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The Franconia Custom Fleece Dress Sheet is both a fitted cooler and travel sheet. Constructed of premium quality 380 gram fleece with a pill-resistant finish, this dress sheet wicks moisture to protect your wet horse from chills. Choose body and trim colors to reflect your personalized style. Allow 3-4 weeks for delivery; 5-6 weeks if you add a monogram.
VELCRO® brand tabs attach to the blanket along with VELCRO® brand closures. Nylon lining.
The Amigo® Stable Blanket has a high-tech ripstop outer shell, 200 grams of fiber fill and a coat-smoothing polyester lining. To ensure your horse or pony's comfort, this stable blanket has the little extras that come from being a member of the Horseware® Ireland family: classic cut, low cross surcingles, dual front closures and tail cord.
A quality turnout at a reasonable price. Waterproof/breathable 600 denier polyester lined in soft cotton with nylon shoulders. 300 grams of fill keeps horse comfortable in cold weather.
Keep your horse comfortable and protected with the Mio Medium Turnout Blanket. 600 denier polyester outer shell, 200 grams of fill, and a nylon lining. As with all of the Amigo turnouts, the Mio is waterproof, breathable, and has taped seams. Features Rambo®'s original fit.
The Weatherbeeta Genero Standard Neck Medium Weight Turnout Blanket is both waterproof and breathable. This mid-weight winter horse blanket has a durable 1200 denier ripstop outer shell, 220 grams of poly fill for warmth and a hygienic 210 denier nylon lining.
The Rider's International Bug Free Fly Sheet in tightly woven mesh protects your horse from flying insects. Bugs can't bite through, yet it's still lightweight and flexible. The full neck cover has no opening for bugs to enter. Lycra® at the neck ensures your horse can graze comfortably.
The Amigo® Pony Hero 6 Original Turnout Sheet from Horseware® Ireland keeps your pony dry in its 600 denier waterproof and breathable polyester outer shell. This pony sheet has a smooth polyester lining and features the classic Horseware cut.
The Custom Anti-Sweat Sheet from Franconia International offers the breathability of a classic Irish Knit with the fun of choosing your own fashionable trim color. Quality materials and construction will let this anti-sweat sheet endure years of use. Allow 3-4 weeks for delivery; 5-6 weeks if you add a monogram.
The Weatherbeeta Original 1200D Standard Neck Pony Turnout Sheet has a new, better-fitting design for ponies. It has a durable 1200 denier ripstop polyester outer shell that is paired with a smooth lining. No fill. Traditional gussets allow freedom of movement.
Cleverly designed for your horse's comfort, this Pessoa® Coolwave Combo Fly Sheet has an attached neck cover for complete protection against flies. It's made of ripstop polyester mesh with a nylon lining and innovative stretch action shoulder gusset that allow freedom of movement. A padded stretch panel at the base of the neck cover allows great range of motion.
The Rambo® Helix Sheet from Horseware® Ireland pleases the serious equestrian with its multi-use functionality and great looks! This sheet is a cooler, stable sheet and travel rug in one. Advanced fabric technology efficiently wicks moisture from the horse to prevent chill.
Engineered for quick-drying performance and comfort, the Rambo® Sport Cooler from Horseware® Ireland is designed to prevent overheating even on hot days. The top section of this cooler is made of super wickable, cotton-rich fabric, and the contoured mesh sides encourage great airflow.
This Amigo Fly Sheet is made of soft-knitted polyester that is both lightweight and breathable. Nylon lining along mane and shoulders prevent rubbing while a removable neck cover allows you to give your horse extra protection from pests.
The Rider's International Supreme Pony Turnout Sheet helps shield your pony from the rain, wind, and mud. It features a waterproof and breathable Teflon®-coated 1200 denier ripstop outer shell that is woven in a grid pattern to prevent tearing and makes it a great candidate for years of wear! No fill.
Waterproof and breathable, the Rider's International Plaid Turnout Rug features a Teflon®-coated outer shell and 340 grams of insulation for warmth. This attractive and comfortable horse blanket stays secure with its deep cut, tail flap, buckle-front closures and surcingles.
The Amigo® Pony Hero 6 Original Blanket from Horseware® Ireland protects your pony from winter elements. This waterproof, breathable pony blanket has 200 grams of insulation and a 600 denier polyester outer shell. Classic Horseware cut and Front Leg Arches™.
Engineered for durability, this WeatherBeeta Ripshield Plus Detach-A-Neck Fly Sheet helps stop tears so your fly sheet lasts longer. This fly sheet has a removable belly flap and detachable neck for versatility in coverage depending on conditions.
The WeatheBeeta Bugbeeta Detach-a-Neck Mesh Sheet in soft, fine and durable polyester mesh has a removable neck cover that lets you adjust coverage as needed. This fly sheet has a large tail flap and closes with an adjustable belly wrap and removable leg straps.
The WeatherBeeta Pony Original 1200D Standard Neck Turnout Blanket has an improved design for ponies. This pony turnout has a tough 1200D ripstop polyester outer, a nylon lining, 220 grams of poly fill and a large tail flap for complete protection from the elements.
Ideal for mild weather, the Amigo® Hero 6 Turnout Sheet from Horseware® Ireland is both waterproof and breathable. It features a tough polyester outer shell and smooth polyester lining. Horseware's classic cut keeps it secure.
The Hug Turnout Sheet from Rider's International features the unique Hug closure system that prevents chafing and keeps bad weather out. This turnout sheet has a 600 denier waterproof/breathable outer shell and a nylon/mesh lining.
The Kensington™ Protective UV/Fly Sheet offers your horse protection from harmful UV rays and flies. It also helps keep your horse cleaner and drier year-round. Constructed of Textilene® mesh that is formulated to withstand fading, mildew, soiling and wear and tear in the most extreme weather conditions.
The Saratoga Oasis Summer Dress Sheet is tailor-made for warm weather. In Oasis lightweight mesh fabric, this dress sheet allows air circulation while it protects your horse's coat from insects, dust and sun-fading rays. This summer sheet is treated with 3M® Scotchgard® to resist stains so it always looks great at ringside. Allow 1-2 weeks for delivery. If you add a monogram, allow an additional 1-2 weeks.
Shield your horse from wet conditions with this Shires Stormbreaker 1200D Turnout Sheet. It has a tough ripstop waterproof and breathable outer, a full tail flap, shoulder gussets and fleece at the withers for great comfort.
The Baker® Tartan Plaid Sheet is a time-tested classic in triple woven acrylic. It can be used as a dress sheet, stable sheet or for layering. Stays secure with contoured cut, bias surcingles and dual front straps.
The Rambo® Optimo Stable Sheet from Horseware® Ireland is updated for the season! Constructed with Rambo split technology, this sheet provides your horse with exceptional comfort and allows full freedom of movement. It has a tough 1000D ripstop polyester outer, a coat-smoothing polyester lining and wither padding for added comfort.
The Pessoa Alpine Turnout Sheet comes with a one-year warranty against tearing. This waterproof and protective turnout features a 1200 denier Ballistic Weave™ fabric that is Teflon®-coated to repel dirt and stains. Extras include a removable tail cover, adjustable leg straps, powder-coated hardware and an ID tag.
Exquisite and warm, the Saratoga Custom Wool Riding Blanket is made in America of luxurious 26 oz. Melton wool. This custom exercise rug includes basic web tail cord and trim. Allow 1-2 weeks for delivery; 3-4 weeks if you add a monogram.
Horseware's Amigo® Hero 6 Medium Weight Turnout Blanket is constructed to keep your horse comfortable. This mid-weight blanket is both waterproof and breathable, and features 200 grams of insulation for warmth. Horseware's classic fit keeps it securely and comfortably in place.
Durable, waterproof and breathable, the Rider's International Heavyweight Turnout Blanket is perfect for colder weather. It a 640 denier ripstop outer shell and 340 grams of fill.
This item is the custom order form only. Click on the PDF below to view custom options.
The Rider's International Supreme Stable Blanket is super warm and super thick. The perfect stable blanket. Breathable, ripstop nylon is durable and helps keep your horse dry. 400 grams of insulation ensures your horse stays warm in the harshest weather.
The Pessoa® Alpine Mid Weight Turnout Blanket keeps your horse warm, dry and looking sharp! This medium-weight horse blanket features a tough 1200 denier Ballistic Weave™ outer, 180 grams of insulation and a moisture-wicking lining.
The Rambo® Newmarket Fleece by Horseware® Ireland is a classic and versatile design. Warm, light and breathable, it stays in place with easy to fasten closures.
The Rambo Fleece Rug is perfect for multiple uses in cool weather. It's made in an anti-pill, high-tech, double-bonded fleece with excellent thermal and wicking properties.
The Amigo® Three-In-One Sheet from Horseware® Ireland eliminates worry that your horse isn't properly covered in fluctuating weather conditions. This unique sheet has a contoured waterproof, breathable top section to protect against heavy rain and mesh section airflow to prevent overheating. Overall, this sheet protects against flies and UV damage.
The Amigo® Bravo 12 Turnout Sheet is horse turnout sheet with the Rambo original cut design and Leg Arch™ for freedom of movement. Waterproof and breathable outer shell so your horse stays dry and comfortable.


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Message Subject
Do The Women Stay Home Cooking and Sewing While The Men Take This Country Back?
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This is pathetic. Let me wake you kids up a little bit, because you all seem to have some pretty silly ideas about the apocalypse.
There is no room for gender roles in basic survival.
Gender roles are a luxury of an organized society.
EACH AND EVERY PERSON, be it man or woman, should know everything they need to survive by themselves. That means cooking, cleaning, sewing, shooting, trapping, everything. In a survival situation, you cannot depend on the luxury of a capable partner. You may be separated, one may fall ill, or straight up die.
If you need another half to make a full set, YOU ARE USELESS AS AN INDIVIDUAL.
If you can shoot a rabbit but can't cook it, or if you can stitch wounds but not chop wood, you are a one-legged bastard in an ass-kicking contest.
Each and every one of us will have to be a force unto ourselves. Our long-term survival depends on it.
Any woman can shoot a gun as well as a man, and any man can sew and cook as well as a woman. There is no excuse not to learn. It's not like we'll forget how to do one if we learn to do another.
And as for gender roles? Fuck you with the man-does-hunting woman-does-sewing bullshit. You're a fool if you think an gender-organized regimen is either necessary or advantageous. You're a bigger fool if you think a survival situation will grant you time to plan who does what. You'd both better be equally capable to do whatever needs doing, whenever it needs doing, at a moment's notice.
I consider a woman who can't hunt and a man who can't sew to be equally huge embarrassments.
Anonymous Coward 26339639
bullshit humans are tribal...everyone (not lazy ass) including the old have a place a job to do. it is about survival of us not one or two..
Anonymous Coward 31984457
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The function of a suction fan is to collect the dirt, debris and allergens into the dirt cup or storage bags. If the fan belt is worn out or broken it will not create the suction pressure to collect the dirt and debris. The good news is that the fan belt can be replaced and it can be done at home without having to take it to a repair center.
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•The first and foremost thing that you need to do is unplug the vacuum cleaner from the outlet and remove the cover of the vacuum. Also remove the bag from inside.
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•Rotating the bristles clockwise and counterclockwise will loosen the dirt. Wipe the inside. Using a tissue paper or a clean rag, wipe the inside edges of the compartment and the sides of the plate.
•If the fan belt has to be replaced, remove it from its hub, clean the area and then put the new one.
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The suction fans and belts can be bought from GoVacuum.com. Check out for replacements of your brand. GoVacuum.com is one portal that has been recommended by thousands of users for the cleaning solutions.
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Woodbine MD
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36534-012: Developing an Integrated Cadastre System for Land Resources Management and Property Right Registration (formerly Development of Cadastral Survey and Land Management in Urban Areas - State Committee on Land Resources)
Project Data Sheet (PDS): Overview
The purpose of the TA is to (i) strengthen the agencies involved in land resources management through cadastre surveys; (ii) work out and introduce optimal land management systems at provincial level, and a centralized land management system at national level. It is expected the following components would be included (i) cadastre surveys of land resources through aerospace photography, topographic, geodesic, agrochemical, geobotanic studies and mapping; (ii) automation of field works through ... Read More
|Project Number
|Type or Modality of Assistance
Technical Assistance
|Public sector (Sovereign)
|Approval Number
TA No. 4218
|Approval Date
|19 Nov 2003
|Geographical Location
Agriculture, natural resources and rural development
|Land-Based Natural Resources Management
Project Data Sheets (PDS) contain summary information on the project or program. Because the PDS is a work in progress, some information may not be included in its initial version but will be added as it becomes available. Information about proposed projects is tentative and indicative.
The Public Communications Policy (PCP) recognizes that transparency and accountability are essential to development effectiveness. It establishes the disclosure requirements for documents and information ADB produces or requires to be produced.
The Accountability Mechanism provides a forum where people adversely affected by ADB-assisted projects can voice and seek solutions to their problems and report alleged noncompliance of ADB's operational policies and procedures.
In preparing any country program or strategy, financing any project, or by making any designation of, or reference to, a particular territory or geographic area in this document, the Asian Development Bank does not intend to make any judgments as to the legal or other status of any territory or area.
View the latest procurement notices for Goods, Works and Related Services, or Consulting Services.


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The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer.
1. An isolated antisense oligonucleotide that hybridizes to a target DNA having the nucleotide sequence of SEQ. ID NO:1 at 5� C. to 25� C. below Tm in aqueous solution at 1 M NaCl;
wherein Tm is the melting temperature of a complementary oligonucleotide hybridized to the target DNA in aqueous solution at 1 M NaCl, wherein the complementary oligonucleotide is exactly complementary to SEQ. ID NO:1 and the same length as the antisense oligonucleotide; and
wherein hybridization of the antisense oligonucleotide to an mRNA encoding hTRT (SEQ. ID NO:1.)inhibits expression of the mRNA.
