Patent Application
20140234359
Mammalian viruses have recently been shown to play a critical role in the development of certain types of tumors in animals or humans. At least six families of viruses appear to be involved in tumor development. These include five families of viruses having DNA genomes, which are referred to as DNA tumor viruses and a single family of tumor viruses referred to as retroviruses. Retroviruses have viral particles with RNA genomes and replicate through the synthesis of a DNA provirus in infected cells. Known tumor causing viruses include Hepatitis B virus (HBV, Liver Cancer), Human Papilloma virus (HPV, cervical and other anogenital cancers), Epstein-barr virus (EBV, Burkitt's Lymphoma and Nasopharyngeal carcinoma), Kaposi's sarcoma-associated herpes virus (Kaposi's sarcoma), Human T-cell Lymphotropic virus (adult T-cell leukemia), and Human Immunodeficiency virus (HIV, aids associated cancers).
Although these viruses have each been linked with cancer it is believed that the tumor viruses work through distinct mechanisms. For instance, HBV is believed to cause chronic tissue damage in the liver which drives the continual proliferation of liver cells resulting in a tumor. SV40 and Polyoma virus are believed to produce factors during lytic infection which stimulate host cell gene expression and DNA synthesis. Since most animal cells are non-proliferating they must be stimulated to divide in order to induce the enzymes needed for viral DNA replication. Cell proliferation stimulated in this way can lead to transformation if the viral DNA becomes stably integrated. One common feature of tumor-causing viruses is that these viruses cause changes to the cells by integrating their genetic material within the host cell DNA. DNA viruses can directly insert the DNA into the host DNA. RNA viruses, however, must first transcribe RNA to DNA and then insert the genetic material into the host cell.
Human papilloma virus (HPV) has been implicated in many tumors. HPV infections often persist for extended periods of time and persistent infections with HPVs have been demonstrated to be the primary cause of cervical cancer. The discovery of HPV as an etiologic agent of many human tumors provided the rationale for the development of a vaccine, now sold as either Gardasil® or Cervarix®, both of which have been reported to prevent cervical and potentially other tumors, such as anal cell carcinoma and genital warts. Gardasil®, sold by Merck, is a prophylactic vaccine designed to avoid the development of cervical and other cancers. Gardasil® does not treat existing infections and must be given prior to HPV infection in order to be effective. Gardasil® is typically provided in three 0.5 ml injections over six months. The second injection is two months after the first and the third injection is four months after the second. Gardasil® is composed of recombinant viral like particles (VLPs) assembled from the L1 proteins of HPV. It has been shown that genes encoding the L1 protein in recombinant form are capable of assembling into HPV VLPs when expressed that are morphologically similar to native HPV virions.
A review article on HPV and therapeutic vaccines (Mo et al. Current cancer therapy reviews, 2010, 6, 81-103), notes that HPV, a non-enveloped double-stranded circular DNA virus, may integrate viral DNA into the host genome.
It has been discovered that plant viruses play an important role in the development of human disease. The invention, in some aspects, is directed to novel prophylactic and therapeutic modalities for treating human disease and related products based on the targeting of plant viruses.
In some aspects the invention is directed to a vaccine of an isolated plant viral antigen, wherein the plant viral antigen is immunogenic, and a pharmaceutically acceptable carrier. In some embodiments the plant viral antigen is an immunogenic peptide. Optionally, the vaccine may include an adjuvant.
In other embodiments the plant viral antigen is a nucleic acid comprising at least one gene encoding a plant viral peptide. The vaccine may be a replication defective vector comprising the nucleic acid, which optionally may be an adenoviral vector. In some embodiments the gene is operably linked to a heterologous promoter and transcription terminator.
The plant viral antigen, in some embodiments, is a plant virus selected from the group consisting of Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic-associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; Kennedya yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus; Odontoglossum ringspot virus; Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; Banana bunchy top virus, and Ribgrass mosaic virus.
In other aspects the invention is a method of modulating gastrointestinal plant viral levels in a subject, by administering to the subject an amount of a plant virus vaccine effective to modulate the plant virus levels in the gastrointestinal tract of the subject. In some embodiments the levels of plant virus in the gastrointestinal system of the subject corresponding to the plant virus vaccine are decreased in the gastrointestinal system of the subject relative to the levels that are observed in the absence of the administration of the plant virus vaccine. In other embodiments the levels of plant virus in the gastrointestinal system of the subject are measured in a fecal sample or a blood sample.
Methods involving administering to a subject at risk of having a plant virus associated cancer, a plant virus vaccine in an effective amount to inhibit infection with the plant virus in the subject are provided according to other aspects of the invention. In some embodiments the subject has been exposed to a plant virus.
The invention also relates to a method for treating a subject, wherein the subject has a disease associated with a plant virus, with an anti-viral compound in an effective amount to reduce infection with the plant virus in the subject.
In other aspects of the invention a method is provided. The method comprises determining whether a subject having a virally caused disease has been exposed to a plant virus that causes the disease, and treating the subject with a compound that is a plant defense mechanism against the plant virus in an effective amount to reduce infection of the subject with the plant virus. The disease may optionally be cancer. The method may also include the step of administering a TLR agonist.
In other embodiments the step of determining whether the subject has been exposed to the plant virus involves analyzing a biological sample of the subject for the presence of the plant virus. The biological sample may be, for instance, a fecal or blood sample.
In some embodiments the compound is a naturally occurring substance found in a plant susceptible to the plant virus or is an analog, homolog, or derivative thereof. In other embodiments the compound is a plant defense mechanism against the plant virus selected from the group consisting of flavonoids, anthocyanins, phytoalexins, medicarpin, rishitin, camalexin, capsaisin, glucosinolate, defensins, alpha-amylase, protease inhibitors, lignin and furanocoumarins.
According to yet other aspects, the invention involves a method for silencing plant virus gene expression in a mammal needing relief from the gene expression. The method involves administering to the mammal an inhibitory nucleic acid that targets the genome of an essential plant virus in an effective amount to reduce infection of the mammal with the plant virus.
In some embodiments the inhibitory nucleic acid comprises double stranded nucleic acid of 15 to 30 nucleotides in length. The double stranded nucleic acid may have a first nucleotide sequence that targets the genome of the essential plant virus and a second nucleotide sequence that is a complement of the first nucleotide sequence.
The inhibitory nucleic acid in some embodiments comprises a nucleotide sequence having sufficient complementarity to a target sequence of about 15 to about 30 contiguous nucleotides in an RNA of a virus for the inhibitory nucleic acid to direct cleavage of the RNA via RNA interference. The virus may be selected from the group consisting of Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic-associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; Kennedya yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus; Odontoglossum ringspot virus; Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; and Ribgrass mosaic virus, wherein the target sequence is in a gene essential for infectivity or replication of the virus. In some embodiments the gene essential for infectivity or replication of the virus is selected from a group consisting of plant virus genome-linked protein (VPg), VPg-Pro, the 3′UTR, the 5′ UTR, zinc finger region of the capsid protein, and tRNA like domain.
A vector composition comprising a nucleic acid encoding an inhibitory nucleic acid that targets the genome of an essential plant virus operably linked to a mammalian promoter is provided according to other aspects of the invention.
A method is also provided for performing a physical analytical step on a biological sample of a subject, identifying the presence of plant virus in the biological sample based on the physical analytical step, and determining a course of treatment for the subject based on the presence of the plant virus. In some embodiments the presence of the plant virus is indicative of a predisposition to cancer. In other embodiments the biological sample is a fecal sample. In yet other embodiments the plant virus is tobacco mosaic virus, Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic-associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; a yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus; Odontoglossum ringspot virus; Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; or Ribgrass mosaic virus.
The method may also involve analyzing the status of inflammation in the subject.
The course of treatment in the method may be the administration of a plant virus vaccine.
