Patent Application
20190085327
First discovered in Caenorhabditis elegans, microRNA (miRNA) have been found in plants and animals including humans. Encoded by genes transcribed from DNA but not translated into protein (non-protein-coding RNA), miRNAs have been found to regulate as much as more than 30% mammalian genes. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and their main function is believed to be modulating gene expression.
With many potential applications of miRNA for therapeutic purposes, however, one major obstacle has been the delivery of miRNA or its precursors into target cells. A new method is needed for safe and predictable administration of miRNAs to animal targets.
The present invention provides bacteria-mediated system for introducing miRNA into a target cell through bacterial infection. In one embodiment, the bacterium contains a miRNA-encoding DNA, and expresses the miRNA in the bacterium through a prokaryotic promoter, or in the target cell using a eukaryotic promoter. In an alternative embodiment, the bacterium contains a DNA that encoding a precursor to the miRNA, and expresses the precursor in the bacterium through a prokaryotic promoter, or in the target cell using a eukaryotic promoter. If the precursor is expressed in the bacterium, it can be processed in the bacterium or in the target cell into the mature miRNA.
In one aspect, the present invention provides a DNA vector that encodes at least a microRNA (miRNA) or a miRNA precursor. The miRNA is capable of modulating, i.e., up-regulating or down-regulating, the expression of at least one eukaryotic, prokaryotic, or viral gene. The miRNA precursor can be a pri-miRNA, a pre-miRNA or a miRNA-duplex. In one embodiment, the vector encodes two miRNAs that have a substantially complementary region—when expressed, the two form a duplex. In one embodiment, the miRNA is a mature or guide miRNA. The vector can further include a prokaryotic or eukaryotic promoter, and can further encode an Hly gene. In one embodiment, the at least one gene targeted by the miRNA is cancer-related.
In one aspect, the present invention provides a bacterium that contains a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding said miRNA or said precursor, said miRNA capable of modulating the expression of at least one eukaryotic, prokaryotic, or viral gene. The bacterium can be a live invasive bacterium or a derivate thereof In one embodiment, the bacterium further contains an enzyme or ribozyme that is capable of processing the miRNA precursor closer to a mature miRNA, such as an endonuclease. In one embodiment, the bacterium further contains at least one of a bacterial RNase III, a Dicer, a Dicer-like enzyme, Drosha and Pasha. The bacterium can further include an enzyme that assists in transporting its genetic materials, upon their release, into the cytoplasm of the target eukaryotic cell. In one embodiment, that enzyme is is listeriolysin O encoded by Hly A gene. The eukaryotic target gene can be an animal, e.g., mammalian or avian gene. In one embodiment, the at least one gene targeted by the miRNA is cancer-related.
In one aspect, the present invention provides a method of delivering a miRNA or a miRNA precursor to an animal cell by infecting the animal cell with the bacterium of the present invention. In an embodiment, the animal cell is a human cell. The method may further include a step of lysing the bacterium after infecting it into the animal cell.
In one aspect, the present invention provides a method of manufacturing a miRNA. In an embodiment, the method includes the step of infecting a bacterium with a prokaryotic vector that encodes at least a miRNA. Alternatively, the method includes the step of infecting a bacterium with a first prokaryotic vector that encodes at least a miRNA precursor, and a second prokaryotic vector that encodes at least one enzyme for processing said miRNA precursor into a miRNA. The method further includes the steps of expressing the miRNA or miRNA precursor and the enzyme, respectively, in the bacterium, and harvesting miRNA from the bacterium.
In one aspect, the present invention provides a method of regulating the expression of at least one target gene in an animal cell. The method includes the steps of: infecting an animal cell with the bacterium of the present invention; and lysing said bacterium to release its content, thereby allowing an miRNA from said content or produced from said content to interact with an mRNA of a target gene, and thereby regulating the expression of said gene. In one feature, the mechanism of regulation is translation repression, mRNA degradation or both.
In one aspect, the present invention provides a method of treating or preventing a disorder in an animal. The method includes regulating the expression of at least one target gene known to be involved in the disorder by infecting the cells of the animal with bacteria comprising a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at least said miRNA or said precursor, said miRNA capable of modulating the expression of at least said gene. The animal can be mammalian or avian.
In one aspect, the present invention provides a method of treating or preventing cancer or a cell proliferation disorder in an animal. The method includes regulating the expression of at least one target gene known to be involved in cell proliferation or in cancer by infecting the cells of the animal with bacteria comprising a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at least said miRNA or said precursor, said miRNA capable of modulating the expression of at least said gene.
In one aspect, the present invention provides a method of treating or preventing a disorder in an animal caused by at least one defective miRNA in the animal. The method includes infecting the cells of the animal with bacteria comprising a functional version of said miRNA, a RNA precursor to said functional miRNA, or a DNA molecule encoding at least said functional miRNA or said precursor.
In one aspect, the present invention provides a method of treating or preventing a disorder in an animal caused by at least one upregulated miRNA in the animal. The method includes infecting the cells of the animal with bacteria comprising an antisense version of said miRNA, a RNA precursor to said antisense version of the miRNA, or a DNA molecule encoding at least said antisense version or said precursor.
In another aspect, the present invention provides a method of discovering or validating a therapeutic target. The method includes the steps of infecting a mammalian cell with the bacterium of the present invention; lysing said bacterium to release its content; and investigating an interaction between a substrate and an miRNA from said content or produced from said content. The substrate may modulate the miRNA activity or the miRNA may modulate the substrate activity.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
As used herein, “modulating” refers to either increasing or decreasing (e.g., silencing), in other words, either upregulating or downregulating. As used herein, “introducing” or “delivering” a microorganism to a target cell, refers to the process of infecting the target cell with the microorganism (e.g., a bacterium), and, in certain cases, releasing the genetic materials inside the microorganism into a desired location of the target cell (e.g., the cytoplasm), possibly through lysing the microorganism.
MicroRNAs (miRNAs) are a class of endogenous, single or double-stranded, about 22 nucleotide-long RNA molecules that regulate as much as 30% of mamalian genes (Czech, NEJM 354:1194-1195 (2006); Mack, Nature Biotech. 25:631-638 (2007); Eulalio, et al, Cell 132:9-14 (2008)). miRNA represses protein production by blocking translation or causing transcript degradation. An miRNA may target 250-500 different mRNAs. miRNA is the product of the Dicer digestion of pre-miRNA, which in turn is the product of primary miRNA (pri-miRNA).
Dicer is a member of the RNase III ribonuclease family. Dicer cleaves long double-stranded RNA (dsRNA) and short hairpin RNA (shRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) about 20-25 nucleotides long, usually with a two-base overhang on the 3′ end. Dicer also cleaves pre-microRNA (miRNA) into miRNA duplex. Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component argonaute is an endonuclease capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.
Referring to
As shown in
Accordingly, in one aspect, the present invention provides a system that delivers miRNA, or miRNA precursor, or a DNA encoding the miRNA or miRNA precursor, or a mixture of any of the above to target cells using bacteria, to effect gene modulation through either translation repression, mRNA degradation or both. The bacteria is preferably non-pathogenic or therapeutic strains with the capability to enter cells. The target cells can be eukaryotic cells. The miRNA of the present invention modulates, e.g., downregulates, genes of interest in target cells. The eukaryotic cells can be mammalian cells or avian cells. The gene of interest can be a mammalian, avian, eukaryotic, bacterial or viral gene. If the molecule delivered is a miRNA precursor, it can be processed into a mature miRNA inside bacteria, or in the target cell using cell's existing processing machinery. In animal cells, enzymes or ribozymes such as Drosha, Pasha, and Dicer, are part of this machinery, which processes the miRNA precursors into a mature form that can guide the multi-enzyme complex, RNA-induced silencing complex (RISC), to the target mRNA.
