Compositions and methods of using transposons

A composition and small molecule technology, applied in biochemical equipment and methods, drug combinations, active ingredients of heterocyclic compounds, etc., can solve the problems of lack of whole genome and functional screening

Inactive Publication Date: 2016-11-09
YALE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While loss-of-function screens of cancer cells using shRNA libraries have been successfully employed to identify synthetic lethal targets, genome-wide gain-of-function screens for negatively selected genes are lacking

Method used

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  • Compositions and methods of using transposons
  • Compositions and methods of using transposons
  • Compositions and methods of using transposons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Embodiment 1: PB obtains functional screening

[0170] Generation of PB transposons containing a doxycycline (Dox)-inducible system to drive overexpression of endogenous genes upon insertion (PB[Mut-tetO-KAT-TETRKRAB], figure 1 ). Mutant cells were also labeled by co-expressing the Katushka (KAT) fluorescent marker. This is applied to KRAS G12V Transformed cell line - AML-RAS, which passes oncogenic KRAS G12V Induction was generated in a patient-derived TSC2-deficient angiomyolipoma cell line, TRI-102. TRI-102 is a slow growing benign tumor cell line that fails to form colonies in soft agar. KRAS G12V Introduction of these cells increases proliferation and allows anchorage-independent growth ( Figure 13A and 13B ) - characteristic of transformation typically exhibited by patient-derived cancer cells with activating RAS mutations. Thus, AML-RAS and TRI-102 provide ideal experimental and control cell lines to screen for RAS synthetic lethal mutations.

[0171] T...

Embodiment 2

[0188] Example 2: Conditional mutagenesis screening based on PB transposons.

[0189] Forward genetic approaches require functional examination of a large number of non-coding genes. Given the complexity of alternative splicing in the human genome and the constrained characterization of noncoding genes, this paper describes a systematic gene activation approach for genome-wide review. A screening method using a conditional expression system based on piggyBac (PB) transposon mutagenesis combined with high-throughput sequencing analysis was developed. Using this approach, a negative selection screen was performed for genes that impair the growth and / or survival of cancer cells expressing oncogenic KRAS. In one round of PB mutagenesis, 18,032 protein-coding genes, 10,362 long noncoding RNAs (lncRNAs), and 8,683 pseudogenes were successfully censored. Interestingly, both protein-coding and non-coding components of the WNT signaling pathway were found to specifically antagonize o...

Embodiment approach

[0200] The listing of elements in any definition of a variable recited herein includes defining that variable to be any single element or combination (or subcombination) of listed elements. Embodiments recited herein include an embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

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Abstract

The present invention provides compositions and methods of using transposons. In one aspect, methods are disclosed that are useful for identifying negatively selected genes in an insertional mutagenesis screen. In another aspect, compositions for reducing proliferation of a tumor cell expressing an oncogenic RAS include an activator of a WNT pathway. Pharmaceutical compositions for reducing proliferation of tumor cells in a subject in need thereof by administering an effective amount of an activator of a WNT pathway to the tumor cells of the subject are also disclosed.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 905,819, filed November 18, 2013, which is hereby incorporated by reference in its entirety. Background of the invention [0003] Recent advances in sequencing technology have enabled the identification of specific mutations in individual tumors, raising the possibility of developing targeted therapies for specific tumors. Functional genomics has proven to be a powerful approach for uncovering the underlying drivers of human biological and disease processes. CRISPR-Cas9 and shRNA libraries provide efficient screening tools to knock out or knock down protein-coding genes. Targeting specific oncogenic alterations and pathways in tumor cells has been found to be highly effective in treating some cancers, including HER2-amplified breast cancer and acute promyelocytic leukemia. However, for many common mutations including ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N2501/724C12N5/0693C12N2510/00A61P35/00A61P35/04A61P43/00A61K31/505A61K31/55A61K38/1709A61K45/06A61K48/00A61K2300/00C12Q1/6886C12Q2600/156C12Q2600/158
Inventor T·许F·钱S·兰德特
Owner YALE UNIV
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