Engineering technology for accessible precision therapeutics and diagnostics
By: Nicole Rose Blumenfeld
Publisher: [publisher not identified]
Published: 2020
Place of publication: [New York, N.Y.?]
Language: eng
Description: Over the last two decades, the concept of precision medicine has remained more of a promise than a reality. While there has been significant advancement in the field in terms of scientific discovery, precision medicine has yet to truly permeate standard clinical practice. There are a few individual examples, such as the treatment of breast cancer, in which the precision medicine approach has been ubiquitously adopted, but for most applications it remains exploratory. This barrier can arguably be attributed to the lack of accessible technology. That is, highly laborious, costly, and time-consuming methods that inhibit the integration of precision medicine techniques into the current clinical paradigm. In this dissertation, we aim to develop new technology, for both therapeutics and diagnostics, that would enable access to precision medicine by considering factors such as scalability, manufacturability, cost, turnaround time and integration. In Aim 1, we developed a direct tissue engineering approach to increase endogenous brown fat for the treatment of obesity.

This method capitalized on the use of brown adipose tissue (BAT), a highly metabolic tissue that expends energy via uncoupled respiration and has been shown to correlate with a lean phenotype and decreased risk of metabolic disease. Existing methods that seek to increase BAT mass include either the use of pharmacologic agents, which often exhibit detrimental off-target effects, or cold exposure, which is obviously unsustainable in practice. Cell therapies that involve the isolation of adipocyte progenitor cells have also been explored but are not easily scaled and are difficult to implement. Here, we developed a method to convert a patient’s own white adipose tissue (WAT) en masse to thermogenic BAT in a single ex vivo step, followed by reimplantation back into the patient. We demonstrated that this method, called exBAT, was able to convert full fragments of WAT to a BAT-like tissue, which sustained its phenotype up to 8-weeks after reimplantation in a mouse model. Further, allogeneic transplantation of exBAT in a diet-induced obesity mouse model exhibited a trend toward weight loss which should further be explored with additional dosing experiments.

This method is highly scalable, patient-specific, and easily implemented with current clinical practice and has the potential to provide a precise method to combat the growing challenge of obesity. In Aim 2, we shifted our focus to the development of a point-of-care (POC) diagnostic device for precision oncology. Here, we developed a device capable of performing a POC liquid biopsy for the detection of resistance mutations in non-small cell lung cancer (NSCLC). While liquid biopsies, which seek to identify tumor fragments in a patient’s blood, hold significant promise and advantages over traditional tissue biopsies, there are still several challenges including long turnaround time, high cost, and challenges with sensitivity. We sought to build a fully integrated device that can reduce the turnaround time for liquid biopsies from 2 weeks to one hour, enabling much higher throughput for important genotyping tests in NSCLC patients, and thereby enabling faster access to treatment. We demonstrated the ability to isolate plasma from undiluted whole blood at the POC, purify and concentrate circulating nucleic acids, and perform detection of low variant allelic frequency (VAF) mutations down to 1% in a microfluidic chip using a low-cost thermocycler.

The device was initially designed to identify the presence or absence of T790M mutations, an important gatekeeper mutation with a clear clinical use case that confers sensitivity toward specific tyrosine kinase inhibitors (TKIs) in advanced NSCLC patients. However, the device can be easily extrapolated toward any type of molecular profiling and has the potential to significantly increase access to precision oncology diagnostics and therapeutics. Finally, in Aim 3, we sought to develop a molecular diagnostic for detection of SARS-CoV-2 that would provide a qualitative result in less than 15 minutes at the POC. As the COVID-19 pandemic has continued to spread rapidly throughout the world, there is still an unmet need for high-throughput, ultrafast diagnostics that are sensitive, specific and accessible to all. To meet this challenge, we developed a molecular diagnostic that performs RT-PCR off of crude lysate from patient specimens in 15 minutes or less. To achieve this, we built upon previously demonstrated photothermal amplification techniques and extended its capabilities to perform ultrafast RT-PCR using a low-power infrared LED.

We also sought to integrate sample preparation methods for both nasopharyngeal (NP) swabs and saliva samples to eliminate the need for labor-intensive RNA extraction and enable full automation for POC testing. Testing of our device using purified SARS-CoV-2 RNA showed high sensitivity and a limit of detection down to 500 copies/mL. We also demonstrated preliminary results showing the ability to detect SARS-CoV-2 RNA in unpurified saliva and further testing of clinical specimens in the POC device is ongoing. With a significantly faster and low-cost test that maintains gold-standard sensitivity and specificity, this device has the potential to drastically increase testing throughput and help contain the spread of COVID-19. Underlying this work is the development of accessible technology for precision medicine. Aim 1 focuses on a simple, patient-specific tissue engineering approach to treating obesity, which is significantly more scalable than other cell and tissue engineering methods. Aim 2 demonstrates the ability to perform a highly sensitive liquid biopsy at the POC down to 1% VAF.

Aim 3 demonstrates a new POC diagnostic for SARS-CoV-2 that provides a result in less than 15 minutes. Both Aims 2 and 3 focus on the development of POC diagnostics and were designed to be user-friendly, scalable, and easily integrated into current clinical paradigms. In Appendix I, we expand the discussion of POC diagnostics and present a design framework based on cost and budget constraints that was used for the development of these POC devices. Overall, the sum of this work illustrates examples of thoughtful engineering for the development of impactful new technologies for precision therapeutics and diagnostics.
Notes: Department: Biomedical Engineering.

Thesis advisor: Samuel K. Sia.

Thesis (Ph.D.)--Columbia University, 2020.