Uncovering the interplay between epigenome editing efficiency and sequence context using a novel inducible targeting system

Epigenome editing is an attractive way to manipulate gene expression. However, editing efficiencies depend on the DNA sequence context in a manner that remains poorly understood. Here we developed a novel system in which any protein can be recruited at will to a GFP reporter. We named it ParB/ANCHOR-mediated Inducible Targeting (PInT). Using PInT, we tested how CAG/CTG repeat size affects the ability of histone deacetylases to modulate gene expression. We found that repeat expansion reduces the effectiveness of silencing brought about by HDAC5 targeting. This repeat-length specificity was abolished when we inhibited HDAC3 activity. Our data guide the use of these histone deacetylases in manipulating chromatin. PInT can be adapted to study the effect of virtually any sequence on epigenome editing

Direct characterization of circulating DNA in blood plasma using μLAS technology

Circulating cell-free DNA (cfDNA) is a powerful cancer biomarker for establishing targeted therapies or monitoring patients' treatment. However, current cfDNA characterization is severely limited by its low concentration, requiring the extensive use of amplification techniques. Here we report that the μLAS technology allows us to quantitatively characterize the size distribution of purified cfDNA in a few minutes, even when its concentration is as low as 1 pg/μL. Moreover, we show that DNA profiles can be directly measured in blood plasma with a minimal conditioning process to speed up considerably speed up the cfDNA analytical chain

Repeat Detector: versatile sizing of expanded tandem repeats and identification of interrupted alleles from targeted DNA sequencing

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and preclinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntington’s disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntington’s disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and in the development of novel therapies

Therapeutic antibodies targeting app-c99

The present invention relates to an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) and to pharmaceutical compositions containing said antibody/ies

Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease

Gamma-secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-beta precursor protein generates the amyloid-beta protein (Abeta) of Alzheimer's disease, partially inhibiting gamma-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of gamma-secretase, but at low resolution (15 A). The limited amount of purified gamma-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four gamma-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active gamma-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme