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A method for highly selective nucleic acid enrichment for sensitive detection of mutations in cancer and genectic conditions.

Description:

Brief Description: 

This technology increases the fraction of clinically relevant, rare mutant alleles (DNA and/or RNA) to enable  high sensitivity  detection with common downstream methods such as capped (XNA or PNA) PCR, ddPCR, and sequencing.

 

Motivation:

Liquid biopsy is a simple, minimally invasive, rapidly developing diagnostic method to analyze cell-free nucleic acid fragments in body fluids to obtain critical information on patient health and disease status, enable personalized therapy, and replace invasive methods such as tissue biopsy. The sensitivity of available tests is, however, insufficient for patients at early disease stage and for cancer screening. Detection of alleles that contain critical clinical information is challenged by their very low concentrations and sequence homologies with abundant wildtype nucleic acids. This invention comprises of a high efficiency assay that reduces the concentrations of wildtype nucleic acids, DNA and/or RNA, and increases the fraction of clinically-relevant mutant alleles (DNA and/or RNA) to enable their downstream detection by a variety of methods.

 

Technology:

This invention utilizes DNA-guided Argonaute (Ago) endonuclease that cleaves guide-complementary DNA and RNA with single nucleotide precision,  greatly increasing the fractions of clinically-relevant rare alleles in a patient sample to enhance the sensitivity of downstream detection methods such as drop digital PCR, sequencing, and clamped (e.g., XNA and PNA) enzymatic amplification, enabling detection of low-frequency (0.01%) mutant alleles. This invention has many advantages over competing technologies such as CRISPR Cas9-based, Ago does not require targets to contain any specific (PAM-like) motifs; is a multi-turnover enzyme; cleaves ssDNA, dsDNA, and RNA targets in a single assay; is DNA-guided (less expensive and more stable than RNA guides), and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

 

Applications:

•       Increase the effectiveness and sensitivity of liquid biopsies

•       Enable detection of specific genetic materials (e.g., mutations associated with various types of cancers and fetal disorders; and strains of pathogens 

 

Advantages:

 •       Improves sensitivity of downstream detection and genotyping methods

•       Capable of enriching both DNA and RNA alleles in the same assay

•       Versatile: does not require targets to contain specific (PAM-like) motifs

•       Turnover enzymes:  reduced enzyme consumption

•       Highly specific: resolves single nucleotide differences between sequences.

•       Can be used as standalone kit for qualitative and quantitative assay to enrich rare sequences or integrated with existing downstream amplification such as capped (RT-) PCR/LAMP and/or sequencing.

•       Amenable for incorporation into point of care devices

 

Stage of Development:

•       Reduction to practice

•       In a proof of concept experiments, this invention enabled detection of cancer-related cell free alleles at fractions as low as 0.1% in blood samples from pancreatic cancer patients, in excellent agreement with tissue biopsy genotyping.

 

Intellectual Property

1.       Provisional Patent Application No. 62642984

2.       Related PCT Filed

a.       International Patent PCT/US2017/013403 (“WO2017123921A1”)        

https://patents.google.com/patent/WO2017123921A1/en

 

References:

      Song et al., 2018,  High Specific Enrichment of Rare Nucleic Acids using Thermus Thermophilus

Argonaute; In review.  Early pre-print available. doi: https://doi.org/10.1101/491738

https://www.biorxiv.org/content/early/2018/12/10/491738

Swarts et al. Nucleic Acids Res, 2015, 43(10) - 5120.

Sheng et al. Proc Natl Acad Sci, 2014, 111(2):652-7.

Makarova et al. Biol Direct. 2009, 4:29.

        

Desired Partnerships:

1.       Licensing

2.       Co-development:

 

 Docket Number 18-8416


Patent Information:
For Information, Contact:
Joshua Jeanson
Associate Director, SEAS/SAS Licensing Group
University of Pennsylvania
jeanson@upenn.edu
Inventors:
Jinzhao Song
Haim Bau
Michael Mauk
Jorrit Hegge
John van der Oost
Keywords: