Joseph Iwasyk

Inspired by the potential of the novel Proximity-Ligation Assay (PLA) to quantify proteins using qPCR, I performed research to investigate whether the PLA assay could be condensed from 3 complicated steps into a single-step, homogeneous assay.

Read Full Research Report PDF Here: "Integration of Proximity Assays with the peakPCR Platform to develop a Sensitive, Homogeneous assay for Point-of-Care Diagnostics”.

Abstract:

Proximity assays present a novel, sensitive biomolecular detection method that could be used to develop a homogeneous point-of-care diagnostic for proteins, toxins, or other biomarkers. Traditional diagnostic approaches for quantifying biomarkers rely on either antibodies (Lateral Flow Assays) or RT-qPCR to detect a target analyte. Shortcomings of these methods include lack of sensitivity, low multiplexing capability, lack of quantitative readout, lack of specificity for target molecules, or complexity of the method. With recent advances in antibody-oligonucleotide conjugate technology, researchers have combined the antigen-targeting power of antibodies with PCR amplification to create proximity-based assays (PLA and PEA) which present multiple advantages for biomarker detection and quantification. This project integrates the novel concept of proximity assays with a portable peakPCR device to enable a homogeneous assay capable of rapid, sensitive detection of proteins by qPCR. First, a PLA protocol from an existing kit was modified to develop a homogeneous PLA assay format in the peakPCR device for detection of serial dilutions of CRP analyte. Cq values from the qPCR amplification curves were compared to assess the assay sensitivity and performance. The first key result focused on the conversion of the multi-step PLA into a single, homogeneous assay by modification of the protocol and master mix components. Thus, a “1-step PLA” was successfully designed and integrated with the peakPCR cartridge, enabling all reagents to be preloaded into the wells. The second key group of results focused on characterizing and optimizing features of the proximity assay, including the impact of temperature, concentration of antibodies, and assay protocol on the sensitivity and dynamic range. A novel approach toward increasing the signal-to-noise ratio of the assay was explored by exploiting hypothesized differences in melting temperature between the signal and background. Notably, it was demonstrated that there is likely a critical temperature for the incubation/ligation or extension phase that enhances signal-to-noise. The final key result transitioned the project towards full independence from premade kits or reagents, using a proximity extension assay (PEA) with custom-designed antibody-oligo probes, PEA master mix, and protocol inspired from relevant literature. Preliminary results of the PEA indicate a reproducible, sensitive assay in the peakPCR device for detection of CRP. Overall, the results indicate that proximity assays can be performed in a homogeneous format with the peakPCR platform to detect proteins such as CRP in as little as 30 minutes. The estimated LOD for detecting CRP was about 500pM for PLA and as low as 0.17pM for PEA. Further development and optimization of the proximity assays with respect to the peakPCR cartridge could improve assay sensitivity, reduce time, enable multiplexing, and simplify the assay approach. Furthermore, product development steps could be taken to properly package and seal all reagents in the wells of the peakPCR cartridge. Thus, this project indicates significant potential for the integration of proximity assays with the peakPCR device to expand the platform’s functionality beyond sole quantification of nucleic acids towards a broader field of point-of-care diagnostics for quantifying proteins, toxin levels, hormones and other relevant biomarkers.

Key Results:

• Quantification of C-Reactive Protein (CRP) in Human Serum samples by qPCR on Diaxxo PCR

• No washing or transfer steps: Just add sample to wells of cartridge

• Dynamic Range between 1-100 ng/mL of CRP (single-well)

• Assay time of about 30-40 minutes

• Multi-Omic capability proven by quantifying RP gene & CRP simultaneously

• Room-temperature stability of the sealed cartridge out to 48hrs

Comparison Between State-of-the-Art & My Research:The state-of-the-art for protein quantification by qPCR is either a PLA or PEA. (Left) the 3 major phases for a PLA are shown as the State-of-the-Art. Each phase requires multiple pipetting and liquid handling steps, ultimately restricting the assay to a lab setting. (Right) The invention proposes a mixture that combines all components of a PLA or PEA and qPCR with a stabilizer. Moisture is removed and the mix can be stored at ambient temperature for subsequent use in a simple, ‘just-add-sample’ assay format

Eager to acquire funding for my research , I pursued a variety of grants in Switzerland. One of the main funding applications drafted was for the Innosuisse Grant | Requesting CHF 1.2M. While we made it past formal checks and to a final review, we did not ultimately receive funding. However, the applications was a highly informative lesson on complete end-to-end grant writing for me.

