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RNA guided eradication of human jc virus and other polyomaviruses

A polyoma virus, virus technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, and resistance to vector-borne diseases

Active Publication Date: 2017-11-28
TEMPLE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, no system has been developed to target JCV with gRNA-guided endonuclease attack

Method used

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  • RNA guided eradication of human jc virus and other polyomaviruses
  • RNA guided eradication of human jc virus and other polyomaviruses
  • RNA guided eradication of human jc virus and other polyomaviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1: Materials and methods

[0116] Using CRISPR / Cas9 technology to detect the effect of Cas9 and gRNA on targeting T-antigen and its function. Design of guide RNA for CRISPR / Cas9 targeting JCV as in Figure 1A and 1B shown.

[0117] cell culture . The human oligodendroglioma cell line TC620 (Wollebo et al., 2011) and SVG-A, which is derived from primary human fetal glial cells transformed with the original defective SV40 expressing SV40 T-Ag Cell line (Major et al., 1985), TC620 and SVG-A were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), as previously described (Wollebo et al., 2011). HJC-2 is a JCV-induced hamster glioblastoma cell line expressing JCV T-Ag (Raj et al., 1995). BsB8 is a mouse cell line derived from tumors of cerebellar neuroectodermal origin generated in transgenic mice expressing the JCV early protein T-Ag (Krynska et al., 2000). SVG-A cells expressing Cas9 and JCV T-antigen gRNA ...

Embodiment 2

[0133] Example 2: Expression of gRNA targeting T-Ag reduces the expression of T-Ag and the expression of T-Ag-stimulated JCV late genes in TC620 cells transfected with T-Ag and Cas9

[0134] In the first set of experiments, it was determined whether the combination of Cas9 with various gRNAs targeting TM1, TM2, and TM3 could suppress T-antigen expression in the human oligodendrocyte cell line TC620. In these experiments, gRNA m1 was complementary to the TM1 target sequence of SEQ ID NO:1; gRNA m2 was complementary to the TM2 target sequence of SEQ ID NO:5; and gRNA m3 was complementary to the TM3 target sequence of SEQ ID NO:9.

[0135] Western blot analysis results from transfection of TC620 cells with plasmids expressing T-antigen alone or in combination with Cas9, and / or in combination with T-Ag expression plasmids targeting gRNA are shown in Figure 2A middle. As shown, the presence of Cas9 together with ml or m2 gRNA significantly reduced the level of T-Ag production in ...

Embodiment 3

[0137] Example 3: Clonal derivatives of SVGA expressing Cas9 and gRNA targeting T-Ag have reduced ability to support JCV infection

[0138]To investigate the effect of gRNA-guided Cas9 on JCV infection, experiments were performed with the SVG-A cell line. This line supports viral gene expression and also allows full productive viral lytic infection. First, a stable clonal cell line was established from SVG-A cells expressing Cas9 or Cas9+gRNA ml. In these experiments, gRNA ml was complementary to the TM 1 target sequence SEQ ID NO:1. Three independent clones were selected and used for JCV infection for 7 days at MOI = 1.0. Viral infection was assessed by Western blot analysis for the presence of viral capsid protein, VP1 and accessory protein, Agno protein, with α-tubulin as a loading control. Quantitative PCR (Q-PCR) was performed simultaneously to determine the level of viral DNA in the culture medium as a marker of DNA replication and thus of virus production. Such as ...

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Abstract

The present invention includes methods and compositions for elimination of polyomaviruses, such as John Cunningham Virus (JVC), from host cells, and the treatment of polyomavirus related diseases, such as progressive multifocal leukoencephalopathy (PML). The compositions include isolated nucleic acid sequences comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target sequence in a polyomavirus.

Description

[0001] Statement Regarding Federally Funded Research [0002] This invention was made with US Government support under Grant No. NIHR01 NS087971 issued to Kamel Khalili and Wenhui Hu by the National Institutes of Health. The U.S. government may have certain rights in the invention. [0003] The Sequence Listing associated with this application is submitted electronically via EFS-Web and is hereby incorporated by reference into this specification in its entirety. The name of the text file containing the sequence listing is 0392-7Temple JCV PCT SeqLst 10 302015_ST25. The size of the text file is 8KB, and the text file was created on October 30, 2015. field of invention [0004] The present invention relates to compositions that specifically lyse target sequences in polyomaviruses, eg, human neurotrophic polyomaviruses such as John Cunningham virus. Such a composition, which may comprise a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonucleas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63
CPCC12N9/22C12N15/63C12N2310/20C12N15/1131Y02A50/30A61K48/005
Inventor K·卡利里W·胡H·沃勒勃
Owner TEMPLE UNIVERSITY
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