Several types of cancers (e.g. pancreatic and brain malignancies) remain extremely lethal despite the introduction of chemotherapy more than 70 years ago. The hallmark of all cancers are somatic mutations which cannot be corrected. In sharp contrast, epigenetic changes (essential contributors to tumorigenesis) are pharmacologically reversible. The ability to remedy detrimental epigenetic alterations are opening up new treatment avenues. Glioblastoma is an example where an epigenetic mark guides treatment. In epigenetics, the Chromatin ImmunoPrecipitation (ChIP) assay is used to determine the genomic location of a given protein and its modification(s) (e.g. histones, transcription factors or epigenetic modifying enzymes). The assay requires small samples of chromatin or DNA be fragmented reproducibly, without contamination or sample degradation. Unfortunately, current technologies suffer from epitope loss and irreproducibility due to time consuming and inconsistent shearing, making the ChIP assay unreliable for clinical use. We have overcome this bottleneck with PIXUL (PIXelated ULtrasound). The device uses ultrasound transducers that treat samples equally in every well of a 96-well microplate. All samples are processed simultaneously and reproducibly in a few minutes (a minimum 10x improvement over existing technologies). With PIXUL, cells are grown in 96-well microplates, and then cross- linked and harvested in situ (15min) and sheared in 2 minutes. Similarly, dozens of tissue samples can also be quickly processed in parallel, all in one microplate (for e.g., toxicity studies). No other commercial system is capable of this. The prepared microplate may further be used directly in the assay, eliminating the need for time consuming manual transfer steps. PIXUL is automated and requires little training. Our workflow breakthrough is a major step towards fast and accurate assays (critically important for drug discovery and personalized medicine), making PIXUL a valuable sample preparation tool in epigenetics, and also in NGS and potentially a variety of other analytical applications (e.g., proteomics, metabolomics). We have excellent results with our current lab prototype (ChIP-qPCR and ChIP-Seq results), and ChIP assay signals are stronger and more reproducible than from commercial competitors. The overarching goal of this project is to advance PIXUL to a commercial prototype for end-user evaluation and feedback.
Two Aims are proposed.
In Aim#1 we will develop a custom microplate that will decrease shearing time and heat generation. The main goal here is maximize epitope integrity.
In Aim#2 we will fabricate commercial prototype PIXUL devices for evaluation and feedback. This information will be used to manufacture market-ready PIXUL instrument. Major epigenetic companies have stated that PIXUL is a game changer and expressed interest in partnering with us to market it.
Personalized medicine requires fast and easy tools to detect, diagnose and treat diseases. Currently, there is a major bottleneck in pre-analytical sample preparations. Our high throughput solution will reduce sample preparation times by an order of magnitude, and improve reproducibility dramatically, an essential step towards realizing the goal of personalized medicine.
| Bomsztyk, Karol; Denisenko, Oleg; Wang, Yuliang (2018) DNA methylation yields epigenetic clues into the diabetic nephropathy of Pima Indians. Kidney Int 93:1272-1275 |