According to other aspects of the invention, a method for treating a plant virus associated cancer is provided. The method involves administering to a subject having a plant virus associated cancer an inhibitor of plant specific RNA dependent RNA polymerase in an effective amount to treat the cancer.
In some embodiments the inhibitor is an RNA dependent RNA polymerase antagonist. The RNA dependent RNA polymerase antagonist may be an inhibitory peptide, such as an antibody. In other embodiments the RNA dependent RNA polymerase antagonist is an inhibitory nucleic acid such as siRNA, shRNA, or miRNA.
A method for identifying an anti-cancer agent is provided according to other aspects of the invention. The method involves performing a physical analytical step on a plant to determine a plant defense mechanism for preventing infection with a plant virus, identifying an association of the plant virus with a mammalian cancer, and selecting the plant defense mechanism as an anti-cancer agent for the mammalian cancer.
A kit including a set of primers for detecting plant viruses, a reagent for processing the primers to detect plant viruses, and instructions for analyzing a human or animal biological sample to detect the presence of plant viruses using the set of primers and reagent is provided in other aspects of the invention.
A method for determining the presence of a plant virus in a human gut capable of inducing a virally caused disease is provided according to yet another aspect of the invention. The method involves conducting an analytic test for such plant virus in the blood or fecal matter of the human using a set of first reagents for detecting plant viruses, and using a second reagent for processing the first reagents to detect plant viruses. In some embodiments the set of first reagents comprises a set of antibodies against a plurality of said plant viruses.
According to other aspects of the invention, a method for treating HIV is provided. The method involves administering to a subject having or at risk of having HIV a plant viral vaccine in an effective amount to treat or prevent HIV infection in the subject. In some embodiments the plant viral vaccine is banana bunchy virus.
In other aspects, a composition for modulating gastrointestinal plant viral levels in a subject is provided. The composition is formulated in amount sufficient for administering to the subject an amount of a plant virus vaccine effective to modulate the plant virus levels in the gastrointestinal tract of the subject, wherein the plant virus vaccine is optionally a vaccine as described herein.
In other aspects a composition of a plant virus vaccine in an effective amount to inhibit infection with the plant virus in a subject at risk of having a plant virus associated cancer is provided.
A composition comprising an anti-viral compound for use in the treatment of a subject having a disease associated with a plant virus is provided according to other aspects of the invention.
A composition comprising a compound that is a plant defense mechanism against a plant virus for use in the treatment of a subject who has been identified as having a virally caused disease, such as cancer, and has been exposed to the plant virus that causes the disease.
A composition comprising an inhibitory nucleic acid that targets the genome of an essential plant virus for use in silencing plant virus gene expression in a mammal needing relief from the gene expression and in an effective amount to reduce infection of the mammal with the plant virus.
A composition comprising an anti-viral compound for use in the treatment of a subject having a plant virus associated cancer, wherein the anti-viral compound is a compound that interferes with viral synthesis.
This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Each of the above embodiments and aspects may be linked to any other embodiment or aspect. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
A group of researchers recently analyzed the enteric RNA viral community present in healthy humans (Zhang et al. PLOS Biology, January 2006, v. 4, p. 108) and discovered that the majority of the viral sequences present in human fecal samples were similar to plant RNA viruses. Upon further analysis of the viruses taken from these samples, it was discovered that these viruses were active and still capable of infecting plants. Traditionally plant viruses were believed to be harmless in humans. Although plant viruses have long been, and are currently, considered non-pathogenic for animals, our discoveries (that lead to the invention) prompt us to consider that plant viruses may infect animal cells and that they may be causally related to human disease.
It has now been discovered that these active viruses present in many human subjects, which were previously thought to be harmless, play critical roles in the development of disease. A number of diseases, including tumors, in humans and animals are associated with plant virus infection. The ability to prevent plant viral infection and/or to treat plant viral infection has profound implications for the treatment of a wide array of diseases. As such, the invention relates to preventative and therapeutic vaccines which are specific for plant viruses as well as compounds that are effective in reducing or eliminating the activity of plant viruses, in order to treat diseases in which plant viruses play a role. The invention also encompasses diagnostic, prognostic and drug discovery based methods.
Plant viruses are structurally similar to mammalian viruses in many respects. Two families of plant viruses are characterized as single-stranded DNA viruses, both having small circular genome components. A single family of plant viruses is categorized as a reverse-transcribing virus, having a single circular double-stranded DNA structure. The replication of the reverse-transcribing virus is through an RNA intermediate. Several plant viruses and many mycoviruses are characterized as double-stranded RNA viruses. A few plant viruses are negative sense single-stranded RNA. They are characterized as such because some or all of their genes are translated into a protein from an RNA strand complementary to that of the genome. Finally, the majority of plant viruses are positive sense single-stranded RNA. Some viruses use host reverse transcriptase or that from co-infectious agents.
Many of the plant viruses reported to be present in the gut or nasal passages are RNA viruses whose genomes encode RNA dependent RNA polymerase that can bind to “permissive” factors or proteins that make a host, a plant or even a mammalian cell, permissive for plant virus infection. In a recent study, investigators reported that Pepper Mild Mottled Virus (PMMV) can infect mammalian cells and the report suggested for the first time that mammalian cells may be hosts to plant (Colson et al. POLF1, v. 5, April 2010, p. 1).
The data presented in the Examples is the first demonstration of a direct link between a plant virus and a mammalian disease, such as cancer. It was discovered that viral DNA from tobacco mosaic virus is stably incorporated into genomic DNA from human bladder cancer. The development of bladder cancer is strongly linked to exposure to smokeless tobacco. The discovery that tobacco mosaic virus is stably incorporated into genomic DNA from human bladder cancer strongly supports the assertion that the virus creates a susceptibility to the development of cancer, similar to the role played by papilloma virus in cervical cancer. Additionally, human bladder cancer cells treated with a plant anti-viral agent showed significantly less proliferation than control (untreated or methanol treated) cells. The data indicate that plant viruses play a role in cancer such as bladder cancer and that treatment of the viral infection can reduce cellular proliferation and, thus, such compounds are useful therapeutics. Additionally, after the priority date of the instant application Li et al (Biosci. Rep. 32, p. 174, 2012) published a study demonstrating that TMV induces autophagy in HeLa cells, confirming Applicant's work.
Although Applicant is not bound by mechanism of action it is believed that the plant virus contributes to mammalian disease by integrating plant viral DNA into the host genome in an oncogenic manner or transcriptionally silent manner or alternatively by remaining independent of the host DNA by altering the function of the host cells by utilizing a mechanism which is similar to RNA interference and can regulate host gene expression. When the viral DNA is integrated in an oncogenic manner it may be integrated into the chromosome near an oncogene or in another site that would cause it to be expressed in a dysregulated fashion. The dysregulated expression of the viral DNA causes increased expression, leading to the proliferation of the host cell. Plant viral DNA that is incorporated in transcriptionally silent manner may also result in the development of cancer or other disease when the host cell is exposed to a trigger event. Once the plant viral DNA is silently integrated into the genome it may lay dormant for a period of time, and later be reactivated under conditions of stress, such as inflammation or TLR activation. The reactivation in response to conditions of stress can activate new gene transcription from the integrated viral DNA sequences, resulting in cellular proliferation. Thus, TLR agonists can be administered together with the vaccines or other therapeutics of the invention in order to activate viral transcription, to enhance the therapy.
“Plant viruses” as used herein refers to a group of viruses that have been identified as being pathogenic to plants. These viruses rely on the host for replication, as they lack the molecular machinery to replicate without the host. Plant viruses include but are not limited to tobacco mosaic virus, Maize chlorotic mottle virus; Maize rayado fino virus; Oat chlorotic stunt virus; Chayote mosaic tymovirus; Grapevine asteroid mosaic-associated virus; Grapevine fleck virus; Grapevine Red Globe virus; Grapevine rupestris vein feathering virus; Melon necrotic spot virus; Physalis mottle tymovirus; Prunus necrotic ringspot; Nigerian tobacco latent virus; Tobacco mild green mosaic virus; Tobacco necrosis virus; Eggplant mosaic virus; Kennedya yellow mosaic virus; Lycopersicon esculentum TVM viroid; Oat blue dwarf virus; Obuda pepper virus; Olive latent virus 1; Paprika mild mottle virus; PMMV; Tomato mosaic virus; Turnip vein-clearing virus; Carnation mottle virus; Cocksfoot mottle virus; Galinsoga mosaic virus; Johnsongrass chlorotic stripe mosaic virus; Odontoglossum ringspot virus; Ononis yellow mosaic virus; Panicum mosaic virus; Poinsettia mosaic virus; Pothos latent virus; or Ribgrass mosaic virus. An extensive listing of plant viruses, which can be treated or prevented according to the invention, is set forth in Brunt, A. A. et al (eds.) (1996), Plant Viruses Online Descriptions and Lists from the VIDE Database. Version: 20 Aug. 1996 (URL:http://biology.anu.edu.au/Groups/MES/vide/) and Dallwitz (1980) and Dallwitz, Paine and Zurcher (1993). These viruses include all of those listed on Appendix A of U.S. Patent Application Ser. No. 61/537,306, to which the instant application claims priority and which is specifically incorporated by reference and in Brunt, A. A. et al (eds.) (1996), Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20 Aug. 1996 (URL:http://biology.anu.edu.au/Groups/MES/vide/) and Dallwitz (1980) and Dallwitz, Paine and Zurcher (1993). Exemplary plant viruses and the plants they infect are presented below in Table 1.
Zea mays
Zea mays
Avena sativa
Sechium edule
Vitis rupetris
Vitis vinifera
Vitis rupestris
Vitis rupestris
Cucumis melo, C. sativus
Physalis mottle tymovirus
Datura (Jimson weed),
Mandragora (mandrake),
belladonna (deadly nightshade),
Lycium barbarum (Wolfberry),
Physalis philadelphica
Capsicum (paprika, chili pepper),
Solanum (potato, tomato,
petunia (Petunioideae)
Prunus necrotic ringspot
Nicotiana glauca, N. tabacum,
Capsicum annum, Eryngium
aquaticum
Nicotiana tobacum,
Chenopodium quinoa, N.
glutinosa
Nicotiana tabacum,
Chenopodium amaranticolor,
Cucumis sativus, N. clevelandii
Chenopodium amaranticolor, C.
quinoa, Cucumis sativus,
Nicotiana clevelandii, N.
glutinosa, eggplant, and tomato
Kennedya yellow mosaic virus
Kennedya rubicunda,
Desmodium triflorum, D.
scorpiurus, Indigofera australis,
Lycopersicon esculentum TVM
Lycopersicon esculentum
Avena sativa, Hordeum vulgare,
Linum usitatissimum
Nicotiano glutinosa,
Chenopodium amaranticolor, N.
tabacum, and pepper
Oleo europaea
Capsicum annuum, Nicotiana
benthamiana, N. clevelandii
Capsicum frutescens, C. annuum
Lycopersicon esculentum
Dianthaus caryophyllus
Avena sativa, Dactylis glomerata,
Hordeium vulgare, Triticum
aestivum, cocksfoot, and wheat
Galinsoga mosaic virus
Galinsoga parviflora
Sorghum halepense
Odontoglossum ringspot virus
Chenopodium quinoa (L),
Nicotiana tabacum cv. Xanthi-nc
Ononis yellow mosaic virus
Ononis repens
Panicum mosaic virus
Panicum vigatum
Euphorbia pulcherrima, E.
fulgens, Nicotiana benthamiana,
E. cyathophora
Pothos latent virus
Nicotiana clevelandii, N.
benthamiana, N. hispens
Plantago lanceolata
The invention relates to the use of novel vaccines to prevent plant viruses from transforming mammalian host cells into cancerous lesions. Additionally, by following the mechanisms of effective plant host defenses, therapeutic modalities for the plant virus-induced tumors may be derived from an understanding of known plant host-defense mechanisms that have evolved to protect the plant from the plant virus. Further stress conditions such as inflammation or TLR activation that would lead to increase viral replication may be monitored and treated in patients that have been exposed to plant viruses.
The methods are useful for treating disease in a subject. As used herein, a subject is a mammal such as a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments human subjects are preferred. A disease treatable according to the methods of the invention is any disease in which a plant virus plays a role in the development, maintenance or advancement of the disease. Such diseases are referred to as disease associated with a plant virus and include, for instance proliferative disorders, such as cancer, and neurodegenerative diseases. A disease associated with a plant virus is not a disease known to be associated with a mammalian virus, such as, for instance, HIV or HBV infection.
It was discovered according to the invention that Tobacco Mosaic Virus (TMV) is present in human bladder cancer cells. Inhibition of the virus using an anti-viral agent resulted in a reduction in proliferation of the infected cancer cells. As a result TMV is implicated in the development and progression of human bladder cancer. In addition to bladder cancer, several serious cancers are linked to the use of tobacco, including cancers of the lung, esophagus, larynx (voice box), mouth, throat, kidney, bladder, pancreas, stomach, and cervix, as well as acute myeloid leukemia. Even smokeless tobacco, including snuff and chewing tobacco, increase the risks of oral, facial, and bladder cancer. Furthermore, tobacco field workers have a significantly higher incidence of bladder and other cancers. Bladder cancers have very distinct morphological appearances and individual tumors appear as “tree-like” growths along the bladder wall.
The incidence of different types of cancer vary based on geographical areas, as do different plant viruses that infect food ingested by humans. For instance, the incidence of stomach cancer is highest in Asia and South America and the incidence of cervical cancer is highest in Latin America, Africa, India and Australia. Cancers with the highest incidence in the more developed countries such as North America and Europe include breast cancer and prostate cancer. Gastrointestinal cancers are highest in Japan and Southeast Asia. In India, the leading cancer, oral maxillo-facial tumors, are significantly linked to chewing leaves of the Betel plant that is frequently infected with the plant virus, badnavirus. These differences may reflect the impact of lifestyle or foods. Importantly, food groups that are ingested in regional areas include plants that are well documented to be infected with plant viruses. Thus, plant viruses are a significant etiologic factor in the majority of cancers, including but not limited to Tobacco Mosaic Virus with bladder and other tobacco-associated tumors; Rice Virus with stomach and gastro-intestinal tumors; Pepper viruses with other regional stomach tumors, etc. One class of virus, found in food, spice and medicine, that is extensively used by humans is Solanaceae. It is believed that the presence of the Cauliflower mosaic virus is associated with gastrointestinal, colon, and head and neck cancers.
The invention involves in some aspects methods of modulating gastrointestinal plant viral levels in a subject by administering to the subject a plant virus vaccine. The level of plant virus in the gastrointestinal tract of a subject can be determined using a number of known techniques in the art. For instance, Zhang et al 2006, supra, describes methods for determining levels of plant virus in human gastrointestinal tracts. Plant virus levels van be determined in human fecal or blood samples, for instance. Exemplary assays are provided below.
The levels of plant virus in the gastrointestinal system may be compared to a control. For instance, the levels may be compared to standard known levels or ranges of levels for normal or diseased subjects. Alternatively, the levels may be compared in the same or different subjects before and/or after vaccine administration. In other embodiments the levels may be compared to prior levels measured in the same subject to assess changes over time.
Additionally, it has been discovered that a plant virus vaccine and other anti-viral therapeutics described herein can be used to treat a subject at risk of having a plant virus associated cancer. A subject at risk of having a plant virus associated cancer as used herein is a subject who is at risk of coming into contact with a plant virus associated with a disease. The subject could come into contact with the plant virus by being exposed to a plant, by residing in or traveling to a geographical region associated with a particular plant, by being in a particular age group that might be exposed to a plant or any other factor determined to be a risk factor for exposure to a plant associated with a virus. In some embodiments the subject has been exposed to a plant virus.
