and cell death.
- these specialized proteins are those known collectively as cecropins.
- the principal cecropins — cecropin A, cecropin B, and cecropin D — are small, highly homologous, basic peptides.
- Boman 's group showed that the amino-terminal half of the various cecropins contains a sequence that will form an amphipathic alpha-helix.
- Antibacterial peptides from pig intestine isolation of a mammalian cecropin," Proc. Natl. Acad. Sci. USA, vol. 86, pp. 9159-9162 (1989).
- Cecropin peptides have been observed to kill a number of animal pathogens other than bacteria. See Jaynes et al. , "In Vitro Cytocidal Effect of Novel Lytic Peptides on Plasmodium falciparum and Trypanosoma cruzi," FASEB, 2878-2883 (1988); Arrowood et al, "Hemolytic properties of lytic peptides active against the sporozoites of Cryptosporidium parvum," J. Protozool, vol. 38, No. 6, pp. 161S-163S (1991); and Arrowood et al, "In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum," Antimicrob.
- Defensins originally found in mammals, are small peptides containing six to eight cysteine residues. Ganz et al, "Defensins natural peptide antibiotics of human neutrophils," J. Clin. Invest., vol. 76, pp. 1427-1435 (1985). Extracts from normal human neutrophils contain three defensin peptides: human neutrophil peptides HNP-1, HNP-2, and HNP-3. Defensin peptides have also been described in insects and higher plants.
- sarcotoxins Slightly larger peptides called sarcotoxins have been purified from the fleshfly Sarcophaga peregrina.
- Okada et al. "Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae," J. Biol. Chem. , vol. 260, pp. 7174-7177 (1985).
- the sarcotoxins presumably have a similar antibiotic function.
- Other lytic peptides have been found in amphibians.
- Zasloff showed that the Xenopus -derived peptides have antimicrobial activity, and renamed them magainins. Zasloff, "Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial DNA sequence of a precursor,” Proc. Natl. Acad. Sci. USA, vol. 84, pp. 3628-3632 (1987).
- the synthetic lytic peptide known as S-l has been shown to destroy intracellular Brucella abortus-, Trypanosoma cruzi-, Cryptosporidium parvum-, and infectious bovine he ⁇ esvirus I (IBR)-infected host cells.
- S-l or Shiva 1
- IBR infectious bovine he ⁇ esvirus I
- Bonamia ostreae the intrahemocytic parasite of the flat oyster Ostrea edulis
- Mol. Mar. Biol, vol. 3, pp. 327-333 (1994) reports the in vitro use of a magainin to selectively reduce the viability of the parasite Bonamia ostreae at doses that did not affect cells of the flat oyster Ostrea edulis.
- Also of interest are the designed peptides disclosed in McLaughlin et al. , "Amphipathic
- Lytic peptides such as are known generally in the art may be used in practicing the present inventions.
- virus-specific mechanisms may also be used to activate a toxin or toxic mechanism.
- the toxic mechanism triggered while preferably a peptide or protein toxin as described above, could also comprise the activation of a host-cell toxin or toxic mechanism, e.g., apoptosis or necrosis.
- Method I it may also be desirable to place a "stop” or “termination” codon upstream of the virus-specific promoter to prevent "read through” and unintended expression of the toxin in the absence of a virus-specific inducer.
- a message could include, in addition to a sequence encoding the toxin, appropriate start and termination signals, spacers, and perhaps caps, polyadenylated tails, and other sequences and modifications known to promote efficient gene expression and nucleic acid stability within the cell.
- virus-specific mechanisms work only in association with certain normal cellular regulatory factors. Some of the cell specificity of viruses is based upon which cell types have these regulatory factors present in their cytoplasm, as they are absolutely essential for some viruses. However, even if a virus factor requires one or more normal cellular factors to function, the mechanism is still considered “virus-specific" within the scope of the present specification and the claims, because the contribution of the virus is essential, and the host cell factors alone are not capable of inducing toxin expression through a construct of the present invention.
- the term “transformed cell” is sometimes taken to imply that exogenous DNA has been integrated into the cell's genome (or is otherwise stably maintained in the cell, e.g., as in an episome), whereas in the present specification and claims the term “transformed cell,” standing alone, carries no implication either way as to whether exogenous DNA or RNA is stably maintained in the cell; the exogenous nucleic acid in a "transformed” cell may be present only transiently.
- the term “transformed cell” has sometimes been used to refer to cancers or immortal cell lines, while the term as used here carries no such implication either way.
- a "transformed cell” is a prokaryotic or eukaryotic cell into which an exogenous genetic construct in accordance with the present invention has been introduced.
- transformed cell is also intended to include progeny and descendants of such cells that retain one or more copies of the introduced genetic construct. Unless context clearly indicates otherwise, a "transformed cell” may be in vivo, ex vivo, or in vitro.