In one embodiment, the present invention provides a prokaryotic vector that encodes at least a miRNA or a miRNA precursor. The miRNA can modulate the expression of at least one eukaryotic, prokaryotic, or viral gene. The miRNA precursor can be (a) pri-miRNA, (b) pre-miRNA or (c) a miRNA duplex. For the miRNA duplex, the vector, which can be a double-strand circular plasmid, encodes two miRNAs that have a complementary region to form the duplex. The plasmid can have one or two promotors. The promoter can be prokaryotic or eukaryotic depending on where the vector is to be expressed. In one embodiment, at least one prokaryotic promoter, such as T7, is provided on the vector as the vector is intended to be expressed inside a carrier bacterium. In another embodiment, at least one eurkaryotic promoter is provided on the vector as the vector is intended to be expressed inside the target eukaryotic cell.
In one embodiment, one or more DNA molecules encoding the miRNA precursor carried by the bacteria is delivered to the eukaryotic cells, transcribed in the eukaryotic cells and then processed to produce a mature miRNA in the eukaryotic cells. The DNA molecules can be under the control of RNA-polymerase II compatible promoters, or RNA-polymerase III compatible promoters (e.g., U6, H1), which usually direct the transcription of small nuclear RNAs (snRNAs) (P. J. Paddison, A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002), T. R. Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002)). A double “Trojan horse” technique can be used with an invasive and auxotrophic bacterium carrying a eukaryotic transcription plasmid. This plasmid is, in turn, transcribed by the target cell to form a miRNA precursor which is further processed into a mature miRNA that triggers the intracellular process of RNAi.
In one advantageous aspect, the present invention provides a method of delivering miRNAs, miRNA precursors, or DNA encoding the miRNAs or precursors to eukaryotic cells by infecting the cells with bacteria, preferably non-pathogenic or therapeutic strains of bacteria, to effect gene modulation in eukaryotic cells. The bacteria comprise (a) a miRNA, (b) a miRNA precursor, or (c) a DNA encoding the miRNA or the precursor.
In an embodiment, the bacterium itself is capable of synthesizing and processing the miRNA precursor into a mature miRNA. In one embodiment, the bacterium has an enzyme or ribozyme to process or digest the precursors. The enzyme can be an endonuclease. The endonuclease can be a member of RNase III family, such as a bacterial RNase III, a Dicer, or Dicer-like enzyme. The enzyme may be Drosha or Pasha. In one embodiment, the enzyme is endogenous to the carrier bacterium. In another embodiment, the enzyme is exogenous to the carrier bacterium and introduced through vectors that express such enzyme, e.g., a dicer-like enzyme, or a Drosha-like enzyme.
The resulting miRNA can modulate, e.g., knockdown or silence, the expression of one or more target genes, which means it is capable of reducing the expression of the gene by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%.
Bacterial delivery is more attractive than viral delivery as it can be controlled by use of antibiotics and attenuated bacterial strains which are unable to multiply. Also, bacteria are much more accessible to genetic manipulation which allows the production of vector strains specifically tailored to certain applications. In one embodiment of the invention, the methods of the present invention are used to create bacteria that cause gene modulation in a tissue specific manner.
The non-virulent bacteria of the present invention may enter a mammalian host cell through various mechanisms. Professional phagocytes actively engulfing bacteria, invasive bacteria strains have the ability to invade non-phagocytic host cells. Naturally occurring examples of such bacteria are intracellular pathogens such as Listeria, Shigella and Salmonella, but this property can also be transferred to other bacteria such as E. coli and Bifidobacteriae, including probiotics through transfer of invasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, C.R.Acad.Sci.Paris 318, 1207 (1995)). In other embodiments of the invention, bacteria used to deliver small RNAs to host cells include Shigella flexneri (D. R. Sizemore, A. A. Branstrom, J. C. Sadoff, Science 270, 299 (1995)), invasive E. coli (P. Courvalin, S. Goussard, C. Grillot-Courvalin, C.R.Acad.Sci.Paris 318,1207 (1995), C. Grillot-Courvalin, S. Goussard, F. Huetz, D. M. Ojcius, P. Courvalin, Nat Biotechnol 16, 862 (1998)), Yersinia enterocolitica (A. Al-Mariri A, A. Tibor, P. Lestrate, P. Mertens, X. De Bolle, J. J. Letesson Infect Immim 70, 1915 (2002)) and Listeria monocytogenes (M. Hense, E. Domann, S. Krusch, P. Wachholz, K. E. Dittmar, M. Rohde, J. Wehland, T. Chakraborty, S. Weiss, Cell Microbiol 3, 599 (2001), S. Pilgrim, J. Stritzker, C. Schoen, A. Kolb-Maurer, G. Geginat, M. J. Loessner, I. Gentschev, W. Goebel, Gene Therapy 10, 2036 (2003)). Any invasive bacterium is useful for DNA transfer into eukaryotic cells (S. Weiss, T. Chakraborty, Curr Opinion Biotechnol 12, 467 (2001)).
In an embodiment, a prokaryotic vector that encodes at least a microRNA (miRNA) or a miRNA precursor is constructed. The miRNA can modulate the expression of at least one eukaryotic, prokaryotic, or viral gene. Then an invasive bacterium is transformed with the vector. The bacterium can be a live one, or a derivate thereof, e.g., a half-dead bacterium or functional particle derived from the bacterium. In one embodiment, the bacterium is capable of expressing and/or processing the miRNA or its precursor. In an alternative embodiment, the bacterium is not capable of expressing and/or processing the miRNA or its precursor, but only acts as a carrier for further expression and processing to take place in the target eukaryotic cell. The miRNA precursor can be (a) pri-miRNA, (b) pre-miRNA, (c) a miRNA duplex, or a mixture thereof.
At this point, the transformed bacterium contains one or more of the following: a microRNA (miRNA), a miRNA precursor, a DNA molecule encoding said miRNA or said precursor, or a mixture of any of the above.
The content of the bacterium is delivered to the target cell via bacterial invasion (“bactofection”), and is liberated within the mammalian target cell after bacterial lysis triggered either by auxotrophy, endosomes, or by timed addition of antibiotics, resulting in modulation of the target genes.
Liberation of bacterial DNAs and RNAs from the intracellular bacteria may occur through active mechanisms. The bacterial DNAs and RNAs may comprise a mixture of miRNAs, miRNA precursors, and/or their encoding plasmids. One mechanism involves the type III export system in S. typhimurium, a specialized multiprotein complex spanning the bacterial cell membrane whose functions include secretion of virulence factors to the outside of the cell to allow signaling towards the target cell, but which can also be used to deliver antigens into target cells. (Rüssmann H. Int J Med Microbiol, 293:107-12 (2003)) or through bacterial lysis and liberation of bacterial contents into the cytoplasm. The lysis of intracellular bacteria is triggered through addition of an intracellularly active antibiotic (tetracycline) or occurs naturally through bacterial metabolic attenuation (auxotrophy) or through cellular endosome or lysosome. After liberation of the eukaryotic transcription plasmid, miRNAs, miRNA precursors, are produced within the target cell and, in turn, trigger the miRNA-based modulation of the targeted gene(s).
The present invention can be performed using the naturally invasive pathogen Salmonella typhimurium. In one aspect of this embodiment, the strains of Salmonella typhimurium include SL 7207 and VNP20009 (S. K. Hoiseth, B. A. D. Stacker, Nature 291, 238 (1981); Pawelek J M, Low K B, Bermudes D. Cancer Res. 57(20):4537-44 (Oct. 15, 1997)).
In another embodiment of the invention, the present invention is performed using attenuated E. coli. In one example of this embodiment, the strain of E. coli is BM 2710 (C. Grillot-Courvalin, S. Goussard, F. Huetz, D. M. Ojcius, P. Courvalin, Nat Biotechnol 16, 862 (1998)). In one feature of this embodiment, the BM 2710 strain is engineered to possess cell-invading properties through an invasion plasmid, e.g., one that encodes the Inv gene. According to another feature of the present invention, the bacterium of the invention contains a vector that has the HlyA (listeriolysine O) gene, as the Hly protein is considered important for genetic materials escape from the entry vesicles. Obviously, that vector could be the same invasion plasmid. Accordingly, in one embodiment, the bacterium has a plasmid that encodes both the Inv and Hly genes. Accordingly, in one embodiment, the bacterium has a plasmid that encodes both the Inv and Hly genes. In one aspect of the invention, this plasmid is pGB2inv-hly. In one example, the E. coli strain used in the present invention is BL21 (DE3) pLysE.