Innosuisse Grant Title: “DiaxxOmic: Integrated therapeutic drug monitoring & model-informed precision dosing platform for improved personalized medicine”

After performing extensive market research, I worked with the team from Diaxxo, Swiss TPH, ETH and clinical collaborators to organize an Innosuisse Grant Application for roughly CHF 1.2M. The application was close to 100 pages in length and contained extensive Business Plans, Value Proposition, Work Package descriptions, risk mitigation analysis, and budgeting. While unfortunately the grant was not awarded, the project was an excellent lesson in market research, planning, and partnership development. We maintained connections with the clinical partners afterward in case of future opportunities.For the proposal, DiaxxOmic proposed to combine drug monitoring & dose modeling into a single platform to reduce toxicity & increase treatment efficacy. Novel assay technology directly links patient data to a continuously updating model-informed precision dosing tool, enabling a data-driven approach to personalized medicine. The full Innosuisse Application PDF is available upon request.

Executive Summary for Innosuisse Application:

Antibiotics & drugs with a narrow therapeutic window require precision dosing to optimize patient outcome. Despite widespread use of therapeutic-drug monitoring (TDM), there is a high prevalence of suboptimal dosing that reduces therapeutic effect, increases toxicity, and exacerbates the growing antimicrobial resistance (AMR) problem. Suboptimal dosing is due to 1) a lack of standardized TDM assays and 2) limited capability for data interpretation. A lack of standardized TDM, stemming from issues of cost /complexity /expertise, yields discrepant results & prevents clinical consensus. Furthermore, existing TDM fails to directly integrate assay data with interpretation or modeling software, creating a barrier for patient-specific modeling. To achieve personalized medicine, there is a need for a standardized TDM assay linked directly to pharmacology insights.

Diaxxo is developing an accessible personalized medicine platform - diaxxOmic. The TDM assay uses a point-of-care, low-cost qPCR device & single-use cartridge to rapidly quantify drugs, proteins, & nucleic acids. Results are sent to a pharmacology dashboard for continuously updating model-informed precision dosing (MIPD). During this project, a proof-of-concept "VancoPod" cartridge will be developed to monitor vancomycin pharmacokinetics (PK), pharmacodynamics (PD) and nephrotoxicity with the goal of reducing Acute-Kidney Injury for patients & optimizing doses to fight AMR.

DiaxxOmic is radically innovative: using qPCR to quantify multiple PK/PD markers & link data directly to MIPD models on a single platform is entirely novel in clinical pharmacology. Accessibility of diaxxOmic enables its disruptive nature, ushering in the next-gen of personalized medicine. Commercialization will expand the platform throughout Switzerland and the EU/US to improve patient outcomes & reduce hospital burden. By adapting to monitor other drugs, diaxxOmic will boost local R&D to create a personalized medicine hub in Switzerland

DiaxxOmic Innosuisse Grant Partnership Overview:

Our team extensively researched and drafted a business case for the project in Point-of-need Therapeutic Drug Monitoring. The Executive Summary & Key Proof-of-Concept data is provided in the document below.

Download the diaxxOmic Executive Summary Here

Abstract:

Cancer treatment, antimicrobials, immunosuppressants, psychoactive drugs, organ transplantation and other procedures require a high degree of precision while dosing to optimize the patient outcome and minimize off target effects. While it has been proven that drug distribution & therapeutic impact are highly variable from patient-to-patient, current dosing strategies still are not personalized. The current “one-sizefits-all” model results in suboptimal therapeutic exposure, increased hospital burden, adverse side-effects, and death. Furthermore, existing TDM methods are complex with long turnaround-time, ultimately reducing availability at the point-of-need. Thus, there is a need for a novel, high-resolution point-of-need TDM tool to enable personalized medicine that improves treatment of patients and reduces hospital burden. diaxxo is developing a rapid, multi-omic Therapeutic Drug Monitoring (TDM) platform – called diaxxOmic - that will serve as a point-of-need tool for health care professionals and drug manufacturers to provide personalized therapeutics.

Proof-of-Concept:

To accomplish multi-omic, point-of need Therapeutic Drug Monitoring (TDM), the diaxxOmic platform combines the specificity of immunoassays with the signal amplification of real-time quantitative polymerase chain reaction (qPCR) to enable rapid, sensitive quantification of relevant biomarkers and drug concentrations for PK & PD analysis. For a proof-of-concept, over the last six months a team of researchers from the Swiss Tropical and Public Health Institute, ETH Zürich and Diaxxo AG have collaborated to develop a point-of-need platform to quantify host-derived inflammation biomarkers linked to infections. Numerous experiments have demonstrated technical feasibility of the platform, with preliminary data shown below for qPCR quantification of a key inflammation biomarker, the C-reactive protein (CRP)1, using Diaxxo’s point-of-care qPCR device. Notably, the assay requires no washing steps or transfer of reagents (homogeneous), is rapid with minimal hands-on time (performed in 30-minutes), sensitive and quantitative. Quantification of other inflammation biomarkers, such as IL-10, have been performed. Testing is in progress for quantifying antimicrobial and oncology drug concentrations in serum. The next phase of development will focus on integrating assays into a single cartridge for measuring key proteins, drugs, nucleic acids and metabolites linked to PK, PD and pharmacogenomics parameters