2. The oligonucleotide of
3. The oligonucleotide of
4. The oligonucleotide of
5. The oligonucleotide of
6. The oligonucleotide of
7. The oligonucleotide of
8. The oligonucleotide of
9. The oligonucleotide of
10. The oligonucleotide of
11. The oligonucleotide of
12. The oligonucleotide of
13. The oligonucleotide of
14. The oligonucleotide of
The present application is a continuation-in-part of U.S. patent application Ser. No. 08/974,549, filed Nov. 19, 1997 now U.S. Pat. No. 6,166,178; and a continuation-in-part of U.S. patent application Ser. No. 08/974,584, filed Nov. 19, 1997, both of which are continuation-in-part applications of U.S. patent application Ser. No. 08/915,503 filed Aug. 14, 1997, now abandoned, U.S. patent application Ser. No. 08/912,951 filed Aug. 14, 1997, and U.S patent application Ser. No. 08/911,312, all filed Aug. 14, 1997 now abandoned, all three of which are continuation-in-part applications of U.S., patent application Ser. No. 08/854,050, filed May 9, 1997 now U.S. Pat. No. 6,261,836, which is a continuation-in-part application of U.S. patent application Ser. No. 08/851,843, filed May 6, 1997 now U.S. Pat. No. 6,093,809, which is a continuation-in-part application of U.S. patent application Ser. No. 08/846,017, filed Apr. 25, 1997 now abandoned, which is a continuation-in-part application of U.S. patent application Ser. No. 08/844,419, filed Apr. 18, 1997 now abandoned, which is a continuation-in-part application, U.S. patent application Ser. No. 08/724,643, filed Oct. 1, 1996 now abandoned. This application is also a continuation-in-part of Patent Convention Treaty Patent Application Serial No.: PCT/US97/17885 and to Patent Convention Treaty Patent Application Serial No.: PCT/US97/17618, both filed on Oct. 1, 1997. Each of the aforementioned applications is explicitly incorporated herein by reference in its entirety and for all purposes.
The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer, with TRT antisense oligonucleotides.
The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section should not be construed as an admission of prior invention.
It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972, Nature New Biol., 239:197; Olovnikov, 1973, J. Theor. Biol., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5′ to 3′. When the RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence or aging of normal human somatic cells in vitro and in vivo. The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity), or the ability of a cell to escape senescence, i.e., to become immortal. The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomeric DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750).
Consistent with the relationship of telomeres and telomerase to the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBO J. 11:1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416). Human telomerase is thus an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer.
The present invention provides TRT antisense polynucleotides, which are useful for detecting, diagnosing, and treating telomerase-related conditions.
In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence that is substantially complementary or substantially identical to a contiguous sequence of an hTRT nucleic acid that has the nucleotide sequence of FIG. 1.
In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence exactly complementary or identical to a contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 2.
In one embodiment, the hTRT polynucleotide comprises a sequence that is exactly complementary or identical to a contiguous sequence of an hTRT nucleic acid having the nucleotide sequence of FIG. 1.
In one embodiment, the polynucleotide is a DNA or an RNA. In one embodiment, the polynucleotide comprises one or more non-naturally occurring, synthetic nucleotides.
In one embodiment, the polynucleotide is identical to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1. In one embodiment, the polynucleotide is exactly complementary to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1.
In one embodiment, the polynucleotide is an antisense polynucleotide. In one embodiment, the polynucleotide is at least about 20 nucleotides in length to at least about 50 nucleotides in length.
In one embodiment, the polynucleotide inhibits telomerase activity by at least about 50% in transformed cells ex vivo, as compared to control cells that are not treated with the polynucleotide. In one embodiment, the polynucleotide inhibits telomerase expression by at least about 50% in vitro, as compared to control expression reactions that lack the polynucleotide. In one embodiment, the polynucleotide is selected from the group consisting of PS-ODN number 3, 4, 7, 8, 16, 21, 25, 26, 27, 28, 29, 33, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 62, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 93, 94, 96, 100, 112, 114, 130, 143, 144, 151, 152, 201, 202, 203, 208, 209, 210, 211, 212, 213, 230, 237, and 241.
FIG. 1 presents the nucleotide sequence of a cDNA (SEQ ID NO:1) encoding a naturally occurring human telomerase reverse transcriptase (hTRT) protein.
FIG. 2 presents the amino acid sequence (SEQ ID NO:2) of a naturally occurring, 1132-residue human telomerase reverse transcriptase (hTRT) protein.
FIG. 3 shows inhibition of hTRT expression in vitro by hTRT sequence-specific antisense phosphorothioate oligonucleotides (PS-ODN). Each bar in the graph represents the in vitro inhibitory activity of a specific oligonucleotide, numbered starting with P,S-ODN #1. The PS-ODN are a series of 30-mers that span the hTRT mRNA and are offset one from the next by fifteen nucleotides. For example, ODN #1 corresponds to positions 16-35 of hTRT and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC. ODN #2 corresponds to positions 31-60 and is GGCATCGCGGGGGTGGCCGGGGCCAGGGCT (SEQ ID NO:4), and so one to the end of the RNA (see the cDNA sequence of FIG. 1, which represents an hTRT RNA sequence). The data are presented as a normalized percentage of the control with no added PS-ODN.
I. Introduction
Telomerase is a ribonucleoprotein complex (RNP) comprising an RNA component and a catalytic protein component. The catalytic protein component of human telomerase, hereinafter referred to as telomerase reverse transcriptase ("hTRT"), has been cloned, and protein, cDNA, and genomic sequences determined. See, e.g., Nakamura et al., 1997, Science 277:955, and copending U.S. patent applications Ser. Nos. 08/912,951 and 08/974,549. The sequence of a full-length native hTRT has been deposited in GenBank (Accession No. AF015950), and plasmid and phage vectors having hTRT coding sequences have been deposited with the American Type Culture Collection, Rockville, Md. (accession numbers 209024, 209016, and 98505). The catalytic subunit protein of human telomerase has also been referred to as "hEST2" (Meyerson et al., 1997, Cell 90:785), "hTCS1" (Kilian et al., 1997, Hum. Mol. Genet. 6:2011), "TP2" (Harrivigton et al., 1997, Genes Dev. 11:3109), and "hTERT" (e.g., Greider, 1998, Curr. Biol. 8:R178-R181). The RNA component of human telomerase (hTR) has also been characterized (see U.S. Pat. No. 5,583,016).
Human TRT is of extraordinary interest and value because, inter alia, telomerase activity in human cells and other mammalian cells correlates with cell proliferative capacity, cell immortality, and the development of a neoplastic phenotype. hTRT antisense polynucleotides, including the exemplary polynucleotides described herein, hybridize to and/or amplify naturally occurring hTRT genes or RNA. Such oligonucleotides are thus useful for diagnostic or prognostic applications to telomerase related conditions, including cancer. The hTRT antisense polynucleotides of the invention are also useful as therapeutic agents, e.g., antisense oligonucleotides, ribozymes, or triplex compositions, for inhibition of telomerase expression and activity (e.g., telomerase catalytic activity, infra).