In an embodiment, a miRNA precursor, e.g., a pre-miRNA, is expressed and processed into miRNA in the bacteria and then delivered into the eukaryotic cells. In another embodiment, a miRNA precursor, e.g., a pre-miRNA, is expressed in the bacteria and then delivered into the eukaryotic cells and processed into miRNA in the eukaryotic cells. In an alternative embodiment, a DNA molecule encoding a miRNA precursor, e.g., a pri-miRNA, is delivered into the eukaryotic cells, and the miRNA precursor is expressed and then processed into miRNA in the eukaryotic cells.
In an embodiment, a miRNA is expressed in the bacteria and then delivered into the eukaryotic cells. In another embodiment, a DNA molecule encoding a miRNA is delivered into the eukaryotic cells, and the miRNA is expressed in the eukaryotic cells.
In an embodiment, the at least one gene is one gene, or more than 2, 4, 8, 16, 32, 64, 100, 200, or 400 genes.
The present invention also provides a method to manufacture microRNA by transforming a bacterium or other host cell with a vector that expresses either the miRNA or a precursor under the control of an appropriate promoter. In the case of bacteria, a prokaryotic promoter, e.g., T7, is provided in the vector. If the vector encodes a miRNA precursor, e.g., a pre-miRNA, vectors that express needed processing enzymes discussed herein above are also transformed into the bacterium.
Wildtype miRNAs serve gene-regulatory functions in cells; some have been associated with various types of human diseases, including cancers. Therefore, miRNAs can be used to study their regulatory target genes and how they themselves are targeted by other molecules, e.g., miRNA inhibitors.
Methods of the invention can be used to study gene function in vitro and in vivo. Vectors of the invention can be used to transfect cultured animal cells as a research tool in drug target/pathway identification and validation. For example, after infecting the host cells with the bacteria of the invention and releasing the bacterial content to provide certain miRNA or precursor that can be processed into miRNA, cells can be observed for phenotypical or morphology changes that suggest some pathway of interest has been affected. Such phenotypical changes can involve numbers of nuclei, nuclei morphology, cell death, cell proliferation, DNA fragmentation, cell surface marker, and mitotic index, etc. In another example, interaction between a molecule/substrate in the host cell and the miRNA transfected into the cell can be isolated or identified to discover potential therapeutic target. That target can be upstream and modulates the miRNA activity, or, the target can be downstream and its activity is modulated by the miRNA.
In terms of in vivo applications, since miRNA can be introduced into a host body by the method of the present invention, a systems biology approach can be adopted in studying the effect of certain miRNA on different cell types and different tissues.
These in vivo and in vitro methods use bacteria with desirable properties (invasiveness, attenuation, steerability). For example, Bifidobacteria and Listeria, are used to perform bacteria-mediated RNAi methods of the present invention. Invasiveness as well as eukaryotic or prokaryotic transcription of one or several miRNAs or miRNA precursors is conferred to a bacterium using plasmids.
The bacteria-mediated miRNA compositions of the present invention can be used for the treatment and or prevention of various diseases, including the diseases summarized in Dykxhoorn, Novina & Sharp. Nat. Rev. Mol. Cell Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184 (2007); de Fougerolles, et al. Nature Rev. Drug Discov. 6:443-453 (2007); Czech, NEJM 354:1194-1195 (2006); and Mack, Nature Biotech. 25:631-638 (2007).
In an embodiment, the present invention can be used as a cancer therapy or to prevent cancer by targeting one or more cancer-related gene. This method is effected by silencing or knockingdown genes involved with cell proliferation or other cancer phenotypes. The bacteria of the present invention used for cancer treatment is preferably bacteria engineered to safely seek out and kill tumors (Forbes, Nature Biotechnology 24:1484-1485 (2006)). The bacteria can be an obligate anaerobe, such as Clostridium novyi—NT, or facultative anaerobe, such as Salmonella typhimurium and Escherichia coli. (ibid.)
Examples of these genes are various oncogenes, such as k-Ras. For example, k-Ras has been shown to be regulated by miRNA Let-7. These oncogenes are active and relevant in the majority of clinical cases. For example, K-Ras is aberrantly active in the majority of human colon cancer, pancreatic cancer, and non small cell lung cancers. K-Ras mutation confers resistance to chemotherapy and current targeted therapy. The bacteria-mediated and miRNA-based gene targeting methods of the present invention can be applied to reach the intestinal tract for colon cancer treatment and prevention. These methods are also used to treat animals carrying xenograft tumors, to treat and prevent cancer in k-RasV12 model of intestinal tumorgenesis, and to prevent and treat tumors in the adenomatous polyposis coli min mouse model (APC-min model).
The methods of the present invention can also be used to create cancer-preventing “probiotic bacteria” for use, especially with the target of GI tract or liver. The methods of the present invention are used as therapy against inflammatory conditions, e.g. inflammatory bowel disease (IBD), including crohn's diseases, ulcerative colitis. These methods are used to silence or knockdown non-cancer gene targets (viral genes, for treatment and prevention of hepatitis B, C; inflammatory genes, for treatment and prevention of inflammatory bowel disease) and others.
The methods of the present invention can be used for delivery of gene silencing to the gut and colon, and for oral application in the treatment of various diseases, namely colon cancer treatment and prevention. In another aspect of this embodiment, delivery of gene silencing is extra-intestinal, such as topical, intravenous.
These bacteria produced and/or delivered miRNA molecules can also be used to silence or knockdown non-cancer gene targets. The RNA molecules of the invention can also be used to treat or prevent ocular diseases, (e.g., age-related macular degeneration (AMD) and diabetic retinopathy (DR)); infectious diseases (e.g. HIV/AIDS, hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), RCV, cytomegalovirus (CMV), dengue fever, west Nile virus); respiratory diseases (e.g., respiratory syncytial virus (RSV), asthma, cystic fibrosis); neurological diseases (e.g., Huntingdon's disease (HD), amyotrophic lateral sclerosis (ALS), spinal cord injury, Parkinson's disease, Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., diabetes); genetic disorders; and inflammatory conditions (e.g., inflammatory bowel disease (IBD), arthritis, rheumatoid disease, autoimmune disorders), dermatological diseases.
Because miRNAs are part of animal cell's regulatory mechanism of many cellular pathways, defective miRNA itself may cause disorders or diseases in animals. Therefore, one therapeutic use of the present invention is simply producing and delivering functional copies or genetically engineered copies of miRNA into an animal to treat or prevent such disorder or disease.
According to the invention, any microorganism which is capable of delivering a molecule, e.g., a miRNA molecule, into the cytoplasm of a target cell, such as by traversing the membrane and entering the cytoplasm of a cell, can be used to deliver miRNA and its precursors to such cells. In a preferred embodiment, the microorganism is a prokaryote. In an even more preferred embodiment, the prokaryote is a bacterium. Also within the scope of the present invention are microorganisms other than bacteria which can be used for delivering RNA to a cell. For example, the microorganism can be a fungus, e.g., Cryptococciis neoformans, protozoan, e.g., Trypanosoma cruzi, Toxoplasma gondii, Leishmania donovani, and plasmodia.