- the introduced genetic construct may or may not be integrated into the genome of the cell; it could, for example, either be present in a plasmid or inco ⁇ orated into a chromosome.
- the introduced construct may comprise linear or circular DNA or RNA, with or without the ability to replicate.
- a "transformed cell” may be a somatic cell or a germ cell.
- a “transgenic” cell or organism is one in which exogenous DNA has been integrated into the genome of the cell or organism, or is otherwise maintained in the cell more than transiently.
- an episome or cDNA might be used to introduce a constmct into skin cells to prevent episodes of he ⁇ es eruption, without actually being integrated into a chromosome. If the episome is maintained more than transiently, then the cell would be considered to be “transgenic" within the scope of this definition.
- transgenic cell is also considered to be “transformed,” but that not all “transformed” cells need be “transgenic.”
- virus permissive cell or a cell that is “permissive” to a virus refers to a cell that can support infection by and replication of a particular virus.
- non-permissive cell refers to a cell that cannot support propagation of that virus.
- a “vims-specific element” is a virus-encoded gene product or nucleic acid sequence that does not naturally occur in the host cell in the absence of viral infection.
- the "specificity" of a “virus-specific element” refers to specificity as compared to products naturally occurring in the uninfected host cell, and does not imply specificity as compared to other viruses.
- one of the strengths of the present invention is that many viral elements tend to be conserved, so that a single construct in accordance with the present invention will protect against multiple viruses.
- Virus-specific elements include for example, but are not limited to, virus-specific inducers, RNA-dependent RNA polymerases, and vims- specific nucleic acid sequences that can act as primers for a DNA polymerase.
- virus-specific promoter is a promoter that requires a virus-specific inducer, or a complex between a virus-specific inducer and host cellular factors, to allow production of a gene product.
- virus-specific inducer is a virus-encoded gene product that can induce or activate a promoter, and that does not naturally occur in the host cell in the absence of viral infection.
- the "specificity" of a “vims-specific inducer” refers to specificity as compared to products naturally occurring in the uninfected host cell, and does not imply specificity as compared to other viruses.
- one of the strengths of the present invention is that viral inducers tend to be conserved, so that a single construct in accordance with the present invention will protect against multiple viruses.
- virus-specific inducer should also be construed to include virus-specific regulatory elements other than conventional inducers, other regulatory elements that can effectively be made to function as inducers. Although such adaptations of other regulatory elements to act as inducers are not, in general, preferred, they should be recognized as equivalent to the use of more conventional "inducers” for pu ⁇ oses of the present invention. Furthermore, there could be specific circumstances in which it is more effective or convenient to use such a specific alternative regulatory element.
- a virus- specific repressor (“Repressor 1”) can be made to function as an inducer of expression of a toxin via the following constmct: The construct contains two genes, Gene 1 and Gene 2. Both Genes 1 and 2 are repressible. The expression of Gene 1 is repressed in the presence of virus- specific Repressor 1; but in the absence of Repressor 1, Gene 1 constitutively expresses
- Repressor 2 is preferably a virus-derived repressor, or is otherwise a repressor that interferes with no normal cellular functions. (Note that Repressor 2 must be different from Repressor 1; and that in general, Repressor 2 should be derived from a virus other than that encoding Repressor 1.) Repressor 2 acts to repress the expression of Gene 2. In the absence of Repressor 2, Gene 2 causes the expression of a toxin that kills the cell. Thus in the absence of vims-specific Repressor 1, Gene 1 causes the constitutive expression of Repressor 2. Repressor 2 in turn represses Gene 2, so no toxin is expressed.
- virus-specific Repressor 1 in the presence of virus-specific Repressor 1, Gene 1 is repressed, no Repressor 2 is expressed, so Gene 2 now constitutively expresses the toxin, thereby killing the cell.
- a virus-specific regulatory element that does not normally function as an inducer can be made to act, in effect, as an inducer for a gene that expresses a toxin.
- Gene 1 and Gene 2 should preferably be on a single construct, to minimize the possibility that Gene 2 might be found in a cell in the absence of Gene 1, with resulting undesirable lethal effects.
- RNA-dependent RNA polymerase is an enzyme or enzyme complex that can function to make a complementary copy of an RNA sequence from an RNA template. Please refer to the definition of RNA replicases, transcriptases, and polymerases in "Classification and Nomenclature of Viruses," Francki et al. Eds., 1991, page 55, each of which is considered to be an “RNA-dependent RNA polymerase” as that term is used in the specification and Claims.
- a "toxin” is a gene product(s) that causes or leads to the destruction or incapacitation of a cell. This definition is intended to include the induction of indigenous events leading to cell death, such as apoptosis or necrosis.