The invention thus provides antisense oligonucleotide reagents, which can be used to detect expression of hTRT or reduce expression and activity of hTRT gene products in vitro, ex vivo, or in vivo. Administration of the antisense reagents of the invention to a target cell results in reduced telomerase activity, and is particularly useful for treatment of diseases characterized by high telomerase activity (e.g., cancers). Detection and inhibition of hTRT expression can be performed in a cell or cell extracts from a human, a mammal, a vertebrate, or other eukaryote.
The antisense polynucleotides of the invention are characterized by their ability to specifically hybridize to naturally occurring and synthetic hTRT nucleic acids, e.g., the hTRT gene, including any upstream, flanking, noncoding, and transcriptional control elements, hTRT pre-mRNA, mRNA, cDNA and the like. The hTRT antisense polynucleotides of the invention are typically at least 7-10 nucleotides in length to typically more 20 nucleotides up to about 100 nucleotides in length, preferably approximately 30 nucleotides in length. Such antisense oligonucleotides are used to detect the presence of hTRT nucleic acid in a biological sample, for diagnosis and/or prognosis of telomerase related conditions, e.g., cancers of any of a wide variety of types, including solid tumors and leukemias, diseases of cell proliferation, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, disease of immune dysfunction, etc.
The antisense polynucleotides of the invention also can be used to inhibit telomerase expression in vitro, to inhibit telomerase expression and activity in cells ex vivo, and can be used in vivo as therapeutic agents for the treatment of telomerase-related conditions listed above, including cancers of a wide variety of types (see, e.g., exemplary cancers listed in U.S. patent application Ser. No. 08/974,549; and U.S. patent application Ser. No. 08/974,584). In one embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression at least by 50% in vitro (see, e.g., the antisense oligonucleotides of FIG. 3). In another embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression and activity at least 50% in transformed cells ex vivo (see, e.g., exemplary antisense hTRT oligonucleotides listed in Table 1).
II. Definitions
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. Use of the term "polynucleotide" includes oligonucleotides (i.e., short polynucleotides). This term also refers to deoxyribonucleotides, ribonucleotides, and naturally occurring variants, and can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogues or modified backbone residues or linkages), such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like, as described herein.
As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of approximately 7 nucleotides or greater in length, and up to as many as approximately 100 nucleotides in length, which can be used as a primer, probe or amplimer. Oligonucleotides are often between about 10 and about 50 nucleotides in length, more often between about 14 and about 35 nucleotides, very often between about 15 and about 30 nucleotides, and the terms oligonucleotides or oligomers can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogies or modified backbone residues or linkages).
A polynucleotide "specifically hybridizes" or "specifically binds" to a target polynucleotide if the polynucleotide hybridizes to the target under stringent conditions. As used herein, "stringent hybridization conditions" or "stringency" refers to conditions in a range from about 5� C. to about 20� C. or 25� C. below the melting temperature (Tm) of the target sequence and a probe with exactly or nearly exactly complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc.; Sambrook et al. (1989) Molecular Cloning,: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory hereinafter, "Sambrook"); and Current Protocols in Molecular Biology (Ausubel et al., eds. through and including the 1997 supplement), incorporated herein by reference). As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, and the like), and the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art, e.g., Sambrook, supra and Ausubel et al. supra. Typically, stringent hybridization conditions are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 30� C. for short nucleic acids (e.g., 7 to 50 nucleotides) and at least about 60� C. for long nucleic acids (e.g., greater than 50 nucleotides). As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.
An "identical" polynucleotide refers to a polynucleotide that has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared. An "exactly complementary" polynucleotide refers to a polynucleotide whose complement has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared.
A "substantially complementary" polynucleotide and a "substantially identical" polynucleotide have the ability to specifically hybridize to a reference gene, DNA, cDNA, or mRNA, e.g., the hTRT nucleotide sequence of FIG. 1 and its exact complement.
An "antisense" polynucleotide is a polynucleotide that is substantially complementary to a target polynucleotide and has the ability to specifically hybridize to the target polynucleotide.
A "telomerase-related condition" refers to a diseases and disease conditions in a patient and/or a cell, characterized by under- or over-expression of telomerase or hTRT gene products. In addition to cancer, which is characterized by over-expression of telomerase, such conditions include diseases of cell proliferation, e.g., hyperplasias, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, etc.
As used herein, "isolated," when referring to a molecule or composition, such as, for example, an oligonucleotide, means that the molecule or composition is separated from at least one other compound, such as other oligonucleotides or other contaminants with which it is associated in vivo or in its naturally occurring state or synthetic state. An isolated composition can also be substantially pure.
A "synthetic" oligonucleotide refers to a polynucleotide synthesized using in vitro chemical methods, e.g., by using a machine that synthesizes polynucleotides using the phosphodiester method, the diethylphosphoramidite method, the phosphotriester methods, the solid support method, and other methods known to those skilled in the art.
As used herein, "recombinant" refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
As used herein, the term "substantially pure," or "substantially purified," when referring to a composition comprising a specified reagent, such as an oligonucleotide, means that the specified reagent is at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the composition (not including, e.g., solvent or buffer). Thus, for example, an antisense oligonucleotide preparation that specifically binds an hTRT gene or mRNA is substantially purified.
"TRT" activity refers to one or more of the activities found in naturally-occurring full-length TRT proteins. These activities include "telomerase catalytic activity" (the ability to extend a DNA primer that functions as a telomerase substrate by adding a partial, one, or more than one repeat of a sequence, e.g., TTAGGG, encoded by a template nucleic acid, e.g., hTR), "telomerase conventional reverse: transcriptase activity" (see Morin, 1997, supra, and Spence et al., 1995, Science 267:988); "nucleolytic activity" (see Morin, 1997, supra; Collins and Grieder, 1993, Genes and Development 7:1364; Joyce and Steitz, 1987, Trends Biochem. Sci. 12:288); "primer (telomere) binding activity" (see, Morin, 1997, supra; Collins et al., 1995, Cell 81:677; Harrington et al., 1995, J. Biol. Chem. 270:8893); "dNTP binding activity" (Morin, 1997, supra; Spence et al., supra); and "RNA (e.g., hTR) binding activity" (see Morin, 1997, supra; Harrington et al., 1997, Science 275:973; Collins et al., 1995, Cell 81:677,).
"TRT" refers to telomerase reverse transcriptase protein, and "hTRT" refers to human telomerase reverse transcriptase protein.
The term "hTRT" is intended to refer to alleles, conservatively modified variants, polymorphic variants, and interspecies homologues of hTRT encoded by nucleic acids that specifically hybridize to the hTRT nucleic acid sequence provided in FIG. 1.
"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art (see, e.g., Creighton (1984) Proteins, W. H. Freeman and Company.
III. How to Make Antisense Polynucleotides
As described herein, the present invention provides antisense polynucleotides, which have the ability to specifically hybridize to hTRT. Without intending to be limited to any particular mechanism, it is believed that antisense oligonucleotides bind to, and interfere with the translation of, the sense hTRT mRNA. Alternatively, the antisense molecule may render the hTRT mRNA susceptible to nuclease digestion, interfere with transcription, interfere with processing, localization or otherwise with RNA precursors ("pre-mRNA"), repress transcription of mRNA from the hTRT gene, or act through some other mechanism. However, the particular mechanism by which the antisense molecule reduces hTRT expression is not critical.
Generally, to assure specific hybridization, the antisense sequence is substantially complementary to the target hTRT mRNA sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence. The antisense polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to hTRT RNA or its gene is retained as a functional property of the polynucleotide.
In one embodiment, the antisense sequence is complementary to relatively accessible sequences of the hTRT mRNA (e.g., relatively devoid of secondary structure). These sequences can be determined by analyzing predicted RNA secondary structures using, for example, the MFOLD program (Genetics Computer Group, Madison Wis.) and testing in vitro or in vivo as is known in the art. FIG. 3 and TAble 1 show examples of oligonucleotides that are useful in cells for antisense suppression of hTRT function and are capable of hybridizing to hTRT (i.e., are substantially complementary to hTRT). Another useful method for identifying effective antisense compositions uses combinatorial arrays of oligonucleotides (see, e.g., Milner et al., 1997, Nature Biotechnology 15:537).
A. Triplex-forming Antisense Polynucleotides
As one embodiment of the antisense molecules described herein, the present invention provides polynucleotides that bind to double-stranded or duplex hTRT nucleic acids (e.g., in a folded region of the hTRT RNA or in the hTRT gene), forming a triple helix-containing, or "triplex" nucleic acid. Triple helix formation results in inhibition of hTRT expression by, for example, preventing transcription of the hTRT gene, thus reducing or eliminating telomerase activity in a cell. Without intending to be bound by any particular mechanism, it is believed that triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules to occur.
Triplex oligo- and polynucleotides of the invention are constructed using the base-pairing rules of triple helix formation (see, e.g., Cheng et al., 1988, J. Biol. Chem. 263: 15110; Ferrin and Camerini-Otero, 1991, Science 354:1494; Ramdas et al., 1989, j. Biol. Chem. 264:17395; Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 9591; each of which is incorporated herein by reference) and the hTRT MnRNA and/or gene sequence. Typically, the triplex-forming oligonucleotides of the invention comprise a specific sequence of from about 10 to at least about 25 nucleotides or longer "complementary" to a specific sequence in the hTRT RNA or gene (i.e., large enough to form a stable triple helix, but small enough, depending on the mode of delivery, to administer in vivo, if desired). In this context, "complementary" means able to form a stable triple helix. In one embodiment, oligonucleotides are designed to bind specifically to the regulatory regions of the hTRT gene (e.g., the hTRT 5′-flanking sequence, promoters, and enhancers) or to the transcription initiation site, (e.g., between −10 and +10 from the transcription initiation site). For a review of recent therapeutic advances using triplex DNA, see Gee et al., in Huber and Carr, 1994, Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N.Y. and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA 94:5854, which are both incorporated herein by reference.
B. Ribozymes
In another embodiment, the present invention provides ribozymes useful for inhibition of hTRT telomerase activity. The ribozymes of the invention bind and enzymatically cleave and inactivate hTRT mRNA. Useful ribozymes can comprise 5′- and 3′-terminal sequences complementary to the hTRT mRNA and can be engineered by one of skill on the basis of the hTRT mRNA sequence disclosed herein (see PCT publication WO 93/23572, supra). Ribozymes of the invention include those having characteristics of group I intron ribozymes (Cech, 1995, Biotechnology 13:323) and others of hammerhead ribozymes (Edgington, 1992, Biotechnology 10:256).
Ribozymes of the invention include those having cleavage sites such as GUA, GUU and GUC. Other optimum cleavage sites for ribozyme-mediated inhibition of telomerase activity in accordance with the present invention include those described in PCT publications WO 94/02595 and WO 93/23569, both incorporated herein by reference. Short RNA oligonucleotides between 15 and 20 ribonucleotides in length corresponding to the region of the target hTRT gene containing the cleavage site can be evaluated for secondary structural features that may render the oligonucleotide more desirable. The suitability of cleavage sites may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assay, or by testing for in vitro ribozyme activity in accordance with standard procedures known in the art.
As described by Hu et al., PCT publication WO 94/03596, incorporated herein by reference, antisense and ribozyme functions can be combined in a single oligonucleotide. Moreover, ribozymes can comprise one or more modified nucleotides or modified linkages between nucleotides, as described above in conjunction with the description of illustrative antisense oligonucleotides of the invention.
C. Synthesis of Antisense Polynucleotides
The antisense nucleic acids (DNA, RNA, modified, analogues, and the like) can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art. In one embodiment, for example, antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning. For example, an antisense RNA that hybridizes to hTRT mRNA can be made by inserting (ligating) an hTRT DNA sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid). Provided that the promoter and, preferably termination and polyadenylation signals, are properly positioned, the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.
The present invention also provides hTRT antisense polynucleotides (RNA, DNA. or modified) that can be produced by direct chemical synthesis. Chemical synthesis is generally preferred for the production of oligonucleotides or for oligonucleotides and polynucleotides containing nonstandard nucleotides (e.g., probes, primers and antisense oligonucleotides). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859 (1981); and the solid support method of U.S. Pat. No. 4,458,066.
Chemical synthesis typically produces a single stranded oligonucleotide, which may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase and an oligonucleotide primer using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is often limited to sequences of about 100 or 150 bases, longer sequences may be obtained by the ligation of shorter sequences or by more elaborate synthetic methods.
It will be appreciated that the hTRT polynucleotides and oligonucleotides of the invention can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired TM). Techniques for rendering oligonucleotides nuclease-resistant include those described in PCT publication WO 94/12633. A wide variety of useful modified oligonucleotides may be produced, including oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al., 1991, Science 254:1497) or incorporating 2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates. Still other useful oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3, where n is from 1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a folate group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Folate, cholesterol or other groups that facilitate oligonucleotide uptake, such as lipid analogs, may be conjugated directly or via a linker at the 2′ position of any nucleoside or at the 3′ or 5′ position of the 3′-terminal or 5′-terminal nucleoside, respectively. One or more such conjugates may be used. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other embodiments may include at least one modified base form or "universal base" such as inosine, or inclusion of other nonstandard bases such as queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
The invention further provides oligonucleotides having backbone analogues such is phosphodiester, phosphorothioate, phosphorothioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, chiral-methyl phosphonates, nucleotides with short chain alkyl or cycloalkyl intersugar linkages, short chain heteroatomic or heterocyclic intersugar ("backbone") linkages, or CH2—NH—O—CH2, CH2—N(CH3)—OCH2, CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones (where phosphodiester is O—P—O—CH2), or mixtures of the same. Also useful are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506).
Useful references include Oligonucleotides and Analogues, A Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan et al., Jul. 9, 1993, J. Med. Chem. 36(14):1923-1937; Antisense Research and Applications (1993, CRC Press), in its entirety and specifically Chapter 15, by Sanghvi, entitled "Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides;" and Antisense Therapeutics, ed. Sudhir Agrawal (Humana Press, Totowa, N.J., 1996).