As used herein, the term “invasive” when referring to a microorganism, e.g., a bacterium, refers to a microorganism which is capable of delivering at least one molecule, e.g., an RNA or RNA-encoding DNA molecule, to a target cell. An invasive microorganism can be a microorganism which is capable of traversing a cell membrane, thereby entering the cytoplasm of said cell, and delivering at least some of its content, e.g., RNA or RNA-encoding DNA, into the target cell. The process of delivery of the at least one molecule into the target cell preferably does not significantly modify the invasion apparatus. In a preferred embodiment, the microorganism is a bacterium. A preferred invasive bacterium is a bacterium which is capable of delivering at least one molecule, e.g., an RNA or RNA-encoding DNA molecule, to a target cells, such as by entering the cytoplasm of a eukaryotic cell. Preferred invasive bacteria are live bacteria, e.g., live invasive bacteria. Invasive microorganisms include microorganisms that are naturally capable of delivering at least one molecule to a target cell, such as by traversing the cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms which are not naturally invasive and which have been modified, e.g., genetically modified, to be invasive. In another preferred embodiment, a microorganism which is not naturally invasive can be modified to become invasive by linking the bacterium to an “invasion factor”, also termed “entry factor” or “cytoplasm-targeting factor”. As used herein, an “invasion factor” is a factor, e.g., a protein or a group of proteins which, when expressed by a non-invasive bacterium, render the bacterium invasive. As used herein, an “invasion factor” is encoded by a “cytoplasm-targeting gene”. Naturally invasive microorganisms, e.g., bacteria, may have a certain tropism, i.e., preferred target cells. Alternatively, microorganisms, e.g., bacteria can be modified, e.g., genetically, to mimic the tropism of a second microorganism.
Delivery of at least one molecule into a target cell can be determined according to methods known in the art. For example, the presence of the molecule, by the decrease in expression of an RNA or protein silenced thereby, can be detected by hybridization or PCR methods, or by immunological methods which may include the use of an antibody. Determining whether a microorganism is sufficiently invasive for use in the present invention may include determining whether sufficient RNA (miRNA or miRNA precursors) or its encoding DNA, was delivered to host cells, relative to the number of microorganisms contacted with the host cells. If the amount of RNA, is low relative to the number of microorganisms used, it may be desirable to further modify the microorganism to increase its invasive potential.
Bacterial entry into cells can be measured by various methods. Intracellular bacteria survive treatment by aminoglycoside antibiotics, whereas extracellular bacteria are rapidly killed. A quantitative estimate of bacterial uptake can be achieved by treating cell monolayers with the antibiotic gentamicin to inactivate extracellular bacteria, then by removing said antibiotic before liberating the surviving intracellular organisms with gentle detergent and determining viable counts on standard bacteriological medium. Furthermore, bacterial entry into cells can be directly observed, e.g., by thin-section-transmission electron microscopy of cell layers or by immunofluorescent techniques (Falkow et al. (1992) Annual Rev. Cell Biol. 8:333). Thus, various techniques can be used to determine whether a specific bacteria is capable of invading a specific type of cell or to confirm bacterial invasion following modification of the bacteria, such modification of the tropism of the bacteria to mimic that of a second bacterium. Bacteria that can be used for delivering RNA according to the method of the present invention are preferably non-pathogenic. However, pathogenic bacteria can also be used, so long as their pathogenicity has been attenuated, to thereby render the bacteria non-harmful to a subject to which it is administered. As used herein, the term “attenuated bacterium” refers to a bacterium that has been modified to significantly reduce or eliminate its harmfulness to a subject. A pathogenic bacterium can be attenuated by various methods, set forth below.
Without wanting to be limited to a specific mechanism of action, the bacterium delivering the RNA or DNA into the eukaryotic cell can enter various compartments of the cell, depending on the type of bacterium. For example, the bacterium can be in a vesicle, e.g., a phagocytic vesicle. Once inside the cell, the bacterium can be destroyed or lysed and its contents delivered to the eukaryotic cell. A bacterium can also be engineered to express a phagosome degrading enzyme to allow leakage of RNA from the phagosome. In some embodiments, the bacterium can stay alive for various times in the eukaryotic cell and may continue to produce RNA (miRNA or miRNA precursors). The RNA or RNA-encoding DNA can then be released from the bacterium into the cell by, e.g., leakage. In certain embodiments of the invention, the bacterium can also replicate in the eukaryotic cell. In a preferred embodiment, bacterial replication does not kill the host cell. The present invention is not limited to delivery of RNA or RNA-encoding DNA by a specific mechanism and is intended to encompass methods and compositions permitting delivery of RNA or RNA-encoding DNA by a bacterium independently of the mechanism of delivery.
Set forth below are examples of bacteria which have been described in the literature as being naturally invasive (section 2.1), as well as bacteria which have been described in the literature as being naturally non-invasive bacteria (section 2.2), as well as bacteria which are naturally non-pathogenic or which are attenuated. Although some bacteria have been described as being non-invasive (section 2.2), these may still be sufficiently invasive for use according to the invention. Whether traditionally described as naturally invasive or noninvasive, any bacterial strain can be modified to modulate, in particular to increase, its invasive characteristics (e.g., as described in section 2.3).
The particular naturally invasive bacteria employed in the present invention is not critical thereto. Examples of such naturally-occurring invasive bacteria include, but are not limited to, Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., and enteroinvasive Escherichia coli. The particular Shigella strain employed is not critical to the present invention.
Examples of Shigella strains which can be employed in the present invention include Shigella flexneri 2a (ATCC No. 29903), Shigella sonnet (ATCC No. 29930), and Shigella disenteriae (ATCC No. 13313). An attenuated Shigella strain, such as Shigella flexneri 2a 2457T aroA virG mutant CVD 1203 (Noriega et al. supra), Shigella flexneri M90T icsA mutant (Goldberg et al Infect Immun., 62:5664-5668 (1994)), Shigella flexneri Y SFLl 14 aroD mutant (Karnell et al. Vacc, 10:167-174 (1992)), and Shigella flexneri aroA aroD mutant (Verma et al Vacc, 9:6-9 (1991)) are preferably employed in the present invention. Alternatively, new attenuated Shigella spp. strains can be constructed by introducing an attenuating mutation either singularly or in conjunction with one or more additional attenuating mutations.
At least one advantage to Shigella RNA vaccine vectors is their tropism for lymphoid tissue in the colonic mucosal surface. In addition, the primary site of Shigella replication is believed to be within dendritic cells and macrophages, which are commonly found at the basal lateral surface of M cells in mucosal lymphoid tissues (reviewed by McGhee, J. R. et al Reproduction, Fertility, & Development 6:369 (1994); Pascual, D. W. et al Immunomethods 5:56 (1994)). As such, Shigella vectors may provide a means to express antigens in these professional antigen presenting cells. Another advantage of Shigella vectors is that attenuated Shigella strains deliver nucleic acid reporter genes in vitro and in vivo (Sizemore, D. R. et al. Science 270:299 (1995); Courvalin, P. et al Comptes Rendus de 1 Academie des Sciences Serie Ill-Sciences de Ia Vie-Life Sciences 318:1207 (1995); Powell, R. J. et al In: Molecular approaches to the control of infectious diseases (1996). F. Brown, E. Norrby, D. Burton and J. Mekalanos, eds. Cold Spring Harbor Laboratory Press, New York. 183; Anderson, R. J. et al Abstracts for the 97th General Meeting of the American Society for Microbiology: E. (1997)). On the practical side, the tightly restricted host specificity of Shigella stands to prevent the spread of Shigella vectors into the food chain via intermediate hosts. Furthermore, attenuated strains that are highly attenuated in rodents, primates and volunteers have been developed (Anderson et al (1997) supra; Li, A. et al Vaccine 10:395 (1992); Li, A. et al Vaccine 11:180 (1993); Karnell, A. et al Vaccine 13:88 (1995); Sansonetti, P. J. and J. Arondel Vaccine 7:443 (1989); Fontaine, A. et al. Research in Microbiology 141:907 (1990); Sansonetti, P. J. et al. (1991) Vaccine 9:416; Noriega, F. R. et al. Infection & Immunity 62:5168 (1994); Noriega, F. R. et al. Infection & Immunity 64:3055 (1996); Noriega, F. R. et al. Infection & Immunity 64:23 (1996); Noriega, F. R. et al. Infection & Immunity 64:3055 (1996); Kotloff, K. L. et al. Infection & Immunity 64:4542 (1996)). This latter knowledge will allow the development of well tolerated Shigella vectors for use in humans.
Attenuating mutations can be introduced into bacterial pathogens using non-specific mutagenesis either chemically, using agents such as N-methyl-N′-nitro-N-nitrosoguanidine, or using recombinant DNA techniques; classic genetic techniques, such as Tn10 mutagenesis, P22-mediated transduction, λ phage mediated crossover, and conjugational transfer; or site-directed mutagenesis using recombinant DNA techniques. Recombinant DNA techniques are preferable since strains constructed by recombinant DNA techniques are far more defined. Examples of such attenuating mutations include, but are not limited to: (i) auxotrophic mutations, such as aro (Hoiseth et al. Nature, 291:238-239 (1981)), gua (McFarland et al Microbiol. Path., 3:129-141 (1987)), nad (Park et al. J. Bact, 170:3725-3730 (1988), thy (Nnalue et al. Infect. Immun., 55:955-962 (1987)), and asd (Curtiss, supra) mutations;
(ii) mutations that inactivate global regulatory functions, such as cya (Curtiss et al. Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al (1987), supra), phoP/phoQ (Groisman et al. Proc. Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et al. Proc. Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phopc (Miller et al. J. Bact, 172:2485-2490 (1990)) or ompR (Dorman et al. Infect. Immun., 57:2136-2140 (1989)) mutations;
(iii) mutations that modify the stress response, such as recA (Buchmeier et al. Mol. Micro., 7:933-936 (1993)), htrA (Johnson et al. Mol. Micro., 5:401-407 (1991)), htpR (Neidhardt et al. Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp (Neidhardt et al. Ann. Rev. Genet, 18:295-329 (1984)) and groEL (Buchmeier et al. Sci., 248:730-732 (1990)) mutations;
(iv) mutations in specific virulence factors, such as IsyA (Libby et al. Proc. Natl. Acad. Sci., USA, 91:489-493 (1994)), pag or prg (Miller et al (1990), supra; and Miller et al (1989), supra), iscA or virG (d'Hauteville et al. Mol. Micro., 6:833-841 (1992)), plcA
(Mengaud et al. Mol Microbiol., 5:367-72 (1991); Camilli et al. J. Exp. Med, 173:751-754 (1991)), and act (Brundage et al. Proc. Natl. Acad. Sci., USA, 90:11890-11894 (1993)) mutations; (v) mutations that affect DNA topology, such as top A (Galan et al. Infect. Immun., 58: 1879-1885 (1990));
(vi) mutations that disrupt or modify the cell cycle, such as min (de Boer et al. Cell, 56:641-649 (1989)).
(vii) introduction of a gene encoding a suicide system, such as sacB (Recorbet et al. App. Environ. Micro., 59:1361-1366 (1993); Quandt et al. Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al. App. Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA (Molin et al. Ann. Rev. Microbiol., 47:139-166 (1993));
(viii) mutations that alter the biogenesis of lipopolysaccharide and/or lipid A, such as rFb (Raetz in Esherishia coli and Salmonella typhimurium, Neidhardt et al, Ed., ASM Press, Washington D.C. p 1035-1063 (1996)), galE (Hone et al. J. Infect. Dis., 156:164-167 (1987)) and htrB (Raetz, supra), msbB (Reatz, supra)
(ix) introduction of a bacteriophage lysis system, such as lysogens encoded by P22 (Rennell et al. Virol, 143:280-289 (1985)), λ murein transglycosylase (Bienkowska-Szewczyk et al. Mol. Gen. Genet., 184:111-114 (1981)) or S-gene (Reader et al. Virol, 43:623-628 (1971)); and
The attenuating mutations can be either constitutively expressed or under the control of inducible promoters, such as the temperature sensitive heat shock family of promoters (Neidhardt et al. supra), or the anaerobically induced nirB promoter (Harbome et al. Mol Micro., 6:2805-2813 (1992)) or repressible promoters, such as uapA (Gorfinkiel et al. J. Biol. Chem., 268:23376-23381 (1993)) or gcv (Stauffer et al. J. Bact, 176:6159-6164 (1994)).
The particular Listeria strain employed is not critical to the present invention. Examples of Listeria strains which can be employed in the present invention include Listeria monocytogenes (ATCC No. 15313). Attenuated Listeria strains, such as L. monocytogenes actA mutant (Brundage et al. supra) or L. monocytogenes plcA (Camilli et al. J. Exp. Med., 173:751-754 (1991)) are preferably used in the present invention. Alternatively, new attenuated Listeria strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above. The particular Salmonella strain employed is not critical to the present invention.
Examples of Salmonella strains which can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated Salmonella strains are preferably used in the present invention and include S. typhi-aroC-aroD (Hone et al. Vacc. 9:810 (1991) and S. typhiinurium-axoA mutant (Mastroeni et al. Micro. Pathol. 13:477 (1992)). Alternatively, new attenuated Salmonella strains can be constructed by introducing one or more attenuating mutations as described fro Shigella spp. above.
The particular Rickettsia strain employed is not critical to the present invention. Examples of Rickettsia strains which can be employed in the present invention include Rickettsia Rickettsiae (ATCC Nos. VR149 and VR891), Ricketsia prowaseckii (ATCC No. VR233), Rickettsia tsutsugamuchi (ATCC Nos. VR312, VR1 50 and VR609), Rickettsia mooseri (ATCC No. VR144), Rickettsia sïbirica (ATCC No. VR151), and Rochalimaea quitana (ATCC No. VR358). Attenuated Rickettsia strains are preferably used in the present invention and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular enteroinvasive Escherichia strain employed is not critical to the present invention. Examples of enteroinvasive Escherichia strains which can be employed in the present invention include Escherichia coli strains 4608-58, 1184-68, 53638-C-17, 13-80, and 6-81 (Sansonetti et al. Ann. Microbiol. (Inst. Pasteur), 132A:351-355 (1982)).
Attenuated enteroinvasive Escherichia strains are preferably used in the present invention and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
Furthermore, since certain microorganisms other than bacteria can also interact with integrin molecules (which are receptors for certain invasion factors) for cellular uptake, such microorganisms can also be used for introducing RNA into target cells. For example, viruses, e.g., foot-and-mouth disease virus, echovirus, and adenovirus, and eukaryotic pathogens, e.g., Histoplasma capsulatum and Leishmania major interact with integrin molecules.
Examples of bacteria which can be used in the present invention and which have been described in the literature as being non-invasive or at least less invasive than the bacteria listed in the previous section (2.1) include, but are not limited to, Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp. It may be necessary to modify these bacteria to increase their invasive potential. The particular Yersinia strain employed is not critical to the present invention.
Examples of Yersinia strains which can be employed in the present invention include Y. enterocolitica (ATCC No. 9610) or Y. pestis (ATCC No. 19428). Attenuated Yersinia strains, such as Y. enterocolitica YeO3-R2 (al-Hendy et al. Infect. Immun., 60:870-875 (1992)) or Y. enterocolitica aroA (O'Gaora et al. Micro. Path., 9:105-116 (1990)) are preferably used in the present invention. Alternatively, new attenuated Yersinia strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Escherichia strain employed is not critical to the present invention. Examples of Escherichia strains which can be employed in the present invention include E. coli H10407 (Elinghorst et al Infect. Immun., 60:2409-2417 (1992)), and E. coli EFC4, CFT325 and CPZ005 (Donnenberg et al. J. Infect. Dis., 169:831-838 (1994)). Attenuated Escherichia strains, such as the attenuated turkey pathogen E. coli 02 carAB mutant (Kwaga et al. Infect. Immun., 62:3766-3772 (1994)) are preferably used in the present invention. Alternatively, new attenuated Escherichia strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Klebsiella strain employed is not critical to the present invention.
Examples of Klebsiella strains which can be employed in the present invention include K. pneumoniae (ATCC No. 13884). Attenuated Klebsiella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Bordetella strain employed is not critical to the present invention.