- a "toxin” may, for example, be a compound that induces conditional lethality, i.e., cell death requires both expression of a conditional toxin gene (for example, thymidine- kinase) and the exogenous administration of a compound (for example, ganciclovir or acyclovir) that together produce a lethal effect.
- a conditional toxin gene for example, thymidine- kinase
- a compound for example, ganciclovir or acyclovir
- Another example is the combination of the gene encoding cytosine deaminase and the prodrug 5-fluorocytosine. It has been suggested that a cell expressing cytosine deaminase will convert 5-fluorocytosine to the cytotoxic compound
- 5-fluorouracil for use in killing tumor cells. See, e.g., J. Harris et al, "Gene therapy for cancer using tumour-specific prodrug activation,” Gene Ther., vol. 1, pp. 170-175 (1994).
- “Negative single-stranded DNA” is a single strand of DNA that cannot be directly transcribed to form messenger RNA capable of translation by ribosomes to synthesize a toxin, but that is complementary to a positive DNA strand that can be transcribed to form messenger RNA, that can in turn be translated by ribosomes to synthesize a toxin.
- a "virus” may be a double-stranded DNA virus, a single-stranded ( + ) or (-) DNA virus, a double-stranded RNA vims, a single-stranded (+) or (-) RNA vims, a retrovirus, a virus containing both RNA and DNA, or a viroid.
- exogenous nucleic acid sequence is a DNA or RNA sequence that is artificially introduced into a cell or organism, and that does not naturally occur in wild type cells or organisms of the same species.
- exogenous is also intended to include copies of such a sequence in the progeny of a cell or the progeny of an organism that is originally transformed with such a sequence.
- a “vector” is a vehicle that can deliver exogenous nucleic acid to a cell.
- a “vector” may or may not be capable of replication.
- a “vector” may include, for example, the free nucleic acid itself.
- An “organism” is a prokaryotic or eukaryotic organism, single-celled or multi-celled, including humans.
- viral promoters useful for regulating the expression of toxins and anti-sense nucleic acid sequences in accordance with the present invention.
- lymphocytic choriomeningitis virus L gene encodes a putative RNA polymerase. Virology 169:377-384.
- liver specific expression of hepatitis B virus is determined by the combined action of the core gene promoter and the enhancer. Journal of Virology 63:919-927.
- Adenovirus containing a deletion of the early region 2A gene allows growth of adeno-associated virus with decreased efficiency.
- adenovims DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated vims promoters. Journal of Virology 64:2103.
- the autonomous parvovirus MVM encodes two nonstructural proteins in addition to its capsid polypeptides.
- Vero cells with adenovirus type 2 mRNA produce authentic viral polypeptide patterns: early mRNA promotes growth of adenovirus-associated virus. Proceedings of the National Academy of Sciences 77:931.
- Adenovirus E1B55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNA's. Journal of Virology 62:206.
- Reovirus protein 83 is a poly ® -dependent poly(G) polymerase. Virology 193:356-366.
- RNA sequence of astrovirus distinctive genomic organization and a putative retro-virus-like ribosomal frameshifting signal that directs the viral replicase synthesis. Proc Natl Acad Sci USA 1993;90: 10539-10543.
- nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. J Virol 1989;63:4653-4664.
- Niesters HGM Strauss JH. Defined mutations in the 5' nontranslated sequence of Sindbis vims RNA. J Virol 1990;64:4162-4168.
- Banerjee AK Barik S. Gene expression of vesicular stomatitis virus genome RNA. Virology 1992; 188:417-428.
- Burkreyev AA Volchkov VE, Blinov VM, Netesov SV.
- the VP35 and VP40 proteins of filoviruses homology between Marburg and Ebola viruses. FEBS Lett 1993;322:41-46.
- Burkreyev AA Volchkov VE, Blinov VM, Netesov SV.
- the GP-protein of Marburg virus contains the region similar to the "immunosuppressive domain" of oncogenic retrovirus P15E proteins. FEBS Lett 1993;323: 183-187.
- Marburg virus a filoviras: homologies with paramyxoviruses and rhabdoviruses. Virology 1992;187:534-547.
- Bunyamwera virus the prototype of the family Bunyaviridae. Virology 1989; 173:426-436.
- Schmaljohn CS Nucleotide sequence of the L genome segment of Hantaan viras. Nucleic Acids Res 1990; 18:6728.
- Schmaljohn CS Jennings GB, Hay J, Dalrymple JM. Coding strategy of the S genome segment of Hantaan virus. Virology 1986;155:633-643.
- Schmaljohn CS Schmaljohn AL
- Dalrymple JM Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order. Virology 1987;157:31-39.