The plant virus vaccine and other anti-viral therapeutics described herein can also be used to treat a subject having a plant virus associated neurodegenerative disease. A subject having a plant virus associated neurodegenerative disease as used herein is a subject who is at risk of or who has come into contact with a plant virus associated with a neurodegenerative disease. Plant virus associated with a neurodegenerative diseases include for instance amytrophic lateral sclerosis (ALS) and Parkinson's disease. A link between consumption of the plant Cycas micronesica, for example by the people of Guam, and the development of ALS/Parkinsonism Demensia Complex has been established (Shen, W. et al, Ann Neurol, 2010; 68, p. 70-80.) Others have proposed an epidemiologic connection between consumption of castor bean plants, which may be infected with viruses such as Olive latent virus 2, and ALS.
In some aspects the invention is directed to a vaccine that is composed of an isolated plant viral antigen. A plant viral “antigen” or “immunogen” as used herein refers to a non-infectious plant virus or immunogenic portion, fragment or derivative thereof. The antigen may be a nucleic acid antigen and/or a peptide antigen and optionally may include lipids, such as those found in viral lipid envelopes. For instance an antigen or immunogen may comprise a viral like particle (VLP), whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a protein, a glycoprotein, a lipoprotein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof.
The plant viral antigen is immunogenic. The term “immunogenic” as used herein refers to the specific biological immune response to a substance i.e. antigen or immunogen in a host animal. An immunogenic peptide is a viral peptide that elicits an immune response specific for the virus or viruses. Immunogenic peptides of viruses are well known in the art. Exemplary plant viral peptides are shown in Example 5. These peptides include but are not limited to SEQ ID NOs 1-429. The immunogenic peptides in some embodiments are the peptides of Example 5, immunogenic variants or fragments thereof.
In some instances the antigen, and thus the vaccine, is composed of attenuated virus. The virus, may be, for instance, heat killed intact virus.
The TMV peptides presented in Example 5 are those identified by Moudallal et al, A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic, EMBO J. 1985 May; 4(5): 1231-1235. Moudallal et al, identified a number of conformation-dependent epitopes in the viral protein. In their assays Moudallal et al, concluded that “virtually the entire sequence of TMVP possessed antigenic activity.”
The plant viral antigen may also be a nucleic acid of at least one gene encoding a plant viral peptide. Examples of nucleic acids encoding plant viruses and plant virus genes are set forth in Example 6. These nucleic acid sequences include but are not limited to SEQ ID NOS: 430-438, as well as fragments and functional variants thereof.
In order to effect expression of the gene the nucleic acid may be delivered in a vector and/or operably linked to a heterologous promoter and transcription terminator. As used herein, a “vector” may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, and virus genomes.
A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
As used herein, a coding sequence and regulatory sequences are said to be “operably joined” when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. As used herein, “operably joined” and “operably linked” are used interchangeably and should be construed to have the same meaning. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably joined to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Often, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
The vector may be a replication defective vector. These types of vectors include but are not limited to adenoviral vectors.
The antigen in the vaccine may be an antigenic determinant. An “antigenic determinant” or “epitope” as used herein refers to a portion of an antigen that contacts a particular antibody. When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
As used herein, the term “vaccine composition” includes at least one immunogenic antigen or immunogen in a pharmaceutically acceptable carrier useful for inducing an immune response in a host. Vaccine compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration. As used herein, the term “host cell” refers to any mammalian cell, whether located in vitro or in vivo. For example, host cells may be located in a transgenic animal.
The vaccine composition may be formulated with or co-administered with an adjuvant. An “adjuvant” as used herein refers to a substance added to a vaccine to increase a vaccine's immunogenicity by stimulating the humoral and/or cellular immune response and/or functioning as a depo. Known vaccine adjuvants include, but are not limited to, oil and water emulsions, oil-in-water emulsions, water-in-oil emulsions, water-in-oil-in-water emulsions, saponin, aluminum hydroxide, dextran sulfate, carbomer, sodium alginate, (N,N-dioctadecyl-N′,N-bis(2-hydroxyethyl)-propanediamine), paraffin oil, muramyl dipeptide, cationic lipids, DMRIE, DOPE, and TLR ligands such as CpG oligonucleotides.
Before the instant invention, plant viruses were utilized as carriers or drug delivery reagents in vaccines. For instance, the prior art has shown the use of inactivated virus like particles derived from plants as carriers for non-plant based antigens in vaccines. These viral like particles can be loaded with DNA encoding foreign peptides which will produce the antigen of interest or they could be loaded with drugs. Modified plant viruses have also been used as smart bombs to deliver chemical payloads. These modified plant viruses have a viral shell with DNA removed leaving a cargo space of 17 nanometers which can be filled with drugs to deliver to cells. The viral shell may be coated in small proteins called signal peptides, which target the complex to a particular tissue. When administered to a subject the virus presumably travels to the target tissue and injects the payload into the cell. These prior art constructs differ from the plant viral vaccines of the invention in several important ways.
The vaccines of the invention are designed such that the antigen is part of the plant virus. In other words the vaccine includes components which elicit a specific immune response against a plant virus in the host. In addition to the plant viral antigen, the vaccine can include other foreign antigens in some embodiments, as long as it includes an immunogenic plant virus antigen. In some embodiments the vaccine does not include any nucleic acid and/or protein other than the plant viral nucleic acid and/or protein. Thus in some embodiments the plant viral antigen is an immunogenic nucleic acid or peptide of a plant virus, and is not a plant viral particle having a foreign peptide or nucleic acid incorporated therein.
Recombinant immunogenic proteins of plant viruses can be assembled into VLPs for use as vaccines. VLPs can be assembled from naturally expressed or recombinantly produced viral proteins. Disulfide bonds, including inter-capsomeric disulfide bonds have been demonstrated to be important for VLPs stability and possibly assembly. Typically, the recombinant proteins can be produced in many different types of host cells. The host cells are transformed with the appropriate genetic constructs and once the proteins are produced, they may be harvested and purified using any known procedures. It is possible that parts of the VLP can be fused to proteins of interest to help increase the immunogenicity of the vaccine.
The invention also relates to a method for treating a subject, wherein the subject has a disease associated with a plant virus, with an antiviral compound in an effective amount to reduce infection of the subject with the plant virus. An effective amount to reduce infection of the subject with the plant virus refers to an amount of an antiviral compound that increases the resistance of the subject to infection with the virus, in other words, decreases the likelihood that the subject will develop the disease resulting from the virus, as well as reducing the viral levels to treat the disease, maintain the viral levels to prevent the disease from becoming worse, or to slow the progressive infection with the virus compared to in the absence of the therapy.
An anti-viral compound, as used herein is any compound that inhibits or interferes with viral development, infectivity or replication. A number of anti-viral compounds are known in the art. For instance, anti-viral compounds include but are not limited to, compounds which interfere with cell entry, compounds that interfere with viral synthesis, compounds that interfere with transcription and translation and compounds that inhibit viral assembly.
Compounds which interfere with cell entry include, for instance, agents which mimic the virus-associated protein (VAP) and bind to the cellular receptors, such as VAP anti-idiotypic antibodies, natural ligands of the receptor and anti-receptor antibodies and agents which mimic the cellular receptor and bind to the VAP, including anti-VAP antibodies, receptor anti-idiotypic antibodies, extraneous receptor and synthetic receptor mimics.
Compounds that interfere with viral synthesis, include but are not limited to agents that block reverse transcription such as nucleotide or nucleoside analogues and inhibitors of RNA dependent RNA polymerase. Inhibitors of RNA dependent RNA polymerase are particularly interesting plant anti-viral compounds. It has previously been shown that replication of a plant virus and infection of the host cell by the virus resulted from the binding of the plant RNA dependent RNA polymerase to a host factor that allowed infection. Our analysis demonstrates that the plant virus host factor has sequence homology to an analogous factor that may be necessary for lysogenic infection with papilloma viruses. The factor may be associated with release from dead cells or conditions of inflammation in the host.
