Noninvasive Prenatal Diagnosis of Trisomy 21 via droplets-based Circulating Fetal Cells Genetic Analysis. – KaryoDrop

Trisomy 21 is the most common chromosomal aberration that causes a birth defect widely known as Down Syndrome. Improvements in technology have allowed development of increasingly sensitive non-invasive tests to calculate the risk of trisomy 21. However, these tests can be prohibitively expensive and potential cases must be confirmed by invasive procedures: today, karyotyping of cells obtained by chorionic villus sampling and amniocentesis, which are both costly and carry a risk of complications such as miscarriage (0.5 -1%), is still the only diagnostic test available.

Our objective is to develop a simple to use, rapid and reliable non-invasive prenatal test that is significantly more rapid and cost-effective than current alternatives, and that will avoid the need for karyotyping in most cases. To achieve this goal, we will use a validated technology (ISET®) to enrich rare circulating fetal cells based on their size from maternal blood. Single cells will then be encapsulated within droplets in a simple microfluidic chip and linear isothermal amplification based fluorescent detection will be used to enumerate chromosome 21. By coupling rare cell enrichment and digital analysis of the chromosomic content of thousands of cells, we will obtain a simple, reliable and inexpensive system for diagnosis of trisomy 21, as well as other fetal aneuploidies.

Circulating fetal trophoblastic cells are extremely rare (a few per ml of blood), and do not present any known specific surface marker. This is why the deployment of assays based on circulating fetal cells are still hampered by the inability of current enrichment techniques to recover highly pure populations of fetal cells. Indeed, the quality of the diagnosis is directly linked to the capacity to count two or three chromosomes 21 per cell, but also to the number of fetal trophoblastic cells isolated and identified. We will overcome all the technical barriers limiting the use of circulating fetal cell-based assays in the clinic. Extra villous trophoblast isolation using ISET is efficient and rapid, recovering 30 to 50 from 10 mL of maternal blood, along with a few thousand maternal cells in liquid format. The enriched cells will be individually compartmentalized in picolitre volume droplets using a user-friendly, self-loading microfluidic chip, forming a two-dimensional droplet array that can be analyzed using fluorescence microscopy, allowing massively parallel analysis at the single-cell level. In each droplet, trophoblastic cells will be identified based on phenotypic markers (e.g. size) and genetic markers (e.g. presence of Y chromosome for male fetuses). Genetic markers will be identified and chromosomes (including chromosome 21) counted using, linear, isothermal padlock (PL) mediated rolling circle amplification (RCA).

Within the period of this funding, we expect to have demonstrated the feasibility of two major products. First, ISET, which has been developed and commercialized for oncology applications, will be adapted and optimized to the specific needs of the project, including the recovery of cells from the filter. Second, a microfluidic chip and a reagent kit will be developed to identified trophoblastic cells and enumerate their chromosomes of in a single-step closed-reaction format. Once all the technologies have been integrated, Karyodrop will be used in a clinical trial to test its efficacy and benchmarked against existing technology such as the cell-free fetal DNA screening and fetal karyotyping. As the KaryoDrop system is based on combining and adapting proven technologies in an innovative manner, we expected it to go rapidly to the market for Down syndrome screening, and its flexible nature will allow it to be adapted to develop diagnostic tools for a wide range of other genetic diseases.