- Equine he ⁇ esvirus type 1 unique short fragment encodes glycoproteins with homology to he ⁇ es simplex viras type 1 gD, gi and gE. J. Gen. Virol. 71 (Pt 12), 2969-2978
- RNA polymerases of poxvirases, prokaryotes, and eukaryotes nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc. Natl. Acad. Sci. U.S.A. 835, 3141-3145
- RNA sequence of astrovirus distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis. Proc. Natl. Acad. Sci. U.S.A. 90 (22), 10539-10543
- Equine arteritis virus is not a togavirus but belongs to the coronaviras- like superfamily. J. Virol. 65 (6), 2910-2920
- BRSV bovine respiratory syncytial virus
- Parvoviridae The viruses and their replication. In Fields Virology
- Retroviridae The viruses and their replication. In Fields Virology.
- HSV as a gene transfer vector for the nervous system. Molecular Biotechnology. 4: 87-99.
- Par-1 a gene required for establishing polarity in C. elegans embryos, encodes a putative ser-thr kinase that is asymmetrically distributed.
- cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringe pv tabaci.
- SEQ. ID NO. 1 is the designed lytic peptide Phor21: KFAKFAKKFAKFAKKFAKFAK
- SEQ. ID NO. 2 is a DNA sequence targeting he ⁇ es viruses.
- the asterisks (***) in the sequence do not denote bases, and do not denote breaks in the continuous nucleic acid sequence. Instead, the asterisks are used to label different portions of the sequence.
- the first "subsequence” i.e. , the portion before the first set of ***
- the second "subsequence” i.e., the portion between the first and second set of ***
- the third “subsequence” is the stop codon TAG
- the fourth "subsequence” is a polyadenylation signal taken from the native Hyalophora cecropia gene for cecropin B.
- SEQ. ID NO. 3 is a (-)RNA sequence targeting the OSU9 rotavirus.
- the asterisks (***) in the sequence do not denote bases, and do not denote breaks in the continuous nucleic acid sequence. Instead, the asterisks are used to label different portions of the sequence.
- the first "subsequence” i.e., the portion before the first set of ***
- the second "subsequence” i.e., the portion between the first and second set of ***
- the third “subsequence” is 26 bp of rotavirus RdRp promoter from rotaviras OSU9, namely, bp 1036-
- SEQ. ID NO. 3 is an RNA sequence. If SEQ. ID NO. 3 were encoded in DNA for transcription as RNA, e.g., as part of a chromosome or a plasmid, the U's in the sequence would be replaced by T's, and the sequence would be flanked by an appropriate promoter (preferably a constitutive promoter) on the 5' end, and by a stop codon on the 3' end.
- an appropriate promoter preferably a constitutive promoter
- SEQ. ID NO. 4 is a (-)DNA sequence targeting canine parvoviras.
- the asterisks (***) in the sequence do not denote bases, and do not denote breaks in the continuous nucleic acid sequence. Instead, the asterisks are used to label different portions of the sequence.
- the first "subsequence” i.e., the portion before the first set of ***
- the second "subsequence” i.e., the portion between the first and second set of ***
- the third "subsequence” is a subunit of the NS1 promoter from accession number M38245, bp 120-274.
- the fourth "subsequence” provides the 3'-OH primer sequence, and is taken from canine parvovirus accession number D26079, bp 4925-5075.
- SEQ. ID NO. 5 is a DNA sequence targeting he ⁇ es viruses. It is identical to SEQ. ID NO. 2, except that the final ttcgaa at the end of the first "subsequence" (i.e., the portion before the first set of ***) is a Hindlll site, and the initial ggtacc at the beginning of the fourth "subsequence" (i.e., the portion following the third set of ***) is a Kpnl site
- SEQ. ID NO. 6 is a DNA sequence designed to be inco ⁇ orated into a cell's genome, where it will cause the constitutive production of mRNA's targeting influenza viruses via Method II.
- the asterisks (***) in the sequence do not denote bases, and do not denote breaks in the continuous nucleic acid sequence. Instead, the asterisks are used to label different portions of the sequence.
- the first "subsequence” i.e., the portion before the first set of ***
- the second "subsequence” is a 6-base BamHl site.
- the third "subsequence” is a ribozyme binding site (recognition sequence) to which the RNA-dependent RNA polymerase will bind. (The ribozyme binding site also contains an in-frame stop codon for the DNA-dependent RNA polymerase.)
- the fourth “subsequence” is a 6-base EcoRI site.
- the fifth “subsequence” encodes the lytic peptide Phor21.
- the sixth “subsequence” is a stop codon for the RNA- dependent RNA polymerase.
- the seventh “subsequence” is a 6-base Bglll site.
- the eighth "subsequence” is a human polymerase I promoter, i.e., a DNA-dependent RNA polymerase.
- the ninth "subsequence” is a 6-base BamHl site.
- the tenth “subsequence” is a pair of bases that allow the restriction enzyme BamHl to function more effectively. (As written here, the fifth "subsequence” appears to be in the positive sense, but its relation to the human polymerase I promoter ensures that it will be transcribed as negative mRNA.)
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