Seneca valley virus based compositions and methods for treating disease

A virus and genome technology, applied in the fields of biochemical equipment and methods, gene therapy, chemical instruments and methods, etc., to achieve the effect of easy-to-manipulate genomes, small and small genomes

Inactive Publication Date: 2009-06-03
纽特罗佩克斯公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Although these newly defined anticancer therapies have certain advantages in the early s

Method used

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  • Seneca valley virus based compositions and methods for treating disease
  • Seneca valley virus based compositions and methods for treating disease
  • Seneca valley virus based compositions and methods for treating disease

Examples

Experimental program
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Embodiment 1

[0295] Virus Amplification and Purification

[0296] Cultivation of Sineica Valley virus in PER.C6 cells: Sineika Valley virus was subjected to a single plaque purification and well-separated plaques were selected and amplified in PER.C6 cells (Fallaux et al. 1998). Crude viral lysates (CVL) were prepared from Sineica Valley virus-infected PER.C6 cells by three freeze-thaw cycles. PER.C6 cells in 50 x 150cm 2 Cultivate in T.C. culture flask, what use is to contain 10% fetal calf serum (Biowhitaker, Walkersvile, MD, USA) and 10mM MgCl 2 (Sigma, St Louis, MO, USA) Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA). After 30 hours of virus infection, the infected cells were harvested when complete CPE was observed and collected by centrifugation at 1500 rpm for 10 minutes at 4°C. Cell pellets were resuspended with cell culture supernatant (30 ml), followed by three freeze-thaw cycles. The resulting CVL was clarified by centrifugation at 1500 rpm for 10...

Embodiment 2

[0298] Electron microscopy

[0299] Sineca Valley virus was embedded on a grid coated with polyvinyl formal carbon (formvar carbon) using the direct application method, stained with uranyl acetate, and then observed with a transmission electron microscope. Representative microscopic images of viral particles were taken at high magnification. For TEM observation, ultrathin sections of Sineca Valley virus-infected PER.C6 cells were excised from the embedding blocks, and the resulting sections were examined under a TEM.

[0300] The purified Sineca Valley virus particles are spherical, with a diameter of about 27nm, and appear as a single presence or a small amount of aggregation on the grid. A representative picture of the Seneca Valley virus is shown in figure 2 shown. Dye-penetrated damaged virus particles or empty capsids can also be seen in some fields of view. Ultrastructural analysis of infected PER.C6 cells revealed intracytoplasmic lens inclusions. image 3 Shown...

Embodiment 3

[0302] Isolation of Sineica Valley Virus Nucleic Acid

[0303] Isolation of RNA: Genomic RNA of Sineica Valley virus was extracted by guanidine thiocyanate and phenol extraction using Trizol (Invitrogeng, Carlsbad, CA). The separation process was performed according to the method recommended by the supplier. Briefly, 250 μl of purified Seneca Valley virus was mixed with 3 volumes of TRIZOL and 249 μl of chloroform. The aqueous phase contained Seneca Valley virus RNA and was precipitated with 600ul of isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in DEPC-treated water. The amount of RNA was estimated by optical density detection at 260 nm. A portion of RNA was taken and analyzed by electrophoresis in a 1.25 denatured agarose gel (CambrexBio Sciences Rockland Inc., Rockland, ME, USA), and the electrophoresis bands were observed and imaged with EB staining ( FIG. 4 ).

[0304] cDNA synthesis: cDNA of the Sineica Valley virus genome was s...

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Abstract

The present invention relates to a novel RNA picornavirus that is called Seneca Valley virus ('SVV'). The invention provides isolated SW nucleic acids and proteins encoded by these nucleic acids. Further, the invention provides antibodies that are raised against the SVV proteins. Because SVV has the ability to selectively kill some types of rumors, the invention provides methods of using SVV and SVV polypeptides to treat cancer. Because SVV specifically targets certain tumors, the invention provides methods of using SW nucleic acids and proteins to detect cancer. Additionally, due to the information provided by the tumor-specific mechanisms of SVV, the invention provides methods of making new oncolytic virus derivatives and of altering viruses to have tumor-specific tropisms.

Description

[0001] This application claims the following priority: US Patent 60 / 664,442, (filed March 23, 2005); US Patent 60 / 726,313 (filed October 13, 2005); US Patent 11 / 335,891 (filed 2006 19 January). These applications are incorporated herein by reference in their entirety. [0002] The content contained in this publication is protected by copyright. The copyright owner has no objection to the facsimile copying by anyone of the patent document or the patent disclosure, as this disclosure is a document or record of the United States Patent and Trademark Office; otherwise, all copyright rights are reserved. [0003] All patent applications, published patent applications, issued and issued patents, texts and articles cited in this specification are hereby incorporated by reference in their entirety in order to more fully describe the state of the art of the present invention. Background technique [0004] Viral therapy holds great promise in cancer treatment. Tumor cytolytic viruses...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07H21/04C12N7/00C12N15/86C07K14/085
Inventor P·哈伦贝克S·R·普利斯L·M·豪尔斯
Owner 纽特罗佩克斯公司
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