Compounds that interfere with transcription and translation include, for instance, agents that block transcription factor binding and inhibitory nucleic acids such as antisense and siRNA.
Compounds that inhibit viral assembly include protease inhibitors.
Exemplary anti-viral compounds include but are not limited to Tenofovir
Disoproxil Fumarate, Abacavir, Emtricitabine, Lamivudine, Zidovudine, Atazanavir Sulfate, Nevirapine, Stavudine, Didanosine, Efavirenz, Lopinavir, Zalcitabine, Entecavir, Apricitabine, Adefovir, Nevirapine, Delavirdine, Etravirine, Rilpivirine, portmanteau inhibitors, and Ritonavir.
Another anti-viral compound useful according to the invention is melittin and analogs thereof. Such compounds are described in Marcos et al PNAS v. 92, p. 12466, 1995. Melittin is a 26 amino acid amphipathic peptide.
A recently developed antiviral strategy, also encompassed by anti-viral compounds according to the invention is double-stranded RNA activated caspase oligomerizer (DRACO) methods. DRACO involves the destruction of dsRNA inside infected cells while sending a signal to the cell to begin apoptosis.
A number of these anti-viral compounds are naturally occurring plant viral defense mechanisms. These are chemicals or other mechanisms developed by plants to avoid infection or treat infection by viruses. Naturally occurring plant viral defense mechanisms include but are not limited to chloroquine, Resistance (R) proteins, salicylic acid, jasmonic acid, inhibitory nucleic acids specific for essential plant genes, such as argonaute (e.g., AGO1, AGO2, flavonoids, anthocyanins, phytoalexins, medicarpin, rishitin, camalexin, capsaisin, glucosinolate, defensins, alpha-amylase, protease inhibitors, lignin and furanocoumarins. Medicinal plants have been described previously. For instance, Mukhtar et al (Virus Research, v. 131, p. 111-120 (2008)) which is incorporated by reference is a review article on medicinal plants having anti-viral activities. Such plants fall within the anti-viral compounds of the invention.
Anti-viral compounds of the invention also include inhibitory nucleic acids that target the plant virus. Previous studies have shown that administration of siRNA in animal models is useful for preventing infection. These same mechanisms are useful in treating plant viruses that have infected mammalian cells. Preferably, the virus is selected from any of the viruses listed in Appendix A of U.S. Patent Application Ser. No. 61/537,306 which is incorporated by reference or Table 1. A target nucleic acid is any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.
The inhibitory nucleic acids target nucleic acids that are part of a viral genome and, in particular, nucleic acids comprising essential genes. More specifically, the inhibitory nucleic acid inhibit expression of the target viral sequence. “Essential genes” refer to genes whose expression is required for infection and/or replication functions of the virus. The viral genome may be selected, for example, from the genomes of a virus noted in Appendix A of U.S. Patent Application Ser. No. 61/537,306 and/or Table 1. Essential genes in the genomes of the viruses noted above are known to the skilled artisan. The gene essential for infectivity or replication of the virus may be for instance plant virus genome-linked protein (VPg), VPg-Pro, the 3′UTR, the 5′ UTR, zinc finger region of the capsid protein, or tRNA like domain.
Thus, the invention also features the use of small nucleic acid molecules, referred to as short interfering nucleic acid (siNA) that include, for example: microRNA (miRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules to knockdown expression of viral proteins. An siNA of the invention can be unmodified or chemically-modified. An siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic siNA molecules capable of modulating gene expression or activity in cells by, for instance, RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Furthermore, siNA having multiple chemical modifications may retain its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic applications.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; and Sproat, U.S. Pat. No. 5,334,711; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′ amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al. Science, 1991, 253, 314317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334 339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al.).
In one embodiment, one of the strands of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence identical to the nucleotide sequence or a portion thereof of the targeted RNA. In another embodiment, one of the strands of the double-stranded siRNA molecule comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of a target RNA or a portion thereof, and the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target RNA. In another embodiment, each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure. By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al, WO 97/26270). Other useful RNA derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2′O-alkylated residues or 2′-O-methyl ribosyl derivatives and 2′-O-fluoro ribosyl derivatives. The RNA bases may also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence may be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated. The bases may also be alkylated, for example, 7-methylguanosine can be incorporated in place of a guanosine residue. Non-natural bases that yield successful inhibition can also be incorporated.
For example the siRNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siRNA molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the siRNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siRNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. The siRNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siRNA molecule does not require the presence within the siRNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siRNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
The siNA are composed of nucleotide sequences that are complementary to nucleotide sequences of a target gene. “Complementarity” as used herein refers to the degree to which a nucleic acid can form hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional bonds. The binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Methods for determining binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
“Perfectly complementary” as used herein means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, an siNA molecule of the invention comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.
The siNA molecules modulate gene expression. The term “modulate” as used herein refers to change in the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits such that it is up regulated or down regulated, and such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
Inhibition of gene expression indicates that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siRNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. A therapeutically or prophylactically significant reduction is about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150% or more compared to a control.
A gene is a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
In some embodiments an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a genomically integrated transgene or a plasmid-based expression vector. Thus, in some embodiments a molecule capable of inhibiting mRNA expression, or microRNA activity, is a transgene or plasmid-based expression vector that encodes a small-interfering nucleic acid. Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems. In some embodiments, transgenes and expression vectors are controlled by tissue specific promoters. In other embodiments transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
In another embodiment, a short interfering nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid-specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters. Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter.
Viral-mediated delivery mechanisms to deliver siNAs to cells in vitro and in vivo have been described in Xia, H. et al. (2002) Nat Biotechnol 20(10):1006). Plasmid- or viral-mediated delivery mechanisms of shRNA may also be employed to deliver shRNAs to cells in vitro and in vivo as described in Rubinson, D. A., et al. ((2003) Nat. Genet. 33:401-406) and Stewart, S. A., et al. ((2003) RNA 9:493-501). Other methods of introducing siNA molecules of the present invention to target cells include a variety of art-recognized techniques including, but not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation as well as a number of commercially available transfection kits (e.g., OLIGOFECTAMINE® Reagent from Invitrogen) (see, e.g. Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA 99:5515-5520; Calegari, F. et al. (2002) Proc. Natl. Acad. Sci., USA Oct. 21, 2002; J-M Jacque, K. Triques and M. Stevenson (2002) Nature 418:435-437).
In another embodiment of the invention, the siNA may be transported or conducted across biological membranes using carrier polymers which comprise, for example, contiguous, basic subunits, at a rate higher than the rate of transport of siNA molecules which are not associated with carrier polymers. Combining a carrier polymer with siNA, with or without a cationic transfection agent, results in the association of the carrier polymer and the siNA. The carrier polymer may efficiently deliver the siNA, across biological membranes both in vitro and in vivo. Accordingly, the invention provides methods for delivery of an siNA, across a biological membrane, e.g., a cellular membrane including, for example, a nuclear membrane, using a carrier polymer. The invention also provides compositions comprising an siNA in association with a carrier polymer.
Other inhibitor molecules that can be used include sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins. Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-Ras mutation in bladder carcinoma cells (Feng et al., Cancer Res. 55(10):2024-8, 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9,1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).
Anti-sense oligonucleotides may be designed to hybridize to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of an viral protein encoded by a given DNA sequence (e.g. either native polypeptide or a mutant form thereof), so that its expression is reduce or prevented altogether. Anti-sense techniques may be used to target a coding sequence; a control sequence of a gene, e.g. in the 5′ flanking sequence, whereby the anti-sense oligonucleotides can interfere with control sequences. Anti-sense oligonucleotides may be DNA or RNA and may be of around 14-23 nucleotides, particularly around 15-18 nucleotides, in length. The construction of antisense sequences and their use is described in Peyman and Uhlmann, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, though total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective sense RNA molecules to hybridize. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene.