D. Labeled Antisense Oligonucleotides
It is often useful to label the antisense polynucleotides of the invention, for example, when the hTRT polynucleotides are to be used for detection of hTRT expression, and for diagnosis and prognosis of telomerase related conditions. The labels may be incorporated by any of a number of means well known to those of skill in the art. Suitable labels are any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as radioactivity, that can be used to quantitate the amount of bound detectable moiety.
The label can be incorporated in or attached to a polynucleotide either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety. For example, the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules.
IV. Exemplary Antisense Polynucleotides
A series of 30-mer antisense oligonucleotides, which span the entire hTRT sequence, are exemplary embodiments of the present invention (see FIG. 3). These oligonucleotides were systematically assayed for the ability to inhibit hTRT expression in vitro. The results of the experiment are presented in FIG. 3 (see also Example I). Any suitable series of hTRT antisense oligonucleotides can be tested in a similar fashion. For example, a series of 20-mer antisense oligonucleotides, offset one from the next by 10 nucleotides can be synthesized and tested in the same manner. A series of 25-mer, 35-mer, or 15-mer oligonucleotides can be examined in the same manner.
Selected oligonucleotides from the series of FIG. 3 were then tested ex vivo for their ability to inhibit hTRT expression in tumor cells (see Example II). The hTRT antisense oligonucleotides active for inhibiting telomerase activity ex vivo in tumor cells were than assayed for their long term cell culture effects on hTRT expression, telomerase activity, telomere dynamics, and cell proliferation (see Example II). The oligonucleotides of Table I represent exemplary oligonucleotides that inhibited telomerase activity ex vivo.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
Inhibition of hTRT Expression in vitro
In this example, inhibition of hTRT expression was examined using an in vitro cell-free expression system. A series of 30-mer antisense phosphorothioate oligonucleotides (PS-ODNs), which span the entire hTRT sequence, was systematically assayed for the ability to block hTRT expression in vitro (see FIG. 3). Co-expression of luciferase was used to normalize the samples and demonstrate the specificity of inhibition.
For inhibition of hTRT expression in vitro, an hTRT transcription/expression plasmid was prepared according to standard methodology for in vitro transcription and translation of hTRT RNA. Coupled transcription-translation reactions were performed with a reticulocyte lysate system (Promega TNT™) according to standard conditions (as performed in Example 7, U.S. patent application Ser. No. 08/974,549). Each coupled transcription/translation reaction included hTRT RNA transcribed from the expression plasmid, and a test antisense polynucleotide at a range of standard test concentrations, as well as the luciferase transcription/translation internal control (see, e.g., Sambrook et al., supra, Ausubel et al., supra). The translation reaction can also be performed with hTRT RNA that is synthesized in vitro in a separate reaction and then added to the translation reaction. 35S-Met was included in the reaction to label the translation products. The negative control was performed without added PS-ODN.
The labeled translation products were separated by gel electrophoresis and quantitated after exposing the gel to a phosphorimager screen. The amount of hTRT protein expressed in the presence of hTRT specific PS-ODNs was normalized to the co-expressed luciferase control. The data are presented in FIG. 3 as a percentage of the control, which is without added PS-ODN.
Inhibition of hTRT Expression Ex Vivo
A. Reagents
Cells: ACHN cells, NCI, catalogue #503755; 293 cells, ATCC; BJ (see, e.g., Kim et al., Science 266: 2011-2015 (1994)); additional cells from the ATCC or NCI.
Media and solutions: RPMI 1640 medium, BioWhitaker; DMEM/M199 medium, BioWhitaker; EMEM, BioWhitaker; Fetal Bovine Serum, Summit (stored frozen at −20� C., stored thawed at 4� C.); Trypsin-EDTA, GIBCO (catalogue #25300-054) (stored frozen at −20� C., stored thawed 4� C.; Isoton II (stored at RT); DMSO (stored at RT); oligonucleotides (see Table 1 and FIG. 3, stored in solution at −20� C.); PBS (Ca++/Mg++ free); TE; 10 mM Tris-HCL, pH 8.0; 1 mM EDTA.
To prepare oligonucleotide stocks: Dissolve oligonucleotide nucleotides (PS-ODNs) in the appropriate amount of TE to make a concentrated stock solution (1-20 mM).
B. Treatment of Cells Ex Vivo with Antisense hTRT Oligonucleotides
1. For plating cells prior to oligonucleotide treatment, stock cultures of cells in log-phase growth (in T75 flask) were used. ACNH, 293, and BJ cells were used in this assay. The media was removed by aspiration, and the cells were rinsed with 2-5 ml of PBS. 1 ml of trypsin-EDTA was added to the cells, swirled to distribute, and incubated for 2 minutes. The trypsin was inactivated with 9 ml of media. The cells were gently triturated with media. 200 μL of the cells were then counted with a Coulter counter and diluted to the appropriate volume and number of cells per well.
2. For 6-well dishes, 1.1�105 cells total per well, 2 ml/well were added. The cells were allowed to settle 4-6 h prior to any treatment with oligonucleotides. The amount of cells can be scaled up or down proportionally for 12-well, 100 mm, or 150 mm dishes. For example, for 12-well dishes, use 4.6�104 cells in 2 ml media; for 100 mm dishes use 6�105 cells in 10 ml media; for 150 mm dishes use 1.7�106 cells in 35 ml media.
3. Oligonucleotides were diluted in media and fed to the cells at a range of standard test concentrations. Serial, sterile dilutions of the ODNs (see, e.g., Table 1) were prepared in sterile, filtered media for feeding the cells. The cells were treated in single, duplicate, or triplicate wells. Control wells were treated with TE diluted in media.
4. The cells were fed daily with freshly diluted PS-ODN-media by aspirating the media and then feeding with 2 ml of freshly diluted oligonucleotide in media.
5. When cells were near 70-80% confluent (3-4 days), the number of cells was determined per well. The media was removed by aspiration, and the cells were rinsed twice with 2 ml PBS. 0.5 ml trypsin-EDTA was added to the cells, swirled, and incubated for 2 minutes. The cells were triturated gently with 2 ml media per well. 200 μL of cells were counted in a Coulter counter. If necessary, the cells are replated at 1.1�105 cells per well, 2 ml media per well, and fed with PS-ODN as described above.
6. Samples of the cells were also harvested for analysis of telomerase activity by TRAP activity. The cells can also be analyzed by isolating RNA and performing RT-PCR, by TRF measurement, or by telomere length measurement (see, e.g., Example section, U.S. patent application Ser. No. 08/974,549 for assay protocols).
7. The cell population doublings (PDLs) were calculated for each timepoint according to the following formula. PDLs (P): Pn=Pn-1+[((ln(Total # cells))−(ln(# cells plated))/ln(2)].
8. Graph PDL vs. time (in days) for the full dose range of each PS-ODN as compared to control untreated cells.
9. Steps 2-8 were repeated for the desired duration (usually 2-4 weeks) or until cell growth was inhibited significantly.