Examples of Bordetella strains which can be employed in the present invention include B. bronchiseptica (ATCC No. 19395). Attenuated Bordetella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Neisseria strain employed is not critical to the present invention. Examples of Neisseria strains which can be employed in the present invention include N. meningitidis (ATCC No. 13077) and N. gonorrhoeae (ATCC No. 19424). Attenuated Neisseria strains, such as N. gonorrhoeae MS11 aro mutant (Chamberlain et al. Micro. Path., 15:51-63 (1993)) are preferably used in the present invention. Alternatively, new attenuated Neisseria strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above. The particular Aeromonas strain employed is not critical to the present invention. Examples of Aeromonas strains which can be employed in the present invention include A. eucrenophila (ATCC No. 23309). Alternatively, new attenuated Aeromonas strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Franciesella strain employed is not critical to the present invention. Examples of Franciesella strains which can be employed in the present invention include F. tularensis (ATCC No. 15482). Attenuated Franciesella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Corynebacterium strain employed is not critical to the present invention. Examples of Corynebacterium strains which can be employed in the present invention include C. pseudotuberculosis (ATCC No. 19410). Attenuated Corynebacterium strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Citrobacter strain employed is not critical to the present invention. Examples of Citrobacter strains which can be employed in the present invention include C. freundii (ATCC No. 8090). Attenuated Citrobacter strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Chlamydia strain employed is not critical to the present invention. Examples of Chlamydia strains which can be employed in the present invention include C. pneumoniae (ATCC No. VR1310). Attenuated Chlamydia strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Hemophilus strain employed is not critical to the present invention. Examples of Hemophilus strains which can be employed in the present invention include H. sornmis (ATCC No. 43625). Attenuated Hemophilus strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Brucella strain employed is not critical to the present invention. Examples of Brucella strains which can be employed in the present invention include B. abortus (ATCC No. 23448). Attenuated Brucella strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Mycobacterium strain employed is not critical to the present invention. Examples of Mycobacterium strains which can be employed in the present invention include M. intracelhilare (ATCC No. 13950) and M. tuberculosis (ATCC No. 27294). Attenuated Mycobacterium strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Legionella strain employed is not critical to the present invention. Examples of Legionella strains which can be employed in the present invention include L. pneumophila (ATCC No. 33156). Attenuated Legionella strains, such as a L. pneumophila mip mutant (Ott, FEMS Micro. Rev., 14:161-176 (1994)) are preferably used in the present invention. Alternatively, new attenuated Legionella strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Rhodococcus strain employed is not critical to the present invention. Examples of Rhodococcus strains which can be employed in the present invention include R. equi (ATCC No. 6939). Attenuated Rhodococcus strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Pseudomonas strain employed is not critical to the present invention. Examples of Pseudomonas strains which can be employed in the present invention include P. aeruginosa (ATCC No. 23267). Attenuated Pseudomonas strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Helicobacter strain employed is not critical to the present invention. Examples of Helicobacter strains which can be employed in the present invention include H. mustelae (ATCC No. 43772). Attenuated Helicobacter strains are preferably used in the present invention, and can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Salmonella stain employed is not critical to the present invention. Examples of Salmonella strains which can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated Salmonella strains are preferably used in the present invention and include S. typhi aroC aroD (Hone et al Vacc, 9:810-816 (1991)) and S. typhimurium aroA mutant (Mastroeni et al. Micro. Pathol, 13:477-491 (1992))). Alternatively, new attenuated Salmonella strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above. The particular Vibrio strain employed is not critical to the present invention.
Examples of Vibrio strains which can be employed in the present invention include Vibrio cholerae (ATCC No. 14035) and Vibrio cincinnatiensis (ATCC No. 35912). Attenuated Vibrio strains are preferably used in the present invention and include V. cholerae RSI virulence mutant (Taylor et al J. Infect. Dis., 170:1518-1523 (1994)) and V. cholerae ctxA, ace, zot, cep mutant (Waldor et al J. Infect. Dis., 170:278-283 (1994)). Alternatively, new attenuated Vibrio strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
The particular Bacillus strain employed is not critical to the present invention. Examples of Bacillus strains which can be employed in the present invention include Bacillus subtilis (ATCC No. 6051). Attenuated Bacillus strains are preferably used in the present invention and include B. anthracis mutant pX01 (Welkos et al Micro. Pathol, 14:381-388 (1993)) and attenuated BCG strains (Stover et al Nat, 351:456-460 (1991)). Alternatively, new attenuated Bacillus strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above. The particular Erysipelothrix strain employed is not critical to the present invention.
Examples of Erysipelothrix strains which can be employed in the present invention include Erysipelothrix rhusiopathiae (ATCC No. 19414) and Erysipelothrix tonsillarum (ATCC No. 43339). Attenuated Erysipelothrix strains are preferably used in the present invention and include E. rhusiopathiae Kg-Ia and Kg-2 (Watarai et al. J. Vet. Med. Sci., 55:595-600 (1993)) and E. rhusiopathiae ORVAC mutant (Markowska-Daniel et al Int. J. Med. Microb. Virol. Parisit. Infect. Dis., 277:547-553 (1992)). Alternatively, new attenuated Erysipelothrix strains can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for Shigella spp. above.
Whether organisms have been traditionally described as invasive or non-invasive, these organisms can be engineered to increase their invasive properties, e.g., by mimicking the invasive properties of Shigella spp., Listeria spp., Rickettsia spp., Salmonella spp, or enteroinvasive E. coli spp. For example, one or more genes that enable the microorganism to access the cytoplasm of a cell, e.g., a cell in the natural host of said non-invasive bacteria, can be introduced into the microorganism.
Examples of such genes referred to herein as “cytoplasm-targeting genes” include genes encoding the proteins that enable invasion by Shigella or the analogous invasion genes of entero-invasive Escherichia, or listeriolysin O of Listeria, as such techniques are known to result in rendering a wide array of invasive bacteria capable of invading and entering the cytoplasm of animal cells (Formal et al. Infect. Immun., 46:465 (1984); Bielecke et al. Nature, 345:175-176 (1990); Small et al. In: Microbiology-1986, pages 121-124, Levine et al. Eds., American Society for Microbiology, Washington, D.C. (1986); Zychlinsky et al. Molec. Micro., 11:619-627 (1994); Gentschev et al (1995) Infection & Immunity 63:4202; Isberg, R. R. and S. Falkow (1985) Nature 317:262; and Isberg, R. R. et al. (1987) Cell 50:769). Methods for transferring the above cytoplasm-targeting genes into a bacterial strain are well known in the art. Another preferred gene which can be introduced into bacteria to increase their invasive character encodes the invasin protein from Yersinia pseudotuberculosis, (Leong et al. EMBO J., 9:1979 (1990)). Invasin can also be introduced in combination with listeriolysin, thereby further increasing the invasive character of the bacteria relative to the introduction of either of these genes. The above genes have been described for illustrative purposes; however, it will be obvious to those skilled in the art that any gene or combination of genes, from one or more sources, that participates in the delivery of a molecule, in particular an RNA or RNA-encoding DNA moleecule, from a microorganism into the cytoplasm of a cell, e.g., an animal cell, will suffice. Thus, such genes are not limited to bacterial genes, and include viral genes, such as influenza virus hemagglutinin HA-2 which promotes endosmolysis (Plank et al. J. Biol. Chem., 269: 12918-12924 (1994)). The above cytoplasm-targeting genes can be obtained by, e.g., PCR amplification from DNA isolated from an invasive bacterium carrying the desired cytoplasm-targeting gene. Primers for PCR can be designed from the nucleotide sequences available in the art, e.g., in the above-listed references and/or in GenBank, which is publicly available on the internet (www.ncbi.nlm.nih.gov/). The PCR primers can be designed to amplify a cytoplasm-targeting gene, a cytoplasm-targeting operon, a cluster of cytoplasm-targeting genes, or a regulon of cytoplasm-targeting genes. The PCR strategy employed will depend on the genetic organization of the cytoplasm-targeting gene or genes in the target invasive bacteria. The PCR primers are designed to contain a sequence that is homologous to DNA sequences at the beginning and end of the target DNA sequence. The cytoplasm-targeting genes can then be introduced into the target bacterial strain, e.g., by using Hfr transfer or plasmid mobilization (Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); Bothwell et al. supra; and Ausubel et al. supra), bacteriophage-mediated transduction (de Boer, supra; Miller, supra; and Ausubel et al. supra), chemical transformation (Bothwell et al. supra; Ausubel et al. supra), electroporation (Bothwel et al. supra; Ausubel et al. supra; and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and physical transformation techniques (Johnston et al. supra; and Bothwell, supra). The cytoplasm-targeting genes can be incorporated into lysogenic bacteriophage (de Boer et al Cell, 56:641-649 (1989)), plasmids vectors (Curtiss et al. supra) or spliced into the chromosome (Hone et al. supra) of the target strain.