Triple helix approaches have also been investigated for sequence-specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83, 1997). Minor-groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have led to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
The diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target, for example, a viral protein of interest.
The siNA that targets a viral target may be a single siNA or multiple siNA. Thus, a mixture of siNAs targeting either the same viral gene or at least 2, 3, 4, 5 or up to at least 10 different viral genes may be used. Each of the siNAs, can be screened for potential off-target effects may be analyzed using, for example, expression profiling. Such methods are known to one skilled in the art and are described, for example, in Jackson et al. Nature Biotechnology 6:635-637, 2003. In addition to expression profiling, one may also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which may have off-target effects. One may initially screen the proposed siNAs to avoid potential off-target silencing using the sequence identity analysis by any known sequence comparison methods, such as BLAST. Design of siNAs is known to the skilled artisan, see for example, Dykxhoorn & Lieberman 2006 “Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs” Annu Rev Biomed Eng.
The dose of the siNA will be in an amount necessary to effect RNA interference, e.g., post translational gene silencing, of the particular target gene, thereby leading to inhibition of target gene expression or inhibition of activity or level of the protein encoded by the target gene. Assays to determine expression of the target sequence are known in the art. In one embodiment, a reporter gene, e.g., GFP, may be fused to the target sequence in a test cell, e.g., in a test animal. Effectiveness of silencing can then be measured by examining the reporter gene expression. Target cells which have been transfected with the siNA molecules can be identified by routine techniques such as immunofluorescence, phase contrast microscopy and fluorescence microscopy. In one embodiment, reduced levels of target gene mRNA may be measured by in situ hybridization (Montgomery et al., (1998) Proc. Natl. Acad. Sci., USA 95:15502-15507) or Northern blot analysis (Ngo, et al. (1998)) Proc. Natl. Acad. Sci., USA 95:14687-14692). Preferably, target gene transcription is measured using quantitative real-time PCR (Gibson et al., Genome Research 6:995-1001, 1996; Heid et al., Genome Research 6:986-994, 1996).
As used herein, “inhibition of target gene expression” includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced. The decrease may be of at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an siNA.
The molecules useful herein are isolated molecules. As used herein, the term “isolated” means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material is occurs naturally (e.g., cytoplasmic or membrane component). The isolated molecules may be substantially pure and essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the molecules are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing. Because an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may comprise only a small percentage by weight of the preparation. The peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems. In some embodiments, the peptide is a synthetic peptide.
The term “purified” in reference to a protein or a nucleic acid, refers to the separation of the desired substance from contaminants to a degree sufficient to allow the practitioner to use the purified substance for the desired purpose. Preferably this means at least one order of magnitude of purification is achieved, more preferably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material. In specific embodiments, a purified thymus derived peptide is at least 60%, at least 80%, or at least 90% of total protein or nucleic acid, as the case may be, by weight. In a specific embodiment, a purified thymus derived peptide is purified to homogeneity as assayed by, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis, or agarose gel electrophoresis.
The therapeutic compounds described herein can be administered in combination with other therapeutic agents and such administration may be simultaneous or sequential. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The administration of the other therapeutic agent, including chemotherapeutics and TLR activators/agonists and the compounds of the invention can also be temporally separated, meaning that the therapeutic agents are administered at a different time, either before or after, the administration of the therapeutics described herein. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
Thus, in some instances, the invention also involves administering another cancer treatment (e.g., radiation therapy, chemotherapy or surgery) to a subject. Examples of conventional cancer therapies include treatment of the cancer with agents such as All-trans retinoic acid, Actinomycin D, Adriamycin, anastrozole, Azacitidine, Azathioprine, Alkeran, Ara-C, Arsenic Trioxide (Trisenox), BiCNU Bleomycin, Busulfan, CCNU, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Cytoxan, DTIC, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, 5-fluorouracil, Epirubicin, Epothilone, Etoposide, exemestane, Erlotinib, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Herceptin, Hydrea, Ifosfamide, Irinotecan, Idarubicin, Imatinib, letrozole, Lapatinib, Leustatin, 6-MP, Mithramycin, Mitomycin, Mitoxantrone, Mechlorethamine, megestrol, Mercaptopurine, Methotrexate, Mitoxantrone, Navelbine, Nitrogen Mustard, Oxaliplatin, Paclitaxel, pamidronate disodium, Pemetrexed, Rituxan, 6-TG, Taxol, Topotecan, tamoxifen, taxotere, Teniposide, Tioguanine, toremifene, trimetrexate, trastuzumab, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, Velban, VP-16, and Xeloda.
Other therapeutics for cancer involve antibodies or other binding proteins conjugated to a cytotoxic agents. The conjugates include an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (i.e., a radioconjugate). Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins and fragments thereof which can be used in the conjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
For selective destruction of the cell, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail.
Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
TLR activation causes plant viral gene transcription. Therefore, the compositions of the invention can be combined with a TLR activation therapy, in order to induce viral transcription. TLR activators or agonists include but are not limited to TLR 3, 7, 8, and 9 agonists.
The term “TLR3 agonist” refers to a molecule that interacts with (directly or indirectly) and is capable of activating a TLR3 polypeptide to induce a full or partial receptor-mediated response (i.e. induces TLR3-mediated signaling). A TLR3 agonist, thus, may or may not bind to a TLR3 polypeptide, and may or may not interact directly with the TLR3 polypeptide. TLR3 agonists include for instance, naturally-occurring double-stranded RNA (dsRNA); synthetic ds RNA; and synthetic dsRNA analogs, such as those described in Alexopoulou et al. (2001) Nature 413:732-738. An exemplary, non-limiting example of a synthetic ds RNA analog is poly(I:C).
“TLR7 agonist” and “TLR8 agonists” include single stranded RNA having specific motifs as well as other molecules that interact with (directly or indirectly) and are capable of activating a TLR7 and/or TLR8 polypeptide to induce a full or partial receptor-mediated response (i.e. induces TLR7 and/or 8-mediated signaling).
A “TLR9 agonist” as used herein is a molecule that interacts with (directly or indirectly) and is capable of activating a TLR9 polypeptide to induce a full or partial receptor-mediated response (i.e. induces TLR9-mediated signaling). TLR9 agonists include but are not limited to CpG oligonucleotides.
The therapeutics of the invention may also be combined with CLIP inhibitors. CLIP inhibitors are described extensively in US2011/0118175 and US2010/0166782, each of which are incorporated by reference. CLIP inhibitors include, for instance, but are not limited to FRIMAVLAS (SEQ ID NO. 439).
The invention also involves combinations of the active agents described herein with compounds that make cells more immunogenic, such as autophagy inhibitors and/or a fatty acid metabolism inhibitors. Thus, in some embodiments the invention involves the co-administration of a vaccine or anti-viral therapy of the invention with an autophagy inhibitor and/or a fatty acid metabolism inhibitor. Autophagy inhibitors and fatty acid metabolism inhibitors have been described extensively in U.S. Provisional Application No. 61/511,289 and U.S. patent application Ser. No. 13/054,147 and WO2010/008554 each of which is incorporated by reference.
When used in combination with the therapies of the invention the dosages of known therapies may be reduced in some instances, to avoid side effects.
Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (56th ed., 2002). In some embodiments, the therapeutic compounds of the invention are formulated into a pharmaceutical composition that further comprises one or more additional anticancer agents.
The compounds of the invention are administered in prophylactically or therapeutically effective amounts. A prophylactically or therapeutically effective amount means that amount necessary to attain, at least partly, the desired effect, or to delay the onset of, inhibit the progression of, prevent the reoccurrence of, or halt altogether, the onset or progression of the viral infection and/or the resultant disease being treated, i.e. cancer. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art; however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reason.
