Unmet Need
It is estimated that, in 2020, 1,806,590 people were diagnosed with cancer and 606,520 people will die from the disease (see NCI). Nevertheless, there are a limited number of molecular diagnostic methods that are routinely deployed in clinical practice. Although the amount of ctDNA is dependent on tumor stage, size, tumor type, among other factors, studies have shown that, using 4.0-7.5 mL of plasma, ctDNA is undetectable in nearly 50% of stage I cancer patients. While the sensitivity of detecting ctDNA in early-stage cancers is woefully low, there is agreement that analysis of ctDNA provides an opportunity to noninvasively detect all stages of cancer, track tumor burden, and monitor response to treatment. The limiting factor in using blood based liquid biopsies for early cancer detection and screening is the volume of blood required to extract a measurable number of ctDNA copies.
An apheresis machine is a device that can draw whole blood, separate the blood components, and infuse the blood components back into the individual. This device provides the opportunity to screen large volumes of plasma without extracting it from the body. However, current DNA capture technologies requires the plasma to be chemically treated before the ctDNA can be capture. The dCas9 capture system is the first technology designed to capture ctDNA from flowing unaltered plasma.
Technology Overview
Inventors at Johns Hopkins University have developed a method that uses dCas9 to capture ctDNA from flowing unaltered plasma. Since the dCas9 capture system does not require the plasma to be chemically treated, the screened plasma can be returned to the patient, which allows for larger volumes of plasma to be screened than can be extracted. Furthermore, since only ctDNA is selectively captured, this method allows for other analytical methods – such as proteomics – to be performed on the same aliquot of plasma.
Stage of Development
The inventors have conducted proof-of-concept experiments by capturing the BRAF (T1799A) DNA mutation spiked into blood plasma. The number of captured mutant BRAF DNA was quantified using qPCR. To test the performance of the dCas9 capture system, a range of mutant allele fractions (MAF) (MAF 35%/150copies, MAF 10%/40 copies, and MAF 1%/4 copies) were captured using the commercially available Zymo cfDNA extraction kit and the dCas9 capture system. Compared to the results of the Zymo cfDNA extraction kit, the dCas9 capture system was able to capture, on average, between 101% and 117% of the mutant BRAF DNA. Furthermore, the dCas9 system enriched the mutant BRAF DNA on average between 1.8 and 3.3-fold higher in comparison to the off-target capture of ACTB. In a similar proof-of-concept experiment, using sgRNA specific to pro-viral HIV and 16S DNA from Staphylococcus aureus, the dCas9 capture system showed capture enrichment of 3.9 and 6.5-fold more than the nonspecific control ACTB DNA. This demonstrates the versatility of the technology beyond cancer detection in the area of infectious disease. Furthermore, the dCas9 capture system, with its current setup, cost less than the commercially available Zymo cfDNA extraction kit ($5.30 with dCas9 capture vs. $6.54 with Zymo cfDNA kit, per sample).
In an experiment to show that the dCas9 system can capture ctDNA in flowing unaltered plasma, 6ml of plasma at MAF 10%, was pumped across the dCas9 system at 6ml/min for 5min, 10min, 20min, 40min, and 80min. It was shown that the number of dCas9 captured mutant BRAF DNA copies doubled with time (slope = -1.035 Ct) and is highly linear (R2= 0.874). To increase the sensitivity of the dCas9 capture system, the inventors are currently optimizing a dCas9 specific microfluidic device.
Publications:
1. ACS Appl. Mater. Interfaces 2022, 14, 21, 24113–24121, - https://doi.org/10.1021/acsami.2c03186