In addition to genetically engineering bacteria to increase their invasive properties, as set forth above, bacteria can also be modified by linking an invasion factor to the bacteria. Accordingly, in one embodiment, a bacterium is rendered more invasive by coating the bacterium, either covalently or non-covalently, with an invasion factor, e.g., the protein invasin, invasin derivatives, or a fragment thereof sufficient for invasiveness. In fact, it has been shown that non-invasive bacterial cells coated with purified invasin from Yersinia pseudotuberculosis or the carboxyl-terminal 192 amino acids of invasin are able to enter mammalian cells (Leong et al. EMBO J. 9:1979 (1990)). Furthermore, latex beads coated with the carboxyl terminal region of invasin are efficiently internalized by mammalian cells, as are strains of Staphylococcus aureus coated with antibody-immobilized invasin (reviewed in Isberg and Trail van Nhieu Ann. Rev. Genet. 27:395 (1994)). Alternatively, a bacterium can also be coated with an antibody, variant thereof, or fragment thereof which binds specifically to a surface molecule recognized by a bacterial entry factor. For example, it has been shown that bacteria are internalized if they are coated with a monoclonal antibody directed against an integrin molecule, e.g., α5β1, known to be the surface molecule with which the bacterial invasin protein interacts (Isberg and Tran van Nhieu, supra). Such antibodies can be prepared according to methods known in the art. The antibodies can be tested for efficacy in mediating bacterial invasiveness by, e.g., coating bacteria with the antibody, contacting the bacteria with eukaryotic cells having a surface receptor recognized by the antibody, and monitoring the presence of intracellular bacteria, according to the methods described above. Methods for linking an invasion factor to the surface of a bacterium are known in the art and include cross-linking.
The present invention provides a method for delivering RNA (or RNA-encoding DNA) to any type of target cell, where the RNA is miRNA or miRNA precursor. As used herein, the term “target cell” refers to a cell which can be invaded by a bacterium, i.e., a cell which has the necessary surface receptor for recognition by the bacterium.
Preferred target cells are eukaryotic cells. Even more preferred target cells are animal cells. “Animal cells” are defined as nucleated, non-chloroplast containing cells derived from or present in multicellular organisms whose taxonomic position lies within the kingdom animalia. The cells may be present in the intact animal, a primary cell culture, explant culture or a transformed cell line. The particular tissue source of the cells is not critical to the present invention. The recipient animal cells employed in the present invention are not critical thereto and include cells present in or derived from all organisms within the kingdom animalia, such as those of the families mammalia, pisces, avian, reptilia.
Preferred animal cells are mammalian cells, such as humans, bovine, ovine, porcine, feline, canine, goat, equine, and primate cells. The most preferred animal cells are human cells.
In a preferred embodiment, the target cell is in a mucosal surface. Certain enteric pathogens, e.g., E. coli, Shigella, Listeria, and Salmonella, are naturally adapted for this application, as these organisms possess the ability to attach to and invade host mucosal surfaces (Kreig et al. supra). Therefore, in the present invention, such bacteria can deliver RNA molecules (miRNA or precursors) or RNA-encoding DNA to cells in the host mucosal compartment.
Although certain types of bacteria may have a certain tropism, i.e., preferred target cells, delivery of RNA or RNA-encoding DNA to a certain type of cell can be achieved by choosing a bacterium which has a tropism for the desired cell type or which is modified such as to be able to invade the desired cell type. Thus, e.g., a bacterium could be genetically engineered to mimic mucosal tissue tropism and invasive properties, as discussed above, to thereby allow said bacteria to invade mucosal tissue, and deliver RNA or RNA-encoding DNA to cells in those sites.
Bacteria can also be targeted to other types of cells. For example, bacteria can be targeted to erythrocytes of humans and primates by modifying bacteria to express on their surface either, or both of, the Plasmodium vivax reticulocyte binding proteins-1 and -2, which bind specifically to erythrocytes in humans and primates (Galinski et al. Cell, 69: 1213-1226 (1992)). In another embodiment, bacteria are modified to have on their surface asialoorosomucoid, which is a ligand for the asilogycoprotein receptor on hepatocytes (Wu et al. J. Biol. Chem., 263:14621-14624 (1988)). In yet another embodiment, bacteria are coated with insulin-poly-L-lysine, which has been shown to target plasmid uptake to cells with an insulin receptor (Rosenkranz et al. Expt. Cell Res., 199:323-329 (1992)). Also within the scope of the present invention are bacteria modified to have on their surface p60 of Listeria monocytogenes, which allows for tropism for hepatocytes (Hess et al. Infect. Immun., 63 :2047-2053 (1995)), or a 60 kD surface protein from Trypanosoma cruzi which causes specific binding to the mammalian extra-cellular matrix by binding to heparin, heparin sulfate and collagen (Ortega-Barria et al. Cell, 67:411-421 (1991)).
Yet in another embodiment, a cell can be modified to become a target cell of a bacterium for delivery of RNA. Accordingly, a cell can be modified to express a surface antigen which is recognized by a bacterium for its targeted entry into the cell, i.e., a receptor of an invasion factor. The cell can be modified either by introducing into the cell a nucleic acid encoding a receptor of an invasion factor, such that the surface antigen is expressed in the desired conditions. Alternatively, the cell can be coated with a receptor of an invasion factor. Receptors of invasion factors include proteins belonging to the integrin receptor superfamily. A list of the type of integrin receptors recognized by various bacteria and other microorganisms can be found, e.g., in Isberg and Tran Van Nhieu Ann. Rev. Genet. 27:395 (1994). Nucleotide sequences for the integrin subunits can be found, e.g., in GenBank, publicly available on the interne.
As set forth above, yet other target cells include fish, avian, and reptilian cells. Examples of bacteria which are naturally invasive for fish, avian, and reptilian cells are set forth below.
Examples of bacteria which can naturally access the cytoplasm offish cells include, but are not limited to Aeromonas salminocida (ATCC No. 33658) and Aeromonas schuberii (ATCC No. 43700). Attenuated bacteria are preferably used in the invention, and include A. salmonicidia vapA (Gustafson et al. J. MoI. Biol., 237:452-463 (1994)) or A. salmonicidia aromatic-dependent mutant (Vaughan et al. Infect. Immun., 61:2172-2181 (1993)).
Examples of bacteria which can naturally access the cytoplasm of avian cells include, but are not restricted to, Salmonella galinarum (ATCC No. 9184), Salmonella enteriditis (ATCC No. 4931) and Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are preferred to the present invention and include attenuated Salmonella strains such as S. galinarum cya crp mutant (Curtiss et al. (1987) supra) or S. enteritidis aroA aromatic-dependent mutant CVL30 (Cooper et al. Infect. Immun., 62:4739-4746 (1994)).
Examples of bacteria which can naturally access the cytoplasm of reptilian cells include, but are not restricted to, Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are preferable to the present invention and include, attenuated strains such as S. typhimuirum aromatic-dependent mutant (Hormaeche et al. supra).
The present invention also provides for delivery of miRNA or its precursors to other eukaryotic cells, e.g., plant cells, so long as there are microorganisms which are capable of invading such cells, either naturally or after having been modified to become invasive. Examples of microorganisms which can invade plant cells include Agrobacterium tumerfacium, which uses a pilus-like structure which binds to the plant cell via specific receptors, and then through a process that resembles bacterial conjugation, delivers at least some of its content to the plant cell.
Set forth below are examples of cell lines to which RNA can be delivered according to the method of this invention.
Examples of human cell lines include but are not limited to ATCC Nos. CCL 62, CCL 159, HTB 151, HTB 22, CCL 2, CRL 1634, CRL 8155, HTB 61, and HTB104.
Examples of bovine cell lines include ATCC Nos. CRL 6021, CRL 1733, CRL 6033, CRL 6023, CCL 44 and CRL 1390. Examples of ovine cells lines include ATCC Nos. CRL 6540, CRL 6538, CRL 6548 and CRL 6546.
Examples of porcine cell lines include ATCC Nos. CL 184, CRL 6492, and CRL 1746.
Examples of feline cell lines include CRL 6077, CRL 6113, CRL 6140, CRL 6164, CCL 94, CCL 150, CRL 6075 and CRL 6123.
Examples of buffalo cell lines include CCL 40 and CRL 6072.
Examples of canine cells include ATCC Nos. CRL 6213, CCL 34, CRL 6202, CRL 6225, CRL 6215, CRL 6203 and CRL 6575.
Examples of goat derived cell lines include ATCC No. CCL 73 and ATCC No. CRL 6270.
Examples of horse derived cell lines include ATCC Nos. CCL 57 and CRL 6583.
Examples of deer cell lines include ATCC Nos. CRL 6193-6196.
Examples of primate derived cell lines include those from chimpanzee's such as ATCC Nos. CRL 6312, CRL 6304, and CRL 1868; monkey cell lines such as ATCC Nos. CRL 1576, CCL 26, and CCL 161; orangautan cell line ATCC No. CRL 1850; and gorilla cell line ATCC No. CRL 1854.
In a preferred embodiment of the invention, the invasive bacteria containing the miRNA or its precursor molecules, and/or DNA encoding such, are introduced into an animal by intravenous, intramuscular, intradermal, intraperitoneally, peroral, intranasal, intraocular, intrarectal, intravaginal, intraosseous, oral, immersion and intraurethral inoculation routes.
Bacteria of the present invention can be lyophilized or made into their spore form.
The amount of the live bacteria of the present invention to be administered to a subject will vary depending on the species of the subject, as well as the disease or condition that is being treated. Generally, the dosage employed will be about 103 to 1015 viable organisms, preferably about 104 to 1012 viable organisms per subject.
The invasive bacteria of the present invention are generally administered along with a pharmaceutically acceptable carrier and/or diluent. The particular pharmaceutically acceptable carrier an/or diluent employed is not critical to the present invention. Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone (Levine et al. J. Clin. Invest, 79:888-902 (1987); and Black et al, J. Infect. Dis., 155:1260-1265 (1987)), or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame (Levine et al. Lancet, 11:467-470 (1988)). Examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-30% (w/v) but preferably at a range of 1-10% (w/v).
Set forth below are other pharmaceutically acceptable carriers or diluents which may be used for delivery specific routes. Any such carrier or diluent can be used for administration of the bacteria of the invention, so long as the bacteria are still capable of invading a target cell. In vitro or in vivo tests for invasiveness can be performed to determine appropriate diluents and carriers. The compositions of the present invention can be formulated for a variety of types of administration, including systemic and topical or localized administration. Lyophilized forms are also included, so long as the bacteria are invasive upon contact with a target cell or upon administration to the subject. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the composition, e.g., bacteria, of the present invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the pharmaceutical compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, 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 composition, e.g., bacteria, and a suitable powder base such as lactose or starch.
The pharmaceutical compositions 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. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The pharmaceutical compositions may also be formulated in rectal, intravaginal or intraurethral compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the bacteria of the present invention are formulated into ointments, salves, gels, or creams as generally known in the art, so long as the bacteria are still invasive upon contact with a target cell.
The compositions may, if desired, be presented in a pack or dispenser device and/or a kit which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The invasive bacteria containing the miRNA/precursors or their encoding DNA to be introduced can be used to infect animal cells that are cultured in vitro, such as cells obtained from a subject. These in vitro-infected cells can then be introduced into animals, e.g., the subject from which the cells were obtained initially, intravenously, intramuscularly, intradermally, or intraperitoneally, or by any inoculation route that allows the cells to enter the host tissue. When delivering RNA to individual cells, the dosage of viable organisms to administer will be at a multiplicity of infection ranging from about 0.1 to 106, preferably about 101 to 104 bacteria per cell.
In yet another embodiment of the present invention, bacteria can also deliver RNA molecules encoding proteins to cells, e.g., animal cells, from which the proteins can later be harvested or purified. For example, a protein can be produced in a tissue culture cell.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references including literature references, issued patents, published patent applications as cited throughout this application are hereby expressly incorporated by reference, but they are not admitted to be prior art to presently claimed invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
For single promoter plasmid, the transkingdom miRNA (TMIR) plasmid was constructed as follows (
The Hly A gene was amplified from pGB2Qinv-hly (provided by C. Grillot-Courvalin) by PCR (Pfx DNA polymerase, Invitrogen Inc.) with the following primers:
and were cloned into the EcoRV site of KSII(+). PstI fragments containing the inv locus of pGB2Ωinv-hly were inserted into the PstI site of KSII(+)/Hly. The Hly-Inv fragment was excised with BamHI and SalI. After blunting, it was ligated into the EcoRV site incorporated within the T7 terminator of pT7miRNA. The resulting TMIR plasmid encoding the Let-7a miRNA or one of its precursors was transformed into E. coli (BL21 (DE3) pLysE) cells. Cells were cultured overnight to allow for expression and miRNA processing.
For double-promoter plasmid, the T7-Therapeutic Pathway Identification and Validation (TPIV®) plasmid was constructed to include an RNAi cassette with two T7 promoters (
For vector construction, T7 Terminators were annealed according to standard molecular biology techniques, and digested with either BamHI/Xbal or XbaI/SalI. Terminators were then cloned into the BamHI/XbaI or XbaI/SalI sites of the TPIV® vector using the following primers:
Hela cells were infected with E. coli (BL21 (DE3) pLysE) cells at an MOI of 1000 for 2 hours at 37° C. Twenty-four hours after infection, cellular RNA was harvested with TRIZOL (Invitrogen) and protein extracted in lysis buffer. Amplification of k-Ras mRNA by RT-PCR was conducted using OneStep RT-PCR kit (Invitrogen) and the following primers to amplify a 278 base pair fragment of k-Ras mRNA:
The resulting PCR product was stained and run on an agrose gel and visualized according to standard molecular biology procedures (
Specifically, western blot analysis to determine k-Ras protein levels was performed using 50 ug cell extract separated on a 12% SDS-PAGE gel. k-Ras protein levels were determined by incubation with monoclonal k-Ras antibody (SantaCruz) and visualized by enhanced chemiluminescence (GE Biosciences) (
A fragment of the human Let-7 miRNA (Let-7a) purchased from Integrated DNA Technologies, Inc. was cloned into the TMIR plasmid as described above.
The Let-7a DNA flanked by restriction enzyme sites (BamHI/SalI) has the following sequence (SEQ ID NO:9):
Results of the mRNA assays are shown in
DNA sequence encoding pri-miRNA sequence of the human miR-155 miRNA is cloned into the TMIR plasmid. Bacterial transformation with the plasmid is carried out. In one example, processing of the pri-miRNA to pre-miRNA occurs in the bacteria with the expression of human Drosha either in the same TMIR plasmid as the pri-miRNA sequence or on a separate bacterial expression vector. In another example, pri-miRNA processing occurs in the infected eukaryotic cells without the need for bacterial Drosha expression.
Hela cells are then infected with the transformed bacteria and Bachl mRNA and protein levels are analyzed using the assays described.
The miR-155 pri-miRNA sequence is as follows:
DNA sequences encoding two strands of a human miR-155 miRNA duplex is cloned into the TPIV® plasmid. Bacterial transformation with the plasmid is carried out. Hela cells are then infected with the transformed bacteria and k-Ras mRNA levels are analyzed using the assays described.
The miR-155 miRNA duplex sequences are as follows:
DNA sequence encoding the human miR-155 miRNA sequence is cloned into the TMIR plasmid. Bacterial transformation with the plasmid is carried out. Hela cells are then infected with the transformed bacteria and Bachl mRNA and protein levels are analyzed using the assays described.
The miR-155 miRNA sequence is as follows:
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.
This application claims priority to and the benefit of U.S. provisional patent application Ser. No. 60/947,311 filed Jun. 29, 2007, the content of which applications is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60947311 | Jun 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12667865 | Dec 2010 | US |
| Child | 16045128 | US |