The term “preventing” or “reducing” or “inhibiting” as used herein refers to preventing plant viral infection in an individual susceptible for infection or re-infection. Accordingly, administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the infection or the resultant disease, such that the disease or infection is prevented or, alternatively, delayed in its progression. Any mode of administration of the therapeutic agents of the invention, as described herein or as known in the art, including topical administration or mucosal administration of the compounds of the instant invention, may be utilized for the prophylactic treatment of the plant infection or resultant disease.
An effective amount for treating precancerous or cancerous tissue may be an amount sufficient to prevent, delay or inhibit the development of a tumor or slow the growth or reverse the growth of a tumor in the subject compared to the levels in the absence of treatment. According to some aspects of the invention, an effective amount is that amount of a compound of the invention alone or in combination with another medicament, which when combined or co-administered or administered alone, results in a biological affect associated with treating the precancerous or cancerous tissue. Prevention or inhibition as used in this context refers to any reduction or delay in tumor formation as a result of the treatment when compared to an untreated subject.
As defined herein, a therapeutically effective amount of an active compound of the invention (i.e., an effective dosage) ranges from about 0.001 to 3000 mg/kg body weight, preferably about 0.01 to 2500 mg/kg body weight, more preferably about 0.1 to 2000 mg/kg body weight, and even more preferably about 1 to 1000 mg/kg, 2 to 900 mg/kg, 3 to 800 mg/kg, 4 to 700 mg/kg, or 5 to 600 mg/kg body weight. In one embodiment, the average adult is 60 kg and is administered about 0.5 to 50 mg, about 1 to 45 mg, about 2 to 40, about 3 to 35 mg, about 4 to 30 mg, about 5 to 25 mg, about 6 to 20 mg of compound. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an active compound can include a single treatment or, preferably, can include a series of treatments.
Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays, animal studies and human studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
Multiple doses of the molecules of the invention are also contemplated. In some instances, when the molecules of the invention are administered with another therapeutic, for instance, an anti-cancer agent a sub-therapeutic dosage of either or both of the molecules may be used. A “sub-therapeutic dose” as used herein refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent.
Pharmaceutical compositions of the present invention comprise an effective amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. The compounds are generally suitable for administration to humans. This term requires that a compound or composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to humans.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The compounds may be sterile or non-sterile.
The agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). In a particular embodiment, intraperitoneal injection is contemplated.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
The agent may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
The compounds of the invention may be administered directly to a tissue. Direct tissue administration may be achieved by direct injection. The compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
According to the methods of the invention, the compound may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers for the compounds of the invention are well-known to those of ordinary skill in the art. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Exemplary pharmaceutically acceptable carriers for peptides in particular are described in U.S. Pat. No. 5,211,657. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
The compounds of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration. The invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as a syrup, an elixir or an emulsion.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the active agent (see, for example, Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
In yet other embodiments, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary biodegradable implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System”, claiming priority to U.S. patent application serial no. 213,668, filed Mar. 15, 1994). PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject. In accordance with one aspect of the instant invention, the agent described herein may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the agents of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
In general, the agents of the invention may be delivered using the biodegradable implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
Bioadhesive polymers of particular interest include biodegradable hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the platelet reducing agent is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Therapeutic formulations of the compounds of the invention or other therapeutic may be prepared for storage by mixing a compounds of the invention having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
The compounds of the invention may be administered directly to a cell or a subject, such as a human subject alone or with a suitable carrier. Alternatively, a peptide may be delivered to a cell in vitro or in vivo by delivering a nucleic acid that expresses the peptide to a cell. Various techniques may be employed for introducing nucleic acid molecules of the invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like.
The invention also relates to assays for identifying therapeutics and therapeutic courses of treatment. The presence of plant viral DNA in a tumor cell may be assessed, for instance, in order to determine an appropriate therapeutic regimen against the tumor. For example one method involves performing a physical analytical step on a biological sample of a subject, identifying the presence of plant virus in the biological sample based on the physical analytical step, and determining a course of treatment for the subject based on the presence of the plant virus. Another method involves identifying an anti-cancer agent, by performing a physical analytical step on a plant to determine a plant defense mechanism for preventing infection with a plant virus, identifying an association of the plant virus with a mammalian cancer, and selecting the plant defense mechanism as an anti-cancer agent for the mammalian cancer.
The expression of plant viral genes in the tumor cell is determined using methods known to the skilled artisan. The detection methods generally involve contacting a plant viral binding molecule with a sample in or from a subject or in an in vitro cell. Preferably, the sample is first harvested from the subject, although in vivo detection methods are also envisioned. The sample may include any body tissue or fluid that is suspected of harboring the cancer cells. For example, the cancer cells are commonly found in or around a tumor mass for solid tumors. The binding molecules are referred to herein as isolated molecules that selectively bind to plant viral DNA, such as DNA, RNA or antibodies.
In aspects of the invention pertaining to cancers, the subject is a human either suspected of having the cancer, or having been diagnosed with cancer. Methods for identifying subjects suspected of having cancer may include physical examination, subject's family medical history, subject's medical history, biopsy, or a number of imaging technologies such as ultrasonography, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography. Diagnostic methods for cancer and the clinical delineation of cancer diagnoses are well known to those of skill in the medical arts.
As used herein, a tissue sample is tissue obtained from a tissue biopsy, a surgically resected tumor, or any other tumor cell mass removed from the body using methods well known to those of ordinary skill in the related medical arts. The phrase “suspected of being cancerous” as used herein means a cancer tissue sample believed by one of ordinary skill in the medical arts to contain cancerous cells. Methods for obtaining the sample from a biopsy include gross apportioning of mass, microdissection, laser-based microdissection, or other art-known cell-separation methods.
Because of the variability of the cell types in diseased-tissue biopsy material, and the variability in sensitivity of the predictive methods used, the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1000, 5000, 10,000, to 50,000 or more cells. The appropriate sample size may be determined based on the cellular composition and condition of the biopsy and the standard preparative steps for this determination and subsequent isolation of the nucleic acid for use in the invention are well known to one of ordinary skill in the art.
The methods may involve the steps of isolating nucleic acids from the sample and/or an amplification step. Typically, a nucleic acid comprising a sequence of interest can be obtained from a biological sample, more particularly from a sample comprising DNA (e.g. gDNA or cDNA) or RNA (e.g. mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Various commercial kits are available such as the High pure PCR Template Preparation Kit (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., Cold Spring Harbor Laboratory Press 1989, Cold Spring Harbor, N.Y., USA; Ausubel et al., Current Protocols in Molecular Biology 2003, John Wiley & Sons).
When the quantity of the nucleic acid is low or insufficient for the assessment, the nucleic acid of interest may be amplified. Such amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, T7-polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).
Polymerase chain reaction (PCR) technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in “PCR Protocols: A Guide to Methods and Applications”, Innis, M. A., et al. Eds. Academic Press, Inc. San Diego, Calif. (1990) which is incorporated herein by reference. Applications of PCR technology are disclosed in “Polymerase Chain Reaction” Erlich, H. A., et al., Eds. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) which is incorporated herein by reference. U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,075,216, which are each incorporated herein by reference describe methods of performing PCR. PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5′ and 3′ primers that hybridize to sequences present in an RNA or DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the nucleotide sequence between the primers with the free nucleotides to produce complementary strand of DNA.
PCR primers can be designed routinely by those having ordinary skill in the art using sequence information. The mRNA or cDNA is combined with the primers, free nucleotides and enzyme following standard PCR protocols. The mixture undergoes a series of temperature changes. If the test gene transcript or cDNA generated therefrom is present, that is, if both primers hybridize to sequences on the same molecule, the molecule comprising the primers and the intervening complementary sequences will be exponentially amplified. The amplified DNA can be easily detected by a variety of well-known means. If no gene transcript or cDNA generated therefrom is present, no PCR product will be exponentially amplified.