10. Table 1 shows exemplary oligonucleotides that were tested using this assay, and which inhibited telomerase expression and activity by approximately 50% or more.
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"A crazy combination of banana, peanut butter, bacon, and chocolate...yeah, hence the name. I was inspired to create this concoction by a burger known as the Elvis which came topped with bacon and peanut butter. Refrigerate leftovers."
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10 slices
8 (1 ounce) squares
semisweet chocolate, or more if needed, chopped
bananas, peeled
5 tablespoons
smooth peanut butter, divided
SO WRONG!!!!! BUT SOOOO GOOD!!!! I sprinkled some crumbled bacon on top of half and kosher salt (sparingly) on the other half before the chocolate hardened, added a little more crunch and garnish. Used thick cut bacon, and cooked it in the oven at 300 degrees for about 25 mins. to make a soft yet crispy bacon. Also used nutella on some instead of peanut butter, even more wrong. Great recipe, I love bacon.
This was a fun, freaky, odd little recipe the Recipe Group chose this week. I only wanted a taste, so I cooked three slices of bacon and cut each into 3 pieces. Then I cut 9 banana slices and topped each one with peanut butter. I plopped them down onto the bacon and stuck a toothpick through the middle. Because I didn't want to fool with a candy thermometer, I just melted some Wilton Candy Melts and spooned on the chocolate. I did top them with little colorful sprinkles. I placed them on wax paper until they were dry. I could taste each ingredient - a sweet and salty little party in my mouth. I probably won't fix this again, but it was fun to try out such a quirky recipe.
16 Ratings
Prepared these for the Recipe Group and our guests enjoyed them this evening after dinner. I didn't have the time or patience for the candy thermometer so I purchased Dolci Microwave Hard Dark Choc Shell Coating. It worked great! I also crumbled the bacon. This eliminated the toothpick and made it easier for me. I spread the PB on the banana, dipped it in the crumbled bacon, pressed lightly, and then drizzled the chocolate over the top and smoothed with a spoon around the sides. Still a little messy but quick and easy. Before the chocolate set-up, I sprinkled Sea Salt, Banana Chips or crumbled bacon on each. This was a fun and different recipe and we were pleasantly surprised with the sweet/smokey/salty flavor. I will make these again. Thanks for sharing!
I made these for the recipe group this week. I will go ahead and say...this was very messy for me and mine looked nothing like the picture. However, I think that should be attributed to my inexperience rather than the recipe. I loved this combo, though- definitely a little bizarre, but sinfully yummy. One error I made was making the bacon too crispy- I disklike soggy fatty bacon but with as crisp as it was it fell apart when I tried to skewer it to the banana. Honestly, in the future...only when I am home alone, of course...I may just make a sort-of banana split out of it and call it good. Anyway, a pleasantly surprising little snack that has all of my favorite foods in one- peanut butter, chocolate, and bacon. Thanks for sharing!
I made these for Recipe Group, and they were sooooo good! Made as written, but I had some cooked, crumbled bacon to use up, so I just 'dipped' the peanut butter bananas in there and that seemed to work out great. once the chocolate hardened, I drizzled them with some more chocolate just to make them look pretty. I don't know how often I would actually make these, but I will definately be making them again! Thanks for sharing such a fun, unique and 'freakishly' delicious recipe. :)
Made this today for a birthday party tomorrow. If I may quote my hubby..."It's like a mouthful of awesome!" I took short cuts (microwave chocolate, precooked bacon warmed up, different coating method) but still...WOW! Thanks for the recipe!
Made these for Recipe Group but I didn't love these as much as I expected - but they were good. I used maple cured bacon which added another flavor. Make sure your banana isn't too ripe or they fall apart. I gave up trying to spread the PB on the banana and just dabbed it on the bacon squares then added the banana. I spooned my chocolate over each, let it set, then turned them over and covered the bottom. I doubt I'll make these again, but loved trying them. Thanks for sharing.
I love all the ingredients in this recipe and the combo of sweet and salty, so when these were choosen as the recipe of the week by Recipe Group I was excited to make them. I only made two because I was the only one interested in trying them. I used a round slice of banana, topped that with the peanut butter then place a small piece of bacon on the peanut butter. I stuck it on a wodden skewer and spooned the melted chocolate over the entire piece. I sprinkled a little sea salt on top too. When I ate it I thought this would be better with a thin, crisp piece of proscuitto in place of the bacon. My bacon was crisp but the texture turned me off from eating the second one, I wanted some more crunch. Thanks for a fun recipe!
* Percent Daily Values are based on a 2,000 calorie diet.
Circus Freaks
Serving Size: 1/30 of a recipe
Servings Per Recipe: 30
Amount Per Serving
Calories: 73
Calories from Fat: 43
Something about the grill's heat brings out the best flavor in the simplest chicken recipe.
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Originally written on Fox Sports | Last updated 11/12/12
Joe Harris scored nine of his 15 points in the opening minutes of the second half Monday night and Virginia rallied to beat Fairfield 54-45 in the NIT Season Tip-off. Akil Mitchell had 12 points and Evan Nolte added 11 for the Cavaliers (1-1), who will play in the second round Tuesday night. Josip Mikulic scored 13 points and Derek Needham and Desmond Wade had 10 each for Fairfield (1-1). The Stags scored only 15 points in the second half, making just 5 of 24 shots. In the first half, they were 12 for 23 with six 3-pointers, including three in as many tries by the 7-foot Mikulic. Harris scored nine of the Cavaliers' first 11 points in the second half, including a 15-foot jumper with 17 minutes left that gave them their first lead at 33-32. Virginia stretched its margin to 42-37 as the Stags went 5 1/2 minutes without a point, but Desmond Wade made a pair of free throws for Fairfield and Colin Nickerson followed several Cavaliers' misses with a layup at the other end, making it a...
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Originally written on CLNS Radio | Last updated 4/22/13
Julio Borbon Claimed by the Cubs The Chicago Cubs made a splash in the waiver pool by claiming Julio Borbon after he got designated for assignment by the Texas Rangers. The Chicago Cubs made another move by designating infielder Alberto Gonzalez for assignment. Borbon, 27, is a career .283 hitter who spent parts of four seasons with the Rangers. He was designated for assignment on April 9 and the Rangers had 10 days to try to trade him. After a trade could not be worked out the Cubs claimed him on waivers. Borbon arrived in Chicago Friday night and ended up pinch-running in the 9th inning. He was thrown out trying to steal second to end the game. Borbon will provide the club with outfield depth and possibly will have a starting role mid-season depending on what the Cubs do at the trade deadline. He is an interesting topic due to him being a highly regarded prospect and has hit at such a high level. The one thing missing is the pop from his bat,
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Tuesday, June 30, 2015 (17:03:43)In this article I will take you step by step through the process of creating a custom, but simple, report on a Kik messenger database using the Forensic Browser for SQLite.
As we work through the process we will chose which columns we think will be useful in our report and modify our report by creating simple visual SQL joins on different tables displaying numerics as timezone adjusted dates and importing pictures saved in external binary PLists.
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With so many nations' rescue authorities depending on volunteers, a bunch of Californian kids have set an example for the rest of the world by getting together to raise money for the Coast Guard Foundation, which itself supports those who often risk their lives saving sailors in trouble at sea.
Kids set the world an example with their lemonade stand
The kids were from the Training Center Petaluma, California, and they set up a lemonade stand to raise funds for The Foundation.
The kids sold lemonade outside of a 3K-5K family run/walk event at 50 cents a pop. Amazingly, they sold 100 cups of lemonade and were able to raise $50 for The Foundation. Master Chief Petty Officer Richard Wolfe helped organize the kids' effort locally and arranged to send the donation to the Foundation.
Coast guard kids and lemonade stand
The lemonade stand sales were a small way of saying 'thanks for all your hard work at the foundation and for all the Coasties who use it,' Wolfe said. The names of the girls who worked the stand are: Hayle, Alyese, Cailen, Jessica and Summer.
Needless to say, the Foundation was touched by the kids' showing their support.
'Every day the Foundation works hard to bolster support and raise money for the men and women of the United States Coast Guard and their families,' said Anne Brengle, president of the Coast Guard Foundation. 'We are touched by the efforts of these children to help us in our mission. Every donation we receive, large or small, makes an impact, and we are truly grateful for their actions and support.'


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Press release from CNW Group
Connacher to Present at CIBC 2010 Energy & Infrastructure Conference
Monday, April 19, 2010
Connacher to Present at CIBC 2010 Energy & Infrastructure Conference12:46 EDT Monday, April 19, 2010CALGARY, April 19 /CNW/ - Connacher Oil and Gas Limited (CLL-TSX) announced today that it will be presenting at the CIBC 2010 Energy & Infrastructure Conference on Tuesday, April 20, 2010 in Toronto, ON at 4:05 PM ET. The presentation will be made by Mr. Richard Gusella, President and Chief Executive Officer and will be derived from the Spring 2010 Investor Presentation that is posted on Connacher's website at www.connacheroil.com. To listen to the live audiocast click the following link:http://events.startcast.com/events6/118/energy2010/Default.aspx?Presentation=62Connacher Oil and Gas Limited is a Calgary-based exploration, development, production, refining and marketing oil company. Our major asset is our 100 percent interest in Great Divide bitumen production projects. Our first 10,000 bbl/d SAGD project ("Pod One") is currently operational. Algar is the company's second 10,000 bbl/d bitumen production project. It was designed for further expansion, upon receipt of regulatory approvals. We also own conventional crude oil and natural gas production in Western Canada and own and operate a 9,500 bbl/d heavy oil refinery located at Great Falls, Montana. Connacher maintains an approximate 20 percent equity stake in Petrolifera Petroleum Limited, a public oil company active in Argentina, Peru and Colombia in South America. Common shares and our outstanding convertible debentures are listed for trading on the Toronto Stock Exchange.For further information: Richard A. Gusella, President and Chief Executive Officer, OR Grant D. Ukrainetz, Vice President, Corporate Development, Phone: (403) 538-6201, Fax: (403) 538-6225, firstname.lastname@example.com, Website: www.connacheroil.com


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9 Easy Baking Recipes for Homemade Ingredients - Free eCookbook!
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Copycat Entenmann's Crumb Cake Recipe
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Now this is what we call breakfast. Copycat Entenmann's Crumb Cake Recipe might not make all of your dreams come true, but it will come pretty close. Copycat breakfast recipes like this don't come around too often, so be sure to take advantage. It only takes a few simple ingredients to create and the finished product is a light and fluffy pastry your entire family will love. Create something to be proud of by baking up this delightful pastry recipe.
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EVENTOct 25, 2014 News 0 comments
EVENT UPCOMING! Please read about this new upcoming event for SSGC!
If you always loved the slender games then you should go here! Get all updates on slender news, and games!
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EVENT UPCOMING! Please read about this new upcoming event for SSGC!
This game Includes all of the X9 Features... Such as Iron Sight's Post Processing Effects, and new weapons!
All Slenderman's games. I add a game when i found a new.
you guys should check out my game Indiedb.com
here is the link to download for Insane: Slender's return. the Slender more frightening to me. enjoy =): Dhc-games.webs.com
Hi guys..here is the newest version of Slender '9 Pages' witch a ******* Hardcore Survival Mode!
Download Link: http : // www . mediafire . com/?d7l2034ao9v2a6d
(Without spaces)
Launch the game and Enjoy it!
Hey bros yesterday i've buied slenderman's shadows if you want i do a gameplay, the map are fantastic and you can customize the glowstick! is a mythical game!!!!!!!!!!!!!
Haunt for download!
Download here:
I got so scared from Slender Mod
Slenderman's shadow is comming out with carnavil this year!
Yeah, it is really creepy! Slender now looks like an egg xD!
Promote Slender Fans *o* on your homepage or blog by selecting a button and using the embed code provided (more).


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Two more contest results are in
Posted by Teresa Hanafin June 7, 2012 10:17 AM
Photographer Paul Marotta has sent me the results of his judging of the "Outdoor Sculptures" entries, and John Blanding, assistant chief photographer for the Globe, sat with me last night and judged "The Color Yellow" contest.
I'll post the Sculpture results today and the Yellow results tomorrow.
Welcome to your community for New England's amateur photographers. Take pictures ... get published ... win money ... have a blast!
The Color Green It's the color of hope, envy, regeneration, relaxation, and money -- as well as the theme of the October contest. Make it the focal point of your best photograph.
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Life and wildlife in Madagascar
Life and wildlife in Madagascar
For years before I started college, I knew I wanted to spend a semester studying abroad, but I wasn't sure where. By my junior year at Brandeis, I made up my mind to travel somewhere off the beaten path, and, of course, Madagascar is pretty far off the beaten path for someone like me....
An essay about Rebirth Workshops
Now that it's been several months since I returned from a week-long Rebirth Workshop in Mississippi, I'm happy to look back and provide an overview of what we did that made it such an intense experience for me as a photographer....
Photography apps for your phone
Thinking of ditching your separate camera and moving to just using your phone for all your photos? What apps should you go for? Instagram made headlines recently after being bought by Facebook for $1 billion. What does it include, and what else is out there?...
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Arenal Nayara, La Fortuna
This collection of colourful casitas gazes up to the crater of Arenal. The country's most-active volcano is surrounded by a national park that's home to 850 species of birds, as well as monkeys and jaguars, making the lodge a prime spot for wildlife fans. The decor reflects the tropical surroundings, with brightly painted walls and furnishings, while the bungalows come with vaulted bamboo roofs, outdoor monsoon showers and balconies with hammocks and Jacuzzis.
Arenal Nayara, La Fortuna, Arenal NP (00 506 2479 1600; arenalnayara.com). Doubles start at US$277 (£185), including breakfast.
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text: Out shopping and was keen on a pizza and beer for lunch, saw the Italian flag, done deal, what a little treasurer, great atmosphere and awesome pizza, margarita with basil perfect thin base with a great tasting tomato base. The size and price is very good for Tokyo. We will be back...... And the... read more