Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA.
Towards clinically translatable in vivo nanodiagnostics
Nature Reviews Materials
Temporally resolved direct delivery of second messengers into cells using nanostraws
LAB ON A CHIP
2016; 16 (13): 2434-2439
Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations.Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use.Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in nonsmall-cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR.The dCas9 method provides important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.
View details for DOI 10.1373/clinchem.2017.278911
View details for PubMedID 29038154
Molecular profiling of single circulating tumor cells from lung cancer patients
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (52): E8379?E8386
Determining the Time Window for Dynamic Nanowire Cell Penetration Processes.
2015; 9 (12): 11667?77
Second messengers are biomolecules with the critical role of conveying information to intracellular targets. They are typically membrane-impermeable and only enter cells through tightly regulated transporters. Current methods for manipulating second messengers in cells require preparation of modified cell lines or significant disruptions in cell function, especially at the cell membrane. Here we demonstrate that 100 nm diameter 'nanostraws' penetrate the cell membrane to directly modulate second messenger concentrations within cells. Nanostraws are hollow vertical nanowires that provide a fluidic conduit into cells to allow time-resolved delivery of the signaling ion Ca(2+) without chemical permeabilization or genetic modification, minimizing cell perturbation. By integrating the nanostraw platform into a microfluidic device, we demonstrate coordinated delivery of Ca(2+) ions into hundreds of cells at the time scale of several seconds with the ability to deliver complex signal patterns, such as oscillations over time. The diffusive nature of nanostraw delivery gives the platform unique versatility, opening the possibility for time-resolved delivery of any freely diffusing molecules.
View details for DOI 10.1039/c6lc00463f
View details for Web of Science ID 000378941700008
View details for PubMedID 27292263
Plasma membrane and actin cytoskeleton as synergistic barriers to nanowire cell penetration.
2014; 30 (41): 12362-12367
Nanowire (NW) arrays offer opportunities for parallel, nondestructive intracellular access for biomolecule delivery, intracellular recording, and sensing. Spontaneous cell membrane penetration by vertical nanowires is essential for these applications, yet the time- and geometry-dependent penetration process is still poorly understood. In this work, the dynamic NW-cell interface during cell spreading was examined through experimental cell penetration measurements combined with two mechanical models based on substrate adhesion force or cell traction forces. Penetration was determined by comparing the induced tension at a series of given membrane configurations to the critical membrane failure tension. The adhesion model predicts that penetration occurs within a finite window shortly after initial cell contact and adhesion, while the traction model predicts increasing penetration over a longer period. NW penetration rates determined from a cobalt ion delivery assay are compared to the predicted results from the two models. In addition, the effects of NW geometry and cell properties are systematically evaluated to identify the key factors for penetration.
View details for DOI 10.1021/acsnano.5b05498
View details for PubMedID 26554425
Bruton's tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib.
Therapeutic advances in hematology
2014; 5 (4): 121-133
Nanowires are a rapidly emerging platform for manipulation of and material delivery directly into the cell cytosol. These high aspect ratio structures can breach the lipid membrane; however, the yield of penetrant structures is low, and the mechanism is largely unknown. In particular, some nanostructures appear to defeat the membrane transiently, while others can retain long-term access. Here, we examine if local dissolution of the lipid membrane, actin cytoskeleton, or both can enhance nanowire penetration. It is possible that, during cell contact, membrane rupture occurs; however, if the nanostructures do not penetrate the cytoskeleton, the membrane may reclose over a relatively short time frame. We show with quantitative analysis of the number of penetrating nanowires that the lipid bilayer and actin cytoskeleton are synergistic barriers to nanowire cell access, yet chemical poration through both is still insufficient to increase long-term access for adhered cells.
View details for DOI 10.1021/la502273f
View details for PubMedID 25244597
Quantification of nanowire penetration into living cells.
2014; 5: 3613-?
Aberrant signaling of the B-cell receptor pathway has been linked to the development and maintenance of B-cell malignancies. Bruton's tyrosine kinase (BTK), a protein early in this pathway, has emerged as a new therapeutic target in a variety of such malignancies. Ibrutinib, the most clinically advanced small molecule inhibitor of BTK, has demonstrated impressive tolerability and activity in a range of B-cell lymphomas which led to its recent approval for relapsed mantle cell lymphoma and chronic lymphocytic leukemia. This review focuses on the preclinical and clinical development of ibrutinib and discusses its therapeutic potential.
View details for DOI 10.1177/2040620714539906
View details for PubMedID 25360238
View details for PubMedCentralID PMC4212313
Bruton tyrosine kinase inhibitors: a promising novel targeted treatment for B cell lymphomas.
British journal of haematology
2013; 163 (4): 436-443
High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.
View details for DOI 10.1038/ncomms4613
View details for PubMedID 24710350
Constitutive or aberrant signalling of the B cell receptor signalling cascade has been implicated in the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors of Bruton tyrosine kinase (BTK), a protein early in this cascade and specifically expressed in B cells, have emerged as a new class of targeted agents. There are several BTK inhibitors, including ONO-WG-307, LFM-A13, dasatinib, CC-292, and PCI-32765 (ibrutinib), in preclinical and/or clinical development of which ibrutinib is currently in phase III trials. Recent clinical data suggest significant activity of ibrutinib as a first in class oral inhibitor of BTK. This review provides an overview of ongoing clinical studies of BTK inhibitors.
View details for DOI 10.1111/bjh.12573
View details for PubMedID 24111579
View details for PubMedCentralID PMC4444436