PCR product may be detected by several well-known means. One method for detecting the presence of amplified DNA is to separate the PCR reaction material by gel electrophoresis and stain the gel with ethidium bromide in order to visual the amplified DNA if present. A size standard of the expected size of the amplified DNA is preferably run on the gel as a control.
In some instances, such as when unusually small amounts of RNA are recovered and only small amounts of cDNA are generated therefrom, it is desirable to perform a PCR reaction on the first PCR reaction product. The second PCR can be performed to make multiple copies of DNA sequences of the first amplified DNA. A nested set of primers are used in the second PCR reaction. The nested set of primers hybridize to sequences downstream of the 5′ primer and upstream of the 3′ primer used in the first reaction.
Branched chain oligonucleotide hybridization may be performed as described in U.S. Pat. No. 5,597,909, U.S. Pat. No. 5,437,977 and U.S. Pat. No. 5,430,138, which are each incorporated herein by reference. Northern blot analysis methods are well known by those having ordinary skill in the art and are described in Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Additionally, mRNA extraction, electrophoretic separation of the mRNA, blotting, probe preparation and hybridization are all well-known techniques that can be routinely performed using readily available starting material.
Hybridization methods for nucleic acids are well known to those of ordinary skill in the art (see, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). The nucleic acid molecules hybridize under stringent conditions to nucleic acid markers expressed in cancer cells. The tissue may be obtained from a subject or may be grown in culture.
In the assays of the invention, the presence of the plant virus may be indicative of a predisposition to cancer. As such, the discovery of the presence of a plant virus may lead to the recommendation for a particular therapeutic regimen to avoid development of a disease such as cancer. Additionally it may lead to a further analysis of the status of inflammation in the subject. It is believed that a triggering event such as the induction of inflammation may lead to the activation of a dormant virus and development of cancer.
The invention also includes articles, which refers to any one or collection of components. In some embodiments the articles are kits. The articles include pharmaceutical or diagnostic grade compounds of the invention in one or more containers. The article may include instructions or labels promoting or describing the use of the compounds of the invention. One kit includes a set of primers for detecting plant viruses, a reagent for processing the primers to detect plant viruses, and instructions for analyzing a human or animal biological sample to detect the presence of plant viruses using the set of primers and reagent.
In one embodiment, a kit comprises antibodies against the starvation markers being measured in a method of the invention. The kit may further comprise assay diluents, standards, controls and/or detectable labels. The assay diluents, standards and/or controls may be optimized for a particular sample matrix.
As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment of infections, cancer, and autoimmune disease.
“Instructions” can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
Thus the agents described herein may, in some embodiments, be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents. In certain embodiments agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
The kit may be designed to facilitate use of the methods described herein by physicians and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflects approval by the agency of manufacture, use or sale for human administration.
The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject. The kit may include a container housing agents described herein. The agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
Methods:
Genomic DNA was extracted from T-24 human bladder cells using the Qiagen DNeasy Blood and Tissue Kit (Cat#69504) according to the manufacturer's directions. 1 μg of DNA, 1 μL of 10 μM forward primer (table below), and 1 μL of 10 μM reverse primer (table below), were used with the USB Taq PCR Master Mix Plus Kit according to the manufacturer's directions. Using a BioRad iCycler thermo cycler, 30 cycles of 1 min at 940 C, 1 min 520 C, 1 min at 720 C. Finally one 10 min elongation at 720 C was performed. PCR products were run on a polyacrylamide gel and analyzed on a Licor Odyssey Infrared Imager.
The following primers corresponding to SEQ ID NOs:486-493 were used in the study:
Results: PCR was performed on T24 bladder cancer cell DNA using TMV primers to detect the presence of plant viral DNA. The data is shown in
Methods:
T-24 Efavirenz Culture:
T-24 human bladder cells were grown in a 12 well plate in a total volume of 2 mL of 10% FBS complete RPMI. Cells were left untreated or treated with 2 μL of methanol (Sigma-Aldrich) or treated with 10 μM efavirenz (Toronto Research Chemicals Cat# E425000). Cells were grown in CO2 incubator at 37° C. for 48 hours. After 48 hours, cells were harvested and counted using trypan blue on a hemocytometer.
MitoTracker Red:
Mitochondrial membrane potential was assessed using Mitotracker Red (CM-H2XROS, Invitrogen). The cells were resuspended in warm (37° C. PBS containing a final concentration of 0.5 μM dye. The cells were incubated for 20 minutes, pelleted, and resuspended in PBS for analysis.
Results:
The human bladder cancer T24 cell line was used to determine the effects of and anti-viral treatment on human tumor cells infected with plant virus. The T24 cells were grown in culture and then treated or not with the anti-reverse transcriptase drug, efavirenz, for twenty four or forty eight hours. Cell death assays were performed in triplicate. Efavirenz was effective in killing a percentage of the cells, presumably the subset of the population that are producing viruses or reverse transcribing. It is expected that treatment of the bladder cancer cells with a TLR activator to activate new virus replication in combination with the anti-viral drug will be useful in increasing cell death further.
Methods:
Total RNA was extracted from T-24 human bladder cells and C57B/6 mouse splenocytes using the Qiagen RNeasy Minit Kit (Cat#74104) according to the manufacturer's directions. cDNA was synthesized with the BioRad iScript cDNA Synthesis Kit (1708891) using a BioRad iCycler thermo cycler according to the manufacturer's directions. The following primer sets were used with iTaq SYBER Green Super Mix with ROX (BioRad 172-5850) on an Agilent Technologies Stratagene Mx3005P real time PCR machine.
Primer sets were used according to Zhou, X. et al. Complete nucleotide sequence and genome organization of tobacco mosaic virus isolated from Vicia faba. Sci. China C Life Sci. 2000 Vol. 43 No. 2.
The primers corresponding to SEQ ID NOs:494-507, 233 and 344 are presented below:
Results:
The impact of TLR activation on viral gene transcription in a human bladder cancer cell was examined. The results are shown in
Methods:
Using the software package ClustalX 2.1, the protein sequences from tobacco mosaic virus (TMV), pepper mild mottled virus (PMMV), rice grassy stunt virus (RGSV), cauliflower mosaic virus (CMV), and banana bunchy top virus (BBTV) were aligned with protein sequences of either known anti-apoptotic proteins from other viruses or human proteins associated with cell death pathways. Homologies are indicated by the bar graphs below the sequence information and indicate significant relationships.
Results:
The ClustalX 2.1 alignment of plant virus protein sequences versus known viruses was generated and the results are shown in
The sequence alignments show striking homology between a number of plant viruses and mammalian viruses, suggesting a possible common origin. The high sequence homology provides a guide for selecting the appropriate plant viral vaccine or anti-viral strategy for a particular disease. Interestingly, the significant homology between HIV and Banana bunchy top virus (BBTV), suggests the use of a new plant viral vaccine for the treatment of HIV infection. The BBTV may be used as a prophylactic or therapeutic vaccine for the treatment of HIV infection.
TMV Examplary Peptides:
Relicase 1a
RNA Polymerase
Charged Protein
CaMV Proteins:
Cauliflower mosaic virus peptides obtained from UniPro (with UniPro accession number; http://www.uniprot.org/uniprot):
CaMV Peptides:
PMMV Protein Sequences:
PPMV Peptides
Oat Blue Dwarf Virus Protein Sequence:
Oat Blue Dwarf Virus (OBDV) Peptides
Rice Grassy Stunt Virus Protein Sequence:
Rice Grassy Stunt Virus Peptides
Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/537,306, entitled “PLANT VIRAL VACCINES AND THERAPEUTICS” filed on Sep. 21, 2011, which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/56621 | 9/21/2012 | WO | 00 | 3/20/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61537306 | Sep 2011 | US |
